Genome-wide association of breast cancer: composite likelihood with imputed genotypes

We describe composite likelihood-based analysis of a genome-wide breast cancer case–control sample from the Cancer Genetic Markers of Susceptibility project. We determine 14 380 genome regions of fixed size on a linkage disequilibrium (LD) map, which delimit comparable levels of LD. Although the numbers of single-nucleotide polymorphisms (SNPs) are highly variable, each region contains an average of ∼35 SNPs and an average of ∼69 after imputation of missing genotypes. Composite likelihood association mapping yields a single P-value for each region, established by a permutation test, along with a maximum likelihood disease location, SE and information weight. For single SNP analysis, the nominal P-value for the most significant SNP (msSNP) requires substantial correction given the number of SNPs in the region. Therefore, imputing genotypes may not always be advantageous for the msSNP test, in contrast to composite likelihood. For the region containing FGFR2 (a known breast cancer gene) the largest χ² is obtained under composite likelihood with imputed genotypes (χ₂² increases from 20.6 to 22.7), and compares with a single SNP-based χ₂² of 19.9 after correction. Imputation of additional genotypes in this region reduces the size of the 95% confidence interval for location of the disease gene by ∼40%. Among the highest ranked regions, SNPs in the NTSR1 gene would be worthy of examination in additional samples. Meta-analysis, which combines weighted evidence from composite likelihood in different samples, and refines putative disease locations, is facilitated through defining fixed regions on an underlying LD map.     

Genetic association of SNPs in the FTO gene and predisposition to obesity in Malaysian Malays

The common variants in the fat mass- and obesity-associated (FTO) gene have been previously found to be associated with obesity in various adult populations. The objective of the present study was to investigate whether the single nucleotide polymorphisms (SNPs) and linkage disequilibrium (LD) blocks in various regions of the FTO gene are associated with predisposition to obesity in Malaysian Malays. Thirty-one FTO SNPs were genotyped in 587 (158 obese and 429 non-obese) Malaysian Malay subjects. Obesity traits and lipid profiles were measured and single-marker association testing, LD testing, and haplotype association analysis were performed. LD analysis of the FTO SNPs revealed the presence of 57 regions with complete LD (D'' = 1.0). In addition, we detected the association of rs17817288 with low-density lipoprotein cholesterol. The FTO gene may therefore be involved in lipid metabolism in Malaysian Malays. Two haplotype blocks were present in this region of the FTO gene, but no particular haplotype was found to be significantly associated with an increased risk of obesity in Malaysian Malays.     

Association of MICA gene polymorphisms with liver fibrosis in schistosomiasis patients in the Dongting Lake region

Major histocompatibility complex class I chain-related A (MICA) is a highly polymorphic gene located within the MHC class I region of the human genome. Expressed as a cell surface glycoprotein, MICA modulates immune surveillance by binding to its cognate receptor on natural killer cells, NKG2D, and its genetic polymorphisms have been recently associated with susceptibility to some infectious diseases. We determined whether MICA polymorphisms were associated with the high rate of Schistosoma parasitic worm infection or severity of disease outcome in the Dongting Lake region of Hunan Province, China. Polymerase chain reaction-sequence specific priming (PCR-SSP) and sequencing-based typing (SBT) were applied for high-resolution allele typing of schistosomiasis cases (N = 103, age range = 36.2-80.5 years, 64 males and 39 females) and healthy controls (N = 141, age range = 28.6-73.3 years, 73 males and 68 females). Fourteen MICA alleles and five short-tandem repeat (STR) alleles were identified among the two populations. Three (MICA*012:01/02, MICA*017 and MICA*027) showed a higher frequency in healthy controls than in schistosomiasis patients, but the difference was not significantly correlated with susceptibility to S. japonicum infection (Pc > 0.05). In contrast, higher MICA*A5 allele frequency was significantly correlated with advanced liver fibrosis (Pc < 0.05). Furthermore, the distribution profile of MICA alleles in this Hunan Han population was significantly different from those published for Korean, Thai, American-Caucasian, and Afro-American populations (P < 0.01), but similar to other Han populations within China (P > 0.05). This study provides the initial evidence that MICA genetic polymorphisms may underlie the severity of liver fibrosis occurring in schistosomiasis patients from the Dongting Lake region.     

