維生素B12依賴型甲基丙二酸血癥一家系臨床表型和基因突變分析及療效評(píng)價(jià)

利婧 孫毅明 歐俐羽 朱瑜齡 王倞 李歡 張成

維生素B12依賴型甲基丙二酸血癥一家系臨床表型和基因突變分析及療效評(píng)價(jià)

利婧 孫毅明 歐俐羽 朱瑜齡 王倞 李歡 張成

目的探討維生素B12依賴型甲基丙二酸血癥家系臨床特點(diǎn)、基因突變和維生素B12治療效果.方法采集一維生素B12依賴型甲基丙二酸血癥家系共4名成員臨床資料,抽取外周靜脈血行血漿氨基酸和?；鈮A譜分析及基因檢測(cè),評(píng)價(jià)維生素B12治療效果.結(jié)果家系中先證者12歲發(fā)病,以學(xué)習(xí)成績(jī)下降、性格改變?yōu)槭装l(fā)癥狀,病程中出現(xiàn)幻覺(jué)、雙下肢無(wú)力;先證者之弟主要表現(xiàn)為易怒、學(xué)習(xí)成績(jī)較差.經(jīng)血漿氨基酸和酰基肉堿譜分析,先證者及其弟血漿丙酰肉堿、丙酰肉堿/乙酰肉堿比值升高,尿液甲基丙二酸水平升高.基因檢測(cè)顯示,先證者及其弟均存在MMACHC基因復(fù)合雜合突變c.482G>A(p.Arg161Gln)和c.609G>A(p.Trp203X),其父攜帶MMACHC基因錯(cuò)義突變c.482G>A(p.Arg161Gln),其母攜帶MMACHC基因無(wú)義突變c.609G>A(p.Trp203X).經(jīng)維生素B12治療后均癥狀好轉(zhuǎn).結(jié)論晚發(fā)型維生素B12依賴型甲基丙二酸血癥系MMACHC基因復(fù)合雜合突變所致,維生素B12治療反應(yīng)良好,早期診斷、及時(shí)治療對(duì)患者預(yù)后意義重大.

甲基丙二酸; 代謝缺陷,先天性; 維生素B12; 表型; 基因; 突變; 系譜

甲基丙二酸血癥(MMA)cblC型系MMACHC基因突變導(dǎo)致其編碼的cblC蛋白缺乏,使L?甲基丙二酸無(wú)法轉(zhuǎn)變?yōu)殓晁岫谘褐行罘e,是臨床最常見(jiàn)的維生素B12代謝障礙性疾病,呈常染色體隱性遺傳,發(fā)病率為1/10萬(wàn)～1/6萬(wàn)[1].根據(jù)發(fā)病年齡,可以分為早發(fā)型(1歲內(nèi)發(fā)病)和晚發(fā)型甲基丙二酸血癥.迄今文獻(xiàn)報(bào)道僅約10%患者為晚發(fā)型甲基丙二酸血癥[2?3].維生素B12依賴型甲基丙二酸血癥臨床罕見(jiàn),通常于兒童期、青少年期或成年期發(fā)病,主要表現(xiàn)為精神癥狀、意識(shí)障礙和神經(jīng)系統(tǒng)癥狀等,經(jīng)及時(shí)治療預(yù)后較好[4?5],屬可治性神經(jīng)系統(tǒng)遺傳性疾病.本研究報(bào)道一晚發(fā)型甲基丙二酸血癥家系,總結(jié)其臨床表現(xiàn)、血液化學(xué)、基因檢測(cè)和維生素B12治療后轉(zhuǎn)歸,探討維生素B12治療MMAcblC型的作用機(jī)制,以期提高兒科和神經(jīng)科醫(yī)師對(duì)甲基丙二酸血癥的認(rèn)識(shí).

對(duì)象與方法

一、研究對(duì)象

本研究MMAcblC型患者于中山大學(xué)附屬第一醫(yī)院神經(jīng)科就診,其家系2代共4名成員(圖1),漢族,其中2例符合常染色體隱性遺傳特點(diǎn).

