Patent Application
20200087360
The present invention is in the field of methods of treating a diarrhea disorder.
Whole exome sequencing (WES) is a powerful approach for the identification of causal mutations of protein-coding sequences in rare human disorders1. However, this approach generally fails to interrogate the remaining non-coding 98% of the human genome, despite strong emerging indications that a significant proportion of disease-associated variants affect non-coding functions2,3. While whole genome sequencing (WGS) is increasingly utilized and can in principle identify both coding and non-coding mutations, it raises the significant difficulty of interpreting non-coding sequence changes for functional relevance. This is a particular challenge for regulatory sequences located distant from known protein-coding genes because the exact positions and in vivo functions of most such distant-acting regulatory sequences in the human genome remain poorly annotated. Furthermore, the in vivo consequences of changes to these sequences are considerably more difficult to predict than those in protein-coding sequences. In contrast to coding mutations, a very limited number of sequence changes affecting human distant-acting regulatory elements associated with severe phenotypes have been identified, and even fewer are understood at the mechanistic level4.
The present invention provides for a recombinant or isolated polypeptide comprising the amino acid sequence of an enhancer polypeptide.
In some embodiments, the amino acid sequence comprises at least 70% identity of SEQ ID NO:1, SEQ ID NO:2, or SEQ ID NO:3.
The amino acid sequence of the mouse enhancer polypeptide is as follows:
The amino acid sequence of the human enhancer polypeptide is as follows:
The amino acid sequence of the longer mouse enhancer polypeptide is as follows:
In some embodiments, the polypeptide comprises one or more of the following amino acid sequences: MAAGVIR (SEQ ID NO: 4), SEEEEEEEEEEEEEE (SEQ ID NO: 5), SPETP (SEQ ID NO: 6), QLLRFSELIS (SEQ ID NO: 7), RYFGRKD (SEQ ID NO: 8), GQDPDA (SEQ ID NO: 9), LYYADLV (SEQ ID NO: 10), PLGPLAELFDYGL (SEQ ID NO: 11), LERKY (SEQ ID NO: 12), HITPM (SEQ ID NO: 13), QRKLPPSFWKEP (SEQ ID NO: 14), PLGLLH (SEQ ID NO: 15), and GTPDFSDLLASWS (SEQ ID NO: 16). In some embodiments, the polypeptide comprises two or more, three or more, four or more, five or more, six or more, seven or more, eight or more, nine or more, ten or more, eleven or more, or twelve or more of amino acid sequences SEQ ID NOs: 4-16. In some embodiments, the polypeptide comprises one or more, two or more, three or more, four or more, five or more, six or more, seven or more, eight or more, nine or more, ten or more, eleven or more, or twelve or more, or all, of the individual and/or consecutive stretches of amino acid residues that are identical between the two sequences indicated with an asterisks (“*”) in
In some embodiments, the amino acid sequence comprises at least 80%, 90%, 95%, or 99% identity of SEQ ID NO:1, SEQ ID NO:2, or SEQ ID NO:3.
The present invention also provides for a nucleic acid encoding the polypeptide of the present invention.
The present invention also provides for a host cell comprising the nucleic acid encoding the polypeptide of the present invention capable of expressing the polypeptide.
The present invention also provides for a method for synthesizing and/or purification/isolation of the polypeptide and/or nucleic acid of the present invention.
The present invention also provides for a transgenic non-human mammal, wherein the mammal is deleted or knocked out for one or more of an intestine-critical region (ICR). In some embodiments, the mammal is a mouse or rat.
The present invention also provides for a pharmaceutical composition comprising the polypeptide of the present invention and a pharmaceutically acceptable carrier.
The present invention also provides for a method of treating or preventing a subject suffering or at risk or suspected of suffering from a diarrhea disease or disorder, the method comprising administrating a pharmaceutical composition of the present invention to a subject in need of such treatment.
In some embodiments, the subject is a mammal. In some embodiments, the mammal is human. In some embodiments, the subject is suffering from a diarrhea disease or disorder. In some embodiments, the subject at risk or suspected of suffering from a diarrhea disease or disorder. In some embodiments, the diarrhea disease or disorder is a congenital diarrhea disorder, or a severe congenital malabsorptive diarrhea.
