全外显子组测序鉴定汉族人群扩张型心肌病新的MYH7基因突变

目的：我们应用全外显子测序筛查一个扩张型心肌病患者家系的致病基因。方法：我们对一位在复旦大学附属中山医院就诊的扩张型心肌病患者及其家族成员采集临床资料,同时采集外周血做全外显子测序,寻找可能的致病基因突变,然后用sanger测序对患者及家系成员验证。结果：通过对家系患者及家族成员基因测序分析,经过多个数据库过滤,我们发现了致病基因突变位点MYH7 c.2458G>C (p.Ala820Pro),随后我们发现在家族成员中携带该突变位点的成员心功能异常。检索数据库发现该位点既往未在汉族人群中报道过,首次发现该突变位点与扩张型心肌病有关。结论：本研究通过全外测序发现了一个扩张型心肌病家系的致病基因,为新的汉族人群扩张型心肌病致病位点。     

二个无虹膜症家系致病基因的鉴定

目的:明确2个呈常染色体显性遗传的无虹膜症家系的临床特征,并找出导致该病的基因突变。方法:2个家系无虹膜症患者接受遗传咨询及眼科检查,并采用候选基因法分别对2个家系成员的人类配对盒基因6(human paired box gene 6,PAX6)进行突变筛查,对未检出突变的家系进成员行外显子捕获及全外显子组测序。结果:家系1患者完全符合无虹膜症的临床特征,家系2患者还出现上睑下垂的表型。突变筛查发现,家系1的PAX6基因存在一个已知的基因突变c.718CT(p.Arg240~*),但家系2未筛查到任何已知的基因突变。结论:家系1中发现一个PAX6基因突变(p.Arg240~*),但在家系2中尚未筛查到致病基因,提示无虹膜症尚存在新的遗传异质性。     

马凡综合征FBN1突变的一个家系分析及产前诊断

目的对一马凡综合征(Marfan's syndrome,MFS)个例进行原纤维蛋白-1基因(FBN1)突变分析并对该家系的1例MFS孕妇进行产前诊断。方法提取先证者及其家族成员外周全血基因组DNA,先证者羊水细胞DNA和培养后羊水细胞的RNA。用PCR和DNA双向测序技术检测存在于FBN1外显子中的潜在突变。RT-PCR扩增RNA检测所发现突变的相应外显子并进行基因测序。结果发现该先证者FBN1基因外显子23错义突变c.2785AC(p.Thr929Pro),其患MFS的父亲和哥哥发现同样突变。该家族其他表型正常的成员该位点未发现突变。胎儿羊水细胞的DNA与羊水培养细胞RNA均未发现该位点的突变。结论 FBN1错义突变c.2785AC(p.Thr929Pro)为该家族的致病原因,该MFS孕妇的胎儿未遗传该FBN1的致病突变。     

一角膜营养不良家系的TGFBI基因突变*

摘要:目的对一角膜营养不良家 系的致病基因进行检测,明确该家系角膜营养不良的致病基因突变位点。方法采集先证者及其家系成员外周血标本，提取基因组DNA,采用全外显子组测序筛查先证者致病基因及突变位点，并通过Sanger测序对 先证者及其家系成员DNA样本进行验证。结果全外显 子测序结果显示，先证者5号染色体上的转化生长因子β诱导基因(TGFBI)4号外显子发生c.370 C>T(p.R124C)杂合突变,8号外显子发生c.1007 A>T(p.E336V)杂合突变。Sanger 测序验证 发现患者父亲及家系其他患者TGFBI基因也发生c.370 C>T(p.R124C)杂合突变,患者母亲及家系其他正常成员未见TGFBI基因发生突变。TGFBI基因c.370 C>T(p.R124C)突变与疾病表型共分离。结论TGFBI 基因e.370 C>T(p.R124C)杂合突变 是导致该家系角膜营养不良的致病原因。     

Miyoshi肌病致病基因的突变分析

目的寻找Miyoshi肌病家系的DYSF基因的分子缺陷.方法用RT-PCR技术和序列分析筛查家系成员的DYSF基因的编码序列,确定患者的基因突变.结果患者DYSF基因的53号外显子存在6429 del G突变.结论 6429 del G突变为一移码突变,它使dysferlin蛋白的合成在2035密码子处提前终止,使其稳定性降低进而导致疾病的发生.     