先證者 女性,13歲,主因性格改變1年、雙下肢無(wú)力2個(gè)月,于2016年11月24日至我院神經(jīng)科門(mén)診就診.患者1年前無(wú)明顯誘因出現(xiàn)易激惹、易哭鬧,學(xué)習(xí)成績(jī)下降,未予重視;2個(gè)月前出現(xiàn)雙下肢無(wú)力,勉強(qiáng)能夠獨(dú)立行走,上樓梯費(fèi)力,無(wú)肢體麻木,雙上肢未見(jiàn)異常;1月余前出現(xiàn)幻覺(jué),以幻視為主,逐漸進(jìn)展至行走不能.1個(gè)月前曾至外院就診,診斷為"可疑線粒體腦肌病或腦炎",予靜脈注射免疫球蛋白(IVIg)和甲潑尼龍等治療(具體劑量不詳),癥狀無(wú)明顯好轉(zhuǎn),為求進(jìn)一步診斷與治療,遂至我院就診.患兒足月順產(chǎn),單胎,生長(zhǎng)發(fā)育和智力發(fā)育正常;父母身體健康,無(wú)相似臨床癥狀,否認(rèn)近親婚配.門(mén)診體格檢查:身高和體重于同齡兒童正常范圍.神經(jīng)系統(tǒng)檢查:高級(jí)智能和腦神經(jīng)檢查未見(jiàn)異常,雙側(cè)小腿肌萎縮,雙下肢近端肌力3級(jí)、遠(yuǎn)端4級(jí),肌張力降低,雙上肢肌力5級(jí),肌張力正常;雙下肢痛觸覺(jué)和關(guān)節(jié)位置覺(jué)減退,四肢腱反射未引出,雙下肢Babinski征陽(yáng)性,腦膜刺激征陰性.實(shí)驗(yàn)室檢查:血尿便常規(guī)、肝腎功能試驗(yàn)、血液化學(xué)、免疫學(xué)指標(biāo)、血氨、血清維生素B12均于正常值范圍,血清同型半胱氨酸(Hcy)165.30 μmol/L(5～20 μmol/L).影像學(xué)檢查:胸部X線未見(jiàn)明顯異常.頭部MRI顯示輕度腦萎縮,尤以白質(zhì)顯著,雙側(cè)側(cè)腦室旁長(zhǎng)T1、長(zhǎng)T2信號(hào),擴(kuò)散加權(quán)成像(DWI)呈顳頂葉放射冠低信號(hào),表觀擴(kuò)散系數(shù)(ADC)升高(圖2).磁共振波譜(MRS)顯示,N?乙酰天冬氨酸(NAA)峰、肌酸(Cr)峰、膽堿(Cho)峰峰值降低(圖3).電生理學(xué)檢查:心電圖未見(jiàn)明顯異常.腦電圖顯示中度異常,以θ波為主要背景波.

患兒Ⅱ2 男性,10歲,先證者之弟,于2016年12月6日至我院神經(jīng)科門(mén)診就診.患兒足月順產(chǎn),單胎,18個(gè)月方可獨(dú)立行走和會(huì)說(shuō)話,智力發(fā)育較同齡正常兒童差,目前讀小學(xué),學(xué)習(xí)成績(jī)較差,易怒,未予重視.門(mén)診體格檢查:身高和體重于同齡兒童正常范圍.神經(jīng)系統(tǒng)檢查:注意力和計(jì)算力較差,語(yǔ)言復(fù)述能力較差,腦神經(jīng)檢查未見(jiàn)明顯異常;四肢肌力5級(jí)、肌張力正常,感覺(jué)系統(tǒng)未見(jiàn)異常,腱反射降低,病理反射未引出,腦膜刺激征陰性.實(shí)驗(yàn)室檢查:血尿便常規(guī)、肝腎功能試驗(yàn)均于正常值范圍,血氨 51 μmol/L(10～47 μmol/L),血清維生素B12于正常值范圍,血清同型半胱氨酸84.20 μmol/L.

