Patent Application
20160145687
The present invention relates to the field of molecular diagnostics. More specifically the present invention relates to means and methods for predicting, before seroconversion, a risk of a subject for Type 1 diabetes (T1D).
By the time of clinical diagnosis of Type 1 diabetes (T1D), an estimated 80-90% of insulin producing pancreatic beta cells have been destroyed by autoimmune mechanisms with immune cells infiltrating the islets. Autoantibodies against beta cell-specific antigens, such as proinsulin and insulin, glutamic acid decarboxylase 65 (GAD-65), islet antigen-2 (IA-2), and zinc transporter 8 (ZnT8), can usually be detected in serum before the clinical onset of the disease, but although the number of circulating autoantibodies is predictive for the development of T1D, predicting the onset of clinical disease remains a challenge.
Susceptibility to T1D is inherited, with the human leukocyte antigen (HLA) class II haplotypes encoding DR3-DQ2 (DRB1*03-DQA1*0501-DQB1*0201) and DR4-DQ8 (DRB1*0401-DQA1*0301-DQB1*0302) molecules conferring an estimated 50% of the total genetic risk. In addition, more than 40 non-HLA genes with more modest effect on disease risk, such as INS, CTLA4, PTPN22, and IL2R, have been discovered. When combined with a suitable genetic background, largely unknown random environmental factors probably contribute to the breakdown of self-tolerance. For instance, dietary factors, such as early exposure to foreign complex proteins and vitamin D deficiency, have been associated with T1D pathogenesis, and viral infections are believed to trigger autoimmunity. T1D prevalence is also associated with westernized lifestyle and level of hygiene.
There is a need for kits and methods for predicting the risk of development of T1D prior to any signs of seroconversion.
In one aspect the present invention provides a method of predicting, before seroconversion, a risk of a subject for Type 1 diabetes (T1D). The method may comprise the steps of a) providing a sample from said subject, b) determining a gene expression profile in said sample, and c) using said gene expression profile for the prediction of the probability of the subject to develop T1D.
In some embodiments, said subject is determined to have an increased risk of developing T1D, if in said gene expression profile at least one gene selected from the group consisting of RNASE2, CPPED1, VNN1, ALPL, PHOSPHO1, EMR3, CREB5, ROPN1L, CLC DEFA1, and PRSS33 is up-regulated as compared to a control gene profile.
In some further embodiments, said subject is determined to have an increased risk of developing T1D, if in said gene expression profile, alternatively or additionally to those mentioned above, at least one gene selected from the group consisting of CEACAM1, IFIT3, IFITM3, OAS1, and VCAN is up-regulated as compared to a control gene profile, and/or PMP22 is down-regulated as compared to a control gene profile.
In some still further embodiments, said subject is determined to have an increased risk of developing T1D, if in said gene expression profile, alternatively or additionally to those mentioned above, at least one gene selected from the group consisting of CTTN, DCAF12, EPB42, GMPR, MBNL3, NFIX, NPRL3, SELENBP1, SLC4A1, SLC6A8, TMEM158, TNS1, TRIM58, and VWCE is up-regulated as compared to a control gene profile.
In another aspect, the present invention provides an in vitro screening for determining, prior to seroconversion, whether an individual, preferably having an HLA-conferred risk, is at risk of developing T1D. The kit may comprise one or more testing agents capable of detecting the expression level of one or more genes selected from any gene profile disclosed herein, in a biological sample obtained from a subject whose risk of developing T1D is to be determined.
Other aspects, specific embodiments, objects, details, and advantages of the invention are set forth in the following drawings, detailed description, examples, and dependent claims.
In the following the invention will be described in greater detail by means of preferred embodiments with reference to the attached [accompanying] drawings, in which
In some implementations, the present disclosure is directed to methods, transcriptome profiles and kits useful for determining, before seroconversion, the risk that an individual will develop Type 1 diabetes (T1D).
As used herein, the term “seroconversion” refers to the first detection of one or several T1D-associated autoantibodies against beta cell-specific antigens in serum. These include islet cell specific autoantibodies (ICA), insulin autoantibodies (IAA), glutamic acid decarboxylase 65 autoantibodies (GADA), islet antigen-2 autoantibodies (IA-2A), and zinc transporter 8 autoantibodies (ZnT8A). In some embodiments, the following cut-off values may be used for determining the presence or absence of the autoantibodies: ICA≧4 JDFU (Juvenile Diabetes Foundation units), IAA≧3.48 RU (relative units), GADA≧5.36 RU, IA-2A≧0.43 RU, and ZnT8A≧0.61 RU. Seroconversion may occur years, e.g. 1 to 2 years, before clinical diagnosis.
As used herein, the term “transcriptome profile” refers to the set of RNA molecules, including but not limited to messenger RNA (mRNA) molecules and non-coding RNA (ncRNA) molecules, such as microRNA (miRNA) and long non-coding RNA (long ncRNA, IncRNA), produced in a given sample or a population of cells. It may also refer to a cDNA profile produced from an mRNA profile. The term may be used interchangeably with the terms “gene expression profile”, “transcriptional signature”, “RNA signature” and the like, as is evident to a person skilled in the art.
