王斐  韩连书  叶军  邱文娟  张雅芬  高晓岚  王瑜  杨艳玲  顾学范

　　【摘要】 目的  检测甲基丙二酸血症（methylmalonic acidemia, MMA）患儿MUT基因突变类型及突变频率，探讨基因型与临床表型之间的关系。方法  依据串联质谱检测血酰基肉碱、气相色谱-质谱检测尿甲基丙二酸以及维生素B12负荷试验等，诊断21例单纯MMA患儿；采用聚合酶链反应和直接测序法对这些患儿进行MUT基因突变检测分析。结果  在21例单纯MMA患儿中14例检测到17种MUT基因突变，其中8种为未报道突变。以c.323G>A (R108H)、c.729_730insTT (D244LfsX39)与c.1630_1631GG>TA (G544X) 较为常见，突变频率分别为14.3%、10.7%及14.3%，以错义突变多见(64.7%)。14例MUT突变患儿中10例为早发型，1例为迟发型，3例由新生儿出生筛查检出；11例为维生素B12无效型，3例为有效型。结论  揭示了中国MMA患儿中MUT基因的部分突变谱，MUT基因突变患儿发病较早，多为维生素B12无效型。上海新华医院儿童内分泌科韩连书

　　【关键词】 甲基丙二酸；甲基丙二酰辅酶A变位酶；基因；突变

　　Analysis of MUT gene mutations in patients with Methylmalonic acidemia*  WANG Fei1, HAN Lian-shu1, YE Jun1, QIU Wen-juan1, ZHANG Ya-fen1, GAO Xiao-lan1, WANG Yu1, ZHANG Yongjun, GU Xue-fan1, YANG Yan-ling2.  1 (Department of Pediatric Endocrinology and Genetic Metabolism, Shanghai Institute for Pediatric Research, Xinhua Hospital, Shanghai Jiaotong University School of Medical, Shanghai, 200092. P. R. China); 2(Department of Pediatric, First Hospital, Beijing Universty, Beijing, 100034. P. R. China)

　　Corresponding author: HAN Lian-shu, Email: xhhanlianshu@Yahoo.com.cn

　　【Abstract 】 Objective  To investigate the types and allele frequencies of MUT gene mutations in methylmalonic acidemia (MMA), and analyze the genotype-phenotype correlations in patients with methylmalonyl-CoA mutase deficiency. Methods  The diagnosis of this disease mainly depends on the measurement of C3 (acylcarnitine),C3/C0 (free carnitine) and C3/C2 (acetylcarnitine) in the blood by tandem mass spectrometry, the detection of methylmalonic acid in the urine by gas-chromatography mass spectrometry, the determination of total homocysteine in the serum, and the loading test of vitamin B12. The entire coding region of MUT gene was screened by polymerase chain reaction (PCR) combined with DNA direct sequencing in 21 isolated MMA patients. Novel mutations were identified by restriction fragment length polymorphism (RFLP) and sequence analysis in 100 controls. Results 17 mutations of MUT gene were detected in 14 of 21 patients, in which 8 mutations were novel. R108H, D244LfsX39 and G544X were more frequent, the frequency was 9.5%, 7.1% and 9.5%, respectively. Most mutations were missence mutations (64.7%), and most of them were in exons 2 and 3 (55.6%). 10 of 14 patients with MUT gene mutations had early-onset disease, while one case had late-onset disease, and 3 cases had been found by newborn screening. 11 of these 14 patients were not responsive to vitamin B12, while 3 cases were responsive to vitamin B12. Conclusion This study revealed a part of MUT gene mutations spectrum in Chinese patients with isolated MMA. The patients carried MUT mutations often had early-onset disease, and most of them were VitB12-non-responsive.

　　【Key words】 methylmalonic acid; methylmalonyl-CoA mutase; gene; mutation

　　Supported by Technology Pillar Program in the Eleventh Five-year Plan Period(2006AB105A05, 2006BA105A07); National High Technology Research and Development Program(2007AA02Z447); Key Subject of Shanghai Municipality(2008ZD001); Scientific Research Foundation Project of Shanghai Municipality(2006043).