一个中国神经性肌强直家系致病基因初步研究简

目的对一遗传性神经性肌强直家系2个已知致病基因进行初步筛查。方法应用外显子高通量测序分析方法对家系患者的KCNAl基因和KCNQ2基因进行DNA测序。结果基因检测未发现KCNAl基因和KCNQ2基因外显子突变。结论该神经性肌强直家系的发病与KCNAl基因和KCNQ2基因无关。     

一个中国神经性肌强直家系致病基因初步研究

目的对一遗传性神经性肌强直家系2个已知致病基因进行初步筛查。方法应用外显子高通量测序分析方法对家系患者的KCNA1基因和KCNQ2基因进行DNA测序。结果基因检测未发现KCNA1基因和KCNQ2基因外显子突变。结论该神经性肌强直家系的发病与KCNA1基因和KCNQ2基因无关。     

一个遗传性出血性毛细血管扩张症家系的致病基因研究

目的:通过对一个遗传性出血性毛细血管扩张症家系进行ENG基因和ALK1基因测序,确定基因突变位点。方法:用RT-PCR方法对家系4代11名成员的ENG基因14个外显子和ALK1基因9个外显子进行扩增;扩增产物纯化后直接测序查找基因突变位点。结果:先证者及家系其他患者ENG基因第4外显子存在无义突变c.447G﹥A,导致149位氨基酸Trp(UGG)变为Stop(UGA),在该家系未发现ALK1基因突变。结论:ENG基因无义突变c.G447A引起氨基酸发生p.Trp149Stop改变,是该家系致病的遗传基础。     

变性高效液相色谱技术在家族性高胆固醇血症低密度脂蛋白受体基因突变检测中的应用

目的 以变性高效液相色谱(DHPLC)，分析检测家族性高胆固醇血症(FH)一汉族家系成员的低密度脂蛋白受体(LDLR)基因突变，以明确诊断。方法 收集临床诊断为家族性高胆固醇血症的汉族一个家系共37名成员，其中30人为一级和二级亲属，7名为亲属配偶作为对照，提取基因组DNA，聚合酶链反应(PCR)方法扩增LDLR基因包含启动子和全部基因编码区(1-18外显子)及临近的内含子序列共21个片段，琼脂糖凝胶电泳鉴定产物。采用DHPLC技术检测了LDLR基因，对洗脱曲线异常者进行核苷酸序列分析。结果 该家系中发现4处变异，其中1处经核苷酸序列测定明确了突变的性质为第3内含子的剪接突变，并在此家系5名成员中得到证实，而对照组中未检出。结论 成功地建立了以DHPLC筛查LDLR基因点突变的方法及技术参数，该方法简便，结果稳定，可作为大样本筛查突变位点的一种便捷可靠手段。     

家族性高胆固醇血症家系低密度脂蛋白受体基因突变分析

目的:对1例临床确诊为纯合型家族性高胆固醇血症(FH)先证者及其3代家系成员进行基因检测和系谱分析,探讨其发病机制.方法:先证者家中收集该家系3代共10例血标本及临床资料.对其家系成员进行血脂测定,酚氯仿法提取患儿及家系成员基因组DNA并鉴定,应用多聚酶链反应-单链构象多态性(PCR-SSCP)分析结合DNA直接测序方法,检测其低密度脂蛋白受体(LDL-R)基因的全部18个外显子和启动子及载脂蛋白B(ApoB100)26外显子,核苷酸序列分析结果与GeneBank比对寻找突变.结果:(1)先证者右锁骨下动脉起始,双侧颈总动脉分叉处,中段内一中膜轻度增厚,左房轻度增大,二尖瓣、三尖瓣及主动脉瓣轻度返流,冠脉血流储备减低;(2)该家系排除ApoB100基因26外显子3500附近位点突变;(3)核苷酸序列分析证实先证者LDL-R基因第13外显子发生D601Y纯合突变,为1864位G→T碱基置换,导致天冬氨酸改变为酪氨酸,先证者父亲和母亲LDL-R基因第13外显子均发生D601Y杂合突变.结论:该先证者LDL-R基因存在D601Y纯合突变,其父母LDL-R基因存在D601Y杂合突变,可能为该家系中FH的致病突变.     

Associations between VHL genotype and clinical phenotype in familial von Hippel-Lindau disease

BACKGROUND: Von Hippel-Lindau (VHL) disease is an autosomal dominant hereditary disorder associated with tumours and cysts in the central nervous system (CNS) and other visceral organs. Germline mutations in the VHL gene on chromosome 3p25-26 are considered the cause of this disease. MATERIALS AND METHODS: We studied six patients with VHL disease and their relatives. Loss of heterozygosity (LOH) was determined by five flanking microsatellite polymorphic markers in the VHL locus. Multiplex ligation-dependent probe amplification (MLPA) and quantitative real-time polymerase chain reaction (qPCR) amplification were used to detect the genomic deletions. Single-strand conformation polymorphism (SSCP) analysis was applied to test for sequence variations. RESULTS: Three germline deletions in the VHL gene (142.9, 53.3 and 3.3 kb) were found by MLPA. These deletions were defined clearly by qPCR analyses. The142.9 kb germline deletion was significantly associated with patients with CNS haemangioblastomas (P < 0.01 by Fisher's exact test), and one missense mutation (Gln209Arg) was detected from a patient with a pancreatic cyst in the same family. LOH was also detected from a patient with bilateral renal cell carcinomas. CONCLUSION: Diverse genetic conditions are associated with the clinical manifestations of VHL disease. Genomic deletions that can be detected by MLPA or qPCR are major causes for this syndrome. Missense mutations and LOH accompanying the disease lead to complex clinical symptoms and genotypic determination can facilitate a clinical diagnosis because of their strong association.     

