Development of a SNP set for human identification: A set with high powers of discrimination which yields high genetic information from naturally degraded DNA samples in the Thai population.

This study describes the development of a SNP typing system for human identification in the Thai population, in particular for extremely degraded DNA samples. A highly informative SNP marker set for forensic identification was identified, and a multiplex PCR-based Invader assay was developed. Fifty-one highly informative autosomal SNP markers and three sex determination SNP markers were amplified in two multiplex PCR reactions and then detected using Invader assay reactions. The average PCR product size was 71 base pairs. The match probability of the 54-SNP marker set in 124 Thai individuals was 1.48×10(-21), higher than that of STR typing, suggesting that this 54-SNP marker set is beneficial for forensic identification in the Thai population. The selected SNP marker set was also evaluated in 90 artificially degraded samples, and in 128 naturally degraded DNA samples from real forensic casework which had shown no profiles or incomplete profiles when examined using a commercial STR typing system. A total of 56 degraded samples (44%) achieved the matching probability (PM) equivalent to STR gold standard analysis (successful genotyping of 44 SNP markers) for human identification. These data indicated that our novel 54-SNP marker set provides a very useful and valuable approach for forensic identification in the Thai population, especially in the case of highly to extremely degraded DNA. In summary, we have developed a set of 54 Thai-specific SNPs for human identification which have higher discrimination power than STR genotyping. The PCRs for these 54 SNP markers were successfully combined into two multiplex reactions and detected with an Invader assay. This novel SNP genotyping system also yields high levels of genetic information from naturally degraded samples, even though there are much more difficult to recover than artificially degraded samples.

Identification of a susceptibility locus for severe adolescent idiopathic scoliosis on chromosome 17q24.3.

Adolescent idiopathic scoliosis (AIS) is the most common spinal deformity, affecting around 2% of adolescents worldwide. Genetic factors play an important role in its etiology. Using a genome-wide association study (GWAS), we recently identified novel AIS susceptibility loci on chromosomes 10q24.31 and 6q24.1. To identify more AIS susceptibility loci relating to its severity and progression, we performed GWAS by limiting the case subjects to those with severe AIS. Through a two-stage association study using a total of ∼12,000 Japanese subjects, we identified a common variant, rs12946942 that showed a significant association with severe AIS in the recessive model (P=4.00 × 10(-8), odds ratio [OR]=2.05). Its association was replicated in a Chinese population (combined P=6.43 × 10(-12), OR = 2.21). rs12946942 is on chromosome 17q24.3 near the genes SOX9 and KCNJ2, which when mutated cause scoliosis phenotypes. Our findings will offer new insight into the etiology and progression of AIS.

Impact of allele copy number of polymorphisms in FCGR3A and FCGR3B genes on susceptibility to ulcerative colitis.

BACKGROUND: Polymorphisms in the Fcγ receptor genes have been implicated in several autoimmune diseases, including ulcerative colitis (UC). However, most of these reports had not taken into account the effect of copy number variation at this region. METHODS: We investigated the combined effect of allele and gene copy number of FCGR3A-158F/V and FCGR3B-NA1/NA2 on susceptibility to UC. Study subjects were composed of a total of 752 Japanese patients with UC and 2062 Japanese control subjects. To estimate allele copy number of the 2 polymorphisms, we integrated the results of PCR-based real-time Invader assay (PCR-RETINA) that measures the allelic ratio and Taqman assay that detects the total copy number. We analyzed the associations of allele copy number with UC using logistic regression model. RESULTS: Gene and allele copy numbers of FCGR3A and FCGR3B were successfully determined in more than 99.5% of the study subjects. Allele copy number of FCGR3A-158F/V demonstrated significant association with susceptibility to UC (P = 0.02), although each single-nucleotide polymorphism and copy number variation alone did not show significant association. Although allele copy number of FCGR3B-NA1/NA2 (P = 0.002) also showed significant association with UC susceptibility, this association seemed to reflect the effect of FCGR3B gene copy number. Subsequent haplotype analyses revealed a strong association of a haplotype FCGR2A-131H/R and copy number of FCGR3B gene (P = 6.5 × 10). CONCLUSIONS: Allele copy number of FCGR3A-158F/V and FCGR3B gene copy number were associated with UC susceptibility. Our results suggest that organizing handling of immune complex by FCGR3A, FCGR3B, and FCGR2A may play a crucial role in the pathogenesis of UC.

Genetic variants in GPR126 are associated with adolescent idiopathic scoliosis.

