慢性肌酸磷酸激酶升高患儿的临床分析暨幼年起病的晚发型Pompe病临床和基因分析

【作者】 仇佳晶；

【导师】 魏珉；

【作者基本信息】 中国协和医科大学 ， 儿科学， 2007， 博士

【摘要】 背景和目的因血清肌酶谱升高而就诊的患儿在临床中并不少见。肌酶谱包括肌酸磷酸激酶(CK)、天门冬氨酸氨基转移酶(AST)、乳酸脱氢酶(LDH)和α羟丁酸脱氢酶HBDH。其中CK主要存在于骨骼肌、心肌和脑细胞的胞浆中,少量存在于线粒体中;而AST、LDH、HBDH等除肌肉组织外还存在于肝脏、肾脏、红细胞等其他组织中。CK是可逆催化ATP和肌酸之间高能磷酸转移的转移酶,血清中的CK 95%以上来源于骨骼肌,因此血清CK的升高对于提示肌肉组织病变最为特异。骨骼肌是机体执行运动和能量代谢的重要器官,肌肉病变在成人中患病率约为1%,但由于不少患者没有及时就诊或被误诊误治,真正的患病率要远远超过这个比例。临床上,与CK增高相关的疾病较多,肌源性、脑源性、代谢性、肿瘤性疾病等需要排除;血清CK升高还可见于许多生理和病理情况,如剧烈运动、感染、药物、中毒、内分泌疾病等。在儿童患者,急性CK升高常与心肌炎、肌炎等感染、免疫性疾病相联系;而慢性持续的CK升高,病因比较复杂:可见于免疫炎症性疾病、遗传代谢性疾病以及一些无症状患儿中,诊断不明确,误诊率高,如何建立一个相对科学的诊断流程,已成为困扰临床医生的一个难题;有些疾病只要及时发现,早期治疗,完全可以治愈;有些及时诊断,早期给予适当的治疗则可改善症状,防止肌肉萎缩等并发症的发生,因此明确持续血清肌酸磷酸激酶升高的儿童患者的病因诊断对治疗和判断预后有非常重要的意义。方法对2005年1月-2007年3月,从协和医院遗传门诊入院的肌酸磷酸激酶持续升高超过3月患儿的临床资料,实验室检查,肌肉活检组织病理资料,酶生化检测及活性测定,基因分析等进行回顾性分析,明确持续血清肌酸磷酸激酶升高的儿童患者的病因诊断,希望对今后建立一个合理的诊断流程提供参考。结果25例患儿,男18例,女7例。年龄从2岁9月-15岁,平均年龄7.8岁。病程3个月-7年。CK值在285 U/L-15720 U/L(18-198U/L)。25例患儿均进行了开放性肌肉活检病理检查,6例进行了皮肤成纤维细胞内溶酶体酸性葡萄糖苷酶活性测定,12例进行了基因分析。病因诊断:16例患儿获得诊断,确诊率64%,诊断分别为:7例dystrophin相关肌营养不良症,1例携带者;2例Pompe病,1例糖原累积症Ⅲ型;1例线粒体肌病,1例戊二酸尿症Ⅱ型,2例炎症性肌肉病,1例脊肌萎缩症。其余9例未能明确诊断。结论血清肌酸磷酸激酶持续增高对儿童期患者多提示神经肌肉疾病或累及肌肉的代谢性疾病,病因复杂,除常规生化检查外,肌电图、肌活检、酶活性测定、基因检测等多种检查的综合应用有助于提高诊断率。背景和目的糖原累积症Ⅱ型(Pompe病)是一种常染色体隐性遗传病,其代谢基础为溶酶体内α-1,4-葡萄糖苷酶的缺陷,糖原不能被分解,正常结构的糖原堆积在溶酶体和胞质中,造成溶酶体的增生和破坏,相应的脏器结构和功能损害。