两个中国常染色体显性遗传性多囊肾病家系的表型与基因型分析

目的研究中国人多囊肾病基因1（polycystic kidney disease 1 gene，PKD1）突变的特点，检测基因突变位点。方法25例多囊肾患者，正常对照16名，扩增PKD1基因的第44、45外显子的基因片段，变性梯度凝胶电泳突变检测系统进行初筛，然后测序。结果发现1个移码突变（12431delCT）、1个无义突变（C12217T）、1个多态性（A50747C），突变检测率为8％（2／25）。结论检测到2个新的可能的致病突变：1个移码突变（12431delCT）、1个无义突变（C12217T）。     

应用变性高效液相色谱技术检测汉族人Ⅰ型多囊肾致病基因突变

目的通过采用变性高效液相色谱（denaturing high-pedormanee liquid chromatogtraphy，DHPLC）技术检测汉族人常染色体显性多囊肾病（autosomal dominant polycystic kidney disease，ADPKD）Ⅰ型致病基因PKD1的突变，建立更为快速、敏感的突变筛查系统。方法以来源于19个ADPKD家系的67名成员血样标本的基因组DNA为模板，通过长链PCR和巢式PCR联合扩增的方法扩增PKD1全编码区，然后采用DHPLC方法进行初步筛查，将存在异常色谱图的扩增产物经核苷酸测序，确定突变的具体位点和类型，并与以往采用单链构象多态性（single strand conformation polymorphism，SSCP）方法检测出的突变结果相比较。结果共检测出14个致病突变，包括10个错义突变、1个插入突变、1个缺失突变、2个无义突变，其中12个突变位点与之前SSCP的检测结果相同，另新发现nt32819G→A和nt37137T→C两个突变位点，突变检出率为73．7％。结论DHPLC方法可以作为更为有效筛查汉族人ADPKD PKD1突变位点的检测途径。     

一成人型多囊肾家系基因突变鉴定及产前诊断

目的 对一常染色体显性遗传性多囊肾病(Autosomal dominant polycystic kidney disease, ADPKD)家系进行致病基因突变鉴定，并对先证者妻子首次妊娠进行产前诊断。方法 用聚合酶链式反应,通过微卫星标记进行基因定位、DNA序列测定，确定基因突变；用AS-PCR对家系其他患者成员进行突变点检测和筛查；联合应用突变检测和连锁分析进行产前诊断。结果 该家系中多囊肾疾病的致病基因为PKD2,突变为外显子5中c.1249C﹥T ( p.R417X)；胎儿产前诊断结果显示未获得致病突变。结论 该家系的致病突变为c.1249C﹥T( p.R417X), 成功进行了产前诊断。     

两例常染色体显性多囊肾患者PKD1基因的突变检测

目的研究两例常染色体显性多囊肾患者的致病原因。方法对常染色体显性多囊肾患者的多囊肾病1基因(PKD1)3′端单拷贝区进行了聚合酶链反应-变性高效液相色谱(PCR-denaturing high-per-formance liquid chromatography,DHPLC)分析,并对有异常峰形的PCR产物进行测序。结果在1例患者中发现第42外显子的C11901A有一个无义突变,导致原丝氨酸3897变为终止密码子;而另一例患者第35外显子的C10737T有一个错义突变,导致原苏氨酸3509变为甲硫氨酸。在正常对照中发现两种同义突变分别为第42外显子的G11824A及C11860T。结论用DHPLC和DNA测序方法对两名患者进行PKD1的突变检测中,发现一个新的无义突变、一个错义突变以及两种同义突变。     

中国人GJB2耳聋基因突变分析

目的 确定常染色体隐性遗传性聋G励基因突变的类型和频率，从分子水平探讨发病机理．方法 收集中国人常染色体隐性遗传性聋4个家系(39名个体)和健康对照组50人的外周血DNA样本。PCR扩增GJB2基因片段，行Apa I酶切和序列分析。结果 检出2个家系4例患者GJB2基因235del C纯合性缺失，导致移码突变，2例患者为235delC和232G→A(Ala78Thr)双重杂合性突变。正常对照组中发现1例235del C携带者。耳聋患者组和健康对照组中均存在79G0→A(V27I)，341A→G(E114G)两种改变。在对照组中这两种改变的等位基因频率分别为30％、21％。结论 两个家系与GJB2基因235del C有关，232G→A是1个新的突变。     

