Single nucleotide polymorphisms and haplotype frequencies of CYP3A5 in a Japanese population

In order to identify single nucleotide polymorphisms (SNPs) and haplotype frequencies of CYP3A5 in a Japanese population, we sequenced the proximal promoter region, all exons, and the surrounding intronic regions using genomic DNA from 187 Japanese subjects. Thirteen SNPs, including seven novel ones: 13108T>C, 16025A>G, 16903A>G, 16993C>G, 27448C>A, 29782A>G, and 31551T>C (A of the translational start codon of GenBank Accession # NG_000004.2 is numbered 1 according to the CYP Allele Nomenclature), were identified. The most common SNP was 6986A>G (key SNP for CYP3A5*3), with a 0.759 frequency. Two novel SNPs, 29782A>G (I456V) and 31551T>C (I488T), as well as 12952T>C (*5 marker) were found, but these alterations were always associated with the *3A marker SNPs, 6986A>G and 31611C>T. Using these 13 SNPs, haplotype analysis was performed and five novel *1 haplotypes (subtypes) (*1e to *1i) and six novel *3 haplotypes (subtypes) (*3d to *3i) were identified. Our findings suggest that CYP3A5*3 is the major defective allele and that other functional exonic SNPs are rare in the Japanese.     

The GABA type A receptor alpha5 subunit gene is associated with bipolar I disorder

Several genetic studies have revealed that bipolar disorders are linked with the chromosomal locus of 15q11-q13, where the gamma-aminobutyric acid (GABA) receptor alpha5 subunit gene (GABRA5) locates. GABA is one of the major neurotransmitters that may be involved in the pathogenesis of bipolar disorder. Five polymorphisms in the GABRA5 gene, -754C>T in the promoter region, IVS1-21G>A, IVS2-26T>A, (*)302C>T in 3'-UTR of exon 5, and a CA repeat polymorphism in the 3' flanking region were examined in a Japanese population. IVS1-21G>A exhibited significant differences in the distribution of the genotype and allele frequency in bipolar I disorder patients but not in bipolar II disorder patients, compared with control subjects. The haplotype analysis showed that IVS1-21G>A/IVS2-26A>T was associated with bipolar I disorder, and the IVS1-21A/IVS2-26T haplotype was a negative risk factor for susceptibility to the disorders (odds ratio: 0.57, 95% confidence interval: 0.44-0.73). These results suggest that the GABRA5 gene may confer susceptibility to bipolar I disorder.     

Polymorphisms in genes that regulate cyclosporine metabolism affect cyclosporine blood levels and clinical outcomes in patients who receive allogeneic hematopoietic stem cell transplantation

In patients who received allogeneic hematopoietic stem cell transplantation (HSCT), we investigated the correlations between single nucleotide polymorphisms (SNPs) in genes that regulate cyclosporine metabolism and clinical outcomes. All patients received sibling-matched HSCT. DNA samples of patients and donors were analyzed for 4 SNPs: MDR1 +1236C>T (rs1128503), +2677G>T>A (rs2032582), +3435C>T (rs1045642), and CYP3A5 +6986G>A (rs776746). A total of 156 patients (median age 40 years) were analyzed. Nineteen patients received HSCT for nonmalignant disease. The CYP3A5 +6986AA genotype was associated with a high cyclosporine blood level after transplantation. However, this genotype was not related to any particular clinical outcome. In contrast, the MDR1 +1236C>T SNP was correlated with specific clinical outcomes. When neither the donor nor the recipient had the CC genotype of MDR1 +1236, patients had lower creatinine levels (P < .001) and less transplantation-related mortality (TRM) (P = .012). These patients also showed longer overall survival (OS) in both univariate (P = .003) and multivariate (P = .003) analyses. Although the CYP3A5 +6986AA genotype was correlated with a high blood cyclosporine concentration, lack of the MDR1 +1236CC genotype in both the donor and recipient was correlated with less TRM and a longer OS in patients who received allogeneic HSCT.     

