一成骨不全家系的COL141基因突变检测

成骨不全(Osteogenesis imperfecta,OI)是一种由于Ⅰ型胶原形成障碍,导致骨脆性增强为主要症状的常染色体显性遗传性疾病。临床上主要表现为骨质脆弱、蓝巩膜、耳聋和中等程度的关节畸形等症状。成骨不全基因分别定位于17q21．31-q22和7q22．1,其致病基因分别为COL1A1和COL1A2。对一常染色体显性遗传的成骨不全家系进行连锁分析,在COL1A1遗传位点发现紧密连锁(LOD=9．31;θ=.00)。突变检测发现在COL1A1基因第26内含子5′端剪接位点处存在一由GT转换为AT的致病突变,该突变引起的异常剪接是导致成骨不全的致病原因之一。     

成骨不全Ⅰ型家系的基因检测和COL1A2基因新突变的致病性鉴定

为了揭示成骨不全(Osteogenesis imperfecta,OI)Ⅰ型家系的分子遗传学发生机制,文章采用PCR-DNA直接测序法,对患儿COL1A1和COL1A2基因共103个外显子(E)进行突变检测。结果显示:患儿COL1A1基因未发现任何病理性突变,而在COL1A2基因E19内发现一新的杂合错义突变(p.G316C),该突变来自其父,而其母正常,其他表型正常的6位亲属也均未发现该突变;通过DHPLC(Denaturing high performance uid chromatography)筛检,发现患儿与其父均有异常双峰,而其母和所有正常对照均为正常单峰;通过ASA(Allele specific amplification)筛检,患儿与其父均有391 bp的特异扩增带,而其母和所有正常对照均未见特异扩增带;保守性分析结果显示,该突变位点所在甘氨酸在进化上具有高度保守性;SIFT和Poly Phen-2软件预测结果显示,新突变造成的结果是"有害的"和"很可能有害"。上述结果均说明COL1A2基因c.946GT/p.G316C新突变是导致OI-Ⅰ型的致病性突变,是引起患儿发病的真正内因。患儿父母若再次孕育,可在孕早期进行产前基因诊断或孕前期进行PGD(Preimplantation genetic diagnosis)予以防患。     

一性连锁Alport综合征中国家系COL4A5基因新突变位点的检测

通过PCR和直接测序的方法,对一性连锁Alport综合征家系17个受检个体的COL4A5基因所有51个外显子及其相邻内含子的DNA序列进行检测。结果发现,在第26外显子2240位点,男患者存在C碱基缺失(2240delc),女患者存在杂合缺失,同时对女患者相应的PCR产物进行克隆和测序以验证PCR测序结果的可靠性,而在正常家系成员和80例对照中均未发现此位点异常,说明2240delc为引起该家系临床病变的突变位点,不是多态性位点。在性连锁Alport综合征中,COL4A5基因的这个单碱基缺失突变位点为首次报道。     

一个Ⅰ型神经纤维瘤家系的基因突变分析

鉴定了一个中国家庭中的常染色体显性遗传病-Ⅰ型神经纤维瘤, 通过连锁分析和NF1基因测序, 发现该家系中NF1疾病的致病基因与NF1基因连锁, 并在NF1基因上发现了一个无义突变G1336X, 该突变导致神经纤维蛋白从C末端截断1 483个氨基酸残基。G1336X突变在该家系中与疾病共分离, 但家系中的正常成员未能检出, 表明NF1基因的G1336X的突变是引起该家族患NF1疾病的原因。该突变是第一次在中国NF1疾病人群中报道。     

一中国脑海绵状血管瘤家系中发现krit1基因新的缺失突变

脑海绵状血管瘤（CCM）是多定位于中枢神经系统的一种脑部血管异常,少数在皮肤和视网膜处有并发症。依据致病基因在染色体上的不同位置分为CCM1、CCM2和CCM33种类型。目前,CCM1、CCM2和CCM3的致病基因已经被克隆,分别为krit1、MGC4607和细胞程序性死亡10基因（PDCD10）。利用连锁分析发现内蒙古的一个家系属于CCM1,突变检测发现患者CCM1基因（krit1）第9内含子和第10外显子拼接位点处存在一“GTA”缺失,该突变导致终止密码子提前出现,产生截短蛋白。实验结果支持krit1为CCM1致病基因。     

