二代测序技术在杜氏肌营养不良症家系中的分子诊断应用

对22例临床拟诊断为杜氏肌营养不良症（DMD）患儿的家系临床资料进行分析,探讨二代测序（NGS）技术在DMD患者分子诊断中的临床应用价值。先证者同时送检遗传性神经肌肉病相关panel行NGS检测和Dystrophin基因多重连接探针扩增术（MLPA）检测,分析两种方法检出的Dystrophin基因外显子缺失/重复突变结果,Dystrophin基因点突变经Sanger测序验证。通过NGS测序,22例先证者均检出Dystrophin基因突变,其中外显子缺失/重复型突变14例,点突变及微小变异8例。MLPA检测结果与NGS检测结果一致。Sanger测序结果显示NGS检测出的点突变及微小变异是正确的。1例错义突变c.6290G>T,1例无义突变c.3487C>T,4例微小缺失导致的移码突变c.1208delG、c.7497_7506delGGTGGGTGAC、c.9421_9422delAA、c.8910_8913delTCTC在人类基因突变数据库中均未见报道,为Dystrophin基因新突变。该研究结果显示NGS技术可全面检测Dystrophin基因外显子缺失/重复、点突变、微小缺失和内含子突变等变异,在DMD患者分子诊断中的临床应用有一定价值,值得推广。     

儿童散发Rett综合征临床特征、基因突变与遗传咨询

目的 寻找散发典型Rett综合征患儿甲基化CpG结合蛋白2基因(MECP2基因)突变,探讨基因型和表型之间的关系,并为遗传咨询提供帮助.方法 提取患者及其父母静脉血白细胞基因组DNA,使用MECP2基因外显子特异引物进行PCR扩增和DNA测序检测.结果 2例患者MECP2基因外显子1、2、3未发现突变,外显子4分别存在一种杂合错义突变,核苷酸变化分别为c.C473T和c.C397T,导致相应的氨基酸变化p.T158M和p.R133C;其父母均无相应突变;c.C397T患者的临床表型较c.C473T轻.结论 典型Rett综合征患者多数存在MECP2基因突变,应行基因检测,阳性结果患者应检测其父母相应变化,为遗传咨询提供依据.     

26例不典型Rett综合征MECP2基因的突变分析

目的 了解不典型Rett综合征患儿MECP2基因的突变频率、突变类型、是否存在突变热点,寻找基因型和表型的相互关系。方法取26例不典型Rett综合征患儿外周静脉抗凝血,采用Miller’s蛋白酶K氯化钠盐析法提取基因组DNA,采用PCR方法扩增MECP2基因的外显子及结合区,1％的琼脂糖凝胶电泳鉴定目的PCR产物,然后进行DNA直接测序。DNA测序结果与人基因组序列（GeneBank AF030876）比较。结果26例不典型Rett综合征患儿中有12例存在突变。突变类型包括错义突变,由于单个碱基缺失导致的移码突变和剪切位点的突变,其中错义突变为最常见类型。c．397C〉T为3例,c．473C〉T、c．916C〉T、c．806delG各为2例,c．397A〉G、c．1005G〉A、c．IVS2-2A〉T各为1例。结论不典型Rett综合征患儿存在MECP2基因突变,R133C、T158M和R306C为其热点突变。基因突变类型和表型之间有一定的相关性。     

应用变性高效液相色谱技术检测结节性硬化症TSC1基因外显子15基因突变的研究

目的探讨结节性硬化症(TSC)TSC1基因外显子15基因突变的特点。方法 1996—2006年采用多聚酶链扩增和变性高效液相色谱(DHPLC)技术,对21个家系59名研究对象进行了TSC1基因外显子15检测,对DHPLC检测异常者再进行多聚酶链扩增产物直接测序方法证实其突变类型。结果对21个家系TSC1基因外显子15共检测出4个家系的3种突变形式,其中c.1708～1709delAG(p.Arg570GlyfsX17)与c.1888～1891delAAAG(p.Lys630GlnfsX22)两种突变为国内尚未报道的小的缺失突变,c.1460CG(p.Ser487Cys)突变为1种罕见的错义突变。TSC1基因外显子15突变基因检出频率为4/21(19%)。在检出突变的4个家系中1个为家系突变,其余3个为散发突变。结论 TSC1基因外显子15的c.1460CG(p.Ser487Cys),c.1708～1709delAG(p.Arg570GlyfsX17)与c.1888～1891delAAAG(p.Lys630GlnfsX22)突变为目前国内首报突变。     

