耳聋相关基因COCH的非同义单核苷酸多态性致聋表型预测

群体凝血因子C同源物基因(Coagulation factor C homology,COCH)是人类发现的第一个伴前庭功能障碍的耳聋基因,位于人类染色体14q12-q13上。迄今,在COCH基因上发现16个位点突变导致常染色体显性遗传非综合征型耳聋DFNA9的发生,其中包括13个非同义单核苷酸多态性(Non-synonymous single nucleotide polymorphisms,nsSNPs)位点。由于该基因其他nsSNPs的基因型与表型关系尚不清楚,因此文章采用生物信息学方法,从COCH基因全部的SNPs中分级筛选,结合已知的致病nsSNPs信息及蛋白三维结构验证,首次预测出由COCH基因编码的cochlin蛋白的vWFA (Von Willebrand factor type A domain)区的8个高风险致病性nsSNPs(I176T、R180Q、G265E、V269L、I368N、I372T、R416C和Y424D)。同时,对位于LCCL (Limulus factor C, cochlin, and late gestation lung protein Lgl1)区域的6个已知致病突变的nsSNPs ( P51S、G87W、I109N、I109T、W117R和F121S)进行了三维结构模拟,发现突变体均发生了环状结构或链状结构的改变。本研究对COCH基因的基因型与表型的相关性研究为遗传性耳聋筛查提供了相应的理论依据,也对该基因所编码的cochlin蛋白的功能研究具有一定的指导意义。     

特发性少精子症和无精子症与Pygo2基因蛋白编码区SNPs的相关性

男性不育常伴随精子数量减少。Pygo2基因在染色质重塑的伸长精细胞中表达, 其功能受损会导致精子形成阻滞和精子生成减少而引发不育。文章旨在检测引起人特发性少精子症和无精子症的Pygo2基因突变。从77例正常生育力男性和195例特发性少精子症和无精子症患者静脉血提取DNA, 采用聚合酶链式反应-测序方法对Pygo2基因3个蛋白质编码区进行测序对比, 非同义单核苷酸多态性(Single nucleotide polymorphisms, SNPs)位点分别用SIFT、Polyphen-2和 Mutation Taster软件进行诱发蛋白质结构和表型改变的检测和分析。结果表明, 195例患者中, 178例(30例轻度或中度少精子症, 57例重度少精子症和91例无精子症)基因序列分析报告完好, 无精子症中3例患者分别在2个位点(rs61758740, rs141722381)发生了非同义突变SNPs, 重度少精子症中1例患者在位点rs61758741发生了非同义突变, 3个突变位点在SNPs基因数据库都已有报道, 轻度或中度少精子症患者以及正常生育力男性中不存在SNPs。rs61758740可使PYGO2蛋白第141位蛋氨酸(M)变为异亮氨酸(I), rs61758741使PYGO2蛋白第261位碱性赖氨酸(K)变为酸性谷氨酸(E), rs141722381使PYGO2蛋白第240位亲水侧链天冬酰胺(N)变为疏水侧链异亮氨酸(I)。软件分析表明, 在所发现的3个SNP非同义突变位点中, rs141722381引起的单个氨基酸改变会导致PYGO2蛋白空间结构破坏和诱发相关疾病。因此, Pygo2基因蛋白质编码序列区SNPs可能是特发性少精子症和无精子症的诱发因素之一, 导致男性不育。     

牙鲆MyoD基因单核苷酸多态性位点的筛选

运用PCR-SSCP技术研究100尾牙鲆(Paralichthys olivaceus)MyoD基因的单核苷酸多态性(Single nucleotide polymorphisms, SNPs), 并将筛选到的突变位点与牙鲆生长性状进行相关性分析。结果表明, 在外显子1和内含子1上存在3个SNPs, 在外显子1 (MyoD2)基因座发现3种基因型AA、AB和BB, G863A突变, 属于同义突变。在内含子MyoD4基因座检测到DD、FF、CD、CE、DE和DF型个体。利用最小二乘法研究MyoD基因多态性位点对牙鲆生长性状的影响。结果表明, 外显子1的SNPs对生长性状无显著影响(P0.05)。内含子1的SNPs对牙鲆的生长性状影响均显著(P0.05)。研究结果为SNPs位点与牙鲆生长性能关联分析奠定了基础。       