在两个X连锁显性腓骨肌萎缩症家系中发现同一GJB1基因突变Glu208Lys

在两个X连锁显性腓骨肌萎缩症(Charcot-Marie-Tooth disease, CMT) 家系中进行了GJB1基因的突变分析。提取基因组DNA, PCR(polymerase chain reaction)反应扩增GJB1基因编码序列, 进行单链构象多态性(single strand conformational polymorphism, SSCP)分析, 对有差异SSCP带型的PCR产物进行测序, 结果在两家系中发现同一GJB1基因c.622G→A (Glu208Lys)突变。所发现的突变位点在国内尚未报道。     

与内含子相邻的核苷酸是tRNA内切酶的识别特征

含有内含子的tRNA前体必须经过剪接反应加工成熟.顺序比较指出与内含子顺序相邻的核苷酸有一定的特异性.用寡核苷酸定点突变技术改变这2个位点的核苷酸,确定这些tRNA前体的剪接效率.结果如下:当37位和38位都是嘌呤核苷酸时,tRNA内切酶能够有效地酶切酵母 tRNA~(phe)前体;如果其中的 1个位点变成嘧啶核苷酸,但另1个位点的核苷酸是野生型的嘌呤核苷酸,tRNA前体的酶切效率将降低.如果2个位点的核苷酸都发生变异,其中1个是嘧啶核苷酸,另1个是变异的嘌呤核苷酸,tRNA前体的酶切效率就会进一步降低.如果2个位点都是嘧啶核苷酸,tRNA前体就难以为tRNA内切酶酶切了.由此提出,与内含子相邻的核苷酸也是tRNA由切酶识别的结构特征.tRNA前体的37位和38位核苷酸的改变可能影响剪接位点之间的距离或它们的精细结构,从而影响tRNA内切酶酶切的效率.     

一中国正常血钾周期性麻痹家系中的SCN4A基因的T704M突变

家族性周期性麻痹(periodic paralysis,PP)是以反复发作骨骼肌迟缓性瘫痪为特征的一组疾病。我们对一个来自湖北省的正常血钾周期性麻痹家系进行致病基因突变检测。应用微卫星标记对该家系进行连锁分析,证实致病基因可能同SCN4A连锁。对SCN4A基因全部外显子测序,发现患者在一个致病突变热点存在碱基替换C2188T。该突变导致编码氨基酸改变Thr704Met,经单链构象多态性分析证明该突变只存在于该家系患者,不存在于家系中健康人和100名无亲缘关系对照中,Thr704Met是该家系的致病突变。     

一种新的非翻译区剪接位点识别方法

为提高非翻译区剪接位点识别的精度,提出一种统计概率与支持向量机相结合的识别方法 .该方法主要分为两个阶段,第一阶段应用统计学方法对非翻译区(UTR)序列进行描述,将序列中各碱基之间的相关性、位置特异性、保守性等特征用概率形式描述,以概率参数作为第二阶段支持向量机的输入向量,第二阶段应用带有多项式核函数的支持向量机(SVM)对剪接位点进行识别.通过对人类5′UTR剪接位点数据集进行测试,结果表明:该方法对非翻译区剪接位点的识别取得了很好的效果.     

脊髓性肌萎缩症SMN1基因2+0基因型携带者的家系研究

脊髓性肌萎缩症(spinal muscular atrophy, SMA)是一种儿童时期较为常见的神经肌肉病,属于常染色体隐性遗传。绝大多数SMA由运动神经元存活基因1 (survival motor neuron 1, SMN1)的纯合缺失突变所致。而SMN1的2+0基因型个体作为一种特殊的SMA携带者,给携带者筛查以及家系的遗传咨询带来了巨大的挑战。已有研究表明,g.27134T>G和g.27706₂7707delAT多态位点变异对于Ashkenazi犹太人群中的2+0基因型个体具有提示作用。为进一步探究这两个多态位点是否在中国人群也具有特异性,本研究纳入了44例家系成员和204例已知SMN1基因拷贝数的对照样本。44例家系成员来自于9个无关的SMN1基因纯合缺失的SMA家系,先证者双亲之一疑似为2+0基因型携带者。利用多重连接探针扩增(multiplex ligation-dependent probe amplification,MLPA)和短串联重复(short tandem repeat, STR)连锁分析进行基因型的鉴定以及多态位点的筛查,最终...     

