四例脂蛋白肾病患者载脂蛋白E基因突变筛查

目的 通过对4例脂蛋白肾病患者及家系成员载脂蛋白E(apoE)基因的分析， 研究脂蛋白肾病的发病机制。 方法 调查患者家系情况，对其中2例患者的家系成员进行尿常规筛查及血肌酐、血脂和血清脂蛋白的检测。PCR法扩增4例患者apoE基因的外显子， DNA测序， 发现突变后，寻找限制性内切酶酶切位点。使用聚合酶链反应-限制性酶切片段长度多态性(PCR-RFLP)的方法筛查正常对照及其家系成员。 结果 4例脂蛋白肾病患者中有2例携带杂合的apoE基因缺失突变 (142-144-0)，2例患者携带杂合的apoE点突变 (Arg25Cys)，2种突变均可见于尿液检查正常的亲属，并均表现为apoE升高。 结论 4例脂蛋白肾病的患者中发现两种apoE基因的突变，apoE Arg25Cys和apoE(142-144-0)。结合既往的研究结果，apoE(142-144-0)同时见于5例(本研究2例，前期研究3例)患者，为中国脂蛋白肾病患者常见的致病性基因突变。患者家系成员中的携带者可以不表现出肾脏病。     

小睑裂综合征家系FOXL2基因的突变研究

目的对一个常染色体显性遗传的小睑裂综合征家系4代12例患者,及家系中12例正常成员和80例正常对照的FOXL2基因进行研究。方法设计合成FOXL2基因特异引物应用聚合酶链反应(PCR)扩增FOXL2基因,PCR产物直接测序。结果该家系所有患者的FOXL2基因892C>T,为无义突变。在正常人的FOXL2基因中未发现突变。结论FOXL2基因的突变可能是该家系中小睑裂综合征重要的致病因素。     

一个Liddle综合征家系上皮钠通道基因突变的研究

目的：对1个Liddle综合征家系成员进行基因突变分析。方法：1个3代均有高血压患者的家系中，1例14岁的成员临床诊断为Liddle综合征。抽取所有存活家系成员的外周血基因组DNA，PCR扩增上皮钠通道β亚单位基因（βENaC)和γ亚单位基因（γENaC)第13外显子，产物直接DNA测序进行基因突变检测。结果：βENaC基因第13外显子的DNA经双向测序显示，先证者及另两例家系成员第616号密码子均存在CCC－TCC（Pro-Ser)杂合错义突变，并且第632号密码子存在GAC－CAC（Asp-His)的变异与之连锁。对150例无关个体进行直接测序未发现GAC－CAC变异，证明这是一新的突变。其他家系成员均未发现这一基因突变，另2例基因突变的成员的临床生化检验结果符合Liddle综合征；所有家系成员中未检测到γENaC基因第13外显子的突变。结论：对临床诊断的Liddle综合征患者及其家属，进行基因突变检测有助于早期筛选出家系中的其他患者。该家系中，第632号密码子检测到一新的突变GAC－CAC（Asp-His)，通过表型分析认为这一新突变有可能与Liddle综合征有关。     

von Hippel-Lindau病家系调查及基因学研究(附二例报告)

目的探讨vonHippel-Lindau病(VHL)基因突变类型及改良的基因诊断方法。方法调查1例VHL病Ⅰ型家系及1例Ⅱ型家系,分别绘制树状图;抽取2个家族共8位成员的外周血,提取基因组DNA。改良方法进行3个外显子翻译区及剪接区的扩增,扩增产物经纯化后直接测序。将所得突变类型与人类基因突变数据库(HGMD)核对。结果VHL病I型家系4代12位家族成员中,有双肾透明细胞癌Ⅱ级合并视网膜血管母细胞瘤1例,中枢神经系统血管母细胞瘤合并视网膜血管母细胞瘤1例,突变基因携带者1例。Ⅱ型家系7位家族成员中,有双肾透明细胞癌Ⅱ级合并双肾多发囊肿1例,肾上腺嗜铬细胞瘤1例。Ⅰ型家系中,5例接受检测者中2例基因测序均见VHL基因第478位与479位核苷酸分别发生C→T及T→C突变,导致第89位编码氨基酸由亮氨酸转变为丝氨酸。Ⅱ型家系3例接受检测者中,2例基因测序见VHL基因第452位核苷酸发生G→T突变,导致第80位编码氨基酸由丝氨酸转变为异亮氨酸。测序获得突变类型与HGMD核对,发现I型家系中VHL基因第478位核苷酸与479位核苷酸的多点突变,为VHL基因第89位氨基酸位点未曾报道过的突变类型。2个家系的基因突变位点均...     

