分子遗传学技术诊断MECP2重复综合征四例分析北大核心CSCD

目的 用多重连接探针扩增技术（MLPA）和微阵列比较基因组杂交技术（array-CGH）研究4例以运动、智力发育落后为主要表现的患儿甲基化CpG结合蛋白2基因（MECP2）基因突变特点.方法 取北京大学第一医院2012年6月至2014年4月收治的4例患儿及其中例2、例4母亲的外周血,提取基因组DNA;先对患儿用MLPA方法进行微缺失和微重复检测,然后用array-CGH进行分析进一步确定重复片段的大小;同时对2例患儿的母亲进行array-CGH和X染色体失活分析（XCI）.结果 4例患儿均表现为严重肌张力低下,运动、智力发育落后和语言发育障碍,除例2之外,另3例患儿婴儿期均反复发生肺炎.MLPA显示4例患儿均存在染色体Xq28重复;array-CGH检测显示4例患儿Xq28区域存在重复,4例患儿重复片段大小分别为14.931 Mb、0.393 Mb、0.482 Mb、0.299 Mb,经与UCSC（http：//genome.ucsc.edu/）数据库比对,4例患儿的重复片段均包含MECP2和宿主细胞因子C1基因（HCFC1）.例2和例4患儿的母亲存在Xq28重复,其中例4患儿母亲的重复片段起止位点和大小与患儿完全相同,例2母亲重复片段为0.343 Mb,小于患儿,近着丝粒断点与患儿不同,远端断点与患儿相同.X染色体失活分析发现母亲二条X染色体活性比例为0∶100,存在重复的一条X染色体完全失活,并且将发生重复的这条X染色体遗传给了患儿.结论 对于运动智力发育落后、肌张力低下、语言发育障碍和反复发生感染的患儿进行MLPA和array-CGH联合检测是诊断MECP2重复综合征的有效且特异的方法.     

Wolf-Hirschhorn综合征4例及文献复习

目的　探讨Wolf-Hirschhorn综合征（WHS）临床特征及基因突变。方法　回顾分析2017-11-20—2018-05-26南京医科大学附属儿童医院收治的4例发育延迟及智力低下患儿的临床资料，临床拟诊为WHS。应用染色体微阵列芯片分析技术进行基因检测，并复习相关文献总结疾病特点。结果　2例男性和2例女性患儿因生后特殊面容（希腊头盔样面容）、智力低下、发育延迟、肌张力低下、癫痫，应用染色体微阵列芯片分析技术发现患儿4p16.3区域2.24～3.8 Mb的缺失，确诊WHS，给予抗癫痫及康复治疗并定期随访。结论　尽早完善染色体芯片技术检查有助于早期诊断WHS，且能判断预后。染色体微阵列芯片分析与传统细胞遗传学分析方法相比，具有高分辨和高准确度的优点，可为产前遗传学诊断提供更详细信息。     

Wolf-Hirschhorn综合征4例及文献复习

目的　探讨Wolf-Hirschhorn综合征（WHS）临床特征及基因突变。方法　回顾分析2017-11-20—2018-05-26南京医科大学附属儿童医院收治的4例发育延迟及智力低下患儿的临床资料，临床拟诊为WHS。应用染色体微阵列芯片分析技术进行基因检测，并复习相关文献总结疾病特点。结果　2例男性和2例女性患儿因生后特殊面容（希腊头盔样面容）、智力低下、发育延迟、肌张力低下、癫痫，应用染色体微阵列芯片分析技术发现患儿4p16.3区域2.24～3.8 Mb的缺失，确诊WHS，给予抗癫痫及康复治疗并定期随访。结论　尽早完善染色体芯片技术检查有助于早期诊断WHS，且能判断预后。染色体微阵列芯片分析与传统细胞遗传学分析方法相比，具有高分辨和高准确度的优点，可为产前遗传学诊断提供更详细信息。     

4号染色体4q21/q22缺失综合征1例报告

目的提高对4q21/q22缺失综合征的临床和基因特征的认识。方法回顾性分析1例有特殊面容及体格智力发育迟缓患儿的临床资料及基因检测结果。结果患儿,女,2个月。表现为喂养困难,发育迟缓,新生儿期肌张力低下,头大,前额及枕部突出,小手足;合并先天性心脏病。应用染色体微阵列芯片分析技术发现患儿4q21.21q22.2区域有一缺失,缺失片段大小约15.26 Mb,至少包含76个基因,确诊为4q21/q22缺失综合征。结论患儿有典型面容特征,发育迟缓,伴其他系统累及时应考虑4q21/q22缺失综合征可能,染色体芯片检测技术有助于明确诊断。     

