Mutation analysis of seven patients with Waardenburg syndrome北大核心CSCD

Objective: To perform genetic analysis for 7 patients with Waardenburg syndrome. Methods: Potential mutation of MITF, PAX3, SOX10 and SNAI2 genes was screened by polymerase chain reaction and direct sequencing. Functions of non-synonymous polymorphisms were predicted with Polyphen2 software. Results: Seven mutations, including c. 649-651delAGA (p. R217del), c. 72delG (p. G24fs), c. 185T>C (p. M62T), c. 118C>T (p. Q40X), c. 422T>C (p. L141P), c. 640C>T (p. R214X) and c. 28G>T (p. G43V), were detected in the patients. Among these, four mutations of the PAX3 gene (c. 72delG, c. 185T>C, c. 118C>T and c. 128G>T) and one SOX10 gene mutation (c. 422T>C) were not reported previously. Three non-synonymous SNPs (c. 185T>C, c. 128G>T and c. 422T>C) were predicted as harmful. Conclusion: Genetic mutations have been detected in all patients with Waardenburg syndrome. © 2016, West China University of Medical Sciences. All rights reserved.     

一个先天性小眼畸形家系的全外显子组测序分析及产前诊断

目的分析1个先天性小眼畸形家系的临床表型及遗传学病因。方法应用高通量测序技术对先证者及其父母进行全外显子组测序,筛选候选致病位点,对其家系进行Sanger测序验证,并通过羊水穿刺和Sanger测序为先证者母亲提供产前诊断。结果全外显子组测序和Sanger测序发现家系中的3例患者均携带OTX2基因c.289C>T(p.R97*)杂合变异,先证者母亲亦携带该变异,但无小眼畸形。先证者的父亲、舅母和胎儿未携带上述变异。结论OTX2基因c.289C>T(p.R97*)杂合变异很可能是该家系的发病原因。上述诊断将有助于该家系的遗传咨询和产前诊断。     

一个Waardenburg综合征Ⅱ型家系SOX10基因的突变分析CSCD

目的对1例Waardenburg综合征Ⅱ型先证者及其家系成员进行SOX10基因的突变分析,探讨其可能的分子生物学致病原因。方法抽提先证者及其家系成员的外周血基因组DNA,芯片捕获高通量测序方法对MITF、PAX3、SOX10、SNA12、END3和ENDRB基因的全部外显子及其侧翼序列进行检测。根据高通量测序结果,对先证者及其父母进行突变位点的Sanger测序验证分析。结果Sanger测序结果显示先证者存在SOX10C．127c〉T（P．R43X）杂合突变,导致SOX10基因第43位编码精氨酸的密码子（CGA）突变为终止密码子（UGA）,产生截短蛋白,影响蛋白质功能的正常发挥。经检索人类基因突变数据库,该突变为未报道过的新突变。患儿父母未检测到该突变。结论先证者SOX10基因C．127c〉T（P．R43X）杂合突变可能是其分子生物学致病原因。     

Germline mutations of the MSR1 gene in prostate cancer families from Germany

The MSR1 gene at 8p22 has been suggested as a candidate gene for hereditary prostate cancer because germline variants have been found to be associated with the disease. Aside from a single nonsense mutation (R293X) that was found repeatedly at low frequencies in several samples, little evidence has been gained by follow-up studies to confirm the gene's relevance for prostate cancer. Prompted by reasonable support for a linkage to 8p22, we sought to determine the mutation spectrum of MSR1 in our family sample. Screening of 139 probands (representing 139 prostate cancer families) revealed 15 novel and a total of 20 sequence variants within the 10 coding exons and their intronic proximities. Aside from the known mutation c.877C>T (R293X) present in two of our families, we identified a second nonsense allele (c.251C>G; S84X) and a splice-site mutation (c.818-1G>A) that results in mRNA instability (each in a single pedigree). The novel missense alleles were c.703C>T (H235Y), c.856C>T (P286S), c.905C>T (P302L), c.1193C>G (A398G), and c.1289A>G (K430R). Of the eight variants that affect the encoded protein (splice site, nonsense, and missense), only R293X as well as the polymorphism c.823C>G (P275A) were additionally present at remarkable frequencies in further samples of sporadic prostate cancer and controls. Of note, carriers of R293X were equally frequent in 367 sporadic prostate cancer cases (1.9%) and in 197 controls (2.0%). To our knowledge, our study is the first to demonstrate further loss of function variants of MSR1 apart from R293X. Nevertheless, the low frequencies of deleterious alleles, in addition to an apparently moderate penetrance, does not support MSR1 as a major susceptibility gene in this family sample.     

