Wiskott-Aldrich综合征10例临床特点与实验室诊断分析

目的探讨10例Wiskott-Aldrich综合征(WAS)患儿的临床及分子特点。方法总结10例拟诊WAS患儿临床资料,包括血常规、免疫功能、骨髓常规和扫描电镜检查及临床表型评分。流式细胞术(FCM)检测10例患儿外周血单个核细胞(PBMC)中WAS蛋白(WASP)表达。PCR扩增WASP基因序列并直接双向测序分析9例患儿及其亲属突变情况。结果本组均男性,多以自幼大便带血丝及皮肤瘀点瘀斑起病。均有血小板减少伴小血小板,湿疹和免疫缺陷表现。临床表型评分3例评5分,3例评4分,4例评3分。具有阳性家族史患儿临床诊断年龄明显早于无家族史者。多数患儿IgA(8/9)、IgE(8/9)和IgG(7/9)升高,除6例CD4~+T比例下降(6/9),其余患儿淋巴细胞亚群分类正常。1例淋巴细胞增殖功能降低(1/3)。骨髓常规缺乏特征性改变。5例患儿淋巴细胞扫描电镜(SEM)均可见典型微绒毛异常。10例患儿WASP均为阴性,9例行WASP基因分析发现7种不同突变,3例为新型突变(168 C>A,T45 K;747-748 del T,I 238 Fs X260;253 Ins A,C73X)。8例位于编码区,1例位于内含子。4...     

1例Wiskott-Aldrich综合征临床特征和致病基因突变分析

目的:分析1例Wiskott-Aldrich综合征患者的临床特征及分子遗传特征。方法:分析患者的临床特征,收集患者及其父母外周血,提取基因组DNA,针对WASP基因所有编码外显子及外显子和内含子交界处进行PCR扩增测序。结果:我们报道的患者具有典型的WAS表型,临床得分为5分;患儿Coomb’s试验,自身抗体中ANA及类风湿因子均阳性,伴有自身免疫性疾病。患儿为WASP基因第7外显子中第665位核苷酸C突变为T(c.665C>T),导致211位密码子发生无义突变,该位置提前出现终止密码(p.R211X);其母亲为此突变基因携带者。结论:这例男性中国Wiskott-Aldrich综合征患儿由于WASP基因突变致病,此基因型患儿(p.R211X)拥有典型的WAS表型且伴有自身免疫性疾病的临床型为国内首次报道。     

一例瓜氨酸血症Ⅰ型患儿的临床特点及ASS1基因突变分析

目的对一例临床怀疑瓜氨酸血症Ⅰ型患儿进行分子遗传学研究,明确致病突变,并对其相关的临床特点、转归进行总结。方法收集患儿的临床资料,提取患儿外周血DNA,应用高通量捕获测序确定患儿ASS1基因突变情况,并用Sanger测序验证测序结果以及ASS1基因的家系分析。结果患儿生后第6天出现体温不升、呻吟、喂养困难、惊厥,血氨明显升高(1240μmol/L),血串联质谱(MS/MS)分析提示血瓜氨酸显著升高(1979.03μmol/L),尿气相色谱/质谱(GC/MS)提示乳清酸升高,予限制蛋白饮食、精氨酸治疗后血氨恢复正常,神经系统损害持续加重,头颅MRI提示脑萎缩、脑软化明显,随访至12月时死亡。基因序列分析发现患儿ASS1基因存在c.910CT和c.1087CT复合杂合突变,其中c.910CT突变来源于父亲、c.1087CT突变来源于母亲。c.910CT突变导致第304号氨基酸由精氨酸变异为色氨酸,c.1087CT突变导致第363号氨基酸由精氨酸变异为色氨酸,为错义突变。结论通过高通量捕获测序和Sanger测序确定了一例瓜氨酸血症Ⅰ型患儿的致病基因,随访发现该复合杂合突变引起的瓜氨酸血症患儿临床表现重,预后极差。     

一例Wiskott-Aldrich综合征致病基因突变分析及产前诊断

目的 研究1例Wiskott-Aldrich综合征的致病基因突变类型,并以此为依据对该患者家庭做定制的产前诊断。方法 采集该家系中患者及正常人血样,提取DNA,用聚合酶链反应扩增WASP基因,并对扩增产物进行直接测序,确定突变位点。患儿母亲再次妊娠12周时,取胎儿绒毛组织,进行定制的产前诊断。 结果 患儿存在WASP基因c.107-108delTT突变,其母亲为杂合子,胎儿不存在该位点的异常。 结论 该患儿发病是由WASP基因突变所致,胎儿不存在此位点异常。     