As used herein, the terms “up-regulation” and “up-regulated” refer to increase in expression of a gene in a sample as compared to a corresponding control sample. More specifically, the terms refer to increased transcription of a specific mRNA. Said increase can be determined qualitatively and/or quantitatively according to standard methods known in the art. The expression is increased if the expression level of the gene in the sample is, for instance, at least about 1.5 times, 1.75 times, 2 times, 3 times, 4 times, 5 times, 6 times, 8 times, 9 times, time times, 10 times, 20 times or 30 times the expression level of the same gene in the control sample.
As used herein, the terms “down-regulation” and “down-regulated” refer to decrease in expression of a gene in a sample as compared to a corresponding control sample. More specifically, the terms refer to decreased transcription of a specific mRNA. Said decrease can be determined qualitatively and/or quatitatively according to standard methods known in the art. The expression is decreased if the expression level of the gene in the sample is, for instance, at least about 1.5 times, 1.75 times, 2 times, 3 times, 4 times, 5 times, 6 times, 8 times, 9 times, time times, 10 times, 20 times or 30 times lower than the expression level of the same gene in the control sample.
As used herein, the term “updown-regulated” refers to three alternative situations: i) said gene is up-regulated in some subjects while down-regulated in other subjects; ii) some alternative probes for a given gene show up-regulation while some other probes for the same gene show down-regulation; and iii) before seroconversion, said gene shows both up-regulation and down-regulation de-pending on the measurement time point used.
The inventors have herein identified a transcriptome profile which exhibits predictive value regarding an individual's risk of developing T1D. Importantly, said prediction can be made prior to any signs of seroconversion. The identified predictive transcriptome profile applies in particular to individuals having a Human Leukocyte Antigen (HLA)-conferred risk for T1D. As used herein, the term “HLA-conferred risk for T1D” refers to a predisposition to T1D as determined on the basis of the individual's HLA profile as generally known in the art. In some embodiments, HLA-conferred susceptibility takes place if the individual carries HLA-DQB1 alleles *02/*0302 or *0302. If the individual whose risk for T1D is to be determined has HLA-conferred susceptibility, then also the control gene profile should be obtained from an individual having a corresponding HLA-conferred susceptibility or from a pool of such individuals.
As described in the experimental part, the present disclosure is based on a genome-wide transcriptomics analysis on a unique series of prospective whole-blood RNA samples from at-risk children collected in the Finnish Type 1 Diabetes Prediction and Prevention (DIPP) study. The samples covered the time span from before the development of autoantibodies (seroconversion) through the diagnosis of diabetes. Healthy, persistently autoantibody-negative children matched for date and place of birth, gender and the HLA-DQB1-conferred genetic risk were chosen as controls.
In one aspect, the present disclosure is directed to a method of predicting, before seroconversion, a risk of a subject for Type 1 diabetes (T1D) comprising the following steps:
a) providing a sample from said subject,
b) determining a gene expression profile in said sample, and
c) using said gene expression profile for the prediction of the probability of the subject to develop T1D. Preferably, the determined gene expression profile is compared to that of a corresponding control.
The above aspect may also be defined differently, i.e. as a method of predicting risk of T1D in an individual before seroconversion, the method comprising:
a) measuring the level of one or more members of one or more predictive gene profiles described herein, in a sample obtained from the individual,
b) identifying, preferably by a processor of a computing device, for the one or more measured levels, a difference between the measured level and a corresponding predetermined control level, and
c) responsive to the identifying, determining, preferably by the processor, a prediction corresponding to a relative risk of the individual developing T1D.
Furthermore, the above aspect may also be defined differently, i.e. as a method of predicting risk of T1D in an individual, the method comprising:
a) measuring one or more gene expression levels of any gene profile described herein, in a sample obtained from the individual,
b) calculating, preferably by a processor of a computing device, a risk assessment score corresponding to a relative risk of the individual developing T1D, wherein the risk assessment score is based in part upon a comparison of the gene expression level and a corresponding, possibly predetermined, control level.
In some embodiments, the risk for developing T1D can be determined based on the up-regulation of one or more genes selected from non-interferon regulated genes listed in Table 1 as compared to a corresponding control level. In some embodiments, a predetermined control level may be used.
Alternatively or additionally, the risk for developing T1D can be determined based on the up-regulation of one or more genes selected from interferon-regulated genes listed in Table 2 and/or on down-regulation of PMP22 (peripheral myelin protein 22, Gene Bank ID 5376) as compared to a corresponding control level. In some embodiments, a predetermined control level may be used.
Alternatively or additionally, the risk for developing T1D can be determined based on the up-regulation of one or more genes selected from erythrocyte-related genes listed in Table 3 as compared to a corresponding control level. In some embodiments, a predetermined control level may be used.