Rett综合征与MECP2基因

Rett综合征是一种严重影响儿童精神运动发育的神经遗传病 ,诊断主要依靠国际上根据临床表现制定的标准 ,包括典型和非典型Rett综合征两类。该病的遗传方式尚无定论。致病基因是位于染色体Xq2 8的MECP2基因 ,编码甲基化结合蛋白 2。推测致病机制为MECP2基因突变导致蛋白结构和功能异常 ,使胚胎发育时期应当转录静止的基因继续转录 ,而这种“转录噪音”对神经系统的生长和发育具有致病作用。典型Rett综合征患儿MECP2基因主要有 8种热点突变。MECP2基因作用的下游靶基因尚不清楚。     

微阵列分析技术检测脑发育迟缓患儿基因拷贝数变异

目的 分析发育迟缓患儿基因拷贝数变异(CNVs)与临床表现的相关性.方法 应用微阵列单核苷酸多态性(SNP array)分析技术对1例发育迟缓患儿及其临床表型正常的父母亲进行全基因组CNVs分析.结果 在患儿chr8p23.3p23.1区域发现7.9 Mb片段缺失,在chr8p23.1p11.23区域发现27.4 Mb片段重复;在患儿父亲chr7q31.1区域发现1.21 Mb重复,chrXp22.33区域发现99 kb缺失;患儿母亲未检测到CNVs改变.结论 SNP array技术有助于进一步明确发育迟缓患儿的遗传机制.     

15q11.2-13.2微重复四倍体综合征1例并文献复习

目的 采用分子遗传学技术分析1例常规染色体核型拟诊为21/22三体的发育迟缓伴孤独症患儿,明确遗传学诊断。方法 收集患儿及其父母的外周血标本,常规提取基因组DNA,应用高分辨染色体核型分析（400-550带）检测患儿及其父母的染色体数目及结构,微阵列比较基因组杂交技术(array-CGH)筛查患儿的全基因组拷贝数变异,以荧光原位杂交技术（FISH）对异常的基因拷贝进行染色体精确定位和定量。结果 女,2岁,发育迟缓伴孤独症样表现。外侧眼角下垂、内眦赘皮。常规染色体核型检查（320带）分别为47,XX,+22和47,XX,+21。高分辨染色体核型分析显示,该患儿携带额外标记染色体（SMC）,核型为47,XX,+mar dn,尚不能确定是否为21/22三体携带者,患儿父亲高分辨率核型染色体分析提示为46,XY,母亲为46,XX,提示患儿携带SMC为新生突变。array-CGH检测显示15q11.2-13.2区域微重复（chr15:22684529-30730543,8.0 Mb,hg19）。FISH验证该SMC来源于15号染色体,由15q11.2-13.2区域二倍体及双着丝粒组成。患儿最终诊断为15q11.2-13.2微重复四倍体综合征。复习文献报道的15q11.2-13.2拷贝数增加病例的临床表型,微重复四倍体综合征的主要表型有智力低下/发育迟缓（100％）、肌张力低下（92.9％）、孤独症/孤独症样表现（71.4％）和癫痫（61.5％）等。结论 15q11.2-13.2微重复四倍体综合征是患儿发生精神发育迟滞伴孤独症的遗传学基础,array-CGH能够快速、准确地检测基因组的微小失衡。     

3例MECP2重复综合征临床分析和文献复习

MECP2重复综合征(MDS)是儿科少见病,主要表现为运动发育落后、语言缺失或落后、反复感染、严重智力障碍、癫癎、孤独症样表现及婴儿早期肌张力低下等。该文3例患儿均为男孩,病例1、2首发表现为运动发育落后、语言缺失或落后,病例3首发表现为反复感染,查体均有肌张力均低下、病理征均阴性。病例1有全身强直-阵挛发作,脑电图示局灶性发作,予奥卡西平、左乙拉西坦、氯硝基安定联合抗癫癎治疗,癫癎发作控制。病例3出现过失神发作1次及点头发作3次,发作间期多次脑电图正常,未予抗癫癎治疗。3例患儿的反复感染随年龄增长得到改善,语言及智力改善不明显。微阵列比较基因组杂交技术(aCGH)检测发现3例患儿的X染色体存在MECP2基因重复,确诊为MDS。对于发育落后伴反复感染、癫癎发作的患儿,应考虑到MDS可能,早期行aCGH检测有助于诊断。     

