Comprehensive genetic screening reveals wide spectrum of genetic variants in monogenic forms of diabetes among Pakistani population.

BACKGROUND: Monogenic forms of diabetes (MFD) are single gene disorders. Their diagnosis is challenging, and symptoms overlap with type 1 and type 2 diabetes. AIM: To identify the genetic variants responsible for MFD in the Pakistani population and their frequencies. METHODS: A total of 184 patients suspected of having MFD were enrolled. The inclusion criterion was diabetes with onset below 25 years of age. Brief demographic and clinical information were taken from the participants. The maturity-onset diabetes of the young (MODY) probability score was calculated, and glutamate decarboxylase ELISA was performed. Antibody negative patients and features resembling MODY were selected (n = 28) for exome sequencing to identify the pathogenic variants. RESULTS: A total of eight missense novel or very low-frequency variants were identified in 7 patients. Three variants were found in genes for MODY, i.e. HNF1A (c.169C>A, p.Leu57Met), KLF11 (c.401G>C, p.Gly134Ala), and HNF1B (c.1058C>T, p.Ser353Leu). Five variants were found in genes other than the 14 known MODY genes, i.e. RFX6 (c.919G>A, p.Glu307Lys), WFS1 (c.478G>A, p.Glu160Lys) and WFS1 (c.517G>A, p.Glu173Lys), RFX6 (c.1212T>A, p.His404Gln) and ZBTB20 (c.1049G>A, p.Arg350His). CONCLUSION: The study showed wide spectrum of genetic variants potentially causing MFD in the Pakistani population. The MODY genes prevalent in European population (GCK, HNF1A, and HNF4a) were not found to be common in our population. Identification of novel variants will further help to understand the role of different genes causing the pathogenicity in MODY patient and their proper management and diagnosis.

Causal variants in Maturity Onset Diabetes of the Young (MODY) - A systematic review.

BACKGROUND: Maturity Onset Diabetes of the Young (MODY) is an autosomal dominant type of diabetes. Pathogenic variants in fourteen genes are reported as causes of MODY. Its symptoms overlap with type 1 and type 2 diabetes. Reviews for clinical characteristics, diagnosis and treatments are available but a comprehensive list of genetic variants, is lacking. Therefore this study was designed to collect all the causal variants involved in MODY, reported to date. METHODS: We searched PubMed from its date of inception to December 2019. The search terms we used included disease names and name of all the known genes involved. The ClinVar database was also searched for causal variants in the known 14 MODY genes. RESULTS: The record revealed 1647 studies and among them, 326 studies were accessed for full-text. Finally, 239 studies were included, as per our inclusion criteria. A total of 1017 variants were identified through literature review and 74 unpublished variants from Clinvar database. The gene most commonly affected was GCK, followed by HNF1a. The traditional Sanger sequencing was used in 76 % of the cases and 65 % of the studies were conducted in last 10 years. Variants from countries like Jordan, Oman and Tunisia reported that the MODY types prevalent worldwide were not common in their countries. CONCLUSIONS: We expect that this paper will help clinicians interpret MODY genetics results with greater confidence. Discrepancies in certain middle-eastern countries need to be investigated as other genes or factors, like consanguinity may be involved in developing diabetes.

Genetic architecture of type 1 diabetes with low genetic risk score informed by 41 unreported loci.

Type 1 diabetes (T1D) patients with low genetic risk scores (GRS) may be non-autoimmune or autoimmune mediated by other genetic loci. The T1D-GRS2 provides us an opportunity to look into the genetic architecture of these patients. A total of 18,949 European individuals were included in this study, including 6599 T1D cases and 12,323 controls. 957 (14.5%) T1D patients were identified with low GRS (GRS < 8.43). The genome-wide association study on these patients identified 41 unreported loci. Two loci with common variants and 39 loci with rare variants were identified in this study. This study identified common SNPs associated with both low GRS T1D and expression levels of the interferon-α-induced MNDA gene, indicating the role of viral infection in T1D. Interestingly, 16 of the 41 unreported loci have been linked to autism spectrum disorder (ASD) by previous studies, suggesting that genes residing at these loci may underlie both T1D and autism.