Association of PTEN gene polymorphisms with liver cancer risk

Objective: To find out if there are any relationship between three single nucleotide polymorphisms (SNPs) of phosphatase and tensin homolog (PTEN) gene (rs1234213, rs1234220, and rs2299939) and the susceptibility of liver cancer. Methods: Genotypes of the three SNPs in the PTEN gene were achieved utilizing polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) method. Comparison of genotypes and alleles distribution differences between the case and the control subjects was accomplished with χ² test. The analysis of linkage disequilibrium (LD) and haplotypes of the three SNPs was performed using SHEsis software. We adopted odds ratios (ORs) with 95% confidence intervals (95% CIs) to show the relative risk of liver cancer. Results: TC genotype and C allele of rs1234220 polymorphism showed much more frequently in cases than in controls, reflecting that the TC genotype and the C allele may be linked to the increased risk of liver cancer (OR=2.225, 95% CI=1.178-4.204; OR=1.941, 95% CI=1.124-3.351). Rs2299939 polymorphism showed an opposite result that the GT genotype probably reduce the risk of liver cancer (OR=0.483, 95% CI=0.259-0.900). Statistical significance was not found in the distribution differences of the genotypes of rs1234213 between two groups. LD and haplotype analysis results of the three SNPs showed that the T-C-G haplotype frequency was much higher in cases than in healthy objects, which proved that the T-C-G haplotype might be a susceptibility haplotype for liver cancer (OR=3.750, 95% CI=1.396-10.077). Conclusions: PTEN gene polymorphisms might relate to liver cancer risk.     

THEMIS and PTPRK in celiac intestinal mucosa: coexpression in disease and after in vitro gliadin challenge

Celiac disease (CD) is an immune mediated, polygenic disorder, where HLA-DQ2/DQ8 alleles contribute around 35% to genetic risk, but several other genes are also involved. Genome-wide association studies (GWASs) and the more recent immunochip genotyping projects have fine-mapped 39 regions of genetic susceptibility to the disease, most of which harbor candidate genes that could participate in this disease process. We focused our attention to the GWAS peak on chr6: 127.99–128.38 Mb, a region including two genes, thymocyte-expressed molecule involved in selection (THEMIS) and protein tyrosine phosphatase, receptor type, kappa (PTPRK), both of which have immune-related functions. The aim of this work was to evaluate the expression levels of these two genes in duodenal mucosa of active and treated CD patients and in controls, and to determine whether SNPs (rs802734, rs55743914, rs72975916, rs10484718 and rs9491896) associated with CD have any influence on gene expression. THEMIS showed higher expression in active CD compared with treated patients and controls, whereas PTPRK showed lower expression. Our study confirmed the association of this region with CD in our population, but only the genotype of rs802734 showed some influence in the expression of THEMIS. On the other hand, we found a significant positive correlation between THEMIS and PTPRK mRNA levels in CD patients but not in controls. Our results suggest a possible role for both candidate genes in CD pathogenesis and the existence of complex, regulatory relationships that reside in the vast non-coding, functional intergenic regions of the genome. Further investigation is needed to clarify the impact of the disease-associated SNPs on gene function.     

