Genetic variation in the IGSF6 gene and lack of association with inflammatory bowel disease.

The immunoglobulin superfamily 6 gene (IGSF6) on chromosome 16p11-p12 has been investigated as a positional and functional candidate for inflammatory bowel disease (IBD) susceptibility. Screening of the six exons of IGSF6 for single nucleotide polymorphisms (SNPs) detected four novel SNPs, and validated three of six SNPs listed in the international SNP database (dbSNP). The seven SNPs in IGSF6 formed five distinct linkage disequilibrium groups. There was no evidence for association of the common SNPs with disease in a large cohort of patients with IBD. The novel SNPs and the linkage disequilibrium map will be a useful resource for the analysis of IGSF6 in other immune disorders.     

Gene-centric association mapping of chromosome 3p implicates MST1 in IBD pathogenesis

Association mapping and candidate gene studies within inflammatory bowel diseases (IBD) linkage regions, as well as genome-wide association studies in Crohn's disease (CD) have led to the discovery of multiple risk genes, but these explain only a fraction of the genetic susceptibility observed in IBD. We have thus been pursuing a region on chromosome 3p21–22 showing linkage to CD and ulcerative colitis (UC) using a gene-centric association mapping approach. We identified 12 functional candidate genes by searching for literature cocitations with relevant keywords and for gene expression patterns consistent with immune/intestinal function. We then performed an association study composed of a screening phase, where tagging single nucleotide polymorphisms (SNPs) were evaluated in 1,020 IBD patients, and an independent replication phase in 745 IBD patients. These analyses identified and replicated significant association with IBD for four SNPs within a 1.2 Mb linkage disequilibrium region. We then identified a non-synonymous coding variant (rs3197999, R689C) in the macrophage-stimulating 1 (MST1) gene (P-value 3.62 × 10^–6) that accounts for the association signal, and shows association with both CD and UC. MST1 encodes macrophage-stimulating protein (MSP), a protein regulating the innate immune responses to bacterial ligands. R689C is predicted to interfere with MSP binding to its receptor, suggesting a role for this gene in the pathogenesis of IBD.     

Genetic Variation at the Chromosome 16 Chemokine Gene Cluster: Development of a Strategy for Association Studies in Complex Disease

The chemokine gene cluster [CCL22, CX3CL1, CCL17] (previously known as [SCYA22, SCYD1, SCYA17]) is a candidate locus for one of the susceptibility genes for inflammatory bowel disease that are located in the peri‐centromeric region of chromosome 16. Screening for sequence variation at this locus led to the detection of 14 single nucleotide polymorphisms (SNPs). An efficient experimental and computational approach was developed to estimate allele frequencies and pairwise linkage disequilibrium relationships between SNPs at this locus, and to test them for association with inflammatory bowel disease. The 12 common SNPs were assigned to 5 distinct linkage disequilibrium groups. Genotyping of one SNP from each linkage disequilibrium group in a large cohort of families with inflammatory bowel disease did not provide convincing evidence of association with either Crohn's disease or ulcerative colitis. We describe an efficient experimental design from SNP screening to association testing. This strategy can be used to test candidate genes for involvement in susceptibility to complex disease.     

Association analysis of the SHC1 gene locus with longevity in the Japanese population

The SHC1 gene encodes a signaling and transforming protein that has been implicated in the aging process in worms and mammals. In this study we examined 230 Japanese centenarians and 180 healthy younger controls and looked at the SHC1 locus as a candidate region that may be associated with longevity. We identified 12 single nucleotide polymorphisms (SNPs) within a 10-kb region encompassing the entire SHC1 gene from the DNA of 30 centenarians and 24 healthy younger controls. Five SNPs, including three nonsynonymous sites, lay within coding elements, six were located within introns, and one was in the 3 untranslated region. All of these SNPs were relatively rare, with a minor allele frequency of less than 5% in our subjects. A pairwise linkage disequilibrium analysis using the r ² statistic showed that two of the SNP pairs are in tight linkage disequilibrium at this locus. We investigated the possible association of SHC1 with longevity using association analyses with allelotypes and haplotypes but found that the SNPs identified in SHC1 had no impact on longevity for Japanese centenarians.Abbreviations LD Linkage disequilibrium - SNP Single nucleotide polymorphism     