The foregoing aspects and others will be readily appreciated by the skilled artisan from the following description of illustrative embodiments when read in conjunction with the accompanying drawings.
Increased mortality of chr17ΔICR/ΔICR compared to wild-type.
Before the present invention is described, it is to be understood that this invention is not limited to particular embodiments described, as such may, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting, since the scope of the present invention will be limited only by the appended claims.
Where a range of values is provided, it is understood that each intervening value, to the tenth of the unit of the lower limit unless the context clearly dictates otherwise, between the upper and lower limits of that range is also specifically disclosed. Each smaller range between any stated value or intervening value in a stated range and any other stated or intervening value in that stated range is encompassed within the invention. The upper and lower limits of these smaller ranges may independently be included or excluded in the range, and each range where either, neither or both limits are included in the smaller ranges is also encompassed within the invention, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in the invention.
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, the preferred methods and materials are now described. All publications mentioned herein are incorporated herein by reference to disclose and describe the methods and/or materials in connection with which the publications are cited.
As used in the specification and the appended claims, the singular forms “a”, “an”, and “the” include plural references unless the context clearly dictates otherwise. Thus, for example, reference to a “polypeptide” includes a single polysaccharide molecule, and a plurality of polysaccharide molecules having the same, or similar, chemical formula, chemical and/or physical properties.
The terms “optional” or “optionally” as used herein mean that the subsequently described feature or structure may or may not be present, or that the subsequently described event or circumstance may or may not occur, and that the description includes instances where a particular feature or structure is present and instances where the feature or structure is absent, or instances where the event or circumstance occurs and instances where it does not.
These and other objects, advantages, and features of the invention will become apparent to those persons skilled in the art upon reading the details of the invention as more fully described below.
It is to be understood that, while the invention has been described in conjunction with the preferred specific embodiments thereof, the foregoing description is intended to illustrate and not limit the scope of the invention. Other aspects, advantages, and modifications within the scope of the invention will be apparent to those skilled in the art to which the invention pertains.
All patents, patent applications, and publications mentioned herein are hereby incorporated by reference in their entireties.
The invention having been described, the following examples are offered to illustrate the subject invention by way of illustration, not by way of limitation.
Distant-acting transcriptional enhancers are a predominant category of non-coding DNA in the human genome. However, the detection and functional interpretation of causative mutations affecting enhancers in human disorders remains challenging. Here are identified microdeletions of a non-coding sequence (intestine-critical region, ICR) on human chromosome 16p13.3 that cause inherited severe and intractable congenital diarrhea in affected infants. Transgenic mouse reporter assays show that the ICR is a transcriptional enhancer active in vivo during development of the gastrointestinal system. Targeted deletion of the ICR enhancer in mice cause symptoms recapitulating all major aspects of the human condition. Transcriptome analyses of human and mouse intestinal tissues reveal that the ICR deletion affects the expression of multiple genes, including strong down-regulation of gastrointestinal hormone peptides. Taken together, these results demonstrate that an enhancer deletion causes a severe congenital disorder and highlight the increasing potential for the discovery of disease-causing non-coding mutations as whole genome sequencing becomes routine in the clinic.
In this Example, it is demonstrated how the identification of non-coding deletions in a small number of patients is coupled to purpose-built mouse models which can be used to elucidate the regulatory basis of an inherited severe disease. It is also shown that mice carrying the non-coding deletion accurately recapitulate molecular and physiological phenotypes of the human disease condition, thus providing an animal model to explore the etiology of the human disorder.