圖1 MMAcblC型家系圖Figure 1 The pedigree of MMAcblC.

二、研究方法

1.血漿氨基酸和?；鈮A譜分析 采集先證者及其弟外周靜脈血各2 ml制備干血濾紙,API3200液質(zhì)聯(lián)用儀(美國(guó)SCIEX公司)采用串聯(lián)質(zhì)譜法行血漿氨基酸和?；鈮A譜分析.采集先證者及其弟尿液制備3張尿液濾紙片,7890/5975C氣相質(zhì)譜聯(lián)用儀(美國(guó)Agilent公司)行尿液氣相色譜質(zhì)譜分析,檢測(cè)尿液各種有機(jī)酸之間的比值.

2.目標(biāo)區(qū)域捕獲測(cè)序與數(shù)據(jù)分析 經(jīng)先證者父母同意,采集該家系4名成員外周靜脈血各2 ml,行甲基丙二酸血癥相關(guān)二代基因測(cè)序(NGS).(1)基因組DNA提取和基因組文庫(kù)制備:2 ml外周靜脈血置于乙二胺四乙酸(EDTA)抗凝管中,采用QIAmp DNA Mini Kit試劑盒(德國(guó)QIAGEN公司)提取基因組 DNA. 取 3 μ g DNA,以 Tris?乙 二 胺 四 乙 酸(EDTA)緩沖液稀釋至30 ng/μl,采用Covris?S220超聲儀(美國(guó)Covaris公司)將樣本DNA片段化為長(zhǎng)度150 bp,采用標(biāo)準(zhǔn)DNA文庫(kù)構(gòu)建試劑盒(北京邁基諾基因科技股份有限公司)進(jìn)行片段末端修復(fù)加"腺嘌呤"和產(chǎn)物純化,經(jīng)聚合酶鏈反應(yīng)(PCR)擴(kuò)增后,采用Nanodrop 2000紫外分光光度計(jì)(美國(guó)Therom公司)進(jìn)行DNA文庫(kù)定量.(2)目標(biāo)區(qū)域捕獲測(cè)序:采用GenCap液相捕獲目標(biāo)基因技術(shù)對(duì)已知的176種代謝性疾病相關(guān)基因進(jìn)行探針捕獲,覆蓋每種基因的外顯子及其上下游內(nèi)含子長(zhǎng)度50 bp區(qū)域,生物素標(biāo)記的探針與文庫(kù)DNA雜交,鏈霉素標(biāo)記的磁珠共價(jià)結(jié)合生物素標(biāo)記的探針,抓取目的基因,磁力架吸附攜帶目的基因的磁珠,洗脫純化,富集目的基因,再采用NextSeq 500高通量測(cè)序儀(美國(guó)Illumina公司)進(jìn)行雙端測(cè)序.(3)數(shù)據(jù)處理與分析:原始數(shù)據(jù)剔除接頭和低質(zhì)量短序列(<40 bp),BWA軟件(http://bio-bwa.sourceforge.net/)將預(yù)處理的數(shù)據(jù)與hg19人類基因組數(shù)據(jù)庫(kù)(http://hgdownload.cse.ucsc.edu/)進(jìn)行比對(duì),GATK3.3.0軟件包(https://www.broadinstitute.org/gatk)校準(zhǔn)并重新匹配至參考序列,單堿基突變采用GATK UnifiedGenotyper分析,單核苷酸多態(tài)性(SNP)和插入/缺失突變采用共識(shí)編碼序列數(shù)據(jù)庫(kù)(CCDS)、hg19人基因組數(shù)據(jù)庫(kù)、單核苷酸多態(tài)性數(shù)據(jù)庫(kù)信息進(jìn)行注釋,確定候選變異位點(diǎn),SIFT預(yù)測(cè)軟件(http://sift.jcvi.org)對(duì)變異位點(diǎn)進(jìn)行生物信息學(xué)分析.