Monogenic Causes in the Type 1 Diabetes Genetics Consortium Cohort: Low Genetic Risk for Autoimmunity in Case Selection.

HYPOTHESIS: About 1% of patients clinically diagnosed as type 1 diabetes have non-autoimmune monogenic diabetes. The distinction has important therapeutic implications but, given the low prevalence and high cost of testing, selecting patients to test is important. We tested the hypothesis that low genetic risk for type 1 diabetes can substantially contribute to this selection. METHODS: As proof of principle, we examined by exome sequencing families with 2 or more children, recruited by the Type 1 Diabetes Genetics Consortium (T1DGC) and selected for negativity for 2 autoantibodies and absence of risk human leukocyte antigen haplotypes. RESULTS: We examined 46 families that met the criteria. Of the 17 with an affected parent, 7 (41.2%) had actionable monogenic variants. Of 29 families with no affected parent, 14 (48.3%) had such variants, including 5 with recessive pathogenic variants of WFS1 but no report of other features of Wolfram syndrome. Our approach diagnosed 55.8% of the estimated number of monogenic families in the entire T1DGC cohort, by sequencing only 11.1% of the autoantibody-negative ones. CONCLUSIONS: Our findings justify proceeding to large-scale prospective screening studies using markers of autoimmunity, even in the absence of an affected parent. We also confirm that nonsyndromic WFS1 variants are common among cases of monogenic diabetes misdiagnosed as type 1 diabetes.

Rare Genetic Variants of Large Effect Influence Risk of Type 1 Diabetes.

Most replicated genetic determinants for type 1 diabetes are common (minor allele frequency [MAF] >5%). We aimed to identify novel rare or low-frequency (MAF <5%) single nucleotide polymorphisms with large effects on risk of type 1 diabetes. We undertook deep imputation of genotyped data followed by genome-wide association testing and meta-analysis of 9,358 type 1 diabetes case and 15,705 control subjects from 12 European cohorts. Candidate variants were replicated in a separate cohort of 4,329 case and 9,543 control subjects. Our meta-analysis identified 27 independent variants outside the MHC, among which 3 were novel and had MAF <5%. Three of these variants replicated with P replication < 0.05 and P combined < P discovery In silico analysis prioritized a rare variant at 2q24.3 (rs60587303 [C], MAF 0.5%) within the first intron of STK39, with an effect size comparable with those of common variants in the INS and PTPN22 loci (combined [from the discovery and replication cohorts] estimate of odds ratio [ORcombined] 1.97, 95% CI 1.58-2.47, P combined = 2.9 × 10-9). Pharmacological inhibition of Stk39 activity in primary murine T cells augmented effector responses through enhancement of interleukin 2 signaling. These findings provide insight into the genetic architecture of type 1 diabetes and have identified rare variants having a large effect on disease risk.

Clonal copy-number mosaicism in autoreactive T lymphocytes in diabetic NOD mice.

Concordance for type 1 diabetes (T1D) is far from 100% in monozygotic twins and in inbred nonobese diabetic (NOD) mice, despite genetic identity and shared environment during incidence peak years. This points to stochastic determinants, such as postzygotic mutations (PZMs) in the expanding antigen-specific autoreactive T cell lineages, by analogy to their role in the expanding tumor lineage in cancer. Using comparative genomic hybridization of DNA from pancreatic lymph-node memory CD4+ T cells of 25 diabetic NOD mice, we found lymphocyte-exclusive mosaic somatic copy-number aberrations (CNAs) with highly nonrandom independent involvement of the same gene(s) across different mice, some with an autoimmunity association (e.g., Ilf3 and Dgka). We confirmed genes of interest using the gold standard approach for CNA quantification, multiplex ligation-dependent probe amplification (MLPA), as an independent method. As controls, we examined lymphocytes expanded during normal host defense (17 NOD and BALB/c mice infected with Leishmania major parasite). Here, CNAs found were fewer and significantly smaller compared to those in autoreactive cells (P = 0.0019). We determined a low T cell clonality for our samples suggesting a prethymic formation of these CNAs. In this study, we describe a novel, unexplored phenomenon of a potential causal contribution of PZMs in autoreactive T cells in T1D pathogenesis. We expect that exploration of point mutations and studies in human T cells will enable the further delineation of driver genes to target for functional studies. Our findings challenge the classical notions of autoimmunity and open conceptual avenues toward individualized prevention and therapeutics.

