儿童脊髓性肌萎缩症的基因诊断

目的探讨儿童脊髓性肌萎缩症(SMA)的特异性基因诊断方法。方法应用聚合酶链反应-限制性片段长度多态性(PCR—RFLP)技术.对19例临床诊断为SMA患儿及21名健康儿童的运动神经元存活(SMN)基因进行检测。结果SMA患儿SMN基因的第7和第8号外显子均缺失,健康儿童SMN基因的第7和第8号外显子均未缺失。结论检测SMN基因第7和第8号外显子缺失的方法可用于SMA的基因诊断,且PCR—RFLP技术对SMA的诊断具有较高的特异性和敏感性。     

儿童脊髓性肌萎缩症的基因学研究

目的：研究我国儿童型脊髓性肌萎缩症(SMA)患者的运动神经元生存 (SMN)基因及神经细胞凋亡抑制蛋白 (NAIP)基因外显子的缺失情况,以探讨此二种基因与SMA表型之间的关系。方法：应用PCR和PCR -酶切法检测15例Ⅰ～Ⅲ型SMA患者(Ⅰ型4例,Ⅱ型3例,Ⅲ型8例)、20例表型正常的SMA直系亲属及30例正常对照的SMN基因的第7,8号外显子和NAIP基因的第5 ,6号外显子缺失情况。结果：7例Ⅰ型和Ⅱ型SMA患者中6例纯合缺失SMN基因外显子7和8,1例纯合缺失外显子7而保留外显子8;8例Ⅲ型SMA患者仅1例有外显子7和8的缺失,余7例均无SMN基因的缺失;15例Ⅰ～Ⅲ型SMA患者均未检测到NAIP基因外显子5和 /或 6的缺失。结论：Ⅰ型、Ⅱ型SMA可通过SMN基因第7,8号外显子的检测进行确诊,方法简便可靠,Ⅲ型SMA患者SMN基因缺失率低,故通过检测SMN基因 7,8外显子进行基因诊断尚需谨慎,NAIP基因在SMA发病中的作用尚不清楚,有待进一步研究。     

疑似脊髓性肌萎缩症患儿338例的运动神经元存活基因分析

目的 研究儿童脊髓性肌萎缩症(SMA)运动神经元存活基因SMN1缺失和诊断的意义.方法 根据国际诊断标准、病例随访和基因分析结果对338例疑似SMA的患儿进行诊断和分型.应用PCR-酶切方法分析患儿SMN1基因外显子7和外显子8的纯合缺失.应用等位基因特异PCR结合变性高效液相色谱分析(DHPLC)方法分析患儿的SMN1基因拷贝数,确定杂合缺失.结果 (1)确诊SMA 267例,其中Ⅰ型143例,Ⅱ型82例,Ⅲ型42例,分别占53.6%、30.7%和15.7%.(2)267例SMA患儿的SMN1基因缺失分析显示:SMN1基因外显子7和8均纯合缺失为183例,占68.5%(183/267),仅外显子7纯合缺失,外显子8不缺失为34例,占12.7%(34/267),外显子7杂合缺失为33例,占12.4%(33/267),非缺失为17例,占6.4%(17/267),未见SMN1基因外显子8的单独缺失.(3)Ⅰ型和Ⅱ型SMN1基因缺失率相近.Ⅲ型SMN1基因纯合缺失率较低于Ⅰ型和Ⅱ型,杂合缺失率较高于Ⅰ型和Ⅱ型.结论 (1)我国儿童SMA的SMN1基因纯合缺失和杂合缺失频率提示,SMN1基因突变存在种族异质性,SMN1基因内微小突变需要研究.(2)SMN1基因诊断具有特异性和无创性,80%SMA患儿通过SMN1基因纯合缺失分析得到诊断.(3)Ⅲ型SMA的临床诊断和基因分析需要进一步研究.     