Congenital diarrhea disorders are a heterogeneous group of inherited diseases of the gastrointestinal tract starting within the first few weeks of life, often immediately after birth5-7. These disorders are often life-threatening, cannot be successfully treated, and affected individuals often depend on life-long parenteral nutrition (
Eight patients from seven unrelated families of common ethnogeographic origin are studied with an autosomal recessive pattern of severe congenital malabsorptive diarrhea7 (
To identify possible structural genomic changes at this locus, all WES data sets, as well as WGS data from one of the patients are further examined. In WES data, an absence of coverage of three consecutive exons of a predicted transcript of C16ORF91 is observed in a subset of patients, suggesting the presence of a deletion (
To explore possible non-coding functions of the ICR sequence, Encyclopedia of DNA Elements (ENCODE) data9 are examined. The interval contains a 400 bp region with high evolutionary conservation across vertebrates that shows CpG island and DNAse hypersensitivity signatures, and encompasses a cluster of multiple binding sites for transcription factors identified by ChIP-seq (
To examine if deletion of the minimal ICR sequence is sufficient to cause the in vivo phenotypes observed in human patients, a 1,512 bp mouse sequence orthologous to the human 1,528 bp ICR from the mouse genome is removed using homologous recombination in embryonic stem cells (
To explore the molecular basis of the phenotypes observed upon ICR deletion, possible changes in gene transcription in human and mouse digestive tract tissues are examined. Such changes may reflect dysregulation of direct target genes of the ICR enhancer, indirect downstream regulatory events, or the absence or general dysfunction of intestinal cell populations. RNA sequencing of duodenal and stomach biopsies obtained from a ΔL/ΔL patient are performed, as well as a non-diseased sibling. Among the genes showing the strongest down-regulation genome-wide in at least one of these tissues, eight encode gastrointestinal peptide hormones secreted by enteroendocrine cells14, and four have other relationships to gastrointestinal function (Table 1). Top 30 upregulated and downregulated genes, constructed with a threshold of X7 up or downregulation. These genes are selected by from a longer list in duodenal and stomach biopsies comparing affected to a sibling wild-type control. The fold changes are calculated as the expression ratio wild type/affected for down regulated genes and affected/wild type for up regulated genes.
Particularly pronounced changes are observed for five peptide hormones: gastric inhibitory polypeptide (GIP), motilin (MLN) and ghrelin (GHRL) in the duodenum and gastrin (GAST) and somatostatin (SST) in the stomach, all of which show >100-fold reduction in expression. In addition MBOAT415,16, a ghrelin-modifying enzyme, and ARX, a transcription factor controlling enteroendocrine celldevelopment17 and associated with syndromic congenital diarrhea18,19 show 20- to 30-fold down-regulation in the ΔL/ΔL small intestine. These results are consistent with abnormal development or function of enteroendocrine cells20. Among the genes showing the largest increase in expression, eight are related to the gastrointestinal tract including gastrokines 1 and 2 (GKN1, GKN2), crucial for homeostasis of gastric epithelial cells and maintenance of gastric mucosa integrity21, pepsin precursor (PGA3) and motilin receptor (MLNR; Table 1). Quantitative RT-PCR of selected candidates including seven gastrointestinal peptide hormones and ARX confirmed their dysregulation in ΔL/ΔL samples. Consistent with these observations in human patients, RNA sequencing of a panel of mouse digestive tract biopsies taken at different stages of development show that nearly all of these genes are dysregulated in chr17ΔICR/ΔICR mice. For the genes shown in Table 1, across all profiled mouse digestive tract tissues 121 of 191 valid comparisons show significant changes in expression (p<0.05), the vast majority of which (105 of 121; 87%) is in the same direction as in human biopsies. Together, these results are consistent with major disruptions of normal intestinal physiology in chr17ΔICR/ΔICR humans and mice and highlight the close resemblance between the human disease condition and the mouse knockout model.
To further explore the pathophysiology associated with ICR deletions, biopsies obtained from two ΔL/ΔL homozygous patients are subjected to immunohistochemical staining with chromogranin A (CHGA), an early marker of enteroendocrine cell development. Increased immunoreactivity, as compared to healthy controls, is seen in the duodenal villi and stomach pyloric mucosae, a hyperplastic change that further supports that ICR deletions cause abnormal development of enteroendocrine cells (
The involvement of distant-acting regulatory regions in human diseases remains poorly understood and few cases of disease-causing variations that affect transcriptional enhancers have been documented22-26. Only one of these examples constitutes a complete deletion of an enhancer24 and it remains unclear if deletion of the homologous sequence in mice produces a phenotype mimicking the human condition. It is shown that a deletion of a developmental enhancer sequence is the cause of a severe, recessively inherited gastrointestinal disease. Enhancer activity is highly tissue-specific, and the tissues with enhancer activity in vivo are consistent with the gastrointestinal disease etiology. The observed molecular and physiological phenotypes suggest that the enhancer deletion affects normal development of enteroendocrine cells and thereby normal enteroendocrine hormone secretion. This is supported by the striking phenotypic similarity between chr17ΔICR/ΔICR mice and mice with an intestinal-specific deletion of Neurog3, a proendocrine transcription factor required for development of enteroendocrine cells27. Since chr17ΔICR/ΔICR mice resemble human patients homozygous for ICR deletions in all disease aspects examined in this study, these mice are likely to provide an accurate model for studying the human condition and exploring therapeutic interventions. Beyond congenital diarrhea, the results highlight the potential role that distant-acting regulatory elements may play in the pathology of other Mendelian diseases. While WGS approaches identify increasing numbers of disease-associated non-coding variants, their functional interpretation remains challenging. This example demonstrates the importance of detailed experimental follow-up of such findings through in vivo models, an approach that will benefit from the emerging suite of highly efficient genome editing tools28.