Mutations in CYP1B1 cause primary congenital glaucoma by reduction of either activity or abundance of the enzyme

Primary congenital glaucoma (PCG) is an autosomal recessive disorder caused predominantly by mutations in the CYP1B1 gene. A total of five frequent single nucleotide polymorphisms (SNPs) have been identified in the coding sequence of CYP1B1: rs10012C>G (p.R48G), rs1056827G>T (p.A119S), rs1056836C>G (p.V432L), rs1056837C>T (p.D449D), and rs1800440A>G (p.N453S). We performed a functional characterization of four common CYP1B1 variants presenting different coding SNP haplotypes (RAVDN, GSLDN, RALDS, and RALDN) and five CYP1B1 mutations reported for PCG patients: c.182G>A (p.G61E), c.608A>G (p.N203S), c.1033_1035del (p.L343del), c.241 T>A (p.Y81N), and c.685G>A (p.E229 K). Each mutation was embedded in its corresponding background SNP haplotype. The common variants revealed variation in enzymatic activity; among them, RAVDN showed the highest activity. Mutants p.G61E, p.N203S, and p.L343del each revealed a residual activity (<10%) of their respective haplotype. The microsomal CYP1B1 abundance relative to total protein also showed variation in common variants and a significant reduction in p.L343del, p.Y81N, and p.E229 K. The free energy of folding (DeltaDeltaG) values suggest that the lower stability of the mutants is one key property leading to the experimentally observed lower protein abundance. Our new measure of relative enzymatic activity (U/mg total protein), which combines activity and abundance values, was significantly lower for all five mutations compared to the corresponding background haplotype. We classified p.Y81N and p.E229 K not as mutations but as hypomorphic alleles, since their relative activity values are intermediate between bona fide mutations and the common variant with the lowest activity (RALDS). We propose that CYP1B1 mutations can act by either reducing enzymatic activity (p.G61E and p.N203S), reducing the abundance of the enzyme (p.Y81N and p.E229 K), or both (p.L343del).     

Identification of variants in NFKBIA and association analysis with hepatocellular carcinoma risk among chronic HBV patients

Human nuclear factor of kappa light chain gene enhancer in B cells inhibitor, alpha (NFKBIA) inhibits the action of NF-kappaB by forming a heterodimer with NF-kappaB, and preventing its translocation to the nucleus. We have sequenced a human NFKBIA full gene including -1000bp promoter region to identify its gene polymorphisms as a potential candidate gene for host genetic study of Hepatocellular Carcinoma (HCC). Nine novel single nucleotide polymorphisms (SNPs) and one GAA deletion were identified; two in promoter region (c.-673A>T, c.-642C>T), two in exon 1 (c.78G>A (Leu26Leu), c.81C>T (Asp27Asp)), three in introns (c.284T>A, c.1952A>G and c.2444C>T) and three in 3'UTR (c.2710-2712delGAA, c.2758G>A and c.3053G>A). Among ten identified variants, six were selected for larger scale genotyping (n=1,750) for association study based on frequencies and location. Haplotypes, their frequencies and linkage disequilibrium coefficients (/D'/) between SNP pairs were estimated. Allele frequencies of each SNPs and haplotypes were compared between patients with HCC and patients without HCC among HbsAg positives by logistic regression. As a conclusion, we could not find any significant association of NFKBIA variants with development of HCC among chronic hepatitis B patients.     

温州地区汉族正常人群CYP1A2基因多态性分布特征

目的检测CYP1A2基因多态性在温州地区汉族正常人群的分布特征。方法采用聚合酶链反应（PCR）产物直接测序法检测108例随机血液样本DNA中CYP1A2基因序列单核苷酸多态性位点（SNP）的分布。对检测到的3个多态位点2159G〉A、3613T〉C、5347C〉T,进一步采用PCR技术分析472例正常人位点的基因型频率和等位基因频率。结果 （1）2159 G〉A位点：G和A等位基因的频率分别为93.8%,6.2%,GG、GA、AA基因型频率分别为87.7%、12.1%、0.2%（χ2=0.325,P〉0.05）;（2）3613 T〉C位点：T和C等位基因的频率分别为97.9%、2.3%,TT、TC、CC基因型频率分别为95.3%、4.4%、0.3%（χ2=0.298,P〉0.05）;（3）5347 C〉T位点：C和T等位基因的频率分别为87.9%、12.1%。CC、CT、TT基因型分布频率分别为77.8%、20.3%、1.9%（χ2=0.742,P〉0.05）;（4）2159 G〉A、5347 C〉T组成的单倍型频率为3.2%。结论温州地区汉族正常人群CYP1A2基因存在2159G〉A、3613T〉C、5347C〉T多态位点。     

Polymorphisms in the annexin A5 gene promoter in Japanese women with recurrent pregnancy loss