Genetic Polymorphisms and Haplotypes of the Human Cardiac Sodium Channel α Subunit Gene (SCN5A) in Japanese and their Association with Arrhythmia

Genetic variations in cardiac ion channels have been implicated not only as the causes of inherited arrhythmic syndromes, but also as genetic risk factors for some acquired arrhythmias. To elucidate the potential roles of genetic polymorphisms of the α subunit of the voltage‐gated sodium channel type V (SCN5A) in cardiac rhythm disturbance, the entire SCN5A coding exons and their flanking introns were sequenced in 166 Japanese arrhythmic patients and 232 healthy controls. We detected 69 genetic variations, including 54 novel ones. Out of the 12 novel nonsynonymous single nucleotide polymorphisms (SNPs), p.Leu1988Arg was found at a frequency of 0.015. The other 11 SNPs were rare (0.001), with 6 found in arrhythmic patients and 5 in healthy controls. The frequency of a novel intronic SNP, c.703+130G>A, was significantly higher in the patients than in the controls, suggesting this SNP is associated with an unknown risk factor for arrhythmia. Following linkage disequilibrium analysis, the haplotype structure of SCN5A was inferred using high‐frequency SNPs. The frequency of the haplotype harbouring both p.Leu1988Arg and the common SNP p.His558Arg (haplotype GG) was significantly lower in the patients than in the controls. This finding suggests that this haplotype (GG) might have been positively selected in the controls because of its protective effect against arrhythmias. This study provides fundamental information necessary to elucidate the effect of genetic variations in SCN5A on channel function and cardiac rhythm in Japanese, and probably in the Asian population.     

Association of caspase-9 and RUNX3 with inflammatory bowel disease

Previous linkage studies have identified a region at 1p36 as the susceptibility locus (IBD7) of inflammatory bowel disease (IBD). The objective of this study was to investigate whether polymorphisms of caspase-9 (CASP9) gene and RUNX3 are associated with IBD susceptibility and clinical phenotypes. We studied 555 Crohn's disease (CD) and 651 ulcerative colitis (UC) patients recruited from a single UK center. A total of 964 healthy Caucasian subjects were recruited as controls from general practitioner well person clinics in Oxfordshire. Fourteen single nucleotide polymorphisms (SNPs) of CASP9 and 11 SNPs of RUNX3 were genotyped using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) (homogenous MassEXTEND, hME, Sequenom?, Sequenom Inc., San Diego, CA). Linkage disequilibrium (LD) and haplotype association analysis were performed using 2ld and phase v2.0 software. No association of individual SNPs of CASP9 or RUNX3 with UC or CD was identified. The rs1052571 of CASP9 was associated with severe UC [P = 0.0034, odds ratio (OR) = 1.957, 95% confidence interval (CI) = 1.240-3.088]. Significant haplotype associations between CASP9 and IBD were identified, while no association of RUNX3 haplotypes with either UC or CD was found. Our findings suggested that CASP9 gene might be another IBD susceptibility gene.     

Associations Between G/A1229, A/G3944, T/C30875, C/T48200 and C/T65013 Genotypes and Haplotypes in the Vitamin D Receptor Gene, Ultraviolet Radiation and Susceptibility to Prostate Cancer