6个SNP位点与环境的交互作用对苗族高血压易感性的影响分析

本研究旨在探讨基因单核苷酸多态性(single nucleotide polymorphisms)与传统危险因素相互作用对苗族高血压发病的影响。选取212例苗族人群作为研究对象,对6个SNP位点(ALDH2-rs671,ATP2B1(rs17249754,rs2681472),CASZ1-rs12046278,MTHFR-rs17367504,ZNF652-rs16948048)进行Snapshot基因分型,并应用逻辑回归分析基因与环境的交互作用。研究结果显示:(1)6个SNPs的基因型分布均符合Hardy-Wenberg遗传平衡定律(p0.05),在高血压实验组与对照组的比较中,6个SNP位点多态性基因频率分布差异无统计学意义;(2)二元逻辑回归分析显示BMI、TG、LDL、血脂异常和年龄可能为苗族高血压患病的危险因素;(3)将潜在危险因素带入逻辑回归模型中,分析发现肥胖与ZNF652-rs16948048、MTHFR-rs173-67504基因位点多态性的交互作用与苗族EH发病存在关联性。综上所述,传统危险因素-肥胖与ZNF652-rs16948048、MTHFR-rs17367504基因多态性的交互作用可能对苗族高血压的发生有一定影响。     

MicroRNA基因及其靶位点的单核苷酸多态性与疾病易感性

微小RNAs(MicroRNAs)是一类内源性19～25个核苷酸大小的非编码RNA分子,能够通过碱基匹配原则识别并结合于靶基因3'非翻译区的靶位点,从而抑制靶基因的翻译和/或促进靶基因降解。近年许多研究表明,单核苷酸多态性(single nucleotide polymorphisms,SNPs)可影响m icroRNA对靶基因的调控过程。SNPs可发生在m icroRNA基因(指在pri-,pre-and mature-miRNA序列中),也可发生在靶基因的3'非翻译区的靶位点。这些SNPs通过影响microRNA对靶基因的调控过程,参与许多疾病如肿瘤、神经系统疾病、肌肥大、心血管疾病以及2型糖尿病的发生发展过程。本文拟对MicroRNAs及其靶mRNA的结合位点SNPs与疾病的相关研究做一综述。     

ERCC5基因miRNA结合部位多态性rs4150390与乳腺癌的易感性分析

本研究旨在探讨切除修复交叉互补基因5(complementation group 5,ERCC5)基因miRNAs结合位点的单核苷酸多态性(single nucleotide polymorphisms,SNPs)与甘肃地区女性乳腺癌易感性间的关系。以甘肃省散发乳腺癌病例101人和乳腺纤维腺瘤病例101人做研究对象;通过在线软件预测的方法,筛选出ERCC5基因rs4150390 SNP(GA)为研究位点,利用PCR-RFLP方法进行基因组DNA rs4150390位点SNP分型分析。研究结果显示rs4150390 A/G基因SNP与甘肃地区女性乳腺癌易感性无相关性,并且和肿瘤组织ER、PR、Her-2、P53的表达也无相关性。但当乳腺癌患者携带rs4150390突变型基因A/A时与肿瘤组织Ki67阴性表达相关(odds ratio,4.947;95%confidence interval,3.311~7.393;p=0.046)。综上所述rs4150390 A/G基因多态性与甘肃地区女性乳腺癌易感性无相关性。     

与疾病相关的非同义单核苷酸多态性预测的研究进展

单核苷酸多态性(single nucleotide polymorphism,SNPs),即在基因组水平上由单个核苷酸的变异而引起的DNA序列多态性变化,具体是指在DNA序列中的单个碱基的变异,其是人类基因组变异种最常见的一种。SNP研究最主要的目的就是对人类表型变异遗传学的理解,尤其是关于人类遗传疾病的研究。而非同义单核苷酸多态性(nsSNPs)是SNPs中的一种,主要是指处于编码区会引起翻译后对应氨基酸序列变化的单核苷酸突变。因为nsSNPs可能会对蛋白质的功能造成影响,被认为是造成人类遗传病的主要原因。因此将与疾病相关的nsSNPs从中性的nsSNPs中区分出来是很重要的。本文根据国内外与疾病相关nsSNPs预测的研究,分析了预测中所涉及到的特征属性,总结了对这些特征进行优化的特征选择方法,并概述了在预测过程中使用的各种分类器。     