　　甲基丙二酸血症(methylmalonic acidemia, MMA)是一种常染色体隐性遗传病，主要是由于甲基丙二酰辅酶A变位酶(methylmalonyl-CoA mutase, MCM)自身缺陷或其辅酶腺苷钴胺素代谢缺陷所致。正常情况下体内异亮氨酸、缬氨酸、苏氨酸、蛋氨酸、胆固醇和奇数链脂肪酸分解代谢生成甲基丙二酰CoA，在MCM催化下生成琥珀酰CoA，进入三羧酸循环。MCM缺陷时导致大量的甲基丙二酰CoA堆积，引起一系列病理生理变化。根据MCM的残余活性可分为mut0型（完全无活性）及mut-型（有残余活性者）[1]。临床可表现为嗜睡、呕吐、抽搐、肌张力低下、运动及智力障碍等。甲基丙二酰辅酶A变位酶编码基因命名为MUT (MIM 251000)，定位于染色体6p21.2-p12，由13个外显子组成，编码750个氨基酸。至今已发现MUT突变170余种，国内仅报道两种突变[2]。MMA包括mut0、mut-、cblA、cblB、cblC、cblD、cblF及cblH八种亚型。其中mut0、mut-、cblA、cblB、cblH缺陷型患者，仅表现甲基丙二酸血症，故称为单纯MMA，而cblC、cblD和cblF缺陷型则合并同型半胱氨酸血症[3]。故对2004~2007年诊断的21例单纯MMA患儿进行MUT基因突变检测。

　　1  对象和方法

　　1.1   对象   单纯MMA患儿21例，19例来自本院，2例来自北京大学第一医院。男12例，女9例。除一对同卵姐妹外，其余患儿相互间无血缘关系。正常对照组为100名无血缘关系的非MMA儿童。

　　1.2  方法

　　1.2.1  诊断：(1) 依据串联质谱检测血中丙酰肉碱(propionylcarnitine, C3)、C3与游离肉碱(free carnitine,C0)比值和C3与乙酰肉碱(acetylcarnitine,C2)比值增高[4]；(2) 气相色谱-质谱检测尿甲基丙二酸水平(与17烷酸比值)升高[5]；(3)血清同型半胱氨酸正常[6]。(4) 维生素B12负荷试验：每次1mg，im，每日1次，连续3天，若血丙酰肉碱或尿甲基丙二酸下降50%，提示为维生素B12有效型。

　　1.2.2  PCR扩增与DNA测序分析：取患儿及部分患儿父母外周静脉血，分离白细胞，提取DNA。根据参考文献[7]设计MUT基因13个外显子PCR引物序列。PCR反应体系为50mL，反应条件为94°C预变性3min，94°C变性30s，48°C~62°C退火30s至1min，72°C延伸30s至1min，35个循环后72°C继续延伸5min。PCR产物纯化后在ABI 3700测序仪上正反双向测序。结合GenBank（序列号:4594）人类MUT基因序列来识别患儿中的突变。

　　1.2.3  突变鉴定：8种未报道的突变中c.68C>G、c.424A>G、c.554C>T和c.1514T>C分别通过限制性片段长度多态性（Restriction Fragment Length Polymorphism, RFLP）分析法鉴定。酶切反应体系为20mL，PCR产物6mL(DNA 1mg)，10×NEB缓冲液2mL，限制性内切酶0.5U，加水至20mL。其他4种未报道突变由于未找到相应位点限制性内切酶，故通过直接测序法鉴定。以上8种未报道突变鉴定采用100名非MMA儿童作为对照，以排除基因多态性。