Next generation sequencing of SNPs for non-invasive prenatal diagnosis: challenges and feasibility as illustrated by an application to β-thalassaemia

β-Thalassaemia is one of the most common autosomal recessive single-gene disorder worldwide, with a carrier frequency of 12% in Cyprus. Prenatal tests for at risk pregnancies use invasive methods and development of a non-invasive prenatal diagnostic (NIPD) method is of paramount importance to prevent unnecessary risks inherent to invasive methods. Here, we describe such a method by assessing a modified version of next generation sequencing (NGS) using the Illumina platform, called ‘targeted sequencing'', based on the detection of paternally inherited fetal alleles in maternal plasma. We selected four single-nucleotide polymorphisms (SNPs) located in the β-globin locus with a high degree of heterozygosity in the Cypriot population. Spiked genomic samples were used to determine the specificity of the platform. We could detect the minor alleles in the expected ratio, showing the specificity of the platform. We then developed a multiplexed format for the selected SNPs and analysed ten maternal plasma samples from pregnancies at risk. The presence or absence of the paternal mutant allele was correctly determined in 27 out of 34 samples analysed. With haplotype analysis, NIPD was possible on eight out of ten families. This is the first study carried out for the NIPD of β-thalassaemia using targeted NGS and haplotype analysis. Preliminary results show that NGS is effective in detecting paternally inherited alleles in the maternal plasma.     

Association between ERAP1 gene polymorphisms and ankylosing spondylitis susceptibility in Han population

Purposes: The present study was designed to investigate the relationship between endoplasmic reticulum amino peptidase 1 (ERAP1) gene polymorphisms and ankylosing spondylitis (AS) in Han population of Shaanxi province. Methods: 100 AS patients and 100 healthy people were enrolled in present study as case and control groups respectively, and the control group was matched with the case group by age and gender. ERAP1 gene rs27434 and rs7711564 polymorphisms were test by TaqMan probe genotyping method. SHEsis software was used to operate linkage disequilibrium (LD) and haplotype analysis between the two single nucleotide polymorphisms (SNPs). χ² test was employed to compare the differences of the genotype, allele and haplotype frequencies between the case and control groups. Relative risk of AS was represented by odds ratios (ORs) and 95% confidence intervals (95% CIs). Results: In ERAP1 rs27434 and rs7711564 polymorphisms, the frequencies of AA and CC genotypes in case group were significantly higher compared to those in control group (P=0.036; P=0.039), and so were the frequencies of A and C alleles (OR=1.589, 95% CI=1.070-2.359, P=0.028; OR=1.535, 95% CI=1.021-2.308, P=0.050). Linkage disequilibrium test and haplotype analysis of the alleles of the two SNPs showed that the frequency of A-C haplotype was higher in case group than that in control group (P=0.005), which indicated that A-C might be the susceptible haplotype to AS. Conclusions: ERAP1 gene rs27434 and rs7711564 polymorphisms may increase the risk of AS.     

No association of VAMP8 gene polymorphisms with glioma in a Chinese Han population

Vesicle-associated membrane protein 8 (VAMP8) gene plays an important role in biological functions like endosomal fusion, sequential granule-to-granule fusion and autophagy. The current research identified VAMP8 acted as a novel oncogene by promoting cell proliferation and therapeutic resistance in glioma. Nevertheless, the association between VAMP8 genes polymorphism and glioma patients has not been well studied. In our study, to explore the association between single nucleotide polymorphisms (SNPs) of VAMP8 gene with glioma risk in the Chinese Han population, we performed a hospital based case-control study (992 cases and 1008 controls). Eight common tagging SNPs of VAMP8 gene were genotyped, while no significant difference in allele or genotype frequency was found between glioma patients and healthy controls. No positive linkage disequilibrium (LD) was detected either. No haplotype distribution was positive. Accordingly, our study suggested that VAMP8 gene variants might not contribute to glioma susceptibility and associated with glioma in the Chinese Han population.     

