Identification of a founder mutation for Pendred syndrome in families from northwest Iran

ObjectiveMutations in the SLC26A4 gene cause both Pendred syndrome and autosomal recessive nonsyndromic hearing loss (ARNSHL) at the DFNB4 locus. The SLC26A4 mutations vary among different communities. Previous studies have shown that mutations in the SLC26A4 gene are responsible for the more common syndromic hereditary hearing loss in Iran. This study assesses the possibility of a founder mutation for Pendred syndrome in northwest Iran.Materials and methodsIn this study, we performed comprehensive clinical and genetic evaluations in two unrelated families from northwest Iran with nine members affected by hearing loss (HL). After testing short tandem repeat (STR) markers to confirm linkage to the SLC26A4 locus, we screened the SLC26A4 gene by Sanger sequencing of all 21 exons, exon–intron boundaries and the promoter region for any causative mutation. We identified the same causative mutation in these two families as we had detected earlier in two other Azeri families from northwest Iran. To investigate the possibility of a founder effect in these four families, we conducted haplotype analysis, and 14 single nucleotide polymorphisms (SNPs) throughout the SLC26A4 gene were genotyped.ResultsPatients in the two families showed the phenotype of Pendred syndrome. A known frameshift mutation (c.965insA, p.N322Fs7X) in exon 8 was identified in the two families, which was the same mutation that we detected previously in two other Azeri families. The results of haplotype analysis showed that all 15 patients from four families shared the founder mutation. Common haplotypes were not observed in noncarrier members.ConclusionsBased on the results of our two studies, the c.965insA mutation has only been described in Iranian families from northwest Iran, so there is evidence for a founder mutation originating in this part of Iran.     

Choosing blindly but wisely: differentially private solicitation of DNA datasets for disease marker discovery

Objective To propose a new approach to privacy preserving data selection, which helps the data users access human genomic datasets efficiently without undermining patients’ privacy.Methods Our idea is to let each data owner publish a set of differentially-private pilot data, on which a data user can test-run arbitrary association-test algorithms, including those not known to the data owner a priori. We developed a suite of new techniques, including a pilot-data generation approach that leverages the linkage disequilibrium in the human genome to preserve both the utility of the data and the privacy of the patients, and a utility evaluation method that helps the user assess the value of the real data from its pilot version with high confidence.Results We evaluated our approach on real human genomic data using four popular association tests. Our study shows that the proposed approach can help data users make the right choices in most cases.Conclusions Even though the pilot data cannot be directly used for scientific discovery, it provides a useful indication of which datasets are more likely to be useful to data users, who can therefore approach the appropriate data owners to gain access to the data.     

Association of <Emphasis Type="Italic">SLC22A4/5</Emphasis> Polymorphisms with Steroid Responsiveness of Inflammatory Bowel Disease in Japan

Purpose We investigated the association between steroid responsiveness and single nucleotide polymorphisms of SLC22A4/A5 located within inflammatory bowel disease 5 locus. Our goal is personalized steroid therapy adjusted to match individual variations in drug responsiveness in each inflammatory bowel disease patient. Methods Unrelated Japanese cohorts of 94 patients with Crohn’s, 94 patients with ulcerative colitis, and 257 healthy control subjects were consecutively enrolled in this study. Genotyping and haplotype analysis focusing on steroid responsiveness was performed by using 15 single nucleotide polymorphisms. Results The G allele of −368T > G in SLC22A5, in which strong linkage disequilibrium was observed and the limited diversity of three haplotypes was estimated, was significantly associated with steroid resistance in Japanese patients with Crohn’s disease (P = 0.016). Haplotype analysis between −446C > T and −368T > G in the SLC22A5 promoter region showed that the CG allele appeared to be a risk haplotype for steroid resistance (CG: odds ratio, 4.13; 95 percent confidence interval, 1.41–12.1; P = 0.016). Conclusions This extensive linkage disequilibrium may form a general risk haplotype for steroid resistance in Crohn’s disease in Japanese. Further analyses of the pharmacogenomics of steroid responsiveness are warranted to achieve the goal of individualized steroid therapy against inflammatory bowel disease. Supported by a grant-in-aid from the Ministry of Health, Labour and Welfare (K.I.), Japan. Address of correspondence: Yoshiaki Arimura, M.D., First Department of Internal Medicine, Sapporo Medical University, S-1, W-16, Chuo-ku, Sapporo, 060-8543, Japan. E-mail: arimura@sapmed.ac.jp     

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.     