3.Sanger測(cè)序驗(yàn)證 據(jù)基因變異位點(diǎn)設(shè)計(jì)上下游引物,由賽默飛世爾科技(中國(guó))有限公司合成,上游引物序列:5'?CCTCCATGACCTTGCTTTTC?3',下游引物序列:5'?GGGATGCAGGTGGTGAGAC?3'.PCR反應(yīng)體系共25 μl,反應(yīng)條件為95℃ 10 min;94℃ 30 s、64℃ 30 s、72℃ 45 s,共3個(gè)循環(huán);94℃ 30 s、62℃ 30 s、72℃ 45 s,共5個(gè)循環(huán);94 ℃ 30 s、60℃ 30s、72℃ 45s,共 10 個(gè)循環(huán);94℃30 s、58 ℃ 30 s、72 ℃ 45 s,共 17 個(gè)循環(huán);最終72℃ 5 min;4℃冷卻備用.然后采用ABI3130xl測(cè)序儀[賽默飛世爾科技(中國(guó))有限公司]將PCR擴(kuò)增產(chǎn)物進(jìn)行毛細(xì)管電泳,測(cè)序結(jié)果與標(biāo)準(zhǔn)參考序列(NM_001330540.1)進(jìn)行比對(duì).

結(jié) 果

一、臨床特征

該家系共2代4名成員,男性2名,女性2名;先證者12歲發(fā)病,主要表現(xiàn)為性格改變、學(xué)習(xí)成績(jī)下降,病程中出現(xiàn)幻覺(jué)、雙下肢無(wú)力癥狀,病程1年;先證者之弟自幼表現(xiàn)為易怒、智力低下,未明顯累及運(yùn)動(dòng)系統(tǒng).

二、血漿氨基酸和?；鈮A譜分析

先證者血漿甲硫氨酸為7.80 μmol/L(8.50～50.00μmol/L),丙二酸0.60μmol/L(0～0.30μmol/L),丙酰肉堿(C3)6.39 μmol/L(0.30～5.00 μmol/L),丙酰肉堿/乙酰肉堿(C2)比值為0.41(0.02～0.25);尿液甲基丙二酸水平為 63.70 μmol/L(0～4 μmol/L).先證者之弟血漿丙酰肉堿5.29 μmol/L,C3/C2比值為 0.59,天冬氨酸為 62.60 μmol/L(18～47 μmol/L),精 氨 酸 95.50 μ mol/L(11～69 μ mol/L),丙 氨 酸576.70 μmol/L(210～545 μmol/L),谷氨酰胺水平為702 μmol/L(249～579 μmol/L);尿液甲基丙二酸為75 μmol/L.

圖2 頭部MRI檢查所見(jiàn) 2a 橫斷面T2WI顯示,輕度腦萎縮,尤以白質(zhì)顯著(箭頭所示),雙側(cè)側(cè)腦室輕度擴(kuò)大 2b 冠狀位FLAIR成像顯示,雙側(cè)側(cè)腦室旁異常高信號(hào)(箭頭所示)2c 橫斷面DWI顯示,雙側(cè)顳枕葉深部側(cè)腦室旁異常低信號(hào)(箭頭所示)2d 橫斷面ADC圖顯示病變呈高信號(hào)(箭頭所示) 圖3 頭部MRS顯示,左側(cè)側(cè)腦室旁NAA峰、Cr峰、Cho峰和NAA/(Cho+Cr)比值降低,考慮白質(zhì)髓鞘化不良,提示代謝性疾病Figure2 Head MRI findings AxialT2WI showed mild encephalatrophy,espically in the white matter(arrows indicate),and slight enlargment of the bilateral ventricles (Panel 2a). Coronal FLAIR image showed abnormal hyperintense signals in bilateral periventricular area(arrows indicate,Panel 2b).Axial DWI showed abnormal hypointense signal in the deep part of bilateral temporo?occipital lobe beside lateral ventricle(arrows indicate,Panel 2c).Axial ADC showed hyperintensities in the lesion(arrow indicates,Panel 2d). Figure 3 MRSshoweddecreaseofNAA,Cr,ChoandNAA/(Cho+Cr)inleft periventricular regions,which reflected of neuronal damage and malformation in white matter myelination and suggested metabolic disease.