Replication of the LINGO1 gene association with essential tremor in a North American population

A marker in the LINGO1 gene, rs9652490, showing significant genome-wide association with essential tremor (ET), was recently reported in an Icelandic population. To replicate this association in an independent population from North America, we genotyped 15 SNPs in the LINGO1 gene in 257 Caucasian ET cases (‘definite,'' ‘probable'' or ‘possible'') and 265 controls enrolled in an epidemiological study at Columbia University. We observed a marginally significant association with allele G of the marker rs9652490 (P=0.0569, odds ratio (OR)=1.33). However, for ‘definite'' or ‘probable'' ET, rs9652490 was significantly associated with ET (P=0.03, OR=1.41). Our subsequent analysis of early-onset ET (age at onset <40 years) revealed that three SNPs, rs177008, rs13313467 and rs8028808, were significantly associated with ET (P=0.028, OR=1.52; P=0.0238, OR=1.54; and P=0.0391, OR=1.55, respectively). These three SNPs represent a 2.3 kb haplotype. Finally, a meta-analysis of three published studies confirms allelic association with rs9652490 and two adjacent SNPs. Our study independently confirms that the LINGO1 gene is a risk factor for ET in a Caucasian population in North America, and further shows that those with early-onset ET are likely to be at high risk.     

Association of FGFR2 gene polymorphisms with the risk of breast cancer in population of West Siberia

Polymorphisms within intron 2 of the FGFR2 gene have been associated with increased risk of breast cancer (BC) in European and Asian populations. The study by Easton et al reported two FGFR2 SNPs, rs2981582 and rs7895676, to be among those most strongly associated with BC risk. Statistical modeling suggested that rs7895676 was the variant responsible for the association observed in the region. In this work, we studied the association between seven FGFR2 SNPs, including rs2981582 and rs7895676, and BC risk in the Russian population of 766 case and 665 control women from Siberia, Russian Federation. In our population, allelic frequencies and the magnitude of linkage disequilibrium (LD) were different from those observed in European and Asian populations. The following three SNPs were significantly associated with BC in our study: rs7895676[C] (odds ratio (OR)=1.28 (1.12–1.43), P=1.7 × 10⁻³), rs2981582[T] (OR=1.46 (1.30–1.62), P=2 × 10⁻⁶) and rs3135718[G] (OR=1.43 (1.27–1.58), P=6 × 10⁻⁶). The latter two SNPs were in strong (r²=0.95) LD in our sample. Maximum likelihood analysis showed that the model, including rs7895676, only explains that the association is significantly (P<0.001) worse than any of the models, including either rs2981582 or rs3135718. Thus, in addition to the confirmation of association of FGFR2 with the BC risk in this new population, our study has suggested that rs7895676 is not likely to represent the causative variant.     

MUTYH-associated polyposis (MAP): evidence for the origin of the common European mutations p.Tyr179Cys and p.Gly396Asp by founder events

MUTYH-associated polyposis (MAP) is an autosomal recessive adenomatous polyposis caused by biallelic germline mutations of the base-excision-repair gene MUTYH. In MAP patients of European origin, the combined allele frequency of the mutations p.Tyr179Cys and p.Gly396Asp ranges between 50 and 82%, while these mutations have not been identified in Far Eastern Asian populations, supporting the hypothesis that a founder effect has occurred at some point in European history. To investigate the natural history of the two common European MUTYH alleles, we genotyped six gene-flanking microsatellite markers in 80 unrelated Italian and German MAP patients segregating one or both mutations and calculated their age in generations (g) by using DMLE+2.2 software. Three distinct common haplotypes, one for p.Tyr179Cys and two for p.Gly396Asp, were identified. Estimated mutation ages were 305 g (95% CS: 271–418) for p.Tyr179Cys and 350 g (95% CS: 313–435) for p.Gly396Asp. These results provide evidence for strong founder effects and suggest that the p.Tyr179Cys and p.Gly396Asp mutations derive from ancestors who lived between 5–8 thousand years and 6–9 thousand years B.C., respectively.     

