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甲基丙二酸血症患儿MUT基因突变分析

发表者:韩连书 人已读

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21例单纯MMA患儿进行MUT基因突变检测。

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。

 

2.1  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<5.0mmol/L);C3/C0:0.89±0.30(参考值<0.2);C3/C2:1.47±1.24(参考值<0.3)。尿气相色谱-质谱结果示甲基丙二酸显著增高,3-羟基丙酸及甲基枸橼酸轻度增高,其检测结果的中位数分别为685.57(119.45~2976.25)、22.89(0.47~570.73)和22.87(1.6~128.21)。血清同型半胱氨酸检测结果均正常。vitb12负荷试验11例患儿无效,3例有效。

2.2        MUT基因检测结果及突变频率分析

21例单纯MMA患儿中14例检测到MUT基因突变(表1),其中13例检测到2个突变,1例检测到1个突变,等位基因突变检出率为96.4%(27/28)。突变类型为错义突变、无义突变、剪切突变及插入突变,其中以错义突变为主,检出率为64.7%(11/17)。9种突变已有报道,其中c.323G>A(R108H)、c.729_730ins TT(D244LfsX39)、c.1630_1631GG>TA (G544X) 较为常见,突变频率分别为14.3%(4/28)、10.7%(3/28)及14.3%(4/28)。此外,8种突变尚未见报道(图1),c.68C>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。

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)

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。

2  14MUT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

C3

参考值<5.0

mmmol/L

 

尿甲基丙二酸

参考值<1.1

基因型

1

未发病

出生筛查

无效

15.18

263.47

1943G>A/1630_1631GG>TA   

2

未发病

出生筛查

无效

16.13

119.45

1943G>A/1630_1631GG>TA

3

7天

嗜睡,纳差,呼吸困难

无效

31.94

2063.87

1061C>T/754-1G>A

4

未发病

出生筛查

有效

7.92

566.23

729_730ins TT/ ?

5

1天

纳差,嗜睡

无效

9.02

593.58

925T>G/1630_1631GG>TA   

6

8月

呕吐,嗜睡,纳差,发育迟缓

有效

13.17

1863.35

278G>A/567T>G

7

9月

呕吐,嗜睡,腹泻

有效

11.21

730.18

323G>A/1514T>C

8

3天

呼吸困难,嗜睡,纳差

无效

19.30

2047.93

554C>T/2062G>T

9

1月

呕吐,抽搐,纳差,嗜睡

无效

26.37

2277.22

68C>G/323G>A

10

3月

呕吐,嗜睡,抽搐

无效

28.97

2976.25

1880A>G/1630_1631GG>TA

11

6岁

呼吸困难,呕吐,发育迟缓

无效

8.57

640.96

323G>A/729_730ins TT

12

1月

呕吐,抽搐

无效

8.47

486.86

433G>A/1677-1G>A

13

2月

抽搐,纳差,呼吸困难,呕吐

无效

58.77

2902.82

323G>A/424A>G

14

3月

发热,呕吐,嗜睡,抽搐

无效

7.48

1850

1880A>G/729_730ins TT

甲基丙二酰辅酶A变位酶有两个主要结构域,即C-端腺苷钴胺素结合区和N-端底物甲基丙二酰辅酶A结合区[7,8],大部分MUT突变发生于这两个结构域中。G717V在美洲黑人和加纳人中常见[7];E117X主要见于日本人群[9,10];R108C见于60%的西班牙患者[8];R369H则在美国、日本、韩国、土耳其和希腊多人群中见到[8,11];N219Y见于法国和土耳其后裔中[12];G427D和G544X在亚洲人群中多见[13]。本研究中c.323G>A(R108H)、c.1630_1631GG>TA(G544X) 及c.729_730insTT (D244LfsX39)较为常见。R108H突变在4例患儿中检测出,其突变位点108位为CpG二核苷酸突变热点,除R108H突变外,也可发生c.322C>G(R108G)和c.322C>T(R108C)突变。G544X突变在4例患儿中发现,突变发生在两个结构域之间的链接区中,导致第544位甘氨酸翻译提前终止,可能影响钴胺素结合区的功能。c.729_730insTT(D244LfsX39)在3例患儿中检出,突变导致从第244位天门冬氨酸发生移码,在第283位密码子处提前终止,可能影响底物结合区和钴胺素结合区功能。

甲基丙二酰辅酶A变位酶蛋白氨基酸序列中氨基酸88-422是N-末端(βα)8TIM barrel结构域,属于底物结合区,578-750是C-末端(βα)5barrel结构域,属于钴胺素结合区,424-577为两个结构域之间链接区,可关闭(βα)8TIM barrel 结构域[7,14]。本研究检测到8种国内外尚未报道的突变,c.68C>G(S23X)突变位于前导序列中,突变造成蛋白在翻译起始AUG密码子处截短,从而使MCM酶活性丧失;c.424A>G(T142A)、c.554C>T(S185P)、c.925T>G(W309G) 及c.1061C>T (S354F)突变位于(βα)8TIM barrel 结构域中,可能影响底物的结合;c.2062G>T(E688X)突变位于(βα)5 钴胺素结合区,可能影响底物、酶与钴胺素结合;c.1514T>C(I505T)突变则位于两个结构域之间的链接区中,从而造成酶蛋白构象改变,可能影响底物与钴胺素之间的结合;c.754-1G>A为剪切突变,可能影响mRNA的稳定性。