A Missense Mutation in the SERPINH1 Gene in Dachshunds with Osteogenesis Imperfecta

Osteogenesis imperfecta (OI) is a hereditary disease occurring in humans and dogs. It is characterized by extremely fragile bones and teeth. Most human and some canine OI cases are caused by mutations in the COL1A1 and COL1A2 genes encoding the subunits of collagen I. Recently, mutations in the CRTAP and LEPRE1 genes were found to cause some rare forms of human OI. Many OI cases exist where the causative mutation has not yet been found. We investigated Dachshunds with an autosomal recessive form of OI. Genotyping only five affected dogs on the 50 k canine SNP chip allowed us to localize the causative mutation to a 5.82 Mb interval on chromosome 21 by homozygosity mapping. Haplotype analysis of five additional carriers narrowed the interval further down to 4.74 Mb. The SERPINH1 gene is located within this interval and encodes an essential chaperone involved in the correct folding of the collagen triple helix. Therefore, we considered SERPINH1 a positional and functional candidate gene and performed mutation analysis in affected and control Dachshunds. A missense mutation (c.977C>T, p.L326P) located in an evolutionary conserved domain was perfectly associated with the OI phenotype. We thus have identified a candidate causative mutation for OI in Dachshunds and identified a fifth OI gene.     

Identification of a novel COL1A1 frameshift mutation, c.700delG,in a Chinese osteogenesis imperfecta family

Osteogenesis imperfecta (OI) is a family of genetic disorders associated with bone loss and fragility. Mutations associated with OI have been found in genes encoding the type I collagen chains. People with OI type I often produce insufficient α1-chain type I collagen because of frameshift, nonsense, or splice site mutations in COL1A1 or COL1A2. This report is of a Chinese daughter and mother who had both experienced two bone fractures. Because skeletal fragility is predominantly inherited, we focused on identifying mutations in COL1A1 and COL1A2 genes. A novel mutation in COL1A1, c.700delG, was detected by genomic DNA sequencing in the mother and daughter, but not in their relatives. The identification of this mutation led to the conclusion that they were affected by mild OI type I. Open reading frame analysis indicated that this frameshift mutation would truncate α1-chain type I collagen at residue p263 (p.E234KfsX264), while the wild-type protein would contain 1,464 residues. The clinical data were consistent with the patients’ diagnosis of mild OI type I caused by haploinsufficiency of α1-chain type I collagen. Combined with previous reports, identification of the novel mutation COL1A1-c.700delG in these patients suggests that additional genetic and environmental factors may influence the severity of OI.     

Identification and molecular characterization of two novel mutations in COL1A2 in two Chinese families with osteogenesis imperfecta

Osteogenesis imperfecta (OI, also known as brittle bone disease) is caused mostly by mutations in two type Ⅰ collagen genes, COL1A1 and COL1A2 encoding the pro-α1 (Ⅰ) and pro-α2 (Ⅰ) chains of type Ⅰ collagen, respectively. Two Chinese families with autosomal dominant OI were identified and characterized. Linkage analysis revealed linkage of both families to COL1A2 on chromosome 7q21.3-q22.1. Mutational analysis was carried out using direct DNA sequence analysis. Two novel missense mutations, c.3350A＞G and c.3305G＞C, were identified in exon 49 of COL1A2 in the two families, respectively. The c.3305G＞C mutation resulted in substitution of a glycine residue (G) by an alanine residue (A) at codon 1102 (p.G1102A), which was found to be mutated into serine (S), argine (R), aspartic acid (D), or valine (V) in other families. The c.3350A＞G variant may be a de novo mutation resulting in p.Y1117C. Both mutations co-segregated with OI in respective families, and were not found in 100 normal controls. The G1102 and Y1117 residues were evolutionarily highly conserved from zebrafish to humans. Mutational analysis did not identify any mutation in the COX-2 gene (a modifier gene of OI). This study identifies two novel mutations p.G1102A and p.Y1117C that cause OI, significantly expands the spectrum of COL1A2 mutations causing OI, and has a significant implication in prenatal diagnosis of OI.     