临床根据发病年龄、主要累及的脏器、进展速度的不同分为早发型和晚发型,早发型又称为经典型,多于生后不久-婴儿期即发病,糖原在心脏、骨骼肌、平滑肌、肾小管上皮,中枢神经系统等全身组织中广泛沉积,多于1-2岁之内死于心脏功能衰竭,或循环合并呼吸衰竭。晚发型在幼年期或成人期起病,以骨骼肌受累为主,表现四肢无力,进展较慢,但呼吸肌受累可合并呼吸功能不全。已证实Pompe病为其基因GAA的突变引起,该基因已被克隆,定位于17q25,全长约20kb,包含20个外显子(其中第一号外显子不翻译),目前国外已发现突变类型100多种,在台湾,婴儿型Pompe病是最为常见的糖原累积病。Pompe病在中国大陆地区从围生期到成人期都有临床病例报道,但对此致病基因及其突变的研究尚少,本研究主要对中国大陆地区幼年起病的晚发型糖原累积症Ⅱ型(Pompe病)患者进行临床和基因突变分析。方法对2例经酶学确诊的幼年型Pompe病人(男,2岁9月;女,9岁)进行临床分析,包括实验室生化检测、肌电图、肌活检病理等,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个编码外显子,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2例病人临床资料:1例表现进行性近端肌肉无力,肌张力低下,1例表现肌容积减少,血清肌酶升高,合并呼吸肌受累,肺功能检查显示限制性通气障碍,随访15月时因肺部感染诱发死亡。2例均有肌酸激酶升高;肌电图提示肌源性损害;肌活检呈空泡性改变、糖原沉积;皮肤成纤维细胞α-1,4-葡萄糖苷酶活性测定明显低下(0.5-2.7nmol/小时/mg蛋白)。突变检测:在编码区未发现缺失,插入,无义突变等突变类型,测序发现4个杂合错义突变,例1患儿为c796C＞T,p.Pro266Ser;c2105 G＞A,p.Arg702His;例2患儿为c2132 C＞G,p.Thr711Arg;c2167 G＞A,p.Va1723Met。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1) 2例幼年起病的晚发型Pompe病患儿在临床表现上存在差异性:可以肌无力为突出表现,或以肌容积减少,肌肉萎缩为主诉,而无力症状轻微。可合并呼吸肌受累。心脏改变不明显。2)实验室检查:血清CK轻度升高,肌活检病理呈空泡样糖原沉积,皮肤成纤维细胞中α-1,4-葡萄糖苷酶活性明显低下。3)幼年起病的晚发型Pompe病预后与病程长短,有无呼吸肌受累相关。4) 2例幼年起病的晚发型Pompe病患儿均在GAA基因发现有突变存在,突变类型为杂合错义突变,未发现插入、缺失、剪切和无义突变类型。5) 4个复合杂合错义突变中:c796 C＞T,p Pro266Ser突变,仅见丁韩国的晚发型病人,在其他人种中未见报道;3个新发现突变,c2105 G＞A,p.Arg702His;c2132 C＞G,p.Thr711Arg;c2167G＞A,p.Val723Met,位于15号外显子上,为致病突变可能性大。更多还原

