中国汉族两个马凡综合征家系FBN1基因的一个新突变

目的 对两个常染色体显性遗传的马凡综合征家系进行基因诊断,并探讨其临床特点。方法 完成家系调查和系谱分析,通过聚合酶链式反应和直接测序的方法对收集到的两个家系中的成员进行原纤维蛋白1(fibrillin 1,FBN1)基因的突变检测。结果 两个家系均呈常染色体显性遗传模式。对两个家系成员进行FBN1基因突变检测发现,在两个家系的患者中发现一个相同的突变位点,即FBN1第27号外显子3463位碱基由G变为A( 3463G＞A),导致原纤维蛋白1第1 155位氨基酸由天冬氨酸变为天冬酰胺(Asp 1155Asn),而两个家系的正常成员及选取的100名健康对照中均未发现该突变位点。结论 先证者均符合Ghent标准诊断为马凡综合征,基因诊断发现两家系中相同的突变位点3463G＞A为中国汉族马凡综合征患者中首次报道。     

TBX5基因在中国人心手综合征患者中的突变分析

目的 检测中国心手综合征患者ＴＢＸ５基因突变。方法 利用单链构象多态性分析和测序方法,对７个心手综合征患者家系进行ＴＢＸ５基因突变检测。结果 发现３个家系患者有单链构象多态性改变,经测证实为３个新位点基因突变：１个心手综合征家系患者为ＴＢＸ５基因单个碱基缺失引起移码突变,２个心手综合征家系为ＴＢＸ５基因碱基替换引起错义突变。结论中国人ＴＢＸ５基因突变可引起心手综合征。     

应用下一代半导体靶向测序技术检测Marfan综合征致病突变

目的 应用Ion Torrent PGM半导体测序仪和Ion AmpliSeqTMInherited Disease Panel对3例马凡综合征(Marfan syndrome,MFS)进行致病基因突变检测,明确其致病突变,并评价下一代半导体靶向测序诊断复杂单基因遗传病的效果.方法 在知情同意的基础上采集3例MFS患者及1名正常志愿者外周血,提取基因组DNA,经多重PCR扩增富集目的基因片段.每个样本用特异性序列标签进行标记后,应用Ion One Touch系统进行模板制备、乳化PCR及磁珠颗粒富集;最后用318半导体测序芯片进行高通量测序.用Ion Torrent Suite 3.2软件进行序列比对及SNPs和Indels提取,再用dbSNP 137数据库过滤得到SNPs和indels,剩余的可疑突变经Sanger法测序验证.结果 用一张318芯片得到855.80Mb的总数据量,4个样本的平均测序深度均达到100×以上,对目的区域的覆盖度在98％以上.数据经软件分析及数据库过滤后,在3例MFS患者中分别得到3个FBN1基因可疑突变,并经Sanger法测序验证,一个为已报道FBN1基因错义突变(p.E1811K),另外两个为新发现的突变,包括一个无义突变(p.E2264X),1个插入突变(p.L871FfsX23).结论 在3例MFS患者中都成功检出FBN1基因致病突变,表明半导体靶向测序可对复杂单基因遗传病进行高效、准确的基因诊断.     

一个X-连锁视网膜色素变性中国家系的RPGR基因的新突变

目的 对中国人X-连锁视网膜色素变性一家系进行分子遗传学检测，报告RPGR基因突变。方法 首先对该家系X染色体进行致病基因的连锁分析，然后用单链构象多态性技术和直接DNA测序方法进行基因突变分析。结果 连锁分析在多态性微卫星遗传标记DXSS012和DXS8025产生正的Lod值分别为2．41（Zmax=2．40，θ=0）和1．26。进一步单倍型分析确定该家系致病基因位于Xp21．1，与RP3连锁。用RPGR基因突变分析，在外显子ORF15＋483－484发现GA缺失，引起阅读框架的改变，该基因缺失突变在家系中共分离。结论 报告了中国人X-连锁视网膜色素变性RPGR基因外显子ORF15＋483-484的GA缺失突变，丰富了中国人RPGR基闪突变谱，为今后研究X-连锁视网膜色素变性的基因奠定基础。     