Adolescent idiopathic scoliosis (AIS) is the most common pediatric skeletal disease. We previously reported a locus on chromosome 10q24.31 associated with AIS susceptibility in Japanese using a genome-wide association study (GWAS) consisting of 1,033 cases and 1,473 controls. To identify additional AIS-associated loci, we expanded the study by adding X-chromosome SNPs in the GWAS and increasing the size of the replication cohorts. Through a stepwise association study including 1,819 cases and 25,939 controls, we identified a new susceptibility locus on chromosome 6q24.1 in Japanese (P = 2.25 × 10(-10); odds ratio (OR) = 1.28). The most significantly associated SNP, rs6570507, was in GPR126 (encoding G protein-coupled receptor 126). Its association was replicated in Han Chinese and European-ancestry populations (combined P = 1.27 × 10(-14); OR = 1.27). GPR126 was highly expressed in cartilage, and the knockdown of gpr126 in zebrafish caused delayed ossification of the developing spine. Our results should provide insights into the etiology and pathogenesis of AIS.

A genome-wide association study of HCV-induced liver cirrhosis in the Japanese population identifies novel susceptibility loci at the MHC region.

BACKGROUND & AIMS: We performed a genome-wide association study (GWAS) of hepatitis C virus (HCV)-induced liver cirrhosis (LC) to identify predictive biomarkers for the risk of LC in patients with chronic hepatitis C (CHC). METHODS: A total of 682 HCV-induced LC cases and 1045 CHC patients of Japanese origin were genotyped by Illumina Human Hap 610-Quad bead Chip. RESULTS: Eight SNPs which showed possible associations (p<1.0 × 10(-5)) at the GWAS stage were further genotyped using 936 LC cases and 3809 CHC patients. We found that two SNPs within the major histocompatibility complex (MHC) region on chromosome 6p21, rs910049 and rs3135363, were significantly associated with the progression from CHC to LC (pcombined=9.15 × 10(-11) and 1.45 × 10(-10), odds ratio (OR)=1.46 and 1.37, respectively). We also found that HLA-DQA1(*)0601 and HLA-DRB1(*)0405 were associated with the progression from CHC to LC (p=4.53 × 10(-4) and 1.54 × 10(-4) with OR=2.80 and 1.45, respectively). Multiple logistic regression analysis revealed that rs3135363, rs910049, and HLA-DQA1(*)0601 were independently associated with the risk of HCV-induced LC. In addition, individuals with four or more risk alleles for these three loci have a 2.83-fold higher risk for LC than those with no risk allele, indicating the cumulative effects of these variations. CONCLUSIONS: Our findings elucidated the crucial roles of multiple genetic variations within the MHC region as prognostic/predictive biomarkers for CHC patients.

A genome-wide association study identifies 2 susceptibility Loci for Crohn's disease in a Japanese population.

BACKGROUND & AIMS: Crohn's disease is an inflammatory bowel disease induced by multiple genetic and environmental factors. Genome-wide association studies have identified genetic factors that affect the risk for Crohn's disease in European populations, but information from other ethnic groups is scarce. We therefore investigated genetic factors associated with Crohn's disease in the Japanese population. METHODS: We performed a genome-wide association study with 372 individuals with Crohn's disease (cases) and 3389 controls, all from the Japanese population. To confirm identified associations, we performed a replication study with an independent panel of 1151 Crohn's disease cases and 15,800 controls. We also performed an association analysis using genome-wide genotype imputation in the discovery cohort. RESULTS: We confirmed associations of Crohn's disease with variants in MHC (rs7765379, P = 2.11 × 10(-59)), TNFSF15 (rs6478106, P = 3.87 × 10(-45)), and STAT3 (rs9891119, P = 2.24 × 10(-14)). We identified 2 new susceptibility loci: on chromosome 4p14 (rs1487630, P = 2.40 × 10(-11); odds ratio, 1.33), and in the SLC25A15-ELF1-WBP4 region on 13q14 (rs7329174 in ELF1, P = 5.12 × 10(-9); odds ratio, 1.27). CONCLUSIONS: In a genome-wide association study, we identified 2 new susceptibility loci for Crohn's disease in a Japanese population. These findings could increase our understanding of the pathogenesis of Crohn's disease.

Whole-genome sequencing of liver cancers identifies etiological influences on mutation patterns and recurrent mutations in chromatin regulators.

Hepatocellular carcinoma (HCC) is the third leading cause of cancer-related death worldwide. We sequenced and analyzed the whole genomes of 27 HCCs, 25 of which were associated with hepatitis B or C virus infections, including two sets of multicentric tumors. Although no common somatic mutations were identified in the multicentric tumor pairs, their whole-genome substitution patterns were similar, suggesting that these tumors developed from independent mutations, although their shared etiological backgrounds may have strongly influenced their somatic mutation patterns. Statistical and functional analyses yielded a list of recurrently mutated genes. Multiple chromatin regulators, including ARID1A, ARID1B, ARID2, MLL and MLL3, were mutated in ∼50% of the tumors. Hepatitis B virus genome integration in the TERT locus was frequently observed in a high clonal proportion. Our whole-genome sequencing analysis of HCCs identified the influence of etiological background on somatic mutation patterns and subsequent carcinogenesis, as well as recurrent mutations in chromatin regulators in HCCs.