癫痫相关基因SCN1A启动子区多态性位点的生物信息学分析

SCN1A是多种神经系统疾病的致病基因,该基因的精确表达对于维持神经系统正常功能非常重要.为了认识SCN1A启动子区多态性位点(single nucleotide polymorphisms,SNPs)的保守性及其功能意义,应用生物信息学方法分析了目前已发表位于SCN1A启动子区的11个SNPs,分别命名S1～S11.分析结果表明,S3、S5和S7等位基因频率没有人种差异性,其余SNP等位基因频率均有人种差异性;接近核心启动子的SNPs要比远离核心启动子SNPs的保守程度高,提示靠近核心启动子的SNPs影响SCN1A基因表达的概率可能越大;大部分SNPs祖传等位基因在哺乳动物中是保守的(S3除外),暗示这些SNPs新生等位基因有可能在人类进化过程起到一定的作用:启动子分析软件预测发现,含S2、S4、S8及S9等4个SNPs不同等位基因的同一序列分别存在不同转录因子结合元件,而含S1和S11不同等位基因的同一序列都只能预测到含其中一个等位基因的序列存在转录因子结合元件,这些差异可能是SNPs影响SCN1A表达的重要原因之一.这些分析将为进一步研究SCN1A启动子区SNPs与神经系统疾病的相互关系打下基础.     

Ex-FABP 作为鸡腹脂性状主要候选基因的研究

细胞外脂肪酸结合蛋白 (extracellular fatty acid-binding protein, Ex-FABP) 基因是脂肪酸结合蛋白 (fatty acid-binding protein, FABP) 家族的另一成员,参与鸡的脂肪酸、肌纤维、骨骼等调控过程 . 利用 PCR-SSCP (single strand conformation polymorphism) 和 DNA 测序的方法,对不同品种的杂交鸡进行了 Ex-FABP 基因 5′调控区部分序列的多态性分析,发现了 3 个单核苷酸多态性 (single nucleotide polymorphisms, SNPs) 位点 . 其中,此片段中的碱基 C (－1000) 的插入和碱基 T → C (－1011) 的突变,导致此处比野生型基因少了 1 个 cap ,多了 1 个 Nkx-2 、 1 个 AhR/Ar 和 2 个 CF1 等转录因子结合位点的产生 . 利用 SAS 软件对 Ex-FABP 基因 5′调控区的 SNPs 与屠体性状的最小二乘分析,发现基因型 BB (突变型) 与腹脂重存在极显著相关 (P<0.01). 研究结果表明,细胞外脂肪酸结合蛋白 (Ex-FABP) 基因是影响脂肪沉积、控制腹脂性状的主要候选基因 .     

DNA 池结合DHPLC 和直接测序技术在江豚SNPs 检测中的应用

选取江豚基因组中的2 个已知单核苷酸多态性(single nucleotide polymorphisms,SNPs)位点,通过PCR 扩增,将PCR 产物按基因频率不同制备成0 ～ 50% 的11 个DNA 池(DNA pool),用于变性高效液相色谱(denaturing high performance liquid chromatography,DHPLC)和直接测序分析,以探讨DNA 池中基因频率的最低要求。结果显示,当稀有等位基因的基因频率不少于5% 时可在DHPLC 检测过程中明显分辨;而利用DNA 池进行直接测序时的基因频率则需达到10% 。这提示,为保证DHPLC 分析的准确性和可靠性,制备DNA 池时等摩尔DNA 混合的个体数最好不超过10 个。DNA 池结合DHPLC 技术的高效性与准确性可在大规模的SNPs 位点筛选中发挥作用。     

Exploration of deleterious single nucleotide polymorphisms in late-onset Alzheimer disease susceptibility genes

