Common and rare genetic variations associated with common variable immunodeficiency (CVID) and methods of use thereof for the treatment and diagnosis of the same

Information

  • Patent Grant
  • 10519501
  • Patent Number
    10,519,501
  • Date Filed
    Tuesday, August 18, 2015
    8 years ago
  • Date Issued
    Tuesday, December 31, 2019
    4 years ago
Abstract
Compositions and methods useful for the diagnosis and treatment of common variable immunodeficiency are disclosed.
Description

This invention relates to the fields of genetics and the diagnosis of common variable immunodeficiency (CVID). More specifically, the invention provides compositions and methods useful for the diagnosis and treatment of CVID.


BACKGROUND OF THE INVENTION

Several publications and patent documents are cited through the specification in order to describe the state of the art to which this invention pertains. Each of these citations is incorporated herein by reference as though set forth in full.


Common variable immunodeficiency (CVID) disorders are manifested by insufficient quantity and quality of immunoglobulin leading to susceptibility to bacterial infections1. CVID is considered to be a primary immunodeficiency disease (PIDD) as it is believed to result from intrinsic deficits affecting immunological functions. CVID is heterogeneous in presentation, presenting either early or later in life and associated with a group of known comorbidities2. Efforts to subcategorize CVID in order to predict outcomes and comorbid conditions both clinically and by immunologic phenotypes are ongoing3. BAFFR4, TACI5,6,7, and certain HLA haplotypes8,9 have been identified as potential gene candidates for susceptibility to CVID. ICOS10,11, CD8112, CD1913,14, CD2015, harbor disease causing mutations that presently explain only a small percentage of cases. The heterogeneous presentations of patients with CVID make management of these conditions difficult and searches for novel genetic predictors of disease causation or susceptibility have been of limited success.


SUMMARY OF THE INVENTION

In accordance with the present invention, methods are provided for the diagnosis and treatment of CVID. An exemplary method entails detecting the presence of at least one CVID associated CNV in a target polynucleotide wherein if said CNV(s) is/are present, said patient has an increased risk for developing CVID.


In one aspect of the present invention, a method for detecting a propensity for developing common variable immunodeficiency disorder (CVID) in a patient in need thereof is provided. An exemplary method entails detecting the presence of at least one SNP containing nucleic acid in a target polynucleotide, said SNP being informative of a the presence of a CVID associated copy number variation (CNV), wherein if said SNP is present, said patient has an increased risk for developing CVID, wherein said SNP containing nucleic acid is provided in Tables 1 to 5. In one embodiment, at least 1, 2, 3, 4, 5, 10, 15, 20 or all of the SNPs provided herein are detected.


In another embodiment of the invention a method for identifying agents which alter immune cell function or signaling is provided. Such a method comprises providing cells expressing at least one nucleic acid comprising the CVID associated CNVs of the invention, (step a); providing cells which express the cognate wild type sequences which lack the CNV (step b); contacting the cells from each sample with a test agent and analyzing whether said agent alters immune signaling or function of cells of step a) relative to those of step b), thereby identifying agents which alter immune cell signaling or function. Methods of treating CVID patients via administration of test agents identified using the methods described herein are also encompassed by the present invention. The invention also provides at least one isolated CVID related SNP-containing nucleic acid selected from the group listed in Tables 1 to 5. In one embodiment, a multiplex SNP panel containing all of the informative SNPs from the tables provided herein is disclosed. Such SNP containing nucleic acids which indicate the presence of CVID associated CNV(s) may optionally be contained in a suitable expression vector for expression in immune cells. Alternatively, they may be immobilized on a solid support. In yet another alternative embodiment, the panel may be provided in silico.


According to yet another aspect of the present invention, there is provided a method of treating CVID in a patient determined to have at least one prescribed single nucleotide polymorphism indicative of the presence of a CVID-associated copy number variation, as described hereinbelow, by administering to the patient a therapeutically effective amount of an agent useful for modulating immune function. This method provides a test and treat paradigm, whereby a patient's genetic profile is used to personalize treatment with therapeutics targeted towards specific gastrointestinal defects found in individuals exhibiting CVID. Such a test and treat model may benefit up to 50% of patients with CVID with greater efficacy and fewer side effects than non-personalized treatment.





BRIEF DESCRIPTION OF THE DRAWINGS


FIGS. 1A-1C. SNP based case:control significance is shown in negative log base 10 genome wide for Discovery (FIG. 1A) and Replication (FIG. 1B and FIG. 1C) inclusive CVID cohorts. Subsequently, CVID cases with specific disease subphenotypes were compared to CVID cases without the subphenotype. The single SNP tests are shown as single points. Multiple neighboring SNPs of similar significance boost confidence in the association as shown in the strong peak on 6p22.1-p21.32 of the Discovery case:control cohort. Conversely the median significance P value is kept low by minimizing population stratification which minimizes the genomic inflation factor.



FIGS. 2A-2B. Frequency of CNV in cases (FIG. 2A) and controls (FIG. 2B). CNVs are called on a single sample basis with deletions and duplications in specific genomic regions based on the genotype and intensity signal of contiguous SNPs. These single sample CNV profiles are plotted as a SNP-based statistic to allow for SNP-based association testing. Lastly, neighboring SNPs with similar significance define a CNVR. Light grey indicates case deletion; Dark grey indicates case duplication; Black indicates control deletion; Medium grey indicates control duplication.



FIG. 3. Duplication of ORC4L Found Exclusive to 15 CVID Cases. Vertical lines indicate the SNP probe coverage. Rectangles delineate regions of copy number variation in individual cases. Three exons of ORC4L are shown duplicated in 15 cases and 0 controls. It is likely that the duplications could extend to a larger region of ORC4L based on coverage.



FIGS. 4A-4K. CNV Validation with Illumina Intensity Data Review and Independent Array Technology, Affymetrix 2.7M



FIG. 5. The core diagnosis of CVID has many clinical progressions with varying frequency. The height and size of subphenotypes signify the frequency of the specific progression. Given significant SNP genotype associations for each CVID subphenotype progression vs. CVID patients without the progression, prediction can be made which may improve clinical outcome.



FIG. 6. Schematic Representation of Observed Data with Classification Divided by a Hyperplane. Binary SVM selects a hyperplane (bold line) that maximizes the width of the ‘gap’ (margin) between the two classes. The hyperplane is specified by ‘boundary’ training instances, called support vectors shown with circles. New cases are classified based on which side of the hyperplane they are mapped.



FIG. 7. SVM Model for CVID Case vs. individual not affected by CVID.



FIG. 8. SVM Model for CVID case with Bronchiectasis versus CVID case without Bronchiectasis.



FIG. 9. SVM Model for CVID case with OSAI (organ specific autoimmunity) versus CVID case without OSAI.



FIG. 10. Pedigree of a family with autosomal dominant common variable immunodeficiency



FIG. 11. SVM Algorithm Hyperplane.



FIG. 12. S535N mutation in IRF2BP2.



FIG. 13. RT-PCR or IRF2BP2.





DETAILED DESCRIPTION OF THE INVENTION

Common variable immunodeficiency disorders (CVID) are a group of uncommon heterogeneous immune defects characterized by hypogammaglobulinemia, failure of antibody production, susceptibility to bacterial infections and an array of serious comorbidities. To address the underlying immunopathogeneses of CVID, we conducted the first genome-wide association study of patients with CVID. 363 patients were genotyped with 610,000 SNPs. Due to the relative rarity of this group of disorders, the study cohort was recruited at four sites. Patients were randomly divided into a discovery cohort of 179 cases in comparison with 1,917 disease-free controls and a replication cohort of 109 cases in comparison with 1,114 controls, controlled for population stratification. Our analyses detected strong association with the MHC region and uncovered a novel association with a cluster of ADAM genes that was replicated in the independent case cohort. Analysis of the same cases for copy number variation (CNV) revealed 16 disease-associated deletions and duplications, as well as numerous unique rare intraexonic deletions and duplications suggesting multiple novel genetic etiologies for individual CVID cases. Analysis of CVID comorbidities identified significantly associated SNP genotypes with the major CVID clinical phenotypes. Taken together, our integrative genome-wide analysis of SNP genotypes and CNVs has uncovered multiple novel susceptibility loci for CVID, both common and rare, consistent with the highly heterogeneous nature of CVID. These results may allow for improved diagnosis of CVID, prediction of the CVID clinical phenotypes, and mechanistic insights into immune diseases based upon these unique genetic variations.


I. Definitions

For purposes of the present invention, “a” or “an” entity refers to one or more of that entity; for example, “a cDNA” refers to one or more cDNA or at least one cDNA. As such, the terms “a” or “an,” “one or more” and “at least one” can be used interchangeably herein. It is also noted that the terms “comprising,” “including,” and “having” can be used interchangeably. Furthermore, a compound “selected from the group consisting of” refers to one or more of the compounds in the list that follows, including mixtures (i.e. combinations) of two or more of the compounds. According to the present invention, an isolated, or biologically pure molecule is a compound that has been removed from its natural milieu. As such, “isolated” and “biologically pure” do not necessarily reflect the extent to which the compound has been purified. An isolated compound of the present invention can be obtained from its natural source, can be produced using laboratory synthetic techniques or can be produced by any such chemical synthetic route.


The term “genetic alteration” as used herein refers to a change from the wild-type or reference sequence of one or more nucleic acid molecules. Genetic alterations include without limitation, base pair substitutions, additions and deletions of at least one nucleotide from a nucleic acid molecule of known sequence.


A “single nucleotide polymorphism (SNP)” refers to a change in which a single base in the DNA differs from the usual base at that position. These single base changes are called SNPs or “snips.” Millions of SNP's have been cataloged in the human genome. Some SNPs such as that which causes sickle cell are responsible for disease. Other SNPs are normal variations in the genome. Frequently SNPs are used to “mark” particular genes and genetic regions, particularly those containing CNVs for example.


A “copy number variation (CNV)” refers to the number of copies of a particular gene or segment thereof in the genome of an individual. CNVs represent a major genetic component of human phenotypic diversity. Susceptibility to genetic disorders is known to be associated not only with single nucleotide polymorphisms (SNP), but also with structural and other genetic variations, including CNVs. A CNV represents a copy number change involving a DNA fragment that is ˜1 kilobases (kb) or larger. CNVs described herein do not include those variants that arise from the insertion/deletion of transposable elements (e.g., ˜6-kb KpnI repeats) to minimize the complexity of future CNV analyses. The term CNV therefore encompasses previously introduced terms such as large-scale copy number variants (LCVs), copy number polymorphisms (CNPs), and intermediate-sized variants (ISVs), but not retroposon insertions. The terminology “duplication-containing CNV” is also used herein below consistent with the CNV definition provided.


“CVID-associated SNP” or “CVID-associated specific marker” is a SNP or marker which is associated with an increased or decreased risk of developing CVID not found normal patients who do not have this disease. Such markers may include but are not limited to nucleic acids, proteins encoded thereby, or other small molecules. Thus, the phrase “CVID-associated SNP containing nucleic acid” is encompassed by the above description.


The term “solid matrix” as used herein refers to any format, such as beads, microparticles, a microarray, the surface of a microtitration well or a test tube, a dipstick or a filter. The material of the matrix may be polystyrene, cellulose, latex, nitrocellulose, nylon, polyacrylamide, dextran or agarose.


The phrase “consisting essentially of” when referring to a particular nucleotide or amino acid means a sequence having the properties of a given SEQ ID NO. For example, when used in reference to an amino acid sequence, the phrase includes the sequence per se and molecular modifications that would not affect the functional and novel characteristics of the sequence.


The phrase “partial informative CNV” is used herein to refer to a nucleic acid that hybridizes to sequences comprising a duplication on a chromosome however, the partial informative CNV may not be identical to the duplication, rather, the CNV may correspond to only a portion of the duplication, but yet is still informative for the presence of the same.


“Target nucleic acid” as used herein refers to a previously defined region of a nucleic acid present in a complex nucleic acid mixture wherein the defined wild-type region contains at least one known nucleotide variation which may or may not be associated with CVID. The nucleic acid molecule may be isolated from a natural source by cDNA cloning or subtractive hybridization or synthesized manually. The nucleic acid molecule may be synthesized manually by the triester synthetic method or by using an automated DNA synthesizer. With regard to nucleic acids used in the invention, the term “isolated nucleic acid” is sometimes employed. This term, when applied to DNA, refers to a DNA molecule that is separated from sequences with which it is immediately contiguous (in the 5′ and 3′ directions) in the naturally occurring genome of the organism from which it was derived. For example, the “isolated nucleic acid” may comprise a DNA molecule inserted into a vector, such as a plasmid or virus vector, or integrated into the genomic DNA of a prokaryote or eukaryote. An “isolated nucleic acid molecule” may also comprise a cDNA molecule. An isolated nucleic acid molecule inserted into a vector is also sometimes referred to herein as a recombinant nucleic acid molecule.


With respect to RNA molecules, the term “isolated nucleic acid” primarily refers to an RNA molecule encoded by an isolated DNA molecule as defined above. Alternatively, the term may refer to an RNA molecule that has been sufficiently separated from RNA molecules with which it would be associated in its natural state (i.e., in cells or tissues), such that it exists in a “substantially pure” form.


By the use of the term “enriched” in reference to nucleic acid it is meant that the specific DNA or RNA sequence constitutes a significantly higher fraction (2-5 fold) of the total DNA or RNA present in the cells or solution of interest than in normal cells or in the cells from which the sequence was taken. This could be caused by a person by preferential reduction in the amount of other DNA or RNA present, or by a preferential increase in the amount of the specific DNA or RNA sequence, or by a combination of the two. However, it should be noted that “enriched” does not imply that there are no other DNA or RNA sequences present, just that the relative amount of the sequence of interest has been significantly increased.


It is also advantageous for some purposes that a nucleotide sequence be in purified form. The term “purified” in reference to nucleic acid does not require absolute purity (such as a homogeneous preparation); instead, it represents an indication that the sequence is relatively purer than in the natural environment (compared to the natural level, this level should be at least 2-5 fold greater, e.g., in terms of mg/ml). Individual clones isolated from a cDNA library may be purified to electrophoretic homogeneity. The claimed DNA molecules obtained from these clones can be obtained directly from total DNA or from total RNA. The cDNA clones are not naturally occurring, but rather are preferably obtained via manipulation of a partially purified naturally occurring substance (messenger RNA). The construction of a cDNA library from mRNA involves the creation of a synthetic substance (cDNA) and pure individual cDNA clones can be isolated from the synthetic library by clonal selection of the cells carrying the cDNA library. Thus, the process which includes the construction of a cDNA library from mRNA and isolation of distinct cDNA clones yields an approximately 10−6-fold purification of the native message. Thus, purification of at least one order of magnitude, preferably two or three orders, and more preferably four or five orders of magnitude is expressly contemplated. Thus the term “substantially pure” refers to a preparation comprising at least 50-60% by weight the compound of interest (e.g., nucleic acid, oligonucleotide, etc.). More preferably, the preparation comprises at least 75% by weight, and most preferably 90-99% by weight, the compound of interest. Purity is measured by methods appropriate for the compound of interest.


The term “complementary” describes two nucleotides that can form multiple favorable interactions with one another. For example, adenine is complementary to thymine as they can form two hydrogen bonds. Similarly, guanine and cytosine are complementary since they can form three hydrogen bonds. Thus if a nucleic acid sequence contains the following sequence of bases, thymine, adenine, guanine and cytosine, a “complement” of this nucleic acid molecule would be a molecule containing adenine in the place of thymine, thymine in the place of adenine, cytosine in the place of guanine, and guanine in the place of cytosine. Because the complement can contain a nucleic acid sequence that forms optimal interactions with the parent nucleic acid molecule, such a complement can bind with high affinity to its parent molecule.


With respect to single stranded nucleic acids, particularly oligonucleotides, the term “specifically hybridizing” refers to the association between two single-stranded nucleotide molecules of sufficiently complementary sequence to permit such hybridization under pre-determined conditions generally used in the art (sometimes termed “substantially complementary”). In particular, the term refers to hybridization of an oligonucleotide with a substantially complementary sequence contained within a single-stranded DNA or RNA molecule of the invention, to the substantial exclusion of hybridization of the oligonucleotide with single-stranded nucleic acids of non-complementary sequence. For example, specific hybridization can refer to a sequence which hybridizes to any CVID specific marker gene or nucleic acid, but does not hybridize to other nucleotides. Also polynucleotide which “specifically hybridizes” may hybridize only to a single specific marker, such as an CVID-specific marker shown in the Tables contained herein. Appropriate conditions enabling specific hybridization of single stranded nucleic acid molecules of varying complementarity are well known in the art.


For instance, one common formula for calculating the stringency conditions required to achieve hybridization between nucleic acid molecules of a specified sequence homology is set forth below (Sambrook et al., Molecular Cloning, Cold Spring Harbor Laboratory (1989):

Tm=81.5° C.+16.6 Log[Na+]+0.41(% G+C)−0.63 (% formamide)−600/# bp in duplex


As an illustration of the above formula, using [Na+]=[0.368] and 50% formamide, with GC content of 42% and an average probe size of 200 bases, the Tm is 57° C. The Tm of a DNA duplex decreases by 1-1.5° C. with every 1% decrease in homology. Thus, targets with greater than about 75% sequence identity would be observed using a hybridization temperature of 42° C.


The stringency of the hybridization and wash depend primarily on the salt concentration and temperature of the solutions. In general, to maximize the rate of annealing of the probe with its target, the hybridization is usually carried out at salt and temperature conditions that are 20-25° C. below the calculated Tm of the hybrid. Wash conditions should be as stringent as possible for the degree of identity of the probe for the target. In general, wash conditions are selected to be approximately 12-20° C. below the Tm of the hybrid. In regards to the nucleic acids of the current invention, a moderate stringency hybridization is defined as hybridization in 6×SSC, 5×Denhardt's solution, 0.5% SDS and 100 μg/ml denatured salmon sperm DNA at 42° C., and washed in 2×SSC and 0.5% SDS at 55° C. for 15 minutes. A high stringency hybridization is defined as hybridization in 6×SSC, 5×Denhardt's solution, 0.5% SDS and 100 μg/ml denatured salmon sperm DNA at 42° C., and washed in 1×SSC and 0.5% SDS at 65° C. for 15 minutes. A very high stringency hybridization is defined as hybridization in 6×SSC, 5×Denhardt's solution, 0.5% SDS and 100 μg/ml denatured salmon sperm DNA at 42° C., and washed in 0.1×SSC and 0.5% SDS at 65° C. for 15 minutes.


The term “oligonucleotide,” as used herein is defined as a nucleic acid molecule comprised of two or more ribo- or deoxyribonucleotides, preferably more than three. The exact size of the oligonucleotide will depend on various factors and on the particular application and use of the oligonucleotide. Oligonucleotides, which include probes and primers, can be any length from 3 nucleotides to the full length of the nucleic acid molecule, and explicitly include every possible number of contiguous nucleic acids from 3 through the full length of the polynucleotide. Preferably, oligonucleotides are at least about 10 nucleotides in length, more preferably at least 15 nucleotides in length, more preferably at least about 20 nucleotides in length.


The term “probe” as used herein refers to an oligonucleotide, polynucleotide or nucleic acid, either RNA or DNA, whether occurring naturally as in a purified restriction enzyme digest or produced synthetically, which is capable of annealing with or specifically hybridizing to a nucleic acid with sequences complementary to the probe. A probe may be either single-stranded or double-stranded. The exact length of the probe will depend upon many factors, including temperature, source of probe and use of the method. For example, for diagnostic applications, depending on the complexity of the target sequence, the oligonucleotide probe typically contains 15-25 or more nucleotides, although it may contain fewer nucleotides. The probes herein are selected to be complementary to different strands of a particular target nucleic acid sequence. This means that the probes must be sufficiently complementary so as to be able to “specifically hybridize” or anneal with their respective target strands under a set of pre-determined conditions. Therefore, the probe sequence need not reflect the exact complementary sequence of the target. For example, a non-complementary nucleotide fragment may be attached to the 5′ or 3′ end of the probe, with the remainder of the probe sequence being complementary to the target strand. Alternatively, non-complementary bases or longer sequences can be interspersed into the probe, provided that the probe sequence has sufficient complementarity with the sequence of the target nucleic acid to anneal therewith specifically.


The term “primer” as used herein refers to an oligonucleotide, either RNA or DNA, either single-stranded or double-stranded, either derived from a biological system, generated by restriction enzyme digestion, or produced synthetically which, when placed in the proper environment, is able to functionally act as an initiator of template-dependent nucleic acid synthesis. When presented with an appropriate nucleic acid template, suitable nucleoside triphosphate precursors of nucleic acids, a polymerase enzyme, suitable cofactors and conditions such as a suitable temperature and pH, the primer may be extended at its 3′ terminus by the addition of nucleotides by the action of a polymerase or similar activity to yield a primer extension product. The primer may vary in length depending on the particular conditions and requirement of the application. For example, in diagnostic applications, the oligonucleotide primer is typically 15-25 or more nucleotides in length. The primer must be of sufficient complementarity to the desired template to prime the synthesis of the desired extension product, that is, to be able anneal with the desired template strand in a manner sufficient to provide the 3′ hydroxyl moiety of the primer in appropriate juxtaposition for use in the initiation of synthesis by a polymerase or similar enzyme. It is not required that the primer sequence represent an exact complement of the desired template. For example, a non-complementary nucleotide sequence may be attached to the 5′ end of an otherwise complementary primer. Alternatively, non-complementary bases may be interspersed within the oligonucleotide primer sequence, provided that the primer sequence has sufficient complementarity with the sequence of the desired template strand to functionally provide a template-primer complex for the synthesis of the extension product.


Polymerase chain reaction (PCR) has been described in U.S. Pat. Nos. 4,683,195, 4,800,195, and 4,965,188, the entire disclosures of which are incorporated by reference herein.


The term “vector” relates to a single or double stranded circular nucleic acid molecule that can be infected, transfected or transformed into cells and replicate independently or within the host cell genome. A circular double stranded nucleic acid molecule can be cut and thereby linearized upon treatment with restriction enzymes. An assortment of vectors, restriction enzymes, and the knowledge of the nucleotide sequences that are targeted by restriction enzymes are readily available to those skilled in the art, and include any replicon, such as a plasmid, cosmid, bacmid, phage or virus, to which another genetic sequence or element (either DNA or RNA) may be attached so as to bring about the replication of the attached sequence or element. A nucleic acid molecule of the invention can be inserted into a vector by cutting the vector with restriction enzymes and ligating the two pieces together.


Many techniques are available to those skilled in the art to facilitate transformation, transfection, or transduction of the expression construct into a prokaryotic or eukaryotic organism. The terms “transformation”, “transfection”, and “transduction” refer to methods of inserting a nucleic acid and/or expression construct into a cell or host organism. These methods involve a variety of techniques, such as treating the cells with high concentrations of salt, an electric field, or detergent, to render the host cell outer membrane or wall permeable to nucleic acid molecules of interest, microinjection, PEG-fusion, and the like.


The term “promoter element” describes a nucleotide sequence that is incorporated into a vector that, once inside an appropriate cell, can facilitate transcription factor and/or polymerase binding and subsequent transcription of portions of the vector DNA into mRNA. In one embodiment, the promoter element of the present invention precedes the 5′ end of the CVID specific marker nucleic acid molecule such that the latter is transcribed into mRNA. Host cell machinery then translates mRNA into a polypeptide.


Those skilled in the art will recognize that a nucleic acid vector can contain nucleic acid elements other than the promoter element and the CVID specific marker nucleic acid molecule. These other nucleic acid elements include, but are not limited to, origins of replication, ribosomal binding sites, nucleic acid sequences encoding drug resistance enzymes or amino acid metabolic enzymes, and nucleic acid sequences encoding secretion signals, localization signals, or signals useful for polypeptide purification.


A “replicon” is any genetic element, for example, a plasmid, cosmid, bacmid, plastid, phage or virus, that is capable of replication largely under its own control. A replicon may be either RNA or DNA and may be single or double stranded.


An “expression operon” refers to a nucleic acid segment that may possess transcriptional and translational control sequences, such as promoters, enhancers, translational start signals (e.g., ATG or AUG codons), polyadenylation signals, terminators, and the like, and which facilitate the expression of a polypeptide coding sequence in a host cell or organism.


As used herein, the terms “reporter,” “reporter system”, “reporter gene,” or “reporter gene product” shall mean an operative genetic system in which a nucleic acid comprises a gene that encodes a product that when expressed produces a reporter signal that is a readily measurable, e.g., by biological assay, immunoassay, radio immunoassay, or by colorimetric, fluorogenic, chemiluminescent or other methods. The nucleic acid may be either RNA or DNA, linear or circular, single or double stranded, antisense or sense polarity, and is operatively linked to the necessary control elements for the expression of the reporter gene product. The required control elements will vary according to the nature of the reporter system and whether the reporter gene is in the form of DNA or RNA, but may include, but not be limited to, such elements as promoters, enhancers, translational control sequences, poly A addition signals, transcriptional termination signals and the like.