染色体1p36缺失综合征2例诊断及随访

目的探讨1p36缺失综合征的临床特征。方法应用染色体微阵列分析技术(CMA)对2例有特殊面容的生长发育落后患儿进行基因检测,并长期随访例1患儿身高和体质量。结果患儿男女各1例,均表现为特殊面容、肥胖、矮小、智力低下,尤其是语言发育落后;应用CMA发现2例患儿1p36.33-1p36.32区域均有缺失,例1女性患儿为1 757kb的杂合缺失,例2男性患儿为2 533 kb的杂合缺失,均确诊为1 p 36缺失综合征。例1女性患儿自7岁起长期随访身高增长,发现其12岁起身高曲线自-2SD向下偏离。结论 1p36缺失综合征有典型面容特征,智力障碍,尤其是语言发育落后,矮小。女性患者可能因青春期中期身高增速减缓而导致终身高矮小。CMA有助于明确诊断。     

5p部分单体3例全基因组拷贝数变异分析

目的 应用全基因组微阵列芯片平台,对染色体核型提示为Cri du chat综合征的新生儿进行全基因组拷贝数变异（CNVs）的检测,以帮助解释基因型与表型的相关性。方法 2009年6月至2010年5月复旦大学附属儿科医院收治的染色体核型提示为Cri du chat综合征的3例新生儿进入研究。采用Cytogenetic Whole Genome芯片筛查全基因组CNVs,针对发现的所有CNVs进行分析,参照国际基因组拷贝数变异多态性数据库除外正常人群多态性CNVs。结合本研究3例与DECIPHER数据库已报道的Cri du chat综合征患儿的临床表型,行5p缺失大小及范围分析,对重复区域行候选基因分析。结果 3例患儿经微阵列芯片检测,均证实并更为精确的定位了5p的缺失范围。例1 5p缺失位于5p15.33-p13.3,例2 缺失位于5p15.33-5p15.1,例3 缺失位于5p15.33-p14.3;此外例2发现9p部分重复,例3发现7p部分重复。结合DECIPHER数据库已报道的5例Cri du chat综合征临床表型,重复区域和候选基因分析显示,临床表型为猫叫样哭声或声音异常：缺失片段重叠区域为5p15.33-15.31内3.86 Mb,覆盖(IRX1和IRX2与胚胎形成相关的基因);临床表型为面容异常：缺失片段重叠区域为5p15.2-15.1内2.51 Mb（覆盖ANKH与颅骨干骺端发育相关的基因）。例3合并有先天性巨结肠。因纳入病例均为新生儿,无法评价是否存在智力低下和生长发育迟缓,无法对相应的关键区域进行分析。结论 本研究提供了微阵列平台罕见潜在致病可能CNVs的分析方法,进一步为建立5p部分缺失表型基因型关联性提供了依据。     

Zespół duplikacji MECP2 – opis przypadku

Microdeletion/microduplication syndromes belong to a group of disorders of chromosomal aberrations, so-called adjacent genes whose detection requires the use of molecular cytogenetic techniques (FISH, MLPA, aCGH). MECP2 duplication syndrome is clinically characterized by psychomotor retardation, minor dysmorphic facial features, hypotonia in infancy and increasing spasticity later in life. Among the other symptoms, autistic traits, disorders of speech development, epilepsy, recurrent infections, gastro-oesophageal reflux and constipations have been reported. The authors present the case of a 4-year-old boy with MECP2 duplication syndrome diagnosed due to the delay psychomotor development and muscle hypotonia. Due to the clinical features and de/dysmyelination changes in MRI inborn errors of metabolism, including mutations in the gene SCO2, was excluded. Diagnosis of proband was estimated on the result of aCGH studies, which concluded duplication of Xq28 region covering the gene MECP2. The authors emphasize that in cases of delayed psychomotor development of unclear etiology, with or without dysmorphic features, diagnosis based on aCGH should be considered.     

