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...     

原发性红斑肢痛症SCN9A基因突变研究

目的鉴定一重型原发性红斑肢痛症家系SCN9A基因的突变情况。方法收集家系临床资料,提取家系及健康对照者外周血DNA,PCR扩增SCN9A基因全部外显子及其侧翼序列并测序。结果发现先证者SCN9A基因9号外显子存在c.1185C→G杂合突变,引起N395K氨基酸改变。先证者的健康母亲和50例无血缘关系的健康对照者中未见此突变。结论 SCN9A基因的这种错义突变是引起患者临床表型的特异突变。     

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。     

GNAS新错义突变导致D/E型短指畸形

目的对我国一个短指家系进行致病基因进行鉴定。方法应用全外显子测序对该短指家系进行基因突变检测,并用Sanger测序对突变位点进行验证。结果该家系共有3名患者,均为短指表型,符合常染色体显性遗传。全外显子检测提示患者均存在GNAS基因(NM_001077488:exon13:c.A1145T:p.D382V)杂合错义突变,Sanger测序验证与全外显子结果一致。结论 GNAS基因第13号外显子上c.A1145T杂合错义突变是该短指家系的致病原因。     

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.     

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     

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。     

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.     

家族性VHL综合征患者家系突变类型及二次打击的分析

目的 分析家族性希佩尔·林道(VHL)综合征患者家系的突变类型、临床特征，探索VHL患者发病的分子遗传学机制，及其二次打击的类型。方法 收集VHL患者家系资料，并在先证者外周血和肾癌标本中提取DNA,通过高通量测序(NGS)检测患者VHL基因胚系突变位点，并通过UCSCXena数据库、甲基化特异性PCR法(MSP)、微卫星稳定性检测来明确患者的第二次打击位点。结果 NGS测序发现胚系突变位点位于第3外显子上的c.500G>A R167Q突变，依据UCSCXena数据库、MSP分析、微卫星稳定检测结果提示患者存在单核苷酸多态性，未见明确杂合性缺失、甲基化、体系突变。结论 第3外显子的胚系突变是该家族性肾癌先证者出现临床特征的基础，第二次打击位点是疾病发生的关键，对家族性肾癌家系的胚系突变和第二次打击位点的研究对患者及患者家系的诊断及治疗有指导意义。数据库的利用能够为家族性肾癌突变、甲基化位点的筛选进行指导。     

汉族人群青少年特发性脊柱侧凸家系中存在复合型杂合DOCK9基因突变

目的探讨青少年特发性脊柱侧凸(AIS)家系中可能的致病基因突变。方法对一个汉族AIS家系的3名成员(先证者及其表型正常的父母)进行全基因组测序,筛选潜在的致病基因突变,并通过Sanger测序验证所有发现的突变。结果在AIS家系中发现DOCK9基因存在复合型杂合基因突变c.3259TC(p.F1087L)和c.2465AG(p.Y822C)。患者的父母是未出现AIS表型的突变基因携带者,父亲携带c2465AG突变,而母亲携带c.3259TC突变。结论复合型杂合DOCK9基因突变可能导致AIS的发生,其在AIS发生机制中的作用有待于进一步探索。     

Multiple epiphyseal dysplasia: A clinical and genetic study of 12 cases in a Swedish 6-generation family

Background Multiple epiphyseal dysplasia (MED) is a common genetically and clinically heterogeneous skeletal dysplasia characterized by early-onset osteoarthritis, mainly in the hip and knee, and mild-to-moderate short stature. Here we report on a 6-generation MED family with 17 affected members.Method The clinical and radiographic data on the 12 affected members still living were scrutinized. A structured inquiry comprising state of health and MED-related symptoms since birth up to the present time and the osteoarthritis outcome (KOOS) questionnaire were sent to all living family members with MED. The 5 known gene loci for autosomal dominant MED were analyzed for linkage, using fluorescence-labeled microsatellite markers. Linkage was ascertained with markers close to the COL9A2 gene, which was analyzed for mutations by sequencing.Results We identified an exon 3 donor splice mutation in the COL9A2 gene in all affected family members. Clinical, radiographic, and questionnaire data from affected family members suggested that MED caused by COL9A2 mutations starts in early childhood with knee pain accompanied by delayed ossification of femoral epiphyses. The disease then either stabilizes during puberty or progresses with additional joints becoming affected; joint surgery might be necessary. The progression of the disease also affects muscles, with increasing atrophy, resulting in muscle fatigue and pain. Muscular atrophy has not been reported earlier in cases with COL9A2 mutations.Interpretation In a patient with clinically suspected or verified MED, it is important to perform DNA-based analysis to identify a possible disease-causing mutation. This information can be used to carry out genetic risk assessment of other family members and to achieve an early and correct diagnosis in the children.     

