Apolipoprotein E Guangzhou(arginine 150 proline):一种新的脂蛋白肾病相关的基因突变

目的通过分析1个包含4个脂蛋白肾病患者的家系所有成员的载脂蛋白E (apoE)基因,寻找脂蛋白肾病相关的apoE基因突变。方法收集该家系全部成员的病史,所有成员进行尿常规、血肌酐、血脂、血清脂蛋白检查。抽提基因组DNA,PCR法扩增所有成员的apoE第4外显子。割胶纯化PCR产物后直接测序,并将纯化的PCR产物亚克隆至pMD 18-T载体,挑菌落测序。结果4例脂蛋白肾病患者和1例无症状直系亲属均携带一种新的apoE点突变的杂合子,150号密码子发生点突变:CGC→CCC(由精氨酸变成脯氨酸),伴有血浆apoE的升高。结论apoE点突变apoE Guangzhou(arginine 150 proline)可能是该家系脂蛋白肾病发病的原因。     

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。     

常染色体显性遗传Alport综合征致病基因 COL4A4杂合剪接突变的致病性分析及基因型-表型关联分析

目的探讨常染色体显性遗传Alport综合征(autosomal dominant Alport syndrome, ADAS)致病基因COL4A4杂合剪接突变的致病机制及基因型与表型的关联, 以加深对COL4A4剪接突变的认识以及对ADAS表型异质性的理解。方法本研究为病例系列分析。从3家医院收集5个ADAS家系先证者及家系成员的临床资料。对从先证者中经全外显子组测序(whole exome sequencing, WES)发现的COL4A4杂合剪接变异, 通过RNA体内剪接或Minigene体外实验分析其对mRNA正常剪接的影响。结果在此5个ADAS家系患者中经WES发现了4个COL4A4杂合剪接变异位点。家系1、家系2、家系3和家系4中多数患者呈孤立性镜下血尿或合并微量蛋白尿, 个别患者合并显性蛋白尿、进入老年期后出现肾功能轻度减退。家系5中4例患者均呈快速肾功能进展, 于28～41岁进展至终末期肾病。家系1、家系2患者携带的c.735+3A>G和家系3患者携带的c.694-1G>C均可引起COL4A4的第12号外显子跳跃导致42 bp核苷酸框内缺失(c.694     

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。     

常染色体隐性遗传性远端肾小管酸中毒患儿ATP6V0A4和ATP6V1B1基因突变分析

目的 分析常染色体隐性遗传性远端肾小管酸中毒(rdRTA)患儿ATP6V0A4和ATP6V1B1基因的突变,进行基因型和表型的相关性研究.方法 PCR扩增基因组DNA,直接测序分析来自3个家系3例患儿的ATP6V0A4和ATP6V1B1基因的突变位点,选取不相关的100例健康人作为对照.结果 1例患儿携带ATP6V0A4基因的1个新的纯合无义突变(p.R194X);1例患儿携带ATP6V1B1基因1个新的杂合无义突变(p.R114X)和1个已经报道过的杂合突变p.I386fsX441;第3例患儿未发现以上2个基因的突变.结论 对中国rdRTA患者基因突变分析有利于了解该类疾病的基因型和表型的相关性,增强临床医生对该类疾病的认识和治疗.     

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。     

强直性脊柱炎一家系全外显子组测序分析

目的： 对一强直性脊柱炎（ankylosing spondylitis,AS）家系进行全外显子测序,筛选该家系的易感基因,为其发病机制提供理论依据。方法： 收集1组AS家系成员的临床资料,其中男性患者2例,年龄分别为48岁和18岁,病程分别为23年和4年。提取相关家系成员外周血DNA进行全外显子测序,测序结果与人类数据库比对,过滤掉同义突变及高频突变,整合家系成员单核苷酸非同义突变,寻找致病基因。结果： 家系成员共得原始数据80 G,数据具有较高质量值,通过对家系患者与正常人测序结果比对分析,同时经过多个生物数据库数据过滤,发现JAK2基因12号外显子上存在的杂合突变c.1709A>G（p.Tyr570Cys）为该家系的可能致病基因突变。另外,该家系MUC3A基因c.1151T>C突变可能是该家系患病成员肠道症状的原因之一。结论： 运用全基因组外显子测序寻找AS易感基因是可行的,JAK2基因c.1709A>G突变可能是导致该家系AS的致病突变及位点。     