Association between genetic variants in sortilin-related receptor 1 (SORL1) and Alzheimer's disease in adults with Down syndrome

Recent reports have suggested that variants in the sortilin-related receptor gene (SORL1) increase the risk of late onset Alzheimer's disease (AD) in Northern European, Hispanic, African–American and Isreali–Arab populations. SORL1 directs trafficking of amyloid precursor protein (APP) and under-expression of SORL1 may lead to over-expression of β amyloid peptides. Adults with Down syndrome (DS) over-express APP and have early onset and high risk for AD. We investigated the relation of seven variants in the gene for SORL1 to age at onset and risk for AD among 208 adults with DS, 45–70 years of age at baseline. Participants were ascertained through the New York State developmental disability service system and followed at 18-month intervals. Information from cognitive assessments, caregiver interviews, medical record review and neurological examination was used to establish the diagnosis of dementia. Homozygosity for the minor T allele in rs556349 and for the minor C allele in rs536360 was associated with later age at onset and reduced risk of AD (HR = 0.26, 95% CI: 0.08–0.86; and HR = 0.40, 95% CI: 0.16–0.98, respectively). Mean age at onset was approximately four years later in individuals who were homozygous for those alleles compared with those who had at least one major allele. These findings indicate a modest association of variants in SORL1 with AD. In addition, we did not observe the same alleles to be associated with AD compared with earlier studies, suggesting that these SNPs are in linkage disequilibrium (LD) with the putative functional variants or that expression of the SORL1 gene and hence its interaction with APP might be modified by the extremely high levels of APP characteristic of Down syndrome. Thus, further studies are needed to identify functional variants that influence risk for AD in this uniquely vulnerable population.     

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     

Next generation sequencing for HLA loci in full heritage Pima Indians of Arizona,Part II: HLA-A, -B,and -C with selected non-classical loci at 4-field resolution from whole genome sequences

While the samples and data from the Pima Indians of the Gila River Indian Community have been included in many international HLA workshops and conferences and have been the focus of numerous population reports and the source of novel alleles at the classical HLA loci, they have not been studied for the non-classical loci. In order to expand our HLA-disease association studies, we typed over 300 whole genome sequences from full Pima heritage members, controlled for first degree relationship, and employed recently developed computer algorithms to resolve HLA alleles. Both classical—HLA-A, -B, and -C— and non-classical— HLA-E, -F, -G, -J, -L, -W, -Y, -DPA2, -DPB2, -DMA, -DMB, -DOA, -DRB2, -DRB9, TAP1— loci were typed at the 4-field level of resolution. We present allele and selected haplotype frequencies, test the genotype distributions for population structure, discuss the issues that are created for tests of Hardy-Weinberg equilibrium over the four sample spaces of high resolution HLA typing, and address the implications for the evolution of non-classical pseudogenes that are no longer expressed in a phenotype subject to natural selection.     

Evidence for association with hepatocellular carcinoma at the PAPSS1 locus on chromosome 4q25 in a family-based study

A region on chromosome 4q25 has recently been highlighted as linked to hepatocellular carcinoma (HCC). In this study, we performed a family-based association analysis with 67 single-nucleotide polymorphisms (SNPs) to map this linkage region in 240 families with HCC, 212 (88.3%) of which were ascertained through hepatitis B virus surface antigen (HBsAg)-positive index cases. Individual SNP analysis with correction for multiple testing identified 10 SNPs in two correlated haplotype blocks, located in or around the 3′-phosphoadenosine 5′-phosphosulfate synthetase-1 (PAPSS1) gene (all P-values: <0.0075). Our linkage data and GIST (Genotype identity-by-descent sharing test) indicate that 6 of these 10 SNPs contributed to the linkage signal. The haplotype block of the strongest association with HCC extended from the intron 5 to the 3′-flanking region of PAPSS1; multiple consecutive three-SNP haplotypes in this region were significant. The most significant haplotype showed odd ratios of 3.41 (95% confidence interval (CI)=1.36–8.53) for homozygous individuals in a case-unaffected sibling analysis. This haplotype also revealed an association with elevated serum α-fetoprotein and with poor survival in familial cases and an independent series of HBsAg-positive cases with small tumor present at the time of hospital admission. These results implicate PAPSS1 as a candidate HCC-susceptibility gene in hepatitis B carriers.     

