朱燕鳳 張 婷,4 陳 揚(yáng) 林 凱 陸鑄今 王曉紅 俞 蕙 陸國(guó)平

·論著·

Wolman病臨床及LIPA基因突變1例

朱燕鳳1,3張 婷1,3,4陳 揚(yáng)2,3林 凱1陸鑄今2王曉紅1俞 蕙1陸國(guó)平2

目的 探討Wolman病的臨床特點(diǎn)及分子診斷的意義。方法 對(duì)1例符合Wolman病臨床表現(xiàn)的患兒進(jìn)行白細(xì)胞溶酶體酸性脂酶LIPA基因測(cè)序，分析其突變的類型。結(jié)果 約5月齡女?huà)?，發(fā)現(xiàn)“皮膚黃染10余天，加重伴發(fā)熱3 d”入院。查體見(jiàn)肝脾顯著腫大，黃疸。實(shí)驗(yàn)室檢查提示貧血，肝功能衰竭，高三酰甘油血癥；X線胸腹片和腹部增強(qiáng)CT均提示特征性雙側(cè)腎上腺增大和廣泛鈣化。骨髓涂片可見(jiàn)海藍(lán)色泡沫狀組織細(xì)胞，PAS染色提示脂質(zhì)沉積。DNA測(cè)序顯示，LIPA基因編碼區(qū)第7外顯子上發(fā)生c.796G>T，p.G266*的純合無(wú)義突變，導(dǎo)致266位甘氨酸(GGA)突變?yōu)榻K止密碼(TGA)(p.G266*)，致溶酶體酸性脂酶缺失。結(jié)論 Wolman病嬰兒期起病，以顯著肝脾腫大、特征性雙側(cè)腎上腺增大和廣泛鈣化、高三酰甘油血癥為特征，相應(yīng)的酶學(xué)分析和LIPA基因檢測(cè)均可確診Wolman病。

Wolman??； 溶酶體酸性脂酶；LIPA基因突變； 肝脾大； 腎上腺鈣化

1 病例資料

女，約5月齡。因“發(fā)現(xiàn)皮膚黃染10余天，加重伴發(fā)熱3 d”收治復(fù)旦大學(xué)附屬兒科醫(yī)院(我院)PICU。入院前12 d被撿拾后發(fā)現(xiàn)其腹脹明顯送外院就診，發(fā)現(xiàn)肝脾腫大伴肝功能異常，遂轉(zhuǎn)入我院。

個(gè)人史、出生史、既往史和家族史均不詳。

入院查體：T 38℃, HR 156·min-1，RR 46·min-1，BP 82/46 mmHg。神志清楚，精神反應(yīng)欠佳。前囟平軟，皮膚鞏膜重度黃染，無(wú)皮疹。頸軟，淺表淋巴結(jié)未觸及明顯腫大。雙肺呼吸音粗，可聞及中濕啰音。心音有力，律齊，胸骨左緣3～4肋間可聞及Ⅳ級(jí)收縮期雜音。腹部明顯膨隆，腹壁靜脈顯露，臍膨出，約1 cm×1 cm，可回納。肝臟右肋下捫及10 cm，劍突下捫及8 cm，質(zhì)韌。脾臟肋下捫及10 cm，質(zhì)韌。腸鳴音3～5·min-1。雙下肢脛前凹陷性水腫。四肢肌張力正常，活動(dòng)可，神經(jīng)系統(tǒng)檢查未見(jiàn)異常。

實(shí)驗(yàn)室檢查：血常規(guī)WBC 3.5×109·L-1，Hb 52 g·L-1，PLT 42×109·L-1。入院后多次查肝功能、血脂以及凝血功能指標(biāo)均明顯異常(表1)。血氨118 μmol·L-1。TORCH篩查陰性，HBV、HCV、HIV、EBV、腸道病毒和梅毒檢測(cè)均陰性。血培養(yǎng)陰性。為了除外先天代謝性疾病，入院第2天行血串聯(lián)質(zhì)譜檢查示肉堿輕度升高，尿有機(jī)酸串聯(lián)質(zhì)譜未見(jiàn)異常。

表1 本文患兒肝功能、血脂和凝血功能檢測(cè)數(shù)據(jù)

影像學(xué)檢查：入院第1天X線胸腹片示肺炎，雙側(cè)腎上腺區(qū)多發(fā)細(xì)小斑片狀鈣化 (圖1A)。入院第2天腹部增強(qiáng)CT示肝臟增大伴彌漫性密度減低，脾大，腎臟密度略減低，雙腎上腺區(qū)廣泛鈣化 (圖1B，C)，腹水。腹部B超示肝腫大，巨脾，少到中等量腹水。超聲心動(dòng)圖示房間隔缺損、心包積液。同位素肝膽顯像示肝臟攝取清除功能差，肝膽排泄受阻。

圖1 本文患兒X線胸腹片及腹部增強(qiáng)CT所見(jiàn)