In some further embodiments, the gene profile to be determined or measured comprises at least one gene selected from the group consisting of ADM, ALPL, ASGR2, BCL6, C19orf59, C3AR1, C4BPA, C5orf4, CACNG6, CBS, CCL8, CEACAM1, CLC, CNN2, CPPED1, CREB5, CTTN, CYSTM1, DCAF12, DEFA1 (includes others), DSC2, DYSF, EMR3, EPB42, EPB49, FAM129A, FAM46C, FOLR3, GMPR, GPR84, HBD, HLA-C, IFIT3, IFITM3, IGF2BP2, IRF5, KIAA1324, KRT1, LILRB3, LRG1, MAFB, MBNL3, MMP9, MX1, MYOF, NFIX, NPCDR1, NPRL3, OAS1, OLIG2, OSBP2, PHOSPHO1, PMP22, PROK2, PRSS33, RAB31, RBPMS2, RFX2, RNASE2, ROPN1L, RRP12, SEC14L1, SELENBP1, SIRPB1, SLC22A4, SLC29A1, SLC4A1, SLC6A8, SMOX, SNCA, SORT1, TGM3, TMEM158, TNS1, TRIM58, TSHZ3, VCAN, VNN1, VWCE, AHI1, C17orf97, C9orf123, CCL4L1/CCL4L2, CENPA, CEP55, COMMD6, COX7B, CYP4F3, DTL, EPSTI1, ERAP2, GBP5, GLT25D2, GNLY, HINT1, HIST1H4A (includes others), HLA-DPB1, HMMR, HOXB2, IFI27, IFI44, IL18RAP, ITGB1BP1, KLRD1, KLRF1, PRSS23, PTER, RPL26, RPS27L, RSL24D1, SAMD9L, SCOC, SETD9, SUB1, TMEM126B, XAF1, ANXA3, CASP5, HERC5, IFI44, IFI44L, IFIT2, OAS3, PI3, and RSAD2.
In some still further embodiments, said individual has an increased risk for developing T1D if in said gene profile at least one gene selected from the group consisting of ADM, ALPL, ASGR2, BCL6, C19orf59, C3AR1, C4BPA, C5orf4, CACNG6, CBS, CCL8, CEACAM1, CLC, CNN2, CPPED1, CREB5, CTTN, CYSTM1, DCAF12, DEFA1 (includes others), DSC2, DYSF, EMR3, EPB42, EPB49, FAM129A, FAM46C, FOLR3, GMPR, GPR84, HBD, HLA-C, IFIT3, IFITM3, IGF2BP2, IRF5, KIAA1324, KRT1, LILRB3, LRG1, MAFB, MBNL3, MMP9, MX1, MYOF, NFIX, NPCDR1, NPRL3, OAS1, OLIG2, OSBP2, PHOS-PHO1, PMP22, PROK2, PRSS33, RAB31, RBPMS2, RFX2, RNASE2, ROPN1L, RRP12, SEC14L1, SELENBP1, SIRPB1, SLC22A4, SLC29A1, SLC4A1, SLC6A8, SMOX, SNCA, SORT1, TGM3, TMEM158, TNS1, TRIM58, TSHZ3, VCAN, VNN1, and VWCE is up-regulated as compared to a control gene profile.
In some even further embodiments, said individual has an increased risk for developing T1D if in said gene profile at least one gene selected from the group consisting of AHI1, C17orf97, C9orf123, CCL4L1/CCL4L2, CENPA, CEP55, COMMD6, COX7B, CYP4F3, DTL, EPSTI1, ERAP2, GBP5, GLT25D2, GNLY, HINT1, HIST1H4A (includes others), HLA-DPB1, HMMR, HOXB2, IFI27, IFI44, IL18RAP, ITGB1BP1, KLRD1, KLRF1, PRSS23, PTER, RPL26, RPS27L, RSL24D1, SAMD9L, SCOC, SETD9, SUB1, TMEM126B, and XAF1 is down-regulated as compared to a control gene profile. In some preferred embodiments, said individual has an increased risk for developing T1D if KLFR1 (Killer cell lectin-like receptor subfamily F, member 1; Gene Bank ID: 51348) is down-regulated as compared to a control gene profile.
In some still further embodiments, said individual has an increased risk for developing T1D if in said gene profile at least one gene selected from the group consisting of ANXA3, CASP5, HERC5, IFI44, IFI44L, IFIT2, OAS3, PI3, and RSAD2 is updown-regulated as compared to control gene profile. As used herein, the term “updown-regulated” refers to three alternative situations: i) said gene is up-regulated in some subjects while down-regulated in other subjects; ii) some alternative probes for a given gene show up-regulation while some other probes for the same gene show down-regulation; and iii) before seroconversion, said gene shows both up-regulation and down-regulation depending on the measurement time point used.
Optionally, the present methods may further comprise determining variations in the subject's gene profile at different time points in order to monitor, prior to seroconversion, any changes in or the development of the risk for T1D.
A preferred control sample or control gene profile for use in the present methods is a case-matched sample or profile. Case-matching may be made, for instance, on the basis of one of more of the following criteria: age, date of birth, place of birth, gender, predisposition for T1D, HLA status and any relevant demographic parameter. In some embodiments, said control sample or profile consists of a pool of, preferably case-matched, relevant control samples or profiles. In some embodiments, said control prolife has been predetermined prior to predicting a risk of T1D in an individual in accordance with the present disclosure.
Optionally, before to be compared with the control gene profile, the expression level of genes, in particular one or more genes in any of the preferred expression profiles set forth above, are normalized using the expression level of an endogenous control gene having a stable expression in different samples, such as GAPDH.