MECP2 triplication in 3 brothers – A rarely described cause of familial neurological regression in boys

Male patients with large duplications of the methyl CpG-binding protein 2 (MECP2) gene have been identified with a characteristic phenotype consisting of infantile hypotonia replaced by spasticity, developmental delay, severe mental retardation and recurrent respiratory infections. Only one patient with MECP2 triplication, with a more severe phenotype has been reported so far. We report three brothers of unrelated parents with MECP2 triplication. Their phenotypic features include macrocephaly with large ears, infantile hypotonia, developmental delay, significant constipation, recurrent severe respiratory tract infections from early childhood, and seizures followed by neurological regression in late childhood. Our cases indicate that MECP2 triplication is similar to or more severe than that of MECP2 duplication syndrome.     

Progressive cerebellar degenerative changes in the severe mental retardation syndrome caused by duplication of MECP2 and adjacent loci on Xq28

Localised duplications, involving the MECP2 locus, at Xq28 have been associated with a syndrome comprising X-linked mental retardation, hypotonia and recurrent infections in males. We now present neuroradiological evidence that progressive cerebellar degenerative changes may also be a consistent feature of this syndrome, emerging in the second decade of life. We report seven affected males, from three different families who, in addition to the previously described clinical findings, have a reduction in the volume of the white matter and mild dilatation of the lateral ventricles. Three of the older patients show a consistent cerebellar degenerative phenotype. Furthermore, we describe the first female affected with the disorder. The female was mildly affected and shows X-inactivation in the ratio of 70:30, demonstrating that X-inactivation cannot be exclusively relied upon to spare the female carriers from symptoms. In conclusion, there is a radiological phenotype associated with Xq28 duplication which clearly demonstrates progressive degenerative cerebellar disease as part of the syndrome.     

Trisomy 21 and Rett syndrome: a double burden

Rett syndrome is a severe neurodevelopmental disorder generally affecting girls. Affected individuals are apparently normal at birth but later pass through a period of regression with loss of hand and communication skills and the development of hand stereotypies and dyspraxia. Mutations in the methyl-CpG binding protein 2 (MECP2) gene, have now been found to cause Rett syndrome in up to 80% of classical cases. We report a girl with Down syndrome, one of three children with birth defects in a family of five. From the age of 18 months she developed symptomatology considered by her primary physician to be very characteristic of Rett syndrome. However, this remained a clinical diagnosis till the age of 12 years. Laboratory confirmation of the dual diagnosis, which includes a R168X mutation in the MECP2 gene in addition to trisomy 21, has now been possible. The presence of one neurological or developmental disorder does not necessarily preclude a diagnosis of Rett syndrome.     

Late onset epileptic spasms is frequent in MECP2 gene duplication: Electroclinical features and long-term follow-up of 8 epilepsy patients

Mutation of the X-linked methyl CpG binding protein 2 (MECP2) has been first identified as the cause of Rett syndrome. More recently, MECP2 gene duplication syndrome has been identified in males. The MECP2 duplication syndrome is characterized by severe mental retardation, infantile hypotonia, progressive spasticity and recurrent infections. Epileptic seizures are inconstant but poorly described. The aim of the study is to describe the electroclinical features of epilepsy in MECP2 duplication patients in order to refine the epilepsy phenotype and its evolution.MethodsWe conducted a retrospective study in four child neurology departments in France. Eight boys with a MECP2 gene duplication and epilepsy were retrospectively studied. We evaluated both clinical and electroencephalographic data before seizure onset, at seizure onset and during the follow-up.ResultsThe patients started seizures at the median age of 6 years (range: 2.5–17 years). Half exhibits late onset epileptic spasms while the other exhibit either focal epilepsy or unclassified generalized epilepsy. Before seizure onset, EEGs were abnormal in all patients showing a slowing of the background or a normal background with fast activities, while EEG performed in epileptic patients, showed a slowing of the background in 6/8 and localized slow or sharp waves in 7/8. Most patients (6/8) have evolved to drug resistant epilepsy.ConclusionAlthough late onset epileptic spasms are common in patients with MECP2 duplication, no specific electroclinical phenotype emerges, probably due to genetic heterogeneity of the syndrome. Further studies are needed to individualize specific epileptic subtype in larger cohort of patients.     

Coinheritance of mutated SMN1 and MECP2 genes in a child with phenotypic features of spinal muscular atrophy (SMA) type II and Rett syndrome.