Ultraviolet radiation (UVR) may protect against prostate cancer via a mechanism involving vitamin D. Thus, the vitamin D receptor (VDR) gene is a susceptibility candidate, though published data are discrepant. We studied the association of prostate cancer risk with five VDR single nucleotide polymorphisms (SNPs): G/A¹²²⁹ (SNP 1), A/G³⁹⁴⁴ (SNP 2), T/C³⁰⁸⁷⁵ (SNP 3), C/T⁴⁸²⁰⁰ (SNP 4) and C/T⁶⁵⁰¹³ (SNP 5), in 430 cancer and 310 benign prostatic hypertrophy (BPH) patients. The SNP 2 GG genotype frequency was lower in cancer than BPH patients (odds ratio = 0.63, 95% CI = 0.41–0.98, p = 0.039). SNPs 1 and 2, and SNPs 4 and 5, were in linkage disequilibrium. Two copies of haplotypes comprising SNPs 1‐2, G‐G (odds ratio = 0.63, p = 0.039), SNPs 2‐3 G‐C (odds ratio = 0.45, p = 0.008) and SNPs 1‐2‐3 G‐G‐C (odds ratio = 0.44, p = 0.006), but not SNPs 1‐3, G‐C (odds ratio = 0.81, p = 0.34), were associated with reduced risk (reference, no copies of the haplotypes) . These associations were observed after stratification of subjects by extent of UVR exposure. These data show that SNP 2 GG genotype mediates prostate cancer risk, complementing studies reporting this allele is protective in malignant melanoma pathogenesis. They further suggest that published associations of risk with SNP 1 may result from linkage disequilibrium with SNP 2.     

Amino-acid substitutions in the IKAP gene product significantly increase risk for bronchial asthma in children

The complex etiology of bronchial asthma (BA), one of the most common inflammatory diseases throughout the world, involves a combination of various genetic and environmental factors. A number of investigators have undertaken linkage and association studies to shed light on the genetic background of BA, but the genetic aspects of this disease are still poorly understood. In the course of a project to screen the entire human genome for single nucleotide polymorphisms (SNPs) that might represent useful markers for large-scale association analyses of common diseases and pharmacogenetic traits, we identified six SNPs within the gene encoding I-κB-associated protein (IKAP), a regulator of the NF-κB signal pathway. Most of these SNPs were in linkage disequilibrium with each other. We observed a strong allelic association between BA in childhood and two of the SNP sites, T3214A (Cys1072Ser) and C3473T (Pro1158Leu); P = 0.000004 for T3214A and P = 0.0009 for C3473T. T3214A was also associated with BA in adult patients (P = 0.000002), but C3473T was not (P = 0.056). To confirm the above results, we compared estimated frequencies of haplotypes of the six SNPs between BA patients and controls. We found a strong association between BA in childhood and a specific haplotype, TGAAAT, that involved two amino-acid substitutions (819T, 2295G, 2446A, 2490A, 3214A, and 3473T; P = 0.00004, odds ratio, 2.94; 95% confidence interval [CI], 2.48–3.4). On the other hand, haplotype TACGTC, which differed from the TGAAAT haplotype in the last five nucleotides, was inversely correlated with the BA phenotype (P = 0.002; odds ratio, 9.83; 95% CI, 8.35–11.31). These results indicated that specific variants of the IKAP gene, or a variant in linkage disequilibrium with the TGAAAT haplotype, might be associated with mechanisms responsible for early-onset BA. Received: November 9, 2000 / Accepted: November 10, 2000     

Genome-wide search in Finnish families with inflammatory bowel disease provides evidence for novel susceptibility loci

Epidemiological and genetic linkage studies have indicated a strong genetic basis for development of inflammatory bowel disease (IBD) which was recently supported by discovery of the Crohn's disease (CD) susceptibility gene termed NOD2/CARD15. We carried out a genome-wide linkage study in Finnish IBD families, providing a particular advantage to map susceptibility genes for ulcerative colitis (UC) within a genetic isolate. Initially, 92 IBD families with 138 affected sib-pairs (ASPs), were genotyped for 429 markers spaced at approximately 10 cM intervals. Next, the loci on chromosomes 2p13-11, 11p12-q13, and 12p13-12 were high-density mapped in the extended family cohort of 130 families with 173 ASPs. In this study, the most significant lod scores were observed for the UC families on chromosome 2p11 (D2S2333), in the vicinity of the REG gene cluster which is strikingly overexpressed in the IBD mucosa. The maximum two-point lod score was 3.34 (dominant model), and the corresponding NPL score 2.61. For UC, the second highest two-point NPL score of 2.00 was observed at proximal 12p13, where also some evidence for linkage disequilibrium emerged (P=0.07 and P=0.007 for the basic and extended IBD cohorts, respectively). The highest two-point NPL score for the CD families was 2.34 at D12S78 (12q23) with significant evidence for linkage disequilibrium (P=0.004), and for the mixed (MX) families 2.07 at D4S406 near the linkage peak reported previously. This study confirmed several of the IBD loci that have previously been reported and gives evidence for new IBD loci on chromosomes 2p11, 11p12-q13, 12p13-12, 12q23, and 19q13.     