The introduced nucleic acid may or may not be integrated (covalently linked) into nucleic acid of the recipient cell or organism. In bacterial, yeast, plant and mammalian cells, for example, the introduced nucleic acid may be maintained as an episomal element or independent replicon such as a plasmid. Alternatively, the introduced nucleic acid may become integrated into the nucleic acid of the recipient cell or organism and be stably maintained in that cell or organism and further passed on or inherited to progeny cells or organisms of the recipient cell or organism. Finally, the introduced nucleic acid may exist in the recipient cell or host organism only transiently.


The term “selectable marker gene” refers to a gene that when expressed confers a selectable phenotype, such as antibiotic resistance, on a transformed cell.


The term “operably linked” means that the regulatory sequences necessary for expression of the coding sequence are placed in the DNA molecule in the appropriate positions relative to the coding sequence so as to effect expression of the coding sequence. This same definition is sometimes applied to the arrangement of transcription units and other transcription control elements (e.g. enhancers) in an expression vector.


The terms “recombinant organism” or “transgenic organism” refer to organisms which have a new combination of genes or nucleic acid molecules. A new combination of genes or nucleic acid molecules can be introduced into an organism using a wide array of nucleic acid manipulation techniques available to those skilled in the art. The term “organism” relates to any living being comprised of a least one cell. An organism can be as simple as one eukaryotic cell or as complex as a mammal. Therefore, the phrase “a recombinant organism” encompasses a recombinant cell, as well as eukaryotic and prokaryotic organism.


The term “isolated protein” or “isolated and purified protein” is sometimes used herein. This term refers primarily to a protein produced by expression of an isolated nucleic acid molecule of the invention. Alternatively, this term may refer to a protein that has been sufficiently separated from other proteins with which it would naturally be associated, so as to exist in “substantially pure” form. “Isolated” is not meant to exclude artificial or synthetic mixtures with other compounds or materials, or the presence of impurities that do not interfere with the fundamental activity, and that may be present, for example, due to incomplete purification, addition of stabilizers, or compounding into, for example, immunogenic preparations or pharmaceutically acceptable preparations.


A “specific binding pair” comprises a specific binding member (sbm) and a binding partner (bp) which have a particular specificity for each other and which in normal conditions bind to each other in preference to other molecules. Examples of specific binding pairs are antigens and antibodies, ligands and receptors and complementary nucleotide sequences. The skilled person is aware of many other examples. Further, the term “specific binding pair” is also applicable where either or both of the specific binding member and the binding partner comprise a part of a large molecule. In embodiments in which the specific binding pair comprises nucleic acid sequences, they will be of a length to hybridize to each other under conditions of the assay, preferably greater than 10 nucleotides long, more preferably greater than 15 or 20 nucleotides long.


“Sample” or “patient sample” or “biological sample” generally refers to a sample which may be tested for a particular molecule, preferably a CVID specific marker molecule, such as a marker described hereinbelow. Samples may include but are not limited to cells, body fluids, including blood, serum, plasma, cerebral spinal fluid, urine, gastric lavage, saliva, tears, pleural fluid and the like.


The terms “agent” and “compound” are used interchangeably herein and denote a chemical compound, a mixture of chemical compounds, a biological macromolecule, or an extract made from biological materials such as bacteria, plants, fungi, or animal (particularly mammalian) cells or tissues. Biological macromolecules include siRNA, shRNA, antisense oligonucleotides, peptides, peptide/DNA complexes, and any nucleic acid based molecule which exhibits the capacity to modulate the activity of the CNV or SNP-containing nucleic acids described herein or their encoded proteins. Agents and compounds may also be referred to as “test agents” or “test compounds” which are evaluated for potential biological activity by inclusion in screening assays described hereinbelow.


The term “modulate” as used herein refers to increasing/promoting or decreasing/inhibiting a particular cellular, biological or signaling function associated with the normal activities of the genetic alteration containing molecules described herein or the proteins encoded thereby. For example, the term modulate refers to the ability of a test compound or test agent to interfere with signaling or activity of a gene or protein of the present invention. Alternatively, the term may refer to augmentation of the activity of such a protein.


II. Methods of Using CVID-Associated CNVs and/or SNPs for Diagnosing a Propensity for the Development of CVID

The present invention provides methods of diagnosing CVID in a patient or methods for identifying a patient having an increased risk of developing CVID. Diagnosis, as used herein, includes not only the initial identification of CVID-associated with the genetic alterations described herein in a patient but confirmatory testing, or screening in patients who have previously been identified as having or likely to have CVID. The methods include the steps of providing a biological sample from the patient, measuring the amount of particular sets (e.g., those with the highest statistical significance), 1, 2, 3, 4, 5, 10, 10, 20 or all of the CVID associated markers (Tables herein) present in the biological sample, preferably a tissue and/or blood plasma sample, and determining if the patient has a greater likelihood of having or developing CVID based on the amount and/or type of CVID marker expression level determined relative to those expression levels identified in patient cohorts of known outcome. A patient has a greater likelihood of having CVID when the sample has a CNV marker expression profile associated with patients previously diagnosed with CVID. The compositions and methods of the invention are useful for the prognosis and diagnosis and management of CVID.


In another aspect, the patient sample may have been previously genotyped and thus the genetic expression profile in the sample may be available to the clinician. Accordingly, the method may entail storing reference CVID associated marker sequence information in a database, i.e., those CNVs statistically associated with a more favorable or less favorable prognosis as described in the tables herein, and performance of comparative genetic analysis on the computer, thereby identifying those patients having increased risk of CVID.


CVID-related CNV or SNP-containing nucleic acids, including but not limited to those listed below may be used for a variety of purposes in accordance with the present invention. CVID-associated CNV or SNP-containing DNA, RNA, or fragments thereof may be used as probes to detect the presence of and/or expression of CVID specific markers. Methods in which CVID specific marker nucleic acids may be utilized as probes for such assays include, but are not limited to: (1) in situ hybridization; (2) Southern hybridization (3) northern hybridization; and (4) assorted amplification reactions such as polymerase chain reactions (PCR).


Further, assays for detecting CVID-associated CNVs or SNPs may be conducted on any type of biological sample, including but not limited to body fluids (including blood, urine, serum, gastric lavage, cerebral spinal fluid), any type of cell (such as brain cells, white blood cells, mononuclear cells, fetal cells in maternal circulation) or body tissue.


Clearly, CVID-associated CNV or SNP-containing nucleic acids, vectors expressing the same, CVID CNV or SNP-containing marker proteins and anti-CVID specific marker antibodies of the invention can be used to detect CVID associated CNVs or SNPs in body tissue, cells, or fluid, and alter CVID CNV or SNP-containing marker protein expression for purposes of assessing the genetic and protein interactions involved in the development of CVID.


In most embodiments for screening for CVID-associated CNVs or SNPs, the CVID-associated CNV or SNP-containing nucleic acid in the sample will initially be amplified, e.g. using PCR, to increase the amount of the templates as compared to other sequences present in the sample. This allows the target sequences to be detected with a high degree of sensitivity if they are present in the sample. This initial step may be avoided by using highly sensitive array techniques that are important in the art.


Alternatively, new detection technologies can overcome this limitation and enable analysis of small samples containing as little as 1 μg of total RNA. Using Resonance Light Scattering (RLS) technology, as opposed to traditional fluorescence techniques, multiple reads can detect low quantities of mRNAs using biotin labeled hybridized targets and anti-biotin antibodies. Another alternative to PCR amplification involves planar wave guide technology (PWG) to increase signal-to-noise ratios and reduce background interference. Both techniques are commercially available from Qiagen Inc. (USA).


Any of the aforementioned techniques may be used to detect or quantify CVID-associated CNV or SNP marker expression and accordingly, diagnose an increased risk for developing the same.


III. Kits and Articles of Manufacture

Any of the aforementioned products can be incorporated into a kit which may contain a CVID-associated CNV or SNP specific marker polynucleotide or one or more such markers immobilized on a solid support or Gene Chip, provided in silico in a database, one or more oligonucleotides, a polypeptide, a peptide, an antibody, a label, marker, reporter, a pharmaceutically acceptable carrier, reagents suitable for performance of PCR with positive and negative control nucleic acids, a physiologically acceptable carrier, instructions for use, a container, a vessel for administration, an assay substrate, or any combination thereof.


IV. Methods of Using CVID-Associated CNVs and/or SNPs for the Development of Therapeutic Agents

Since the CNVs and SNPs identified herein have been associated with the etiology of CVID, methods for identifying agents that modulate the activity of the genes and their encoded products containing such CNVs and/or SNPs should result in the generation of efficacious therapeutic agents for the treatment of this disorder.


Several regions of the human genome provide suitable targets for the rational design of therapeutic agents. Small nucleic acid molecules or peptide molecules corresponding to these regions may be used to advantage in the design of therapeutic agents that effectively modulate the activity of the encoded proteins.


Molecular modeling should facilitate the identification of specific organic molecules with capacity to bind to the active site of the proteins encoded by the CNV or SNP-containing nucleic acids based on conformation or key amino acid residues required for function. A combinatorial chemistry approach will be used to identify molecules with greatest activity and then iterations of these molecules will be developed for further cycles of screening. Molecules available for testing in this screening assay, include without limitation, Apilimod Mesylate (STA-5326), an oral small-molecule compound that selectively inhibits the production of the IL-12 family of proteins Inhibition of IL-production may improve the gastrointestinal manifestations of CVID. Apilimod mesylate selectively inhibits this pathway and reduces over-production of IL-12 and IL-23. Other agents that may have therapeutic utility for treating CVID symptoms include the ACVR2A antagonist Sotaercept and the ACVR antibody PF3446962 as ACVR2A exhibits a significant CNV duplication associated with CVID. PEG-interleukin-2 and B-lymphocyte stimulators may also be screened for therapeutic utility in the treatment of CVID. The polypeptides or fragments employed in drug screening assays may either be free in solution, affixed to a solid support or within a cell. One method of drug screening utilizes eukaryotic or prokaryotic host cells which are stably transformed with recombinant polynucleotides expressing the polypeptide or fragment, preferably in competitive binding assays. Such cells, either in viable or fixed form, can be used for standard binding assays. One may determine, for example, formation of complexes between the polypeptide or fragment and the agent being tested, or examine the degree to which the formation of a complex between the polypeptide or fragment and a known substrate is interfered with by the agent being tested.


Another technique for drug screening provides high throughput screening for compounds having suitable binding affinity for the encoded polypeptides and is described in detail in Geysen, PCT published application WO 84/03564, published on Sep. 13, 1984. Briefly stated, large numbers of different, small peptide test compounds, such as those described above, are synthesized on a solid substrate, such as plastic pins or some other surface. The peptide test compounds are reacted with the target polypeptide and washed. Bound polypeptide is then detected by methods well known in the art.


A further technique for drug screening involves the use of host eukaryotic cell lines or cells (such as described above) which have a nonfunctional or altered CVID associated gene. These host cell lines or cells are defective at the polypeptide level. The host cell lines or cells are grown in the presence of drug compound. Altered immune signaling or function of the host cells is measured to determine if the compound is capable of regulating this function in the defective cells. Host cells contemplated for use in the present invention include but are not limited to bacterial cells, fungal cells, insect cells, mammalian cells, and plant cells. However, mammalian cells, particularly immune cells are preferred. The CVID-associated CNV or SNP encoding DNA molecules may be introduced singly into such host cells or in combination to assess the phenotype of cells conferred by such expression. Methods for introducing DNA molecules are also well known to those of ordinary skill in the art. Such methods are set forth in Ausubel et al. eds., Current Protocols in Molecular Biology, John Wiley & Sons, NY, N.Y. 1995, the disclosure of which is incorporated by reference herein.


A wide variety of expression vectors are available that can be modified to express the novel DNA sequences of this invention. The specific vectors exemplified herein are merely illustrative, and are not intended to limit the scope of the invention. Expression methods are described by Sambrook et al. Molecular Cloning: A Laboratory Manual or Current Protocols in Molecular Biology 16.3-17.44 (1989). Expression methods in Saccharomyces are also described in Current Protocols in Molecular Biology (1989).


Suitable vectors for use in practicing the invention include prokaryotic vectors such as the pNH vectors (Stratagene Inc., 11099 N. Torrey Pines Rd., La Jolla, Calif. 92037), pET vectors (Novogen Inc., 565 Science Dr., Madison, Wis. 53711) and the pGEX vectors (Pharmacia LKB Biotechnology Inc., Piscataway, N.J. 08854). Examples of eukaryotic vectors useful in practicing the present invention include the vectors pRc/CMV, pRc/RSV, and pREP (Invitrogen, 11588 Sorrento Valley Rd., San Diego, Calif. 92121); pcDNA3.1/V5&His (Invitrogen); baculovirus vectors such as pVL1392, pVL1393, or pAC360 (Invitrogen); and yeast vectors such as YRP17, YIPS, and YEP24 (New England Biolabs, Beverly, Mass.), as well as pRS403 and pRS413 Stratagene Inc.); Picchia vectors such as pHIL-D1 (Phillips Petroleum Co., Bartlesville, Okla. 74004); retroviral vectors such as PLNCX and pLPCX (Clontech); and adenoviral and adeno-associated viral vectors.


Promoters for use in expression vectors of this invention include promoters that are operable in prokaryotic or eukaryotic cells. Promoters that are operable in prokaryotic cells include lactose (lac) control elements, bacteriophage lambda (pL) control elements, arabinose control elements, tryptophan (trp) control elements, bacteriophage T7 control elements, and hybrids thereof. Promoters that are operable in eukaryotic cells include Epstein Barr virus promoters, adenovirus promoters, SV40 promoters, Rous Sarcoma Virus promoters, cytomegalovirus (CMV) promoters, baculovirus promoters such as AcMNPV polyhedrin promoter, Picchia promoters such as the alcohol oxidase promoter, and Saccharomyces promoters such as the gal4 inducible promoter and the PGK constitutive promoter, as well as neuronal-specific platelet-derived growth factor promoter (PDGF), the Thy-1 promoter, the hamster and mouse Prion promoter (MoPrP), and the Glial fibrillar acidic protein (GFAP) for the expression of transgenes in glial cells.


In addition, a vector of this invention may contain any one of a number of various markers facilitating the selection of a transformed host cell. Such markers include genes associated with temperature sensitivity, drug resistance, or enzymes associated with phenotypic characteristics of the host organisms.


Host cells expressing the CVID-associated CNVs and/or SNPs of the present invention or functional fragments thereof provide a system in which to screen potential compounds or agents for the ability to modulate the development of CVID. Thus, in one embodiment, the nucleic acid molecules of the invention may be used to create recombinant cell lines for use in assays to identify agents which modulate aspects of cellular metabolism associated with CVID and aberrant glutaminergic function. Also provided herein are methods to screen for compounds capable of modulating the function of proteins encoded by CNV and SNP-containing nucleic acids.


Another approach entails the use of phage display libraries engineered to express fragment of the polypeptides encoded by the CNV or SNP-containing nucleic acids on the phage surface. Such libraries are then contacted with a combinatorial chemical library under conditions wherein binding affinity between the expressed peptide and the components of the chemical library may be detected. U.S. Pat. Nos. 6,057,098 and 5,965,456 provide methods and apparatus for performing such assays.


The goal of rational drug design is to produce structural analogs of biologically active polypeptides of interest or of small molecules with which they interact (e.g., agonists, antagonists, inhibitors) in order to fashion drugs which are, for example, more active or stable forms of the polypeptide, or which, e.g., enhance or interfere with the function of a polypeptide in vivo. See, e.g., Hodgson, (1991) Bio/Technology 9:19-21. In one approach, discussed above, the three-dimensional structure of a protein of interest or, for example, of the protein-substrate complex, is solved by x-ray crystallography, by nuclear magnetic resonance, by computer modeling or most typically, by a combination of approaches. Less often, useful information regarding the structure of a polypeptide may be gained by modeling based on the structure of homologous proteins. An example of rational drug design is the development of HIV protease inhibitors (Erickson et al., (1990) Science 249:527-533). In addition, peptides may be analyzed by an alanine scan (Wells, (1991) Meth. Enzym. 202:390-411). In this technique, an amino acid residue is replaced by Ala, and its effect on the peptide's activity is determined. Each of the amino acid residues of the peptide is analyzed in this manner to determine the important regions of the peptide.


It is also possible to isolate a target-specific antibody, selected by a functional assay, and then to solve its crystal structure. In principle, this approach yields a pharmacore upon which subsequent drug design can be based.


One can bypass protein crystallography altogether by generating anti-idiotypic antibodies (anti-ids) to a functional, pharmacologically active antibody. As a mirror image of a mirror image, the binding site of the anti-ids would be expected to be an analog of the original molecule. The anti-id could then be used to identify and isolate peptides from banks of chemically or biologically produced banks of peptides. Selected peptides would then act as the pharmacore.


Thus, one may design drugs which have, e.g., improved polypeptide activity or stability or which act as inhibitors, agonists, antagonists, etc. of polypeptide activity. By virtue of the availability of CNV or SNP-containing nucleic acid sequences described herein, sufficient amounts of the encoded polypeptide may be made available to perform such analytical studies as x-ray crystallography. In addition, the knowledge of the protein sequence provided herein will guide those employing computer modeling techniques in place of, or in addition to x-ray crystallography.


In another embodiment, the availability of CVID-associated CNV or SNP-containing nucleic acids enables the production of strains of laboratory mice carrying the CVID-associated SNPs or CNVs of the invention. Transgenic mice expressing the CVID-associated CNV or SNP of the invention provide a model system in which to examine the role of the protein encoded by the CNV or SNP-containing nucleic acid in the development and progression towards CVID. Methods of introducing transgenes in laboratory mice are known to those of skill in the art. Three common methods include: 1. integration of retroviral vectors encoding the foreign gene of interest into an early embryo; 2. injection of DNA into the pronucleus of a newly fertilized egg; and 3. the incorporation of genetically manipulated embryonic stem cells into an early embryo. Production of the transgenic mice described above will facilitate the molecular elucidation of the role that a target protein plays in various cellular metabolic processes, including: aberrant antibody production and function, altered immunoreceptor ligand signaling and aberrant responses to bacterial and viral infections. Such mice provide an in vivo screening tool to study putative therapeutic drugs in a whole animal model and are encompassed by the present invention.


The term “animal” is used herein to include all vertebrate animals, except humans. It also includes an individual animal in all stages of development, including embryonic and fetal stages. A “transgenic animal” is any animal containing one or more cells bearing genetic information altered or received, directly or indirectly, by deliberate genetic manipulation at the subcellular level, such as by targeted recombination or microinjection or infection with recombinant virus. The term “transgenic animal” is not meant to encompass classical cross-breeding or in vitro fertilization, but rather is meant to encompass animals in which one or more cells are altered by or receive a recombinant DNA molecule. This molecule may be specifically targeted to a defined genetic locus, be randomly integrated within a chromosome, or it may be extrachromosomally replicating DNA. The term “germ cell line transgenic animal” refers to a transgenic animal in which the genetic alteration or genetic information was introduced into a germ line cell, thereby conferring the ability to transfer the genetic information to offspring. If such offspring, in fact, possess some or all of that alteration or genetic information, then they, too, are transgenic animals.


The alteration of genetic information may be foreign to the species of animal to which the recipient belongs, or foreign only to the particular individual recipient, or may be genetic information already possessed by the recipient. In the last case, the altered or introduced gene may be expressed differently than the native gene. Such altered or foreign genetic information would encompass the introduction of CVID-associated CNV or SNP-containing nucleotide sequences.


The DNA used for altering a target gene may be obtained by a wide variety of techniques that include, but are not limited to, isolation from genomic sources, preparation of cDNAs from isolated mRNA templates, direct synthesis, or a combination thereof.


A preferred type of target cell for transgene introduction is the embryonal stem cell (ES). ES cells may be obtained from pre-implantation embryos cultured in vitro (Evans et al., (1981) Nature 292:154-156; Bradley et al., (1984) Nature 309:255-258; Gossler et al., (1986) Proc. Natl. Acad. Sci. 83:9065-9069). Transgenes can be efficiently introduced into the ES cells by standard techniques such as DNA transfection or by retrovirus-mediated transduction. The resultant transformed ES cells can thereafter be combined with blastocysts from a non-human animal. The introduced ES cells thereafter colonize the embryo and contribute to the germ line of the resulting chimeric animal.


One approach to the problem of determining the contributions of individual genes and their expression products is to use isolated CVID-associated CNV or SNP genes as insertional cassettes to selectively inactivate a wild-type gene in totipotent ES cells (such as those described above) and then generate transgenic mice. The use of gene-targeted ES cells in the generation of gene-targeted transgenic mice was described, and is reviewed elsewhere (Frohman et al., (1989) Cell 56:145-147; Bradley et al., (1992) Bio/Technology 10:534-539).


Techniques are available to inactivate or alter any genetic region to a mutation desired by using targeted homologous recombination to insert specific changes into chromosomal alleles. However, in comparison with homologous extrachromosomal recombination, which occurs at a frequency approaching 100%, homologous plasmid-chromosome recombination was originally reported to only be detected at frequencies between 10−6 and 10−3. Nonhomologous plasmid-chromosome interactions are more frequent occurring at levels 105-fold to 102 fold greater than comparable homologous insertion.


To overcome this low proportion of targeted recombination in murine ES cells, various strategies have been developed to detect or select rare homologous recombinants. One approach for detecting homologous alteration events uses the polymerase chain reaction (PCR) to screen pools of transformant cells for homologous insertion, followed by screening of individual clones. Alternatively, a positive genetic selection approach has been developed in which a marker gene is constructed which will only be active if homologous insertion occurs, allowing these recombinants to be selected directly. One of the most powerful approaches developed for selecting homologous recombinants is the positive-negative selection (PNS) method developed for genes for which no direct selection of the alteration exists. The PNS method is more efficient for targeting genes which are not expressed at high levels because the marker gene has its own promoter. Non-homologous recombinants are selected against by using the Herpes Simplex virus thymidine kinase (HSV-TK) gene and selecting against its nonhomologous insertion with effective herpes drugs such as gancyclovir (GANC) or (1-(2-deoxy-2-fluoro-B-D arabinofluranosyl)-5-iodou-racil, (FIAU). By this counter selection, the number of homologous recombinants in the surviving transformants can be increased. Utilizing CVID-associated CNV or SNP-containing nucleic acid as a targeted insertional cassette provides means to detect a successful insertion as visualized, for example, by acquisition of immunoreactivity to an antibody immunologically specific for the polypeptide encoded by CVID-associated CNV or SNP nucleic acid and, therefore, facilitates screening/selection of ES cells with the desired genotype.


As used herein, a knock-in animal is one in which the endogenous murine gene, for example, has been replaced with human CVID-associated CNV or informative fragment thereof or SNP-containing gene of the invention. Such knock-in animals provide an ideal model system for studying the development of CVID.


As used herein, the expression of a CVID-associated CNV or SNP-containing nucleic acid, partial informative CNV fragment thereof, or an CVID-associated fusion protein in which the CNV or SNP is encoded can be targeted in a “tissue specific manner” or “cell type specific manner” using a vector in which nucleic acid sequences encoding all or a portion of an CVID-associated CNV or SNP are operably linked to regulatory sequences (e.g., promoters and/or enhancers) that direct expression of the encoded protein in a particular tissue or cell type. Such regulatory elements may be used to advantage for both in vitro and in vivo applications. Promoters for directing tissue specific proteins are well known in the art and described herein.


Methods of use for the transgenic mice of the invention are also provided herein. Transgenic mice into which a nucleic acid containing the CVID-associated CNV or SNP or its encoded protein have been introduced are useful, for example, to develop screening methods to screen therapeutic agents to identify those capable of modulating the development of CVID.


V. Pharmaceutical and Peptide Therapies

The elucidation of the role played by the CVID associated CNVs and SNPs described herein in immunoreceptor ligand signaling and function facilitates the development of pharmaceutical compositions useful for treatment and diagnosis of CVID. These compositions may comprise, in addition to one of the above substances, a pharmaceutically acceptable excipient, carrier, buffer, stabilizer or other materials well known to those skilled in the art. Such materials should be non-toxic and should not interfere with the efficacy of the active ingredient. The precise nature of the carrier or other material may depend on the route of administration, e.g. oral, intravenous, cutaneous or subcutaneous, nasal, intramuscular, intraperitoneal routes.