Fig 1 The results of chest X-ray and contrast-enhanced abdominal CT scan

Notes A: Chest X-ray showed calcified adrenal glands (arrows). B: Reconstruction of contrast-enhanced abdominal CT scan demonstrated enlarged, high-signal-intensity adrenal glands (arrows), indicative of calcifications. The liver (arrow head) had low attenuation relative to that of the spleen (*). C: Contrast-enhanced abdominal CT scan revealed hepatosplenomegaly. The liver (arrow head) had low attenuation relative to that of the spleen (*). Enlarged, heavily calcified adrenal glands (arrows) were present

入院診斷：嬰兒肝病綜合征，急性肝功能衰竭，肺炎，先天性心臟??？臍疝。

診治經(jīng)過(guò)：入院后完善上述相關(guān)輔助檢查，監(jiān)測(cè)生命體征。先后予保肝、止血、降血氨、促肝細(xì)胞生長(zhǎng)等對(duì)癥治療肝功能衰竭。以頭孢曲松和奧硝唑抗感染。間斷給予白蛋白、血漿、少漿血、冷沉淀和血小板等血制品補(bǔ)充支持。患兒入院當(dāng)天X線胸腹片及上腹部CT均提示腎上腺?gòu)V泛鈣化，血脂指標(biāo)升高，肝脾腫大，肝功能衰竭，入院后第2和4天肝病科會(huì)診認(rèn)為患兒可能存在少見(jiàn)或罕見(jiàn)的遺傳代謝性貯積性疾病，通過(guò)文獻(xiàn)檢索考慮Wolman病可能，行LIPA基因檢測(cè)，同時(shí)建議骨髓檢查。入院第7天行骨髓穿刺，骨髓涂片見(jiàn)海藍(lán)色泡沫狀組織細(xì)胞，胞質(zhì)內(nèi)多個(gè)脂質(zhì)空泡(圖 2A, B)，PAS染色陰性(圖 2C)。入院第9天患兒出現(xiàn)少尿伴明顯氣急，繼而出現(xiàn)心率和血氧飽和度下降，先后予利尿劑、腎上腺素、多巴胺和碳酸氫鈉，予胸外按壓、心肺復(fù)蘇等各種搶救措施，但患兒口鼻腔涌出較多鮮血，無(wú)自主心率及呼吸，臨床死亡。死亡診斷：嬰兒肝病綜合征(Wolman??？)，多器官功能衰竭(肝臟、血液、呼吸、循環(huán))，肺炎，先天性心臟病(房間隔缺損)，臍疝。

圖2 本文患兒骨髓涂片見(jiàn)PAS染色陰性的泡沫狀細(xì)胞

Fig 2 Histological study of bone marrow aspirate showing histiocytes with vacuolated cytoplasm

Notes A: Multiple foamy, sea-blue histiocytes with vacuolated cytoplasm (hematoxylin-eosin staining,×100 oil immersion lens); B: Multiple foamy, sea-blue histiocytes with vacuolated cytoplasm (hematoxylin-eosin staining,×100 oil immersion lens); C: vacuolated cytoplasm with negative periodic acid schiff (PAS staining, ×100 oil immersion lens)

LIPA基因測(cè)序分析：以患兒入院第4天抗凝外周血提取基因組DNA，PCR擴(kuò)增LIPA全基因并測(cè)序，測(cè)序結(jié)果經(jīng)DNAMAN軟件序列對(duì)比分析顯示，在LIPA基因編碼區(qū)第7外顯子發(fā)現(xiàn)純合無(wú)義突變(c.796G>T)(圖3)，導(dǎo)致266位甘氨酸(GGA)突變?yōu)榻K止密碼(TGA)(p.G266*)，使LIPA基因編碼的溶酶體酸性脂酶(lysosomal acid lipase, LAL)蛋白質(zhì)合成提前終止，不能合成正常的LAL。

圖3 本文患兒LIPA基因第7外顯子測(cè)序圖

Fig 3 Sequencing analysis of exon 7 ofLIPA

Notes A: control; B: patient. DNA sequencing analysis of all coding exons demonstrated homozygote nonsense mutation (c.796G>T) of exon 7, resulting in a termination codon (GGA-TGA) at the position amino acid 266 (frame)

2 討論

Wolman病(MIM#278000)由以色列Abramov和 Wolman醫(yī)生在1956年首先描述并發(fā)現(xiàn)[1]，屬罕見(jiàn)的常染色體隱性遺傳性溶酶體貯積病(Lysosomal storage disease, LSD)，檢索PubMed數(shù)據(jù)庫(kù)，至2012年11月報(bào)道的病例不足100例。本病是由LAL缺陷導(dǎo)致膽固醇酯和三酰甘油降解障礙[2, 3]。Wolman病發(fā)病率約為1∶350 000活產(chǎn)嬰兒，以猶太人多見(jiàn)[4]。中國(guó)香港和臺(tái)灣分別于1978[5]和1989年各報(bào)道1例[6]，中國(guó)大陸地區(qū)在2012年報(bào)道了首例Wolman病[7]，可能與該病罕見(jiàn)以及臨床對(duì)該病的認(rèn)識(shí)不足有關(guān)。