【Abstract】 objective Elevation of serum creatine kinase(CK)levels are related to a variety of diseases including muscle injury,infections,toxins,endocrine diseases,etal.,but persistently increased serum CK levels were considered a hallmark of neuromuscular disease.Different causes have different treatment and prognosis.The aim of our study is to establish the diagnosis of juvenile patients with chronic elevation of serum creatine kinase(CK) levels.Methods We made a retrospective evaluation of 25 juvenile patients with hyperCKemia over three months in the genetic clinic of our department between January 2005 and March 2007.Clinical data,neurological examination and laboratory examinations were analysized.All the patients underwent open muscle biopsy.Biochemical and genetic investigation were added in selected cases.Results There were 7 females and 18 males aged between 2 year 9 month and 15 years [mean 7.8y].The disease duration were beween 3 months and 7 years. Serum CK leverls were between 285 and 15720 U/L(18-198).We made a diagnosis in 16 patients.The diagnosis included dystrophinopathies in 8 patients;Pompe disease in 2 patients;glycogenosis typeⅢin 1 patient;mitochondrial myopathies in 1 patient;glutaric aciduria typeⅡin 1 patient;spinal muscular atrophy in 1 patients;inflammatory myopathies in 2 patients.Diagnosis were not able to confirm in 9 patients, pathological but not conclusive muscle changes were found in 5 patients. Conclusion Neurological and metabolic myopathies disease were the main causes of pesistent hyperckemia in children,muscle biopsy as well as biochemical and genetic investigations were essential to the diagnosis. Backgrounds and ObjectiveGlycogen-storage disease typeⅡ(GSDⅡ,Pompe’s disease) is an autosomal recessive disorder caused by a functional deficiency of acid alpha-glucosidase(GAA) that leads to glycogen accumulation within lysosomes in most tissues.The GAA gene is located to human chromosome 17q25 and contains 20 exons,19 of which are coding, Clinically,patients with the severe infantile form of GSDⅡhave muscle weakness and cardiomyopathy eventually leading to death before the age of two years.Patients with the juvenile or the adult form of GSDⅡpresent with myopathy with a slow progression over several years or decades,symptoms may start at any age and are related to progressive dysfunction of skeletal muscles.With disease progression,patients become confined to wheelchairs and require artificial ventilation. Respiratory failure is the cause of significant morbidity and mortality in this form of disease.The age of death varies from early childhood to late adulthood,depending on the rate of disease progression and the extent of respiratory muscle involvement.A broad genetic heterogeneity has been described in GSDⅡin Europe,South Africa,USA,Japan and Korea, hower,the investigation has not been performed in the patients from the mainland of China.In this study,clinical analysis and mutations detection were done on Chinese patients.MethodsTwo unrelated juvenile patients with late onset GSDⅡ(one boy,3 year old and one girl,9 year old)were included in the study with the informed consents.The diagnosis was confirmed byα-glucosidase determination in cultured fibroblasts.In addition,their clinical presentation,laboratory findings,electrophysiologic studies and muscle biopsy findings were analyzed in detail.Genomic DNA samples were extracted from fibroblasts of the probands,Genomic DNA samples were extracted from peripheral bloods of their parents and 50 unrelated,normal individuals.All the coding 19 exons and exon-intron boundaries of GAA were detected in the proband by polymerase chain reaction(PCR),direct sequencing.Resultsone patient presented decrease of muscle strength,limb-girdle hypotonia,the other patient presented reduced size of muscle volumes and respiratory problems.Both had increased serum creatinine phosphokinase(CPK) value,myopathic pattern on EMG;vacuoles on muscle biopsy,and deficiency of 1,4-alpha-glucosidase activity.After 1 year follow up,the girl died after pneumonia at 10 year old.One patient was found to be compound heretozygote for the novel mutation Arg702His,and Pro266Ser,previously reported in korean population, with the late-onset phenotype.Two novel missense mutations Thr711Arg, Va1723Met were found on the other patients.As for three new mutations, we were not able to observe the same changes among 100 control chromosomes.ConclusionsThe two cases of juvenile Pompe patients in our study had different characters in the clinical,the involvement of respiratory muscle seems a pronostic factor.We did not find the most commont mutation in the late-onset patients in the white people,and the most prevalent mutation Asp654Glu in the infantile form found in Taiwan district of China were not detected in our patients.The three mutations identified in the patients were new missense mutations causing late onset GSDⅡ,which had not been reported elsewhere before.There is currently no treatment other than supportive care for Pompe disease.With enzyme replacement therapy likely to become available in the near future and other therapies on the horizon,early disease recognition is increasingly important so the patients can receive prompt and appropriate therapy.更多还原