Comparative high-resolution analysis of linkage disequilibrium and tag single nucleotide polymorphisms between populations in the vitamin D receptor gene

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.     

Protective effect of the APOE-e3 allele in Alzheimer's disease

Although several alleles of susceptibility to Alzheimer''s disease (AD) have been studied in the last decades, few polymorphisms have been considered as risk factors for the disease. Among them, the APOE-e4 allele appears to be the major genetic risk factor for the onset of the disease. However, it is important to confirm the potential susceptibility of these genetic variants in different populations in order to establish a genetic profile for the disease in specific communities. This study analyzed the APOE polymorphisms regarding susceptibility to AD in a sample of 264 individuals (primarily Caucasians; 82 cases and 182 controls) in the population from Vitória, ES, Brazil, by PCR restriction fragment length polymorphism (PCR-RFLP) methods. The patients were selected according to clinical criteria for probable AD. Whereas the e4 allele showed statistically significant positive association with susceptibility to AD (OR = 3.01, 95%CI = 1.96-4.61; P < 0.0001), the e2 allele did not. The results of the e4 allele confirm the role of this polymorphism as a risk factor for AD in the sample studied as observed in other populations. Although the e3 allele has been considered neutral in several studies, our results suggest that it acts as a protective factor against AD in the population studied (OR = 0.46, 95%CI = 0.30-0.67; P < 0.0001). This study may provide a new insight into the role of the APOE-e3 allele in the etiology of AD and might help to estabilish a profile of risk for AD in the population from Vitória, ES.     

Tag-SNP analysis of the GFI1-EVI5-RPL5-FAM69 risk locus for multiple sclerosis

A recent genome-wide association study conducted by the International Multiple Sclerosis Genetic Consortium (IMSGC) identified, among others, a number of putative multiple sclerosis (MS) susceptibility variants at position 1p22. Twenty-one SNPs positively associated with MS were located at the GFI-EVI5-RPL5-FAM69A locus. In this study, we performed an analysis and fine mapping of this locus, genotyping eight Tag-SNPs in 732 MS patients and 974 controls from Spain. We observed an association with MS in three of eight Tag-SNPs: rs11804321 (P=0.008, OR=1.29; 95% CI=1.08–1.54), rs11808092 (P=0.048, OR=1.19; 95% CI=1.03–1.39) and rs6680578 (P=0.0082, OR=1.23; 95% CI=1.07–1.41). After correcting for multiple comparisons and using logistic regression analysis to test the addition of each SNP to the most associated SNPs, we observed that rs11804321 alone was sufficient to model the association. This Tag-SNP captures two SNPs in complete linkage disequilibrium (r²=1), both located within the 17th intron of the EVI5 gene. Our findings agree with the corresponding data of the recent IMSGC study and present new genetic evidence that points to EVI5 as a factor of susceptibility to MS.     

HLA imputation in an admixed population: An assessment of the 1000 Genomes data as a training set

Methods to impute HLA alleles based on dense single nucleotide polymorphism (SNP) data provide a valuable resource to association studies and evolutionary investigation of the MHC region. The availability of appropriate training sets is critical to the accuracy of HLA imputation, and the inclusion of samples with various ancestries is an important pre-requisite in studies of admixed populations. We assess the accuracy of HLA imputation using 1000 Genomes Project data as a training set, applying it to a highly admixed Brazilian population, the Quilombos from the state of São Paulo. To assess accuracy, we compared imputed and experimentally determined genotypes for 146 samples at 4 HLA classical loci. We found imputation accuracies of 82.9%, 81.8%, 94.8% and 86.6% for HLA-A, -B, -C and -DRB1 respectively (two-field resolution). Accuracies were improved when we included a subset of Quilombo individuals in the training set. We conclude that the 1000 Genomes data is a valuable resource for construction of training sets due to the diversity of ancestries and the potential for a large overlap of SNPs with the target population. We also show that tailoring training sets to features of the target population substantially enhances imputation accuracy.