FBN1新生突变引起的马凡综合征及其基因型-表型的关联研究

 目的: 本研究对2个不同马凡综合征(Marfan syndrome)的小家系进行致病基因FBN1的编码区和剪切位点突变检测,以寻找致病的突变,并初步探索马凡综合征基因型-表型的关联。方法: 通过临床检查、实验室检查及心脏超声检查确诊2个无血缘关系的家庭中原疑似为马凡综合征的3例患者。运用新一代测序对家系1的疑似患者行FBN1基因的全外显子组测序,并对检出的致病性遗传变异进行Sanger验证及在所有家系成员中验证;对于家系2的存活成员,本研究直接进行PCR扩增FBN1基因的所有编码区及剪切位点,对产物进行直接Sanger测序。另外在50个正常对照中对新发现的突变位点进行基于PCR产物的测序分析,以排除多态性;并对实验结果行生物信息学分析。结果: 所有存活的疑似患者均确诊为马凡综合征。在家系1中,我们检测到了一个FBN1基因数据库中尚未报道的新突变c.4685G>A(p.Cys1562Tyr),并且患者父母和同胞姐姐均未检测到此变异,故此突变为一个新生突变。该错义突变使第1562位上极性中性的含硫的半胱氨酸被极性中性的含羟苯基的酪氨酸所替代,影响了fibrillin-1蛋白一个TGF-β结合结构域,导致蛋白质的二级结构发生改变。家系2含父母及一对同卵双胎患者,其中一患者已去世。我们在存活患者检测到1个FBN1基因的已报道致病突变c.3706T>C(p.Cys1236Arg),该突变在患者父母中不存在,故也为新生突变。结论: 本文报道了一例FBN1基因的新突变及另一例由FBN1基因已知突变引起的马凡综合征,二者皆为新生突变,并在家系中进行了基因型-表型的比较,表明家系1的新突变可能与经典马凡综合征的表型相关,而家系2的已知突变确和新生儿重症马凡综合征表型相关。     

马凡综合征两种新的原纤维蛋白-1基因突变

目的对9例马凡综合征(Marfansyndrome,MFS)患者的原纤维蛋白-1(fibrillin-1,FBN1)基因进行突变筛查,以发现新的FBN1基因突变。方法应用变性高效液相色谱法对MFS患者FBN1基因65个外显子中的35个进行突变筛查,对变性高效液相色谱图形异常的PCR扩增片段用DNA测序鉴定突变位置及性质,并用等位基因特异性PCR以及限制性片段长度多态性分析等方法进一步证实突变。结果在两例MFS患者中发现两种新的FBN1基因突变。其中一种为第34外显子4307～4308位4个碱基TCGT的插入突变(4307insTCGT),另一种为第43外显子5309位的点突变5309G>A。结论FBN1基因移码突变(4307insTCGT)与点突变(5309G>A)分别是这两例MFS患者的发病原因。     

一种新的鸟氨酸氨基甲酰转移酶基因突变E122G的鉴定

目的：测定1个迟发型鸟氨酸氨基甲酰转移酶（ornithine transcarbamylase,OTC)缺乏症的中国汉族家系的基因突变及患者OTC基因外显子序列。方法：用聚合酶链反应－单链构象多态性结合PCR产物直接测，鉴定其突变类型。结果：在该家系患者OTC基因中确认了1个新的错义突变E122G，位于第4外显子的保守残基（GAA→GGA）。结论：OTC基因中E122G突变引起迟发型OTC缺乏症，是1个尚未报道的突变。     