淮海地区汉族苯丙酮尿症患儿苯丙氨酸羟化酶基因突变分析北大核心CSCD

目的研究淮海地区汉族苯丙酮尿症（PKU）人群中苯丙氨酸羟化酶（PAH）基因突变特征。方法选择2003年1月至2013年12月在徐州市妇幼保健院确诊的101例PKU患者,应用聚合酶链反应．单链构象多态性（PCR-SSCP）分析及基因测序方法,对础H基因13个外显子及其两侧部分内含子进行突变的筛查和确定。结果1．在202个PAH等位基因中共检测出24种不同的突变类型,将这24种突变资料提交美国国立生物技术信息中心（NCBI）dbSNP数据库,获得24个序列号SS#2137543837～SS#2137543860。2．PAH基因突变集中在第7外显子及其两侧部分内含子。最常见的突变是c．728G〉A（P．R243Q）（18．317％）,其他较常见突变有C．721C〉T（P．R241C）（8．416％）,C．1155G〉C（P．1385L）（4．950％）,C．1068C〉A（P．Y356X）（3．960％）,c．-71A〉C（-71A〉C）（3．465％）,C．60＋62C／T（IVSl＋62C〉T）（2．970％）。3．在中国汉族PKU人群朋日基因发现了2个新的突变：c．60＋62C〉T（IVSl＋62C〉T）和c．782G〉T（P．R261L）。结论淮海地区PAH基因大部分突变类型和比例与中国其他地区相似,少部分存在明显差异。淮海地区汉族PKU人群PAH基因突变谱呈地域特异性。     

幼年起病的2例晚发型糖原贮积症Ⅱ型/Pompe病临床和基因分析

目的对中国大陆地区幼年起病的晚发型糖原贮积症Ⅱ型（GSDⅡ,Pompe病）患者进行临床和基因突变分析。方法对2例幼年起病的Pompe病患儿进行临床分析,提取患儿皮肤成纤维细胞及其父母的外周血DNA,应用聚合酶链反应（PCR）扩增α-1,4-葡萄糖苷酶的19个外显子,测序,进行突变检测。同时对50例健康对照100个等位基因进行15号外显子的PCR扩增,测序,确定突变特征。结果1例临床表现为进行性近端肌肉无力,肌张力低下;1例表现为肌容积减少,呼吸肌受累;2例均有肌酸磷酸肌酶升高,肌电图提示肌源性损害,肌活检呈空泡性改变,皮肤成纤维细胞α-1,4-葡萄糖苷酶活性测定明显低下。突变检测：测序发现4个杂合错义突变,例1为c796C〉T,p．266Pro〉Ser;c2105G〉A,p．702Arg〉His;例2为c2132C〉G,p．711Thr〉Arg;c2167G〉A,p．723Val〉Met。其中后3个为新突变,50例健康对照100个等位基因测序未发现同样突变。结论幼年起病的Pompe病患儿临床表现存在差异,呼吸肌受累情况影响其预后;检测到3个错义新突变,可能为致病突变。     

多重PCR技术及定量PCR技术在缺失型Duchenne型肌营养不良患儿及携带者诊断中的应用研究

目的　建立一种简便、可靠的缺失型Duchenne型肌营养不良(DMD)患儿及携带者的基因检测方法,为DMD产前诊断的开展奠定基础。方法　首先采用1 8对引物多重PCR反应体系,确定2 0 0 1年3月至2 0 0 4年8月就诊于中国医科大学附属第二医院遗传门诊的48例DMD患儿dystrophin基因缺失情况,然后应用定量PCR方法对1 5例缺失型患儿母亲进行携带者检测。结果　48例DMD患儿中2 5例存在基因缺失,缺失率52 % ( 2 5/ 4 8) ,共涉及1 2个外显子,主要集中在44～52外显子,其中外显子49和50缺失频率最高;1 5例缺失型患儿母亲中检出8例携带者,其中肯定携带者检出率1 0 0 % ( 4 / 4 ) ,可疑携带者检出率3 6% ( 4 / 1 1 )。结论　多重PCR结合定量PCR方法能有效地检出缺失型DMD患儿及携带者,可为产前诊断提供十分重要的信息。     