Non-synonymous single nucleotide polymorphisms (nsSNPs) are considered as biomarkers to disease susceptibility. In the present study, nsSNPs in CLU, PICALM and BIN1 genes were screened for their functional impact on concerned proteins and their plausible role in Alzheimer disease (AD) susceptibility. Initially, SNPs were retrieved from dbSNP database, followed by identification of potentially deleterious nsSNPs and prediction of their effect on proteins by PolyPhen and SIFT. Protein stability and the probability of mutation occurrence were predicted using I-Mutant and PANTHER respectively. SNPs3D and FASTSNP were used for the functional analysis of nsSNPs. The functional impact on the 3D structure of proteins was evaluated by SWISSPDB viewer and NOMAD-Ref server. On analysis, 3 nsSNPs with IDs rs12800974 (T158P) of PICALM and rs11554585 (R397C) and rs11554585 (N106D) of BIN1 were predicted to be functionally significant with higher scores of I-Mutant, SIFT, PolyPhen, PANTHER, FASTSNP and SNPs3D. The mutant models of these nsSNPs also showed very high energies and RMSD values compared to their native structures. Current study proposes that the three nsSNPs identified in this study constitute a unique resource of potential genetic factors for AD susceptibility.     

Screening and structural evaluation of deleterious Non-Synonymous SNPs of ePHA2 gene involved in susceptibility to cataract formation

Age-related cataract is clinically and genetically heterogeneous disorder affecting the ocular lens, and the leading cause of vision loss and blindness worldwide. Here we screened nonsynonymous single nucleotide polymorphisms (nsSNPs) of a novel gene, EPHA2 responsible for age related cataracts. The SNPs were retrieved from dbSNP. Using I-Mutant, protein stability change was calculated. The potentially functional nsSNPs and their effect on protein was predicted by PolyPhen and SIFT respectively. FASTSNP was used for functional analysis and estimation of risk score. The functional impact on the EPHA2 protein was evaluated by using SWISSPDB viewer and NOMAD-Ref server. Our analysis revealed 16 SNPs as nonsynonymous out of which 6 nsSNPs, namely rs11543934, rs2291806, rs1058371, rs1058370, rs79100278 and rs113882203 were found to be least stable by I-Mutant 2.0 with DDG value of > -1.0. nsSNPs, namely rs35903225, rs2291806, rs1058372, rs1058370, rs79100278 and rs113882203 showed a highly deleterious tolerance index score of 0.00 by SIFT server. Four nsSNPs namely rs11543934, rs2291806, rs1058370 and rs113882203 were found to be probably damaging with PSIC score of ≥ 2. 0 by Polyp hen server. Three nsSNPs namely, rs11543934, rs2291806 and rs1058370 were found to be highly polymorphic with a risk score of 3-4 with a possible effect of Non-conservative change and splicing regulation by FASTSNP. The total energy and RMSD value was higher for the mutant-type structure compared to the native type structure. We concluded that the nsSNP namely rs2291806 as the potential functional polymorphic that is likely to have functional impact on the EPHA2 gene.     

Functional genomics based prioritization of potential nsSNPs in EPHX1, GSTT1, GSTM1 and GSTP1 genes for breast cancer susceptibility studies

In the present study, nsSNPs in EPHX1, GSTT1, GSTM1 and GSTP1 genes were screened for their functional impact on concerned proteins and their plausible role in breast cancer susceptibility. Initially, SNPs were retrieved from dbSNP, followed by identification of potentially deleterious nsSNPs using PolyPhen and SIFT. Functional analysis was done with SNPs3D, SNPs&GO and MutPred methods. Prediction and evaluation of the functional impact on the 3D structure of proteins were performed with Swiss PDB viewer and NOMAD-Ref servers. On analysis, 13 nsSNPs were found to be highly deleterious and damaging to the protein structure, of which 6 nsSNPs, rs45549733, rs45506591 and rs4986949 of GSTP1, rs72549341 and rs148240980 of EPHX1 and rs17856199 of GSTT1 were predicted to be potentially polymorphic. It is therefore hypothesized that the 6 identified nsSNPs may alter the detoxification process and elevate carcinogenic metabolite accumulation thus modifies the risk of breast cancer susceptibility in a group of women.     