用PCR-STR、ASPCR、PCR-SSCP技术对PKU患者进行产前基因诊断

为探讨苯丙酮尿症的产前诊断途径，应用３种基因诊断方法从不同角度对１６个苯丙酮尿症家系的苯丙氨酸羟化酶基因进行了分析。（１）聚合酶链反应－短串联重复序列连锁分析：通过扩增苯丙氨酸羟化酶基因内含子３中含短串联重复序列的ＤＮＡ区域，并进行扩增片段长度多态性分析，结果表明：能进行准确产前基因诊断者占６２．５％，能进行５０％排除诊断的家系占３７．５％，１例风险胎儿的产前基因诊断和另一家系生后１５天婴儿的症前诊断获得成功；（２）多重等位基因特异性聚合酶链反应，对苯丙氨酸羟化酶基因中两种最常见的突变点分析结果为：Ａｒｇ２４３Ｇｌｕ检出率为１９％，Ａｒｇ４１３Ｐｒｏ检出率为１２％；（３）应用聚合酶链反应－单链构象多态性分析，不仅检出３例已知的基因突变，还发现２例可能为新的基因突变，诊断率达到４０％。为苯丙酮尿症的产前基因诊断提供了依据。     

神经丝轻链基因在腓骨肌萎缩症中的突变分析

目的：探讨神经丝轻链基因（neurofilament-light gene,NF-L）在中国人腓骨肌萎缩症（Charcot-Marie-Tooth disease,CMT）中的突变特点。方法：应用聚合酶链反应－单链构象多态性技术结合DNA序列分析方法，对32个来自全国5省汉族的CMT家系先证者进行了NF－L基因的突变分析。结果：32例先证者中只有1例患者出现异常条带，经DNA测序证实该患者在NF－L基因的外显子3发生了1329C→T碱基改变，由于编码的氨基酸未改变，均为酪氨酸（Tyr），为一种同义突变。结论：NF－L基因突变可能在中国人的腓骨肌萎缩症患者中少见。     

汉族FBN1基因突变所致马凡综合征一家系心血管表现的性别差异

 目的 通过分析一汉族马凡综合征家系多名患病成员的心血管系统受累表现与原纤维蛋白-1（FBN1）基因突变位点,提高对马凡综合征临床表现性别差异的认识。方法 收集一马凡综合征家系成员的心血管系统受累资料。在知情同意基础上采集外周血,提取基因组DNA,PCR扩增FBN1基因全部65个外显子及外显子内含子交界区,产物纯化后行Sanger法测序。结果 该家系共13名患者,所有9名存活患者均携带有c.5431G>A (p.Glu1811Lys)突变,而家族中正常个体无此突变。9名男性中4例猝死,另4名存在升主动脉扩张,1名表现为扩张性心肌病;4名女性中因心力衰竭死亡1例,另3名存在严重二尖瓣病变而无升主动脉扩张。结论 FBN1突变所致马凡综合征心血管系统受累即使是在同一家系中亦可表现为显著的差异。     

Comprehensive molecular screening of the FBN1 gene favors locus homogeneity of classical Marfan syndrome

In order to estimate the contribution of mutations at the fibrillin-1 locus (FBN1) to classical Marfan syndrome (MFS) and to study possible phenotypic differences between patients with an FBN1 mutation vs. without, a comprehensive molecular study of the FBN1 gene in a cohort of 93 MFS patients fulfilling the clinical diagnosis of MFS according to the Ghent nosology was performed. The initial mutation screening by CSGE/SSCP allowed identification of an FBN1-mutation in 73 patients. Next, sequencing of all FBN1-exons was performed in 11 mutation-negative patients, while in nine others, DHPLC was used. This allowed identification of seven and five additional mutations, respectively. Southern blot analysis revealed an abnormal hybridization pattern in one more patient. A total of 23 out of the 85 mutations identified here are reported for the first time. Phenotypic comparison of MFS patients with cysteine-involving mutations vs. premature termination mutations revealed significant differences in ocular and skeletal involvement. The phenotype of the eight patients without proven FBN1 mutation did not differ from the others with respect to the presence of major cardiac, ocular, and skeletal manifestations or positive familial history. Most likely, a portion of FBN1-mutations remains undetected because of technical limitations. In conclusion, the involvement of the FBN1-gene could be demonstrated in at least 91% of all MFS patients (85/93), which strongly suggests that this gene is the predominant, if not the sole, locus for MFS.     