Comparing the Efficacy of SNP Filtering Methods for Identifying a Single Causal SNP in a Known Association Region

Genome‐wide association studies have successfully identified associations between common diseases and a large number of single nucleotide polymorphisms (SNPs) across the genome. We investigate the effectiveness of several statistics, including p‐values, likelihoods, genetic map distance and linkage disequilibrium between SNPs, in filtering SNPs in several disease‐associated regions. We use simulated data to compare the efficacy of filters with different sample sizes and for causal SNPs with different minor allele frequencies (MAFs) and effect sizes, focusing on the small effect sizes and MAFs likely to represent the majority of unidentified causal SNPs. In our analyses, of all the methods investigated, filtering on the ranked likelihoods consistently retains the true causal SNP with the highest probability for a given false positive rate. This was the case for all the local linkage disequilibrium patterns investigated. Our results indicate that when using this method to retain only the top 5% of SNPs, even a causal SNP with an odds ratio of 1.1 and MAF of 0.08 can be retained with a probability exceeding 0.9 using an overall sample size of 50,000.     

Elements of the surname structure of Austria

Background: Aldehyde dehydrogenase-2 (ALDH2) degrades acetaldehyde metabolized from ethanol. Its encoding gene ALDH2 has a functional polymorphism, ALDH2 Glu487Lys associated with low enzyme activity.

Aim: Since Glu487Lys of this locus is fixed for the functional subunit in all non-East Asian populations, this polymorphism was examined along with G-357A promoter (SacI) and four other intronic loci to identify informative markers to study the role of this gene in Indian populations.

Subjects and methods: A total of 397 males belonging to six ethnic populations, from four linguistic groups of India were included in the present study. No test was performed to detect the phenotype of alcoholism. Genotype of ALDH2*E487K and G-357A promoter site along with four non-coding single nucleotide polymorphisms (SNPs) in the upstream of this polymorphism were determined by PCR and sequencing.

Results: All of the subjects were found to have the common homozygous genotype (ALDH2*1/ALDH2*1) for the E487K site. Allele frequencies of non-coding SNPs varied among populations but genetic variance (F_st) indicated little variation among populations. Four major SNP-defined haplotypes accounted for almost all chromosomes in all populations. The ancestral haplotype was found in high frequency in all populations and linkage disequilibrium was strong and highly significant between all sites (p < 0.05).

Conclusion: The small number of haplotypes in this region is suggesting the strong linkage disequilibrium across the region and confirms the global long-range linkage disequilibrium around the ALDH2 locus. This study provides a baseline for future research into the role of the ALDH2 locus in alcoholism in Indian populations.     

Single-nucleotide polymorphisms in the class II region of the major histocompatibility complex in Japanese patients with immunoglobulin A nephropathy

Immunoglobulin A nephropathy (IgAN) is a form of chronic glomerulonephritis of unknown etiology and pathogenesis. Immunogenetic studies have not conclusively indicated that human leukocyte antigen (HLA) is involved. As a first step in investigating a possible relationship between HLA class II genes and IgAN, we analyzed the extent of linkage disequilibrium (LD) in this region of chromosome 6p21.3 in a Japanese test population and found extended LD blocks within the class II locus. We designed a case-control association study of single-nucleotide polymorphisms (SNPs) in each of those LD blocks, and determined that SNPs located in the HLA-DRA gene were significantly associated with an increased risk of IgAN (P = 0.000001, odds ratio = 1.91 [95% confidence interval 1.46–2.49]); SNPs in other LD blocks were not. Our data imply that some haplotype of the HLA-DRA locus has an important role in the development of IgAN in Japanese patients. Received: May 28, 2002 / Accepted: July 4, 2002     