Suitable samples for use in accordance with the present disclosure include, but are not limited to, tissue samples (e.g. pancreatic samples) and blood samples including whole blood, serum, plasma, peripheral blood mononuclear cells and any purified blood cell type. In essence, any biological sample which contains RNA, preferably mRNA or any other RNA species which represents the genes of interest, may be used for the present purposes. In some embodiments, the sample to be analyzed is extracted total whole-blood RNA or, if desired, the sample may consist of isolated mRNA or any other RNA species representing the genes of interest.
In some embodiments, the subject is a human subject, preferably a child or an adolescent. In some more preferred embodiments, said subject does not show any signs of seroconversion. As used herein, the terms “subject” and “individual” are interchangeable.
The expression level of a gene may be determined by a variety of techniques. In particular, the expression level of a gene may be determined by measuring the quantity of RNA, preferably mRNA or any other RNA species representing the gene of interest, using methods well known in the art. Non-limiting examples of suitable methods include real time (RT) quantitative or semi-quantitative PCR. Primers suitable for these methods may be easily designed by a skilled person.
Other non-limiting ways of measuring the quantity of RNA, preferably mRNA or any other RNA species representing the gene of interest, include transcriptome approaches, in particular DNA microarrays. Generally, when it is the quantity of mRNA that is to be determined, test and control mRNA samples are reverse transcribed and labelled to generate cDNA probes. The probes are then hybridized to an array of complementary nucleic acids immobilized on a solid support. The array is configured such that the sequence and position of each member of the array is known. Hybridization of a labelled probe with a particular array member indicates that the sample from which the probe was derived expresses that gene. Non-limiting examples of commercially available microarray systems include Affymetrix GeneChip™ and Illumina BeadChip. Next Generation Sequencing (NGS) methods may also be used.
If desired, the quantity of RNA, preferably mRNA any other RNA species representing the gene of interest, may also be determined or measured by conventional hybridization-based assays such as Northern blot analysis.
In another aspect, the present disclosure relates to an in vitro kit for predicting, before seroconversion, a risk of a subject T1D. The kit may be used in any one of the methods of the present disclosure. Typically, the kit may comprise a pair of primers and/or a probe specific to one or more genes in any of the preferred expression profiles set forth above, preferably in the expression profile of Table 1 and/or Table 2. A skilled person can easily design suitable primers and/or probes taking into account specific requirements of a technique to be applied.
The kit may further comprise means for detecting the hybridization of the probes with nucleotide molecules, such as mRNA or cDNA, representing genes expressed in a test sample and/or means for amplifying and/or detecting the nucleotide molecules representing the genes expressed in the test sample by using the pairs of primers.
Other optional components in the kit include a compartmentalized carrier means, one or more buffers (e.g. block buffer, wash buffer, substrate buffer, etc.), other reagents, positive or negative control samples, etc.
The kit may also comprise a computer readable medium comprising computer-executable instructions for performing any method of the present disclosure.
All children studied were participants in the Finnish Type 1 Diabetes Prediction and Prevention Project (DIPP). Subjects identified as at risk for T1D based on their HLA haplotype were followed prospectively from birth. The protocol of the DIPP study has been approved by the Ethical Committees of the University Hospitals of Turku, Oulu and Tampere, and a written consent according to the Declaration of Helsinki was obtained from all study subjects and/or their parents. At the time of sample collection, 2.5 ml of venous blood was drawn into PAXgene Blood RNA tubes (PreAnalytix) at the DIPP study clinic, in Turku, Finland. The samples were incubated for 2 hours at room temperature and then stored at −70° C. until analyzed. Islet cell autoantibodies (ICA), insulin autoantibodies (IAA) and autoantibodies to glutamic acid decarboxylase 65 (GAD-65), islet antigen-2 (IA-2), and zinc transporter 8 (ZnT8) were measured from all individuals. Of 28 subjects in this study, nine were sampled starting before or at the time of the appearance of autoantibodies (seroconversion) (S1-5 and S7-10, Table 4); cases S3, S6, S7, and S10 progressed to T1D and the other six did not. The remaining 18 children (D1-18, Table 4) were all sampled starting after seroconversion and all progressed to T1D (D1-18).
Total whole-blood RNA was extracted from the samples using PAXgene Blood RNA kit (Qiagen) according to manufacturer's instructions. RNA quality and quantity was determined using NanoDrop ND-1000 (Thermo Scientific) and Experion Automated Electrophoresis System (Bio-Rad Laboratories).
For all samples, 50 ng of total RNA was processed to cDNA with Ovation RNA amplification system v2, including the Ovation whole blood reagent (NuGEN Technologies). The amplified cDNA was subsequently biotin-labelled and fragmented with Encore biotin module (NuGEN Technologies). Samples were hybridized to GeneChip Human Genome U219 array plate with specific protocols for using the GeneTitan Hybridization, Wash and Stain Kit for 3′ IVT Array Plates (Affymetrix). GeneTitan MC Instrument was used to hybridize, wash, stain and scan the arrays. Affymetrix GeneChip Command Consol 3.1 was used to control GeneTitan hybridization process and in summarizing probe cell intensity data (CEL file generation).