Fibrillin gene (FBN1) mutations in Japanese patients with Marfan syndrome

Marfan syndrome (MFS; MIM #154700) is a connective tissue disorder characterized by cardiovascular, skeletal, and ocular abnormalities. The fibrillin-1 gene (FBN1; MIM no. 134797) on chromosome 15 was revealed to be the cause of Marfan syndrome. To date over 137 types of FBN1 mutations have been reported. In this study, two novel mutations and a recurrent de-novo mutation were identified in patients with MFS by means of single-strand conformational polymorphism (SSCP) analysis. The two novel mutations are a 4-bp deletion at nucleotide 2820-2823 and a G-to-T transversion at nucleotide 1421 (C474F), located on exon 23 and exon 11, respectively. A previously reported mutation at the splicing donor site of intron 2 (IVS2 G + 1A), which is predicted to cause exon skipping, was identified in a sporadic patient with classical MFS. Received: November 1, 1999 / Accepted: November 9, 1999     

An FBN1 Deep Intronic Mutation in a Familial Case of Marfan Syndrome: An Explanation for Genetically Unsolved Cases?

Marfan syndrome (MFS) is caused by mutations in the FBN1 (fibrillin‐1) gene, but approximately 10% of MFS cases remain genetically unsolved. Here, we report a new FBN1 mutation in an MFS family that had remained negative after extensive molecular genomic DNA FBN1 testing, including denaturing high‐performance liquid chromatography, Sanger sequencing, and multiplex ligation‐dependent probe amplification. Linkage analysis in the family and cDNA sequencing of the proband revealed a deep intronic point mutation in intron 56 generating a new splice donor site. This mutation results in the integration of a 90‐bp pseudo‐exon between exons 56 and 57 containing a stop codon, causing nonsense‐mediated mRNA decay. Although more than 90% of FBN1 mutations can be identified with regular molecular testing at the genomic level, deep intronic mutations will be missed and require cDNA sequencing or whole‐genome sequencing.     

Identification of 29 novel and nine recurrent fibrillin-1 (FBN1) mutations and genotype-phenotype correlations in 76 patients with Marfan syndrome

Marfan syndrome (MFS) is an autosomal-dominant disorder of the fibrous connective tissue that is typically caused by mutations in the gene coding for fibrillin-1 (FBN1), a major component of extracellular microfibrils. The clinical spectrum of MFS is highly variable and includes involvement of the cardiovascular, skeletal, ocular, and other organ systems; however, the genotype-phenotype correlations have not been well developed. Various screening methods have led to the identification of about 600 different mutations (FBN1-UMD database; www.umd.be). In this study we performed SSCP and/or direct sequencing to analyze all 65 exons of the FBN1 gene in 116 patients presenting with classic MFS or related phenotypes. Twenty-nine novel and nine recurrent mutations were identified in 38 of the analyzed patients. The mutations comprised 18 missense (47%), eight nonsense (21%), and five splice site (13%) mutations. Seven further mutations (18%) resulted from deletion, insertion, or duplication events, six of which led to a frameshift and subsequent premature termination. Additionally, we describe new polymorphisms and sequence variants. On the basis of the data presented here and in a previous study, we were able to establish highly significant correlations between the FBN1 mutation type and the MFS phenotype in a group of 76 mutation-positive patients for whom comprehensive clinical data were available. Most strikingly, there was a significantly lower incidence of ectopia lentis in patients who carried a mutation that led to a premature termination codon (PTC) or a missense mutation without cysteine involvement in FBN1, as compared to patients whose mutations involved a cysteine substitution or splice site alteration.     