一个眼牙指发育不良家系的基因突变分析

目的 旨在报道一个罕见的中国汉族眼牙指发育不良家系,并对该家系进行基因突变分析.方法 采用PCR及测序方法检测该家系先证者GJA1基因编码区及其侧翼序列的突变,然后在家系成员中验证,并通过Weblogo软件对可疑变异位点进行保守性分析;同时利用PCR、电泳及测序分析方法,检测HOXD13基因突变,排除患者由HOXD13基因突变致病的可能.结果 该家系内患者均携带GJA1基因的杂合突变c.605 C>T,正常个体均无此突变;该突变导致Cx43蛋白第202位氨基酸由精氨酸变成组氨酸(p.R202H);HOXD13基因未见异常.结论GJA1基因c.605 C>T(p.R202H)突变是该中国汉族眼牙指发育不良家系的致病突变,该突变为中国人群中首次报道.     

Clinical features and genetic analysis of seven patients with congenital hyperinsulinismCSCD

Objective: To analyze clinical characteristics, genetic mutation and therapeutic effect of seven patients diagnosed with congenital hyperinsulinism(CHI). Methods: Clinical data for the patients was retrospectively analyzed. Results: All patients presented with hyperinsulinism(serum insulin:2. 0-58. 4 mU/L),even after hypoglycemia (blood glucose: 0. 7-2. 39 mmol/L)has developed. Mutations were identified in 4 patients (57. 1%), which included a heterozygous c. 262C>T(p. R88C) mutation in exon 4 of the UCP2 gene, a heterozygous c. 1495C>A(p. G499C) mutation in exon 12 of the GLUD1 gene, a heterozygous c. 1493C>T(p. S498L) mutation in exon 1 of the GLUD1 gene, and a heterozygous c. 4432G > A (p. G1478R) mutation in exon 37 of the ABCC8 gene. The patient carrying a maternally inherited ABCC8 mutation was treated with cornstarch and had his blood glucose kept normal. All other patients responded well to diazoxide. Conclusion: A genetic diagnosis was attained for 51. 7% of patients in this study. Mild CHI patients can have their blood glucose controlled by giving cornstarch. Diazoxide is safe and effective for most CHI patients. © 2018 MeDitorial Ltd. All rights reserved.     

一个婴儿神经轴索营养不良家系的基因变异分析

目的对1个临床拟诊婴儿神经轴索营养不良(infantile neuroaxonal dystrophy,INAD)家系的患儿及父母进行基因变异分析,明确其致病原因为遗传咨询及产前诊断提供依据。方法应用高通量测序方法对患儿相关致病基因进行初步筛查,再通过直接测序对患儿及父母可疑致病基因进行验证,寻找可能的致病突变位点,采用SIFT及PolyPhen-2生物信息学软件对变异位点进行致病性预测。结果高通量测序筛查显示患儿PLA2G6基因第5和第16外显子存在可疑致病突变;Sanger测序结果显示患儿PLA2G6基因第5外显子c.668C>A(p.Pro223Gln)和第16外显子c.2266C>T(p.Gln756Ter)复合杂合变异,父亲携带c.668C>A杂合变异,母亲携带c.2266C>T杂合变异。c.668C>A变异导致PLA2G6基因编码的第223位脯氨酸被谷氨酰胺替代,为已报道的致病变异;c.2266C>T变异导致编译第756位谷氨酰胺的密码子变为终止密码,使肽链合成提前终止,该变异尚未见文献报道,蛋白功能预测为有害变异;患儿的复合杂合变异分别来自父母。结论PLA2G6基因第5外显子c.668C>A(p.Pro223Gln)和第16外显子c.2266C>T(p.Gln756Ter)变异可能是患儿的致病原因,新变异的发现丰富了PLA2G6基因变异谱。     

Mutational screening of the coding region of growth differentiation factor 9 gene in Indian women with ovarian failure