三、基因檢測(cè)

先證者存在MMACHC基因復(fù)合雜合突變,包括錯(cuò)義突變c.482G>A(p.Arg161Gln)和無(wú)義突變c.609G>A(p.Trp203X),符合MMAcblC型的診斷(圖4a).先證者之弟亦存在MMACHC基因復(fù)合雜合突變,包括錯(cuò)義突變c.482G>A(p.Arg161Gln)和無(wú)義突變c.609G>A(p.Trp203X,圖4b).進(jìn)一步對(duì)其父母行MMACHC基因檢測(cè),提示錯(cuò)義突變c.482G>A(p.Arg161Gln)源自其父(圖4c)、無(wú)義突變c.609G>A(p.Trp203X)源自其母(圖4d).2例患者最終明確診斷為MMAcblC型合并高同型半胱氨酸血癥.

四、治療

先證者明確診斷后即予甲鈷胺1 mg/d肌肉注射,左卡尼汀2 g/d靜脈滴注,以及利培酮0.50 g/次、2次/d口服控制幻視,共住院13 d,出院時(shí)幻視消失,雙下肢肌力4級(jí)、肌張力降低,攙扶可行走.出院后予以甲鈷胺1 mg/次、3次/d和左卡尼汀1 g/次、2次/d口服,并囑低蛋白飲食.出院后3個(gè)月門(mén)診隨訪,臨床癥狀好轉(zhuǎn),雙下肢肌力5-級(jí),可獨(dú)立行走,未再出現(xiàn)幻覺(jué),復(fù)查血漿氨基酸和酰基肉堿譜分析,丙酰肉堿降至正常值范圍,C3/C2比值0.27.先證者之弟明確診斷后予甲鈷胺1 mg/次、3次/d和左卡尼汀1 g/次、2次/d口服,囑低蛋白飲食.3個(gè)月后門(mén)診隨訪,語(yǔ)言復(fù)述能力較前好轉(zhuǎn),注意力和計(jì)算力欠佳.

討 論

甲基丙二酸血癥是維生素B12代謝障礙性疾病,可以根據(jù)維生素B12在細(xì)胞內(nèi)代謝障礙累及環(huán)節(jié)分型(圖5[4],表1).人體從食物中攝取的游離維生素B12在體內(nèi)與內(nèi)因子結(jié)合形成穩(wěn)定復(fù)合物,在回腸遠(yuǎn)端與腸黏膜細(xì)胞膜受體結(jié)合(維生素B12轉(zhuǎn)運(yùn)蛋白復(fù)合體)而被吸收.維生素B12進(jìn)入細(xì)胞后自其轉(zhuǎn)運(yùn)蛋白復(fù)合體釋放至細(xì)胞質(zhì)內(nèi),在cblC蛋白催化下形成Co(Ⅱ)cbl蛋白(即帶2價(jià)電子的氧化態(tài)cbl蛋白),一部分轉(zhuǎn)運(yùn)至線粒體內(nèi)轉(zhuǎn)化為腺苷鈷胺素(AdoCbl),參與線粒體內(nèi)支鏈氨基酸和奇數(shù)鏈脂肪酸代謝,人體必需的支鏈氨基酸和奇數(shù)鏈脂肪酸在分解代謝過(guò)程中生成甲基丙二酸,在甲基丙二酰輔酶A變位酶(MUT)和AdoCbl共同催化下生成琥珀酰輔酶A,參與三羧酸循環(huán),若MUT或AdoCbl代謝異常,可以導(dǎo)致甲基丙二酸、丙酸等中間代謝產(chǎn)物堆積,引起單純型甲基丙二酸血癥,包括cblA型、cblB型和mut型3種亞型;另一部分在細(xì)胞質(zhì)內(nèi)轉(zhuǎn)化為甲鈷胺(MeCbl),參與催化同型半胱氨酸轉(zhuǎn)化為甲硫氨酸[4],若同時(shí)出現(xiàn)AdoCbl和MeCbl合成不足,可以影響同型半胱氨酸和甲基丙二酸的代謝,使甲基丙二酸和同型半胱氨酸蓄積[1],導(dǎo)致甲基丙二酸血癥合并高同型半胱氨酸血癥,包括cblC型、cblD型、cblF型和cblJ型[6?7],尤以cblC型常見(jiàn)[2,8].