中国人恶性高热家系蓝尼定受体-1基因的筛查

目的 筛查中国人恶性高热(MH)家系的蓝尼定受体-1(RYR1)基因.方法 提取确诊为MH的先证者及家系其他成员外周血白细胞的基因组DNA,采用PCR扩增先证者RYR1基因的部分外显子,并进行直接测序,采用Fok Ⅰ限制酶分析验证先证者及家系其他成员基因突变情况.结果 先证者RYR1基因第6 724位碱基C突变为T(c.6724 C＞T),所编码第2 206位氨基酸由苏氨酸变为甲硫氨酸(p.T 2206 M),此突变在白种人已报道;先证者的4个子女中有2个为该错义突变携带者,为MH易感者.结论 中国人MH易感者携带与白种人MH易感者相同的RYR1基因突变.     

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.     

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.     

Lipoprotein glomerulopathy in China

Lipoprotein glomerulopathy (LPG), a rare renal disease, is mainly reported in Japan and China. Chinese cases of LPG showed similar clinical and pathological features as reports from other countries. Three types of APOE mutation have been detected in those patients: APOE Maebashi (142Arg-144Leu-0) and APOE Kyoto (Arg25-Cys) were initially reported, and APOE Guangzhou (Arg150-Pro) is a novel mutation in Chinese patients with LPG. Asymptomatic carriers of all three mutations exist in families, but serum lipid and apolipoprotein E (apoE) levels are markedly elevated. In most of Chinese patients with LPG, long-term treatment with statins or bezafibrates appears to decrease proteinuria. LPG provides a disease model by which to explore pathogenic roles of apoE in common diseases.     

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.     

Association of a novel 3-amino acid deletion mutation of apolipoprotein E (Apo E Tokyo) with lipoprotein glomerulopathy.

Lipoprotein glomerulopathy (LPG) is a newly recognized renal disease characterized by abnormal lipoprotein deposition in the glomeruli, dysbetalipoproteinemia, and a high level of plasma apolipoprotein (apo) E. We identified a novel apo E mutation in a 56-year-old Japanese male with LPG. Although the plasma cholesterol and triglyceride levels were normal, the levels of intermediate-density lipoprotein cholesterol and apo E were elevated to 13 mg/dl (0.336 mmol/l; 4.2+/-2.9 mg/dl, mean +/- SD, in 12 normolipidemic controls) and 9.2 mg/dl, respectively. Biochemical analysis revealed an unusual apo E phenotype (E1/3). Apo E genotyping using DNA digested by a restriction enzyme (HhaI) identified a 66-bp fragment which was not seen with any of the common alleles. Sequence analysis of the amplified genomic DNA fragments showed a 9-bp deletion in exon 4 of the apo E gene resulting in a 3-amino acid deletion (residues 141-143). This novel mutation involves the region of the apo E molecule known to be critically involved in binding to its receptor, and this may well transform the apo E molecule, an inefficient ligand, to its receptor(s). How this mutations causes glomerular damage remains to be determined.     

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。     

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     

Menin mutations in the diagnosis and prediction of multiple endocrine neoplasia type 1

Introduction: Multiple endocrine neoplasia type 1 (MEN1) is an autosomal dominant disorder characterized by the development of multiple endocrine adenomas, typically in the pancreas, anterior pituitary, and parathyroid glands. The disease is associated with germ-line mutations of the menin gene, a putative tumor-suppressor gene located on human chromosome 11q13. Methods: To facilitate the diagnosis and prediction of MEN1 in patients and their relatives, we developed a molecular two-step strategy to screen for menin gene mutations. DNA fragments covering the entire menin coding sequence are generated from patient cDNA by polymerase reaction (PCR) and subsequently analyzed by single-strand conformational polymorphism electrophoresis (SSCP). Fragments with aberrant SSCP migration are DNA-sequenced to directly characterize menin mutations. In a second diagnostic step, genomic DNA of healthy relatives of the corresponding MEN1 index patient is analyzed by PCR, with only the specific exon amplified harboring the family-specific mutation. Mutation-specific restriction enzyme digestion of this PCR product finally allows the identification of mutation carriers through pathological restriction fragment patterns. Results: Using this approach, we identified an in-frame deletion mutation (Δ Tyr Met) located in menin exon 4 (codon 227 – 228) that co-segregates with the disease phenotype in a large MEN1 family from Southern Germany. Conclusion: It is likely that the direct molecular analysis of menin gene mutations will replace the genetic and biochemical screening tests currently used in the clinical management of MEN1 families. In addition, these studies may provide clues to the tumor biology of both sporadic and MEN1-associated endocrine adenomas. Received: 2 January 1998