A Nursing Intervention Increases Quality of Life and Self-Efficacy in Migraine: A 1-Year Prospective Controlled Trial.

OBJECTIVES: To compare the impact of a combined nursing and medical approach to a medical follow-up only on headache outcomes, quality of life, and self-efficacy in a cohort of migraineurs. BACKGROUND: Interdisciplinary approaches have been proposed for migraine management. A nursing intervention could improve patient outcomes. METHODS: We prospectively studied new patients referred to our tertiary headache center for migraine. The control group was followed by a physician; the active group was also followed by a nurse with a personalized intervention including adaptation of the lifestyle. RESULTS: Two hundred patients (176 women and 24 men, mean age 40 years old) were included and classified according to headache frequency. Each group was followed for 12 months with daily headache diaries. One hundred and sixty-two completed the study. There were no significant differences between groups for the decrease in headache days, the percent of chronic patients reverting to episodic status or the cessation of medication overuse. Patients in the control group were more likely to find a successful prophylaxis (55.6 vs 27.7%, P = .002). Despite this, the mean decrease in HIT-6 scores at month 8 was 5.23 ± 9.18 for the active group compared with a decrease of 2.10 ± 9.27 for the control group (P = .030, clinically significant difference of 3.13). Headache Management Self-Efficacy Scale (HMSE) scores, representing the feeling of self-efficacy, increased by 14.35 ± 18.41 for the active group vs 4.69 ± 21.22 in the control group (P = .002). CONCLUSION: A nursing intervention can lower the impact of migraines on the patient's life. The improvement in the HIT-6 score in this study was correlated with improvements in self-efficacy.

Neurostimulation for Refractory Cervicogenic Headache: A Three-Year Retrospective Study.

BACKGROUND: Occipital nerve stimulation (ONS) has been used for the treatment of neuropathic pain conditions and could be a therapeutic approach for refractory cervicogenic headache (CeH). AIM: The aim of this study is to assess the efficacy and safety of unilateral ONS in patients suffering from refractory CeH. METHODS: We conducted a retrospective chart review on patients implanted from 2011 to 2013 at CHUM. The primary outcome was a 50% reduction in headache days per month. Secondary outcomes included change in EuroQol Group Visual Analog Scale rating of health-related quality of life (EQ VAS), six item headache impact test (HIT-6) score, hospital anxiety and depression scale (HADS) score, work status, and medication overuse. RESULTS: Sixteen patients fulfilled the inclusion criteria; they had suffered from daily moderate to severe CeH for a median of 15 years. At one year follow-up, 11 patients were responders (69%). There was a statistically significant improvement in the EQ VAS score (median change: 40 point increase, p = 0.0013) and HIT-6 score (median change: 17.5 point decrease, p = 0.0005). Clinically significant anxiety and depression scores both resolved amongst 60% of patients. At three years, six patients were responders (37.5%). Out of the 11 responders at one-year post implantation, five had remained headache responders (R-R) and one additional patient became a responder (NR-R). There was a statistically significant improvement in the EQ VAS score (median change: 15 point increase, p = 0.019) and HIT-6 score (median change: 7.5 point decrease, p = 0.0017) compared with baseline. Clinically significant anxiety and depression scores both, respectively, resolved among 22.5% and 33.9% of patients. Five out of seven disabled patients were back to work. CONCLUSION: ONS may be a safe and effective treatment modality for patients suffering from a refractory CeH. Further study may be warranted.