Whether it is a polypeptide, antibody, peptide, nucleic acid molecule, small molecule or other pharmaceutically useful compound according to the present invention that is to be given to an individual, administration is preferably in a “prophylactically effective amount” or a “therapeutically effective amount” (as the case may be, although prophylaxis may be considered therapy), this being sufficient to show benefit to the individual.


The following materials and methods are provided to facilitate the practice of the present invention.


Patients


The diagnosis of CVID was established in concordance with existing diagnostic criteria16,38. All patients were enrolled in institutionally approved research protocols to enable genetic analysis and collection of clinical data. Subsets of the patients reported here have been previously included in published studies2,22,39.


Illumina Infinium Assay for SNP Genotyping and CNV Discovery


We performed high-throughput, genome-wide SNP genotyping, using the InfiniumII HumanHap550 BeadChip technology (Illumina San Diego, Calif.), at the Center for Applied Genomics at CHOP. The genotype data content together with the intensity data provided by the genotyping array provides high confidence for CNV calls. Importantly, the simultaneous analysis of intensity data and genotype data in the same experimental setting establishes a highly accurate definition for normal diploid states and any deviation from the norm. To call CNVs, we used the PennCNV algorithm12, which combines multiple sources of information, including Log R Ratio (LRR) and B Allele Frequency (BAF) at each SNP marker, along with SNP spacing and population frequency of the B allele to generate CNV calls. Rare recurrent CNVs were the focus of our study.


CNV Quality Control


We calculated Quality Control (QC) measures on our HumanHap550 GWAS data based on statistical distributions to exclude poor quality DNA samples and false positive CNVs. The first threshold is the percentage of attempted SNPs which were successfully genotyped. Only samples with call rate >98% were included. The genome wide intensity signal must have as little noise as possible. Only samples with the standard deviation (SD) of normalized intensity (LRR) <0.35 were included. All samples must have Caucasian ethnicity based on principle components analysis and all other samples were excluded. Furthermore, case and control matching was insured by calculating a genomic inflation factor between groups. Wave artifacts roughly correlating with GC content resulting from hybridization bias of low full length DNA quantity are known to interfere with accurate inference of copy number variations. Only samples where the GC corrected wave factor of LRR −0.02<x<0.02 were accepted. If the count of CNV calls made by PennCNV exceeds 100, it is suggestive of poor DNA quality, and those samples were excluded. Thus, only samples with CNV call count <100 were included. Any duplicate samples (such as monozygotic twins or repeats on the same patient) were identified and as a result one sample was excluded.


Statistical Analysis of CNVs


CNV frequency between cases and controls was evaluated at each SNP using Fisher's exact test. We only considered loci that were nominally significant between cases and controls (p<0.05) for which patients had the same variation, that were observed in multiple cohorts or were not observed in any of the control subjects, and were validated with an independent method. We report statistical local minimums to narrow the association in reference to a region of nominal significance including SNPs residing within 1 Mb of each other. Resulting nominally significant CNVRs were excluded if they met any of the following criteria: i) residing on telomere or centromere proximal cytobands; ii) arising in a “peninsula” of common CNV arising from variation in boundary truncation of CNV calling; iii) genomic regions with extremes in GC content which produces hybridization bias; or iv) samples contributing to multiple CNVRs. Three lines of evidence establish statistical significance: independent replication p<0.05, permutation of observations, and no loci observed with control enriched significance. We used DAVID (Database for Annotation, Visualization, and Integrated Discovery)42 to assess the significance of functional annotation clustering of independently associated results into InterPro categories.


The following examples are provided to illustrate certain embodiments of the invention. They are not intended to limit the invention in any way.


Example 1

Genome wide single nucleotide polymorphism (SNP) arrays have enabled high throughput typing of genomic DNA with tagging of the whole genome based on linkage disequilibrium. Given both intensity and genotype content, copy number variations (CNVs) can also be detected using the same GWAS platforms. Both SNPs and CNVs have a significant difference in frequency between cases and controls in a variety of diseases. We genotyped 363 CVID cases and 3,031 healthy controls on the Illumina Infinium HumanHap550K BeadChip. We sought to associate SNPs and CNVs with CVID16, as well as with the characteristic clinical and immunologic phenotypes of this syndrome, to address disease heterogeneity. We additionally compared the CVID results with those obtained from Inflammatory Bowel Disease (IBD), another disease with an immunologic etiology but without susceptibility to infection, to serve as a “disease control” cohort and define associations unique to CVID subjects. Included in the cohort were 29 subjects with known mutations in TACI including 10 C104R, 8 A181E, 6 V220A and 1 occurrence of each of C104R/S144X, C104R/S194X, A181E/L171R, R72H, and S194X heterozygous.


Our CVID case cohort was composed of 223 patients from Mount Sinai School of Medicine, 76 patients from University of Oxford, 37 patients from The Children's Hospital of Philadelphia, and 27 patients from University of South Florida. The diagnosis in each case was validated against the ESID/PAGID diagnostic criteria16 and only patients confirmed by these criteria were included.


We first evaluated quality and suitability of the data for a case control study. The primary parameters were sample based call rates and population based stratification. Seven samples had call rates lower than 98% and were excluded. Based on principle components analysis by Eigenstrat17, the cases were stratified into three clusters: 179 cases were of confirmed European ancestry and another 109 Caucasian cases clustered separately, but very close to the other Caucasian cluster, whereas 30 samples were significant outliers from Caucasian and were omitted from this analysis. We also observed comparable clustering of the 179 cases to the 1,917 Caucasian controls. However, there were relatively few Caucasian controls that clustered well with the second Caucasian case cluster. To perform a more comprehensive search for additional control individuals that clustered with the latter case cohort, we ran 18,000 controls in 19 batches together with our cases through Eigenstrat. This identified over 1,000 control samples clustering similarly to the second Caucasian case cohort. We then reran those identified samples together with cases to yield a robustly stratified cluster with a total of 1,114 controls. This yielded a discovery cohort of 179 cases and 1,917 controls along with a replication cohort of 109 cases and 1,114 controls. We also excluded individuals with observed cryptic relatedness of genotypes as well as three known and two newly discovered 22q11 deletions. For CNV calling, samples with high noise (standard deviation of Log R Ratio >0.35) in intensity signal were additionally removed.


First, a genome wide association analysis (GWAS) was performed using the SNP genotype data. Genotype frequencies were compared between cases versus controls and a chi-square test statistic applied in Plink18 for SNPs with at least 90% call rate and 1% minor allele frequency. The discovery cohort of 179 cases and 1,917 controls yielded a low genomic inflation factor of 1.02783 (FIG. 1A). The 1,000 most significant SNPs were evaluated for enrichment in genomic regions to boost confidence in significance based on neighboring SNPs also with significance in the top 1,000. We found 87 such regions with multiple significant SNPs composed of 344 SNPs in total, the remainder of which we excluded as spurious signal (Table 1). The most significant association was the major histocompatibility complex (MHC) with 110 neighboring SNPs showing significance in the top 1,000, the most significant SNP rs3117426 having P=8.62×10−10. A total of 62 regions were supported by two SNPs, and 25 regions were supported by 3 or more SNPs, with 47 regions directly impacting genes while 40 did not.









TABLE 1







Genomic Regions with Multiple SNPs in the 1000 most Significant Associations


















Distance







Region


From
CountSNPs
Best
Best




Genomic Span
Gene
Distance
Exon
Top1000
P
SNP
F_A
F_U



















chr6: 27236785-



MHC

, Many


0

0
110
 8.62E−10
rs3117426
0.3268
0.1907



32521295












chr8: 23746576-

ADAM28, ADAM7, ADAMDEC1,

0

0
4
6.24E−6
rs4872262
0.03911
0.01069



24681608

STC1









chr6: 42776382-
CRIP3, CUL7, GNMT,
0
0
4
1.22E−5
rs422717
0.2961
0.1982


43502348
KLC4, KLHDC3,










MEA1, MRPL2, PARC, PEAS,










PEX6, PPP2R5D, PRPH2,










PTCRA, PTK7, RPL7L1,










SLC22A7, SRF, TBCC,












TNRC5
, TTBK1, UNQ1934,











ZNF318









chr14: 39744450-
BX248273
584047
584047
4
1.69E−5
rs6571989
0.1927
0.115


39909619











chr4: 189671181-


AK095968


0

18764
2
1.78E−5
rs1606234
0.2961
0.4124



189676119











chr16: 6120908-
A2BP1
0
110914
4
1.85E−5
rs12924882
0.1313
0.06915


6137836










chr2: 224812598-
FAM124B
110570
110570
4
1.89E−5
rs4264554
0.3184
0.4353


224841089










chr2: 45599872-
PRKCE, SRBD1
0
0
7
2.12E−5
rs17322265
0.3408
0.4576


45895169











chr10: 73083830-


CDH23, KIAA1812


0

7287
2
2.45E−5
rs7087554
0.5894
0.4729



73084583











chr2: 74996818-
AK125960
7650
7650
2
3.25E−5
rs3771781
0.3184
0.4319


75001224










chr20: 55079605-


BMP7


95559
95559
2
4.44E−5
rs6127923
0.2318
0.1498


55083403











chr7: 4270929-



SDK1



0

0
5
4.70E−5
rs895710
0.2905
0.4003



4287047











chr20: 15025030-
C20orf133
0
50997
4
5.15E−5
rs6043091
0.2151
0.1367


15074507










chr8: 8804291-


MFHAS1


15750
15750
3
6.80E−5
rs400404
0.3715
0.2726


8808564










chr1: 158072814-
CR625159, RP11-
0
0
2
7.31E−5
rs10494349
0.2067
0.1312


158076453
190A12.4, SLAMF8









chr18: 25213638-
CDH2
1202449
1202449
2
7.53E−5
rs9950880
0.2821
0.1943


25220939










chr3: 106535466-


ALCAM
, CBLB, Nbla00127

0
0
5
8.57E−5
rs13062596
0.4358
0.3328


107174255










chr6: 85682448-
TBX18
151830
151830
2
9.07E−5
rs9444253
0.05028
0.1187


85684185











chr1: 20369369-


FLJ32784, UBXN10


0

0
7
9.09E−5
rs7514144
0.2654
0.1808



20490791












chr10: 27709830-


PTCHD3


0

0
7
9.77E−5
rs506659
0.4022
0.3026



27728115











chr12: 123259607-
FAM101A
64652
64652
4
1.01E−4
rs7972182
0.3855
0.2874


123275011










chr13: 68136436-
BC042673
195237
195237
2
1.09E−4
rs287355
0.2219
0.3213


68138181










chr16: 69996550-


CALB2


14714
14714
2
1.09E−4
rs12102284
0.1676
0.1015


70004357










chr11: 25128661-
LUZP2
67899
67899
2
1.10E−4
rs11028465
0.3352
0.2426


25130289










chr12: 49729991-
DKFZp586A011, LETMD1
0
214
2
1.22E−4
rs4768959
0.1397
0.08033


49731928










chr4: 25702025-
LOC389203
161493
161493
5
1.34E−4
rs2048507
0.257
0.1755


25756263










chr7: 13858986-
ETV1
31148
31148
4
1.35E−4
rs12532319
0.09218
0.04617


13866233











chr1: 59987116-


FLJ10986, RP11-242B9.1


0

2458
2
1.52E−4
rs11207520
0.3994
0.3026



59993733











chr6: 12070732-


HIVEP1


47026
47026
2
1.54E−4
rs12193434
0.09777
0.05034


12073684










chr19: 40814281-
MGC10433
0
134
2
1.55E−4
rs2285415
0.352
0.4559


40815243










chr6: 139472753-
HECA
20371
20371
2
1.56E−4
rs17304375
0.2318
0.1549


139477571











chr12: 80435676-


PPFIA2


0

59900
2
1.63E−4
rs2400955
0.4134
0.3159



80444630











chr12: 69717561-
TSPAN8
85073
85073
2
1.63E−4
rs6581986
0.2905
0.2053


69720071











chr11: 102069786-


MMP27


0

0
2
1.69E−4
rs17099394
0.1229
0.0687



102079454











chr3: 188168001-


ST6GAL1


0
3593
2
1.71E−4
rs12495023
0.2765
0.1934


188168026











chr16: 71592439-


ATBF1


0

40845
2
1.80E−4
rs8056528
0.3687
0.2754



71594134












chr8: 124729628-


C8ORFK36


0

0
4
2.05E−4
rs4871402
0.06425
0.1325



124734756











chr22: 42908147-
KIAA1644, PARVG
0
0
8
2.27E−4
rs80303
0.3994
0.5013


43159207










chr20: 44622587-
NADC3, SLC13A3
0
0
5
2.49E−4
rs393990
0.09218
0.1664


44638545










chr1: 9459963-
SLC25A33
54611
54611
2
2.57E−4
rs10746490
0.1453
0.2292


9467504











chr3: 7364204-


GRM7


0

40865
4
2.96E−4
rs12491592
0.1536
0.0939



7369287











chr11: 46472478-
ARHGAP1,
0
0
4
3.19E−4
rs2306029
0.3603
0.4593


46855347
CKAP5, CR612190, F2,










LRP4, MEGF7, ZNF408,










coagulation factor II









chr2: 181999958-
AK125001
27433
27433
2
3.22E−4
rs16867404
0.09497
0.05008


182002374










chr7: 121184414-


PTPRZ1


106504
106504
2
3.24E−4
rs1196493
0.5251
0.4266


121193891











chr3: 8378739-


BC020876


0

129448
2
3.51E−4
rs359030
0.1648
0.1033



8381919












chr6: 25570803-


LRRC16


0

0
2
3.61E−4
rs4320355
0.3799
0.2898



25574868











chr4: 131889955-
BC041448
793982
793982
2
3.68E−4
rs2125639
0.2905
0.2094


131893245










chr15: 58159939-
FOXB1
74505
74505
2
3.71E−4
rs7168491
0.1648
0.1035


58162165











chr6: 153354039-


MTRF1L


0

0
2
3.81E−4
rs9322400
0.4469
0.3526



153355207











chr5: 172969660-
FAM44B
0
0
4
3.83E−4
rs258873
0.2849
0.3798


172992688










chr6: 55433518-
HMGCLL1
0
21202
2
3.86E−4
rs9382494
0.2179
0.3078


55442340










chr9: 15192417-
C9orf52
0
0
2
3.87E−4
rs693196
0.3603
0.2723


15195014










chr4: 20207250-
SLIT2
0
0
2
4.29E−4
rs573118
0.243
0.169


20210955











chr3: 284363-



CHL1



0

19083
2
4.31E−4
rs17273893
0.148
0.09081



285747











chr11: 45198534-
PRDM11
0
650
2
4.36E−4
rs12417962
0.06983
0.1351


45198959










chr3: 138891290-
SOX14
74205
74205
2
4.37E−4
rs12637203
0.1844
0.12


138892064











chr8: 121001793-


DEPDC6


0

0
2
4.41E−4
rs869340
0.324
0.2402



121010709











chr18: 74518781-
SALL3
315148
315148
2
4.72E−4
rs2931060
0.1257
0.07381


74526115










chr4: 143965929-
INPP4B
0
14945
2
4.81E−4
rs10000770
0.1788
0.1158


143971867










chr3: 115619659-
ZBTB20
0
29017
2
5.19E−4
rs2718419
0.2598
0.1844


115623404











chr2: 65518304-


FLJ16124


0

784
2
5.20E−4
rs1194849
0.3659
0.4614



65520560











chr5: 68236398-
AK128486
55041
55041
2
5.27E−4
rs7718291
0.1313
0.2081


68245085











chr21: 27713547-


BC043580


0

19690
2
5.51E−4
rs469709
0.1704
0.1095



27722878











chr3: 5866648-
EDEM1
630006
630006
2
5.91E−4
rs2572690
0.1145
0.06602


5866856










chr16: 7470020-
A2BP1
0
33910
2
5.93E−4
rs2191388
0.2709
0.1948


7474240










chr6: 88936647-


CNR1


4366
4366
2
6.35E−4
rs9344757
0.2179
0.3041


88941540










chr4: 32018838-
PCDH7
1265004
1265004
3
6.41E−4
rs2130904
0.2458
0.3343


32029980










chr1: 202125395-
SNRPE
18492
18492
2
6.48E−4
rs12145634
0.1369
0.08346


202130221










chr7: 38733082-
HVPS41, VPS41
0
0
2
6.67E−4
rs10255854
0.2179
0.3038


38736264










chr11: 120260865-
GRIK4
0
5023
2
6.67E−4
rs12577638
0.2011
0.1356


120269428










chr11: 91278373-
FAT3
444245
444245
2
6.97E−4
rs10501763
0.03911
0.09207


91283755










chr16: 20151678-
GP2
77031
77031
2
7.14E−4
rs9921767
0.0419
0.09572


20152281










chr1: 221871750-


CAPN2


84031
84031
3
7.29E−4
rs3856154
0.4134
0.3254


221882793










chr10: 130929688-


MGMT


224087
224087
2
7.33E−4
rs538186
0.05307
0.1104


130931369










chr5: 170727300-


NPM1


12168
12168
2
7.52E−4
rs7707008
0.1173
0.1894


170734557










chr10: 63071553-
C10orf107
12553
12553
2
7.69E−4
rs10994852
0.1061
0.06051


63080172










chr21: 31427707-
TIAM1
0
0
2
7.93E−4
rs2833297
0.2095
0.1435


31432285










chr4: 42434168-
ATP8A1
80521
80521
2
8.09E−4
rs6812482
0.2709
0.1965


42438752










chr6: 161106705-
PLG
12377
12377
2
8.78E−4
rs9295131
0.2151
0.2986


161109328










chr17: 12323188-
AX747308, BC122562
68477
68477
2
9.02E−4
rs8069430
0.2584
0.186


12325534










chr17: 50125991-
TOM1L1, tom1-like
205624
205624
2
9.20E−4
rs6504930
0.03371
0.08325


50127579










chr16: 77330083-


WWOX


0
305933
2
9.21E−4
rs1364290
0.1844
0.1231


77334376










chr11: 5493737-
HBG2, UBQLNL
0
0
4
9.28E−4
rs2047456
0.3436
0.2624


5511794










chr3: 70644812-
LOC401072
96929
96929
2
9.91E−4
rs17790790
0.5196
0.4293


70648510










chr3: 1292289-


CNTN6


0
0
2
1.06E−3
rs6799262
0.3571
0.2733


1302261










chr3: 64977356-
BC040632
5173
5173
2
1.15E−3
rs1517927
0.2318
0.1643


64985877










chr5: 14818187-
ANKH
0
0
2
1.15E−3
rs17251715
0.3955
0.3113


14825245









We next sought to replicate these SNPs and the direction of the difference in allele frequency, based on our replication cohort of 109 cases and 1114 controls with a genomic inflation factor of 1.04789 (FIG. 1B). Four SNPs displayed significance (P<0.05) and allele frequency in the same direction as the discovery cohort, including rs11207520, rs1194849, rs17790790, and rs4872262. The associated chromosomal region demonstrating replication that impacted characterized genes was 8p21.2, harboring ADAM28, ADAM7, ADAMDEC1, and STC1. When all SNPs contributing to the discovery region were queried in replication rather than just the most significant SNP, 8 (four additional) SNPs displayed significance (P<0.05) and allele frequency in the same direction as discovery. There were three additional regions with directly affected genes: the MHC, UBXN10, and SDK1 (Table 2); others did not overlap genes directly.









TABLE 2







Most Significant Associated Regions Based on Genotype Association





















Count
Dis-











Region

SNPs
tance





P
SNP
F_A
F_U


Genomic

Top
From





Repli-
Repli-
Repli-
Repli-


Span
Gene
1000
Exon
P
SNP
A1
F_A
F_U
cation
cation
cation
cation






















chr6: 27236785-


MHC


110
0
 8.62E−10
rs3117426
T
0.3268
0.1907
0.0004
rs2156875
0.5734
0.4475


32521295














chr8: 23746576-
ADAM28,
4
0
6.24E−6
rs4872262
A
0.0391
0.0107
0.0314
rs4872262
0.0321
0.0135


24681608


ADAM7
,















ADAMDEC1,














STC1













chr7: 4270929-


SDK1


5
0
4.70E−5
rs895710
T
0.2905
0.4003
0.0235
rs4720301
0.3991
0.4793


4287047














chr1: 20369369-
FLJ32784,
7
0
9.09E−5
rs7514144
C
0.2654
0.1808
2.25E−8
rs6426636
0.4679
0.2857


20490791


UBXN10















chr1: 59987116-
FGGY
2
2458
1.52E−4
rs11207520
G
0.3994
0.3026
0.0015
rs11207520
0.2569
0.1706


59993733














chr2: 65518304-
FLJ16124
2
784
5.20E−4
rs1194849
G
0.3659
0.4614
0.0119
rs1876518
0.3853
0.4744


65520560





Bold underline: Known immunological function genes, A1: allele 1 which is the minor allele, F_A: Frequency of allele 1 in affected individuals, F_U: Frequency of allele 1 in unaffected individuals.


See additional detail inTable 11.






We had access to a large IBD cohort that had been genotyped previously19. Since this is also a heterogeneous immunological condition but without increased infectious susceptibility, it was considered that this group would be appropriate as a disease control group. Analysis of the 2,413 IBD cases and 6,197 controls revealed a number of signals in the IBD cohort that were distinct from both controls and the CVID cohort (including 19 CVID subjects who had been diagnosed with IBD). These included a signal in DEPDC6 (P<0.05) with allele frequency in the same direction as well as the MHC locus. In addition, TIAM1, SLAMF8, GRIK4, 6p21.1, PPFIA2, SLC25A33, A2BP1, and UBXN10 were also observed in the IBD cohort only (P<0.05). Importantly, however, the majority of associations in CVID identified in comparison to controls were not observed in the IBD cohort, suggesting that that they were not a general feature of an immunologic disease, but specific to the CVID phenotype. The non-overlapping loci between CVID and IBD included FLJ16124, ADAM28-ADAM7-ADAMDEC1-STC1, ANKH, FLJ109860-RP11-242B9.1, and WWOX, which are therefore more likely to lead to loss of B cell and antibody function.


We next performed a CNV analysis, where rare (<1%) CNVs were called, using PennCNV2° and SNP-based CNV frequency in cases and controls; these were compared for associations. We analyzed deletion and duplication CNV frequency of 311 cases and 2,766 controls (FIG. 2). To limit bias due to population stratification, the contribution of patients from the discovery Caucasian cluster defined in the genotype GWAS was required to establish confidence. We also monitored the contribution of each sample subgroup from the four different sample collection sites. We found 5 deletions and 11 duplications to be recurrent and significantly enriched in the patients as compared to controls (Table 3). In addition, a total of 15 regions were exclusive to CVID cases. Duplications of 2q23.1 associated with multiple exons of ORC4L were observed to be exclusive to 15 cases (P=8.66×10−16) (FIG. 3). We also observed large CNVs with at least 10 SNPs exclusive to single CVID cases totaling 84 deletions and 98 duplications in CNVs. (Table 4). No significant overrepresentation was observed in the controls in this frequency range. Of clinical relevance, 2 patients were newly identified with deletion in the 22q11 region suggesting an alternative presentation of the well-characterized PIDD associated with this microdeletion. Of 5 deletions and 23 duplications significantly enriched in CVID, 4 deletions and 21 duplications significant in CVID were not significant in IBD. 10 regions of homozygous deletion were observed exclusively in CVID cases (Table 5). To further prioritize resulting significant loci for potential functional impact, we calculated distance of CNVs from exons to highlight loci impacting exons directly, a method applied previously21. We examined overall CNV burden in cases compared to controls for significant results, exonic CNVs, and large (100 kb) rare (<1%) CNVs and found deletions to be significantly enriched in cases when considering CNV observations on all loci genome wide (Table 6). All but 5 of the 182 CNVs exclusive to the CVID cohort were intraexonic. To assess the reliability of our CNV detection method, we reviewed the BAF (genotype) and LRR (intensity) values of our Illumina data and experimentally validated all the significant CNVRs using an independent method, the Affymetrix Cytogenetics Whole-Genome 2.7M Array which provides high resolution with 400,103 SNP and 2,387,595 CN probes (FIG. 4).