For seroconverted case-control pairs 1-6, 500 ng of total RNA was amplified and reverse transcribed with RiboAmp OA 1 round RNA amplification kit (Applied Biosystems/Arcturus). cRNA was biotinylated during the overnight (14 h) IVT reaction with Illumina total prep RNA amplification kit (Applied Biosystems/Ambion). cRNA was purified with RiboAmp OA 1 round RNA amplification kit. For seroconverted case-control pairs 7-10, 100 ng of total RNA was amplified and reverse transcribed with Ovation RNA amplification system v2, including the Ovation whole blood reagent (NuGEN Technologies). 5 μg of cDNA was labelled according to NuGEN's Illumina solution protocol, application note #2. cDNA concentration and quality was controlled with Experion automated electrophoresis system.
1.5 μg of each sample was hybridized to Sentrix human WG6 v2 expression bead chips (Illumina) at 58° C. overnight (18 h) according to Illumina whole-genome gene expression direct hybridization protocol, revision A. Hybridizations were detected with 1 μg ml−1 Cyanine3-streptavidine (General Electric Healthcare Life Sciences). Chips were scanned with Illumina bead array reader. The expression intensities were extracted with Bead Studio version 2.3.41 or 3.3 without any normalization or background subtraction.
For all T1D progressor case-control pairs, 100 ng of total RNA was amplified to cDNA with Ovation RNA amplification system v2, including the Ovation whole blood reagent (NuGEN Technologies). The amplified cDNA was labelled according to NuGEN's Illumina solution protocol, application note #2. 750 ng of each cDNA was hybridized to Human HT-12 Expression BeadChips, version 3 (Illumina) at 58° C. overnight (18 h) according to Illumina whole-genome gene expression direct hybridization protocol, revision A. Hybridizations were detected with 1 μg ml−1 Cyanine3-streptavidine (General Electric Healthcare Life Sciences). Chips were scanned with Illumina bead array reader, BeadScan software version 3.5. The expression intensities were extracted with GenomeStudio2008 v1 without any normalization or background subtraction.
The gene expression microarray data were preprocessed using the RMA procedure for the Affymetrix data and quantile normalization for the Illumina data using R/Bioconductor. Differentially expressed genes were identified by comparing subjects who had seroconverted to autoantibody positivity or progressed to clinical T1D with their matched healthy controls. Since the follow-up series are not fully synchronized in time between the different individuals, we first applied a similar approach as in Elo, et al 2010 and Huang, et al 2001 that avoids the need of a direct alignment. More specifically, for each individual, the expression intensity value of a particular probe/probeset x at each time point was given a z-score, defined as z=(x−m)s−1, where m is the mean and s is the standard deviation calculated using all the time points in the matched control time series. Such z-scores penalize those probes/probesets that have high variability in the control series. To identify significant up- or down-regulation across the individuals, the rank product algorithm was applied to the case-wise maximum/minimum z-scores. Genes with false discovery rate (FDR) below 0.05, estimated as the percentage of false positive predictions (pfp), were considered as differentially regulated. To focus on T1D- or seroconversion-specific changes, the same procedure was applied to the data after swapping the case and control pairs. Only those probes were retained that did not pass the criterion FDR<0.05 in the swapped analysis. Additionally, we required that the genes were determined as present in more than 50% of all individuals (at least in one sample per individual) using a two-component Gaussian Mixture model for the Affymetrix data and detection P values for the Illumina data, as recommended by Painter, et al 2011. Finally, the findings from Affymetrix and Illumina arrays were compared, and genes showing concordant changes were regarded as final, validated findings (data not shown).
The functional annotation tool DAVID (Database for Annotation, Visualization and Integrated Discovery) was used to identify enriched biological pathways (BBID, BioCarta, KEGG, Panther, Reactome) among the regulated genes. Pathways with FDR below 20% were reported (modified Fisher's exact test).
Differentially Expressed Genes with Functions Related to the Innate Immunity Responses
The human genes with literature-annotated function in the innate immunity were downloaded from www.innatedb.com. The enrichment of these genes among our differentially expressed genes was calculated using Fisher's exact test.
Overrepresented transcription factor binding sites among the regulated genes were identified using the transcription factor target sets in the Molecular Signatures Database (MSigDB). Each target set in the database contains those genes that share a transcription factor binding site defined in the TRANSFAC (version 7.4) database and is annotated by a TRANSFAC record. Binding sites with P value below 0.05 were reported (hypergeometric test).
A module based method modified from Chaussabel, et al 2008 was used to survey coordinately expressed sets of genes (modules) functionally annotated with literature search. The gene symbols belonging to each module were downloaded from www.biir.net/modules, and compared to the differentially expressed genes. The genes not belonging to any of the modules presented in Chaussabel, et al 2008 were excluded from the analysis. The significance of overlap of each module with each list of differentially expressed genes was calculated using Fisher's exact test. Modules up- or down-regulated with the Benjamini-Hochberg corrected P value below 0.05 were visualized in the module maps with red or blue (up- and down-regulation, respectively), the intensity of the color corresponding to the percentage of the regulated genes in the module.