圖4 MMACHC基因檢測(cè)所見(jiàn) 4a 先證者存在MMACHC基因復(fù)合雜合突變,包括錯(cuò)義突變c.482G>A(p.Arg161Gln,箭頭所示,左圖)以及無(wú)義突變c.609G>A(p.Trp203X,箭頭所示,右圖)4b 先證者之弟存在MMACHC基因復(fù)合雜合突變,包括錯(cuò)義突變c.482G>A(p.Arg161Gln,箭頭所示,左圖)以及無(wú)義突變c.609G>A(p.Trp203X,箭頭所示,右圖) 4c 先證者之父親存在MMACHC基因錯(cuò)義突變c.482G>A(p.Arg161Gln,箭頭所示) 4d 先證者之母存在MMACHC基因無(wú)義突變c.609G>A(p.Trp203X,箭頭所示)Figure 4 Analysis of MMACHC gene test Proband had compound heterozygous mutation in MMACHC gene,including missense mutation c.482G>A(p.Arg161Gln,arrow indicates,left panel)and nonsense mutation c.609G>A(p.Trp203X,arrow indicates,right panel,Panel 4a).Proband's younger brother had the same compound herterozygous mutation as the proband(arrows indicate,Panel 4b).The proband's father canrried MMACHC gene missense mutation c.482G>A(p.Arg161Gln,arrow indicates,Panel 4c).The proband's mother carried MMACHC gene nonsense mutation c.609G>A(p.Trp203X,arrow indicates,Panel 4d).

大多數(shù)甲基丙二酸血癥為早發(fā)型,1歲內(nèi)發(fā)病,表現(xiàn)為喂養(yǎng)困難、生長(zhǎng)發(fā)育遲滯、癲發(fā)作、肌張力降低、酮癥酸中毒等,除神經(jīng)系統(tǒng)癥狀外,還有肝腎功能障礙表現(xiàn)等,若無(wú)早期干預(yù),病死率較高[9?10],因此,國(guó)外和國(guó)內(nèi)部分地區(qū)已將甲基丙二酸血癥作為新生兒出生缺陷的常規(guī)篩查項(xiàng)目[8,11].晚發(fā)型甲基丙二酸血癥患者少見(jiàn),臨床表現(xiàn)多樣,既往有文獻(xiàn)報(bào)道,1歲至學(xué)齡期前發(fā)病的患兒以溶血性尿毒綜合征(HUS)和肺動(dòng)脈高壓為主要表現(xiàn),而青少年期或成年期發(fā)病的患者則以不典型神經(jīng)精神癥狀為主要表現(xiàn)或僅表現(xiàn)為學(xué)習(xí)困難、情緒障礙等,還可合并共濟(jì)失調(diào)、厭食、癲發(fā)作等,臨床明確診斷困難,常延誤治療[12?13].本研究家系中先證者以學(xué)習(xí)成績(jī)下降、性格改變?yōu)槭装l(fā)癥狀,病程中出現(xiàn)幻覺(jué),逐漸出現(xiàn)雙下肢無(wú)力,體格檢查可見(jiàn)雙下肢肌力下降、下肢深淺感覺(jué)對(duì)稱性減弱、腱反射減退或消失等周?chē)窠?jīng)損害體征,均支持晚發(fā)型甲基丙二酸血癥的診斷,進(jìn)一步行遺傳代謝性疾病的血漿氨基酸和?；鈮A譜分析,明確診斷.與既往文獻(xiàn)報(bào)道的晚發(fā)型甲基丙二酸血癥相似,先證者之弟僅出現(xiàn)學(xué)習(xí)成績(jī)差、易怒等癥狀,一直未引起重視,待先證者明確診斷后,行基因檢測(cè)才確定亦存在MMACHC基因突變.先證者頭部MRI突出表現(xiàn)為腦萎縮、白質(zhì)髓鞘化不良,與既往文獻(xiàn)報(bào)道的甲基丙二酸血癥典型影像學(xué)改變相符[13?16].