TABLE 3





Most Significant Associated Regions Based on CNV Association







A) Deletions


















Count
Distance From
P
Cases
Controls




Com-


CNVR Deletion
SNPs
Exon
Deletion
Del
Del
Gene
MSSM
Oxford
USF
bined





chr11: 85365857-
5
0
0.01
2
0


PICALM
, PICALM variant

0
2
0
2


85381622





protein






chr20: 57735790-
6
5902
0.026
3
4
PHACTR3
2
1
0
3


57741780












chr22: 17396663-
270
0
0.029
2
1
ARVCF, COMT, TBX1,
2
0
0
2


18417315





others






chr4: 10256682-
4
2915
0.029
2
1


CLNK


0
1
1
2


10264316












chr10: 46003146-
8
0
0.029
2
1
DKFZp566K0524,
2
0
0
2


46042543





PTPN20A, PTPN20B










B) Duplications


















Count
Distance From
P
Cases
Controls




Com-


CNVR Duplication
SNPs
Exon
Dup
Dup
Dup
Gene
MSSM
Oxford
USF
bined





chr2: 148396730-
6
0
8.66E−16
15
0


ACVR2A
,

10
5
0
15


148433180







ORC4L








chr15: 35053039-
6
23146
5.72E−13
19
10
MEIS2
17
2
0
19


35063531












chr7: 91789778-
4
0
1.20E−09
12
4
ANKIB1
9
3
0
12


91801963












chr2: 163316298-
3
17100
1.00E−07
7
0
KCNH7
6
1
0
7


163316595












chr7: 87180702-
4
202
7.41E−07
7
1
RPIB9
5
2
0
7


87191931












chr19: 9256584-
4
0
0.001
3
0
ZNF699
2
0
1
3


9277749












chr4: 39190766-
6
0
0.004
3
1
UGDH
1
2
0
3


39201960












chr7: 18903915-
4
23171
0.009
3
2


HDAC9


1
2
0
3


18905725












chr5: 170531269-
4
374
0.01
2
0
DKFZp666P032,
1
1
0
2


170536993





RANBP17






chr7: 34685030-
4
4517
0.01
2
0
AAA1, NPRS1
1
1
0
2


34686172












chr1: 170400944-
4
26274
0.01
2
0
DNM3
0
1
1
2


170403742










Additional detail in Table 12.









TABLE 4







Large Genic CNVs Impacting Single CVID Cases not Observed in Controls












Distance




Count
From



CNVR Del Singleton
SNPs
Exon
Gene










A) Deletions Impacting Immune Function Genes










chr4: 86650109-87684765
209
0
ARHGAP24, BC038746, MAPK10


chr16: 75457204-76988778
78
0
ADAMTS18, AK026469, BC035731, CLEC3A, KIAA1576, MON1B, WWOX


chr3: 74573112-74866049
66
0


CNTN3




chr19: 56625454-56761712
64
0
LOC729767, SIGLEC12, SIGLEC6, SIGLEC8


chr4: 103671292-104700248
51
0
AK093356, BDH2, CENPE, CENPE variant





protein, CR604221, LOC133308, MANBA, NFKB1, NHEDC1, UBE2D3, UNQ6308, ZCD2


chr14: 43997374-46152755
45
0
AX748292, BC038722, C14orf106, C14orf155, C14orf28, FANCM, FKBP3, KIAA0423,





KLHL28, PRPF39


chr6: 29844580-30045812
44
0
AK097625, BC035647, HLA-A, HLA-A*0226, HLA-G, HLA-







G2.2
, LOC554223, LOC642032, NR_001317, NR_001318, NR_002139



chr2: 86695685-87945980
40
0
ANAPC1, BC066991, CD8A, CD8B, DQ576041, LOC285074, MGC4677, PLGLB1, PLGLB2,





RGPD1, RGPD2, RMND5A, RNF103


chr6: 22380732-22525808
40
0


PRL




chr6: 31465923-33432505
34
0
AF075059, AGER, AGPAT1, AIF1, AK057104, AL050203, APOM, ATP6V1G2, B3GALT4,







BAT1
, BAT2, BAT3, BAT4, BAT5, BF, BRD2, BTNL2, C2, C4A, C4A variant






protein, C6orf10, C6orf21, C6orf25, C6orf26, C6orf27, C6orf31, C6orf47, C6orf48, CFB, CLIC1,





COL11A2, CREBL1, CS266662, CSNK2B, CYP21A2, DAXX, DDAH2, DKFZp313H139,





DKFZp547I194, DKFZp779M0311, DOM3Z, EGFL8, EHMT2, FKBPL, G18.1a, G6E, G6e, G7c,





G8, GPSM3, HCP5, HLA-DMA, HLA-DMB, HLA-DOA, HLA-DOB, HLA-DPA1, HLA-







DPB1, HLA-DPB2, HLA-DQA1, HLA-DQA2, HLA-DQB1, HLA-DQB2, HLA-DRA, HLA-









DRB1, HLA-DRB5,
HSD17B8, HSPA1A, HSPA1B, HSPA1L, LOC401252, LSM2, LST1, LTA,








LTB, LY6G5B
, LY6G5C, LY6G6C, LY6G6D, MCCD1, MEGT1, MICA, MICB, MSH5, NCR3,






NEU1, NFKBIL1, NG36/G9a, NOTCH4, NR_002742, NR_002745, NR_002812, NR_002971,





NR_003140, PBX2, PFDN6, PP199, PPT2, PRRT1, PSMB8, PSMB9, RAGE, RDBP, RGL2,





RING1, RNF5, RP, RPS18, RXRB, SKIV2L, SLC39A7, SLC44A4, STK19, TAP1, TAP2,







TAPBP, TNF, TNFa
, TNXB, VARS, VPS52, WDR46, ZBTB12, ZBTB22



chr2: 70616413-71523501
30
0
ADD2, AF090102, ANKRD53, ATP6V1B1, CD207, CLEC4F, FIGLA, KIAA1155, MCEE,





MPHOSPH10, N-Acetylglucosamine kinase, NAGK, NP220, OR7E91P, TEX261, TGFA, VAX2,





ZNF638


chr8: 95257759-96161427
28
0
AF086017, AX747981, C8orf38, CCNE2, CDH17, DPY19L4, FSBP, GEM, INTS8, KIAA1429,





RAD54B, RBM35A, TP53INP1, p53DINP1


chr1: 184639488-185615061
24
0
C1orf27, PDC, PLA2G4A, PTGS2


chr4: 124486894-125137357
21
0
AL833449, BC053945, SPRY1


chr1: 226522770-226673588
20
0


KIAA1556, KIAA1639, OBSCN
, TRIM11, TRIM17



chr21: 14806346-14824849
17
0


HACS1, SAMSN1




chr7: 84542383-84633646
16
0


SEMA3D




chr4: 162635366-163113553
15
0


FSTL5




chr19: 48834611-49626859
15
0
AK090553, AK098175, AK131520, AX748287, BC041923, BC045755, CADM4, CR593740,





DKFZp564H1322, FLJ12886, HZF19, HZF6, IRGC, KCNN4, LYPD5, PLAUR ZNF155,





ZNF221, ZNF222, ZNF223, ZNF224, ZNF225, ZNF226, ZNF227, ZNF228, ZNF229,





ZNF230, ZNF233, ZNF234, ZNF235, ZNF284, ZNF285A, ZNF404, ZNF45


chr4: 90603695-91299621
14
0
AK123890, CR605611, KIAA1680, MGC48628, MMRN1, SNCA


chr6: 138020053-138532047
14
0
AK124173, PERP, TNFAIP3


chr7: 30225447-30798217
14
0
AK096056, AK096687, AK097240, AL137445, BC016976, BC041636, BX648714, C7orf24,





CR595731, CRH2R, CRHR2, FLJ22374, GARS, INMT, NOD1, NR_002186, ZNRF2


chr12: 55685282-56515644
14
0
ARHGAP9, AVIL, B4GALNT1, BC019026, BC033961, BC073932, CDK4, CENTG1,





CR625050, CS444342, CTDSP2, CYP1, CYP27B1, DCTN2, DDIT3, DTX3, FAM119B, G43318,





GEFT, GLI1, INHBC, INHBE, KIAA0286, KIAA1002, KIF5A, LRP1, MARCH9, MARS,





MBD6, METTL1, MYO1A, NAB2, NDUFA4L2, NXPH4, OS9, PIP5K2C, R3HDM2, RGL1,





SHMT2, SLC26A10, STAC3, STAT6, TAC3, TSFM, TSPAN31, ZBTB39, ZNEUROK1


chr16: 86267434-86992013
14
0
AK126852, AX747795, BANP, CA5A, DKFZp434G0522, FLJ00104, JPH3, KLHDC4, SLC7A5


chr2: 179266611-179839804
13
0
AK123298, CR624402, FLJ39502, SESTD1, TTN


chr3: 95106214-95125111
12
0


PROS1




chr2: 36745429-37640775
11
0
AK001814, BC017652, CCDC75, CEBPZ, EIF2AK2, HEATR5B, KIAA1414, PRKD3, PRO1853,





QPCT, STRN, SULT6B1, VIT


chr4: 151504432-151570906
11
0


DKFZp686K03100, LRBA




chr11: 127094060-128138864
11
0
AX747861, BC039676, DKFZp686D0662, ETS1, FLI1


chr1: 31912222-32647867
10
0
AK096192, BAI2,





BAI2, BC069257, BC111382, BSDC1, C1orf90, C1orf91, CCDC28B, COL16A1, CR601100,





DCDC2B, DKFZp564C2082, DKFZp686B09139, EIF3S2, HDAC1, IQCC, KHDRBS1, KPNA6,







LCK
, LOC339483, MARCKSL1, PTP4A2, SPOCD1, TMEM39B, TSSK3, TXLNA, UNQ548



chr3: 106447305-107177994
10
0


ALCAM, CBLB, Nbla00127




chr6: 161278798-161959701
10
0
AGPAT4, C6orf59, LPAAT-delta, MAP3K4, MTK1, PARK2, parkin


chr7: 24205709-24955079
10
0
DFNA5, ICERE-1, KIAA0704, MPP6, NPY, OSBPL3







B) Deletions Not Impacting Known Immune Function Genes










chr3: 163785970-165636507
94
0
BC073807, LOC647107


chr2: 78159130-78549747
86
0
BC024248, BC030125


chr3: 7053179-7177909
49
0
GRM7


chr11: 109142987-110423998
48
0
AK094117, AK124179, ARHGAP20, DKFZp434I0812, FDX1, KIAA1726, NR_001287, RDX


chr12: 27002588-28111983
44
0
AK000807, ARNTL2, BC043511, DKFZp564O1863, FGFR1OP2, KIAA0965, KIAA1230,





KLHDC5, LOC728858, MDS023, MRPS35, PPFIBP1, PTHLH, REP15, STK38L, SURB7,





TM7SF3


chr6: 97170276-97625708
39
0
C6orf66, FHL5, GPR63, KIAA1900, KLHL32


chr6: 163188894-163315053
35
33591
PACRG


chr16: 9624870-10467886
35
0
ATF7IP2, DKFZp666E123, DQ587956, DQ595173, GRIN2A, U07199


chr7: 64220284-64795508
34
0
AK057766, DQ596928, FLJ25037, LOC441242, ZNF92


chr5: 90035491-90386715
33
0
GPR98, KIAA0686, VLGR1


chr3: 37578530-37705809
32
0
ITGA9


chr6: 38413748-38630608
32
0
BTBD9


chr20: 51844911-51931244
28
0
NR_002189


chr15: 57055702-57917444
26
0
BNIP2, CCNB2, DKFZp761D081, FAM81A, GCNT3, GTF2A2, LDHAL6B, MYO1E, RNF111


chr15: 54225066-54550705
24
0
MNS1, TEX9


chr17: 3128134-3194433
23
0
OR3A1, OR3A2, OR3A4


chr7: 7480856-7819889
22
0
AK027125, COL28, COL28A1, FLJ20323, RPA3


chr10: 94222227-94754640
22
0
EXOC6, HHEX, IDE, KIF11


chr14: 91502061-91569101
21
0
TRIP11, Trip230


chr2: 42398844-43092929
19
0
COX7A2L, EML4, HAAO, KCNG3, MTA3, OXER1


chr14: 26535543-27350537
19
0
BX538073


chr14: 92744837-93367366
19
0
BTBD7, C14orf130, COX8C, KIAA1409, PRIMA1


chr2: 14622841-14924411
18
0
AX747684, FAM84A


chr12: 10589522-10635183
17
0
KLRA1


chr3: 99229541-99419735
16
0
CR749263, OR5AC2, OR5H1, OR5H14, OR5H15


chr2: 132326867-132449751
15
0
FLJ41821


chr4: 9136858-9881191
15
0
DKFZp586I2219, DQ581767, DQ583133, DQ584082, DQ585713, DQ589421, DRD5, SLC2A9,





WDR1


chr4: 128805691-128920529
15
0
INTU, SLC25A31


chr9: 16987947-17478111
15
0
C9orf39, RP11-340N12.1


chr14: 59952568-60075378
15
0
C14orf39, SIX6


chr18: 5682313-5922670
15
0
AF301223, TTMA


chr19: 23304120-23389292
15
0
AK022793, BC038574, BC043213, ZNF91


chr12: 39980210-40809427
14
0
GLT8D3, PDZRN4


chr19: 19759149-20368239
14
0
CR593334, CR614976, DKFZp761G18121, FLJ44894, NR_003128, ZNF253, ZNF486, ZNF506,





ZNF682, ZNF90, ZNF93


chr2: 98228529-98252357
13
0
DKFZp434J0326, DKFZp451I0318, MGC26733


chr2: 168309981-169429627
13
0
AL080192, B3GALT1, BC035245, LASS6, NOSTRIN, STK39


chr4: 22663503-22803604
13
0
CR607430


chr5: 107695465-109245567
13
0
AK021888, BC034788, FBXL17, FER, MAN2A1, PJA2


chr4: 20984915-21185852
12
0
KCNIP4


chr5: 178315216-178514465
12
0
ADAMTS2, BX648737, CR598488, GRM6, ZNF354C, ZNF454, mGluR6


chr17: 55759021-56436171
12
0
APPBP2, BCAS3, C17orf64, L32131, PPM1D, USP32


chr18: 60137564-60597211
12
0
BC036306


chr5: 38305919-38317501
11
11209
EGFLAM


chr6: 12643097-13219417
11
0
PHACTR1, RPEL


chr4: 69405358-69494103
10
0
TMPRSS11E


chr9: 40497792-40617437
10
0
AK024257


chr11: 102824056-102856185
10
0
DYNC2H1


chr11: 124360372-124420328
10
0
CCDC15


chr12: 47817725-47931507
10
0
TUBA1A, TUBA1B, TUBA1C


chr14: 62671250-62971495
10
0
GPHB5, PPP2R5E, RHOJ


chr15: 91046103-91378387
10
0
CHD2, DKFZp781D1727, LOC400451







C) Duplications Impacting Immune Function Genes










chr19: 5773490-12007370
827
0


ACP5
, ACSBG2, ADAMTS10, AF019226, AF075036, AF161365, AK056073, AK097685,






AK124717, ALKBH7, ANGPTL4, ANGPTL6, ANKRD25, ANKRD47, AP1M2, ARHGEF18,





ASAH3, ATG4D, AX747405, AX747599, AX748210, AY203940, BC007593, BC014506,





BC029904, BC033124, BC039523, BC042816, BGR, C19orf39, C19orf45, C19orf52,







C19orf59
, C3, CAPS, CARM1, CCL25, CD209, CD320, CD70, CDC37, CDKN2D,








CLEC4G
, CLEC4M, CLPP, CNN1, COL5A3, CR598956, CRB3, CTXN1, DENND1C,






DKFZp547H118, DKFZp564K0223, DKFZp564O1762, DKFZp666A071,





DKFZp667O2312, DKFZp761J1410, DKFZp761K0816, DNM2, DNMT1,





DOCK6, ECSIT, EDG5, EDG8, EIF3S4, ELAVL1, ELAVL3, ELOF1, EMR1,







EMR4
, EPOR, EVI5L, FBN3, FBXL12, FCER2, FLJ00153, FLJ11286, FLJ12949,






FLJ20079, FLJ22184, FLJ25758, FUT3, FUT5, FUT6, GPR108, GTF2F1, HNRPM, HSZFP36,







ICAM1
, ICAM3, ICAM4, ICAM5, ILF3, INSR, KEAP1, KHSRP, KIAA0521, KIAA1395,






KIAA1518, KIAA1543, KIAA1588, KIAA1776, KIAA1978, LASS4, LDLR, LOC126075,





LOC162993, LOC388503, LOC401898, LOC440508, LOC55908, LPPR2, LRRC8E, MAP2K7,





MARCH2, MBD3L1, MBD3L2, MCOLN1, MGC19604, MGC20983, MGC33407, MKK7,





MLLT1, MRPL4, MUC16, MYO1F, MYO1F variant





protein, NDUFA11, NDUFA7, NRTN, OLFM2, OR1M1, OR2Z1, OR7D2, OR7D4, OR7E24,





OR7G1, OR7G2, OR7G3, P2RY11, PCP2, PDE4A, PEX11G, PIN1, PNPLA6, PPAN, PPAN-





P2RY11, PRAM1, PRKCSH, PSPN, QTRT1, RAB11B, RAB3D, RANBP3, RAVER1, RDH8,







RETN
, RFX2, RGL3, RPS28, SH2D3A, SLC25A23, SLC25A41, SLC44A2, SMARCA4,






SNAPC2, SPC24, STXBP2, TIMM44, TMED1, TNFSF14, TNFSF9, TRAPPC5, TRIP10,





TSPAN16, TUBB4, TYK2, UBL5, UNQ2443, UNQ501, VAV1, VMAC, XAB2, YIPF2,





ZNF121, ZNF177, ZNF266, ZNF317, ZNF358, ZNF414, ZNF426, ZNF433, ZNF439,





ZNF440, ZNF441, ZNF491, ZNF557, ZNF558, ZNF559, ZNF560, ZNF561, ZNF562,





ZNF627, ZNF653, ZNF69, ZNF699, ZNF700, ZNF763, pp10122


chr14: 92664804-95787550
448
0
AK125038, ASB2, BC016484, BC037859, BC038791, BDKRB2, BTBD7, BX247990,





C14orf109, C14orf130, C14orf132, C14orf139, C14orf142, C14orf152, C14orf48, C14orf49,





CLMN, COX8C, CR611440, DDX24, DICER1, FAM14A, FAM14B, GIG24, GLRX5, GSC,





IFI27, KIAA0928, KIAA1409, KIAA1622, MOAP1, NM_207443,





NR_001459, NR_003002, OTUB2, P27, PRIMA1, SERPINA1, SERPINA10, SERPINA11,





SERPINA12, SERPINA13, SERPINA3, SERPINA4, SERPINA5, SERPINA6, SERPINA9,







TCL1A
, TCL1B, TCL6, TCL6a1, TCL6d1, TML1



chr11: 7678446-10337351
315
0


ADM
, AK000908, AK055772, AK090613, ASCL3, AX747224, BC017787, BC027619,






BC068088, BC073899, BCA3, C11orf16, C11orf17, CMT4B2, CR598129,





DKFZp451C1317, DKFZp686I18166, EIF3S5, IPO7, KIAA0298, KIAA1766,





LMO1, MGC10850, NLRP10, NRIP3, NR_002580, NR_002962,





NR_002977, OR10A3, OR10A6, OR5E1P, OR5P2, OR5P3, OVCH2, RAB6IP1, RIC3,





RPL27A, SBF2, SCUBE2, ST5, STK33, SWAP70, TMEM41B, TMEM9B, TUB, U80769,





WEE1, ZNF143


chr13: 100600344-103567030
197
0
AK093430, AK096424, AK125748, AX747578, BIVM, C13orf27, CR616826, DKFZp434L2319,





ERCC5, FGF14, ITGBL1, KDELC1, LOC196541, RP11-430M15.2-





003, SLC10A2, TPP2, UNQ1910, VGCNL1


chr14: 65357663-66177823
159
0
CR594591, GPHN, MGC88374


chr4: 119628184-120591177
158
0
AK000709, AK024248, AK097701, AK098126, BC035733, BC070391, CEP170L, DQ574659,





DQ575011, DQ575856, DQ576410, DQ582480, DQ599872, FABP2, KIAA0755, KIAA1627,





LOC401152, MYOZ2, Myopodin, SEC24D, SYNPO2, USP53


chr8: 106761674-108458326
156
0
ABRA, ANGPT1, CR602836, Nbla00307, OXR1, ZFPM2


chr4: 113049611-113759418
129
0
ALPK1, C4orf16, C4orf21, C4orf32, CR590073, DKFZp434C0927, FLJ00302, KIAA1527,





LOC91431, NEUROG2, TIFA


chr15: 40145534-40608559
90
0


CAPN3
, GANC, KIAA0770, PLA2G4D, PLA2G4F, SNAP23, TMEM87A, VPS39, ZFP106



chr14: 43658461-45064383
87
0
AX748292, BC038722, C14orf106, C14orf155, C14orf28, FANCM, FKBP3, KIAA0423,





KLHL28, PRPF39


chr10: 67399581-69536663
75
0
CTNNA3, DNAJC12, HERC4, LRRTM3, MYPN, SIRT1


chr3: 107041527-108030375
71
0


CBLB




chr9: 137454076-139460602
70
0
ABCA2, AF161442, AGPAT2, AK023162, AK054908, AK055547, AK090585, AK096249,





AK098241, AK128153, AK128864, AL832276, ANAPC2, AX747706, AY952890, BC015688,





BC032375, BC034456, BC042667, BC043225, BC061888, BC064596, BC092490, BC101937,





BTBD14A, C8G, C9orf116, C9orf139, C9orf140, C9orf142, C9orf163, C9orf167, C9orf75,





C9orf86, CAMSAP1, CARD9, CLIC3, COBRA1, CR614579, CR619051, DKFZp434B205,





DKFZp564M173, DKFZp762I052, DPP7, EDF1, EGFL7, ENTPD2, ENTPD8, FAM69B, FBXW5





FLJ20433, FLJ45224, FUT7, GBDR1, GLT6D1, GPSM1, GRIN1, HBE269, INPP5E, KCNT1,





KIAA0310, KIAA0649, KIAA1062, KIAA1422, KIAA1984, LCN1, LCN10, LCN12, LCN6,





LCN8, LCN9, LHX3, LOC389813, LOC389816, LOC401565, LOC441476, LOC728489,





MAMDC4, MAN1B1, MGC14327, MGC59937, MGC61598, MRP-





S2, MRPS2, NDOR1, NOTCH1, NOXA1, NPDC1, NPTIIc, NR1, NR_002958,





NR_002975, OBP2A, PAEP, PHPT1, PMPCA, PTGDS, QSCN6L1, RNF208,





RP11-100C15.2, RP11-413M3.10, SDCCAG3, SLC34A3, SNAPC4, SOHLH1, SSNA1,





TMEM141, TRAF2, TUBB2C, UAP1L1, UBADC1, UNQ2492, UNQ2541, UNQ747,





hNMDAR1-3b, pp8875, ve-statin


chr18: 73989470-76116152
60
0
AK056304, ATP9B, AX746671, BC016878, BC017654, BC040056, BX537710, C18orf22,





CTDP1, DKFZp434A042, KCNG2, NFATC1, PARD6G, PQLC1, SALL3, TXNL4A, ZNF508,





hdim1+


chr9: 134048500-135886847
54
0
ABO, ADAMTS13, ADAMTSL2, AJ011378, AK123314, AX748058, BARHL1, C9orf166,





C9orf7, C9orf9, C9orf96, C9orf98, CEL, CELL, CR592591, CR593670, DBH, DDX31,





EEF1A1, FLJ46082, GBGT1, GFI1B, GTF3C4, GTF3C5, KIAA1308, LOC389827,





NR_002783, NTNG2, OBP2B, RALGDS, REXO4, RPL7A, SARDH, SETX,







SLC2A6
, SURF1, SURF2, SURF4, SURF5, SURF6, TSC1, TTF1, UNQ2513, VAV2,






XPMC2H, vWF-CP


chr1: 2182293-3539057
50
0


ACTRT2
, AK021767, AK124865, ARHGEF16, BC114358, C1orf93, CR749717, DQ601700,






FAM79A, FLJ42875, HES5, MEGF6, MMEL1, MORN1, PANK4, PEX10, PLCH2, PRDM16,





RER1, RP3-395M20.1, RP3-395M20.10, RP4-740C4.2, SKI, TNFRSF14, WDR8


chr1: 245537326-245976637
49
0
AB120962, AK130400, BC034303, C1orf150, CIAS1, NLRP3, OR13G1, OR2B11, OR2C3,