Three novel mutations of the fibrillin-1 gene and ten single nucleotide polymorphisms of the fibrillin-3 gene in Marfan syndrome patients

Marfan syndrome (MFS) is an autosomal dominant disorder of the extracellular matrix. Allelic variations in the gene for fibrillin-1 (FBN1) have been shown to cause MFS. To date, over 550 mutations have been identified in patients with MFS and related connective tissue diseases. However, about a half of MFS cases do not possess mutations in the FBN1 gene. These findings raise the possibility that variants located in other genes cause or modify MFS. To explore this possibility, firstly we analyzed FBN1 allelic variants in 12 Japanese patients with MFS, and secondly we analyzed fibrillin-3 gene (FBN3) in patients without FBN1 mutations using conformation sensitive gel electrophoresis (CSGE) and direct sequencing analysis. We identified three novel FBN1 mutations and ten FBN3 single nucleotide polymorphisms (SNPs). In this report, we could not detect a responsible mutation of the FBN3 gene for MFS. Although the number of the cases in this report is small, at least these results suggest that disease-causing mutations in exon regions of the FBN3 gene are very rare in MFS.Nucleotide sequence data reported are available in the DDBJ/EMBL/GenBank databases under the accession numbers: AB177797, AB177798, AB177799, AB177800, AB177801, AB177802, AB177803     

Detection of thirty novel FBN1 mutations in patients with Marfan syndrome or a related fibrillinopathy

Marfan syndrome (MFS) is a disorder of the extracellular matrix caused by mutations in the gene encoding fibrillin-1 (FBN1). Recent studies have illustrated the variability in disease severity and clinical manifestations of MFS. Useful genotype-phenotype correlations have been slow to emerge. We screened 57 unrelated patients with MFS or a Marfan-like phenotype using a combination of SSCP and/or DHPLC. We detected 49 different FBN1 mutations, 30 (62%) of which were novel. The mutations comprised 38 substitutions (78%), 10 deletions (20%), and one duplication (2%). There were 28 missense (57%), nine frameshift (18%), eight splice site (16%), and four nonsense mutations (8 %). Genotype-phenotype analysis revealed that patients with an identified FBN1 mutation were more likely to have ectopia lentis and cardiovascular complications compared to those without an identifiable mutation (relative risks of 4.6 and 1.9, respectively). Ectopia lentis was also found to be more prevalent in patients whose mutations involved a cysteine substitution (relative risk 1.6) and less prevalent in those with premature termination mutations (relative risk 0.4). In our hands, we achieved 93% mutation detection for DHPLC analysis of patients who fulfilled the Ghent criteria. Further analysis of detailed clinical information and mutation data may help to anticipate the clinical consequences of specific FBN1 mutations.     

Eight novel mutations of the FBN1 gene found in Japanese patients with Marfan syndrome

Marfan syndrome (MFS), an autosomal dominant connective tissue disorder, is caused by mutations in the gene encoding fibrillin 1 (FBN1). The clinical spectrum and severity of MFS disorder varies greatly both between and within families. Since there have been only a few reports on the relationship between FBN1 genotypes and clinical phenotypes in Japanese patients, the FBN1 gene was analyzed in 27 Japanese patients diagnosed with MFS. The nucleotide sequence of the 65 exons of the FBN1 gene was analyzed by PCR and direct sequencing. We have identified six polymorphisms and nine mutations including: four missense mutations (C1652Y, Q2054P, D2127Y, C2221R) in six patients, three nonsense mutations (R215X, S813X, R2220X) in three patients, and two frameshift mutations (2567insT, 7790insT) in three patients. Six of these nine mutations were in the calcium-binding epidermal growth factor-like domains all causative mutations detected except for C2221R were novel. It has been reported that the severe phenotypes of infantile MFS correlate with mutations in the mid region of FBN1, however, mutations were not detected in this region in the population analyzed in this study. Our results suggest that the location of the mutation is not the sole determinant of phenotypic severity; rather there is some difference in the genetic basis of MFS between Japanese and Caucasian populations.