进行性假性类风湿性发育不良症的临床诊断及WISP3基因突变分析

目的 从临床及影像学角度识别进行性假性类风湿性发育不良症(PPD);了解PPD的基因突变类型.方法 3例表现为关节疼痛、肿胀及运动障碍的患儿(9～16岁)纳入本研究.根据临床表现、骨骼影像学及实验室检查进行PPD的诊断;采用PCR及直接测序法对PPD家系进行WISP3基因突变分析.结果 3例患儿符合PPD的临床诊断:表现为非炎性的多关节(包括指趾关节)膨大,影像学表现为扁平椎、椎体前缘呈鸟嘴或子弹头样改变、股骨头及外周大小关节骨骺(干骺端)扩大等,血沉、C反应蛋白、类风湿因子等实验室指标正常;3例患儿中发现了3种未见报道的WISP3基因突变,突变类型分别为c.624_625insA/c.729_735delGAGAAAA、c.624_625insA/c.866_867insA及c.866_867 insA/c.866_867insA,均为插入或缺失突变,突变位于外显子4及外显子5,各占50%(3/6).结论 PPD临床特点是进行性、非炎性的多关节(包括指趾关节)膨大及运动障碍,类风湿因子等实验室指标正常;全身骨骼(包括脊柱)的影像学检查对诊断十分重要,主要特征为扁平椎、关节骨骺(干骺端)扩大及关节腔狭窄.发现了3种WISP3基因新变异.     

Becker/Duchenne肌营养不良患儿临床表型与基因关联性预测分析

目的 探讨Becker/Duchenne肌营养不良（BMD/DMD）患儿的临床表型与基因型的关联性，为疾病的管理、基因治疗及产前诊断提供理论依据。方法 回顾性分析52例患儿的临床资料及基因检测结果，对52例患儿均采用多重连接探针扩增（MLPA）的方法检测DMD基因，对MLPA检测未发现基因异常的患儿采用外显子芯片捕获结合高通量测序技术（NGS）进行筛查；并对20例先证者的母亲进行了测序验证。结果 结合MLPA和NGS测序技术检测到50例患儿携带BMD/DMD致病基因，检出率为96%。其中，基因缺失36例（69%）、重复7例（13%）、微小突变7例（13%）。在43例存在基因缺失/重复的患儿中，DMD 32例，BMD 11例；37例（86%）符合阅读框架原则，其中DMD 27例（96%），BMD 10例（67%）。7例微小突变均为DMD。结论 阅读框架原则对DMD有极高预测价值，对BMD预测有限。     

Menkes病患儿ATP7A基因突变分析

目的分析并确定2例Menkes病（MD）患儿的ATP7A基因突变及遗传特征。方法收集并分析2例Menkes病患儿的临床资料，提取其外周静脉血DNA，采用PCR法扩增ATPTA基因的23个外显子及其与内含子的连接区，并行DNA直接测序，异常者用DNA限制性内切酶酶切PCR扩增片段方法进行验证。结果发现2个半合子突变，1例在第12外显子检测到不连续的多处碱基缺失c．2589delTGAAGGA＋c．2598delTT＋c．2601delT＋c．2603delT＋c．2605delGTAGATGA（P．E864fsX883），导致移码突变。另1例在第15外显子检测到单个碱基缺失c．3045delT（p．T1016fsX1018），并发现移码突变。2例患儿的母亲均为相应突变但表型正常的携带者。结论发现2个国际上尚未见报道的新ATPTA基因突变（p．E864fsX883与p．T1016fsX1018），首次在国内明确了2例Menkes病患儿的基因诊断。     

Deletion analysis of DMD/BMD children in Singapore using multiplex polymerase chain reaction (PCR) technique.