OR2G2, OR2G3, OR2W5, OR5AY1, OR6F1, ZNF496


chr14: 21582956-21976908
47
0
AK093552, AK125397, AV1S3A1T, AV25S1, AV4S1, TCR-[alpha] V 33.1, TCR-







alpha, TCRA, TCRAV14.1a, TCRAVN1, TCRD, TRA@, V alpha









immunoglobulin
, av27s1, hADV14S1, hADV23S1, hADV29S1, hADV38S2, hDV102S1



chr7: 157944418-158676623
46
0
AX746826, BC041429, BC042556, FAM62B, KIAA1228, NCAPG2, PTPRN2, VIPR2, WDR60


chr19: 48498399-48562727
46
0


CD177




chr22: 34946081-35041308
41
0


APOL1
, APOL2, AX747758, MYH9



chr1: 113185538-113437683
40
0
AFARP1, AX748125, BC023568, BC037540, BC047723, BX648855, LRIG2, SLC16A1


chr2: 187993955-189286647
39
0
CALCRL, CED-6, GULP1, TFPI


chr11: 382079-1386192
38
0
AK094678, AK126635, AP2A2, AX747537, AX748330, BC031953, BC048998, BC066355,





BRSK2, C11orf35, CD151, CEND1, CHID1, DEAF1, DKFZp434K249, DRD4, EFCAB4A,





EPS8L2, HRAS, IRF7, KIAA0899, KIAA1542, LRDD, LRRC56, MG1, MUC5AC,







MUC2
, MUC5AC, MUC5B, MUC6, MUCDHL, NR_002585, PDDC1, PEN11B, PKP3,






PNPLA2, POLR2L, PTDSS2, RASSF7, RNH1, RPLP2, SCT, SIGIRR, SLC25A22,





TALDO1, TMEM16J, TMEM80, TOLLIP, TSPAN4, tollip


chr12: 98815353-99473749
37
0
ACTR6, ANKS1B, AX746635, BC048272, BC062763, DEPDC4, DKFZp434M0331, DQ579381,





DQ583972, DQ595598, DQ598729, EB-





1, GOLGA2L1, KIAA0701, NR1H4, SCYL2, SLC17A8, hArpX


chr4: 7568079-8105372
36
0
ABLIM2, AFAP1, AJ431609, BC043614, KIAA1808, LOC389199, SORCS2


chr14: 23995185-24062459
30
0
AK056368, CMA1


chr14: 103352857-104756910
30
0


ADSS
, ADSSL1, AK057986, AK094143, AKT1, AX721091, AX746996, BRF, BRF1,






C14orf151, C14orf173, C14orf2, C14orf78, C14orf79, CDCA4, CR602005,





DKFZp434N0820, DKFZp434N178, DKFZp686J02145, GPR132, JAG2, KIAA0284,





KIAA0771, KIF26A, LOC374569, LOC400258, MGC23270, NUDT14, PLD4, PPP1R13B,





SIVA1, TDRD9, TMEM179, UGPP


chr17: 73662981-73765190
23
0
AFMID, BC036810, BIRC5, EPR-1, SYNGR2, TK1, UNQ464/PRO809, survivin-3B


chr13: 45066410-45918722
22
0
AK095119, AK124928, C13orf18, CPB2, FLJ32682, KIAA0853, LCP1, LOC220416,





LOC283514, RP11-139H14.4-001, RP11-351K3.2-001, SPERT, ZC3H13


chr19: 46758119-46863746
21
0


CEACAM21
, CEACAM4, UNQ3098



chr8: 6849317-6889488
20
0
AF355799, DEFA3


chr20: 60320976-62223928
17
0
AK056267, AK128329, AL137301, ARFGAP1, ARFRP1, AX747649, AY940852, BC002534,





BC025345, BC069708, BC127852, BHLHB4, BIRC7, C20orf11, C20orf135, C20orf149,





C20orf151, C20orf166, C20orf195, C20orf20, C20orf58, C20orf59, CABLES2, CHRNA4,





COL20A1, COL9A3, DIDO1, DNAJC5, EEF1A2, FLJ00084, FLJ00118, FLJ30313, GATA5,





GMEB2, HRIHFB2281, KCNQ2, KIAA1088, KIAA1269, KIAA1510, LAMA5, LIME1,





LOC198437, NPBWR2, NR_003244, NR_003245, NTSR1, OATP-E,





OGFR, OK/SW-cl.69, 0PRL1, PRIC285, PRPF6, PRR17, PTK6, RGS19, RP4-





697K14.11, RPS21, RTEL1, SAMD10, SLC2A4RG, SLCO4A1, SOX18, SRMS, STMN3, Si-





1-2-





19, TCEA2, TCFL5, TNFRSF6B, TPD52L2, UCKL1, URKL1, YTHDF1, ZBTB46, ZGPAT,





ZNF512B


chr2: 86924985-87026840
16
0


CD8B
, RGPD1, RMND5A



chr6: 128245227-128538250
16
0


C6orf190
, PTPRK



chr1: 111628132-111661435
15
0


CHIA
, RP11-165H20.1



chr7: 80041654-80110504
13
0


CD36




chr12: 6085895-6134080
11
0


VWF




chr12: 17854127-19338878
11
0


CAPZA3
, FLJ22655, PIK3C2G, PLCZ1, PLEKHA5








D) Duplications Not Impacting Known Immune Function Genes










chr8: 430424-977192
189
0
AK128400, BC022082, BC038783, C8orf42, DQ584928, ERICH1, LOC389607


chr10: 2682656-3123648
157
0
PFKP


chr19: 21575835-22802826
152
0
BC030765, CR936832, FKSG70, ZNF100, ZNF208, ZNF257, ZNF43, ZNF492, ZNF676, ZNF99


chr6: 63118894-64091007
116
0
GLULD1, LGS


chr2: 126935385-127317174
98
0
GYPC


chr5: 169763949-170518986
96
0
DKFZp666P032, GABRP, KCNIP1, RANBP17


chr6: 1977258-2424512
92
0
AJ420566, AK023629, AK091028, CR598484, GMDS


chr1: 145196592-145539979
65
0
BC036212, BCL9, CHD1L


chr3: 62813828-63789968
63
0
BC043407, C3orf49, CADPS, FLJ44379, SYNPR


chr5: 172824934-173106866
62
0
BC033564, FAM44B


chr3: 83912925-84849961
54
0
BC068246


chr11: 106523381-106702850
54
0
CWF19L2


chr7: 50911090-51127086
50
0
COBL


chr9: 6557841-7007391
41
0
AK098534, BC042976, GLDC, JMJD2C, KIAA0780


chr17: 793231-917163
40
0
ABR, NXN, TIMM22


chr3: 148425802-149211699
39
0
AK098763, ZIC1, ZIC4


chr13: 62536330-64097876
36
0
AK057471, AK097490, AK098560, BC128161, NR_002171


chr4: 92669000-94351161
34
0
GRID2, KIAA1680


chr3: 11041670-11154453
33
0
SLC6A1


chr4: 141709869-141939520
33
0
TBC1D9


chr13: 113032851-113140550
30
0
ADPRHL1, GRTP1


chr2: 148947731-149053581
29
0
KIAA1461, MBD5


chr3: 141649121-141911988
28
0
CLSTN2, TRIM42


chr12: 20901315-21459060
27
0
IAPP, LST-3TM12, LST3, SLCO1A2, SLCO1B1, SLCO1B3


chr12: 130676799-131128244
27
0
EP400, KIAA1498, KIAA1818, MMP17, NR_002979, PUS1, SFRS8, ULK1


chr7: 34919485-35862821
26
0
AJ011981, BC049371, BC084560, CR593784, CR595224, DPY19L1, DPY19L2P1, HERPUD2,





KIAA0877, SEPT7, T8X20


chr1: 30940148-31013899
25
0
BC044253, LAPTM5, MATN1


chr4: 63433227-65099386
25
0
SRD5A2L2


chr5: 12838251-13150705
22
0
AY328033


chr6: 138653005-138759016
22
0
KIAA1244


chr1: 103111860-103157198
21
0
COL11A1


chr6: 34836231-34976532
21
0
ANKS1A, C6orf107, SNRPC, TAF11


chr15: 27167056-28188067
21
0
APBA2, BC043570, BC070492, BC071630, BC071855, DQ572986, DQ573498, DQ575284,





DQ575742, DQ577333, DQ578370, DQ578838, DQ582641, DQ582940, DQ590322, DQ592322,





DQ595055, DQ596303, DQ596319, DQ597873, KIAA0574, NDNL2, TJP1, hXIIL


chr2: 3287465-3327227
20
0
TSSC1


chr10: 98101269-98152781
19
0
TLL2, TMEM10


chr1: 79037280-79973037
18
0
ELTD1


chr4: 68749536-70089270
18
0
AK123556, TMPRSS11B, TMPRSS11E, UGT2A3, UGT2B10, UGT2B15, UGT2B17, UGT2B7,





YTHDC1


chr2: 27584444-28424525
17
0
AK124439, BC041993, BC048132, BRE, C2orf16, CCDC121, GCKR, MRPL33, RBKS,





SLC4A1AP, SUPT7L, XAB1, ZNF512


chr2: 172356838-172528561
17
0
HAT1, SLC25A12


chr2: 180123158-180216026
16
0
ZNF533


chr4: 15529521-15552238
15
0
FGFBP1


chr18: 14805527-14905835
15
0
ANKRD30B


chr5: 1601764-1621442
14
0
CR749689


chr10: 88722456-89266679
14
0
AK091716, BC036645, BC047063, BC065757, BC082979, BC092519, CR609725, CR614919,





FAM35A, GLUD1, KIAA1975, KIAA2020, MINPP1


chr1: 225308870-225407413
13
0
CDC42BPA


chr7: 74674968-74738702
13
0
LOC441257, PMS2L14


chr1: 193808000-194531039
12
0
KCNT2, SLICK


chr2: 165344115-165435694
12
0
COBLL1, KIAA0977


chr6: 123847128-124726881
12
0
TCBA1, TRDN


chr16: 26961662-26994169
12
0
TNT


chr6: 102029392-102048325
11
11862
GRIK2


chr8: 11397047-11414199
11
0
BLK


chr8: 27689983-27745887
11
0
ESCO2, PBK


chr2: 77709215-78761206
10
0
BC024248, BC030125


chr11: 5444172-5454375
10
11939
HBE1, HBG2, OR51B5


chr11: 19571323-19595111
10
96377
NAV2


chr18: 68553733-69070964
10
0
BC013370, BC034583, NETO1


chr19: 34552727-34574348
10
0
AK094793


chr4: 2059850-2960297
10
0
AB000464, AB000465, AB000466, ADD1, AK054619, BC010180, BC032331, C4orf10, C4orf15,





C4orf8, CR622423, GRK4, KIAA1643, MXD4, Mad4, NOL14, POLN, RNF4, SH3BP2,





TETRAN, TNIP2, ZFYVE28
















TABLE 5







Homozygous Deletions Observed Exclusive to Cases












Cases


Distance From


CNVR
Loss
Gene
Distance
Exon














chr3: 100430538-100430538
2
CLCP1, DCBLD2
327315
327315


chr2: 118777060-118778863
1
INSIG2
192993
192993


chr3: 146130742-146141275
1
DQ595575
883692
883692


chr5: 29455914-29488308
1
AK098570
247692
247692


chr5: 141998044-142000692
1


FGF1


0
16953


chr6: 32525108-32530999
1


HLA-DRA


4306
4306


chr6: 62122152-62144592
1
G43499
251536
251536


chr6: 77159914-77159914
1
IMPG1
320859
320859


chr6: 128018101-128024702
1


C6orf190


46336
46336


chr12: 17277311-17282429
1
LMO3, Nbla03267
625020
625020
















TABLE 6





Genome Global CNV Burden

















Significant Including Common CNV Exonic














Locus

Locus






Exonic
Locus Non-
Exonic
Locus Non-





Significant
Exonic
Significant
Exonic





Case
Case
Control
Control





Enriched
Enriched
Enriched
Enriched
P
DifFreq





Deletion
21
61
8
67
0.022628
0.149431


Duplication
43
173
12
12
0.003336
−0.30093


















Case CNV

Control





Case CNV
Calls Non-
Control CNV
CNV Calls





Calls Exonic
Exonic
Calls Exonic
Non-Exonic
P
DifFreq












Rare CNV Exonic













Deletion
527
1149
3970
10290
0.002138
0.036038


Duplication
813
1785
5539
5997
2.45E−55
−0.16722









Rare CNV >100 KB Exonic













Deletion
136
83
1012
653
0.768312
0.013197


Duplication
278
146
2263
546
2.46E−11
−0.14996












Samples w Rare CNV >100 KB (Impacting gene or not)















Case

Control





Case with
without
Control with
without





large rare
large rare
large rare
large rare





CNV
CNV
CNV
CNV
P
DifFreq





Deletion
131
180
1156
1610
0.951669
0.00329


Duplication
186
125
1391
1375
0.001504
0.095178









Finally, to address the heterogeneity of the CVID phenotype and comorbid clinical manifestations that affect subsets of patients, we compared CVID patients with common sub-phenotypes to patients without any additional comorbidities. Based on recent phenotypic characterization22, we established 16 distinct clinical subtracks within CVID to study: cancer, lymphoma, lymphadenopathy, nodular regenerative hyperplasia of the liver (NRH), lymphoid interstitial pneumonitis (LIP), bronchiectasis, biopsy proven granuloma, GI enteropathy, malabsorption, splenectomy, cytopenias, organ specific autoimmunity (OSAI), low IgM (<50 mg/dL), low IgA (<10 mg/dL), low B cells (CD19+ cells <1%), and young age of symptom onset (<10 yrs) (FIG. 5 and Table 7). The case and control labels within the CVID cohort were based on previously defined criteria. Differences in genotype frequencies were assigned significance and regions with multiple significant SNPs were then scored. Significant SNP associations were made for all subsets (Table 8 and FIG. 1C). The most significant association was observed with NRH on AK096081-AK124028 (P=2.29×10−10). Lymphoma was associated with KIAA0834, PFTK1 and HAVCR1, with corresponding P values ranging from P=1.69×10−8-3.62×10−8. An association was also observed between LIP and FGF14 and ZNF81, with corresponding P values of P=5.76×10−8 and P=7.70×10−8, respectively. OSAI also associated with SNX31 (P=6.89×10−8) and low IgM levels (<50 mg/dL) in the case subjects were associated with LDLRAP1 (P=6.02×10−8). Patients with CVID manifesting enteropathy and their resulting subphenotype significance were additionally queried against the IBD significance observations to assess if specific loci contributed to a common etiology. The SNP rs12889533 was significant in both CVID+enteropathy and IBD with allele frequency difference in the same direction (Table 9).









TABLE 7







CVID Subphenotypes and Sample Sizes











CVID





Subphenotype
Cases
Controls














Cancer
13
249



Lymphoma
9
253



Lymphadenopathy
30
232



NRH
13
249



LIP
10
252



Bronchiectasis
78
184



Granuloma
34
228



GIEnteropathy
19
243



Malabsorption
13
249



Splenectomy
40
222



Cytopenias
27
235



OSAI
75
187



Low IgM (<50 mg/dL)
206
56



Low IgA (<10 mg/dL)
160
102



CD19 (<1%)
11
251



AgeOnsetSx (<10 yrs)
42
220
















TABLE 8







Most Significant Associated Regions Based on Subphenotype Genotype Association


















Significant






Count


Distance



Region
P-value
SNP
A1
F_A
F_U
OR
SNPs
Gene
Distance
From Exon
Subphenotype





















chr1: 68153970-
2.29E−10
rs1926283
C
0.2308
0.01807
16.3
3
AK096081,
0
33408
NRH


68161019







AK124028





chrX: 132092989-
5.50E−10
rs5977837
C
0.2778
0.02174
17.31
2
TFDP3
82912
82912
Lymphoma


132095451













chr10: 58755852-
1.36E−09
rs16910534
T
0.2222
0.01383
20.37
3
IPMK
858286
858286
Lymphoma


58767334













chr20: 55901032-
3.21E−09
rs8124301
T
0.4231
0.07631
8.877
5
C20orf85
174030
174030
Malabsorption


55985359













chr7: 90245135-
1.69E−08
rs975004
G
0.3333
0.03953
12.15
2
KIAA0834,
0
12544
Lymphoma


90245280









PFTK1







chr5: 156402979-
3.62E−08
rs10038271
T
0.7222
0.1877
11.25
2


HAVCR1


0
651
Lymphoma


156407976













chrX: 22422160-
5.71E−08
rs5925651
A
0.3462
0.1386
3.291
3
ZNF645
219665
219665
Bronchiectasis


22428888













chr13: 101641990-
5.76E−08
rs1336698
G
0.65
0.168
9.197
6
FGF14
0
69139
LIP


101673539













chr1: 25735669-
6.02E−08
rs2065970
G
0.06553
0.2411
4.529
5
LDLRAP1
0
0
Low IgM


25753653













chrX: 143116309-
6.77E−08
rs6649722
A
0.3
0.0377
10.94
3
UBE2NL
320286
320286
LIP


143119321













chr8: 101728102-
6.89E−08
rs7815950
G
0.2133
0.05615
4.559
2
SNX31
0
2334
OSAI


101728344













chr8: 13834782-
7.62E−08
rs2682665
C
0.1818
0.01394
15.71
3
SGCZ
149368
149368
Low B cells


13842376













chrX: 47642545-
7.70E−08
rs12387999
A
0.2
0.016
15.38
2
ZNF81
0
2262
LIP


47651320













chrX: 152295068-
9.84E−08
rs5987017
A
0.2143
0.04773
5.442
4
ZNF275
24829
24829
Young age


152303019













chr12: 2531014-
1.21E−07
rs4765961
C
0.4737
0.142
5.439
3


CACNA1C


0
0
GI Enteropathy


2538733













chrX: 30733771-
1.44E−07
rs11095197
T
0.1469
0.3431
3.034
2


MAP3K7IP3


18876
18876
Low IgA


30736604













chrX: 37803525-
1.54E−07
rs5918500
C
0.4878
0.2123
3.533
3
SYTL5
0
0
Young age


37832251













chrX: 53035117-
1.90E−07
rs10127016
G
0.3833
0.125
4.351
2
TMEM29
0
1409
Lymphade-


53039801










nopathy


chr7: 107306707-
2.16E−07
rs9690688
A
0.3636
0.06375
8.393
2
DLD
4709
4709
Low B cells


107314113













chr21: 18188480-
2.84E−07
rs7280675
G
0.1923
0.02008
11.62
2
CHODL
2636
2636
NRH


18192815













chr1: 55704352-
2.85E−07
rs356086
G
0.1111
0.00395
31.5
2
FLJ45337
112975
112975
Lymphoma


55706323













chr21: 45476504-
3.23E−07
rs4592938
A
0.1154
0.00602
21.52
2
C21orf89
1777
1777
NRH


45476918













chr14: 23168471-
4.57E−07
rs222723
C
0.6154
0.1968
6.531
2
DHRS2
0
0
Cancer


23169215













chr2: 109118795-
4.62E−07
rs375099
C
0.25
0.02976
10.87
2
POSH2
0
5794
LIP


109120561













chr12: 30583074-
4.82E−07
rs1905675
C
0.2
0.5453
0.209
2
IPO8
74732
74732
Lymphade-


30598457










nopathy


chrX: 39933689-
5.18E−07
rs2948491
A
0.18
0.04545
4.61
4
BCOR
12163
12163
OSAI


39956544













chr14: 64723761-
8.09E−07
rs4299072
A
0.1667
0.02802
6.938
4
BX161428
0
0
Lymphade-


64783067










nopathy


chr7: 117377201-
9.94E−07
rs17140937
C
0.2692
0.04418
7.971
2
CTTNBP2
76404
76404
Malabsorption


117382435





OR: Odds Ratio Additional detail in Table 13.













TABLE 9







CVID Patients with GI Enteropathy vs CVID Patients without Genotype Association Replication in IBD Based on Single SNP Sigificance






















P_CVID +











SNP
Gene
Distance
GIEnteropathy(IBD)
Allele1
F_A
F_U
OR
P_IBD
Allele1
F_A
F_U
OR






















rs6532122
GPRIN3
3497
0.001204
A
0.2632
0.0947
3.42
9.90E−8
A
0.0786
0.1072
0.7109


rs17041264
COLQ
196
0.000368
T
0.1579
0.0350
5.17
3.15E−6
T
0.0049
0.0135
0.3608


rs12889533
C14orf143
19903
0.000561
T
0.1316
0.4156
0.21
1.28E−4
T
0.3563
0.3878
0.8736





rs12889533 replicates with allele frequency in the same direction while rs6532122 and rs17041264 are significant in the opposite direction of allele frequency.






Finally, to identify potential functional biases specific to CVID, we evaluated clustering into specific INTERPRO categories using DAVID23: Database for Annotation, Visualization, and Integrated Discovery. We found two significant INTERPRO categories with genes contributing from several of our different analysis methods. First, “Interleukin 1/heparin-binding growth factor” P=9.60×10−3 including FGF1, FGF23, FGF6, and FGF14 from subphenotype analysis of lymphadenopathy, cytopenias, LIP, and low IgA. Secondly, “Immunoglobulin I-set” P=5.40×10−2 including CHL1, CNTN4, LINGO2, SDK1 from overall GWAS analysis, cytopenias subphenotype, and duplication CNV.


Discussion

CVID as a clinical phenotype was described more than 50 years ago, but aside from a small number of recessively inherited genes in a few families, and the more prevalent but poorly understood contribution of mutations in TNFRSF13B5,6,7,24, other causes have remained obscure. CVID has thus been hypothesized to represent a diverse collection of genetic lesions resulting in a similar immunologic phehotype. The MHC region has been associated with a myriad of complex diseases25 including immune-related conditions26 and CVID8,9. MHC was robustly associated with this CVID in our study, confirming the prior work that was not performed at the genome-wide level. However, the SNP genotype association analysis presented here also revealed novel genes, with the most significant CVID associations outside of the MHC region with a locus encompassing ADAM28, ADAM7, ADAMDEC1 and STC1 (P=6.24×10−6). The ADAM family proteins are zinc metalloproteases involved in diverse biologic processes, including immune responses. Interestingly, the metallopeptidase MMP27 was also associated with CVID (P=1.69×10−4). Proteins of the matrix metalloproteinase (MMP) family are involved in the breakdown of extracellular matrix to promote routine physiological processes, but may also facilitate disease pathogenesis. Related genes have demonstrated immunological function involved in the regulation of cytokine release, Th2 immune responses and specific inflammatory processes. ADAM28 is also known as the lymphocyte metalloprotease MDC-L, expressed on the lymphocyte cell surface27. As such, it has been defined as a ligand for α4β1 integrin which enables the adhesion of other leukocytes expressing this integrin. STC1 in the same region, is a gene that encodes stanniocalcin 1 protein, involved in regulation of calcium including in antioxidant pathways of marcophages28. In this light, calcium regulation within immune cells has been previously identified as aberrant in certain CVID patient samples29. UBX10 may be a compelling candidate potentially involved in immunopathogenesis of primary antibody failure in that it encodes an Ubiquitin-like protein, which in addition to phosphorylation, has been shown to regulate NF-κB activity30. SDK1 (Sphingosine-dependent protein kinase-1) is important in the survival of alveolar macrophages31. DEPDC6 is a negative regulator of mTORC signaling pathways and RNA expression levels were found to be significantly different between those mice resistant to H5N1 influenza virus when compared to those that were susceptible32. No significant associations in the region of TACI were observed, and subjects with TACI mutations were not separately identified. We reviewed the reported amino acid changes and looked up their corresponding non-synonymous SNP IDs (Table 10). No imputation reference files including these SNPs were available to infer genotypes.