In order to identify transcripts active in different phases of the autoimmune process, the rank product method (R/Bioconductor RankProd package) was applied to find differentially expressed genes between the cases and their controls in five time-windows: before seroconversion, at seroconversion, 6-18 months after seroconversion, 1-2 years before clinical T1D diagnosis, and close to clinical T1D (
In addition to comparing seroconverted children with their controls, we did an additional comparison between those seroconverted children (cases S3, S7 and S10) who had later progressed to T1D, and those who have not been diagnosed with T1D to-date. An unpaired two group rank product analysis was performed to compare the affected and the unaffected cases. The analysis was performed for three time-windows: before seroconversion, at seroconversion, and 6-18 months after seroconversion.
Affymetrix and Illumina data were combined using the gene symbols provided by IPA. To combine the data from the Illumina WG-6 v2 and HT-12 arrays, the probes between the array types were matched based on their sequence similarity. 70% of the probes in Human WG-6 v2 array had remained completely identical to the corresponding probes in the Human HT-12 arrays. In addition, we mapped 6% of the probes using the following criteria: At least 24 consecutive bases (out of 50) were required to be totally identical between the probes. One different base was allowed in the middle if the sequences were otherwise identical at the length of 25 consecutive bases. The probes also had to target the same gene according to annotation. The remaining 24% of the probes were excluded from the analyses, for which the WG-6 v2 and HT-12 data needed to be combined.
Genomic DNA was extracted from whole blood using the salting-out method in the Immunogenetics laboratory, University of Turku (Finland). Thereafter, samples were processed at the Department of Genetics, University Medical Centre Groningen (The Netherlands). The DNA quality and concentration was measured by fluorescence in the Synergy HT Multi-Mode Microplate Reader (Biotek) using the PicoGreen® dsDNA quantitation assay (Invitrogene) according to the manufacturer's instructions. Samples were diluted appropriately to a final concentration of 50 ng/μl and measured with NanoDrop 2000c (Thermo Scientific) prior to hybridization 200 ng of genomic DNA was amplified, enzymatically fragmented and hybridized onto the HumanImmuno BeadChips (Illumina) at 48° C. overnight (20 h) according to the Illumina's Infinium HD Ultra Assay protocol. The staining of the arrays was performed using the Freedom EVO 100 robot (Tecan) and scanned with the iScan Reader (Illumina). Bead intensity data was analysed using the Genotyping Module version 1.9.4 of Genome Studio version 2011.1 (Illumina). NCBI build 36 (hg18) mapping was used (Illumina manifest file Immuno_BeadChip_11419691_B.bpm) for SNP mapping. A cluster set based on 196,524 autosomal and X-chromosome variants was applied to all samples. Quality control of the data and linkage disequilibrium (LD) pruning was performed using PLINK v1.07. First, non-polymorphic markers (N-17216) and markers with duplicated rs identifiers (N-864) were removed. Next, samples with a call rate below 95%, as well as SNPs with a call rate below 98% were excluded. Markers were excluded when they deviated from Hardy-Weinberg equilibrium in controls (P<0.0001). Due to our sample size we decided to focus on common markers only, thus SNPs with minor allele frequency below 10% were removed (N=80,259). Finally we pruned our dataset based on LD between markers if their r2 was greater than 0.8. Our final dataset comprised 30,463 SNPs. The effects of SNPs on gene expression were surveyed within 250 kb to both directions from each differentially expressed gene, using Affymetrix gene coordinates. A linear model was fit for genotype˜gene expression and a p-value was calculated for the null hypothesis that genotype has no effect on gene expression (slope=0). A Benjamini-Hochberg correction was applied for the p-values and effects with FDR<0.05 were considered significant (data not shown). These cis eQTL effects were then validated in a larger study with 5311 human whole-blood samples by Westra et al. (Nature Genetics 2013). The SNPs that had an eQTL effect on our differentially expressed genes were searched for associations with autoimmune diseases in GWA studies, as listed in Ricaño-Ponce and Wijmenga, Annual Review of Genomics and Human Genetics 2013 and/or T1 Dbase (Burren, et al 2011). The proxies (r2>0.8) for these SNPs were found based on HapMap3 (release 2) and 1000 Genomes in the CEU population panel by using SNP annotation and proxy search (http://www.broadinstitute.org/mpg/snap/Idsearch.php).
The gene expression data discussed in this publication are accessible through GEO SuperSeries accession number GSE30211 (http://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE30211).