New pharmacogenetic test for detecting an HLA-A*31: 01 allele using the InvaderPlus assay.

BACKGROUND & AIMS: Carbamazepine (CBZ) is widely used for the treatment of epilepsy and other neurological disorders. However, 3-5% of CBZ-treated individuals suffer from cutaneous adverse drug reactions (cADRs). Recently, in a genome-wide association study, HLA-A*31:01 has been reported to be a strong genetic marker for CBZ-induced cADRs in both Japanese and European populations. As most of the available methods for HLA genotyping are laborious, the development of a simple and rapid genotyping method for HLA-A*31:01 is desirable from the viewpoint of a clinical pharmacogenetic test. METHODS: More than 1700 sequences for HLA-A alleles were obtained from the MHC database of the National Center for Biotechnology Information (dbMHC). Several HLA-A*31:01-discriminating single-nucleotide polymorphisms were selected. These SNPs were used for sequence-specific primer PCR (SSP-PCR) and for the target site of the Invader reaction. By combining SSP-PCR with a target-specific Invader reaction, we designed two sets of primers/probes for HLA-A*31:01 allele detection. The performance of both sets was evaluated using 90 Asian HapMap samples. Further evaluation was carried out using another 376 Japanese samples and 90 CEU (European) and 90 YRI (African) HapMap samples. RESULTS: Our assay specifically detected an HLA-A*31:01 allele in a total of 466 individuals of the Asian population. Furthermore, the assay correctly identified HLA-A*31:01-positive carriers from the CEU and the YRI population, respectively, implying that the assay has potential for application to other ethnic groups. CONCLUSION: We developed a new HLA-A*31:01-detecting method by a combination of SSP-PCR with target-specific InvaderPlus technology. As our assay is rapid and accurate, it is hoped that this method will be used in a pharmacogenetic test in a clinical setting to avoid CBZ-induced cADRs.

Genetic differences in the two main groups of the Japanese population based on autosomal SNPs and haplotypes.

Although the Japanese population has a rather low genetic diversity, we recently confirmed the presence of two main clusters (the Hondo and Ryukyu clusters) through principal component analysis of genome-wide single-nucleotide polymorphism (SNP) genotypes. Understanding the genetic differences between the two main clusters requires further genome-wide analyses based on a dense SNP set and comparison of haplotype frequencies. In the present study, we determined haplotypes for the Hondo cluster of the Japanese population by detecting SNP homozygotes with 388,591 autosomal SNPs from 18,379 individuals and estimated the haplotype frequencies. Haplotypes for the Ryukyu cluster were inferred by a statistical approach using the genotype data from 504 individuals. We then compared the haplotype frequencies between the Hondo and Ryukyu clusters. In most genomic regions, the haplotype frequencies in the Hondo and Ryukyu clusters were very similar. However, in addition to the human leukocyte antigen region on chromosome 6, other genomic regions (chromosomes 3, 4, 5, 7, 10 and 12) showed dissimilarities in haplotype frequency. These regions were enriched for genes involved in the immune system, cell-cell adhesion and the intracellular signaling cascade. These differentiated genomic regions between the Hondo and Ryukyu clusters are of interest because they (1) should be examined carefully in association studies and (2) likely contain genes responsible for morphological or physiological differences between the two groups.

Novel deletion mutations of OPTN in amyotrophic lateral sclerosis in Japanese.

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease characterized by selective motor neuron death in the brain and spinal cord. Many disease genes for ALS have been identified; however, each disease gene is responsible for very small fractions of ALS. Recently, mutations of the gene encoding optineurin (OPTN) are reported in familial and sporadic ALS. OPTN is also responsible for a small number of ALS, 3.8% of familial and 0.29% of sporadic ALS in Japanese. The low prevalence may be an underestimation due to incomplete screening of the mutation. To examine OPTN mutations more extensively, we screened the OPTN deletions using a quantitative PCR system. We examined 710 Japanese ALS subjects who had previously been found to have no OPTN mutations by a screening using a PCR-direct sequence strategy. We identified 3 kinds of deletions in 5 patients; one was homozygous, and the remaining were heterozygous. All deletions occurred due to the Alu-mediated recombination and are expected to result in null alleles. Our results suggest that the OPTN deletion mutation in ALS is not infrequent and the prevalence of the OPTN mutation in Japanese sporadic ALS is considerably high.