TABLE 10







TACI Protein TNFRSF13B Gene Amino Acid Variant and Corresponding


nsSNP rs ID















Clinical Report


nsSNP
DNA position
Alleles
AA Change
(bold-in reports)





rs34562254
16783716
C/T
P (CCC) --> L (CTC)
P251L


rs72553886
16783732
G/T
V (GTC) --> F (TTC)
V246F


rs56063729
16783809
A/G
V (GTG) --> A (GCG)

V220A



rs56248318
16784408
A/C
Q (CAG) --> H (CAT)
Q196H


rs72553885
16784417
A/C
C (TGC) --> * (TGA)
C193X


rs72553883
16784454
A/C
A (GCG) --> E (GAG)

A181E



rs72553882
16784504
A/C
Y (TAC) --> * (TAA)
Y164X


rs72553881
16784541
A/G
G (GGG) --> E (GAG)
G152E


rs72553880
16792777
A/G
A (GCT) --> T (ACT)
A149T


rs72553879
16792911
A/G
C (TGT) --> Y (TAT)
C104Y


rs34557412
16792912
C/T
C (TGT) --> R (CGT)

C104R



rs72553877
16792962
A/T
I (ATC) --> N (AAC)
I87N


rs72553876
16792986
A/G
Y (TAT) --> C (TGT)
Y79C


rs55916807
16793007
C/T
R (CGC) --> H (CAC)

R72H



rs67951770
16796563
C/G
D (GAT) --> H (CAT)
D41H


rs67951769
16796563
—/G
frameshift
D41fx


rs72553874
16796566
C/T
W (TGG) --> R (CGG)
W40R


rs72553884
16784425-16784424
—/G
Frameshift
D191G


rs72553878
16792925-16792924
—/T
Frameshift
Q99H


rs34182967
16793004-16793003
—/C
Frameshift
K73R


rs72553875
16793018-16793017
—/A
Frameshift
S68S
















TABLE 11





Most Significant Associated Regions Based on Genotype Association

























Count










SNPs
Distance







Region

Top
From







Genomic Span
Gene
1000
Exon
BestP
BestSNP
Allele1
F_A
F_U





chr6: 27236785-


MHC



110

0
 8.62E−10
rs3117426
T
0.3268
0.1907


32521295










chr8: 23746576-
ADAM28,

4

0
6.24E−6
rs4872262
A
0.0391
0.0107


24681608


ADAM7
,











ADAMDEC1,










STC1









chr4: 189671181-
AK095968
2
18764
1.78E−5
rs1606234
T
0.2961
0.4124


189676119










chr10: 73083830-
CDH23
2
7287
2.45E−5
rs7087554
A
0.5894
0.4729


73084583










chr7: 4270929-


SDK1



5

0
4.70E−5
rs895710
T
0.2905
0.4003


4287047










chr1: 20369369-
FLJ32784,

7

0
9.09E−5
rs7514144
C
0.2654
0.1808


20490791


UBXN10











chr10: 27709830-
PTCHD3

7

0
9.77E−5
rs506659
T
0.4022
0.3026


27728115










chr1: 59987116-
FGGY
2
2458
1.52E−4
rs11207520
G
0.3994
0.3026


59993733










chr12: 80435676-
PPFIA2
2
59900
1.63E−4
rs2400955
T
0.4134
0.3159


80444630










chr11: 102069786-
MMP27
2
0
1.69E−4
rs17099394
T
0.1229
0.0687


102079454










chr16: 71592439-
ZFHX3
2
40845
1.80E−4
rs8056528
C
0.3687
0.2754


71594134










chr8: 124729628-
KLHL38

4

0
2.05E−4
rs4871402
A
0.0643
0.1325


124734756










chr3: 7364204-
GRM7

4

40865
2.96E−4
rs12491592
A
0.1536
0.0939


7369287










chr3: 8378739-
BC020876
2
129448
3.51E−4
rs359030
C
0.1648
0.1033


8381919










chr6: 25570803-
LRRC16A
2
0
3.61E−4
rs4320355
T
0.3799
0.2898


25574868










chr6: 153354039-
MTRF1L
2
0
3.81E−4
rs9322400
T
0.4469
0.3526


153355207










chr3: 284363-


CHL1


2
19083
4.31E−4
rs17273893
T
0.148
0.0908


285747










chr8: 121001793-
DEPDC6
2
0
4.41E−4
rs869340
C
0.324
0.2402


121010709










chr2: 65518304-
FLJ16124
2
784
5.20E−4
rs1194849
G
0.3659
0.4614


65520560










chr21: 27713547-
BC043580
2
19690
5.51E−4
rs469709
A
0.1704
0.1095


27722878




















P
F_A
F_U








Replication
Replication
Replication
BestP
BestSNP
F_A
F_U



Region
(Discovery
(Discovery
(Discovery
Repli-
Repli-
Repli-
Repli-



Genomic Span
SNP)
SNP)
SNP)
cation
cation
cation
cation






chr6: 27236785-
0.3634
0.1697
0.1468

0.0004

rs2156875
0.5734
0.4475



32521295










chr8: 23746576-

0.03135

0.0321
0.01346

0.0314

rs4872262
0.0321
0.0135



24681608










chr4: 189671181-
0.4646
0.4266
0.4524
0.4646
rs1606234
0.4266
0.4524



189676119










chr10: 73083830-
0.4591
0.5092
0.4829
0.4591
rs7087554
0.5092
0.4829



73084583










chr7: 4270929-
0.4551
0.3578
0.3835

0.0235

rs4720301
0.3991
0.4793



4287047










chr1: 20369369-
0.3018
0.1560
0.1311

2.25E−8

rs6426636
0.4679
0.2857



20490791










chr10: 27709830-
0.9879
0.2798
0.2793
0.6291
rs493965
0.2844
0.3001



27728115










chr1: 59987116-

0.00148

0.2569
0.1706

0.0015

rs11207520
0.2569
0.1706



59993733










chr12: 80435676-
0.8687
0.3899
0.3842
0.5347
rs10746192
0.4306
0.4526



80444630










chr11: 102069786-
0.6947
0.0734
0.06643
0.6947
rs17099394
0.0734
0.0664



102079454










chr16: 71592439-
0.3698
0.3670
0.3368
0.3698
rs8056528
0.367
0.3368



71594134










chr8: 124729628-
0.7201
0.1009
0.1088
0.3161
rs7463896
0.1697
0.1979



124734756










chr3: 7364204-
0.5051
0.0963
0.08318
0.1237
rs7617297
0.3073
0.2592



7369287










chr3: 8378739-
0.1446
0.1147
0.08535
0.1139
rs358994
0.1055
0.0754



8381919










chr6: 25570803-
0.2219
0.2844
0.2469
0.1896
rs301396
0.445
0.3993



25574868










chr6: 153354039-
0.454
0.3945
0.3688
0.454
rs9322400
0.3945
0.3688



153355207










chr3: 284363-
0.6342
0.0963
0.08678
0.6342
rs17273893
0.0963
0.0868



285747










chr8: 121001793-
0.7402
0.3073
0.2966
0.682
rs4871793
0.3113
0.2978



121010709










chr2: 65518304-

0.01381

0.3899
0.4771

0.0119

rs1876518
0.3853
0.4744



65520560










chr21: 27713547-
0.9615
0.0963
0.09532
0.6677
rs17631106
0.0688
0.0769



27722878





Bold underline: Known immunological function Gene













TABLE 12





Most Significant Associated Regions Based on CNV Association







A) Deletions



















Distance










CNVR
Count
From
P
Cases
Controls




Com-


Deletion
SNPs
Exon
Deletion
Del
Del
Gene
MSSM
Oxford
USF
bined





chr11: 85365857-
5
0
0.01
2
0


PICALM
, PICALM variant protein

0
2
0
2


85381622












chr20: 57735790-
6
5902
0.026
3
4
PHACTR3
2
1
0
3


57741780












chr22: 17396663-
270
0
0.029
2
1
ARVCF, C22orf25, C22orf29,
2
0
0
2


18417315





CDC45L, CLDN5, CLTCL1,














COMT
, CR618542, CR625276,













DGCR14, DGCR2, DKFZp761P1121,












DKFZp781E0833, GNB1L, GP1BB,












GSCL, HIRA, KIAA1647, L77561,












LOC128977, MRPL40, SEPT5, SLC25A1,














TBX1
, TRXR2A, TSSK2, TXNRD2,













U84523, UFD1, UFD1L






chr4: 10256682-
4
2915
0.029
2
1


CLNK


0
1
1
2


10264316












chr10: 46003146-
8
0
0.029
2
1
DKFZp566K0524, PTPN20A,
2
0
0
2


46042543





PTPN20B










B) Duplications



















Distance










CNVR
Count
From

Cases
Controls




Com-


Duplication
SNPs
Exon
P Dup
Dup
Dup
Gene
MSSM
Oxford
USF
bined





chr2: 148396730-
6
0
8.66E−16
15
0


ACVR2A
, ORC4L

10
5
0
15


148433180












chr15: 35053039-
6
23146
5.72E−13
19
10
MEIS2
17
2
0
19


35063531












chr10: 53044735-
4
79826
9.63E−13
12
0
PRKG1
12
0
0
12


53045426












chr8: 77719805-
3
27377
1.05E−09
10
1
BC037827
10
0
0
10


77720240












chr7: 91789778-
4
0
1.20E−09
12
4
ANKIB1
9
3
0
3


91801963












chr2: 163316298-
3
17100
1.00E−07
7
0
KCNH7
6
1
0
7


163316595












chr7: 87180702-
4
202
7.41E−07
7
1
RPIB9
5
2
0
7


87191931












chr12: 72825720-
3
0
3.04E−06
7
2
BC061638
7
0
0
7


72832667












chr2: 203004035-
5
20466
6.58E−05
6
3
BMPR2
6
0
0
6


203017311












chr19: 9256584-
4
0
0.001
3
0
ZNF699
2
0
1
0


9277749












chr4: 39190766-
6
0
0.004
3
1
UGDH
1
2
0
3


39201960












chr9: 112207473-
3
553
0.004
3
1
SVEP1
3
0
0
3


112207708












chr7: 18903915-
4
23171
0.009
3
2


HDAC9


1
2
0
3


18905725












chr13: 64053258-
15
505114
0.01
2
0
AK057471, AK098560, BC128161
2
0
0
2


64056530












chr14: 35408995-
5
0
0.01
2
0
BRMS1L
0
2
0
2


35422074












chr5: 170531269-
4
374
0.01
2
0
DKFZp666P032, RANBP17
1
1
0
2


170536993












chr4: 91413829-
3
33381
0.01
2
0
KIAA1680, MGC48628
2
0
0
2


91415037












chr7: 34685030-
4
4517
0.01
2
0
AAA1, NPSR1
1
1
0
2


34686172












chr3: 176427638-
7
5118
0.01
2
0
NAALADL2
2
0
0
2


176429297












chr1: 170400944-
4
26274
0.01
2
0
DNM3
0
1
1
2


170403742












chr2: 167031393-
4
0
0.01
2
0
Na+ channel, SCN7A
2
0
0
2


167045981












chr9: 28683005-
3
21420
0.01
2
0


LINGO2


2
0
0
2


28687679












chr9: 105860023-
9
27434
0.01
2
0
OR13C3, OR13C4, OR13C5, OR13C8,
2
0
0
2


105868928





OR13F1, SMC2, hCAP-E
















TABLE 13







Most Significant Associated Regions Based on Subphenotype Genotype Association



























Distance









Count


From


Significant Region
P-value
SNP
A1
F_A
F_U
OR
SNPs
Gene
Distance
Exon










A) Cancer

















chr14: 23168471-
4.57E−7
rs222723
C
0.6154
0.1968
6.531
2
DHRS2
0
0


23169215












chr6: 140225308-
1.53E−6
rs12111348
T
0.4231
0.1064
6.157
2
BC039503
2009
2009


140225386

















B) Lymphoma

















chrX: 132092989-
 5.50E−10
rs5977837
C
0.2778
0.02174
17.31
2
TFDP3
82912
82912


132095451












chr10: 58755852-
1.36E−9
rs16910534
T
0.2222
0.01383
20.37
3
IPMK
858286
858286


58767334












chr7: 90245135-
1.69E−8
rs975004
G
0.3333
0.03953
12.15
2
KIAA0834, PFTK1
0
12544


90245280












chr5: 156402979-
3.62E−8
rs10038271
T
0.7222
0.1877
11.25
2


HAVCR1


0
651


156407976












chr1: 55704352-
2.85E−7
rs356086
G
0.1111
0.003953
31.5
2
FLJ45337
112975
112975


55706323












chr1: 106430611-
1.55E−6
rs11184786
T
0.3333
0.05336
8.87
2
BC043293
467531
467531


106438079

















C) Lymphadenopathy

















chrX: 53035117-
1.90E−7
rs10127016
G
0.3833
0.125
4.351
2
TMEM29
0
1409


53039801












chr12: 30583074-
4.82E−7
rs1905675
C
0.2
0.5453
0.2085
2
IPO8
74732
74732


30598457












chr14: 64723761-
8.09E−7
rs4299072
A
0.1667
0.02802
6.938
4
BX161428
0
0


64783067












chr5: 141916656-
1.13E−6
rs17706715
A
0.2333
0.05603
5.127
2


FGF1


27191
27191


141926115












chr11: 101265604-
1.55E−6
rs17097290
T
0.15
0.02371
7.267
2
ANGPTL5
513
513


101266102

















D) Nodular regenerative hyperplasia of the liver (NRH)

















chr1: 68153970-
 2.29E−10
rs1926283
C
0.2308
0.01807
16.3
3
AK096081, AK124028
0
33408


68161019












chr21: 18188480-
2.84E−7
rs7280675
G
0.1923
0.02008
11.62
2
CHODL
2636
2636


18192815












chr21: 45476504-
3.23E−7
rs4592938
A
0.1154
0.006024
21.52
2
C21orf89
1777
1777


45476918












chr8: 143046590-
1.18E−6
rs12676273
G
0.3462
0.07229
6.794
3
TSNARE1
235455
235455


143055893












chrX: 27453340-
1.29E−6
rs5971431
C
0.3077
0.05823
7.188
3
AK057304
55692
55692


27462759

















E) LIP

















chr13: 101641990-
5.76E−8
rs1336698
G
0.65
0.168
9.197
6
FGF14
0
69139


101673539












chrX: 143116309-
6.77E−8
rs6649722
A
0.3
0.0377
10.94
3
UBE2NL
320286
320286


143119321












chrX: 47642545-
7.70E−8
rs12387999
A
0.2
0.016
15.38
2
ZNF81
0
2262


47651320












chr2: 109118795-
4.62E−7
rs375099
C
0.25
0.02976
10.87
2
POSH2
0
5794


109120561












chrX: 30122360-
2.52E−6
rs5927496
C
0.7
0.2341
7.633
2
MAGEB2
17927
17927


30125674

















F) Bronchiectasis

















chrX: 22422160-
5.71E−8
rs5925651
A
0.3462
0.1386
3.291
3
ZNF645
219665
219665


22428888












chr2: 200959722-
2.39E−6
rs13019534
G
0.5064
0.2908
2.503
2
AOX1, DNAPTP6,
0
0


201175137







KCTD18, SGOL2




chr8: 15782386-
2.92E−6
rs1563297
A
0.5321
0.3152
2.47
2
TUSC3
116020
116020


15788801












chr19: 51765478-
6.30E−6
rs6509286
C
0.4167
0.2228
2.491
2
AK094504
0
27862


51768118












chrX: 68569536-
1.04E−5
rs7056340
A
0.3141
0.1467
2.663
2
TMEM28
68076
68076


68573727












chr6: 32508322-
1.12E−5
rs2027856
T
0.02564
0.163
0.1351
2


HLA-DRA


4942
4942


32510683

















G) Granuloma

















chr11: 12182328-
4.83E−6
rs16910765
T
0.2206
0.05921
4.497
3
KIAA0750, MICAL2,
0
0


12186013







MICAL2PV1, MICAL2PV2




chr1: 33674005-
1.26E−5
rs12751162
A
0.3824
0.1601
3.248
2
PHC2
4803
4803


33683227












chr6: 88453628-
2.04E−5
rs2250276
G
0.1765
0.04386
4.671
2


AKIRIN2


0
5428


88454474












chrX: 27453340-
3.18E−5
rs5971431
C
0.1912
0.05263
4.255
2
AK057304
55692
55692


27462759

















H) GI Enteropathy

















chr12: 2531014-
1.21E−7
rs4765961
C
0.4737
0.142
5.439
3


CACNA1C


0
0


2538733












chr10: 26643326-
2.90E−6
rs7903552
G
0.3684
0.107
4.869
2


GAD2


9833
9833


26646318












chr5: 11592622-
6.26E−6
rs2727602
T
0.6053
0.2613
4.334
2
CTNND2
0
21030


11597026












chr21: 42551877-
9.95E−6
rs3787986
T
0.3684
0.1152
4.479
4
ABCG1
0
0


42552623












chr9: 4033590-
1.13E−5
rs676472
C
0.2895
0.07613
4.944
3
GLIS3
0
68391


4039110












chr4: 33014145-
1.43E−5
rs6846113
A
0.1053
0.01029
11.32
2
AK093205
552926
552926


33017023

















I) Malabsorption

















chr20: 55901032-
3.21E−9
rs8124301
T
0.4231
0.07631
8.877
5
C20orf85
174030
174030


55985359












chr7: 117377201-
9.94E−7
rs17140937
C
0.2692
0.04418
7.971
2
CTTNBP2
76404
76404


117382435












chr11: 124071934-
1.25E−6
rs1784539
G
0.6154
0.2068
6.136
2


SPA17


2037
2037


124079438












chr10: 30255454-
5.18E−6
rs11007812
T
0.3077
0.06426
6.472
2
CR626438
85918
85918


30255817












chr12: 31276546-
6.76E−6
rs12819069
C
0.3846
0.09839
5.727
2
OVOS2
26191
26191


31281817












chr8: 6374232-
8.56E−6
rs2515477
T
0.4231
0.1185
5.456
2


ANGPT2
, MCPH1

0
1213


6376048

















J) Splenectomy

















chr9: 130092787-
1.03E−6
rs7026795
A
0.425
0.1802
3.363
2
C9orf119
1698
1698


130095025












chr3: 72044083-
1.11E−6
rs7648163
C
0.55
0.2748
3.226
2
AK097190
115138
115138


72052222












chr1: 73821234-
2.23E−6
rs4606267
G
0.325
0.1194
3.552
3
BC041341
244086
244086


73888127












chr9: 70762083-
2.46E−6
rs2993008
T
0.225
0.06306
4.313
2
AK057188, PIP5K1B
0
0


70815910












chr8: 137069094-
3.13E−6
rs6985828
C
0.0625
0.002252
29.53
2
KHDRBS3
340064
340064


137070609












chr15: 66415767-
3.78E−6
rs6494736
C
0.175
0.04054
5.02
2
ITGA11
0
0


66419741












chr7: 141314419-
2.60E−5
rs11761774
A
0.475
0.2455
2.781
2
TAS2R38
4247
4247


141314653

















K) Cytopenias

















chr12: 3711033-
4.28E−6
rs241964
C
0.6111
0.3
3.667
6
C12orf4, C12orf5, CCND2,
0
0


4573405







DYRK4, EFCAB4B, FGF23,












FGF6, PARP11, RAD51AP1




chr8: 2721609-
3.52E−5
rs341672
C
0.2593
0.0812
3.961
2
CSMD1, KIAA1890
59615
59615


2723174












chr3: 2628577-
0.000337
rs1020997
G
0.2407
0.4979
0.3198
2


CNTN4


0
40335


2629597












chr2: 1783619-
0.000426
rs6548056
G
0.1852
0.434
0.2963
2
MYT1L
0
514


1783961












chr11: 2240035-
0.000533
rs17659078
A
0.4444
0.2286
2.699
2
ASCL2
5138
5138


2241166












chr10: 1236883-
0.000603
rs10794730
T
0.4815
0.2596
2.649
2
ADARB2
0
583


1237022












chr9: 1588885-
0.000866
rs1923928
T
0.1852
0.4191
0.315
2
SMARCA2
409607
409607


1595735












chr1: 4222578-
0.000884
rs966321
C
0.2593
0.4979
0.353
2
AX748168
146394
146394


4225577

















L) organ specific autoimmunity (OSAI)

















chr8: 101728102-
6.89E−8
rs7815950
G
0.2133
0.05615
4.559
2
SNX31
0
2334


101728344












chrX: 39933689-
5.18E−7
rs2948491
A
0.18
0.04545
4.61
4
BCOR
12163
12163


39956544












chr3: 109180117-
6.27E−6
rs709477
A
0.2733
0.4893
0.3926
2
BC101231
50156
50156


109186346












chr1: 37438130-
6.96E−6
rs6426015
A
0.2667
0.1096
2.953
3
GRIK3
165699
165699


37446852












chr15: 84701881-
1.19E−5
rs1431234
C
0.3
0.5108
0.4105
2
AGBL1
0
20894


84720707

















M) Low IgM (<50 mg/dL)

















chr1: 25735669-
6.02E−8
rs2065970
G
0.06553
0.2411
4.529
5
LDLRAP1
0
0


25753653












chr4: 59393803-
1.77E−5
rs2899130
A
0.2257
0.4286
2.573
2
BC034799
1366894
1366894


59398745












chr5: 76127981-
2.17E−5
rs615986
T
0.4927
0.2679
0.3767
2


F2RL1


21647
21647


76128963

















N) Low IgA (<10 mg/dL)

















chrX: 30733771-
1.44E−7
rs11095197
T
0.1469
0.3431
3.034
2


MAP3K7IP3


18876
18876


30736604












chrX: 145696682-
3.48E−6
rs6626815
C
0.5656
0.3578
0.4279
5
CXorf1
977620
977620


145751200












chr15: 47024768-
6.75E−6
rs17469978
C
0.2313
0.4167
2.375
2
SHC4
0
16198


47025722












chrX: 32356174-
1.25E−5
rs699457
G
0.2656
0.1078
0.3342
2
DMD
0
0


32388539












chr1: 3915418-
2.69E−5
rs10737395
A
0.1
0.2353
2.769
2
AK124708
236
236


3917931












chr13: 101257602-
3.22E−5
rs1322702
A
0.04375
0.1471
3.768
2
FGF14
0
60734


101258342

















O) Low B cells (CD19+ cells <1%)

















chr8: 13834782-
7.62E−8
rs2682665
C
0.1818
0.01394
15.71
3
SGCZ
149368
149368


13842376












chr7: 107306707-
2.16E−7
rs9690688
A
0.3636
0.06375
8.393
2
DLD
4709
4709


107314113












chr9: 33103066-
1.40E−6
rs12379501
T
0.3182
0.05578
7.9
2


B4GALT1


0
0


33103970












chr13: 37798435-
2.25E−6
rs4943583
G
0.2273
0.02988
9.549
7
UFM1
0
0


37846851

















P) Young age of symptom onset (<10 yrs)

















chrX: 152295068-
9.84E−8
rs5987017
A
0.2143
0.04773
5.442
4
ZNF275
24829
24829


152303019












chrX: 37803525-
1.54E−7
rs5918500
C
0.4878
0.2123
3.533
3
SYTL5
0
0


37832251












chr19: 7505735-
2.31E−6
rs604959
C
0.4762
0.2273
3.091
2
PNPLA6
0
0


7508421









The CNV association analysis also uncovered novel genes associated with either the immunopathogenesis of antibody deficiency or the development of specific complications of CVIDs. In fact, 84 CNV deletions and 98 duplications were identified in one or more CVID patients but were not found in any controls. Most were intra-exonic and thus likely to exert impact. Some of the genes potentially affected by the identified CNV were also discovered in the GWAS part of this study. Many others have direct or potential relevance to the immune system and many were unique to individual patients, thus underscoring the great mechanistic diversity that is likely to underlie this collection of disorders, also reflected in the variability in clinical presentation and disease natural history1. Among those, we noted a highly significant number of subjects with duplications in ORC4L, a gene previously associated with B-Cell lymphoproliferative disorders33. This gene is essential for initiation of DNA replication, and potentially in rapidly proliferating immune cells.


We then performed a SNP genotype association to the particular features of CVID to define potential common mechanistic threads amongst the specific clinical and immumological variants to enable the prediction of CVID clinical phenotypes. Significant individual associations were made with all CVID variables studied (Table 8) including cancer (1), lymphoma (5), lymphadenopathy (3), NRH (3), LIP (4), bronchiectasis (1), granulomatous disease (0), enteropathy (1), malabsorption (2), splenomegaly (0), cytopenias (0), autoimmunity (2), low IgM (1), low IgA (1), low B cells (2), and early age of onset (2). In this regard, PFTK1 is a member of the CDC2-related protein kinase family found constitutively expressed at high levels in B cell lymphomas34, and also found associated with lymphoma in the studied subjects with this complication. Interestingly, HAVCR1, allele frequency also found enriched in the same subjects, plays a role in Th cell development and the regulation of asthma and allergic diseases35. FGF14, associated with LIP and low IgA, is a member of the fibroblast growth factor (FGF) family, which has crucial roles in embryonic development, cell growth, morphogenesis, tissue repair, tumor growth and invasion36. SNX31, associated with organ-specific autoimmunity, is a sorting nexin which may be involved in protein trafficking; SNX family proteins are subunits required for CD28 mediated T cell costimulation37. LDLRAP1, associated with lower serum IgM, encodes a cytosolic protein which contains a phosphotyrosine binding (PTD) domain which interacts with the cytoplasmic tail of the LDL receptor, and is associated with hypercholesterolaemia. In addition to the potential of these findings to provide mechanistic insight into how these subphenotypes arise, they may also allow for the prediction of associated comorbidity at the time of diagnosis. This has the potential to greatly improve the clinical management of the CVIDs.


REFERENCES



  • 1. Chapel H, Cunningham-Rundles C. Update in understanding common variable immunodeficiency disorders (CVIDs) and the management of patients with these conditions. Br J Haematol. 2009 June; 145(6): 709-27. Epub 2009 Mar. 30.

  • 2. Cunningham-Rundles C, Bodian C. Common Variable Immunodeficiency: Clinical and Immunological Features of 248 Patients Clinical Immunology Volume 92, Issue 1, July 1999, Pages 34-48.

  • 3. Wehr C et al. The EUROclass trial: defining subgroups in common variable immunodeficiency. Blood. 2008 Jan. 1; 111(1):77-85. Epub 2007 Sep. 26.