肺泡微石症1例

目的报道1例北京协和医院诊治的肺泡微石症(PAM)病例,并总结该病的临床表现和遗传学特点。方法报道本例的临床表现、影像学表现以及SLC34A2基因扩增及测序结果分析突变类型。结果患者为35岁男性,以咳嗽、咳痰起病,逐渐气短,胸部CT、支气管肺泡灌洗液及肺病变组织病理均符合肺泡微石症病理表现,基因检测发现SLC34A2基因第8外显子纯合子突变(c.A910T)。结论肺泡微石症是一种罕见的遗传性疾病,临床表现不特异,但有着典型的影像学表现。外显子8的c.910AT(Lys304Ter)突变在亚洲人群中最为常见,可能为我们日后的基因筛查或基因治疗提供了潜在的靶点。     

新生儿短链酰基辅酶A脱氢酶缺乏症4例临床特征及基因突变分析

目的探讨青岛地区新生儿短链酰基辅酶A脱氢酶缺乏症(SCADD)患儿的临床特征及基因突变特点。方法利用串联质谱技术检测283 104名新生儿干血滤纸片中酰基肉碱水平,对筛查出的疑似SCADD患儿通过尿有机酸检测、短链酰基辅酶A脱氢酶(ACADS)突变检测进行确诊。结果共确诊4例短链酰基辅酶A脱氢酶缺乏症患儿,患病率1. 4/10万(1/70 776)。患儿临床表现无明显异常,串联质谱检测显示血丁酰基肉碱(C4)及其与乙酰肉碱(C2)、丙酰肉碱(C3)比值均增高。对4例患儿进行尿有机酸分析,乙基丙二酸均增高[8. 41～36. 34 mg/g肌酐(正常值0～6. 20 mg/g肌酐)],还有2例伴乳酸增高,1例伴丙酮酸增高。基因测序共发现7种ACADS突变,4种为已知突变,3种未报道突变,均为错义突变。4例患儿均为复合杂合突变,分别为:c. 1031AG/c. 989GA; c. 1186GA/c. 1195CT; c. 1031AG/c. 445AT; c. 1130CT/c. 1157GA。常见突变为c. 1031AG(25%),ACADS基因型与乙基丙二酸以及C4浓度水平无明显相关。对患儿进行饮食指导,随访均未出现临床症状,体格及智力发育正常。结论通过血串联质谱筛查配合基因测序可以对SCADD明确诊断,早期确诊的新生儿无临床症状,预后较好。     

一个先天性肌营养不良1A型家系的临床、分子病理及遗传学研究

目的 分析并确立1个先天性肌营养不良1A型(congenital muscular dystrophy type 1A,MDC1A)家系的临床、分子病理及遗传学特征.方法 收集该家系患儿及父母的临床资料,对患儿进行腓肠肌活检,采用特异抗体行免疫组织化学染色,包括merosin抗体、抗α抗肌萎缩相关糖蛋白(α-dystroglycan,α-DG)糖链抗体ⅡH6、抗β抗肌萎缩相关糖蛋白(β-dystroglycan,β-DG)抗体及抗肌萎缩蛋白(dystrophin)C末端(Dys-C)抗体;提取患儿及其父母外周血基因组DNA,PCR扩增LAMA2基因的65个外显子,以琼脂糖凝胶电泳鉴定PCR产物,PCR产物纯化后DNA直接测序,确定基因突变的类型,分析基因型和表型的关系.结果 患儿自幼运动发育落后,肌病面容,肌酶中度升高,头颅MRI提示脑白质异常信号,临床诊断为先天性肌营养不良1A型.通过活检肌肉组织免疫学染色提示merosin完全缺失,dystrophin和DG表达正常.基因检测显示先证者LAMA2基因第5外显子c.817A>T纯合突变,其父母分别为此位点杂合突变.结论 本次研究进一步明确了MDC1A患儿的临床特点,通过分子遗传学分析发现该患儿为LAMA2基因c.817A>T(p.R273X)纯合无义突变,其突变基因分别来自父母,符合先天性肌营养不良1A型常染色体隐性遗传的规律,可确诊为先天性肌营养不良1A型.     