Pregabalin for the management of cervicogenic headache: a double blind study.

OBJECTIVES: The primary objective was to evaluate the effects of pregabalin relative to placebo in patients with chronic unilateral cervicogenic headache. Primary and secondary end points: To assess the change from baseline in the frequency of cervicogenic headache days per 28-day period between placebo and treatment group. To assess the change from baseline in the intensity of headache, and health outcome measures. STUDY DESIGN: This was a double-blind, randomized, placebo-controlled, parallel-group study, evaluating the efficacy and safety of pregabalin in patients with cervicogenic headache. PROCEDURES: The study consisted of two phases. A baseline of -28 days and a double-blind placebo-controlled phase: with an escalation and maintenance phase, during which patients remained at their highest dose until the end of the study, at Day 86. RESULTS: Forty one patients were randomized, predominantly females, with a mean age of 52 years old. At screening, both groups had, on average, 26 headache-days per month. By the final phase of the study, the number of headache days dropped to 16 per month for the pregabalin group while remaining stable for the placebo group (p=0.037). No serious adverse events were reported during the study. CONCLUSION: In this study, primary objectives were achieved with a statistically significant change of ten days in frequency of headache days; with minor side effects that were well tolerated.

Functional characterization of the Thr946Ala SNP at the type 1 diabetes IFIH1 locus.

The Thr allele at the Thr946Ala non-synonymous single-nucleotide polymorphism (nsSNP) in the IFIH1 gene confers risk for type 1 diabetes (T1D). IFIH1 binds viral double-stranded RNA (dsRNA), inducing a type I interferon (IFN) response. Reports of this nsSNP's role in IFIH1 expression regulation have produced conflicting results and a study evaluating transfected Thr946Ala protein alleles in an artificial system overexpressing IFIH1 shows that the SNP does not affect IFH1 function. In this study, we examine the effects of the Thr946Ala polymorphism on IFN-α response in a cell line that endogenously expresses physiological levels of IFIH1. Eleven lymphoblastoid cell lines (LCLs) homozygous for the major predisposing allele (Thr/Thr) and 6 LCLs homozygous for the minor protective allele (Ala/Ala) were electroporated with the viral dsRNA mimic, poly I:C, in three independent experiments. Media were collected 24 hours later and measured for IFN-α production by ELISA. Basal IFN response is minimal in mock-transfected cells from both genotypes and increases by about 8-fold in cells treated with poly I:C. LCLs with the Ala/Ala genotype have slightly higher IFN-α levels than their Thr/Thr counterparts but this did not reach statistical significance because of the large variability of the IFN response, due mostly to two high outliers (biological, not technical). A larger sample size would be needed to determine whether the Thr946Ala SNP affects the poly I:C-driven IFN-α response. Additionally, the possibility that this nsSNP recognizes viral dsRNA specificities cannot be ruled out. Thus, the mechanism of the observed association of this SNP with T1D remains to be determined.

Genome-wide search for exonic variants affecting translational efficiency.

The search for expression quantitative trait loci has traditionally centred entirely on the process of transcription, whereas variants with effects on messenger RNA translation have not been systematically studied. Here we present a high-throughput approach for measuring translational cis-regulation in the human genome. Using ribosomal association as proxy for translational efficiency of polymorphic messenger RNAs, we test the ratio of polysomal/non-polysomal messenger RNA level as a quantitative trait for association with single nucleotide polymorphisms on the same messenger RNA transcript. We identify one important ribosomal distribution effect, from rs1131017 in the 5'-untranslated region of RPS26, that is in high linkage disequilibrium with the 12q13 locus for susceptibility to type 1 diabetes. The effect on translation is confirmed at the protein level by quantitative western blots, both ex vivo and after in vitro translation. Our results are a proof-of-principle that allelic effects on translation can be detected at a transcriptome-wide scale.

Differential expression pattern of ZAC in developing mouse and human pancreas.