Contribution of the IL-2 and IL-10 genes to inflammatory bowel disease (IBD) susceptibility

Although the importance of genetic susceptibility to IBD has been established by epidemiological studies, the genes involved remain poorly characterized. Important candidate genes include those encoding the immunoregulatory cytokines IL-2 and IL-10. The aim of this study was to assess the contribution of the IL-2 and IL-10 genes to IBD susceptibility. One hundred and ninety-eight pairs of siblings with IBD were genotyped at dinucleotide repeat polymorphisms within the IL-2 and IL-10 genes, and data analysed by the affected sib-pair method of linkage analysis and the transmission disequilibrium test (TDT). A subset of 89 affected sibling pairs was genotyped at markers flanking the IL-2 gene as part of a genome-wide search. The IL-2 polymorphism showed no linkage to IBD overall, but modest evidence for linkage to the ulcerative colitis (UC) data set (P = 0.028). A microsatellite 4 cM distal to the IL-2 gene showed a similar distortion in the ulcerative colitis subgroup (P = 0.006). The TDT showed some distortion of allelic transmission for the IL-2 polymorphism in the UC group, but this did not reach statistical significance (P = 0.09). Results for the IL-10 polymorphism were not significant. Thus the gene encoding IL-2 may contribute to UC susceptibility, but the effect is modest and must await replication in other data sets. The IL-10 gene does not appear to contribute to the risk of developing UC or Crohn's disease.     

Linkage disequilibrium and haplotype analysis among ten single-nucleotide polymorphisms of interleukin 11 identified by sequencing of the gene

Interleukin 11 (IL11) is a member of the interleukin 6 (IL6)-related cytokine subfamily, which stimulates T cell-dependent development of immunoglobulin-producing B cells. IL11 is also an important paracrine regulator of bone metabolism that induces formation of osteoclasts. In the work reported here, we sequenced the entire IL11 structural gene of 48 alleles in a Japanese test population. These experiments identified ten single-nucleotide polymorphisms (SNPs) and determined their allelic frequencies. One polymorphism was identified upstream of exon 1, one in exon 3, four in intron 4 and four in the 3′ untranslated region (3′UTR) of exon 5. Based on the genotype data, we constructed six haplotypes in the tested population. Two-way comparisons of SNPs revealed two combinations in complete linkage disequilibrium, one with SNPs at nucleotide positions 2753, 3644, 5154, and 5568, and another with SNPs at positions 3686, 5141, and 5734. These results will be useful in disease-association studies where a contribution of the human IL11 gene has been suspected, especially in disorders affecting immune response and bone metabolism. Received: March 23, 2001 / Accepted: May 2, 2001     

Linkage disequilibrium and haplotype analysis among eight novel single-nucleotide polymorphisms in the human tissue-type plasminogen activator (t-PA) gene

Tissue-type plasminogen activator (t-PA), a serine protease, activates the conversion of plasminogen to the fibrinolytic protein, plasmin. The t-PA gene, mapped to chromosome 8p12-p11.2, contains 14 exons. An Alu insertion/deletion (I/D) polymorphism in this gene has been associated with an increased risk for myocardial infarction. In the work reported here we sequenced 11 kilobases (kb) of genomic DNA from 50 normal Japanese volunteers (100 alleles), to include all 14 exons of the t-PA gene, flanking intronic sequences, and 6 kb of the 5′ sequence. These experiments identified eight novel single-nucleotide polymorphisms (SNPs), in addition to the known Alu I/D polymorphism, from which genotypic data we constructed 12 haplotypes in the tested population. Two-way comparisons of SNPs and the Alu polymorphism revealed strong linkage disequilibrium between the Alu site and SNPs at positions 20,209 (χ² = 92.263) and 27,555 (χ² = 47.53), and between SNPs at positions 27,849 and 28,902 (χ² = 66.331). A phylogenic tree was constructed to infer a process of genome construction that would reflect the sequence variations we observed. Our results help to explain the lack of agreement among results of various disease-association studies in which a contribution of the human t-PA gene has been suspected but not always confirmed. Received: January 22, 2001 / Accepted: March 26, 2001     