Multiple epiphyseal dysplasia – report of two families

Multiple epiphyseal dysplasia (MED) is a relatively uncommon inherited disorder of epiphyseal maturation. Affected individuals may have a degree of short-limbed dwarfism, short stubby digits, and stiff or painful joints. We report two families of MED and emphasize the variations of joint involvement. Ten out of 34 members in family A and 13 out of 39 members in family B were suspected of having MED by questionnaire. Radiological examination was done for 3 out of the 10 members in family A and 6 out of the 13 members in family B. In both families, the epiphyseal disturbances in the skeleton were bilaterally symmetric and involved several joints. Apparent dwarfism, short stubby digits and spinal involvement were not observed. The degree and the pattern of affected joints were different in the two families and even among members of the same family. In family A, the knee joint was commonly affected, followed by the ankle joint. The deformity of the joints was mild and caused only slight disability. No apparent hip lesion was present. In family B, the hip joint was predominantly affected, followed by the knee and ankle joints, and the deformity was severer than that in family A. These observations suggest that MED is a group of heterogeneous disorders. Received: 5 July 1999     

常染色体隐性遗传Alport 综合征一家系COL4A3及COL4A4基因突变分析

目的 通过对有近亲婚配史的Alport综合征一家系Ⅳ型胶原α3和α4链的 COL4A3/COL4A4 基因分析，明确常染色体隐性遗传Alport综合征的基因突变,为该病的基因诊断和家系遗传咨询提供更为全面的理论基础｡ 方法 PCR扩增先证者DNA COL4A3/COL4A4 基因的共98个外显子，经直接测序，寻找突变位点，对有意义的突变经限制性内切酶AvaⅡ酶切在家系中分析验证｡ 结果 在该患者中共发现1个错义突变和10个序列变异｡其中在COL4A3 基因上发现一个位于42号外显子上的错义突变 G3725A,导致蛋白质Gly1242Asp的突变｡错义突变在患者中是纯合子，携带者中是杂合子，其他正常家系成员及筛查100条正常人染色体，未发现该突变｡10个序列变异为单核苷酸多态性改变｡ 结论 报道了一个国内较少见的常染色体隐性遗传Alport 综合征家系，同时经基因突变筛查发现Ⅳ型胶原α3链的一个新的致病性的基因突变｡     

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。     

Metatropic Dysplasia of Nonlethal Variant in a Chinese Child – A Case Report

Metatropic dysplasia (MD), is a rare skeletal dysplasia occurring predominantly in infants characterized by a distinctive long torso and short limbs; it is as a result of mutations in the TRPV4 gene. However, a clear distinction between various forms of skeletal dysplasias caused by the transient receptor potential vanilloid 4 (TRPV4) gene is difficult but could be achieved by a combination of gene sequencing, medical and radiological criteria. We hereby report a case of a 14‐month old girl who presented with an abnormal stature. The diagnosis of nonlethal MD was confirmed by X‐ray with dumbbell‐shaped long bones, platyspondyly, and delayed carpal ossification, as well as broadened pelvis with marginally widened ilia, epiphyseal plates, and slightly flattened acetabula. Furthermore, gene sequencing confirmed gene mutation on exon 15 of the TRPV4 gene with a heterozygous missense mutation (c.2396C > T), but no mutation was present in her parents. Our findings recorded metatropic dysplasia with the c.2396C > T mutation in the TRPV4 gene in China. This mutation caused changes in amino acid of TRPV4, which can induce growth retardation in children.     