播散性浅表性光线性汗孔角化症DSAP1位点的精细定位和候选基因的突变检测

播散性浅表性光线性汗孔角化症(DSAP)是一种以多个浅表的角化性皮损,边缘轻微嵴状角化性隆起为特征的少见的慢性角化性皮肤病,呈常染色体显性遗传。以往的研究将该病基因定位于12q23．2—24．1区域(DSAP1)和15q25．1-26．1区域(DSAP2)。本研究对2个无关的六代DSAP家系进行了全基因组扫描和连锁分析,结果显示,这2个DSAP家系在D12窝4位点的最高累积LOD值为8．28(θ=0．00)。单倍型分析结果显示,这2个DSAP家系致病基因位于12q24．1-q24．2(D12S330和D12S354)之间8．0cM的区域内。该区域与DSAP1的致病区域部分重叠。对重叠区域内6个候选基因(CRY1,PWP1,ASCL4,PRDM4,KIAA0789和CMKLR1)的编码区进行序列分析,在DSAP病人中未发现突变位点。提示该6个候选基因可能与这2个DSAP家系的发病机理无关。     

Smith—Fineman—Myers综合征与GRIA3基因的连锁和突变分析

探讨GRIA3基因与中国山东Smith-Fineman-Myers综合征的连锁关系,并分析SFMS患者GRIA3基因突变。采用PCR结合变性聚丙烯酰胺凝胶电泳方法,分析GRIA3基因内多态位点与致病基因之间的连锁关系。采用PCR扩增结合PCR产物直接测序方法检测GRIA3基因开放性阅读框架区域基因突变。GRIA3基因内多态位点分析表明,GRIA3基因与中国山东SFMS家系致病基因紧密连锁,但在该基因开放性阅读框架区域内并未检测到导致疾病的基因突变。中国山东SFMS家系患者不是由于GRIA3基因编码区域基因突变所致。     

基因芯片技术检测拉美夫定相关的HBV变异

美夫定(Lamivudine)是新一代核苷类似物,能够迅速降低慢性乙型肝炎病人的血清HBV DNA水平,但是Lamivudine治疗12个月后,14%～39%的患者都会出现抗药性[1～3],病情会有反复,用药不再有效.已有的研究表明,通过检测患者血液中的病毒突变情况,轮流使用干扰素,Lamivedine,FamcicLovir丙种球蛋白等,可以得到很好的治疗效果[4].本研究中,我们采用拉美夫定耐药位点基因芯片,对44例慢性乙型肝炎病人进行了HBV突变检测,并应用传统的DNA测序的方法进行验证,结果报告如下.     

人肝癌组织中的一个Lis1基因的阅读框移码突变

将ＧＳＴ融合蛋白表达与蛋白质截短检测法（ＰＴＴ）法相结合,检测Ｌｉｓ１基因在肝癌组织中的阅读框移码突变。即从肝癌组织中通过ＲＴ－ＰＣＲ扩增的Ｌｉａ１基因克隆人ＧＳＴ融合蛋白表达载体ｐＧＥＸ０１,并于大肠杆菌ＤＨ５α中进行表达。通过ＳＤＳ－ＰＡＧＥ电泳发现肝癌组织中有截短的ＧＳＴ－Ｌｉｓ１融合蛋白,大小为３３ＫＤ,而全长融合蛋白大小应为７１ＫＤ。经测序验证,发现产生截短蛋白的Ｌｉａ１基因在第１６３位