Recent findings have raised the possibility that polymorphisms within the annexin A5 gene (ANXA5) promoter contribute to the etiology of recurrent pregnancy loss (RPL). In our present study, 243 Japanese women who had suffered more than three fetal losses and a group of 119 fertile controls were genotyped for four ANXA5 gene promoter single-nucleotide polymorphisms (SNPs; SNP1-4: g.-467G >A, g.-448A>C, g.-422T>C, g.-373G>A) previously reported to be associated with this disorder. An additional two SNPs located within the 5'-untranslated region of the ANXA5 (SNP5 and 6: g.-302T>G, g.-1C>T) were also evaluated. Our case--control study revealed that the minor allele was significantly more frequent in the RPL group than controls for all six of these SNPs, among which SNP5 showed the highest significance (P= 0.002). As with the M2 haplotype for SNP1-4 (A-C-C-A) for a western population in previous reports, a haplotype comprising all of the minor alleles for SNP1-6 (A-C-C-A-G-T), the third major haplotype in the Japanese population, showed a significantly higher frequency in our current RPL subjects than in controls (P= 0.025). In addition, the second major haplotype (G-A-T-G-G-C) was found to confer a significant risk of RPL (P= 0.036), implicating SNP5 as a major risk determinant for this disease. Our present findings support the hypothesis that genomic variations within the ANXA5 gene upstream region impact upon the disease susceptibility to RPL. Our data indicate that SNP5 is a novel risk factor for this disease in the Japanese population.     

Confirmation of associations between ion channel gene SNPs and QTc interval duration in healthy subjects

Population-based association studies have identified several polymorphic variants in genes encoding ion channel subunits associated with the electrocardiographic heart-rate-corrected QT (QTc) length in healthy populations of Caucasian origin (KCNH2 rs1,805,123 (K897 T) and rs3,815,459, SCN5A rs1,805,126 (D1,819D), 1,141-3 C>A, rs1,805,124 (H558R), and IVS24+116 G>A, KCNQ1 rs757,092, KCNE1 IVS2-128 G>A and rs1,805,127 (G38S), and KCNE2 rs2,234,916 (T8A)). However, few of these results have been replicated in independent populations. We tested the association of SNPs KCNQ1 rs757,092, KCNH2 rs3,815,459, SCN5A IVS24+116 G>A, KCNE1 IVS2-128 G>A and KCNE2 rs2,234,916 with QTc length in two groups of 200 subjects presenting the shortest and the longest QTc from a cohort of 2,008 healthy subjects. All polymorphisms were in Hardy-Weinberg equilibrium in both groups. The minor allele SCN5A IVS24+116 A was more frequent in the group of subjects with the shortest QTc, whereas the minor alleles KCNQ1 rs757,092 G and KCNH2 rs3,815,459 A were more frequent in the group with the longest QTc. There was no significant difference for KCNE1 IVS2-128 G>A and KCNE2 rs2,234,916 between the two groups. Haplotype analysis showed a twofold increased risk of QTc lengthening for carriers of the haplotype, combining alleles C and A of the two common KCNE1 SNPs, IVS2-129 C>T (rs2,236,609) and rs1,805,127 (G38S), respectively. In conclusion, our study confirms the reported associations between QTc length and KCNQ1 rs757,092 and KCNH2 rs3,815,459.     

Two common founder mutations of the fanconi anemia group G gene FANCG/XRCC9 in the Japanese population

Fanconi anemia (FA) is a rare autosomal recessive disorder of hematopoiesis with eight complementation groups (FA-A, B, C, D1, D2, E, F and G). To date, seven of the FA genes have been identified. Although extensive analyses in Western countries revealed that the subgroup prevalence and mutational spectrum vary depending on the ethnic background, not much data is available on Asian populations. In the present study, 45 unrelated FA families in Japan were screened for FA gene mutations and 10 families with biallelic pathogenic mutations of FANCG/XRCC9, the gene for FA-G, were identified. A splice mutation IVS3+1G>C was detected in all 9 Japanese families, among whom 4 were homozygous and 5 were heterozygous. Among the heterozygotes, three carried 1066C>T in the second allele. In addition, a family homozygous for 1066C>T with Korean ethnicity was identified. Haplotype analysis by means of 9 microsatellite markers spanning the FANCG locus indicates that IVS3+1G>C and 1066C>T are in complete association with distinct ancestry haplotypes. Our data suggest that IVS3+1G>C arose in the Japanese ancestors at a relatively early time, whereas 1066C>T later on migrated from Korea. The two founder mutations with distinct origins account for most of FANCG mutant alleles in the Japanese population.     