Twenty-three children suffering from Duchenne/Becker muscular dystrophy (DMD/BMD) in Singapore were analysed using the multiplex polymerase chain reaction (PCR) technique. Deletions were found in 14 cases. One rare case of total deletion of all nine exons was observed. This is the first DMD/BMD deletion analysis on South East Asian children. This technique for screening deletions was informative in 61 per cent of the local cases and would be useful for rapid diagnosis of deletion cases of DMD/BMD.     

Intragenic deletions in the dystrophin gene in 211 Pakistani Duchenne muscular dystrophy patients

Background: Deletions of single or multiple exonic regions within the dystrophin gene can be detected using current molecular methods in approximately 65% of the patients with X‐linked recessive neuromuscular disorder, Duchenne/Becker muscular dystrophy (DMD/BMD). Population‐based variations in frequency and distribution of dystrophin gene deletions have been reported in DMD/BMD patients. In the present study, the first in the Pakistani population, frequency and distribution of deletions of 18 exons clustered in two hot spots within the dystrophin gene in 211 unrelated DMD patients were analyzed. Methods: A total of 211 patients suffering from DMD were ascertained, and intragenic deletions within the dystrophin gene were detected on polymerase chain reaction amplification of the genomic DNA using 18 primer sets clustered within two major deletion hot spots. lovd v.1.1.0 software from the Leiden Muscular Dystrophy website has been used to predict in‐frame and out‐of‐frame deletions. Results: Intragenic deletions were detected in 86 patients (40.75%): 35 patients (40.69%) had deletions within the proximal hot spot, and 51 patients (59.30%) had deletions confined to the distal deletion hot spot of the dystrophin gene. The most frequently deleted exons were 50, 6, 47, 13 and 52 with deletion frequencies of 15.11%, 12.79%, 10.46%, 8.13%, and 4.65%, respectively. lovd v.1.1.0 predicted out‐of‐frame deletions in 67 DMD patients and in‐frame deletions in 19 DMD patients. Conclusions: The observed proportion of intragenic deletions in the Pakistani population is relatively low, which is comparable with most of the Asian data. Also, deletions in 67 patients (77.9%) are in agreement with the frame‐shift rule.     

Functional significance of dystrophin positive fibres in Duchenne muscular dystrophy.

The age when boys lose the ability to walk independently is one of the milestones in the progression of Duchenne muscular dystrophy (DMD). We have used this as a measure of disease severity in a group of 30 patients with DMD and six patients with intermediate Duchenne/Becker dystrophy (D/BMD). Dystrophin analysis was performed on tissue sections and western blots of muscle biopsy specimens from these patients and the relationships that were found between clinical severity and abundance of dystrophin labelling are reported. All patients with intermediate D/BMD had dystrophin labelling that was detected on sections and blots. Weak dystrophin labelling was found in sections from 21/30 DMD cases and on blots in 18/30 cases. Two non-exclusive patterns of dystrophin labelling were observed on sections: very clear labelling on a small percentage of fibres (usually < 1%) or very weak labelling on a much higher proportion (about 25%). The mean age at loss of mobility among the DMD patients with no dystrophin labelling on tissue sections was 7.9 years (range 6.3-9.5) while the mean age among those with some labelling was 9.9 years (range 8.0-11.9); this is a significant difference. Quantitative estimates of dystrophin abundance were obtained from densitometric analysis of dystrophin bands on blots. In the whole group of 36 patients, a significant positive relationship was found between the abundance of dystrophin and the age at loss of independent mobility. It is concluded that even the very low concentrations of dystrophin found in DMD patients may have some functional significance.     

Dystrophin Abnormality in Progressive Muscular Dystrophy -A Review Article-

Duchenne muscular dystrophy (DMD) is a fatal X-linked recessive disorder of muscle in children, with an incidence of approximately 1 in 3,300 male births. In about a third of affected boys, the disease is due to a new mutation, and most patients die in their early 20s. Over the last few years, the genetic, biochemical and histopathological basis of DMD has been elucidated greatly. In particular, the discovery of "dystrophin," the protein product of the DMD gene is truly an epoch-making success in the history of muscular dystrophy research. Dystrophin is now thought to be a cytoskeletal protein underlying the plasma membrane (known in muscle as the sarcolemma) of normal muscle fiber, and is undetectable or greatly reduced in DMD. In this review article, dystrophin in normal skeletal muscle and various neuromuscular diseases including DMD/BMD (Becker muscular dystrophy), and its carrier is discussed.     