一家系6例遗传性多发性骨软骨瘤基因诊断及文献复习

目的研究一家系中国人遗传性多发性骨软骨瘤基因突变状况,并对该家系中无临床症状的患儿进行诊断。方法收集、整理、分析该家系临床资料,应用PCR反应及扩增产物直接测序技术对先证者及其家系成员进行EXT2基因检测,并对相关文献进行回顾。结果家系中6人均发现了EXT2基因c.514CT突变,其中5例患者有症状,1例患儿生后29小时尚无表现,该突变在中国人中首次发现。该突变位于第2外显子,为无义突变。结论 EXT2基因c.514CT突变是导该家系发生HME的致病基因。     

一个罕见的羧化全酶合成酶缺乏症家系的临床分析与基因诊断

目的对罕见的羧化全酶合成酶缺乏症患儿进行临床诊治和基因突变分析。方法结合临床表现,同时运用血串联质谱、尿气相色谱等,对1例婴儿期起病的疑似羧化全酶合成酶缺乏症/生物素酶缺乏症患儿进行筛查与初步诊断,并提取家系全部成员外周血DNA,应用聚合酶链反应(PCR)扩增生物素酶基因(BTD)的4个外显子和羧化全酶合成酶基因(HLCS)的12个外显子,直接测序,进行先证者及其他家系成员的基因突变检测,以明确患儿基因型与表型之间的关系。结果家系中患儿的发病时间均较早(第一胎男孩为2月+,第二胎女孩为1月),临床主要表现为出生后早期(1-2周)开始头部出现散在湿疹、脓疱疹,3个月时全身发红伴弥漫性浸润性丘疹和脱屑,营养不良貌,毛发稀黄,呼吸急促等。实验室检查发现轻度贫血、代谢性酸中毒,血串联质谱检测发现3-羟基异戊酰基肉碱(C5-OH)水平显著升高,尿有机酸分析显示尿乳酸、丙酮酸、羟基丙酸、丙酰甘氨酸、甲基巴豆酰甘氨酸等显著升高,符合羧化全酶合成酶缺乏症/生物素酶缺乏症的表现。突变检测:家系基因测序BTD基因未发现致病性突变,而在患儿HLCS基因的第9和第10号外显子上发现了致病性的突变位点:c.1522CT杂合突变和c.1711GA杂合突变,均为错义突变,50例健康人对照100个等位基因测序未发现这两个位点的突变。因此,患儿诊断明确。经生物素20mg/d补充治疗与营养干预后,患儿全身情况逐渐好转,2周后皮疹愈合,营养状况也逐渐得到改善。结论羧化全酶合成酶缺乏症以神经系统及皮肤损害、代谢性酸中毒为特征。串联质谱和气相色谱分析、基因突变分析有助于鉴别诊断和确诊,口服生物素疗效显著。本研究对一个羧化全酶合成酶缺乏症家系进行了临床分析和基因诊断,明确了患儿发病的遗传学病因,为这个家庭后面的产前诊断,打下了坚实的基础。     

对MITF基因突变导致Waardenburg TypeⅡ综合征患者临床表型和遗传学分析

目的探讨1例虹膜色素沉着异常,感音神经性耳聋,皮肤白斑患者的遗传学病因。方法应用单核苷酸多态性芯片(single nucleotide polymorphism array,SNP array)检测患者染色体微小异常。用全外显子测序(whole exome sequencing,WES)分析及Sanger测序验证,对患者进行遗传学分析。结果 SNP array检测结果正常。全外显子测序结果及OMIM数据库分析结合患者临床表型得出MITF基因c.775CT突变,259位置氨基酸由精氨酸突变为终止密码子。Sanger测序结果全外显子检测一致,显示患者MITF基因c.775CT(p.Arg259)杂合突变。结论本例患者异常表型是由于MITF基因c.775CT(p.Arg259)杂合突变导致的,并表现为Waardenburg syndrome Ⅱ型,对其家系有遗传指导意义。     

Clinical and Molecular Characterization of Thai Patients with Wiskott–Aldrich Syndrome

Wiskott–Aldrich syndrome (WAS) is an X‐linked recessive primary immunodeficiency disorder caused by mutations in the gene encoding the WAS protein (WASP). Classic WAS is characterized by thrombocytopenia with small‐sized platelets, recurrent infections, eczema and increased susceptibility to autoimmune diseases and haematologic malignancies. Here, we reported on seven unrelated Thai individuals with classic WAS. In addition to clinical and immunologic characterization, mutation analysis by PCR‐sequencing the entire coding region of WASP was performed. Recurrent and novel mutations were successfully identified. A nonsense mutation, the c.55C>T (p.Q19X), has not been previously described, expanding the mutational spectrum of WASP. The patient with this newly described mutation developed cow's milk allergy manifesting as angioedema and urticaria and had cytomegalovirus infection that was successfully treated with long‐term ganciclovir. This study reported long‐term follow‐up of seven patients with molecular confirmation of WAS and infrequent features in the patient with classic WAS carrying the novel nonsense mutation.     