圖5 維生素B12在細(xì)胞內(nèi)代謝障礙累及環(huán)節(jié)以及甲基丙二酸血癥各亞型[4]:cblA型、cblB型和mut型僅累及AdoCbl或MUT蛋白,引起單純甲基丙二酸血癥;cblC型、cblD型和cblF型導(dǎo)致AdoCbl和MeCbl蛋白合成障礙,引起甲基丙二酸血癥合并高同型半胱氨酸血癥Figure 5 Links and different subtypes of MMA caused by intracellular metabolism disorder of vitamin B12[4].cblA type,cblB type and mut type only affect AdoCbl and MUT protein,and cause simple MMA.cblC type,cblD type and cblF type induce the disorder of anabolism of AdoCbl and MeCbl protein and result in MMA complicated with hyperhomocysteinemia.

表1 甲基丙二酸各亞型相關(guān)基因Table 1. Different type of MMA and corresponding gene

MMAcblC型致病基因?yàn)镸MACHC基因,定位于1p34.1,共包含5個(gè)外顯子,全長(zhǎng)10 800 bp,編碼282個(gè)氨基酸殘端蛋白即cblC蛋白,該蛋白在催化維生素B12向AdoCbl和MeCbl轉(zhuǎn)化過(guò)程中發(fā)揮重要作用[2].本研究家系中先證者及其弟經(jīng)基因檢測(cè)明確診斷為MMAcblC型,系MMACHC基因復(fù)合雜合突變所致.其中,c.609G>A為無(wú)義突變,可使第203位色氨酸轉(zhuǎn)錄提前終止,該變異位點(diǎn)為東亞地區(qū)最常見(jiàn)變異位點(diǎn),占40%～60%[8,17?19],該位點(diǎn)純合突變多與早發(fā)型甲基丙二酸血癥有關(guān)[19];c.482G>A為錯(cuò)義突變,可使第161位氨基酸由精氨酸突變?yōu)楣劝滨０?Liu等[17]報(bào)告8例攜帶c.482G>A突變的患者,僅1例于4歲前發(fā)病,余均于4歲后發(fā)病,提示攜帶錯(cuò)義突變c.482G>A的患者臨床表型多為晚發(fā)型.

本研究家系中先證者一經(jīng)明確診斷即予甲鈷胺、左卡尼汀治療,早期即有效控制癥狀,治療3個(gè)月后復(fù)查血漿丙酰肉堿和C3/C2比值明顯下降,提示為MMAcblC型.既往研究顯示,多數(shù)晚發(fā)型MMAcblC型為維生素B12依賴型,建議早期肌肉注射或皮下注射維生素B12(1 mg/d),同時(shí)予低蛋白飲食(每日蛋白質(zhì)攝入量<0.80 g/kg),補(bǔ)充左旋肉堿[50～100 mg/(kg.d)],提高蛋氨酸水平、降低同型半胱氨酸和甲基丙二酸水平,改善臨床癥狀[4,20?21].

晚發(fā)型甲基丙二酸血癥臨床罕見(jiàn),但屬可治性遺傳性疾病,對(duì)維生素B12治療反應(yīng)良好,如果能夠及時(shí)識(shí)別其不典型臨床表現(xiàn),通過(guò)特異性生物學(xué)指標(biāo)和基因檢測(cè)早期診斷、及時(shí)治療,對(duì)患者預(yù)后意義重大.