Subjects:
IDIS patients are recruited at Schneider and Sheba medical centers in Israel. The study is conducted in accordance with the Declaration of Helsinki, and all subjects and their family members had given informed consent for genetic testing and reproduction of patient photos.
Exome Sequencing and Variants Identification:
Exome sequencing is performed using Agilent SureSelect Human All Exon technology (Agilent Technologies, Santa Clara, Calif.). The captured regions are sequenced using Genome Analyzer IIx (Illumina, Inc. San Diego, Calif.). The resulting reads are aligned to the reference genome (build 37) using the Burrows-Wheeler Alignment (BWA) tool29. 70× coverage, where a base is considered covered if ≥5 reads spanned the nucleotide is obtained. Genetic differences relative to the reference genome are identified by the SAMtools variant calling program30, which identifies both single nucleotide variants and small insertion-deletions (indels). Finally, the Sequence Variant Analyzer software (SVA)31 is used to annotate all identified variants. For comparison to controls 1000 samples are subjected to exome or whole genome sequencing at the Center for Human Genome Variation (CHGV, Duke University, NC, USA), dbSNP, 1000 genomes, and NHLBI GO Exome-sequencing Project.
Whole Genome Sequencing:
WGS of individual 2.1 is performed at CHGV, using the Illumina HiSeq platform (Illumina, Inc. San Diego, Calif.) and analyzed as described for exome data. 275 CHGV whole-genome sequenced, unrelated samples are used as controls. To detect copy number variants from WGS the Estimation is used by read depth with single-nucleotide variants (ERDS) tool32.
Biopsy Collection:
Subjects underwent gastro-duodenoscopy following Institutional Review Board (IRB) approval (No. 9881-12-SMC) at Sheba Medical Center, and written informed consent of the patients and family members.
RNA Extraction from Biopsies:
RNA isolation from frozen biopsies is performed using TRI Reagent® method (Sigma-Aldrich Inc.) according to the manufacturer's instructions or by Qiagen RNeasy Mini Kit (Qiagen, Valencia, Calif., USA). Integrity of the samples is measured for concentration and purity using NanoDrop® Spectrophotometer (Nanodrop Technologies, Wilmington, Del., USA).
RNA Sequencing of Human Samples:
Total RNA is prepared according to the Illumina RNA-seq protocol: briefly, globin reduction, polyA enrichment, chemical fragmentation of the polyA RNA, cDNA synthesis, and size selection of 200 bp cDNA fragments are performed. Next, the size-selected libraries are used for cluster generation on the flow cell and prepared flow cells are run on the Illumina HiSeq2000 (Illumina, Inc. San Diego, Calif.). A total of 74.18 million paired-end reads of a 100 bp are obtained for the affected sample and 72.53 million reads to the healthy sample. Reads are aligned to the human genome (NCBI37/hg19) using Tophat v2.0.432 with the default parameters. Gene expression quantification is performed with cuffdiff33 using the Illumina iGenome project UCSC annotation file as a reference.