OBJECTIVE: To establish the risk associated with mutations in the coding region of GDF9 gene in Indian women with ovarian failure. DESIGN: This case-control study was designed for mutational analysis of the GDF9 coding region in a cohort of women with premature ovarian failure (n = 127), primary amenorrhea (n = 58), and secondary amenorrhea (n = 10) compared with controls (n = 220). RESULTS: This case-control study revealed eight mutations in the GDF9 gene, including five novel mutations: c.1-8C>T, c.199A>C (p.Lys67Glu), c. 205C>T, c.646G>A (p.Val216Mat), and c.1353C>T, and three documented mutations: c.398-39C>G, c.447C>T, and c.546G>A. Missense mutation c.199A>C was present in 4 of 127 premature ovarian failure (POF) cases and 1 of 10 secondary amenorrhea cases. The c.646G>A mutation was present in two POF cases. Both missense mutations were absent in controls. Genotype distribution of c.447C>T was significantly different in POF cases than controls (chi(2) = 5.93, P = 0.05). We chose two frequent single-nucleotide polymorphisms (c.398-39C>G, c.447C>T) for haplotyping and found that the C-T haplotype was significantly higher in patients (P = 0.03), whereas the C-C haplotype was representative of the control group. CONCLUSIONS: We report two rare missense mutations, c.199A>C and c.646G>A, which are associated with ovarian failure. The presence of the c.447>T mutation might indicate a higher risk for POF. Haplotype C-T was significantly associated with ovarian failure, whereas the C-C haplotype was representative of the control group.     

GPNMB基因纯合变异导致色素异常性皮肤淀粉样变性一家系

目的:对1个色素异常性皮肤淀粉样变性家系的3例患者的 GPNMB基因进行变异分析,明确其致病原因。 方法:对先证者行高通量测序,采用生物信息学方法寻找致病基因变异,并通过Sanger测序对家系内成员是否携带变异进行验证。结果:先证者及其哥哥、妹妹 GPNMB基因的第5外显子均存在c.5...     

Novel MC1R variants in Ligurian melanoma patients and controls

Several variant forms of the melanocortin-1 receptor gene (MC1R) have been associated with red hair, fair skin and an increased risk for melanoma. Their involvement in melanoma susceptibility is apparently linked both to skin sensitivity and to non-pigmentary pathways. We investigated the frequency of the MC1R variants in the Italian region of Liguria, where the occurrence and penetrance of melanoma are low and primary susceptibility is characterized by prevalence of the CDKN2A c.301G>T [p.G101W] founder mutation. Additionally, we attempted to establish the frequency of the red hair/fair skin phenotype in our region. As predicted by anecdotal evidence, the frequency of red hair/phototype I was very low (0.7%). Screening of 17 red-haired individuals and their red-haired relatives, 207 controls and 214 melanoma patients unselected for hair color but all of Ligurian descent, led to the detection of 8 novel substitutions (c.133T>C [p.F45L], c.248C>T [p.S83L], c.332C>T [p. A111V], c.479G>A [p.R160Q], c.637C>T [p.R213W], c.793G>A [p. V265I], c.923C>T [p. T308M], c.943T>C [p.C315R]), 1 novel deletion (c.520_523delGTC [p.V174del]) and 3 novel synonymous variants (c.366G>C [p. V122V], c.684G>A [p. Q228Q], c.726C>T [p.T241T]). Preliminary genotype/phenotype correlation seems to indicate that other genes involved in the regulation of human pigmentation may mask the recessive action of high-penetrance MC1R alleles, thus determining the low frequency of at-risk phototypes and of incidence and/or penetrance of melanoma in Liguria.     

一个遗传性牙本质发育不全家系的分子遗传研究

目的 研究一个常染色体显性牙本质发育不全家系发病的遗传基础.方法 通过对一个DSPP家系临床检查和家族史调查,连锁分析和DSPP基因的突变检测,以及限制性片段长度多态分析方法,分析该家系发病的分子基础.结果 连锁分析发现,该疾病致病基因与微卫星标记D4S1534完全连锁,对位于该区域的DSPP基因进行测序分析,发现一个新的致病突变(c.49C→T,p.Pro17Ser),该突变位于DSPP基因的第1外显子.该家系所有患者中都检测到了这一致病突变,但家系中的正常个体和100个无亲缘关系的正常人中未发现这个突变.结论 p.Pro17Ser是牙本质发育不全Ⅱ型致病基因DSPP的一个新的致病突变.我们的研究进一步拓展了对牙本质发育不全疾病分子遗传基础的认识.     