细胞毒T淋巴细胞相关抗原4基因多态性与汉族溃疡性结肠炎患者的相关性研究

目的　研究细胞毒T淋巴细胞相关抗原4(CTLA- 4)基因外显子1的49位点A/G和启动子- 318位点C/T多态性与溃疡性结肠炎(UC)的相关性。方法　采用序列特异性引物聚合酶链反应(PCR -SSP)方法,检测82例中国湖北汉族溃疡性结肠炎患者(UC)以及204 例健康对照者CTLA- 4 基因外显子1的49位点A/G和启动子-318位点C/T的基因型和单倍型。结果　UC患者CTLA -4 A+49G和C- 318T基因型与正常对照组间差异无统计学意义(P>0.05),且与性别无关。在单倍型分析中,UC患者CTLA -4单倍型2,3(C-318 G49/T-318 A49)显著低于正常人群(26%比41%,P<0.05,OR=0.4918,95%CI:0.2784~0.8688)。结论　UC患者CTLA- 4 基因A+49G和C -318T单倍型2,3 与UC呈负相关。     

西藏阿里地区细粒棘球蚴人体分离株nad1基因多态性分析

目的了解西藏阿里地区细粒棘球蚴人体分离株的优势基因型及遗传变异情况,为细粒棘球绦虫的溯源及阿里地区棘球蚴病预防控制策略的制定提供支持。方法收集阿里地区某医院2017年棘球蚴病患者病灶切除样品,提取DNA,PCR扩增线粒体NADH脱氢酶1(nad1)基因,扩增产物测序后用BLAST、ClustalX 1.83和MEGA 7.0软件进行同源性和系统进化分析。下载全国细粒棘球蚴人体分离株的nad1基因,利用DnaSP6分析单倍型;利用NetWork软件制作中国细粒棘球绦虫nad1基因的单倍型网络图。结果共收集80例细粒棘球蚴病患者病灶切除样品,其中38份样品PCR扩增出约550 bp的特异性条带。测序分析结果显示,4份样品为细粒棘球绦虫G6基因型,与蒙古人来源的序列(MH300971.1)一致性为99.8%;其余34份样品均为细粒棘球绦虫G1基因型,与标准序列(AF297617.1)相比,其535位的C碱基突变为G碱基,与阿尔及利亚人来源序列(MG672293.1)的一致性为100%。MEGA 7.0软件分析38份样品的序列,结果显示,T、C、A和G碱基占比依次为44.3%~46.5%、7.7%~9.1%、19.8%~21.5%和25.7%~26.0%。我国已报道的细粒棘球绦虫nad1基因共有9个单倍型,单倍型多样性为0.47,核酸多样性为0.19。单倍型网络图显示,H4为我国细粒棘球绦虫nad1基因主要的单倍型。结论细粒棘球绦虫G1、G6基因型是西藏阿里地区的主要基因型,资料分析表明H4为我国流行的细粒棘球绦虫nad1基因的主要单倍型。     

ADAM33基因单核苷酸多态性及单体型与慢性阻塞性肺疾病的关联性研究

目的 探讨中国华北地区汉族人群ADAM33基因S1、S2位点单核苷酸多态性及单体型与慢性阻塞性肺疾病(COPD)及肺功能的关联性.方法 应用DNA直接测序的方法,对90例COPD患者和90名健康对照者的ADAM33基因S1、S2位点基因型进行检测;应用SHEsis在线软件构建单体型并进行单体型关联分析.结果 ①病例组和对照组中S1位点基因型及等位基因频率分布比较差异无统计学意义(P＞0.05),S2位点基因型及等位基因频率分布比较差异有统计学意义(P ＜0.05).②Logistic回归分析表明:ADAM33基因S1位点不同基因型COPD发生的相对危险度比较差异无统计学意义(P＞0.05);S2位点不同基因型COPD发生的相对危险度比较,差异有统计学意义(P＜0.05),其中G/G+C/G基因型的OR值为2.364(95％CI 1.251～4.466).③COPD病例组S2位点基因型与肺功能相关临床指标的关系显示:3种基因型FEV1％预计值比较差异无统计学意义而FEV1/FVC比较差异有统计学意义,其中G/G基因型与C/C、C/G基因型相比FEV1/FVC下降更明显.④SHEsis在线软件对S1、S2位点进行单体型分析结果显示,单体型CG在COPD组和对照组中比较差异有统计学意义(P＜0.05).结论 在中国华北地区汉族人群中,ADAM33基因与COPD的易感性有关,但与疾病的严重程度无关.     