Quantitative Real-Time Reverse Transcriptase Polymerase Chain Reaction (qPCR):
RNA extracted from the biopsies is used for qPCR expression analyses. qPCR is performed using TaqMan® Gene Expression Assays (Applied Biosystems, Foster City, Calif., USA) using the Applied Biosystems StepOnePlus (Applied Biosystems). From 1 μg of biopsy RNA, cDNA is synthesized using the SuperScript® First-strand Synthesis System for RT-PCR (Invitrogen, Carlsbad, Calif., USA) according to the manufacturer's instructions. A total of 20 μl of cDNA is added with 30 μl of water to 50 μp of TaqMan® universal PCR Master Mix (Applied Biosystems) and the resulting 100 μl reaction mixtures are loaded onto a 96-well PCR plate. 14 different TaqMan® Gene Expression Assay are used including three housekeeping genes with the following assays IDs: Hs00757713_m1 (MLN), Hs01074053_m1 (GHRL), Hs00175048_m1 (NTS), Hs00356144_m1 (SST), Hs00174945_m1 (PYY), Hs01062283_m1 (GAST), Hs00292465_m1 (ARX), Hs00174937_m1 (CCK), Hs00175030_m1 (GIP), Hs00219734_m1 (GKN1), Hs00699389_m1 (GKN2).
The housekeeping genes are HMBS (Hs00609297_m1), ACTB (Hs99999903_m1) and GAPDH (Hs99999905_m1). Reference cDNA samples are synthesized using 200 ng of RNA from RNA extracted from stomach and duodenum tissues of two healthy controls (BioCat GmbH, Heidelberg, Germany) for use in the normalization calculations. Quantitative RT-PCR for expression analysis on the missing exons in C16ORF91 is done using cDNA extracted from the Human Digestive System MTC™ Panel (Clontech Laboratories, Inc. Mountain View, Calif.).
Serum Collection:
Whole blood is withdrawn into a Vacutainer serum tube without anti-coagulant. The blood is immediately treated with 1 μM AEBSF (protease inhibitor) and remains at room temperature for 30 min to clot before centrifugation (15 min at 2500 rpm at 4° C.).
ELISA:
Serum hormone levels are determined using sandwich ELISA technique performed by the following commercial kits according to the manufacturer's instructions. Human Ghrelin (Total) ELISA COLD PACKS (Millipore, USA), Human PYY (Total) ELISA Kit (Millipore), and Human gastric inhibitory polypeptide (GIP) ELISA Kit (ENCO).
Linkage Analysis and Homozygosity Mapping:
Genome-wide SNP genotyping from DNA of 6 affected children and 22 relatives from families 1-5 is performed using the Illumina HumanCytoSNP-12v2-1_H, according to the manufacturer's recommendations (Illumina, Inc. San Diego, Calif.) in conjunction with SNP genotypes retrieved from whole exome data. For linkage studies 35,845 informative equally spaced SNP markers are chosen after filtering for Mendelian errors and unlikely genotypes. Genotypes are examined with the use of a multipoint parametric linkage analysis and haplotype reconstruction for an autosomal recessive model with complete penetrance and a disease allele frequency of 0.001 as previously described34. Homozygosity mapping is performed using PLINK35 with the default parameters (length 1000 kb, SNP(N) 100, SNP density 50 kb/SNP, largest gap 1000 kb).
Deletion Analysis:
Boundaries for the two deletion alleles are determined by PCR using amplified DNA and Sanger sequencing. The specific primers are used amplifying across both deletions and inside the overlap region for the two deletions are reported in Table 2. In parallel, polymorphic markers are used that are identified by electronically screening genomic clones located on Chr16 0.86-2.8 Mb. Primers are designed with the Primer3 software (website for: frodo.wi.mit.edu/cgi-bin/primer3/primer3_www.cgi/ from the Whitehead Institute, Massachusetts Institute of Technology, and Cambridge, Mass.). The specific primers used are reported in Table 3. Amplification of the polymorphic markers is performed in a 25-μl reaction containing 50 ng of DNA, 13.4 ng of each primer, and 1.5 mM dNTPs in 1.5 mM MgCl2 PCR buffer with 1.2 U Taq polymerase (Bio-Line, London, UK). After an initial denaturation of 5 minutes at 95° C., 30 cycles are performed (94° C. for 2 minutes, 56° C. for 3 minutes, and 72° C. for 1 minute), followed by a final step of 7 minutes at 72° C. PCR products are electrophoresed on an automated genetic analyzer (Prism 3100; Applied Biosystems, Inc. [ABI], Foster City, Calif.). The breakpoints coordinates are: ΔL—chr16: 1475365-1482378, ΔS—chr16:1480850-1483951, with an overlapping region at chr16: 1480850-1482378 (ICR).