聚合酶链反应-单链构象多态性和限制性酶切分析法对脊髓性肌萎缩症的基因诊断

目的 对脊髓性肌萎缩症（SMA）的早期诊断提供基因学特征和可靠的辅助检测手段。方法 用PCR-单链构象多态性(SSCP)和PCR-限制性酶切分析法对首都儿科研究所附属儿童医院神经科门诊的30个SMA家系和50名入托查体正常儿童运动神经元存活基因(SMN)的第7和8外显子进行缺失检测。结果 SMN基因外显子7和8缺失检测结果：在30例SMA患儿中，22例（73.3%，22/30）同时缺失SMN1基因外显子7和外显子8，4例（13.3%，4/30）显示单纯SMN1基因外显子7纯合缺失，4例（13.3%，4/30）SMN1基因外显子7或8均未见缺失，未见单纯SMN1基因外显子8纯合缺失。1例SMAⅠ型患儿父亲为SMN1基因外显子7和8的纯合缺失。1名正常儿童有SMN2基因外显子7和8的纯合缺失。经过PCR 限制性酶切法检测不伴有缺失的2例SMA Ⅲ型患儿及其家系SMN1基因外显子8 SSCP电泳图中出现了异常条带。结论 PCR-限制性酶切和PCR-SSCP分析法对SMN1基因外显子7和8缺失进行检测是诊断SMA的有效辅助手段,两者联合应用可以相互验证、互为补充；SMN1基因外显子7或8的缺失检测对SMA进行基因诊断是一种简便、特异的诊断方法，并且由于其为一种无创性检查，易被家长接受，是SMA临床症状前诊断、鉴别诊断和临床确诊的重要辅助手段。     

脊髓性肌萎缩的基因诊断和产前基因诊断研究

目的探讨中国人脊髓性肌萎缩(SMA)基因诊断和产前基因诊断的可行性。方法应用复合聚合酶链反应-限制性片段长度多态(PCR-RFLP)方法对31例SMA患儿进行神经元存活基因(SMN)第7外显子缺失分析,并对2例有SMA阳性家族史的家系进行了产前基因诊断。结果96.8％(30／31)SMA患儿携有SMN基因第7外显子缺失。2例产前基因诊断的病例均无SMN基因第7外显子缺失。结论SMN基因缺失检测技术可用于SMA患儿的基因诊断和产前基因诊断。     

中国脊髓性肌萎缩症患儿的SMN基因学研究

目的：明确中国人各型脊髓性肌萎缩症(SMA)患儿SMN1基因外显子7和8缺失,SMN基因转变及微小突变情况。方法：对106例患者,采用PCR-RFLP检测基因缺失,RFLP筛查基因转变,并对PCR产物进行测序分析。基因转变率比较采用Fisher精确检验法进行统计学分析。结果：SMA患儿纯合SMN1外显子7和/或8缺失比率为91.5%,发现1例SMA中存在SMN1外显子7保留,而SMN1非编码外显子8缺失。在SMN1外显子7缺失而无外显子8缺失的患者中,各型间基因转变率差别无统计学意义,在SMN1外显子7缺失的SMA中,其基因转变率为8.3%。SMN第7外显子附近未发现基因微小突变。结论：SMN1基因外显子7和/或8缺失为中国SMA患儿的主要病因。SMA中存在SMN基因转变现象。SMN1基因外显子8的单独缺失可能致病。SMN1基因外显子7附近可能不是此病微小突变的热点区域。［中国当代儿科杂志,2010,12（7）：539-543］     

同时检测两种运动神经元存活基因第7外显子缺失的简便方法及其临床应用

目的：建立同时检测运动神经元存活基因SMNT和SMNC第7外显子缺失的简便方法，用于儿童期起病的脊髓性肌萎缩症（SMA）的临床诊断。方法：应用聚合酶链反应（PCR）－限制性酶切技术，用一对引物同时扩增55例正常成年人和5例SMA患儿及其父母的SMN基因2个成员的第7外显子中的高度保守区，扩增产物经限制性内切酶酶切及非变性聚丙烯酰胺凝胶电泳。结果：此法可检测2个SMN基因第7外显子的缺失情况，经检测，55例正常成人均无SMNT基因第7外显子缺失，SMNC基因第7外显子缺失仅3例，5例SMA患儿的SMNT基因第7外显子均有缺失，患儿父母无SMNT基因和SMNC基因第7外显子缺失，结论：此法对检测2个SMN基因第7外显子的缺失提供了简便，特异，且适合临床特别是基层医院应用，优于PCRSSCP分析的方法。     