甲基丙二酰辅酶A变位酶缺陷型是MMA婴幼儿期发病最常见的类型。本研究的14例患儿中10例1岁内发病。其中 病例1和2为双胞胎患儿,基因型为1943G>A(G648D)/1630_1631GG>TA(G544X),VitB12负荷试验示无效型,血尿代谢指标表现异常,但并无临床症状,至今未经治疗状况良好,原因尚不明。病例12基因型为433G>A/1677-1G>A,VitB12治疗无效,生后不久死亡。14例中仅1例发病较迟,基因型为c.323G>A/c.729_730insTT,VitB12治疗无效。3例VitB12治疗有效的患者基因型分别为c.278G>A/c.567T>G、c.1514T>C/c.323G>A和c.729_730insTT。由此可见MUT基因突变患儿发病早,多为VitB12无效型。

随着串联质谱联合气相色谱-质谱技术的推广应用,甲基丙二酸血症诊断率较前显著提高[15],MUT基因突变检测有助于甲基丙二酸血症的类型鉴别,并可为先证者家系提供产前诊断及高危携带者基因检测。

参  考  文  献

1         Deodato F, Boenzi S, Santorelli FM, et al. Methylmalonic and propionic aciduria. Am J Med Genet C Semin Med Genet, 2006, 142:104-112.

2         Chang HB, Wu JX, Liu ZW, et al. Molecular analysis of two novel mut gene mutations in Chinese patient with methylmalonic acidemia. Chin J Neuro, 2006, 39:742-744. [常会波, 吴建新, 刘哲伟, 等. 甲基丙二酸血症患儿mut基因两个新遗传突变的发现和鉴证. 中华神经科杂志, 2006, 39:742-744.]

3         Wang F, Han LS, Hu YH, et al. Analysis of gene mutations in Chinese patients with Methylmalonic acidemia and homocystinemia. Chin J Pediat, 2009, 47(3):189-193.[王斐, 韩连书, 胡宇慧, 等. 甲基丙二酸血症伴同型半胱氨酸血症患儿基因突变分析. 中华儿科杂志, 2009,47(3):189-193.]

4         Han LS, Ye J, Qiu WJ, et al. Selective screening for Inborn Errors of Metabolism on Clinical Patients Using Tandem Mass Spectrometry in China: a four-year report. J Inherit Metab Dis, 2007, 30:507-514.

5         Schmedes A, Brandslund I. Analysis of methylmalonic acid in plasma by liquid chromatography-tandem mass spectrometry. Clin Chem, 2006, 52:754-757.

6         Wang F, Han LS. Progresses of diagnosis and therapy in methylmalonic acidemia. J Clin Pediatr, 2008, 26:724-727. [王斐, 韩连书. 甲基丙二酸血症诊治研究进展. 临床儿科杂志, 2008, 26:724-727.]

7         Acquaviva C, Benoist JF, Pereira S, et al. Molecular basis of methylmalonyl-CoA mutase apoenzyme defect in 40 European patients affected by mut0 and mut- forms of methylmalonic acidemia: identification of 29 novel mutation in the MUT gene. Hum Mutat, 2005, 25:167-176.

8         Worgan LC, Niles K, Tirone JC, et al. Spectrum of Mutations in mut Methylmalonic Acidemia and Identification of a Common Hispanic Mutation and Haplotype. Hum Mutat, 2006, 27: 31-43.

9         Sakamoto O, Ohura T, Matsubara YM, et al. Mutation and haplotype analyses of the MUT gene in Japanese patients with methylmalonic acidemia.J Hum Genet, 2007, 52:48-55.

10     Kobayashi A, Kakinuma H, Takahashi H. Three novel and six common mutations in 11 patients with methylmalonic acidemia. Pediatrics International, 2006, 48:1-4.

11     Junga JW, Hwangb IT, Parka JE, et al. Mutation analysis of the MCM gene in Korean patients with MMA . Mol Genet Metab, 2005, 84: 367-370.

12     Acquaviva C, Benoist JF. N219Y, a new frequent mutation among mut0 forms of methylmalonic acidemia in Caucasian patients. Eur J Hum Genet, 2001, 9:577-582.

13     Keeratichamroen S, Cairns JR, Sawangareetrakul P, et al. Novel mutations found in two genes of thai patients with isolated methylmalonic acidemia. Biochem Genet, 2007, 45:421-430.

14     Martinez MA, Rincon A, Desviat LR, et al. Genetic analysis of three genes causing isolated methylmalonic acidemia: identification of 21 novel allelic variants. Mol Genet Metab, 2005, 84:317-325.

15     Han LS, Ye J, Qiu WJ, et al. Diagnosis of inborn errors of metabolism using tandem mass spectrometry and gas chromatography mass spectrometry. Nat Med J Chin, 2008, 88:2122-2126. [韩连书, 叶军, 邱文娟, 等. 串联质谱联合气相质谱检测遗传性代谢病. 中华医学杂志, 2008, 88:2122-2126.]

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