中国Alport综合征一家系的基因突变检测与临床表型分析

目的:应用高通量测序技术,检测一个中国遗传性肾病家系的致病基因突变,探讨靶区域捕获和高通量测序方法在遗传性肾病基因筛查中的可行性。方法:收集家系临床资料和外周血样本;分析先证者的临床资料,并观察肾穿组织病理,采用目标区域捕获和高通量测序技术,对先证者355个遗传性肾病相关基因的外显子进行突变筛查;应用Sanger测序,在其他家庭成员中进行突变位点验证及突变-表型共分离分析,并对突变位点进行多物种的保守性分析。结果:结合临床检验结果和肾活检病理观察,先证者符合慢性肾小球肾炎,不排除Alport综合征的可能。基因筛查发现,该家系可确诊为X染色体显性遗传Alport综合征,家系中所有女性患者均为X染色体COL4A5基因c.3641GA(p.Gly1214Glu)杂合突变,而男性患者均为该位点的半合子。且该位点在多个物种中具有高度的序列保守性。结论:该遗传性肾病家系是X染色体显性遗传Alport综合征,致病突变位点为COL4A5 c.3641GA(p.Gly1214Glu)。靶区域捕获和高通量测序技术成本低、高通量、准确性高,适用于遗传性肾病家系的基因突变筛查。     

脂蛋白肾病载脂蛋白E基因序列分析

目的报道2例脂蛋广1肾病（LPG）及其载脂蛋白E（apoE）的基因测序分析，探讨apoE基因突变与LPG发病之间的关系。方法①采用直接测序法分析2例经肾活检证实为LPG患者的apoE基因序列；②荟萃分析国内已发表的有关LPG患者apoE基因测序结果并复习国内外相关文献。结果2例LPG；患者中，1例携带一个已被报道过的apoE基因点突变，DNA序列为编码区C606T杂合子突变，致相应的氨基酸发生改变（Arg158Cys），另一例未发现apoE基因突变。结论LPG患者部分存在apoE基因编码区突变，部分未发现与国内外报道有关的apoE基因突变类型，说明LPG的发病机制可能不仅仅与apoE基因编码区突变有关。     

迟发性家族性局灶节段肾小球硬化的足细胞分子基因突变研究

目的 了解迟发性家族性局灶节段肾小球硬化（FSGS）的足细胞分子基因致病突变特点。 方法 研究对象为上海瑞金医院肾脏科1997年9月至2007年10月收集的31个迟发性家族性FSGS家系。诊断标准：（1）成员年龄大于12岁；（2）1个家系中有2例或2例以上患者经肾活检证实为FSGS，或家系成员中有1例肾活检证实为FSGS，另有1例成员有蛋白尿或肾功能不全。100例健康人为对照组。外周血基因组DNA 经PCR扩增后直接对NPHS2、ACTN4、TRPC6基因行测序分析。 结果 发现ACTN4基因新错义突变L316P，该家系患病成员起病年龄平均（38.7±7.4）岁，肾功能损害进展相对缓慢，家系3例患病成员均为突变杂合子。发现TRPC6基因新杂合错义突变Q889K，该家系患者起病年龄平均（38.0±4.2）岁，肾功能损害进展也较缓慢，家系中临床表现存在个体差异，家系中3例患病成员均为突变杂合子。发现TRPC6静止突变G467G。所有家系中未发现NPHS2致病突变。健康对照组200条染色体亦未发现以上突变。 结论 在31例迟发性FSGS家系中发现2个家系携带致病相关突变：ACTN4新突变L316P和TRPC6新突变Q889K。在中国人群家族性迟发性FSGS中，ACTN4及TRPC6基因突变是致病原因之一，尚未发现NPHS2相关致病突变。     