脊髓性肌萎缩症SMN1和SMN2基因拷贝数变异分析

目的:探讨运动神经元存活（SMN）1和SMN2基因拷贝数变异与脊髓性肌萎缩症（SMA）患儿临床表型的关系。方法:以2011年10月至2012年12月在复旦大学附属儿科医院临床诊断SMA患儿为研究对象,采用基因组DNA多重连接探针扩增（MLPA）技术进行SMN1基因缺失和SMN2基因拷贝数变异检测,探讨拷贝数变异与SMA临床分型的关系。结果:41例临床诊断SMA患儿行基因检测,其中SMN1基因第7和（或）8外显子缺失37例（90.2％）进入分析,男女之比为1∶0.8,发病年龄为（7.5±7.0）个月。Ⅰ型20例（54.1％）,Ⅱ型15例（40.5％）,Ⅲ型2例（5.4％）,发病年龄分别为（2.9±1.8）、（10.7±1.9）和（30.0±8.5)个月。37例SMN1基因第7和（或）8外显子缺失患儿中,18例SMN2基因第7和8外显子拷贝数为2个,其中13例（72.2％）为Ⅰ型,5例（27.8％）为Ⅱ型;19例SMN2基因第7和8外显子拷贝数增加（拷贝数3或4）,其中7例（36.8％）为Ⅰ型,10例（52.6％）为Ⅱ型,2例（10.5％）为Ⅲ型,两组差异有统计学意义。5例患儿父母行SMN1基因检测,共检出杂合缺失9例,其中4例患儿父母均为SMN1基因第7和8外显子杂合缺失,1例患儿父亲为SMN1基因第7和8外显子杂合缺失,母亲未检测到纯合或杂合缺失。结论:SMN1基因第7和（或）8外显子纯合缺失是SMA致病主要原因,SMN2基因拷贝数增加与SMA表型严重程度呈负相关。     

31例儿童脊髓性肌萎缩症临床与基因分析

目的探讨脊髓性肌萎缩症(SMA)的临床表型及基因型特点。方法回顾分析2014年2月至2019年5月确诊的31例脊髓性肌萎缩症患儿的临床资料。结果 31例SMA患儿中,男女比例1.8:1;6月龄内起病12例(38.7%),～18月龄起病17例(54.8%),18月龄后起病2例(6.5%)。首发症状为肌张力低下13例(41.9%),肌力下降9例(29.0%),步态异常5例(16.1%),生长迟缓4例(12.9%)。肌无力主要以近端受累为主,下肢重于上肢,腱反射减弱或消失。在感觉或认知方面均无变化。采用MLPA行基因检测,31例患儿中,SMN1基因外显子7和外显子8纯合缺失29例(93.5%),仅外显子7缺失2例(6.5%),均为2型患儿。不同类型SMA的临床表型与SMN1基因缺失类型之间差异无统计学意义(P0.05)。2型和3型SMA患儿的SMN2基因拷贝数高于1型,3型SMA患儿的SMN2基因拷贝数明显高于2型,不同SMA临床表型与SMN2拷贝数分布差异有统计学意义(P0.05)。30例(96.8%)患儿的父母亲明确诊断为SMN1基因杂合缺失;1例患儿父亲明确诊断为SMN1杂合缺失,母亲未检测到。结论 SMN1基因的检测和分析对SMA患儿具有诊断意义。SMA临床表型的严重程度与SMN2基因拷贝数增加呈反比。     

儿童型脊肌萎缩症SMN基因缺失与微突变检测

目的：研究儿童型脊肌萎缩症(SMA)患者中运动神经元生存基因缺失与微突变情况。方法：收集经临床和肌肉活检确诊的SMA I~III型25例,其中I型5例,II型3例,III 17例及直系亲属24例。采用PCR-RFLP检测SMNt缺失情况,对无SMNt缺失的患者及SMA直系亲属,应用PCR-SSCP结合DNA序列分析的方法,进行SMN基因微突变分析。结果：5例I型和3例II型SMA患者均见SMNt缺失,缺失率100%,6例III型见缺失,缺失率35%(6/17)。11例无缺失的SMA III型患者的gDNA编码区域未发现微突变;24例SMA的直系亲属中未发现SMN基因缺失及突变。结论：①检测到SMNt外显子7缺失可作为SMA的确诊手段,有望替代肌电图和肌活检等有创检查;②对无SMNt外显子7缺失的III型SMA患者,要结合临床进行诊断;③该组无SMNt缺失的III型患者未发现微突变,提示存在遗传异质性。[中国当代儿科杂志,2005,7(6):489-492]     