Rett综合征MECP2基因突变分析

目的 分析我国典型的Rett综合征患儿甲基化CpG结合蛋白－2基因（methyl-CpG-binding protein 2,MECP2）突变。方法 使用PCR扩增、单链构象多态性分析、PCR产物克隆和DNA测序的方法。检测分析了26例Rett综合征患儿、其父母和其中2例患儿的妹妹MECP2基因3个外显子的基因突变。结果 26例Rett综合征患儿中发现14例有9种类型MECP2基因的杂合性突变，突变均位于第3外显子。其中7例有3种错义突变：C473T（T158M)4例,C674G(P225R)1例，C916T（R306C）2例；4例有3种无义突变：C502T（R168X）2例，C763T（R255X）1例，C880T（R294X）1例；2种由于缺失导致的突变：1例为1152del 44bp和1例1158－1167／1171－1186del 26bp；1鲍由于碱基插入导致的移码突变：874insA。1158－1167／1171－1186del 26bp和874insA突变为首次报千，突变均为新生突变。此外，新发现了一种源于你亲的错义变异1141G（P381A）。结论 我国Rett综合征患儿存在MECP2基因突变，典型的Rett综合征MECP2基因突变率大于50％。     

珊瑚状常染色体显性遗传白内障家系相关基因的定位和筛选

目的　明确一个延续 4代具有珊瑚状表型的常染色体显性遗传性白内障家系的基因缺陷。方法　家系成员的基因组 DNA进行全基因组扫描和连锁分析 ;利用 L INKAGE5 .1软件计算两点 L OD值 ;用直接测序法对候选基因进行突变检测。结果　 38个家系成员中有 13个患有遗传性白内障。基因组扫描发现 ,D2 S32 5引物在最大重组率为 0 .1时 ,其两点最大 L OD值 >3,提示与该家系连锁。对人类γ-晶状体蛋白基因簇的 4个基因进行突变检测发现 ,此家系患者 γ- D晶状体蛋白 (γ- D crystallin,CRYGD)基因第 2外显子有 1个 C→A突变 ,此突变导致蛋白第 2 3位的脯氨酸被苏氨酸取代 (P2 3T)。结论　珊瑚状白内障表型与C→ A错义突变的 CRYGD基因密切相关 ,且此突变完全相同于最近报道的与一层状白内障表型共分离的突变。发现相同的基因缺陷引起的白内障浑浊可位于晶状体截然不同的部位 ,其病理机理还需进一步的研究。     

遗传性出血性毛细血管扩张症基因突变分析

目的 分析遗传性出血性毛细血管扩张症(hereditary hemorrhagic telangiectasia,HHT)家系ENG、ACVEL1和SMAD4基因突变.方法 收集4个HHT家系临床资料并分析其临床特点,应用直接测序和多重连接探针扩增技术对11例临床确诊及可疑患者的ENG、ACVRL1和SMAD4基因进行突变分析,将结果与HHT基因突变数据库进行对比.结果 家系2先证者及2个妹妹的ENG基因发生了第2外显子c.207G＞A(p.L69L)同义突变、第8外显子c.1004A＞T(p.Q335L)错义突变、ACVRL1基因第7外显子c.817C＞T(L273L)同义突变;家系3先证者及其母亲和弟的ENG基因发生了第8外显子c.1004A＞T(p.Q335L)突变;也检测到家系4先证者及其兄的ENG基因第8外显子c.1004A＞T(p.Q335L)突变.家系1先证者及其他HHT患者,未检测到基因突变.其中ENG基因第8外显子c.1004A＞ T(p.335Q＞L)为新突变,在200名正常对照中也未检测到该突变.结论 HHT具有遗传异质性,ENG基因第8外显子c.1004A＞T(p.Q335L)为HHT新的致病突变.     

应用变性高效液相色谱检测31例家族性腺瘤性息肉病家系结肠腺瘤性息肉病基因突变

目的 应用变性高效液相色谱(denaturing high performance liquid chromatography,DHPLC)技术检测我国家族性腺瘤性息肉病(familial adenomatons polyposis,FAP)家系的结肠腺瘤性息肉病(adenoinatous pelyposis coli,APC)基因变异特征,研究其病因机制.方法 采集31个家系的先证者、患者和家系成员的外周血淋巴细胞,抽提DNA并以降落式PCR扩增APC基因各外显子和启动子.基因突变检测先由DHPLC进行筛选,发现异常峰者进行测序鉴定并TA克隆鉴定,结果与网络数据进行比对.结果 31个家系中共有15个家系检出了12种不同的突变类型,FAP家系APC基因的突变检出率为48.39%.发现了4种新的突变及3例不同的内含子突变.4个新的突变分别位于255、677、1192、1403密码子,均为移码突变.证明了DHPLC能检出APC基因的突变.在APC基因的突变中,移码突变占86.67%,无义突变占13.33%,说明移码突变是中国人APC基因突变的主要方式.在突变位点上,第15外显子突变最常见,约占86.67%.结论 FAP家系APC基因的突变检出率为48.39%,发现了4种新的导致蛋白编码改变的突变.证实中国人FAP家系中APC基因突变位点以第15外显子最常见,类型以移码突变为主.     

