The main problems in drawing causal inferences from epidemiological case-control studies are confounding by unmeasured extraneous factors, selection bias and differential misclassification of ...exposure. In genetics the first of these, in the form of population structure, has dominated recent debate. Population structure explained part of the significant +11.2% inflation of test statistics we observed in an analysis of 6,322 nonsynonymous SNPs in 816 cases of type 1 diabetes and 877 population-based controls from Great Britain. The remainder of the inflation resulted from differential bias in genotype scoring between case and control DNA samples, which originated from two laboratories, causing false-positive associations. To avoid excluding SNPs and losing valuable information, we extended the genomic control method by applying a variable downweighting to each SNP.
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As part of an ongoing search for genes associated with type 1 diabetes (T1D), a common autoimmune disease, we tested the biological candidate gene
IL2RA (
CD25), which encodes a subunit (IL-2Rα) of ...the high-affinity interleukin-2 (IL-2) receptor complex. We employed a tag single-nucleotide polymorphism (tag SNP) approach in large T1D sample collections consisting of 7,457 cases and controls and 725 multiplex families. Tag SNPs were analyzed using a multilocus test to provide a regional test for association. We found strong statistical evidence in the case-control collection (
P
=
6.5×
10
−8) for a T1D locus in the
CD25 region of chromosome 10p15 and replicated the association in the family collection (
P
=
7.3×
10
−3; combined
P
=
1.3×
10
−10). These results illustrate the utility of tag SNPs in a chromosome-regional test of disease association and justify future fine mapping of the causal variant in the region.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
Genome-wide linkage disequilibrium (LD) mapping of common disease genes could be more powerful than linkage analysis if the appropriate density of polymorphic markers were known and if the genotyping ...effort and cost of producing such an LD map could be reduced. Although different metrics that measure the extent of LD have been evaluated, even the most recent studies have not placed significant emphasis on the most informative and cost-effective method of LD mapping-that based on haplotypes. We have scanned 135 kb of DNA from nine genes, genotyped 122 single-nucleotide polymorphisms (SNPs; approximately 184,000 genotypes) and determined the common haplotypes in a minimum of 384 European individuals for each gene. Here we show how knowledge of the common haplotypes and the SNPs that tag them can be used to (i) explain the often complex patterns of LD between adjacent markers, (ii) reduce genotyping significantly (in this case from 122 to 34 SNPs), (iii) scan the common variation of a gene sensitively and comprehensively and (iv) provide key fine-mapping data within regions of strong LD. Our results also indicate that, at least for the genes studied here, the current version of dbSNP would have been of limited utility for LD mapping because many common haplotypes could not be defined. A directed re-sequencing effort of the approximately 10% of the genome in or near genes in the major ethnic groups would aid the systematic evaluation of the common variant model of common disease.
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IFIH1 (interferon induced with helicase C domain 1), also known as MDA5 (melanoma differentiation-associated protein 5), is one of a family of intracellular proteins known to recognise viral RNA and ...mediate the innate immune response. IFIH1 is causal in type 1 diabetes based on the protective associations of four rare variants, where the derived alleles are predicted to reduce gene expression or function. Originally, however, T1D protection was mapped to the common IFIH1 nsSNP, rs1990760 or Thr946Ala. This common amino acid substitution does not cause a loss of function and evidence suggests the protective allele, Ala(946), may mark a haplotype with reduced expression of IFIH1 in line with the protection conferred by the four rare loss of function alleles. We have performed allele specific expression analysis that supports this hypothesis: the T1D protective haplotype correlates with reduced IFIH1 transcription in interferon-β stimulated peripheral blood mononuclear cells (overall p = 0.012). In addition, we have used multiflow cytometry analysis and quantitative PCR assays to prove reduced expression of IFIH1 in individuals heterozygous for three of the T1D-associated rare alleles: a premature stop codon, rs35744605 (Glu627X) and predicted splice variants, rs35337543 (IVS8+1) and rs35732034 (IVS14+1). We also show that the nsSNP, Ile923V, does not alter pre-mRNA levels of IFIH1. These results confirm and extend the new autoimmune disease pathway of reduced IFIH1 expression and protein function protecting from T1D.
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Expression of the CTLA-4 gene is absolutely required for immune homeostasis, but aspects of its molecular nature remain undefined. In particular, the characterization of the soluble CTLA-4 (sCTLA-4) ...protein isoform generated by an alternatively spliced mRNA of CTLA4 lacking transmembrane-encoding exon 3 has been hindered by the difficulty in distinguishing it from the transmembrane isoform of CTLA-4, Tm-CTLA-4. In the current study, sCTLA-4 has been analyzed using novel mAbs and polyclonal Abs specific for its unique C-terminal amino acid sequence. We demonstrate that the sCTLA-4 protein is secreted at low levels following the activation of primary human CD4(+) T cells and is increased only rarely in the serum of autoimmune patients. Unexpectedly, during our studies aimed to define the kinetics of sCTLA-4 produced by activated human CD4(+) T cells, we discovered that Tm-CTLA-4 is associated with microvesicles produced by the activated cells. The functional roles of sCTLA-4 and microvesicle-associated Tm-CTLA-4 warrant further investigation, especially as they relate to the multiple mechanisms of action described for the more commonly studied cell-associated Tm-CTLA-4.