两个遗传性并多指(趾)家系致病基因突变分析

目的探讨2个并多指(趾)畸形(SPD)家系的致病基因。方法收集2019年1月、2020年12月就诊于临沂市人民医院的2个SPD家系的临床资料, 采集先证者及家系成员静脉血样本, 提取基因组DNA, 对先证者行全外显子组测序筛选候选基因变异;采用Sanger测序对2个家系成员验证其突变位点;采用生物信息学软件PolyPhen-2和PROVEAN对突变位点的致病性进行预测分析, 结合美国医学遗传学与基因组学学会(ACMG)指南对突变位点进行致病性判断。结果家系1三代成员中共有5例患者(男2例、女3例), 先证者为8岁女性, 表现为右手第3、4指并指, 指蹼融合和远端指甲融合, 其余手指活动自如, 双脚未见异常;家系2三代成员中共有4例患者(均为女性), 先证者为4岁女性, 表现为双手第3、4指并指, 示指侧弯。全外显子组测序分别在2个SPD家系中检出同源盒D13(HOXD13)基因c.917G>A和c.917G>T突变, 且2个突变均呈现基因型-表型共分离, 其中HOXD13基因c.917G>T突变未见数据库收录, 为新发杂合错义突变。生物信息学软件预测这2个突变位点均为...     

一个多发性骨骺发育不良大家系的临床特点及致病基因分析

目的通过对一个5代疑似多发性骨骺发育不良(multiple epiphyseal dysplasia,MED)的大家系(患者17例)进行临床特征分析和致病基因的筛查,为遗传咨询和产前分子诊断提供实验依据。方法采集家系成员病史,一般体检、关节、髋部X线片资料;收集该家系外周血样,提取样本DNA,靶向基因高通量测序方法对先证者DNA临床全外显子进行测序,使用Next Gene软件对测序序列进行比对分析,并进一步利用Ingenuity软件对存在的突变进行功能注释,寻找先证者致病突变。针对可疑突变,PCR和Sanger测序对家系其他成员DNA样本进行验证。结果该家系共5代,现存家系成员38人,系谱分析符合常染色体显性遗传特征。家系共有患者17例,其临床表现为:幼时出现走路姿势异常,后出现髋关节及膝关节疼痛,X线有典型骨骺发育不良病理改变。高通量测序及数据分析后,筛选出先证者(Ⅳ-3)软骨低聚物基质蛋白(cartilage oligomeric matrix protein,COMP)基因c.1153G>A(p.Asp385Asn)错义杂合突变,该突变导致其编码蛋白的第385位天冬氨酸被天冬酰胺替代。先证者家系其他成员符合基因型与表型共分离。结论COMP基因c.1153G>A错义杂合突变是导致该MED家系患者发病的分子机制,该突变首次在大家系中被报道,进一步明确了COMP基因c.1153G>A突变的致病性,有利于家系患者的进一步的诊治,也为产前诊断提供了实验依据。     

成人经典型Bartter综合征家系CLCNKB基因突变的研究

目的 分析1个成人经典型Bartter综合征家系CLCNKB基因突变特点。 方法 用PCR方法对先证者CLCNKB基因19个外显子及侧翼序列进行扩增,PCR产物纯化后直接测序或构建T-A克隆测序检测其基因变异。 结果 先证者CLCNKB基因表现为G433E和cDNA 753delG复合杂合突变。家系分析表明,杂合错义突变G433E来自父亲,杂合缺失突变cDNA 753delG来自母亲。患者弟弟携G433E,其妹妹正常。正常对照100条染色体中未发现同样变异。 结论 在1个成人迟发经典型Bartter综合征家系中发现了CLCNKB基因2个突变位点,经检索文献及人类基因突变库（HGMD）,cDNA 753delG为新发现突变位点。     

Role of apolipoprotein E variants in lipoprotein glomerulopathy and other renal lipidoses

Lipoprotein glomerulopathy (LPG) is a new renal lipidosis entity, characterized by peculiar histology and abnormal lipoprotein profiles mimicking type III hyperlipoproteinemia. Recently, it has been clarified that LPG is associated with novel apolipoprotein E (apoE) mutations. In particular, ApoE-Sendai, which substitutes arginine 145 with proline, is observed in most Japanese patients with LPG, although isoelectric focusing polyacrylamide gel electrophoresis has shown that it is consistent with the apoE2 isoform. To confirm the etiological role of these apoE mutations, we established an animal model of LPG. The model was created by the introduction of recombinant adenovirus containing human apoE-Sendai into apoE knockout mice. Both clinical and experimental findings indicate that LPG is caused not only by hyperlipoproteinemia but also by an in situ interaction between apoE variants and glomerular elements. In addition, several studies suggest that the apoE2 mutation is responsible for the development of diabetic nephropathy and IgA nephropathy, as well as renal lipidosis with type III hyperlipoproteinemia. In this review, we present the clinical and histological features of LPG and its pathogenesis, and discuss the role of apoE abnormalities, especially apoE-Sendai and apoE2, in LPG and other renal lipidoses. Received: August 27, 2001 / Accepted: September 11, 2001     

A novel apolipoprotein E mutation, E2 (Arg25Cys), in lipoprotein glomerulopathy.