Adiponectin and resistin gene polymorphisms in association with their respective adipokine levels

Single nucleotide polymorphisms (SNPs) at the adiponectin and resistin loci are strongly associated with hypoadiponectinemia and hyperresistinemia, which may eventually increase risk of insulin resistance, type 2 diabetes (T2DM), metabolic syndrome (MS), and cardiovascular disease. Real-time PCR was used to genotype SNPs of the adiponectin (SNP+45T>G, SNP+276G>T, SNP+639T>C, and SNP+1212A>G) and resistin (SNP-420C>G and SNP+299G>A) genes in 809 Malaysian men (208 controls, 174 MS without T2DM, 171 T2DM without MS, 256 T2DM with MS) whose ages ranged between 40 and 70 years old. The genotyping results for each SNP marker was verified by sequencing. The anthropometric clinical and metabolic parameters of subjects were recorded. None of these SNPs at the adiponectin and resistin loci were associated with T2DM and MS susceptibility in Malaysian men. SNP+45T>G, SNP+276G>T, and SNP+639T>C of the adiponectin gene did not influence circulating levels of adiponectin. However, the G-allele of SNP+1212A>G at the adiponectin locus was marginally associated (P= 0.0227) with reduced circulating adiponectin levels. SNP-420C>G (df = 2; F= 16.026; P= 1.50×10(-7) ) and SNP+299G>A (df = 2; F= 22.944; P= 2.04×10(-10) ) of the resistin gene were strongly associated with serum resistin levels. Thus, SNP-420C>G and SNP+299G>A of the resistin gene are strongly associated with the risk of hyperresistinemia in Malaysian men.     

Comprehensive evaluation of the association between prostate cancer and genotypes/haplotypes in CYP17A1, CYP3A4, and SRD5A2

Genes involved in the testosterone biosynthetic pathway - such as CYP17A1, CYP3A4, and SRD5A2 - represent strong candidates for affecting prostate cancer. Previous work has detected associations between individual variants in these three genes and prostate cancer risk and aggressiveness. To more comprehensively evaluate CYP17A1, CYP3A4, and SRD5A2, we undertook a two-phase study of the relationship between their genotypes/haplotypes and prostate cancer. Phase I of the study first searched for single-nucleotide polymorphisms (SNPs) in these genes by resequencing 24 individuals from the Coriell Polymorphism Discovery Resource, 92-110 men from prostate cancer case-control sibships, and by leveraging public databases. In all, 87 SNPs were discovered and genotyped in 276 men from case-control sibships. Those SNPs exhibiting preliminary case-control allele frequency differences, or distinguishing (ie, 'tagging') common haplotypes across the genes, were identified for further study (24 SNPs in total). In Phase II of the study, the 24 SNPs were genotyped in an additional 841 men from case-control sibships. Finally, associations between genotypes/haplotypes in CYP17A1, CYP3A4, and SRD5A2 and prostate cancer were evaluated in the total case-control sample of 1117 brothers from 506 sibships. Family-based analyses detected associations between prostate cancer risk or aggressiveness and a number of CYP3A4 SNPs (P-values between 0.006 and 0.05), a CYP3A4 haplotype (P-values 0.05 and 0.009 in nonstratified and stratified analysis, respectively), and two SRD5A2 SNPs in strong linkage disequilibrium (P=0.02). Undertaking a two-phase study comprising SNP discovery, haplotype tagging, and association analyses allowed us to more fully decipher the relation between CYP17A1, CYP3A4, and SRD5A2 and prostate cancer.     

一例β-酮硫解酶缺乏症患儿 ACAT1基因变异分析

目的:探讨1例经新生儿疾病筛查拟诊为β-酮硫解酶缺乏症(β-ketothiolase deficiency,BKD)患儿的致病基因变异特点,明确其致病原因。方法:通过多重探针杂交富集患儿 ACAT1基因的全部编码区及其侧翼区序列进行高通量测序,确定可疑变异后应用Sanger测序进行变异位点验证。采用多种在线...     