白介素-1β基因多态性与晚期膝骨关节炎易感性的关联性研究

目的探讨IL-1β基因的单核苷酸多态性和单体型与中国汉族人晚期膝骨关节炎（KOA）的易感性相关。方法采用病例对照研究,纳入120例中国汉族膝OA患者和130例年龄、性别匹配的健康对照者,用酶联免疫吸附测定法（ELISA）测定血清中IL-1β的水平,用聚合酶链反应和限制性片段长度多态性（PCR—RFLP）方法对IL-1β基因的-511C／T（rs16944）、＋3954C／T（rs114363）和-31C／T（rs1143627）位点进行单核苷酸多态性进行分析,并测序验证酶切结果。结果膝骨性关节炎患者组血清中白细胞介素1β水平明显高于健康对照组（t=-8．26,P〈0．01）,单核苷酸多态性分析显示：在膝OA患者组和健康对照组之间IL-1β-31C／T位点基因型分布和等位基因频率没有明显差异,该研究中没有发现IL-1β＋3954Tr基因型。膝OA患中的IL-1β-511TC、IL-1β＋3954CT基因型频率较健康对照组增加（P〈0．05,P〈0．05）,通过Logistic回归分析,IL-1β-511TC、IL-1β＋3954CT基因型与膝OA高风险发病率具有相关性（IL-1β-511TC,OR=1．842,95％CI=1．021—3．327,P〈0．05;IL-1β＋3954CT,OR=2．372,95％CI=1．022—5．509,P〈0．05）。此外,单体型分析显示与单体型TCC相比,单体型TCT和CCC与膝OA高风险发病率具有更强的相关性（TCT．OR=3．24,95％CI：1．50—7．00,P〈0．01;CCC．OR=6．07,95％CI：2．20—16．07,P〈0．01）。结论白细胞介素-1β基因的-511C／T（rs16944）和＋3954C／T（rs114363）位点多态性与中国汉族人膝OA的易感性相关。     

Discrimination of haplotype in mitochondrial DNA mixtures using LNA-mediated PCR clamping

Locked nucleic acid (LNA) has been widely used for various genetic analyses, and has many benefits, in terms of the specificity or sensitivity of amplification, because LNA-containing primers/probes form more stable duplexes with template DNA than probes lacking LNA. Here, we developed a new method for discriminating HV1 haplotypes from mitochondrial DNA (mtDNA) mixtures by applying PCR clamping using LNA. PCR clamping is based on the selective inhibition of amplification using LNA-containing probes, which can discriminate single-nucleotide differences. Before designing probes, we selected 171 sequences with single-nucleotide variations from the HV1 region, and evaluated the specificity of LNA-containing probes for them by predicting Tm values. The differences of Tm between mismatched and exactly matched probe–template duplexes depended markedly on the type of LNA nucleotides for discriminating single-nucleotide differences, and the cytosine LNA nucleotide at the site of variations in the probes was most effective to discriminate these differences. For mixture analysis, each probe targeted one or two variations (16209C, 16217C, 16257A/16261T, 16297C/16298C, 16304C, 16362C, or 16362T) that are particularly common in the Japanese population, and seven designed probes completely inhibited the amplification of exactly matched templates. We prepared mixed samples by mixing DNA from two individuals at a ratio of 1:9, 1:4, 1:1, 4:1, or 9:1, and then performed Sanger sequencing analysis after PCR clamping with each probe. Our method distinguished each haplotype at lower ratios from two-person mixtures, and enabled sensitive detection at 12 pg of total DNA including 600 copies of mtDNA. Moreover, we analyzed three-person mixtures with representative sequences, and detected the minor haplotype of one individual present at a rate of 10% by adding two selected probes. The ability to discriminate haplotypes in mixed samples by using LNA-mediated PCR clamping indicates the potential value of mtDNA analysis in criminal investigations.