BACKGROUND: Lipoprotein glomerulopathy (LPG) is characterized by intraglomerular lipoprotein thrombosis and high plasma concentrations of apolipoprotein (apo) E. An apo E variant, apo E2 (Arg145Pro) Sendai, was recently identified in three patients with LPG. We detected a novel point mutation in the apo E gene in a patient with LPG, and we characterized the mutant apo E. METHODS: The propositus was a 32-year-old male patient on maintenance hemodialysis because of LPG. The mutation was detected by sequencing of genomic DNA from the patient and was confirmed by restriction fragment length polymorphism (RFLP) with Aor51HI. Recombinant apo E2 (Arg25Cys) Kyoto and normal apo E3 were expressed from COS-1 cells. Low-density lipoprotein (LDL) receptor-binding activities of the variants were determined in an in vitro competition assay. RESULTS: The propositus had the apo E phenotype E2/E4, as determined by isoelectric focusing, and the genotype epsilon3/epsilon4, as determined by RFLP with HhaI. Sequence analysis of amplified DNA showed a C to T transition, changing the codon for residue 25 from arginine to cysteine. The proband was a heterozygous carrier for apo E2 (Arg25Cys) Kyoto. A family study showed that the mother was a heterozygous carrier of apo E2 Kyoto and had dysbetalipoproteinemia, but no LPG. The pathophysiological effect of this mutation was investigated in vitro by binding studies of recombinant apo E2 Kyoto to LDL receptors on human fibroblasts. The ability of recombinant apo E2 Kyoto to displace LDL was reduced to 10% compared with recombinant apo E3. CONCLUSIONS: Apo E2 (Arg25Cys) Kyoto is a novel mutation of apo E that is etiologically related to LPG. However, our case indicates that the development of LPG may involve other genetic or environmental factors. Furthermore, our data suggest that arginine-25 of apo E plays an important functional role by influencing the receptor-binding ability of apo E.     

A novel 18-amino acid deletion in apolipoprotein E associated with lipoprotein glomerulopathy.

BACKGROUND: Lipoprotein glomerulopathy (LPG) is a novel disease characterized by proteinuria, lipoprotein thrombi in glomeruli, and an increased concentration of plasma apolipoprotein (apo) E. Previous studies have shown that a genetic disorder of apo E may be associated with the genesis of this disease. METHODS: An apo E mutation was analyzed in a 57-year-old Japanese male with LPG and systemic atherosclerotic complications. Apo E phenotypes were analyzed by isoelectric focusing and immunoblotting. Apo E genotypes were determined by restriction fragment isotyping with HhaI. Polymerase chain reaction (PCR) products of apo E coding region exons 3 and 4 were cloned into pT7Blue-T-vector and were sequenced. RESULTS: A novel apo E mutation was identified in this patient and his family. There was a discrepancy between an apo E phenotype (E1/3) and genotype (E3/3). Sequence analysis showed a 54 bp deletion in exon 4 of the apo E gene, causing the 18-amino acid deletion (Gln 156-Gly 173-->0). This deletion mutation was further confirmed by the detection of a short fragment of PCR-amplified DNA using polyacrylamide gel electrophoresis. The patient was a heterozygote with apo E1, and this mutation was determined to be the structural basis for the apo E1 phenotype. One of two daughters was a heterozygous carrier of apo E1, although she did not have proteinuria or atherosclerotic diseases. CONCLUSIONS: Apo E1 (Gln 156-Gly 173-->0) is a novel mutation of apo E that may be etiologically related to LPG and to the development of atherosclerosis. The result of this family study suggests that the occurrence of LPG may involve other genetic or environmental factors.     

Apolipoprotein E mutations: a comparison between lipoprotein glomerulopathy and type III hyperlipoproteinemia

Apolipoprotein E (ApoE) serves as a ligand for the low-density lipoprotein (LDL) receptor and cell surface receptors of the LDL receptor gene family. More than 10 different causative apoE mutations associated with lipoprotein glomerulopathy (LPG) have been reported. ApoE polymorphisms including three common phenotypes (E2, E3, E4), and a variety of rare mutations can affect blood cholesterol and triglyceride levels. The N-terminal domain of apoE is folded into a four-helix bundle of amphipathic α-helices, and contains the receptor-binding domain in which most apoE mutations that cause LPG or dominant mode of type III hyperlipoproteinemia (HL) are located. No single apoE mutation has been reported that causes both LPG and the dominant mode of type III HL.     