ZAC is a transcription factor and cofactor, a strong candidate for transient neonatal diabetes mellitus (TNDM). TNDM involves impaired beta-cell development and is probably due to a double dose of ZAC, which is normally expressed only from the paternal copy. ZAC and Zac1 (its mouse orthologue) are strongly expressed in the proliferating progenitor/stem cells in many systems and also in some differentiated sites in human and mouse, suggesting a dual role in cell proliferation and differentiation control. Little is known about its expression in developing pancreas, the organ affected in TNDM. In this study, we examined ZAC/Zac1 expression in developing mouse and human pancreas by real-time PCR and dual in situ hybridization and immunofluorescence. Overall pancreatic expression drastically declined during gestation and early post-natal life in the mouse, and between the second trimester and adult in the human. Zac1 was predominantly expressed in mesenchyme in the mouse embryo, while ZAC was specifically expressed in islets of the human fetus. Thus, ZAC/Zac1 may play different roles in mouse and human pancreas development. The specific expression of ZAC in the human fetal beta-cells supports it as the gene involved in TNDM and the different expression pattern of Zac1 in mice from human may explain the much milder phenotype in the mouse model of ZAC double dose.

Study of transcriptional effects in Cis at the IFIH1 locus.

BACKGROUND: The Thr allele at the non-synonymous single-nucleotide polymorphism (nsSNP) Thr946Ala in the IFIH1 gene confers risk for Type 1 diabetes (T1D). The SNP is embedded in a 236 kb linkage disequilibrium (LD) block that includes four genes: IFIH1, GCA, FAP and KCNH7. The absence of common nsSNPs in the other genes makes the IFIH1 SNP the strongest functional candidate, but it could be merely a marker of association, due to LD with a variant regulating expression levels of IFIH1 or neighboring genes. METHODOLOGY/PRINCIPAL FINDINGS: We investigated the effect of the T1D-associated variation on mRNA transcript expression of these genes. Heterozygous mRNA from lymphoblastoid cell lines (LCLs), pancreas and thymus was examined by allelic expression imbalance, to detect effects in cis on mRNA expression. Using single-nucleotide primer extension, we found no difference between mRNA transcripts in 9 LCLs, 6 pancreas and 13 thymus samples, suggesting that GCA and FAP are not involved. On the other hand, KCNH7 was not expressed at a detectable level in all tissues examined. Moreover, the association of the Thr946Ala SNP with T1D is not due to modulation of IFIH1 expression in organs involved in the disease, pointing to the IFIH1 nsSNP as the causal variant. CONCLUSIONS/SIGNIFICANCE: The mechanism of the association of the nsSNP with T1D remains to be determined, but does not involve mRNA modulation. It becomes necessary to study differential function of the IFIH1 protein alleles at Thr946Ala to confirm that it is responsible for the disease association.

Follow-up analysis of genome-wide association data identifies novel loci for type 1 diabetes.

OBJECTIVE: Two recent genome-wide association (GWA) studies have revealed novel loci for type 1 diabetes, a common multifactorial disease with a strong genetic component. To fully utilize the GWA data that we had obtained by genotyping 563 type 1 diabetes probands and 1,146 control subjects, as well as 483 case subject-parent trios, using the Illumina HumanHap550 BeadChip, we designed a full stage 2 study to capture other possible association signals. RESEARCH DESIGN AND METHODS: From our existing datasets, we selected 982 markers with P < 0.05 in both GWA cohorts. Genotyping these in an independent set of 636 nuclear families with 974 affected offspring revealed 75 markers that also had P < 0.05 in this third cohort. Among these, six single nucleotide polymorphisms in five novel loci also had P < 0.05 in the Wellcome Trust Case-Control Consortium dataset and were further tested in 1,303 type 1 diabetes probands from the Diabetes Control and Complications Trial/Epidemiology of Diabetes Interventions and Complications (DCCT/EDIC) plus 1,673 control subjects. RESULTS: Two markers (rs9976767 and rs3757247) remained significant after adjusting for the number of tests in this last cohort; they reside in UBASH3A (OR 1.16; combined P = 2.33 x 10(-8)) and BACH2 (1.13; combined P = 1.25 x 10(-6)). CONCLUSIONS: Evaluation of a large number of statistical GWA candidates in several independent cohorts has revealed additional loci that are associated with type 1 diabetes. The two genes at these respective loci, UBASH3A and BACH2, are both biologically relevant to autoimmunity.