Functional analysis of 13 GBA mutant alleles identified in Gaucher disease patients: Pathogenic changes and "modifier" polymorphisms

Gaucher disease, the most prevalent sphingolipidosis, is caused by the deficient activity of acid beta-glucosidase, mainly due to mutations in the GBA gene. Over 200 mutations have been identified worldwide, more than 25 of which were in Spanish patients. In order to demonstrate causality for Gaucher disease, some of them: c.662C>T (p.P182L), c.680A>G (p.N188S), c.886C>T (p.R257X), c.1054T>C (p.Y313H), c.1093G>A (p.E326K), c.1289C>T (p.P391L), c.1292A>T (p.N392I), c.1322T>C (p.I402T), and the double mutants [c.680A>G; c.1093G>A] ([p.N188S; p.E326K]) and [c.1448T>C; c.1093G>A] ([p.L444P; p.E326K]), were expressed in Sf9 cells using a baculovirus expression system. Other well-established Gaucher disease mutations, namely c.1226A>G (p.N370S), c.1342G>C (p.D409H), and c.1448T>C (p.L444P), were also expressed for comparison. The levels of residual acid beta-glucosidase activity of the mutant enzymes produced by the cDNAs carrying alleles c.662C>T (p.P182L), c.886C>T (p.R257X), c.1054T>C (p.Y313H), c.1289C>T (p.P391L), and c.1292A>T (p.N392I) were negligible. The c.1226A>G (p.N370S), c.1322T>C (p.I402T), c.1342G>C (p.D409H), c.1448T>C (p.L444P), and [c.1448T>C; c.1093G>A] ([p.L444P; p.E326K]) alleles produced enzymes with levels ranging from 6 to 14% of the wild-type. The three remaining alleles, c.680A>G (p.N188S), c.1093G>A (p.E326K), and [c.680A>G; c.1093G>A] ([p.N188S; p.E326K]), showed higher activity (66.6, 42.7, and 23.2%, respectively). Expression studies revealed that the c.1093G>A (p.E326K) change, which was never found alone in a Gaucher disease-causing allele, when found in a double mutant such as [c.680A>G; c.1093G>A] ([p.N188S; p.E326K]) and [c.1448T>C; c.1093G>A] ([p.L444P; p.E326K]), decreases activity compared to the activity found for the other mutation alone. These results suggest that c.1093G>A (p.E326K) should be considered a "modifier variant" rather than a neutral polymorphism, as previously considered. Mutation c.680A>G (p.N188S), which produces a mutant enzyme with the highest level of activity, is probably a very mild mutation or another "modifier variant."     

A proposed molecular diagnostic flowchart for myophosphorylase deficiency (McArdle disease) in blood samples from Spanish patients

McArdle disease is a metabolic myopathy due to molecular defects in the myophosphorylase gene (PYGM), usually diagnosed in muscle biopsy. The aims of this study were to characterize genetically a large series of patients and to establish a protocol of molecular diagnosis on blood samples. We studied 55 Spanish unrelated patients with McArdle disease. Screening for the three more frequent mutations in the PYGM gene in the Spanish population (c.148C>T, p.R50X; c.613G>A, p.G205S; and c.2392T>C, p.W798R) were performed with polymerase chain-reaction and restriction fragment length polymorphism (PCR-RFLP) methods. To identify other mutant alleles, the coding region of PYGM gene was sequenced. The p.R50X mutation was observed in 38 patients, the p.G205S substitution in eight, and the p.W798R change in nine. Nine novel mutations, five missense (c.247A>T, p.I83F; c.521G>A, p.G174D; c.1094C>T, p.A365V; c.1468C>T, p.R490W; and c.1730A>G, p.Q577R), one nonsense mutation (c.2352C>A, p.C784X), three frameshift (c.402del, p.N134KfsX161; c.212_218dup, p.Q73HfsX7; c.1470dup, p.R491AfsX7), and nine previously reported mutations were found. In addition, we also updated the molecular data of 95 unrelated patients with McArdle disease studied thus far in our center. Of these patients, 56 were either homozygous or compound heterozygous for the p.R50X, p.G205S, or p.W798R mutation. By including in the molecular diagnosis protocol sequencing of the exons 1, 14, 17 and 18 of the PYGM gene, 16 further patients were characterized, and therefore we were able to detect the molecular defect in 72 out of 95 patients. A proposed molecular diagnosis protocol of the disease based on blood DNA would avoid muscle biopsy in 75.8% [95% confidence interval (95% CI): 62.1%-78.6%] of patients with McArdle disease.     