A genome-wide map of single nucleotide polymorphisms (SNP) and a pattern of linkage disequilibrium (LD) between their alleles are being established in three main ethnic groups. An important question ...is the applicability of such maps to different populations within a main ethnic group. Therefore, we have developed high-resolution SNP, haplotype and LD maps of vitamin D receptor gene region in large samples from five populations. Comparative analysis reveals that the LD patterns are identical in all four European populations tested with two small regions of 1.3 and 5.7 kb at which LD is disrupted completely resulting in three block-like regions over which there is significant and extensive LD. In an African population the pattern is similar, but two additional LD-breaking spots are also apparent. This LD pattern suggests combined action of recombination hotspots and founder effects, but cannot be explained by random recombination and genetic drift alone. Direct comparison indicates that the tag SNPs selected in one European population effectively predict the non-tag SNPs in the other Europeans, but not in the Gambians, for this region.
Analysis of the Vitamin D Receptor Gene Sequence Variants in Type 1 Diabetes
Sergey Nejentsev ,
Jason D. Cooper 1 ,
Lisa Godfrey 1 ,
Joanna M.M. Howson 1 ,
Helen Rance 1 ,
Sarah Nutland 1 ,
Neil M. ...Walker 1 ,
Cristian Guja 2 ,
Constantin Ionescu-Tirgovişte 2 ,
David A. Savage 3 ,
Dag E. Undlien 4 ,
Kjersti S. Rønningen 5 ,
Eva Tuomilehto-Wolf 6 ,
Jaakko Tuomilehto 6 7 ,
Kathleen M. Gillespie 8 ,
Susan M. Ring 9 ,
David P. Strachan 10 ,
Barry Widmer 11 ,
David Dunger 11 and
John A. Todd 1
1 Juvenile Diabetes Research Foundation/Wellcome Trust Diabetes and Inflammation Laboratory, Cambridge Institute of Medical
Research, University of Cambridge, Cambridge, U.K
2 Clinic of Diabetes, Institute of Diabetes, Nutrition and Metabolic Diseases “N. Paulescu,” Bucharest, Romania
3 Department of Medical Genetics, Queen’s University, Belfast, U.K
4 Institute of Medical Genetics, Ulleval University Hospital, University of Oslo, Oslo, Norway
5 Laboratory of Molecular Epidemiology, Division of Epidemiology, Norwegian Institute of Public Health, Oslo, Norway
6 Diabetes and Genetic Epidemiology Unit, National Public Health Institute, Helsinki, Finland
7 Department of Public Health, University of Helsinki, Helsinki, Finland
8 Department of Clinical Science at North Bristol, Division of Medicine, University of Bristol, Bristol, U.K
9 Avon Longitudinal Study of Pregnancy and Childhood (ALSPAC), University of Bristol, Bristol, U.K
10 Department of Community Health Sciences, St. George’s Hospital Medical School, London, U.K
11 Department of Paediatrics, University of Cambridge, Cambridge, U.K
Address correspondence and reprint requests to Sergey Nejentsev, MD, PhD, JDRF/WT DiabetesInflammation Laboratory, Cambridge
Institute for Medical Research, University of Cambridge, WT/MRC building, Addenbrooke’s Hospital, Cambridge, CB2 2XY, U.K.
E-mail: sergey.nejentsev{at}cimr.cam.ac.uk
Abstract
Vitamin D is known to modulate the immune system, and its administration has been associated with reduced risk of type 1 diabetes.
Vitamin D acts via its receptor (VDR). Four single nucleotide polymorphisms (SNPs) of the VDR gene have been commonly studied,
and evidence of association with type 1 diabetes has been reported previously. We sequenced the VDR gene region and developed
its SNP map. Here we analyzed association of the 98 VDR SNPs in up to 3,763 type 1 diabetic families. First, we genotyped all 98 SNPs in a minimum of 458 U.K. families with two
affected offspring. We further tested eight SNPs, including four SNPs associated with P < 0.05 in the first set and the four commonly studied SNPs, in up to 3,305 additional families from the U.K., Finland, Norway,
Romania, and U.S. We only found weak evidence of association ( P = 0.02–0.05) of the rs4303288, rs12721366, and rs2544043 SNPs. We then tested these three SNPs in an independent set of 1,587
patients and 1,827 control subjects from the U.K. and found no evidence of association. Overall, our results indicate that
common sequence variation in the VDR gene has no major effect in type 1 diabetes in the populations tested.