Identification of five novel WASP mutations in Chinese families with Wiskott-Aldrich syndrome

The Wiskott-Aldrich Syndrome (WAS) is an X-linked recessive immunodeficiency caused by mutation in the gene encoding WAS protein (WASP). The disease is characterized by eczema, thrombocytopenia and severe immunodeificency and is associated with extensive clinical heterogeneity. Mutation studies indicated that the mutated genotypes are also highly variable. In this study, we performed PCR-direct sequencing analysis of the WAS gene in six unrelated Chinese families. Five novel mutations identified, included two nonsense mutations (506C-->T, 1388-->T), a small insertion (685-686insCGCA) and two single-base deletions (384delT, 984delC). All of the mutations are predicted to lead to premature translational termination of WASP.     

ED1基因新突变导致无汗型外胚层发育不良

目的鉴定一个无汗型外胚层发育不良(HED)家系ED1基因的突变及探讨基因型与表型之间的关系,为该病的诊断,产前诊断及遗传咨询提供实验依据。方法对一个HED家系进行调查,临床资料收集及采集外周血,抽取基因组DNA;设计ED1基因外显子引物,行先证者DNA PCR扩增及序列测定,发现候选变异后对先证者的父母及120名匹配正常人进行突变位点序列分析;推导的该基因氨基酸序列(突变位点)用Clustal W软件进行多物种对比。结果先证者发现ED1基因c.158T>G(p.Leu53Arg)纯合突变,母亲为c.158T>G(p.Leu53Arg)杂合突变;先证者父亲及120例正常对照的序列分析结果未检测出相应位置突变。讨论 ED1基因突变检测是直接诊断HED有效手段之一,发现的c.158T>G(p.Leu53Arg)为新致病突变。     

榆次地区非综合征性耳聋患者GJB2基因的检测分析

目的通过对榆次地区100q,J非综合征性耳聋患者GJB2基因突变位点的筛查,了解该地区耳聋基因的突变特点,为耳聋的早期诊断提供依据。方法收集榆次地区100例非综合征性耳聋患者的外周血标本,提取全血基因组DNA后,用聚合酶链反应（PCR）对目的片段进行扩增并测序,结果在GenBank上比对分析。结果100例耳聋患者中,检测到90例患者发生基因突变,其中有3,k位点未见报道：c．54C〉A（2．5％）、c．319h〉G（0．5＊／0）、c．512-5l3insAhCG（O．5％）;4个位点突变发生率较高：C．79G〉A（26．50％）、c．235delC（12．00％）、c．341A〉G（20．50％）、c．765T〉C（13．50％）;另外还检测出8个突变发生率较低的位点：c．109G〉A（1．5％）、C．176-19ldell6（1．00％）、C．223C〉T（1．00％）、c．253T〉C（0．50％）、c．299-300delAT（3．50％）、C．328delG（0．50％）、c．368C〉h（0．50％）、C．608T〉C（2．00％）。结论通过对榆次地区耳聋患者GJB2基因的检测分析,可以明确耳聋患者的病因,了解该地区的耳聋基因突变情况,为今后的耳聋患者的病因学诊断提供参考。     

吕梁地区遗传性耳聋GJB2基因和mtDNA1555位点的突变筛查

目的通过对吕梁地区96例耳聋患者的GJB2基因和mtDNA1555位点突变筛查,了解该地区的基因突变情况及热点突变位点。方法采用聚合酶链式反应（PCR）扩增96例标本的GJB2基因和mtDNA1555位点所在区段,产物酶切、测序分析。结果96例标本共计检出10个GJB2基因突变位点,与编码连接蛋白的非综合征耳聋突变数据库（http：／／davinci．crg．es／deafness／index．php？seccion=mut_db＆db=nonsynd）比对,8个位点已见报道,其中包括4个多肽位点c．79G〉A、C．341A〉G、c．608T〉C、C．457G〉A和4个致病位点C．235delC、e．109G〉A、c．176-C．191dell6和C．299-c．300delAT,其中,c．235delC是主要突变方式,携带率为6．25％（12／192）;2个位点（c．IVS1—35G〉T和c．88A〉G）属首次报道。酶切发现1例患者携带mt．1555纯合突变,后经测序发现还携带有mt．1438A〉G突变位点。结论吕梁地区耳聋患者以GJB2C．235delC为主要致病位点,本次研究结果为吕梁地区的耳聋预防奠定了基础,同时为今后临床医师诊断、治疗及遗传咨询提供了参考。     

A second-site mutation in the initiation codon of WAS (WASP) results in expansion of subsets of lymphocytes in an Wiskott-Aldrich syndrome patient