Loss of the polycystic kidney disease (PKD1) region of chromosome 16p13 in renal cyst cells supports a loss-of-function model for cyst pathogenesis.

It is not known whether mutations in the PKD1 gene cause autosomal dominant polycystic kidney disease (PKD) by an activating (gain-of-function) or an inactivating (loss-of-function) model. We analyzed DNA from cyst epithelial cells for loss of heterozygosity (LOH) in the PKD1 region of chromosome 16p13 using microsatellite markers. 29 cysts from four patients were studied. Five cysts from three patients had chromosome 16p13 LOH. Four of the cysts had loss of two chromosome 16p13 markers that flank the PKD1 gene. In two patients, microsatellite analysis of family members was consistent with loss of the wild-type copy of PKD1 in the cysts. In the third patient, 16p13 LOH was detected in three separate cysts, all of which showed loss of the same alleles. Chromosome 3p21 LOH was detected in one cyst. No LOH was detected in four other genomic regions. These results demonstrate that some renal cyst epithelial cells exhibit clonal chromosomal abnormalities with loss of the wild-type copy of PKD1. This supports a loss-of-function model for autosomal dominant PKD, with a germline mutation inactivating one copy of PKD1 and somatic mutation or deletion inactivating the remaining wild-type copy.     

Familial amyloidosis in a large Spanish kindred resulting from a D38V mutation in the transthyretin gene

BACKGROUND: Transthyretin amyloidosis, also known as familial amyloidotic polyneuropathy, is an autosomal dominant disorder that results from a mutation in the gene encoding plasma transthyretin (TTR). Distinct clinical presentations of the disease have been related so far to different point mutations, polyneuropathy being the predominant clinical feature in the majority of cases. Nevertheless, misdiagnosis of familial forms of amyloidosis is still common. MATERIALS AND METHODS: A 71-year-old man was admitted to our hospital for heart failure. He had been previously diagnosed of AL amyloidosis with predominant polyneuropathic, cardiac and laryngeal involvement on the basis of clinical data and amyloid deposition in tissue specimens. During admission, suspicion of transthyretin amyloidosis was raised due to the absence of renal involvement, monoclonal protein and plasma cell dyscrasia. Complete clinical evaluation and sequence analysis of the TTR gene of the patient and his family were performed. RESULTS: Gene sequence analysis revealed a rare A-to-T transition in exon 2 resulting in the substitution of aspartic acid by valine at position 38 (D38V) in the index case and in two other members of the family. Clinical study of the kindred showed a predominant late-onset heart involvement with variable polyneuropathy. CONCLUSIONS: Here we report a large pedigree from Spain with three members affected by a severe late-onset form of amyloidosis due to a rare D38V TTR mutation. The variations on the natural history of this form of amyloidosis may have important consequences on genetic counselling, follow-up, and therapeutic approaches for these patients.     

小脑性共济失调症患者谷氨酸受体δ2基因12号外显子突变研究

目的探讨小脑性共济失调症患者谷氨酸受体82（GluRδ2）基因12号外显子突变的情况。方法采用PCR、琼脂糖凝胶电泳及DNA测序方法检测24例小脑性共济失调症患者（有家族史者17例、散发者7例）及其16名无症状家系成员和10名正常人的GIuRδ2基因12号外显子突变情况。结果经PCR扩增和琼脂糖凝胶电泳后,24例患者、16名无症状家系成员及10名正常人12号外显子均可见一长度为222bp片段,未见该外显子纯合缺失突变。DNA测序结果显示,24例患者未发现类似h05J小鼠的突变碱基缺失及Lurcher小鼠的突变碱基置换。结论小脑性共济失调症患者中不存在GluRδ2基因12号外显子纯合缺失突变,也不存在碱基突变或缺失,提示GIuRδ2基因12号外显子与本病发病机制可能无关。     