Identification of novel SNPs in the interleukin 6 receptor gene (IL6R)

The human interleukin-6 receptor (IL6R) is responsible for signal transduction of IL6 that might have associations with immune diseases and various infectious diseases. We have sequenced all 10 exons including the putative splicing site to identify single nucleotide polymorphisms in IL6R. Seven novel single nucleotide polymorphisms (SNPs) were identified; one SNP in the promoter region (-183G>A), one synonymous SNP in exon 2 (24013G>A: Ala31Ala), one non-synonymous SNP in exon 9 (48892A>C: Asp358Ala) and four in introns (29753A>C, 42700T>C, 48869T>A and 59818C>T). The frequencies of each SNP in the Korean population (n=300) were 0.48 (-183 G>A), 0.13 (24013 G>A), 0.41 (29753A>C), 0.41 (42700T>C), 0.1 (48869T>A), 0.42 (48892A>C), and 0.07 (59818C>T), respectively. Haplotypes and their frequencies were estimated by the EM algorithm. Linkage disequilibrium coefficients (/D'/) between each SNP pair were also calculated. The information on SNPs and haplotypes in IL6R would be useful for genetic studies of this gene.     

Identification of 25 new mutations in 40 unrelated Spanish Niemann-Pick type C patients: genotype-phenotype correlations

To better characterize Niemann-Pick type C (NPC) in Spain and improve genetic counselling, molecular analyses were carried out in 40 unrelated Spanish patients. The search identified 70/80 alleles (88%) involving 38 different NPC1 mutations, 26 of which are described for the first time. No patient with NPC2 mutations was identified. The novel NPC1 mutations include 14 amino acid substitutions [R372W (c.1114C>T), P434L (c.1301C>T), C479Y (c.1436G>A), K576R (c.1727G>A), V727F (c.2179G>T), M754K (c.2261T>A), S865L (c.2594C>T), A926T (c.2776G>A), D948H (c.2842G>C), V959E (c.2876T>A), T1036K (c.3107C>A), T1066N (c.3197C>A), N1156I (c.3467A>T) and F1224L (c.3672C>G)], four stop codon [W260X (c.780G>A), S425X (c.1274C>A), C645X (c.1935T>A) and R1059X (c.3175C>T)], two donor splice-site mutations [IVS7+1G>A (g.31432G>A) and IVS21+2insG (g.51871insG)], one in-frame mutation [N961_F966delinsS (c.2882del16bpins1bp)] and five frameshift mutations [V299fsX8 (c.895insT), A558fsX11 (c.1673insG), C778fsX10 (c.2334insT), G993fsX3 (c.2973_78delG) and F1221fsX20 (c.3662delT)]. We also identified three novel changes [V562V (c.1686G>A), A580A (c.1740C>G) and A1187A (c.3561G>T)] in three independent NPC patients and five polymorphisms that have been described previously. The combination of these polymorphisms gave rise to the establishment of different haplotypes. Linkage disequilibrium was detected between mutations C177Y and G993fsX3 and specific haplotypes, suggesting a unique origin for these mutations. In contrast, I1061T mutation showed at least two different origins. The most prevalent mutations in Spanish patients were I1061T, Q775P, C177Y and P1007A (10, 7, 7 and 5% of alleles, respectively). Our data in homozygous patients indicate that the Q775P mutation correlates with a severe infantile neurological form and the C177Y mutation with a late infantile clinical phenotype.     

Human vitamin K-dependent GAS6: gene structure, allelic variation, and association with stroke

The product of the growth arrest-specific gene 6 (GAS6), a ligand for the Axl, Sky, and Mer tyrosine kinase receptors, is a vitamin K-dependent protein, structurally related to anticoagulant protein S. Gas6-deficient mice are protected against thrombosis, demonstrating the importance of this protein in the cardiovascular system. The present study was aimed at determining the human GAS6 intron-exon structure and analyzing the gene for the presence of allelic variants that could be associated with atherothrombotic disease. Online analyses allowed us to localize 15 GAS6 exons and to determine the sequence of their intron-flanking regions, in a chromosome 13 region spanning 43.8 kb of DNA. SSCP analysis of PCR-amplified GAS6 exons with their intron-flanking regions from a minimum of 12 control DNA samples, revealed the presence of eight different variants, which were confirmed to be single nucleotide polymorphisms (SNPs). Three of them (c.1263G>C, c.1332C>T, and c.1869T>C) are localized in exons 11, 12, and 14, and appear to be neutral since they do not modify the encoded amino acid. The other SNPs (c.280+170C>G, c.712+26G>A, c.713-155C>T, c.834+7G>A, and c.1478-94C>G) are in introns 3, 7, 8, and 12. A preliminary analysis of five of these SNPs in a group of 110 healthy controls and 188 patients with atherothrombotic disease has revealed statistically significant differences between controls and stroke patients in the allelic distributions of one of these variants (c.834+7G>A in intron 8). The SNP identification in GAS6 reported here would be very useful in future association studies aimed at determining the physiologic role of GAS6 in stroke and other human diseases.