When the transcriptional profiles of S1-10 (Table 1) were compared to their matched healthy controls, 109 unique genes were differentially expressed (false discovery rate, FDR<0.05; Table S2), with 66% of genes being up-regulated and 34% being down-regulated. Genes residing in the genomic TID susceptibility loci (T1Dbase) included the up-regulated oncostatin M (OSM) and the down-regulated killer cell lectin-like receptor subfamily B member 1 (KLRB1) (
Altogether, 12 unique transcription factor genes were identified as being differentially regulated in the seroconverted cases. Analysis of known gene and protein interactions among these transcription factors and other genes in the dataset using the Ingenuity Pathway Analysis (IPA) network neighborhood tool showed that interconnected interferon regulatory factors 5 and 7 (IRF5 and IRF7) had several co-regulated network partners (
A comparison of the samples obtained from the 18 T1D progressors (D1-18, Table 4) to their matched controls revealed 472 differentially expressed genes (FDR<0.05; data not shown). Notably, gene expression was suppressed, as 73% of the differentially regulated genes in the T1D individuals were down-regulated. The up-regulated genes residing in the genomic TID susceptibility loci (T1 Dbase) included the TRAF-type zinc finger domain containing 1 (TRAFD1) and the signal transducer and activator of transcription 2 (STAT2). In contrast, the T1D-associated grancalcin (GCA) and the von Hippel-Lindau binding protein 1 (VBP1) were down-regulated (
We identified upregulation of transcription factors promyelocytic leukemia (PML) and nuclear receptor coactivator 7 (NCOA7), as well as down-regulation of myoneurin (MYNN) both in the seroconverted individuals and in the T1D progressors. Of these, the best studied factor is the PML, which is the major structural component of nuclear PML bodies involved in various cellular activities including protein degradation, DNA damage and apoptosis. PML expression is induced by type I and II interferons and it is known to inhibit viral replication both in the nucleus and cytoplasm. Subsequently, numerous DNA and RNA viruses use PML nuclear body dissociation as their survival mechanism. In addition, PML acts as a cofactor for the promyelocytic leukemia zinc finger (PLZF, also known as ZBTB16) transcription factor in order to induce a specific set of interferon stimulated genes, such as OAS1, STAT1, and radical S-adenosyl methionine domain containing 2 (RSAD2) upon interferon stimulus. Interestingly, also the binding sites of many interferon regulated transcription factors were enriched in the promoters of the differentially expressed genes (data not shown). For instance, IRF7 and IRF8 binding sites were enriched among the up-regulated genes in both the seroconverted cases (P=0.000309 and 0.00173) and the T1D progressors (P=0.0184 and 0.00106). The up-regulated genes identified from the seroconverted cases were also enriched for IRF1 (P=0.0000329) and IRF2 (P=0.0268) motifs, as well as for the interferon stimulated response element (ISRE, P=0.000274), which is bound and regulated by interferon-stimulated gene factor (ISGF3), a heterotrimeric transcription factor complex consisting of IRF9, STAT1 and STAT2 protein subunits.
To identify transcripts that are active in different phases of the T1D autoimmune process, we divided and analyzed the data separately within five time-windows (
A modular data mining strategy that identifies coordinately expressed transcripts, often functionally related, was recently utilized to identify disease-specific transcriptional fingerprints in whole-blood analysis identifying the over-represented interferon module in systemic lupus erythematosus (SLE) and tuberculosis. By utilizing these predefined and literature-annotated modules, we observed that the changes in transcriptional signatures were dynamic relative to different stages of T1D pathogenesis (
Finally, we performed a separate time-window analysis for the seroconverted cases who later progressed to clinical T1D (cases S3, S6, S7 and S10, Table 4) compared to seroconverted cases who have remained nondiabetic for at least 68 months. For cases S3, S7, and S10, data before, at, and 6-18 months after seroconversion were available.
Before seroconversion, nine genes were identified as differentially regulated (FDR<0.05; data no shown). At seroconversion and 6-18 months after seroconversion the difference had increased to 13 and 54 genes, respectively. One of the genes showing constant and high upregulation in the progressors was Ribonuclease, RNase A family, 2 (RNASE2, also known as eosinophil-derived neurotoxin, EDN,
In order to study the effect of possible single nucleotide polymorphisms (SNP) as the cause for the detected expression differences, we utilized a custom SNP array (Immunochip, Illumina) for genotyping. Using representative (LD pruned) SNP markers residing inside a window of +−250 kb around the gene coordinates, 118 differentially expressed genes had a cis eQTL (cis acting expressed quantitative trait loci) effect with 1-54 SNPs per gene (FDR<0.05, data not shown). Comparison to eQTLs recently identified in whole-blood from 5311 individuals validated 27% of the detected cis effects (Table S7). 14 of the identified eQTL genes (the identified SNPs or their proxies r2>0.8), such as Histone cluster 1 H2bd (HIST1H2BD), have also been associated with autoimmune diseases in GWAS, as listed in Ricaño-Ponce and Wijmenga, Annual Review of Genomics and Human Genetics 2013and/or T1 Dbase (data not shown). Interestingly, eQTL effects were found with SNPs adjacent to IRF5, OAS1, OAS2, DDX58, Transporter 2 ATP-binding cassette sub-family B (MDR/TAP) (TAP2), Indoleamine 2,3-dioxygenase 1 (IDO1) and Leukocyte immunoglobulin-like receptor subfamily A (with TM domain) member 5 (LILRA5), which were identified as the core of the central interferon stimulatory network in the seroconverted individuals (
This study provides a comprehensive analysis of transcriptional changes that occur over time in children at risk for or developing T1D, and provide a unique resource for new hypotheses explaining T1D biology. Our results demonstrate that T1D-specific changes are evident in the whole-blood transcriptome, affecting hundreds of genes. As the gene expression analysis was performed on whole-blood samples, the contribution of each blood cell subtype remains unclear. However, the module-based tool may suggest which cell types are important in different phases of T1D pathogenesis (
Taken together, our results indicate the activation of type 1 interferon mediated innate immune system responses throughout the course of T1D development, and importantly even prior to seroconversion. The induction is temporal, as expected for this systemic response, but some individuals show several signature peaks during the follow-up period (
In addition to viral antigens, bacterial DNA, LPS, and flagellin are efficient triggers of TLR signaling and the interferon response, providing an intriguing link between the gut immune system and autoimmunity. Both preclinical and established T1D patients exhibit increased gut permeability, leading to enhanced immune responses in the intestinal tissues. Also, a preliminary study has shown that gut microbiome diversity is reduced in children progressing towards T1D compared to healthy controls. Moreover, the interferon response can be activated without infection by bacteria or viruses; for example, in SLE, IFNα production is driven by TLR7-mediated signaling and is induced by autoantibody-protein-RNA complexes derived from apoptotic cells. Also, endogenous TLR ligands such as RNASE2 that was identified to be up-regulated in the progressors could play a role in the induction of interferons. It has been shown that spontaneous interferon production drives autoimmune diabetes in NOD mice. Although interferons prepare cells for efficient antiviral defense during infection, these cytokines have also wider effects on immune cells, such as promoting dendritic cell maturation and activation, B cell survival and Ig isotype switching, and suppression of T regulatory cells.