  • 4. Bacchelli C, Buckridge S, Thrasher A J, Gaspar H B. Translational mini-review series on immunodeficiency: molecular defects in common variable immunodeficiency. Clin Exp Immunol 2007 September; 149(3):401-9.

  • 5. Salzer U et al. Mutations in TNFRSF13B encoding TACI are associated with common variable immunodeficiency in humans. Nat Genet. 2005 August; 37(8):820-8.

  • 6. Castigli E et al. TACI is mutant in common variable immunodeficiency and IgA deficiency. Nat Genet. 2005 August; 37(8):829-34.

  • 7. Pan-Hammarstr∂m Q, et al Reexamining the role of TACI coding variants in common variable immunodeficiency and selective IgA deficiency. Nat Genet. 2007 April; 39(4):429-30.

  • 8. Volanakis J E et al. Major histocompatibility complex class III genes and susceptibility to immunoglobulin A deficiency and common variable immunodeficiency. J Clin Invest. 1992 June; 89(6):1914-22.

  • 9. Olerup O, Smith C I, Björkander J, Hammarström L. Shared HLA class II-associated genetic susceptibility and resistance, related to the HLA-DQB1 gene, in IgA deficiency and common variable immunodeficiency. Proc Natl Acad Sci USA. 1992 Nov. 15; 89(22):10653-7.

  • 10. Grimbacher B et al. Homozygous loss of ICOS is associated with adult-onset common variable immunodeficiency Nature Immunology 4, 261-268 (2003).

  • 11. Salzer U et al. ICOS deficiency in patients with common variable immunodeficiency Clinical Immunology. Volume 113, Issue 3, December 2004, Pages 234-240.

  • 12. van Zelm M C et al. CD81 gene defect in humans disrupts CD19 complex formation and leads to antibody deficiency. J Clin Invest. 2010 April; 120(4):1265-74. doi: 10.1172/JCI39748. Epub 2010.

  • 13. van Zelm M C et al. An antibody-deficiency syndrome due to mutations in the CD19 gene. N Engl J Med. 2006 May 4; 354(18):1901-12.

  • 14. Kanegane H et al. Novel mutations in a Japanese patient with CD19 deficiency. Genes and Immunity (2007) 8, 663-670; doi:10.1038/sj.gene.6364431; published online 20 Sep. 2007.

  • 15. Kuijpers T W, et al. J Clin Invest. 2010 January; 120(1):214-22. Epub 2009 Dec. 21. CD20 deficiency in humans results in impaired T cell-independent antibody responses.

  • 16. Conley M E, Notarangelo L D, Etzioni A. Diagnostic criteria for primary immunodeficiencies. Representing PAGID (Pan-American Group for Immunodeficiency) and ESID (European Society for Immunodeficiencies). Clin Immunol. 1999 December; 93(3):190-7.

  • 17. Price A L et al. Principal components analysis corrects for stratification in genome-wide association studies. Nat Genet. 2006 August; 38(8):904-9. Epub 2006 Jul. 23.

  • 18. Purcell S et al. PLINK: a toolset for whole-genome association and population-based linkage analysis. American Journal of Human Genetics, 81 (2007).

  • 19. Imielinski M, Baldassano R N, Griffiths A et al.: Common variants at five new loci associated with early-onset inflammatory bowel disease. Nat. Genet. 41,1335-1340 (2009).

  • 20. Wang, K. et al. PennCNV: an integrated hidden Markov model designed for high-resolution copy number variation detection in whole-genome SNP genotyping data. Genome Res. 17, 1665-1674 (2007).

  • 21. Bucan M et al. Genome-wide analyses of exonic copy number variants in a family-based study point to novel autism susceptibility genes. PLoS Genet. 2009 June; 5(6):e1000536. Epub 2009 Jun. 26.

  • 22. Chapel H et al. Common variable immunodeficiency disorders: division into distinct clinical phenotypes. Blood. 2008 Jul. 15; 112(2):277-86. Epub 2008 Mar. 4.

  • 23. Dennis G, Jr, et al. (2003) DAVID: Database for Annotation, Visualization, and Integrated Discovery. Genome Biol 4:3.

  • 24. Zhang L et al. Transmembrane activator and calcium-modulating cyclophilin ligand interactor mutations in common variable immunodeficiency: clinical and immunologic outcomes in heterozygotes. J Allergy Clin Immunol. 2007 November; 120(5): 1178-85.

  • 25. Shiina T, Inoko H, Kulski J K. An update of the HLA genomic region, loci information and disease associations. Tissue Antigens 2004, 64:631-649.

  • 26. International MHC and Autoimmunity Genetics Network et al. Mapping of multiple susceptibility variants within the MHC region for 7 immune-mediated diseases. Proc Natl Acad Sci USA. 2009 Nov. 3; 106(44):18680-5.

  • 27. Bridges L C et al. The lymphocyte metalloprotease MDC-L (ADAM 28) is a ligand for the integrin alpha4beta1. J Biol Chem. 2002 Feb. 1; 277(5):3784-92. Epub 2001 Nov. 27.

  • 28. Sheikh-Hamad D. Mammalian stanniocalcin-1 activates mitochondrial antioxidant pathways: new paradigms for regulation of macrophages and endothelium. Am J Physiol Renal Physiol. 2010 February; 298(2): F248-54. Epub 2009 Aug. 5.

  • 29. Foerster C. et al. B Cell Receptor-Mediated Calcium Signaling Is Impaired in B Lymphocytes of Type Ia Patients with Common Variable Immunodeficiency. J Immunol 2010.

  • 30. Karin M, Ben-Neriah Y. Phosphorylation Meets Ubiquitination: The Control of NF-κB Activity. Annual Review of Immunology Vol. 18: 621-663 (April 2000).

  • 31. Monick M M et al. Cooperative prosurvival activity by ERK and Akt in human alveolar macrophages is dependent on high levels of acid ceramidase activity. J Immunol. 2004 Jul. 1; 173(1):123-35.

  • 32. Boon A C et al. Host genetic variation affects resistance to infection with a highly pathogenic H5N1 influenza A virus in mice. J Virol. 2009 October; 83(20):10417-26. Epub 2009 Aug. 12.

  • 33. Radojkovic M et al. Novel ORC4L Gene Mutation in B-Cell Lymphoproliferative Disorders The American Journal of the Medical Sciences: 2009—Volume 338—Issue 6—pp 527-529.

  • 34. Saltis M and Davidson W. CDC2-related kinase and metabolic regulation in B cell malignancies. The Journal of Immunology, 2007, 178, 49.19.

  • 35. Umetsu S E et al. TIM-1 induces T cell activation and inhibits the development of peripheral tolerance. Nat Immunol. 2005 May; 6(5):447-54. Epub 2005 Mar. 27.

  • 36. Broadley K N et al. Monospecific antibodies implicate basic fibroblast growth factor in normal wound repair. Lab Invest. 1989 November; 61(5):571-5.

  • 37. Badour K et al. Interaction of the Wiskott-Aldrich syndrome protein with sorting nexin 9 is required for CD28 endocytosis and cosignaling in T cells. Proc Natl Acad Sci USA. 2007 Jan. 30; 104(5):1593-8. Epub 2007 Jan. 22.

  • 38. Bonilla F A et al. Practice parameter for the diagnosis and management of primary immunodeficiency. Ann Allergy Asthma Immunol. 2005 May; 94(5 Suppl 1):S1-63.

  • 39. Yong P L, Orange J S, Sullivan K E. Pediatric common variable immunodeficiency: Immunologic and phenotypic associations with switched memory B cells. Pediatr Allergy Immunol. 2010 Mar. 19.



Example 2
Prediction of CVID Status and Development of Complications Based on a Targeted SNP Panels

A Support Vector Machine (SVM) is a decision-based prediction algorithm which can classify data into several groups. SVM is based on training data mapped to a higher dimensional space and separated by a plane defining the two classes of data, followed by a testing stage where status is predicted based on the model and compared to known status to test the accuracy (V. Vapnik. The Nature of Statistical Learning Theory. Springer-Verlag, New York, NY, 1995). The implementation of SVM used is LIBSVM software (Chih-Chung Chang and Chih-Jen Lin, LIBSVM: a library for support vector machines, 2001. Software available at http://www.csie.ntu.du.tw/˜cjlin/libsvm; C. W. Hsu, C. C. Chang, C. J. Lin. A practical guide to support vector classification, 2003). Genome-wide SNP microarrays provide 610,000 genotypes which tag the genome in an unbiased way without need for prior hypothesis concerning candidate genes. Classical genome-wide association methods evaluate single SNP significance for difference in allele frequency. Although successful, the reality that many common complex diseases are polygenic in etiology with epistasis of multiple gene combinations makes an integrative model utilizing more of the data content more incisive. The application of SVM to genome-wide SNP data provides a robust framework for clinical disease prediction to evaluate risk and enact preventative measures, as we have demonstrated previously for type 1 diabetes (Wei Z, et al., From disease association to risk assessment: an optimistic view from genome-wide association studies on type 1 diabetes. PLoS Genet. 2009 October; 5(10):e1000678. Epub 2009 Oct. 9).


There are two components to the data: the target value which is the classification of the subject and the corresponding attributes which are more granular characteristics. In the application to CVID, we sought to predict target Values (Class Labels): CVID (yes/no), Bronchiectasis (yes/no), and OSAI (yes/no). This prediction is based on the attributes (features or observed variables): Genotypes for the top 1,000 SNPs previously identified from case:control genome-wide association (top 0.16% of original data set and all P<0.0015).


Phenotype labels were scaled to +1 for affected and −1 for unaffected. Genotype data is scaled with AA=0, AB=0.5 and BB=1. Data is mapped to high dimensional space by the LIBSVM algorithm to differentiate populations. Iterations of a coarse grid are followed by finer grids to avoid excessive time expenditure of complete grid search. The SVM is based principally on two variables: cost C (the penalty parameter of SVMs) and γ=1/number of variables. The SVM type in our application was C-SVM classification which runs through iterations of minimizing error functions while maintaining a hyperplane with maximum margin to avoid overfitting (FIG. 6). The kernal function maps attribute data into a higher dimensional space to improve distinguishing characteristics. The kernel type in this application was a radial basis function (RBF) which is based on distance from an origin. Once the SVM model is established through training and validated for accuracy with an independent dataset in testing, additional data can be mapped and prediction made based on the model.


The Support vector machine (SVM) approach is a machine learning approach that can map combinations of genotypes in high dimensional space. The top 1000 significant SNPs from the discovery and replication case:control genotype association studies we used as observed characteristics to form a designation decision between CVID (common variable immunodeficiency) case and an individual not affected by CVID (Table 14). The dataset available was separated into a training set of 179 cases and 1,917 controls which the SVM model is based on. In this training set, the case/control label and genotype attributes guide model formation. In the testing set of independent 109 cases and 1,114 controls, only the genotype attributes are supplied to the model established during training which then distinguishes cases or controls based on the genotype profile. The simple diagnostic yes or no question is: based on this person's genotypes do they have CVID? The prediction accuracy turned out well: training data: 98.7% and testing data: 90.5% (FIG. 7).


Similarly, the top 1000 significant SNPs for Bronchiectasis (Table 15) and OSAI (organ specific autoimmunity) (Table 16) as input to the SVM. Training: 107 no Bronchiectasis and 59 Bronchiectasis. Testing: 77 no Bronchiectasis and 19 Bronchiectasis. The model had 100% accuracy in the training stage and 97.9% accuracy in testing stage (FIG. 8). Evaluating our cohort for OSAI yielded Training: 117 no OSAI and 49 OSAI. Testing: 70 no OSAI and 26 OSAI. The model had 100% accuracy in the training stage and 99.0% accuracy in testing stage (FIG. 9).


To demonstrate poor model accuracy based on the same SVM methods, we ran the training stage with randomized testing data. The Bronchiectasis model applied to random affected labels and random genotypes resulted in a prediction accuracy of 27.1%. The Bronchiectasis model applied to random affected labels and correct genotypes resulted in a prediction accuracy of 77.1%. The OSAI model applied to random affected labels and correct genotypes resulted in a prediction accuracy of 67.7%. Given these markedly poorer accuracy results from randomized data compared to our observed data bolsters confidence that our high success rate is less likely due to a confounding factor, bias, or error.


This study represents the first genome-wide population based study of CVID. The use of the relatively large cohorts assembled here was essential, both to discover and to confirm the findings and demonstrates the potential of genome-wide association in complicated polygenic rare diseases. This type of unbiased study has discovered many novel targets that may underlie the development of CVID and provide clues to the pathogenesis of the heterogeneous clinical complications and subtypes of CVID, providing a solid foundation for further studies to understand the mechanism, interplay, and clinical manifestations of CVID, with the immediate potential of improving clinical management. Finally the great diversity identified with regards to unique CNV substantiates the hypothesis that CVIDs are a collection of diverse mechanisms leading to complex phenotypes.









TABLE 14





SNP Panel of 1994 SNPs for classifying CVID case versus individual without CVID
























rs3117426
rs2239914
rs9382495
rs139234
rs11132547
rs1391772
rs2107202
rs3763338
rs649647
rs7677584


rs2523535
rs366178
rs4269167
rs17631106
rs11135816
rs1400438
rs2112040
rs3763822
rs6504204
rs7690236


rs3130931
rs17304375
rs3130933
rs13019329
rs11138566
rs1411439
rs2113616
rs3766566
rs6505069
rs7716554


rs200968
rs3129871
rs9295768
rs7085343
rs11143520
rs1419183
rs2121289
rs3772054
rs6510040
rs7761966


rs879882
rs1634719
rs967005
rs12270763
rs11157606
rs1421240
rs2137512
rs3775574
rs6516091
rs7762279


rs3130564
rs4654849
rs2395174
rs12447026
rs11180576
rs1422122
rs214833
rs3781216
rs6538408
rs7772160


rs7767008
rs2400955
rs2931060
rs10940184
rs11183395
rs1438935
rs2153875
rs3788111
rs6549225
rs7774197


rs720831
rs6581986
rs10000770
rs139240
rs11206396
rs1443365
rs215393
rs3788317
rs6555424
rs7774567


rs4713208
rs2429485
rs10020322
rs3802888
rs11237730
rs1444467
rs2159318
rs3796504
rs656070
rs7781284


rs9266689
rs3129975
rs12636521
rs17790790
rs11247770
rs1445358
rs2168963
rs3801332
rs6577933
rs7789182


rs13194504
rs17099394
rs139136
rs4871793
rs1157548
rs1447398
rs217180
rs3804350
rs6583700
rs7808907


rs6926142
rs2395173
rs9380006
rs2279529
rs11669334
rs1452077
rs2175835
rs3810925
rs6586116
rs7815122


rs4872262
rs12495023
rs10476080
rs2902858
rs11698275
rs1461713
rs220309
rs3814585
rs6590124
rs7822769


rs12680982
rs10259703
rs358994
rs4720301
rs11717880
rs1466633
rs2213215
rs3818409
rs6656611
rs7834603


rs6456785
rs2505323
rs12573587
rs17372123
rs11786911
rs1477246
rs2236861
rs3847985
rs6657001
rs783756


rs2517532
rs2505327
rs139135
rs6679430
rs11819553
rs1481150
rs2237236
rs3863380
rs6688151
rs7838893


rs2156875
rs2484173
rs3095340
rs6485702
rs1182865
rs1497431
rs2242660
rs3913305
rs6688807
rs7857243


rs3130501
rs3132685
rs11087123
rs3134792
rs11848008
rs149990
rs2256726
rs3935608
rs6694182
rs7857474


rs422717
rs2523608
rs9266440
rs1377611
rs11862051
rs1523138
rs2262808
rs4082339
rs6727285
rs7867936


rs393034
rs3094122
rs2718419
rs9380120
rs11873322
rs1525745
rs2277210
rs4082846
rs6737948
rs7871753


rs3117326
rs8056528
rs1194849
rs6799262
rs11906
rs1544488
rs2292263
rs4146035
rs6743415
rs7887141


rs3129882
rs879484
rs7718291
rs12495026
rs11915523
rs1545692
rs2292307
rs4147525
rs6759577
rs7904468


rs3130837
rs2517549
rs12458578
rs2523989
rs11924324
rs1566579
rs2295330
rs420095
rs6764451
rs791033


rs8321
rs10734912
rs6426636
rs7945342
rs11933720
rs1569583
rs2299395
rs4236541
rs6794896
rs7928480


rs6571989
rs2523987
rs469709
rs950210
rs1194491
rs1571272
rs2306810
rs4238992
rs6807774
rs7937011


rs9261290
rs3130932
rs9329172
rs1406977
rs11959994
rs1574342
rs2343468
rs4320356
rs6865181
rs7939415


rs858985
rs2210313
rs13104213
rs720465
rs11963652
rs1582761
rs2358483
rs4324798
rs6877998
rs7964472


rs1606234
rs3135338
rs1876518
rs11870286
rs11977312
rs1592593
rs2373146
rs4353961
rs6886088
rs7970177


rs3129791
rs3770266
rs2572690
rs9257802
rs11998428
rs16181
rs2389789
rs4384353
rs6903608
rs8015024


rs12924882
rs149900
rs2191388
rs2517403
rs12001157
rs1619092
rs2406233
rs4409432
rs6904596
rs8015785


rs4264554
rs6457327
rs3094127
rs7103569
rs12047230
rs1629896
rs2425628
rs4486000
rs6910095
rs8020688


rs17322265
rs4871402
rs386843
rs12374025
rs12049377
rs1652500
rs2427399
rs4497887
rs6917366
rs8038569


rs1453666
rs2517448
rs17099425
rs10746192
rs12060746
rs1654774
rs2448396
rs4512403
rs6917419
rs8050560


rs7087554
rs2902982
rs310319
rs10254238
rs120960
rs1658102
rs2455826
rs4525114
rs6921388
rs8055387


rs853676
rs493965
rs2733393
rs139235
rs1209633
rs16891725
rs2473700
rs4531492
rs6923139
rs8057776


rs3771781
rs80303
rs1539120
rs3749530
rs12146261
rs16899857
rs2492460
rs454127
rs6928830
rs8061733


rs203888
rs9295730
rs2844494
rs2844635
rs12147922
rs16908361
rs2497295
rs4541737
rs6933251
rs8068923


rs3094551
rs200969
rs133826
rs1517927
rs12148329
rs16949286
rs2514861
rs4578658
rs6938076
rs8077733


rs1634718
rs1028308
rs9344757
rs17251715
rs12149564
rs16956295
rs2517646
rs4596286
rs6939576
rs8081687


rs3094550
rs3130350
rs1214751
rs9313540
rs12188351
rs16957467
rs2523454
rs4617771
rs6947841
rs8100750


rs6127923
rs2254556
rs2130904
rs4354950
rs12190473
rs16959820
rs2535319
rs464904
rs6959814
rs8105356


rs1064191
rs17293544
rs2730306
rs6785581
rs12201890
rs16971464
rs2554675
rs4671614
rs6960076
rs8109860


rs2517452
rs393990
rs12145634
rs2454873
rs12204145
rs17043526
rs2555575
rs4689343
rs6961642
rs8138344


rs9257809
rs16851846
rs4350445
rs5009448
rs12238437
rs17052298
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rs7250113
rs9784568


rs2702671
rs1353583
rs10822856
rs10984477
rs1281747
rs17548847
rs2703130
rs4861993
rs725579
rs9809600


rs1893306
rs7962880
rs6542736
rs10984490
rs12827322
rs17627259
rs2703960
rs4864553
rs726558
rs9809773


rs315995
rs6714893
rs9844737
rs1100185
rs12882217
rs17654772
rs274646
rs4865815
rs7272481
rs982511


rs12155045
rs7171256
rs1316471
rs11014455
rs12885228
rs1765886
rs2755176
rs4885371
rs728034
rs9835081


rs6128300
rs1003802
rs236410
rs11022509
rs1289501
rs17685418
rs2763419
rs4901523
rs729154
rs9848911


rs11666885
rs13214001
rs473585
rs11033019
rs12982321
rs17692241
rs2802884
rs4915723
rs729648
rs9861236


rs2685515
rs316021
rs2420175
rs11059821
rs12990534
rs17709684
rs2817200
rs4920566
rs731809
rs9874349


rs6748817
rs4972265
rs3886771
rs11128687
rs12996858
rs17726892
rs2821236
rs4921593
rs7321884
rs988158


rs2279351
rs1650817
rs2025633
rs11131055
rs13023918
rs17734487
rs2822850
rs4933679
rs734434
rs988179


rs11059258
rs7265772
rs6012463
rs11143230
rs13035689
rs17761855
rs2825673
rs494469
rs7349465
rs9904523


rs4130446
rs7724262
rs3849699
rs11165460
rs1304065
rs17796406
rs2852424
rs4950989
rs739107
rs9912125


rs7084512
rs251039
rs12188691
rs11186642
rs13081891
rs17817943
rs2877498
rs4976254
rs740096
rs9913538


rs627830
rs2057904
rs9325837
rs11194816
rs13086978
rs17829645
rs2887022
rs4978774
rs7415038
rs9919607


rs2052701
rs12608393
rs2010464
rs11218881
rs13128635
rs178377
rs2904295
rs4981149
rs7428676
rs9966000


rs1475784
rs2475793
rs13394087
rs1124359
rs13132576
rs1790024
rs2914079
rs4984626
rs752278
rs9989335


rs6419277
rs2455308
rs11237690
rs11250154
rs13142179
rs1833755
rs2924264
rs500293
rs7539255



rs6769238
rs1808378
rs2152174
rs1146229
rs13189435
rs1861089
rs2940930
rs536299
rs7544671



rs7306734
rs4521737
rs4947602
rs1152986
rs13203110
rs186493
rs296200
rs542988
rs7553961



rs3125050
rs547491
rs6437079
rs11547160
rs13224666
rs1866714
rs2962270
rs556439
rs7554157



rs2174559
rs1477602
rs7609626
rs1156250
rs13229792
rs1885831
rs2972418
rs568789
rs7555638



rs6661074
rs7792461
rs722938
rs11576361
rs13238853
rs1889356
rs2974617
rs587263
rs7574670
