YWHAH基因在肾癌中的表达研究

目的分析YWHAH基因在肾癌中的表达特征,以期探讨YWHAH基因与肾癌发生发展的关系。方法利用第二代测序技术对10例肾癌及癌旁组织进行测序,生物信息学分析后,进行30例样本的RT-PCR和QPCR验证。结果在10例测序结果发现YWHAH在肾癌中过表达,肾癌组织中表达量是癌旁组织的3.79倍,QPCR验证结果是肾癌组织中表达量是癌旁组织4.34倍。结论 YWHAH在肾癌中呈过表达,可能与肾癌的发生发展有关系,有望为临床治疗提供了理论基础和治疗依据。     

Familial hypobetalipoproteinemia in a Turkish family with hereditary spastic paraplegia

A 24-year-old male presented with progressive gait disturbance and was diagnosed with hereditary spastic paraplegia. His brother and possibly one uncle also had the condition. Routine biochemical testing found that the patient had unusually low plasma concentrations of low density lipoprotein (LDL) cholesterol and apolipoprotein (apo) B, the hallmark of familial hypobetalipoproteinemia. DNA sequencing showed that he, along with other family members (n = 5; mean LDL cholesterol 0.8 mmol/L, apoB 0.31 g/L), were heterozygous for a single nucleotide deletion in exon 26 of the APOB gene. This mutation is predicted to form a truncated apoB species of 3545 amino acids, which we have designated apoB-78.2.     

1例植物固醇血症的家系调查及致病基因突变研究

目的探讨1例植物固醇血症家系及其致病基因突变。方法对1例诊断为植物固醇血症的患者及其家系成员进行家系调查;通过PCR扩增先证者及其家系成员基因组DNA中ABCG5及ABCG8基因的所有外显子及其侧翼序列,采用Sanger测序法对PCR产物进行基因测序;采用Polyphen2及Mutation Taster生物信息学软件预测突变的致病性。结果 Sanger测序法发现先症者及家系成员中存在多个基因突变,其中ABCG5基因发现3个突变,分别为外显子1 c.64CT(p.Q22X)杂合无义突变、外显子10 c.1336CT(p.R446X)杂合无义突变、外显子13 c.1810CG(p.Q604E)杂合错义突变;ABCG8基因发现4个突变,分别为(ATG前)-19TG纯合突变、外显子2 c.161AG(p.Y54C)纯合错义突变、外显子13 c.1895TC(p.V632A)纯合错义突变、外显子4和5间的内含子g.12902TC纯合突变。Polyphen2及Mutation Taster软件预测ABCG5基因中c.64CT及c.1336CT为致病突变,其他基因突变均为非致病性的多态性位点。结论 ABCG5基因c.64CT及c.1336CT复杂杂合突变是该植物固醇血症家系的基因发病机制。     

Immunoassay for wild-type protein in lymphocytes predicts germline mutations in patients at risk for hereditary colorectal cancer

Using colorectal cancer (CRC) as an example, we present the hypothesis that quantitative immunoassays for wild-type (full-length) proteins can be used to identify carriers of traits for hereditary diseases. In the case of hereditary CRC, this involves identifying individuals with germline mutations in a mismatch-repair (MMR) gene (mainly hMSH2 or hMLH1) or in the adenomatous polyposis coli (APC) gene. Because expression of wild-type protein should reflect wild-type gene dosage, we predicted that individuals harboring a germline mutation will have a reduction of approximately 50% in expression in lymphocytes of the corresponding full-length protein. In this pilot study, we tested lymphoblastoid cell lines that had been established from controls and individuals with, or at high risk for, hereditary CRC: 9 lines from healthy, unaffected individuals; 4 from affected members in familial adenomatous polyposis families (with known germ-line APC mutation); 42 from CRC patients in our Familial CRC Registry (increased risk of hereditary nonpolyposis colon cancer as assessed by family history, age at adenoma or carcinoma diagnosis, and other clinical criteria). For MSH2 and MLH1 we used western blots; for APC we used immunoprecipitation. All familial adenomatous polyposis lines had about 50% less immunoprecipitable full-length APC protein. Some cell lines (7 of 42) from Familial CRC Registry patients showed on western blots a reduction (mean 46%) in either MSH2 or MLH1 (relative to the other protein). All 7 subsequently were proved to contain a germline MMR mutation. We conclude that (1) because most of the expected CRC-causing germ line mutations are truncation-causing, immunoassays for wild-type protein should be able to identify most individuals with hereditary CRC-causing traits; (2) these assays, which are more practical and inexpensive than current mutation-detecting tests for hereditary CRC traits, have the potential for commercial development into broad-based population screens of high-risk patients and their families and the potential to save both lives and health-care dollars; (3) this strategy may be useful for other hereditary cancers and even other hereditary diseases; (4) our approach has the potential to greatly benefit public-health programs for cancer control.