A novel susceptibility locus for type 1 diabetes on Chr12q13 identified by a genome-wide association study.

OBJECTIVE: In stage 1 of our genome-wide association (GWA) study for type 1 diabetes, one locus at 16p13 was detected (P = 1.03 x 10(-10)) and confirmed in two additional cohorts. Here we describe the results of testing, in these additional cohorts, 23 loci that were next in rank of statistical significance. RESEARCH DESIGN AND METHODS: Two independent cohorts were studied. The Type 1 Diabetes Genetics Consortium replication cohort consisted of 549 families with at least one child diagnosed with diabetes (946 total affected) and DNA from both parents. The Canadian replication cohort consisted of 364 nuclear family trios with one type 1 diabetes-affected offspring and two parents (1,092 individuals). RESULTS: One locus at 12q13, with the highest statistical significance among the 23, was confirmed. It involves type 1 diabetes association with the minor allele of rs1701704 (P = 9.13 x 10(-10), OR 1.25 [95% CI 1.12-1.40]). CONCLUSIONS: We have discovered a type 1 diabetes locus at 12q13 that is replicated in an independent cohort of type 1 diabetic patients and confers a type 1 diabetes risk comparable with that of the 16p13 locus we recently reported. These two loci are identical to two loci identified by the whole-genome association study of the Wellcome Trust Case-Control Consortium, a parallel independent discovery that adds further support to the validity of the GWA approach.

A genome-wide association study identifies KIAA0350 as a type 1 diabetes gene.

Type 1 diabetes (T1D) in children results from autoimmune destruction of pancreatic beta cells, leading to insufficient production of insulin. A number of genetic determinants of T1D have already been established through candidate gene studies, primarily within the major histocompatibility complex but also within other loci. To identify new genetic factors that increase the risk of T1D, we performed a genome-wide association study in a large paediatric cohort of European descent. In addition to confirming previously identified loci, we found that T1D was significantly associated with variation within a 233-kb linkage disequilibrium block on chromosome 16p13. This region contains KIAA0350, the gene product of which is predicted to be a sugar-binding, C-type lectin. Three common non-coding variants of the gene (rs2903692, rs725613 and rs17673553) in strong linkage disequilibrium reached genome-wide significance for association with T1D. A subsequent transmission disequilibrium test replication study in an independent cohort confirmed the association. These results indicate that KIAA0350 might be involved in the pathogenesis of T1D and demonstrate the utility of the genome-wide association approach in the identification of previously unsuspected genetic determinants of complex traits.

The IRF5 polymorphism in type 1 diabetes.

The interferon regulatory factor 5 gene (IRF5) has been shown to play a crucial role in harmful immune responses by induction of proinflammatory cytokines. Functional genetic variants associated with increased IRF5 expression of specific isoforms are associated with systemic lupus erythematosus (SLE) and it is possible that they may also predispose to other autoimmune disorders. We tested the association of two IRF5 SNPs, correlated with IRF5 expression and SLE risk, in 947 nuclear family trios type 1 diabetes (T1D) using the transmission disequilibrium test. Our results suggest that the functional IRF5 variations do not confer an obvious risk for T1D.

Evaluation of polymorphic splicing in the mechanism of the association of the insulin gene with diabetes.