TABLE 15





SNP Panel of 1000 SNPs for classifying CVID case with Bronchiectasis versus CVID case without Bronchiectasis
























rs3101219
rs1866236
rs9837945
rs7700961
rs6950759
rs7021834
rs6590583
rs8011776
rs6508240
rs5925649


rs4908463
rs1349159
rs13083177
rs297884
rs2191714
rs1536864
rs6590584
rs7174078
rs2120663
rs5925651


rs1725262
rs1448917
rs960353
rs7735038
rs2330055
rs11142904
rs6590694
rs11858397
rs16946189
rs5926027


rs10864368
rs12612313
rs7629105
rs1050975
rs11238193
rs7037278
rs546381
rs17227996
rs1350552
rs5926032


rs2071414
rs6436652
rs6443582
rs1890366
rs296308
rs2378383
rs11054372
rs12439272
rs621834
rs7882511


rs12034719
rs9288624
rs1522262
rs1933650
rs11766378
rs7025226
rs2071163
rs4924613
rs2433396
rs1527808


rs2982380
rs1515928
rs1994855
rs6904644
rs10499751
rs12238088
rs2239176
rs1712426
rs2847593
rs5943997


rs17038468
rs2556097
rs12487104
rs1331489
rs10270164
rs10491791
rs901528
rs3825786
rs7241842
rs12557438


rs1812242
rs1561466
rs4602335
rs1571827
rs13240999
rs7874528
rs10841413
rs12439521
rs16977580
rs16988109


rs4970535
rs13033944
rs4472002
rs9391956
rs12698917
rs17428350
rs2417862
rs7178085
rs1942159
rs12014989


rs12126652
rs830994
rs4234788
rs3812179
rs258702
rs7871887
rs2203494
rs11855605
rs3753067
rs1419850


rs12131682
rs831007
rs7435615
rs3812178
rs2888019
rs10818955
rs11045417
rs2306335
rs11152012
rs5973335


rs272561
rs831011
rs4697075
rs7760489
rs1557785
rs1413294
rs1861710
rs1993865
rs11663936
rs728149


rs1051648
rs6707969
rs2167955
rs966840
rs1921598
rs2114012
rs10492367
rs1026695
rs10516006
rs7064305


rs3444
rs16862458
rs5002502
rs13196069
rs1526491
rs3847303
rs7307357
rs4776191
rs17638216
rs5972346


rs6667593
rs6433615
rs7665747
rs12665403
rs7781914
rs328875
rs2388962
rs1904109
rs7251154
rs2124748


rs2354463
rs13032587
rs12186184
rs1905212
rs2188508
rs7852829
rs10844021
rs17183491
rs10416824
rs228379


rs10493140
rs17362588
rs1377347
rs9476488
rs17166818
rs1385143
rs1666235
rs3809539
rs6508999
rs2180648


rs10888636
rs2290517
rs13116757
rs9358858
rs1015882
rs4979619
rs10743775
rs1865930
rs7254214
rs5917579


rs2806405
rs12465639
rs7349609
rs6904130
rs2158137
rs12236795
rs12319134
rs11858355
rs1594895
rs1800321


rs2764687
rs10204810
rs4527518
rs2524099
rs10275909
rs7020797
rs995342
rs12914140
rs2217672
rs5917584


rs12728521
rs4667046
rs10034992
rs9501626
rs7777145
rs10481656
rs1389134
rs8032153
rs8113456
rs11266207


rs17124275
rs785240
rs1000226
rs3135338
rs3735258
rs7049083
rs1498712
rs11639228
rs6509286
rs5917593


rs4512683
rs12613687
rs2622604
rs2027856
rs234
rs7041855
rs10877894
rs4436753
rs689292
rs5964215


rs11208446
rs6740981
rs1708670
rs2395173
rs17356935
rs1249904
rs11174573
rs4534816
rs3746660
rs2239455


rs12407601
rs10445792
rs583908
rs154978
rs7455060
rs2225067
rs3936640
rs6497041
rs6084506
rs10854983


rs6663109
rs4673837
rs4693223
rs958423
rs10272242
rs10818337
rs3864455
rs11635007
rs998132
rs5919551


rs7522367
rs6739563
rs10049681
rs804829
rs6466735
rs10984619
rs4763120
rs768399
rs945767
rs5919560


rs11208834
rs11687313
rs10516451
rs1109798
rs4726499
rs11790238
rs11175217
rs8024991
rs6139278
rs5936698


rs1415974
rs4672726
rs4833233
rs3807045
rs2001942
rs6478430
rs962415
rs12591805
rs6076598
rs7056340


rs3738168
rs2043769
rs1155135
rs2257082
rs12703419
rs1860665
rs10506554
rs13380379
rs6516091
rs6625489


rs787492
rs13019534
rs1480902
rs941967
rs855740
rs2297454
rs10506762
rs4965678
rs6054459
rs5936708


rs7541725
rs12053340
rs1383518
rs2268718
rs1111467
rs10818649
rs919993
rs7177883
rs2206423
rs1327347


rs6691042
rs1527944
rs13125153
rs4391265
rs1079060
rs10988617
rs2468358
rs9944290
rs2050104
rs5936488


rs617196
rs1527947
rs12503254
rs1377392
rs2952648
rs11103479
rs7300004
rs11640138
rs8120907
rs5936735


rs263463
rs16836294
rs1456266
rs10948750
rs11777063
rs10905076
rs10777720
rs415595
rs2223565
rs6418420


rs263495
rs17450826
rs951850
rs9342394
rs6558946
rs12781427
rs3782518
rs949429
rs1033470
rs5936510


rs12409961
rs12472818
rs4569797
rs701699
rs7816614
rs10752197
rs2293055
rs12918743
rs4813897
rs5980665


rs2295330
rs16858496
rs939689
rs493265
rs1526371
rs7092558
rs11615235
rs7404377
rs6040808
rs5936518


rs11184300
rs12464171
rs6536370
rs9351817
rs2527748
rs7897007
rs4760566
rs238848
rs2009018
rs5936520


rs11184318
rs10933001
rs6858744
rs199623
rs13254568
rs11816696
rs2129663
rs2521477
rs6110058
rs5936795


rs2765283
rs12479209
rs2047633
rs12207816
rs7836269
rs12254001
rs7134809
rs2910848
rs17272940
rs5936796


rs7516108
rs1400795
rs13140600
rs16881643
rs2736020
rs749631
rs7300570
rs8045557
rs6079923
rs5936524


rs401904
rs17371273
rs2706740
rs10485242
rs1563297
rs2793345
rs12422489
rs763763
rs6046129
rs3811372


rs1748383
rs6711657
rs2706727
rs16881894
rs2720611
rs7071467
rs1334958
rs3935714
rs7267722
rs749789


rs7523184
rs12463466
rs2660404
rs10944450
rs4831774
rs7911971
rs9510802
rs257868
rs6049003
rs7879974


rs1986860
rs7355380
rs6841955
rs16881936
rs10503553
rs4256883
rs4771142
rs9926100
rs6121337
rs6615046


rs4233394
rs11685503
rs2082317
rs7754169
rs822249
rs2138566
rs1336628
rs7199018
rs228838
rs12012447


rs4540634
rs2645779
rs11729502
rs2518321
rs822304
rs10822031
rs4771033
rs7188866
rs6126431
rs5958896


rs1986385
rs1705805
rs11725347
rs12212740
rs11203812
rs10996997
rs903777
rs8049176
rs6069746
rs6621735


rs2766085
rs17709863
rs6818908
rs1858234
rs11204097
rs10762038
rs1952994
rs8047279
rs438363
rs5985075


rs12062372
rs356418
rs11733679
rs4371861
rs12545707
rs2894317
rs391933
rs16959476
rs160396
rs5942366


rs10800150
rs6769422
rs10024068
rs6913350
rs7841346
rs890687
rs398655
rs12449237
rs1151621
rs5942534


rs10918281
rs9875884
rs7698980
rs1933755
rs2942202
rs1550023
rs9603418
rs4458015
rs6062292
rs5941436


rs4472738
rs11129323
rs2280664
rs9492657
rs2685320
rs1684543
rs4346095
rs8057941
rs8129104
rs1570995


rs6680341
rs1015856
rs2692596
rs2249821
rs11136019
rs4547043
rs9526255
rs17296283
rs443823
rs6522689


rs10489199
rs9837352
rs1288536
rs2807278
rs7833986
rs2497304
rs4534729
rs13338146
rs2407255
rs638376


rs2205851
rs33454
rs6849285
rs2024692
rs7822429
rs10736156
rs17069883
rs17712938
rs2825809
rs12833867


rs232293
rs3821477
rs11724261
rs9403904
rs4397440
rs10884480
rs9534596
rs11648482
rs2825817
rs6644233


rs1933539
rs971866
rs957792
rs4870376
rs7824497
rs10748948
rs1323537
rs8055798
rs2825819
rs12556735


rs6593937
rs715382
rs1444127
rs9322536
rs924740
rs10749189
rs9535080
rs16955356
rs2826213
rs5910956


rs12061474
rs7647357
rs16892616
rs12174424
rs1545824
rs690870
rs777771
rs2317835
rs236044
rs4373692


rs11579791
rs6810180
rs4869452
rs2770104
rs2128105
rs7073467
rs9598119
rs378138
rs5992088
rs204330


rs10489951
rs6445254
rs6883671
rs1118242
rs7832751
rs3011399
rs2137512
rs1946482
rs2535707
rs203490


rs892975
rs11130981
rs1466011
rs9654570
rs7015186
rs3011408
rs7988149
rs8081951
rs361893
rs16309


rs6426156
rs1283532
rs2910468
rs751873
rs6472384
rs7070793
rs3013580
rs7219019
rs849357
rs201637


rs4653662
rs7373042
rs2910469
rs9456433
rs11778612
rs1898463
rs4884738
rs17762680
rs1297593
rs149908


rs973252
rs13100924
rs1423131
rs4368835
rs17824659
rs11248595
rs7997966
rs7215084
rs1807510
rs2046394


rs2808611
rs6548962
rs2910475
rs10214534
rs6472770
rs11244515
rs9317784
rs7503953
rs738456
rs683335


rs2808614
rs7610023
rs2961838
rs2457571
rs7461233
rs4962566
rs2325194
rs4792143
rs392277
rs546562


rs11122322
rs6548984
rs1862526
rs9365646
rs5017296
rs12098358
rs1446385
rs4393623
rs5752634
rs5932370


rs2814536
rs2136715
rs1309824
rs9365647
rs3735912
rs7124824
rs1249772
rs12451200
rs427736
rs5932394


rs2644441
rs4855469
rs10940096
rs9355453
rs13272532
rs903209
rs1334825
rs8067882
rs5762430
rs6637420


rs1874400
rs6549411
rs2937715
rs9355454
rs17700155
rs2283257
rs1112466
rs1555142
rs470105
rs6529359


rs3011577
rs9310222
rs10064609
rs916368
rs9969608
rs2270024
rs2325364
rs8073784
rs5762461
rs5932624


rs4443876
rs2322165
rs13164793
rs6915442
rs4602925
rs4569005
rs9600257
rs2075053
rs5750726
rs1324149


rs4658702
rs2322162
rs33003
rs221725
rs7823271
rs869377
rs9573480
rs11079868
rs4821862
rs5975531


rs12407427
rs11718674
rs6874731
rs221723
rs6983626
rs11030196
rs9565288
rs747039
rs2014881
rs5954074


rs1039010
rs11921763
rs4703545
rs7789861
rs1550856
rs4923526
rs4635230
rs17637472
rs2076101
rs12687312


rs384526
rs12714781
rs2656984
rs12540575
rs3019885
rs924714
rs9517921
rs11869714
rs5758991
rs644210


rs6542651
rs6765942
rs1505000
rs4719266
rs10464861
rs1216155
rs11069386
rs739924
rs9615362
rs5919819


rs2872977
rs1319094
rs448840
rs6460807
rs7816758
rs10897108
rs9805437
rs11651374
rs9627450
rs5965846


rs934615
rs7616034
rs7733888
rs6970647
rs4593504
rs4930431
rs12428930
rs9892374
rs4459004
rs1337635


rs2110965
rs11718781
rs1449227
rs136
rs12674562
rs7118125
rs9521350
rs9907787
rs1026162
rs596987


rs7562836
rs10049211
rs4869255
rs142
rs1768869
rs7946537
rs12886280
rs11653824
rs4641225
rs580628


rs13427136
rs11128275
rs1974789
rs135
rs10511649
rs567236
rs11156803
rs17462688
rs7064826
rs995895


rs7601672
rs6808996
rs10062935
rs7783383
rs7045369
rs487345
rs12884688
rs11870700
rs1266320
rs1547727


rs10490207
rs9869897
rs10478040
rs4722417
rs10757394
rs12807746
rs1058010
rs11655981
rs6641094
rs6877


rs9309192
rs4680967
rs256249
rs6966737
rs10966093
rs7481199
rs1952198
rs7220430
rs1731475
rs12008689


rs10184594
rs1561162
rs329312
rs38488
rs824230
rs621310
rs11625855
rs7230740
rs1731470
rs1882713


rs11885364
rs729942
rs329317
rs10272887
rs928484
rs11021014
rs11159087
rs1402630
rs5955621
rs5925038


rs13427078
rs959132
rs2069882
rs739981
rs1115553
rs11021018
rs10483899
rs1940615
rs5909069
rs17253949


rs6545738
rs635268
rs17169344
rs7790537
rs3780169
rs1791459
rs1112573
rs12185468
rs6633148
rs5970118


rs1503236
rs6439419
rs2347577
rs10250368
rs17520103
rs1625749
rs12432856
rs9947725
rs2283712
rs5924705


rs6704741
rs1466827
rs10040966
rs12701041
rs495259
rs11216930
rs8008554
rs930926
rs5955648
rs5925082


rs1517862
rs2052832
rs152523
rs2392151
rs605683
rs17748
rs17259779
rs8083849
rs2122
rs4828733


rs11887934
rs347965
rs6881655
rs2598105
rs662975
rs11216943
rs1286496
rs12961249
rs6633552
rs2071122


rs4549098
rs10513320
rs1025489
rs12701773
rs567490
rs4936507
rs12893100
rs206416
rs16982185
rs3747309


rs7558702
rs391398
rs302401
rs2119053
rs985654
rs7130937
rs17099646
rs3816822
rs6653666
rs5940453


rs1459166
rs6793037
rs10516032
rs4720500
rs11142769
rs4282990
rs4906007
rs11083004
rs5925647
MitoG12373A
















TABLE 16





SNP Panel of 1000 SNPs for classifying CVID case with OSAI (organ specific autoimmunity) versus CVID case without OSAI
























rs2297829
rs16838906
rs4274923
rs7718152
rs3912067
rs2927572
rs6589426
rs1012016
rs2850451
rs5951587


rs2275875
rs1876110
rs9291598
rs10059157
rs7805441
rs1536950
rs11214983
rs7156281
rs9949880
rs5926216


rs6699993
rs10489999
rs187196
rs7719533
rs4730345
rs957903
rs7116557
rs1840790
rs11150900
rs12011998


rs11580688
rs7562525
rs4351004
rs2400176
rs2371366
rs784790
rs7942551
rs1379230
rs11661755
rs6653559


rs1005301
rs1431900
rs10025742
rs17640419
rs17156818
rs10759209
rs4936297
rs12885991
rs12605082
rs5985835


rs12023073
rs1406665
rs10939265
rs2277025
rs2106277
rs7031681
rs7112940
rs1587351
rs12461534
rs3997122


rs533808
rs1147148
rs2687968
rs412805
rs2213979
rs10978723
rs2298767
rs4774966
rs1428752
rs5985846


rs2997447
rs1226906
rs2249563
rs394378
rs6972034
rs7871882
rs743632
rs11632793
rs10413521
rs1921501


rs3008418
rs1147155
rs2732063
rs10516028
rs17153071
rs784683
rs4937230
rs7179813
rs3786521
rs5985847


rs12069719
rs438027
rs1706023
rs868103
rs11772805
rs784676
rs7933438
rs11637130
rs6509637
rs1500727


rs2783711
rs4476347
rs1812922
rs4640812
rs1404228
rs784667
rs7948482
rs11637277
rs1366257
rs7065783


rs17257107
rs10203019
rs10518221
rs10038486
rs7777183
rs7034018
rs10790952
rs2917821
rs2561008
rs5971516


rs1325281
rs1010491
rs6857026
rs248230
rs7797250
rs7034341
rs11221388
rs12915827
rs7245993
rs2692983


rs12136144
rs6707140
rs4271959
rs9393002
rs10488631
rs13287637
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Example 3
Exome Sequencing Identifies Missense IRF2BP2 Mutation in a Family with Autosomal Dominant Common Variable Immunodeficiency (CVID)

Common Variable Immunodeficiency (CVID) is among the most frequently diagnosed forms of primary immunodeficiency disorders (PIDD), and is the most frequent requiring clinical intervention. Defined clinically by low quantity of two immunoglobulin classes (including IgG) and poor specific antibody production, it has been thought of as an “umbrella diagnosis” due to the heterogeneity of its onset and co-morbidities, including autoimmune disease and risk of malignancy. Though thought to be polygenic, roughly 10% of CVID is familial, and in recent years, 11 gene causes and/or associations with CVID have been described (Table 17).









TABLE 17







CVID Gene Associations









Function
Gene
Ref.





BCR function
CD19
Van Zelm, MC, et al. NEJM 2006



CD20
Kuijpers TW, et al. JCI 2010



CD21
Thiel J, et al. JACI 2012



CD81
Van Zelm MC, et al. JCI 2010.


B-cell
TACI
Martinez-Gallo M, et al. JACI 2013


co-stimulation
ICOS
Grimbacher B, et al. Nat Immunol 2003



BAFFR
Warnatz H, et al. Proc Natl Acad Sci 2009



CD27
Van Montfrans J, et al. JACI 2012


DNA repair/
MSH5
Sekine H, et al. Proc Natl Acad Sci 2007


VDJ




recombination




B-cell survival/
LRBA
Lopez-Herrera G, et al. Am J Hum Genet 2012


differentiation




TCR/BCR
PLCγ2
Ombrello MJ, et al. NEJM 2012


signaling









As discussed above in the previous examples, a recent multi-institutional genome-wide array study of CVID showed unique associations with specific single nucleotide polymorphisms (SNP) and copy number variants (CNV), with intraexonic duplications in ORC4L being most highly associated with disease. Beyond individual associations, CVID has a unique pattern of SNP and CNV, as suggested by the successful use of a Support Vector Machine (SVM) algorithm to identify this pattern in CVID patients and controls. SVM can be trained with a variety of data, and produces a “hyperplane” for subsequent classification. In the studies described above, the CVID SVM hyperplane successfully classified cases with an accuracy of 91%, PPV 100%, and NPV 96%.


Though the use of SVM supports the polygenic nature of CVID, a remaining question was whether monogenic CVID would lack the genetic fingerprint of the more common polygenic disease. If this were the case, SVM classification of microarray data would incorrectly classify these individuals as not affected, and could therefore be a useful screening method for monogenic CVID, most notably in familial cases. Accordingly additional studies were undertaken.


Patients


Patients were enrolled in research protocols approved by the host institutional review board to allow genetic analysis and clinical data collection.


Clinical History


The proband is a young woman who experienced recurrent sinopulmonary infections beginning in early adolescence. Evaluation at age 19 years showed IgG 620 mg/dl (normal range 635-1775 mg/dL), undetectable IgM and IgA (normal ranges 71-237 and 70-486 mg/dL respectively), and poor specific antibody production following vaccination to S. pneumoniae (1 of 14 serotypes >1.3 mcg/ml) and N. meningitidis (0 of 4 strains protective). Lymphocyte subsets were normal in number with CD19+ 388 cell/mcl (normal range 100-570), though switched memory B-cells (CD19+IgDCD27+) were low at 0.4% (normal >0.5%). She has been maintained on subcutaneous immunoglobulin with good control of infections, and has no autoimmune disease.


Her family history is notable for CVID in both her father as well as her older brother (FIG. 11). Her father also has a history of psoriasis, and her brother has type I diabetes. Her paternal grandparents, aunts, and their families are unaffected, as is her maternal family.


Genetic Studies


High throughput SNP genotyping was performed with the Infinitium HumanHap610 Beadchip, and the PennCNV algorithm was used for CNV calls. A support vector machine algorithm was trained with data from 179 CVID cases and 1917 controls, utilizing the 658 most significantly associated variants from the 2011 study, and subsequently tested on the patient.


Whole-exome sequencing using the Agilent SureSelect Human All Exon 50 Mb kit was performed on the patient and her family. Variants were matched to disease segregation (which suggested a heterozygous, autosomal dominant pattern), and further narrowed by exclusion of synonymous mutations, in silico analysis of mutation impact, exclusion of SNPs in public databases (1000 Genomes and NHLBI 5400 exomes Project), tissue expression pattern (BioGPS.org), and ties to known immunologic pathways.


RT-PCR and Immunoblotting


RNA was isolated from whole blood obtained from the patient and controls via Trizol reagent (Applied Biosystems, Grand Island, N.Y.) and RNEasy kit (Qiagen, Germantown, Md.). cDNA was produced via high capacity Reverse Transcriptase kit (Applied Biosystems), and custom cDNA primers for IRF2BP2 (both total and isoform 2) and GAPDH were created. RT-PCR performed via SYBR Green core reagents on a QPCR 7900HT system. Gene dose was calculated via DDCT method.


Protein immunoblotting was performed on lymphoblasts derived from peripheral blood mononuclear cells via treatment with phytohemagglutanin (2 ug/ml) and IL-2 (500 IU/ml). Antibodies against IRF2BP2 and TATA binding protein (Abcam, Cambridge, Mass.) were used.


Support Vector Machine Hyperplane Classification


Analysis of the top 658 SNP/CNVs by the pre-trained CVID SVM algorithm predicted that the patient was not affected (FIG. 11).


Whole Exome Sequencing


Sequencing of the proband revealed 12 non-synonymous, rare variants, of which 5 were predicted to be damaging via in silico analysis (SIFT, polyphen2). Three variants (AMBP, IRF2BP2, and PIK3C2G) had been tied to immunologic pathways. Sanger sequencing of IRF2BP2 in the proband and family confirmed that a heterozygous S535N mutation was present in all affected family members, and none of those who were unaffected. This mutation is in the RING domain of the protein, which has been described as the region of interaction with IRF2 and NFAT1 [3]. See FIG. 12.


RT-PCR of IRF2BP2


Quantitative PCR of IRF2BP2 from patient and control peripheral blood showed a lower transcript quantity in the patient versus normal control (FIG. 13). In the normal control, Isoform 1 was predominant, with comparatively less transcripts of Isoform 2 (which differs by exon 1 length). In the patient, isoform expression was roughly equal.


These results reveal that learning algorithms such as SVM provide a valuable method of screening for monogenic diseases within polygenic populations by searching for differences in the genetic pattern of SNP and rare CNV. In the field of PIDD, this is particularly useful for screening for monogenic causes of early-onset autoimmune disease such as type I diabetes or inflammatory bowel disease.


Moreover, these studies reveal that the presence of a SNP in IRF2BP2 on chromosome 1 at position 234742995 is indicative of a new CVID-associated candidate gene.


Example 4

The information herein above can be applied clinically to patients for diagnosing an increased susceptibility for developing CVID, and therapeutic intervention. A preferred embodiment of the invention comprises clinical application of the information described herein to a patient. Diagnostic compositions, including microarrays, and methods can be designed to identify the genetic alterations described herein in nucleic acids from a patient to assess susceptibility for developing CVID. This can occur after a patient arrives in the clinic; the patient has blood drawn, and using the diagnostic methods described herein, a clinician can detect one or more indicative SNPs or CNVs described in the Tables hereinabove. The information obtained from the patient sample, which can optionally be amplified prior to assessment, will be used to diagnose a patient with an increased or decreased susceptibility for developing CVID. Kits for performing the diagnostic method of the invention are also provided herein. Such kits comprise a microarray comprising at least one of the SNPs provided herein in and the necessary reagents for assessing the patient samples as described above.


The identity of CVID-involved genes and the patient results will indicate which variants are present, and will identify those that possess an altered risk for developing CVID. The information provided herein allows for therapeutic intervention at earlier times in disease progression that previously possible. For example, agents such as STA-5326, sotatercept, PF3446962, PEG-interleukin-2 and B-lymphocyte stimulators can be administered as appropriate.


Also as described herein above, several new genes involved in CVID pathogenesis have been identified which provide novel targets for the development of new therapeutic agents efficacious for the treatment of CVID.


While certain of the preferred embodiments of the present invention have been described and specifically exemplified above, it is not intended that the invention be limited to such embodiments. It will be apparent to one skilled in the art that various changes and modifications can be made therein without departing from the scope of the present invention, as set forth in the following claims.

Claims
  • 1. A solid support for detecting at least one common variable immunodeficiency (CVID)-associated copy number variation (CNV) in a sample comprising immobilized nucleic acid probes or primers of 15 or more nucleotides in length, wherein each of said probes or primers present on said solid support specifically hybridize to CVID specific nucleic acids in chromosomal regions consisting of chr11: 85365857-85381622, chr20: 57735790-57741780, chr22: 17396663-18417315, chr4: 10256682-10264316, and chr10: 46003146-46042543, chr2: 148396730-148433180, chr15: 35053039-35063531, chr7: 91789778-91801963, chr2: 163316298-163316595, chr7: 87180702-87191931, and, optionally, one or more of chr19: 9256584-9277749, chr4: 39190766-39201960, chr7: 18903915-18905725, chr5: 170531269-170536993, chr7: 34685030-34686172, and chr1: 170400944-170403742.
  • 2. A kit comprising the solid support of claim 1 and reagents for detecting hybridization of nucleic acids to said solid support.
Parent Case Info

This application is a continuation in part application of PCT/US2011/051385 filed Sep. 13, 2011 which in turn claims priority to U.S. Provisional Application 61/382,231 filed Sep. 13, 2010, which is incorporated herein by reference as though set forth in full.

US Referenced Citations (1)
Number Name Date Kind
20010053519 Fodor Dec 2001 A1
Non-Patent Literature Citations (6)
Entry
Wang, K. et al. Genome Research, 17:1665-1674 (2007).
LePinchon J.-B. et al. American Journal of Medical Genetics Part A (2010), pp. 1300-1304.
DbSNP entry output for Reference SNP (refSNP) Cluster Report: rs2307394, from www.ncbi.nlm.nih.gov, 2 printed pages, printed on Jun. 5, 2018 (Year: 2018).
Orange, J., et al. “Genome-wide association identifies diverse causes of common variable immunodeficiency.” J Allergy Clin Immunol. Apr. 17, 2011;127(6):1360-7.
Uleand, T., et al. “Increased levels of biochemical markers fo bone turnover in relation to persistent immune activation in common variable immmunodeficiency.” European Journal of Clinical Investigation. (2001) 31, 72-78.
Sheridan, C., et al. Can ‘double blockbuster’ strengthen Amgen's backbone? Nature Biotechnology (Apr. 2008) 26, 4, 361-363.
Related Publications (1)
Number Date Country
20160046996 A1 Feb 2016 US
Provisional Applications (1)
Number Date Country
61382231 Sep 2010 US
Divisions (1)
Number Date Country
Parent 13802142 Mar 2013 US
Child 14829312 US
Continuation in Parts (1)
Number Date Country
Parent PCT/US2011/051385 Sep 2011 US
Child 13802142 US