Mouse Transgenic Assays:
The candidate sequence containing the expected enhancer (chr 16: 1479875-1480992) is PCR amplified from human genomic DNA and, using Gateway (Invitrogen) cloning, is cloned into an Hsp68-lacZ vector containing a minimal Hsp68 promoter coupled to a lacZ reporter gene. The construct is microinjected into fertilized FVB/N mouse oocytes, which are implanted into pseudopregnant foster females and embryos are collected at E11.5 through E14.5. Enhancer reporter activity is determined by X-gal staining to detect 3-galactosidase activity. Only patterns observed in at least three different embryos resulting from independent transgenic events are considered reproducible positive enhancers.
Generation of Enhancer Null Mice:
Homologous arms are generated by PCR (see Suppl. Table S5 for primers) and cloned into ploxPN2T vector, which contains neomycin resistant cassette flanked by loxP for positive selection, and an HSV-tk cassette for negative selection. Constructs are linearized and electroporated (20 μg) into W4/129S6 mouse embryonic stem cells (Taconic). The electroporated cells are selected under G418 (150 μg/ml) and 0.2 μM FIAU for a week. Surviving colonies are picked and expanded on 96-well plates, screened both by PCR and sequencing with primers outside but flanking the homologous arm. Clones that are correctly targeted are electroporated with 20 μg of the Cre recombinase-expressing plasmid TURBO-Cre. TURBO-Cre is provided by Dr. Timothy Ley of the Embryonic Stem Cell Core of the Siteman Cancer Center, Washington University Medical School.
Clones positive for Neo removal are screened by PCR and checked for G418 sensitivity. PCR products covering the deleted region and part of homologous arms are gel purified and sequenced to confirm the deletion of the ICR enhancer.
Correctly targeted clones are subsequently injected into C57BL/6J blastocyst stage embryos. Chimeric mice are then crossed to C57BL/6J mice (Charles River) as well as 129S6/SvEvTac (Taconic) to generate heterozygous enhancer null mice, followed by breeding of heterozygous littermates to generate homozygous enhancer null mice.
Genotyping of Enhancer Null Mice:
Genomic DNA is extracted from a 0.2 to 0.3-cm section of tail that is incubated overnight in lysis buffer (containing 100 mM Tris-HCl pH 8.5, 5 mM EDTA, 0.2% SDS, 200 mM NaCl and 50 μg Proteinase K) at 55° C. Genotyping is carried out using standard PCR techniques (see Table 4 for primers). One to two microliters of 50- to 100-fold diluted tail lysate is used in a 20 μl PCR containing 200 μM dNTP, 1.5 mM MgCl2, 5 pmole of each forward and reverse primer and 0.5 U of Taq polymerase.
RNA Sequencing of Mouse Tissues:
Total RNA is extracted from different intestinal regions and stomach of mice at E11.5, P1, P5, P10, P15 and P20 using TRIzol® Reagent (Invitrogen). RNAseq libraries are then constructed using Illumina TruSeq Stranded Total RNA Sample Preparation Kit following manufacture's recommendation. The libraries are sequenced using a 50 bp single end strategy with four samples per lane on an Illumina HiSeq instrument and data is analyzed using the same protocols as described for human, though with the mm9 mouse reference and Illumina iGenome project mouse genome annotation data.
16S Amplicon Analysis (iTags) of Microbial Community Diversity:
Feces and gut content samples are collected from chr17ΔICR/ΔICR mice and wt littermates. DNA is extracted from these samples using PowerFecal® DNA Isolation Kit (MO Bio Laboratories). V4 16S regions are amplified from the DNA samples using barcoded primers and 5 PRIME™ HotMasterMix™ (Fisher Scientific) as previously described36. Amplicons are pooled in equal amount, purified with AMPureXP® magnetic beads (Beckman Coulter), and sequenced.