Structure analysis of deleterious nsSNPs in human PALB2 protein for functional inference

Partner and Localizer of BRCA2 or PALB2 is a typical tumor suppressor protein, that responds to DNA double stranded breaks through homologous recombination repair. Heterozygous mutations in PALB2 are known to contribute to the susceptibility of breast and ovarian cancer. However, there is no comprehensive study characterizing the structural and functional impacts of SNPs located in the PALB2 gene. Therefore, it is of interest to document a comprehensive analysis of coding and non-coding SNPs located at the PALB2 loci using in silico tools. The data for 1455 non-synonymous SNPs (nsSNPs) located in the PALB2 loci were retrieved from the dbSNP database. Comprehensive characterization of the SNPs using a combination of in silico tools such as SIFT, PROVEAN, PolyPhen, PANTHER, PhD-SNP, Pmut, MutPred 2.0 and SNAP-2, identified 28 functionally important SNPs. Among these, 16 nsSNPs were further selected for structural analysis using conservation profile and protein stability. The most deleterious nsSNPs were documented within the WD40 domain of PALB2. A general outline of the structural consequences of each variant was developed using the HOPE project data. These 16 mutant structures were further modelled using SWISS Model and three most damaging mutant models (rs78179744, rs180177123 and rs45525135) were identified. The non-coding SNPs in the 3'' UTR region of the PALB2 gene were analyzed for altered miRNA target sites. The comprehensive characterization of the coding and non-coding SNPs in the PALB2 locus has provided a list of damaging SNPs with potential disease association. Further validation through genetic association study will reveal their clinical significance.     

Computational and Structural Investigation of Deleterious Functional SNPs in Breast Cancer BRCA2 Gene

In this work, we have analyzed the genetic variation that can alter the expression and the function in BRCA2 gene using computational methods. Out of the total 534 SNPs, 101 were found to be non synonymous (nsSNPs). Among the 7 SNPs in the untranslated region, 3 SNPs were found in 5′ and 4 SNPs were found in 3′ un-translated regions (UTR). Of the nsSNPs 20.7% were found to be damaging by both SIFT and PolyPhen server among the 101 nsSNPs investigated. UTR resource tool suggested that 2 SNPs in the 5′ UTR region and 4 SNPs in the 3′ UTR regions might change the protein expression levels. The mutation from asparagine to isoleucine at the position 3124 of the native protein of BRCA2 gene was most deleterious by both SIFT and PolyPhen servers. A structural analysis of this mutated protein and the native protein was made which had an RMSD value of 0.301 nm. Based on this work, we proposed that this most deleterious nsSNP with an SNPid rs28897759 is an important candidate for the cause of breast cancer by BRCA2 gene.     

Targeted genome-wide investigation identifies novel SNPs associated with diabetic nephropathy

Loci contributing to complex disease have been identified by focusing on genome-wide scans utilising non-synonymous single nucleotide polymorphisms (nsSNPs). We employed Illumina's HNS12 BeadChip (13,917 high-value SNPs) which was specifically designed to capture nsSNPs and ideally complements more dense genome-wide association studies that fail to consider many of these putatively functional variants. The HNS12 panel also includes 870 tag SNPs covering the major histocompatibility region. All individuals genotyped in this study were Caucasians with (cases) and without (controls) diabetic nephropathy. About 449 individuals with type 2 diabetes (203 cases, 246 controls) were genotyped in the initial study. 1,467 individuals with type 1 diabetes (718 cases, 749 controls) were genotyped in the follow up study. 11,152 SNPs were successfully analysed and ranked for association with diabetic nephropathy based on significance (P) values. The top ranked 32 SNPs were subsequently genotyped using MassARRAY iPLEX(?) and TaqMan technologies to investigate association of these polymorphisms with nephropathy in individuals with type 1 diabetes. The top ranked nsSNP, rs1543547 (P?=?10(-5)), is located in RAET1L, a major histocompatibility class I-related gene at 6q25.1. Of particular interest, multiple nsSNPs within the top ranked (0.2%) SNPs are within several plausible candidate genes for nephropathy on 3q21.3 and 6p21.3.     