Spinal muscular atrophy (SMA) is a neuromuscular autosomal recessive disease characterized by progressive muscle weakness and atrophy combined with motor neuron degeneration caused by mutations in the SMN 1 gene locus (5q11.2-13.2). Rett syndrome (RS) is an X-linked dominant neurodevelopmental disorder caused by mutations in MECP2 (Xq28) and characterized by normal development until 6-12 months of age, followed by regression with loss of acquired skills, gradual onset of microcephaly, stereotypic hand movements and psychomotor delay. We report a 6-year-old girl who, at 2 years of age, presented with hypotonia, psychomotor delay, amyotrophy and areflexia of the lower extremities. Molecular DNA analysis (PCR-RFLP's) for SMA type II revealed that both exons 7 and 8 of SMN 1 gene were deleted. Over the past 4 years, onset of stereotypic hand-washing movements, epileptic seizures, microcephaly, hyperventilation/breath-holding attacks and severe psychomotor delay raised the suspicion of the coexistence of RS. DNA analysis (DGGE and sequencing) identified the hotspot missense mutation R306C (c.916C>T) in exon 4 of the MECP2 gene. The coinheritance of SMA and RS, two rare monogenic syndromes in the same patient, has not been previously reported. Thorough clinical evaluation in combination with DNA analysis, allowed accurate diagnosis, providing valuable information for the genetic counseling of the family.     

长片段PCR-DNA测序方法在Rett综合征诊断中的应用

目的探讨利用长片段PCR—DNA测序方法检测Rett综合征（RTT）患儿MECP2基因突变的可行性及临床意义。方法对40例临床诊断的RTT患儿用盐析法从外周血提取基因组DNA,采用长片段PCR同时扩增MECP2基因的第3和第4外显子,用1．5％的琼脂糖凝胶鉴定扩增目的片段的大小,进行DNA直接测序。结果在40例RTT患儿中有33例患儿MECP2基因存在突变：无义突变16例;错义突变14例;缺失突变3例,其中有一例为314bp的大片段基因缺失。突变以p．T158M最为多见,占21％（7／33）,其后依次为p．R255X,占12％（4／33）,p．R168X和p．R106W各占9％（3／33）,p．R270X和p．Y141X各占6％（2／33）,p．R133C、p．D156H、p．F157L、p．P225R、p．Q244X、p．Q262X、p．R294X、p．R306C、P322L、c．1005delG、c．1005—1318del314bp和c．1127—1179del53bp各占3％（1／33）。结论长片段PCR方法鉴定了83％（33／40）的RTT患儿存在MECP2基因突变,目前是一种简单、方便、快速、准确的基因诊断方法,能同时发现常见突变和基因大片段的缺失,有助于RTT的诊断。     

Apneic crises: A clue for MECP2 testing in severe neonatal hypotonia-respiratory failure

Males with methyl-CpG-binding protein 2 (MECP2) mutations may present with neonatal encephalopathy. We report on an infant with a MECP2 mutation who exhibited complex constellation of symptoms, including severe hypotonia, respiratory failure, and apneic episodes. In the neonatal period these symptoms are common to other disorders, including Ondine syndrome. Our observation confirms that the triad of severe hypotonia, apneic episodes, and respiratory failure may be caused by MECP2 mutations. Neonatologist and neuropediatricians must be alert to the presence of these symptoms to exclude this rare but severe disorder. Clinical suspicion and molecular confirmation of MECP2 mutation is of great importance for defining the diagnosis of this rare affection.     

Semi-dominant X-chromosome linked learning disability with progressive ataxia,spasticity and dystonia associated with the novel MECP2 variant p.V122A: Akin to the new MECP2 duplication syndrome?

A novel X-chromosome linked phenotype is reported. Three affected males had learning disability in early childhood and subsequently developed progressive ataxia, dystonia, and spasticity with death at ages 9, 14 and 19 years. Two female obligate carriers had learning difficulties with psychosis in one case. A third, possible carrier had learning difficulties and epilepsy. A family study indicates that this inherited syndrome is most likely due to an unreported MECP2 variant, p.V122A, located in the methyl binding domain of the MECP2 protein. The clinical features are similar to those present in the newly reported MECP2 duplication syndrome. Non-progressive neuropsychiatric symptoms in female relatives of a male child with learning disability, ataxia and progressive spasticity may constitute a clue to inherited, MECP2 pathogenesis.     

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。