Histological Analysis of Human Biopsies:
FFPE blocks are sectioned at a thickness of 4 μm and a positive control is added on the right side of the slides. All immunostainings are fully calibrated on a Benchmark XT staining module (Ventana Medical Systems Inc., USA). Briefly, after sections are dewaxed and rehydrated, a CC 1 Standard Benchmark XT pretreatment for antigen retrieval (Ventana Medical Systems) is selected for all immunostainings: Chromogranin A (1:500, Dako, Denmark), and Synaptophysin, (1:200, Life Technologies, Invitrogen, USA). Detection is performed with iView DAB Detection Kit (Ventana Medical Systems Inc., USA) and counterstained with hematoxylin (Ventana Medical Systems Inc., USA). After the run on the automated stainer is completed, slides are dehydrated in ethanol solutions (70%, 96%, and 100%) for one minute each. Sections are then cleared in xylene for 2 minutes, mounted with Entellan and cover slips are added. Chromogranin A and Synaptophysin show cytoplasmic staining.
Generation of Induced Pluripotent Stem Cells (iPSCs) from Patient Lymphocytes:
Whole blood is isolated by routine venipuncture from patient 2.1 and two healthy siblings (2.3-heterozygous carrier, 2.4-unaffected WT) at Sheba Medical Center in Israel, in preservative-free 0.9% sodium chloride containing 100 U/mL heparin. Blood is then shipped overnight to Cincinnati Children's Hospital Medical Center for iPS cell generation. Peripheral blood mononuclear cells (PBMCs) are isolated from whole blood by Ficoll centrifugation as previously described37 and are used to derive iPSCs. Briefly, PBMCs are cultured for 4 days in DMEM containing 10% FCS, 100 ng/ml SCF, 100 ng/ml TPO, 100 ng/ml IL3, 20 ng/ml IL6, 100 ng/ml Flt3L, 100 ng/ml GM-CSF, and 50 ng/ml M-CSF (Peprotech). Transduction using a polycistronic lentivirus expressing Oct4, Sox2, Klf4, cMyc and dTomato is performed38 following the second day of culture in this media. Transduced cells are then cultured for an additional 4 days in DMEM containing 10% FCS, 100 ng/ml SCF, 100 ng/ml TPO, 100 ng/ml IL3, 20 ng/ml IL6, and 100 ng/ml Flt3L. Media is changed every other day. PBMCs are then plated on 0.1% gelatin-coated dishes containing 2×104 irradiated MEFs/cm2 (GlobalStem, Rockville, Md.), and is cultured in hESC media containing 20% knockout serum replacement, 1 mM L-glutamine, 0.1 mM β-mercaptoethanol, 1× non-essential amino acids, and 4 ng/ml bFGF until iPSC colony formation. Putative iPSC colonies are then manually excised and re-plated in feeder free culture conditions consisting of matrigel (BD BioSciences, San Jose, Calif.) and mTeSR1 (STEMCELL Technologies, Vancouver, BC). Lines exhibiting robust proliferation and maintenance of stereotypical human pluripotent stem cell morphology are then expanded and cryopreserved before use in experiments. Standard metaphase spreads and G-banded karyotypes are determined by the CCHMC Cytogenetics Laboratory.
Differentiation of iPSCs into Intestinal Organoids:
The differentiation of induced human pluripotent stem cells is performed as previously described39-41 with minor modifications. Briefly, two clonal iPSC lines from each donor are dispase passaged into a matrigel coated 24 well tissue culture plate and cultured for 3 days in mTeSR1. Following definitive endoderm differentiation, the monolayers are treated for 4 days with RPMI medium 1640 (Gibco) containing 2% defined fetal calf serum, 1× non-essential amino acids, 3 μM CHIR99021 (Stemgent) and 500 ng/mL rhFGF4 (R&D Systems) to induce hindgut spheroid morphogenesis. After the 4th day, “day 0” HIOs are collected and embedded in matrigel matrix and cultured in Advanced DMEM/F12 (Gibco) containing 100 U/mL penicillin/streptomycin (Gibco), 2 mM L-Glutamine (Gibco), 15 mM HEPES (Gibco), N2 Supplement (Gibco), B27 Supplement (Gibco), and 100 ng/mL rhEGF (R&D Systems) for up to 42 days, splitting, passaging, and changing the media periodically.