CARD15/NOD2 Risk Alleles in the Development of Crohn's Disease in the Australian Population

We have previously reported strong evidence for linkage between IBD1 and Crohn's disease (CD) in Australian Crohn's disease families. Three risk alleles for Crohn's disease, (Arg₇₀₂Trp (C /T), Gly₉₀₈Arg (G /C) and 980_fs981 (‐ /C), were recently identified in the CARD15/NOD2 gene on chromosome 16, implicating this as the IBD1 locus. Using a novel diagnostic PCR‐RFLP, we have examined the frequency of these alleles in 205 multiplex IBD families, 107 sporadic Crohn's disease cases and 409 normal individuals. We demonstrate that the three risk alleles are more frequent in Crohn's disease, than in controls, with allelic frequencies of 0.11, 0.02 and 0.07 respectively. Heterozygosity for individual variants conferred a three‐fold increase in risk for Crohn's disease while substantially higher risks were associated with being homozygous or compound heterozygous. Despite a significantly lower population allele frequency for the frameshift mutation than reported by other groups, we see a similar contribution by this allele to the risk of developing Crohn's disease. While the three risk alleles influence susceptibility to Crohn's disease in Australia, we show that these alleles do not fully explain the linkage evidence and suggest that there are very likely additional IBD1 susceptibility alleles yet to be described in Australian CD at the NOD2 locus. We also show a second linkage peak in Australian CD that provides some support for a second disease susceptibility locus on chromosome 16.     

SNP Subset Selection for Genetic Association Studies

Association studies for disease susceptibility genes rely on the high density of SNPs within candidate genes. However, the linkage disequilibrium between SNPs imply that not all SNPs identified in the candidate region need be genotyped. Here we develop several approaches to SNP subset selection, which can substantially reduce the number of SNPs to be genotyped in an association study. We apply clustering algorithms to pairwise linkage disequilibrium measures, with SNP subsets determined for different cut‐off values of Δ using nearest and furthest neighbour clusters. Alternatively, SNP subsets may be determined by the proportion of haplotypes they identify. We also show how power calculations, based on the average power to identify a SNP as the disease susceptibility mutation using haplotype‐based or logistic regression based statistical analyses, can be used to choose SNP subsets. All these methods provide a ranking method for subsets of a specific size, but do not provide criteria for overall choice of SNP subset size. We develop such criteria by incorporating power calculations into a decision analysis, where the choice of SNP subset size depends on the genotyping costs and the perceived benefits of identifying association. These methods are illustrated using eleven SNPs in the MMP2 gene.     

Comparative study of the haplotype structure and linkage disequilibrium of chromosome 1p36.2 region in the Korean and Japanese populations

The patterns of linkage disequilibrium (LD) in the human genome provide important information for disease gene mapping. LDs may vary depending on chromosomal regions and populations. We have compared LD and haplotypes defined by SNPs in the chromosome 1p36.2 region of the Korean and Japanese populations. Fifty-eight SNPs in about 418 kb ranging from tumor necrosis factor receptor 2 (TNFR2:TNFRSF1B) to procollagen-lysine, 2-oxoglutarate 5-dioxygenase (PLOD) gene were examined in 96 healthy Koreans and Japanese each by direct sequencing and fluorescence correlation spectroscopy combined with the PCR-sequence specific primer method (PCR-SSP-FCS), respectively. Upon pair-wise LD analysis, a total of 25 and 16 out of 58 SNPs greater than MAF 10% were included in LD blocks, encompassing almost 81 kb and 55 kb in total, in Koreans and Japanese, respectively. Both similarities and differences were observed in LD strength and haplotype frequencies between the populations. Considerable similarities were observed in the telomeric region where a long-range block of approximately 80 kb including three genes was found to have strong LDs in both Koreans and Japanese. Significant difference in LD strength was present near the TNFR2 region between the Japanese and Korean populations.