X-连锁迟发性脊椎骨骺发育不良家系SEDL基因突变及基因诊断

目的 确定一个X-连锁迟发性脊椎骨骺发育不良(X-linked spondyloepiphyseal dysplasia tarda,SEDL)家系的基因突变类型;建立一种快速基因诊断方法.方法 采用体格检查、影像学检查及家系分析进行临床诊断.针对SEDL基因的第3～6外显子及其侧翼序列设计4对引物,建立基于PCR的变性高效液相色谱技术(denaturing high performance liquid chromatography,DHPLC)快速基因分型方法.常规酚-氯仿法从该家系3代18名成员的外周血中提取基因组DNA,经PCR/DHPLC分析,筛查出SEDL基因突变所在的片段,对该片段进行序列分析以确定突变位点及类型.结果 DHPLC分析发现该家系的SEDL基因突变位点在第4外显子片段,序列分析证实为c.218C》T突变,导致氨基酸序列S73L改变.在该家系的18名成员中,3例男性患者,5例女性肯定携带者和2例未婚女性携带者均带有该突变,其余表型正常的8名成员中未检测到这一突变.各成员的DHPLC峰型所代表的基因型与表型结果相吻合.结论 首次报告中国人SEDL基因c.218C》T突变,丰富了中国人SEDL基因的突变谱.采用的技术快速、可靠,能对SEDL进行快速基因分型和产前诊断.     

一个结节性硬化症家系的临床特征及基因突变分析

目的分析一个中国人结节性硬化症家系的临床特征,并探讨其发病的分子机制。方法收集先证者及其家系成员的临床资料,采用全外显子组测序技术对先证者外周血DNA的TSC1和TSC2基因变异进行鉴定。经生物信息学分析后,对发现的潜在致病变异采用Sanger测序法对父母进行验证。结果先证者及其母亲均携带TSC2基因新的c.4183C>T(p.Q1395X)杂合变异,生物信息学分析提示该变异为潜在的致病变异。先证者母亲同样诊断为结节性硬化症,但症状轻于患者。另外4名未患病的家系成员未发现上述突变。结论TSC2基因新的c.4183C>T(p.Q1395X)杂合变异可能是该家系的致病原因。上述发现扩大了TSC2基因的突变谱。先证者症状重于其母亲考虑与表型异质性有关。     

Characterization of three myotonia-associated mutations of the CLCN1 chloride channel gene via heterologous expression

Two novel mutations of the human CLCN1 chloride channel gene, c.592C>G (p.L198V) and c.2255A>G (p.K752R), are described, occurring coincidentally in the one myotonic patient. These individual mutations and a construct with both mutations in the one cDNA were transcribed and expressed in Xenopus oocytes where channel gating parameters were extracted from chloride currents recorded under voltage clamp. We found that the p.L198V mutation has its major effects on the common (or slow) gate of the chloride channel, as do other dominant ClC-1 mutations, and may therefore be causative of the patient's symptoms (when co-expressed with wild-type human ClC-1, the p.L198V mutation exerts a dominant negative effect on common gating) but the p.K752R mutation appears to be innocuous and may be a benign polymorphism. A third mutant, the recently described c.2795C>T (p.P932L), was expressed in HEK 293 cells. Despite the severity of the disease associated with this mutation, chloride currents in cells expressing p.P932L were not significantly different from those of cells expressing wild-type ClC-1.     

Mutation spectrum in Taiwanese patients with phenylalanine hydroxylase deficiency and a founder effect for the R241C mutation

The spectrum of phenylalanine hydroxylase (PAH) gene mutations was determined in 25 families of hyperphenylalaninemia identified by a neonatal screening program in Taiwan. The coding sequence and exon-flanking intron sequences of PAH gene were amplified and sequenced. Mutations were identified in forty-five of the 50 chromosomes. R241C was the most common mutation (36% of the chromosomes), followed by R408Q (14% of the chromosomes). The remaining mutations were rare and seven mutations have not been reported before: p.F233L (c.697T>C), p.R252Q (c.756G>A), p.E286K (c.856G>A), p.G312V (c.935G>T), p.P314T (c.940C>A), p.I95del (c.284_286delTCA), and p.T81fsX6 (c.241_256del). Both p.R241C and p.R408Q are classified as mild phenylketonuria (PKU) or mild hyperphenylalaninemia (MHP) mutation, which may explain the fact that classical PKU is very rare in Taiwan (n=4, or one in 413,035). This strong founder effect for the p.R241C mutation has been described neither in the Caucasian populations, nor in other reports from Chinese. Since most of the populations in Taiwan are derived from Southeastern China, the spectrum of PAH gene mutations in Southeastern China should be different from other Chinese populations. This report not only disclose a specific spectrum of PAH gene mutation in Taiwan, but may also give clues to the movement of populations in Mainland China.