Wiskott-Aldrich syndrome (WAS) is caused by mutations in the gene encoding WAS protein (WASP ). Recently, somatic mosaicism caused by reversions or second-site mutations has been reported in some inherited disorders including WAS. In this article, we describe somatic mosaicism in a 15-year-old WAS patient due to a second-hit mutation in the initiation codon. The patient originally had a single-base deletion (c.11delG; p.G4fsX40) in the WAS (WASP) gene, which resulted in a frameshift and abrogated protein expression. Subsequently, a fraction of T and natural killer (NK) cells expressed a smaller WASP, which binds to its cellular partner WASP-interacting protein (WIP). The T and NK cells were found to have an additional mutation in the initiation codon (c.1A>T; p.M1_P5del). The results strongly suggest that the smaller WASP is translated from the second ATG downstream of the original mutation, and not only T cells but also NK cells carrying the second mutation acquired a growth advantage over WASP negative counterparts. To our knowledge, this is the first report describing somatic mosaicism due to a second-site mutation in the initiation codon of any inherited disorders.     

中国福建遗传性乳腺癌BRCA1基因突变分析

目的研究福建遗传性乳腺癌患者BRCA1基因突变位点及携带情况。方法对20例遗传性乳腺癌患者血液标本进行检测,对其BRCA1基因第11号外显子全序列进行DNA测序。结果20例标本中检出5患者存在共计9种BRCA1基因突变,其中3个为新发现位点（错义突变1159T〉C,4071A〉C;同义突变4122C〉T）;其它6个已报道位点中2个（2201C〉T,2430T〉C）为同义突变,其余4个（2685T〉C,2731C〉T,3232A〉G,3667A〉G）属错义突变,本研究中BRCA1突变率为25%。结论福建遗传性乳腺癌患者BRCA1基因突变具有地域性特征,开展BRCA1基因突变检测有助于本地区女性患癌风险评估和早期诊断。     

忻州地区耳聋患者常见耳聋基因GJB2、GJB3和线粒体DNA 12S rRNA 1555A〉G相关性分析

目的通过对忻州地区83例耳聋患者常见基因GJB2、GJB3和线粒体DNA 12S rRNA 1555A〉G测序分析,从分子水平研究该地区人群聋病的遗传病因和特点,为临床防聋治聋提供策略、依据。方法收集忻州地区83例耳聋患者外周血样本,提取DNA后对目的基因扩增并进行测序分析。结果83例耳聋患者中GJB2基因检测到57例发生突变,9个突变位点,与编码连接蛋白的非综合症耳聋突变数据库（http：／／davinci．crg．OS／deafneSS／index．php？Seccion=mut_db＆db=nonsynd）比对,8个位点已见报道,其中包括3个多态位点c．79G〉A、c．341A〉G、c．368C〉A和5个致病位点c．235delC、c．30-35delC、c．109G〉A、c．176-c．191dell6和c．299-c．300delAT,其中,c．79G〉A和c．341A〉G是主要突变方式,携带率为30．12％（42／174）和23．49％（39／166）。新发现1例未见报道的突变位点c．186C〉T;患者均未检测出GJB3和线粒体DNA12SrRNA1555A〉G基因突变位点。结论通过对忻州地区常见耳聋基因突变位点的研究,了解忻州地区该基因突变谱,为后续国内耳聋基因型分布提供数据支持,同时也为耳聋的早期诊断、治疗提供理论依据。     

Two novel mutations identified in the Wiskott-Aldrich syndrome protein gene cause Wiskott-Aldrich syndrome and thrombocytopenia

Wiskott-Aldrich syndrome (WAS) and X-linked thrombocytopenia (XLT) are rare X-linked genetic disorders caused by mutations of the Wiskott-Aldrich syndrome protein (WASP) gene. Both disorders are clinically characterized by chronic thrombocytopenia of small platelets. WAS is a more severe form of the disorder and also courses with eczema, and immune dysfunction. In the present study, we investigated two novel mutations of the WASP gene in two Spanish families with patients clinically diagnosed as having XLT and WAS, respectively. In one of the families a missense mutation in exon 12 (1488A>G), resulting in the highly conserved glutamic residue changing to glycine at position 485 (D485G), was identified in several members. Notably, a female of this family, with clinical signs of XLT, was determined as the carrier of the mutation and showed a skewed pattern of X-inactivation, preferentially inactivating the X-chromosome carrying the wild-type allele. In the case of the second family, we describe a WAS patient with a single base deletion in exon 2 (266-267delA), resulting in a frameshift (at codon 78) that creates a stop codon at amino acid 127. As a consequence, there was no WASP expression.