The association of type 1 diabetes with the insulin gene (IDDM2 locus) has been mapped to a short haplotype encompassing two single nucleotide polymorphisms (SNPs) in perfect linkage disequilibrium (r(2) = 1) with each other and with the two allele classes at the variable number of tandem repeats (VNTR) polymorphism upstream of the transcription site. Although it is believed that the genetic effect is mediated through transcriptional effects of the VNTR, an alternative mechanism has been recently proposed: In transfected cells, the common A allele at one of the SNPs (-23A-->T, in relation to the translation-initiation codon) weakens the splicing of intron 1, resulting in a minor ( approximately 15% of total RNA) transcript with a longer 5' untranslated region and sixfold enhanced translational efficiency. The purpose of our study was to confirm these findings in RNA from normal human pancreas and thymus. We report that pancreas does contain the alternative transcript in an allele-dependent manner but at a very low proportion (<5% of total INS mRNA). We believe that this level would have a minor, if any, biological effect involved in the mechanism of the IDDM2 locus.

No association of type 1 diabetes with a functional polymorphism of the LRAP gene.

AIMS/HYPOTHESIS: In a recent linkage analysis of genome-wide gene-expression patterns in lymphoblastoid lines, the gene encoding the leukocyte-derived arginine aminopeptidase (LRAP) was identified as having its expression levels modulated by one of the most pronounced effects in cis, mapping to a single-nucleotide polymorphism (rs2762). As this enzyme has an important role in processing antigenic peptides for the HLA I molecules, a variant that drastically modulates its expression levels might affect risk of autoimmunity. This study was designed to confirm that LRAP expression in B-cell derived lines is controlled by a haplotype marked by rs2762 and to see whether this would be the basis of an association with type 1 diabetes (T1D). METHODS: Single-nucleotide primer extension (SNuPE) was adapted to determine the haplotype-specific expression. Genetic association was tested in 892 nuclear families with one T1D-affected offspring and two parents (2676 individuals). RESULTS: All nine heterozygous RNA samples showed an eight-fold higher level of one haplotype over the other (7.97+/-0.99, p=1.33x10(-9)). However, no association of rs2762 with T1D was found by the transmission disequilibrium test (transmission ratio A/G=377/388, p=0.69). CONCLUSIONS/INTERPRETATION: The genetically determined LRAP expression does not play significant roles in T1D. However, this dramatic genetic effect on LRAP expression justifies further investigation of association with other phenotypes, especially autoimmune and related to host defense to specific pathogens.

Genetic control of alternative splicing in the TAP2 gene: possible implication in the genetics of type 1 diabetes.

The transporter 2, ATP-binding cassette, subfamily B (TAP2) is involved in the transport of antigenic peptides to HLA molecules. Coding TAP2 polymorphisms shows a strong association with type 1 diabetes, but it is not clear whether this association may be entirely due to linkage disequilibrium with HLA DR and DQ. Functionally, rat Tap2 nonsynonymous single-nucleotide polymorphisms (nsSNPs) confer differential selectivity for antigenic peptides, but this was not shown to be the case for human TAP2 nsSNPs. In the human, differential peptide selectivity is rather conferred by two splicing isoforms with alternative carboxy terminals. Here, we tested the hypothesis that alleles at the coding SNPs favor different splicing isoforms, thus determining peptide selectivity indirectly. This may be the basis for independent contribution to the type 1 diabetes association. In RNA from heterozygous lymphoblastoid lines, we measured the relative abundance of each SNP haplotype in each isoform. In isoform NM_000544, the G (Ala) allele at 665 Thr>Ala (rs241447) is more than twice as abundant as A (Thr) (GA = 2.2 +/- 0.4, P = 1.5 x 10(-4)), while isoform NM_018833 is derived almost exclusively from chromosomes carrying A (AG = 18.1 +/- 5.6, P = 2.04 x 10(-7)). In 889 Canadian children with type 1 diabetes, differential transmission of parental TAP2 alleles persisted (P = 0.011) when analysis was confined to chromosomes carrying only DQ*02 alleles, which mark a conserved DR-DQ haplotype, thus eliminating most of the variation at DR-DQ. Thus, we present evidence of TAP2 association with type 1 diabetes that is independent of HLA DR-DQ and describe a plausible functional mechanism based on allele dependence of splicing into isoforms known to have differential peptide selectivities.