Crouzon综合征基因突变检测

目的 研究1个Crouzon综合征家系及1例散发的Crouzon综合征患者的成纤维生长因子受体2(fibroblast growth factors receptor 2,FGFR2)基因突变情况.方法 在1个Crouzon综合征家系的10名成员,和另一例散发者的外周血提取基因组DNA,PCR扩增FGFR2基因的第8和10外显子(部分家族成员仅扩增第8外显子),产物纯化后直接进行DNA测序检测突变.结果 家系中3名成员及另1例散发者FGFR2基因第8外显子的833位核苷酸发生G→T的转换突变,该突变为错义突变,使该位点所编码的氨基酸由半胱氨酸变为苯丙氨酸(C278F).该突变为杂合子突变.结论 FGFR2基因突变是Crouzon综合征致病原因.     

Mutation analysis in PKD1 of Japanese autosomal dominant polycystic kidney disease patients

Autosomal dominant polycystic kidney disease (ADPKD) is a common genetic renal disorder (incidence, 1:1,000). The mutation of PKD1 is thought to account for 85% of ADPKD. Although a considerable number of studies on PKD1 mutation have been published recently, most of them concern Caucasian ADPKD patients. In the present study, we examined PKD1 mutations in Japanese ADPKD patients. Long-range polymerase chain reaction (LR-PCR) with PKD1-specific primers followed by nested PCR was used to analyze the duplicated region of PKD1. Six novel chain-terminating mutations were detected: three nonsense mutations (Q2014X transition in exon 15, Q2969X in exon 24, and E2810X in exon 23), two deletions (2132del29 in exon10 and 7024delAC in exon 15), and one splicing mutation (IVS21-2delAG). There was also one nonconservative missense mutation (T2083I). Two other potentially pathogenic missense mutations (G2814R and L2816P) were on the downstream site of one nonsense mutation. These three mutations and a following polymorphism (8662C>T) were probably the result of gene conversion from one of the homologous genes to PKD1. Six other polymorphisms were found. Most PKD1 mutations in Japanese ADPKD patients were novel and definitely pathogenic. One pedigree did not link to either PKD1 or PKD2.     

Genetics and phenotypic characteristics of autosomal dominant polycystic kidney disease in Finns

Autosomal dominant polycystic kidney disease (ADPKD) is the most common inherited kidney disease, leading to renal insufficiency and renal transplantation. Mutation screening in the major gene for ADPKD, the polycystic kidney disease type 1 (PKD1) gene, has often been incomplete because of multiple homologous copies of this gene elsewhere on chromosome 16. Furthermore, there are only a few studies investigating genotype–phenotype correlations in patients with ADPKD. In this study, we screened the entire coding region of the PKD1 and PKD2 genes in 17 Finnish families with ADPKD via long-range polymerase chain reaction, single-strand conformation polymorphism analysis, and direct sequencing. We were able to identify mutations co-segregating with ADPKD in all 16 families linked to PKD1 by haplotype analysis. Of these mutations, six were insertions/deletions, five nonsense mutations, and five missense mutations. In the only PKD2-linked family, we found a missense mutation, R322Q. With the exception of one mutation (L845S in PKD1), all mutations were novel. Mutations and their location did not have a strong correlation with the phenotype with the exception of subarachnoidal hemorrhage or brain aneurysm, where mutations were located more often at the 5 end of the PKD1 gene than at the 3 end of the PKD1 gene.Electronic Supplementary Material Supplementary material is available for this article at .     