Epilepsy in Duchenne and Becker muscular dystrophies.

Duchenne and Becker muscular dystrophies are X-linked allelic disorders in which the association of central nervous system dysfunction, typically in the form of mental retardation, is a well recognized feature. They are both due to mutations in the dystrophin gene, whose corresponding protein products are expressed both in the muscle and central nervous system. We have observed an increased frequency of epilepsy in children with Duchenne and Becker muscular dystrophy attending our clinic. Out of 254 boys with this condition (201 Duchenne and 53 Becker), eight children, four in the Duchenne and four in the Becker group, had a confirmed diagnosis of epilepsy (cumulative incidence 3.14%, with a subgroup incidence of 1.99% in the Duchenne and 7.54% in the Becker group). Statistical analysis indicated that only the incidence of epilepsy in Becker muscular dystrophy was significant (p < 0.007). Our data suggests that epilepsy may be a rare associated feature in children with muscular dystrophy secondary to dystrophin deficiency.     

Deletional mutations of dystrophin gene and carrier detection in eastern India

Objective  

To determine the pattern of deletions of the dystrophin gene, the major class of mutations among the Duchenne and Becker muscular dystrophy patients of eastern India and to analyze the carrier frequency of the female members of the proband’s family.     

Deletion analysis and clinical correlations in patients with Xp21 linked muscular dystrophy

We carried out molecular deletion analysis on 142 patients with Duchenne/Becker muscular dystrophy which covered 25 exons of the dystrophin gene. We also evaluated the results by comparing with the clinical findings and examples in the literature. A deletion ratio of 63.7% was achieved. Exon 46 was the most frequently affected region. Interestingly we also observed four cases with muscle promoter (Mp) region deletions which have been rarely reported in the literature.     

Functional significance of dystrophin positive fibres in Duchenne muscular dystrophy

The age when boys lose the ability to walk independently is one of the milestones in the progression of Duchenne muscular dystrophy (DMD). We have used this as a measure of disease severity in a group of 30 patients with DMD and six patients with intermediate Duchenne/Becker dystrophy (D/BMD). Dystrophin analysis was performed on tissue sections and western blots of muscle biopsy specimens from these patients and the relationships that were found between clinical severity and abundance of dystrophin labelling are reported. All patients with intermediate D/BMD had dystrophin labelling that was detected on sections and blots. Weak dystrophin labelling was found in sections from 21/30 DMD cases and on blots in 18/30 cases. Two non-exclusive patterns of dystrophin labelling were observed on sections: very clear labelling on a small percentage of fibres (usually < 1%) or very weak labelling on a much higher proportion (about 25%). The mean age at loss of mobility among the DMD patients with no dystrophin labelling on tissue sections was 7.9 years (range 6.3-9.5) while the mean age among those with some labelling was 9.9 years (range 8.0-11.9); this is a significant difference. Quantitative estimates of dystrophin abundance were obtained from densitometric analysis of dystrophin bands on blots. In the whole group of 36 patients, a significant positive relationship was found between the abundance of dystrophin and the age at loss of independent mobility. It is concluded that even the very low concentrations of dystrophin found in DMD patients may have some functional significance.     

Becker muscular dystrophy in a patient with Hodgkin's disease

Genetic disease often predisposes to neoplastic processes because of either alterations in both oncogenes and tumor suppressor genes or imbalances in the cell cycle. The authors present the case of a 15-year-old boy with Hodgkin's disease associated with Becker muscular dystrophy. Becker muscular dystrophy is an X-linked neuromuscular disease due to in-frame mutations in the dystrophin gene, and it is considered a milder variant of Duchenne muscular dystrophy. In most patients with Becker muscular dystrophy, the myopathic process affects the myocardium, leading to cardiomyopathy.