Next generation sequencing exome data analysis aids in the discovery of SNP and INDEL patterns in Parkinson's disease

Whole exome sequencing is an adept method to reveal novel and disease-related SNPs and INDELs as it screen the actionable areas of the genome. We evaluated the exome sequenced datasets of patients with Parkinson's disease (PD) in South African ethnic origin. The primary focus of this study was to discover the SNPs and INDELs patterns responsible for PD. The variant discovery was performed with genome analysis tool kit best practices variant detection pipelines. The SNPs were linked to the genes and categorized based on the filter-based annotation from ANNOVAR. We identified a total of 7955 SNPs and 9952 INDELs in all seven datasets together. A total of 130 missense nsSNPs were prioritized based on its damaging effect predicted from SIFT and Polyphen2 annotation. We noticed a novel nsSNP rs111655870 in gene LRRK2 that shows the mutation of a Leucine to Phenylalanine at position 208 which can alter the protein function. The study also filtered seven nsSNPs in genes NAGA, SULT4A1, MYH8, FLNA, TPM3, ATP13A1, CLN8 that have potentially deleterious effects predicted by various computational tools. This analysis suggested that the above filtered nsSNPs and INDELs have a functional impact and provide the footing for genetic studies related to PD. Further screening of these variations provides deeper insight for molecular mechanism of disease progression.     

The ApoE gene of Alzheimer's disease (AD)

The ApoE gene responsible for the Alzheimer's disease has been examined to identify functional consequences of single-nucleotide polymorphisms (SNPs). Eighty-eight SNPs have been identified in the ApoE gene in which 31 are found to be nonsynonymous, 8 of them are coding synonymous, 33 are found to be in intron, and 3 are in untranslated region. The SNPs found in the untranslated region consisted of two SNPs from 5′ and one SNP from the 3′. Twenty-nine percent of the identified nsSNPs have been reported as damaging. In the analysis of SNPs in the UTR regions, it has been recognized that rs72654467 from 5′ and rs71673244 from 5′ and 3′ are responsible for the alteration in levels of expression. Both native and mutant protein structures were analyzed along with the stabilization residues. It has been concluded that among all SNPs of ApoE, the mutation in rs11542041 (R132S) has the most significant effect on functional variation.     

Computational detection of deleterious SNPs and their effect on sequence and structural level of the <Emphasis Type="Italic">VHL</Emphasis> gene

In this work we have analyzed the genetic variation that can alter the expression and the function of the VHL gene using computational methods. Of 110 single nucleotide polymorphisms (SNPs), 33 were found to be nonsynonymous (nsSNPs) and 23 SNPs were found in untranslated regions. Of the 33 nsSNPs investigated, 36.3% were found to be deleterious by both SIFT and PolyPhen servers. An untranslated region (UTR) resource tool suggested that two SNPs in the 5' UTR region and six SNPs in the 3' UTR region might change the protein expression levels. It was found by both SIFT and PolyPhen servers that a mutation from histidine to arginine at position 115 of the native protein of the VHL gene was most deleterious. A structural analysis of this mutated protein and the native protein was performed and had a root mean square deviation (RMSD) of 2.78 A. Based on this work, we propose that the nsSNP with a SNPid of rs5030812 is an important candidate for the cause of von Hippel-Lindau syndrome via the VHL gene.     

In silico analysis of structural and functional consequences in p16INK4A by deleterious nsSNPs associated CDKN2A gene in malignant melanoma

In this study, we identified the most deleterious nsSNP in CDKN2A gene through structural and functional properties of its protein (p16INK4A) and investigated its binding affinity with cdk6. Out of 118 SNPs, 14 are nsSNPs in the coding region and 17 SNPs were found in the untranslated region (UTR). FastSNP suggested that 7 SNPs in the 5' UTR might change the protein expression levels. Sixty-four percent of nsSNPs are found to be damaged in PolyPhen server among the 14 nsSNPs investigated. With this effort, we modeled the mutant p16INK4A proteins based on these deleterious nsSNPs, out of which three nsSNPs associated p16INK4A had RMSD values of greater than 3.00 A with native protein. From a comparison of total energy of these three mutant proteins, we identified that the major mutation is from Aspartic acid to Tyrosine at the residue position of 84 of p16INK4A. Further, we compared the binding efficiency of both native and mutant p16INK4A with cdk6. We found that mutant p16INK4A has less binding affinity with cdk6 compared to native type. This is due to ten hydrogen bonds and eight salt bridges which exist between the native type and cdk6, whereas the mutant type makes only nine hydrogen bonds and five salt bridges with cdk6. Based on our investigation, we propose that the SNP with the ID rs11552822 could be the most deleterious nsSNP in CDKN2A gene, causing malignant melanoma, as it was well correlated with experimental studies carried out elsewhere.