Autosomal dominant polycystic kidney disease: comprehensive mutation analysis of PKD1 and PKD2 in 700 unrelated patients

Autosomal dominant polycystic kidney disease (ADPKD), the most common inherited kidney disorder, is caused by mutations in PKD1 or PKD2. The molecular diagnosis of ADPKD is complicated by extensive allelic heterogeneity and particularly by the presence of six highly homologous sequences of PKD1 exons 1-33. Here, we screened PKD1 and PKD2 for both conventional mutations and gross genomic rearrangements in up to 700 unrelated ADPKD patients--the largest patient cohort to date--by means of direct sequencing, followed by quantitative fluorescent multiplex polymerase chain reaction or array-comparative genomic hybridization. This resulted in the identification of the largest number of new pathogenic mutations (n = 351) in a single publication, expanded the spectrum of known ADPKD pathogenic mutations by 41.8% for PKD1 and by 23.8% for PKD2, and provided new insights into several issues, such as the population-dependent distribution of recurrent mutations compared with founder mutations and the relative paucity of pathogenic missense mutations in the PKD2 gene. Our study, together with others, highlights the importance of developing novel approaches for both mutation detection and functional validation of nondefinite pathogenic mutations to increase the diagnostic value of molecular testing for ADPKD.     

Kidney enlargement and multiple liver cyst formation implicate mutations in PKD1/2 in adult sporadic polycystic kidney disease

Distinguishing autosomal‐dominant polycystic kidney disease (ADPKD) from other inherited renal cystic diseases in patients with adult polycystic kidney disease and no family history is critical for correct treatment and appropriate genetic counseling. However, for patients with no family history, there are no definitive imaging findings that provide an unequivocal ADPKD diagnosis. We analyzed 53 adult polycystic kidney disease patients with no family history. Comprehensive genetic testing was performed using capture‐based next‐generation sequencing for 69 genes currently known to cause hereditary renal cystic diseases including ADPKD. Through our analysis, 32 patients had PKD1 or PKD2 mutations. Additionally, 3 patients with disease‐causing mutations in NPHP4, PKHD1, and OFD1 were diagnosed with an inherited renal cystic disease other than ADPKD. In patients with PKD1 or PKD2 mutations, the prevalence of polycystic liver disease, defined as more than 20 liver cysts, was significantly higher (71.9% vs 33.3%, P = .006), total kidney volume was significantly increased (median, 1580.7 mL vs 791.0 mL, P = .027) and mean arterial pressure was significantly higher (median, 98 mm Hg vs 91 mm Hg, P = .012). The genetic screening approach and clinical features described here are potentially beneficial for optimal management of adult sporadic polycystic kidney disease patients.     

多囊肾病的临床实践指南

多囊肾病(polycystic kidney disease,PKD)是由基因突变所导致的一类遗传性肾病,按其遗传方式又分为常染色体显性多囊肾病(autosomal dominant polycystic kidney disease,ADPKD)和常染色体隐性多囊肾病(autosom al recessive polycystic kidney disease,ARPKD)。该病的主要病理特点是肾脏囊肿进行性增大、增多,破坏正常的肾脏结构,最终导致终末期肾病(end stage renal disease,ESRD),患者只能依靠透析或肾移植维持生命。我们在参考国内外本领域的基础研究、临床研究和相关指南共识的基础上,结合中国人群的实际情况编写了该项指南,旨在总结多囊肾病的医学遗传学知识和临床处置要点,以提高临床医师的认识水平,为该病的诊治提供规范化建议。     

PKD2 gene mutation analysis in Korean autosomal dominant polycystic kidney disease patients using two-dimensional gene scanning

Autosomal dominant polycystic kidney disease (ADPKD) is genetically heterogeneous and is caused by mutations in the PKD1 or PKD2 genes. ADPKD caused by PKD2 mutations is characterized by a longer survival and a later onset of end-stage renal disease than ADPKD caused by PKD1 mutations. PKD2 encodes a 2.9-kb messenger RNA and is derived from 15 exons. Two-dimensional gene scanning (TDGS) is more efficient in detecting mutations in genes such as PKD2 because it can scan the whole coding regions simultaneously. In order to determine the prevalence of Korean PKD2 patients, all the coding sequences of PKD2 were screened using TDGS and direct sequencing in 46 randomly selected ADPKD patients (group 1). Another 45 ADPKD patients (group 2), who were presumed to be PKD2 patients, were screened in order to identify the type of mutation in the Korean PKD2 patients. Eight novel different mutations and three known mutations in the PKD2 gene were detected in 17 patients: 6 patients (13.0%) in group 1 and 11 patients (24.4%) in group 2. Considering the sensitivity of TDGS, the prevalence of PKD2 in Korean population might be greater than 18.6%. Both known and novel mutations were identified by TDGS in Korean PKD2 patients. Overall, these results showed that TDGS might be useful for diagnosing PKD2.