Deletion analyses of SMN1 and NAIP genes in Malaysian spinal muscular atrophy patients

BACKGROUND: The survival motor neuron 1 (SMN1) gene has been recognized to be responsible for spinal muscular atrophy (SMA) because it is homozygously deleted in more than 90% of SMA patients, irrespective of their clinical severity, whereas the neuronal apoptosis inhibitory protein (NAIP) gene is now considered to be a modifying factor of the severity of SMA. In Malaysia, it remains to be elucidated whether deletion of the SMN1 gene is also a main cause of SMA or whether deletion of the NAIP gene is found in the SMA patients. METHODS: To clarify the pathogenesis of SMA in Malaysia, a deletion analysis of the SMN1 and NAIP genes was performed in 24 Malaysian SMA patients. Deletion analysis of exons 7 and 8 of the SMN1 gene was performed according to the method described by van der Steege et al., while deletion analysis of exon 5 of the NAIP gene was performed according to a method described by Roy et al. RESULTS: Homozygous deletion of SMN1 exon 7 and exon 8 were identified in 19 out of 24 patients (79%). As to the NAIP gene, deletion of exon 5 was detected in six out of 24 patients (25%). NAIP gene deletion was correlated with severity of the disease. CONCLUSIONS: Deletion of the SMN1 exon 7 is a major cause of SMA in Malaysia, and NAIP gene deletions are not rare in type I SMA in Malaysia. The lower percentage of the SMN1 gene deletion may be due to the possibility that the present study included some patients without SMN1 gene abnormality and/or some patients with non-deletion type mutations in the SMN1 gene.     

Molecular analysis of the SMN and NAIP genes in Iranian spinal muscular atrophy patients

Background:  Spinal muscular atrophy (SMA) is an autosomal recessive neuromuscular disorder characterized by degeneration of spinal cord anterior horn cells, leading to muscular atrophy. SMA is clinically classified into three subgroups based on the age of onset and severity. The majority of patients with SMA have homozygous deletions of exons 7 and 8 of the survival motor neuron ( SMN ) gene. The purpose of the present study was to determine the frequency of SMN and neuronal apoptosis inhibitory protein ( NAIP ) gene deletions in Iranian SMA patients. Experience in prenatal diagnosis of SMA in this population is also reported.
Methods:  To study the frequency of deletions of SMN and NAIP genes in an Iranian sample group, 75 unrelated SMA patients (54 type I, eight type II and 13 type III) were analyzed according to the methods described by van der Steege et al and Roy et al .
Results:  Homozygous deletion of SMN1 exons 7 and/or 8 were identified in 68 out of 75 patients (90%). Deletion of exon 5 of the NAIP gene was found in 40/54 of type I, 2/8 of type II and 1/13 of type III patients.
Conclusions:  Deletion of the SMN1 gene is a major cause of SMA in Iran, and NAIP gene deletions were common in the present patients with type I SMA. Also, the incidence of NAIP deletion is higher in more severe SMA.     

Identification of T274I mutation in the SMN1 gene in a patient with spinal muscular atrophy

Proximal spinal muscular atrophy (SMA) is an autosomal recessive neuromuscular disorder characterised by degeneration of motor neurones in the spinal cord. The symptoms of the disease are determinated by mutations of SMN1 gene. About 98% of SMA patients show homozygous absence of exon 7 SMN1 gene, the rest carry small intragenic mutations. Molecular analysis of the presence of exon 7 SMN1 gene deletion is considered as the screening test for SMA. We present a case report of a 9 years old girl with progressive muscular weakness of limbs and trunk. Clinical examination followed by electromyography and muscle biopsy was interpreted as a diagnostic of SMA 3. Molecular analysis did not reveal deletion of exon 7 SMN1 gene. Extended molecular diagnostics using direct sequencing showed missence mutation T2741. Thus, the absence of homozygous deletion of exon 7 SMN1 gene does not exclude SMA diagnosis. All patients fulfilling the diagnostic criteria for SMA, as defined by the International SMA Consortium, without deletion of exon SMN1 gene, should be analysed using direct sequencing.