Fabry病家系的α-半乳糖苷酶A基因突变研究

目的 通过检测3个Fabry病家系基因突变类型明确基因诊断,并进行家系成员的基因型检测.方法 通过PCR和直接测序的方法,对3个Fabry家系的先证者及部分家系成员外周血DNA进行α-半乳糖苷酶A编码GLA基因7个外显子及其相邻内含子的DNA序列检测.结果 （1）先证者1的GLA基因7号外显子内1142位点发生碱基缺失（1142delG）,1142位碱基G的缺失导致蛋白质翻译在390位氨基酸提前终止,该突变国内外均未见报道;（2）先证者2的GLA基因6号外显子内902位点存在1个错义突变,碱基G被A取代,导致其编码的第301位氨基酸由精氨酸变为谷氨酰胺（902G＞A,R301Q）;（3）先证者3的GLA基因3号外显子内484位点存在1个错义突变,碱基T被C取代,导致其编码的第142位氨基酸由半胱氨酸变为精氨酸（484T＞C,C142R）.在3个家系的部分成员中进行基因检测,检出GLA突变基因携带者共6例,其中男性半合子1例,女性杂合子5例,突变类型均与相应先证者符合.100条正常X染色体对照中均未发现上述位点异常.结论 本研究在3个Fabry病家系中检出3种GLA基因突变,其中1142delG为新发现的突变,并在3个家系的部分家系成员中检出男性半合子1例,女性杂合子5例.     

经典型Bartter综合征家系CLCNKB基因突变分析

目的 研究一个经典型Bartter综合征家系CLCNKB基因突变情况。 方法 提取该家系各成员患者外周血淋巴细胞基因组DNA,应用PCR扩增CLCNKB基因全部外显子及侧翼序列,并直接测序检测突变。选取50例无亲缘关系的健康人作为对照。 结果 在患者中检测到1个杂合（错义）突变,其第4号外显子,第482位碱基T→G突变,造成第161位氨基酸由亮氨酸变为精氨酸（482T>G,L161R）;家系中母亲为杂合突变（L161R杂合突变）,父亲未发现突变;查阅国内外文献及人类基因突变数据库,L161R未见报道,属新发现的突变。 结论 发现了一种新的CLCNKB基因突变：L161R。     

Identification of the CLCN7 gene mutations in two Chinese families with autosomal dominant osteopetrosis (type II)

Here we report the identification of two different mutations in chloride channel 7 gene in two unrelated patients with autosomal dominant osteopetrosis type II. We determined that one patient (a 32-year-old woman) carried a heterozygous gene for a R767W mutation in exon 24, and another patient (a 17-year-old boy) carried a heterozygous gene for a novel frameshift mutation (Glu798FS) in exon 25. Recent studies have reported loss-of-function mutations in the chloride channel 7 (CLCN7) gene as a cause of autosomal dominant osteopetrosis type II (ADO-II). The identification of gene mutations in Chinese with ADO has not been reported previously. In this study, we identified mutations of the CLCN7 gene in two unrelated Chinese families with ADO-II. Two probands with ADO-II were diagnosed based on their bone characteristics on X-rays and their laboratory results. All 25 exons of the CLCN7 gene, including the exon–intron boundaries, were sequenced. We found in family 1 that the proband (a 32-year-old woman) was heterozygous for a CLCN7 mutation. The nonsynonymous mutation consisted of a heterozygous C/T transition at codon 2327 in exon 24, which resulted in an arginine (CGG)-to tryptophan (TGG) substitution at position 767 (R767W). The same heterozygous mutation (C/T) was determined in her father and son, who were asymptomatic with normal skeleton radiography. In family 2, we found that the proband (a 17-year-old boy) carried a novel frameshift mutation (Glu798FS) resulting from a G insertion between codon 60 and codon 61 in exon 25. The heterozygous –/G insertion is predicted to elongate the peptide of CLCN7 by 120 amino acids after position 797 amino acids. Similarly, some individuals of this family carried the same heterozygous mutation, but they are all asymptomatic. Furthermore, the R767W and Glu798FS mutations were not found in 100 unrelated controls. Our present findings suggest that the novel Glu798FS mutation in exon 25 and R767W in exon 24 in the CLCN7 gene were responsible for ADO-II in these Chinese patients.