HIOs collected for immunofluorescence analysis are fixed in 4% paraformaldehyde for 1-2 h at room temperature, washed overnight at 4° C. in PBS, and embedded in O.C.T. Compound (Sakura). Sections 8-10p thick are incubated with primary antibodies overnight at 4° C. in 10% normal donkey serum/0.05% Triton X-100-PBS solution and subsequently incubated with secondary antibodies for 1 h at room temperature. The primary antibodies used are: FoxA2 (1:500; Novus), E-Cadherin (1:500; R&D Systems), Synaptophysin (1:1000; Synaptic Systems), CDX2 (1:500; Biogenex), Pd×1 (1:5000; Abcam; data not shown). All secondary antibodies (AlexaFluor; Invitrogen) are used at 1:500 dilution. Confocal microscopy images are captured with a 20× plan apo objective on a Nikon A1Rsi Inverted, using settings of 0.5 pixel dwell time, 1024 resolution, 2× line averaging, and 2.0× A1 plus scan.
Total RNA is extracted from HIOs using a NucleoSpin RNA II kit (Macherey-Nagel), and cDNA is synthesized with SuperScript VILO (Invitrogen) using 300 ng RNA. qPCR analysis is performed with TaqMan Fast Advanced Master Mix and custom designed TaqMan Array 96-Well FAST Plates (Applied Biosystems) consisting of the following targets: 18S—Hs99999901_s1; GAPDH—Hs999999905_m1; ARX—Hs00292465_m1; CHGA—Hs00900370_m1; SYP—Hs00300531_m1; NTS—Hs00175048_m1.
Clinical Phenotypes of Congenital Diarrhea Disorders:
Congenital diarrhea disorders comprise a heterogeneous group of diseases composed of rare enteropathies related to specific etiology and pathogenesis including: (i) defects in absorption and transport of nutrients and electrolytes; (ii) maintenance and differentiation of enterocytes; (iii) differentiation and function of enteroendocrine cells (EECs) and (iv) modulation of the intestinal immune response7. This potentially life threatening condition in young infants and children is defined as congenital, severe, non-infectious diarrhea lasting more than two weeks, with consequent malabsorption, multiple food intolerance and failure to thrive5,6. Since this condition cannot be successfully treated, affected individuals depend on life-long Parenteral Nutrition (PN) and in some cases small bowl transplantation8.
Origins and Relationships of Patients:
Eight patients from seven different families of Jewish Iraqi origin with an apparent autosomal recessive pattern of malabsorptive diarrhea, originally defined as having intractable diarrhea of infancy syndrome (IDIS)7 are studied. Identity By Descent (IBD) analysis confirm the family relations and indicated that the closest cross-family relationship had IBD=0.040.
Mapping of Deletions in Patients:
Exome sequencing analysis of 5 patients (
Whole Genome Sequencing Controls:
Whole-genome sequencing for patient 2.1 confirmed the ΔL attributes and showed that it is the only homozygous genomic deletion in the linked region. None of the deletions are present in 200 ethnically matched Iraqi control chromosomes as well as in either 122 in-house Caucasians WGS samples. In addition, >3000 WGS of diverse sources in the KAVIAR dataset42 are searched and no deletions overlapping are found. Further, 1092 individuals from the 1000 Genome Project43 are scanned within the integrated variant calls file (ALL.wgs.integrated_phasel_v3.20101123.snps_indels_sv.sites.vcf), seeking overlaps with the ⊗L and ⊗S regions, and no such are observed. Searching the Database of Genomic Variants44,45 for large deletions that span the ⊗L and ⊗S regions identified several heterozygous deletions with combined allele frequency <0.004.
Mouse Microbiome Dysbiosis:
The fecal samples of knockout mice exhibit considerably reduced microbial diversity with respect to WT feces (
While the present invention has been described with reference to the specific embodiments thereof, it should be understood by those skilled in the art that various changes may be made and equivalents may be substituted without departing from the true spirit and scope of the invention. In addition, many modifications may be made to adapt a particular situation, material, composition of matter, process, process step or steps, to the objective, spirit and scope of the present invention. All such modifications are intended to be within the scope of the claims appended hereto.
This application claims priority to U.S. Provisional Patent Application Ser. No. 62/675,099, filed on May 22, 2018, which is hereby incorporated by reference.
The invention was made with government support under Contract No. DE-AC02-05CH11231 awarded by the U.S. Department of Energy and Grant No. HG003988 awarded by the National Institutes of Health. The government has certain rights in the invention.
| Number | Date | Country | |
|---|---|---|---|
| 62675099 | May 2018 | US |