　　2  结  果

　　2.1  MUT突变患儿临床资料及辅助检查

　　14例患儿检测到MUT基因突变，即为甲基丙二酰辅酶A变位酶缺陷患儿。其中3例为串联质谱新生儿出生筛查确诊患儿，10例于1岁内起病，主要表现为反复呕吐、嗜睡、抽搐、纳差、智力落后、肌张力低下及发育迟缓。1例患儿于6岁发病，表现为呼吸困难伴有呕吐。14例患儿血串联质谱检测结果为C3:15.55±7.91mmol/L（参考值G经BspHI酶切后患儿和母亲样品为527/403/124bp带，突变来源于母亲；c.424A>G经AciI酶切后患儿为568/502/66bp带；c.554C>T经BspHI酶切后患儿和父亲为582/381/201bp带，突变来源于父亲；c.1514T>C经MfeI酶切后患儿和母亲为310/201/109bp带，正常对照及父亲为201/109bp带，突变来源于母亲。以上突变在100名正常对照组中未发现。其他4种突变经直接测序证实c.754-1G>A来源于母亲，c.925T>G来源于父亲，c.1061C>T来源于父亲，c.2062G>T来源于母亲，这4种突变在100名正常对照组中未检测到。

　　表1  14例患儿MUT基因突变检测结果分析

　　突变位点   核苷酸改变        氨基酸改变   等位基因数                 突变频率                       突变类型      结构域定位

　　 (外显子)                                                                           (%) 

　　2              c.68C>Ga                   S23X               1             3.6%          无义突变          前导肽区

　　2              c.278G>A                  R93H               1             3.6%          错义突变         (βα)8 barrel

　　2              c.323G>A                  R108H              4             14.3%         错义突变         (βα)8 barrel

　　3              c.424A>Ga                 T142A              1             3.6%          错义突变         (βα)8 barre

　　3              c.433G>A                  G145S              1             3.6%          错义突变         (βα)8 barrel

　　3              c.554C>Ta                  S185P              1             3.6%          错义突变          (βα)8barrel

　　3              c.567T>G                  N189K             1             3.6%          错义突变         (βα)8 barrel

　　3              c.729_730ins TT         D244LfsX39     3             10.7%         插入突变         (βα)8 barrel

　　               c.754-1G>Aa                                      1             3.6%          剪接突变          剪切位点

　　5              c.925T>Ga                 W309G            1             3.6%          错义突变         (βα)8 barrel

　　5              c.1061C>Ta                S354F              1             3.6%          错义突变         (βα)8 barrel

　　8              c.1514T>Ca                I505T               1             3.6%          错义突变            链接区

　　9              c.1630_1631GG>TA  G544X             4             14.3%         无义突变            链接区

　　               c.1677 -1G>A                                    1             3.6%          剪接突变          剪切位点

　　11             c.1880A>G                 H627N             2             7.1%          错义突变     (βα)5Cbl-结合区

　　11             c.1943G>A                 G648D             2             7.1%          错义突变     (βα)5Cbl-结合区

　　12             c.2062G>Ta                E688X              1             3.6%          无义突变     (βα)5Cbl-结合区

　　合计           17                                                    27            96.4%

　　a为国内外尚未报道的突变

　　     c.68C>G(S23X)              c.424A>G(T142A)                   c.554C>T(S185P)              c.925T>G(W309G)

　　c.754-1G>A                     c.1061C>T(S354F)                 c.1514T>C(I505T)           c.2062G>T(E688X)

　　图1  MUT基因8种新突变测序图谱

　　2.3     MUT基因突变序列结构分析

　　分析本研究检测到的17种突变在MCM结构中的定位[7]，可知 c.68C>G(S23X)突变位于前导序列中；9种突变即c.278G>A(R93H)、c.323G>A(R108H)、c.424A>G(T142A)、c.433G>A(G145S)、c.554C>T(S185P)、c.567T>G(N189K)、c.729_730insTT(D244LfsX39)、c.925T>G(W309G) 及c.1061C>T(S354F)位于(βα)8TIMbarrel结构域中；3种突变即c.1880A>G(H627N)、c.1943G>A (G648D)和c.2062G>T(E688X) 位于(βα)5钴胺素结合区中；c.1514T>C(I505T)及c.1630_1631GG>TA(G544X)突变则位于两结构域之间的链接区中；c.754-1G>A和 c.1677-1G>A则为剪切位点突变。

　　2.4  基因型与临床表型结果

　　14例患儿基因型与临床表型结果见表2。

　　表2  14例MUT基因突变患儿基因型与临床表型结果

　　编号

　　发病年龄

　　临床症状

　　VitB12

　　治疗

　　效果

　　血C3

　　参考值TA