IL, interleukin
SNP, single nucleotide polymorphism
VDR, vitamin D receptor
Footnotes
Additional information for this article can be found in an online appendix available at
http://diabetes.diabetesjournals.org .
Accepted June 24, 2004.
Received April 2, 2004.
DIABETES
Analysis of Polymorphisms of the Interleukin-18 Gene in Type 1 Diabetes and Hardy-Weinberg Equilibrium Testing
Jeffrey S. Szeszko ,
Joanna M.M. Howson ,
Jason D. Cooper ,
Neil M. Walker ,
Rebecca ...C.J. Twells ,
Helen E. Stevens ,
Sarah L. Nutland and
John A. Todd
From the Juvenile Diabetes Research Foundation/Wellcome Trust Diabetes and Inflammation Laboratory, Cambridge Institute for
Medical Research, University of Cambridge, Cambridge, U.K
Address correspondence and reprint requests to John A. Todd, University of Cambridge, Cambridge Institute for Medical Research,
Wellcome Trust/MRC Building, Addenbrooke’s Hospital, Cambridge CB2 2XY, U.K. E-mail: john.todd{at}cimr.cam.ac.uk
Abstract
Recently, the interleukin-18 cytokine gene ( IL18 ) was reported to be associated with type 1 diabetes. In the present report, we calculated that the reported genotypes of
the two 5′ region/promoter single nucleotide polymorphisms (SNPs), −607 (C→A) (rs1946518) and −137 (G→C) (rs187238), were
not in Hardy-Weinberg equilibrium (HWE). We therefore investigated the association of the −607 and −137 SNPs in a U.K. type
1 diabetic Caucasian case-control collection (1,560 case and 1,715 control subjects tested at −607 and 4,323 case and 4,610
control subjects tested at −137) as well as a type 1 diabetic Caucasian collection comprised of families of European ancestry
(1,347 families tested at −137 and 1,356 families tested at −607). No evidence for association with type 1 diabetes was found,
including for the −607 A/A and C/A genotypes. To evaluate whether common variation elsewhere in the gene was associated with
disease susceptibility, we analyzed eight IL18 tag SNPs in a type 1 diabetic case-control collection (1,561 case and 1,721 control subjects). No evidence for association
was obtained ( P = 0.11). We conclude that common allelic variation in IL18 is unlikely to contribute substantially to type 1 diabetes susceptibility in the populations tested and recommend routine
application of tests for HWE in population-based studies for genetic association.
HWE, Hardy-Weinberg equilibrium
IIPGA, Innate Immunity Programs for Genomic Applications
IL, interleukin
MAF, minor allele frequency
SNP, single nucleotide polymorphism
Footnotes
Additional information for this article can be found in an online appendix at http://diabetes.diabetesjournals.org .
Accepted November 9, 2005.
Received June 29, 2005.
DIABETES
Linkage and congenic strain analyses using the nonobese diabetic (NOD) mouse as a model for human type 1 autoimmune diabetes (T1D) have identified several NOD mouse Idd (insulin dependent diabetes) ...loci, including Slc11a1 (formerly known as Nramp1). Genetic variants in the orthologous region encompassing SLC11A1 in human chromosome 2q35 have been reported to be associated with various immune-related diseases including T1D. Here, we have conducted association analysis of this candidate gene region, and then investigated potential correlations between the most T1D-associated variant and RNA expression of the SLC11A1 gene and its splice isoform.
Nine SNPs (rs2276631, rs2279015, rs1809231, rs1059823, rs17235409 (D543N), rs17235416 (3'UTR), rs3731865 (INT4), rs7573065 (-237 C → T) and rs4674297) were genotyped using TaqMan genotyping assays and the polymorphic promoter microsatellite (GT)n was genotyped using PCR and fragment length analysis. A maximum of 8,863 T1D British cases and 10,841 British controls, all of white European descent, were used to test association using logistic regression. A maximum of 5,696 T1D families were also tested for association using the transmission/disequilibrium test (TDT). We considered P ≤ 0.005 as evidence of association given that we tested nine variants in total. Upon identification of the most T1D-associated variant, we investigated the correlation between its genotype and SLC11A1 expression overall or with splice isoform ratio using 42 PAXgene whole blood samples from healthy donors by quantitative PCR (qPCR).
Using the case-control collection, rs3731865 (INT4) was identified to be the variant most associated with T1D (P = 1.55 × 10-6). There was also some evidence of association at rs4674297 (P = 1.57 × 10-4). No evidence of disease association was obtained at any of the loci using the family collections (PTDT ≥ 0.13). We also did not observe a correlation between rs3731865 genotypes and SLC11A1 expression overall or with splice isoform expression.
We conclude that rs3731685 (INT4) in the SLC11A1 gene may be associated with T1D susceptibility in the European ancestry population studied. We did not observe a difference in SLC11A1 expression at the RNA level based on the genotypes of rs3731865 in whole blood samples. However, a potential correlation cannot be ruled out in purified cell subsets especially monocytes or macrophages.
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