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Analysis of clinical phenotype and genetic mutation with outcome evaluation in one family of vitamin B12?dependent methylmalonic aciduria

LI Jing1,SUN Yi?ming2,OU Li?yu1,ZHU Yu?ling1,WANG Liang1,LI Huan1,ZHANG Cheng11Department of Neurology,2Department of Health Care Clinic,the First Affiliated Hospital,Sun Yat?sen University,Guangzhou 510080,Guangdong,China

LI Jing and SUN Yi?ming contributed equally to this study

ZHANG Cheng(Email:zhangch6@mail.sysu.edu.cn)

ObjectiveTo explore the clinicalfeatures,genetic mutation and vitamin B12therapeutive effectiveness in vitamin B12?dependent methylmalonic acidemia(MMA).MethodsClinical data in a pedigree of 4 members with vitamin B12?dependent MMA was collected.Peripheral blood samples were collected for plasma amino acids and acylcarnitines and gene muatation analysis.The therapeutic efficacy ofvitamin B12was evaluated.ResultsThe initialpresentations ofthe proband were underachievement and personality changes in 12?year old,and accompanied by visual hallucinations and weakness of lower limbs during the course of disease.The younger brother of the proband presented with bad?temper and lower acheivement.The analysis of plasma amino acid and acylcarnitine showed that proband and his younger brother's plasma propionylcarnitine and propionylcarnitine/acetylcarnitine ratio,and the level of methylmalonic acid in urine were increased significantly. Compound heterozygeous mutation of c.482G>A(p.Arg161Gln)and c.609G>A(p.Trp203X)in MMACHC gene were seen in the proband and her younger brother.Her father carried MMACHC gene missense mutation c.482G>A(p.Arg161Gln),while her mother carried MMACHC gene nonsense mutation c.609G>A(p.Trp203X).Symptoms of the proband were improved after vitamin B12therapy.ConclusionsThe late?onset vitamin B12?dependent MMA is caused by compound heterozygote mutation of MMACHC gene.It had good responsive to vitamin B12therapy.Early diagnosis and timely treatment may play a critical role for the outcomes of patients with this disease.

Methylmalonic acid; Metabolism,inborn errors; Vitamin B12; Phenotype;Genes; Mutation; Pedigree

10.3969/j.issn.1672?6731.2017.07.009

國(guó)家自然科學(xué)基金資助項(xiàng)目(項(xiàng)目編號(hào):81471280);廣東省廣州市2015年產(chǎn)學(xué)研專項(xiàng)項(xiàng)目(項(xiàng)目編號(hào):1561000153);國(guó)家自然科學(xué)基金青年科學(xué)基金資助項(xiàng)目(項(xiàng)目編號(hào):81601087);廣東省科學(xué)技術(shù)廳2014年度公益研究與能力建設(shè)專項(xiàng)資金資助項(xiàng)目(項(xiàng)目編號(hào):2014A020212130);國(guó)家自然科學(xué)基金資助項(xiàng)目(項(xiàng)目編號(hào):81271401);國(guó)家自然科學(xué)基金-廣東省聯(lián)合基金重點(diǎn)資助項(xiàng)目(項(xiàng)目編號(hào):U1032004)

利婧、孫毅明并列為本文第一作者

510080廣州,中山大學(xué)附屬第一醫(yī)院神經(jīng)科[利婧、歐俐羽(現(xiàn)在廣西中醫(yī)藥大學(xué)附屬瑞康醫(yī)院神經(jīng)內(nèi)科,郵政編碼:530011)、朱瑜齡、王倞、李歡、張成],保健科(孫毅明)

張成(Email:zhangch6@mail.sysu.edu.cn)

This study was supported by the National Natural Science Foundation of China(No.81471280,81271401),2015 Production,Study and Research Special Project of Guangzhou,Guangdong Province,China(No.1561000153),the National Natural Science Foundation of China for Young Scientists(No.81601087),Non?Profit Study and Capability Building Special Fund Support Project of Guangdong Provincial Department of Science and Technology,China in the Year 2014(No.2014A020212130),and Joint Fund of National Natural Science Foundation of China and Natural Science Foundation of Guangdong Province,China(No.U1032004).

2017?06?12)