In this study, the matching of the cases and controls was based on the HLA-DQB1 genotype. To take into account further susceptibility alleles and alterations affecting the transcriptional changes, genome-wide SNP detection was performed for combinatorial analysis of the genome and transcriptome. This revealed that approximately 10% of the genes differentially expressed between the cases and controls were affected in cis by the genetic variation. The affected variants and genes have previously been linked to several autoimmune diseases, highlighting the shared genetic basis for these disorders. For example in the case of SLE, where strong type 1 interferon signature is also observed, the associated variations in IRF5 have been shown to control cytokine responses upon TLR ligation. This pathway could be genetically linked also to T1D, since only a small part of the genetic factors conferring susceptibility to T1D have been identified. Alternatively, common gene variants influencing gene expression levels, in combination with additional factors, may contribute to the development of the disease. The majority of the expression changes, however, appeared not to be affected by the genetic variation between the individuals suggesting that the majority of the observed differences in gene expression reflect additional levels of regulation, such as epigenetic modifications. Ancillary measurements, such as proteomics and metabolomics data, could be used in combination with transcriptomics results to identify a combination of candidate molecules that may aid in the prediction of progression toward overt T1D or monitoring the activity of the clinical disease. In addition, the correlation of gene expression profiles to markers of virus infections in susceptible children could make it possible to explore the mechanisms leading to the activation of the interferon system.
Another cohort of seroconverted and T1D DIPP children were analyzed (unpublished). Samples were collected as in Example 1. Of 16 subjects in this study, all were sampled starting before the time of the appearance of autoantibodies (seroconversion). Out of these, three children progressed to T1D. A persistently autoantibody negative control child was matched with each case, based on the date and place of birth, gender and HLA-DQB1 genotype (data not shown). In all, 183 blood samples were processed for genome-wide transcriptional analysis with GeneChip Human Genome U219 array plates as in Example 1. Data was analyzed as in Example 1, focusing on the time-window before seroconversion.
The publicly available dataset of Ferreira et al. was preprocessed and analyzed for differential expression similarly as in Example 1. A total of 19,310 transcripts with a gene symbol in Ingenuity Knowledge base were identified from the dataset after removing multiple transcripts mapping to the same gene by selecting the representative transcript with largest IQR across all samples.
Each case child with samples before seroconversion (n=20) was matched with a healthy control child with the same gender and time of birth. For each gene, the maximum and minimum signal log ratio (SLR) between case samples before seroconversion and controls was calculated using linear inter/extrapolation. Differentially expressed genes were identified using rank product test corrected for multiple testing. Using a false discovery rate (FDR) cutoff <0.05 and SLR of 1 and −1 (at least 2-fold change resulted in 23 up-regulated and 1 down-regulated genes that overlapped with the results of Example 1 preceding seroconversion.
Microarray data preceding seroconversion (Example 1) were clustered hierarchically by Pearson correlation analysis independently for SLR data and expression intensity data. The number of clusters was estimated using elbow method. Each gene cluster was further analyzed using linear discriminant method to find the best scoring clusters that discriminates seroconverted cases from the controls. The gene lists of top scoring clusters were compared with the gene lists of Ferreira et al. 2014. Similarly, gene lists in the time-window before seroconversion in Example 2 were compared with the clusters.
Finally, the up-regulated genes common between all compared gene lists were selected and divided into three functionally different groups of interferon-regulated, non-interferon regulated, and erythrocyte-related gene sets shown in Tables 1, 2 and 3, respectively. Owing to their up-regulation in two or three datasets, the listed genes are particularly suitable for predicting, prior to seroconversion, the development of T1D in an individual.
It will be obvious to a person skilled in the art that, as the technology advances, the inventive concept can be implemented in various ways. The invention and its embodiments are not limited to the examples described above but may vary within the scope of the claims.
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/FI2014/050522 | 6/25/2014 | WO | 00 |
| Number | Date | Country | |
|---|---|---|---|
| 61839123 | Jun 2013 | US |