A novel VCP mutation as the cause of atypical IBMPFD in a Chinese family

IntroductionInclusion-body myopathy (IBM) with Paget's disease of bone (PDB) and frontotemporal dementia (FTD), designated as IBMPFD, is a rare, autosomal dominant disorder (MIM 605382). IBMPFD is caused by mutations in the gene that encode valosin-containing protein (VCP). We investigated a Chinese family in which multiple members were diagnosed with PDB and suffered from weakness of the limbs. However, no members of this family were diagnosed with FTD. We made a preliminary diagnosis of PDB, but failed to identify an SQSTM1 mutation in any of the patients. We used whole-exome sequencing to identify the pathogenic gene mutation affecting the Chinese male proband.Materials and methodsAltogether, 254 subjects, including one 56-year-old male proband, four affected, related individuals and additional nine family members from a non-consanguineous Chinese family, and 240 healthy donors were recruited and genomic DNA was extracted. All eight exons and the exon–intron boundaries of the SQSTM1 gene were amplified by polymerase chain reaction (PCR) and directly sequenced in five patients (II13, II4, II5, II8, II9). Using whole-exome sequencing, we identified a novel mutation in VCP as the disease-causing mutation. We confirmed the result by sequencing a 500-bp region of the promoter and the coding region of VCP in all 254 of the participants using Sanger sequencing.ResultsNo mutation in the SQSTM1 gene was identified in the five patients examined using direct Sanger sequencing. However, through whole-exome sequencing we were able to identify a novel missense mutation in exon 3 of the VCP gene (p.Gly97Glu) in the Chinese male proband. This mutation was confirmed using Sanger sequencing. The proband, four affected individuals and three unaffected individuals carried this mutation. We were able to correctly diagnose the patients with atypical IBMPFD. Structural analysis of the p.Gly97Glu mutation in the VCP protein showed that the affected amino‐acid is located in the interface of the protein. This abnormality may therefore interfere with protein function.ConclusionsThis is the first report of a family from China with IBMPFD. A novel VCP mutation was found as the cause of atypical IBMPFD in a Chinese family. Our findings confirm that VCP gene mutations can be a pathogenic cause of IBMPFD.     

X-linked spondyloepiphyseal dysplasia tarda: molecular cause of a heritable disorder associated with early degenerative joint disease

Spondyloepiphyseal dysplasia tarda (SEDT) is a genetically heterogeneous disorder often associated with the early onset of osteoarthrosis. The X-linked recessive form (SEDL) affects men and is characterized by reduced height, arm span exceeding total height, and barrel chest deformity. The radiographic phenotype comprises a hump-shaped deformity of vertebral bodies and mild epiphyseal dysplasia of the femoral head associated with early signs of hip arthrosis. The disorder is caused by mutations in the SEDL (or sedlin) gene on Xp22.12-p22.31. In 4 male patients from a German family, we identified a new nonsense mutation in SEDL exon 4 (C74A). The carrier status for the mutation could be confirmed in 2 women of the family, both of whom show no obvious signs of bone and joint diseases. SEDT should be kept in mind as a differential diagnosis in men with early "primary" bilateral osteoarthrosis.     

X-linked spondyloepiphyseal dysplasia tardaMolecular cause of a heritable disorder associated with early degenerative joint disease

Spondyloepiphyseal dysplasia tarda (SEDT) is a genetically heterogeneous disorder often associated with the early onset of osteoarthrosis. The X-linked recessive form (SEDL) affects men and is characterized by reduced height, arm span exceeding total height, and barrel chest deformity. The radiographic phenotype comprises a hump-shaped deformity of vertebral bodies and mild epiphyseal dysplasia of the femoral head associated with early signs of hip arthrosis. The disorder is caused by mutations in the SEDL (or sedlin) gene on Xp22.12-p22.31. In 4 male patients from a German family, we identified a new nonsense mutation in SEDL exon 4 (C74A). The carrier status for the mutation could be confirmed in 2 women of the family, both of whom show no obvious signs of bone and joint diseases. SEDT should be kept in mind as a differential diagnosis in men with early "primary" bilateral osteoarthrosis