LAL存在于體內(nèi)各種細(xì)胞內(nèi)的溶酶體中，其功能主要為水解溶酶體中的膽固醇酯(膽固醇在體內(nèi)的存儲(chǔ)形式)和三酰甘油，其代謝產(chǎn)物為膽固醇、一酰甘油、二酰甘油和游離脂肪酸，這些代謝產(chǎn)物轉(zhuǎn)至胞漿后參與再酯化、儲(chǔ)積、膜構(gòu)造、激素合成、膽酸合成和供能等各種生理活動(dòng)。如LAL缺乏，大量的膽固醇酯和三酰甘油不能被正常水解，從而沉積在細(xì)胞內(nèi)，以網(wǎng)狀內(nèi)皮系統(tǒng)、小腸黏膜及腎上腺皮質(zhì)病變最為顯著，大量的親脂性脂肪沉積在腎上腺皮質(zhì)導(dǎo)致皮質(zhì)壞死和鈣化[8, 9]。

在大多數(shù)報(bào)道的病例中，先證者往往來(lái)自一個(gè)有血緣關(guān)系的帶有純合突變的家系[10～12]。編碼該酶的基因?yàn)長(zhǎng)IPA，由36 kb核苷酸系列和10個(gè)外顯子組成，位于10q23.2～23.3[10,11,13]。LIPA突變的類型包括錯(cuò)義突變(L179P, G321W, G60V)[13～15]，無(wú)義突變(T22X, Q277X, Y303X, S106X, W140X, G266X)[10, 11, 14, 16, 17]，插入突變(634insT, 351insA)[15, 18]和缺失突變(159～166, 435～436, exon 8, exon 4, exon 3)[13, 19, 20]。LIPA基因缺失、插入和無(wú)義突變都會(huì)導(dǎo)致LAL活性缺失或非常低下，致體內(nèi)大量的膽固醇酯和三酰甘油不能被正常水解，從而沉積在體內(nèi)各個(gè)組織和細(xì)胞內(nèi)，繼而引起明顯的臨床表現(xiàn)，常在生后數(shù)周內(nèi)起病，病情進(jìn)展迅速，預(yù)后不良，通常1歲內(nèi)死亡，即Wolman病。相比之下常見(jiàn)的局限于第10外顯子的錯(cuò)義和缺失突變引起LAL蛋白羧基端小缺失，LAL仍有部分活性，即膽固醇酯儲(chǔ)積病(Cholesteryl ester storage disease, CESD)。CESD由于有部分LAL活性，因此起病較晚，臨床癥狀較輕。在兒童期常表現(xiàn)為肝腫大，有發(fā)展為肝纖維化可能，其他臨床表現(xiàn)多樣，成人以肝纖維化、高膽固醇血癥(可合并高三酰甘油血癥)以及早發(fā)性動(dòng)脈粥樣硬化為特征。

根據(jù)本文病例和黃永蘭等[7]報(bào)道，證實(shí)國(guó)內(nèi)人群中存在LIPA基因突變的現(xiàn)象，推測(cè)國(guó)內(nèi)人群中同一基因突變的輕型表現(xiàn)CESD應(yīng)有一定的發(fā)病，但至今國(guó)內(nèi)尚未有報(bào)道，可能是由于尚未被認(rèn)識(shí)和重視而被漏診，提示以肝纖維化、高脂血癥以及早發(fā)動(dòng)脈粥樣硬化的病例，特別是成人需警惕CESD可能。就目前中國(guó)的人口規(guī)模，運(yùn)用基因測(cè)序技術(shù)檢測(cè)符合Wolman病和CESD臨床表現(xiàn)的病例，會(huì)識(shí)別出更多的病例。結(jié)合目前分子檢測(cè)在國(guó)內(nèi)的應(yīng)用現(xiàn)狀，基因測(cè)序特異性好，但相對(duì)于酶功能的檢測(cè)費(fèi)用高，有一定技術(shù)難度，檢測(cè)時(shí)間較長(zhǎng)，尚不能普遍開(kāi)展。

Wolman病應(yīng)與其他以脂質(zhì)代謝障礙的LSD如尼曼-匹克氏病(鞘磷脂酶活性減低)及戈謝病(β-葡萄糖苷酶缺乏)[21]等相鑒別，尤其是尼曼-匹克氏病骨髓象中有典型的泡沫細(xì)胞即尼曼-匹克細(xì)胞，與Wolman病骨髓象中的泡沫細(xì)胞相似很難鑒別，但尼曼-匹克氏病骨髓涂片PAS染色呈弱陽(yáng)性[22]，而Wolman病PAS染色陰性。結(jié)合相應(yīng)的臨床癥狀，檢測(cè)相應(yīng)的酶活性對(duì)臨床診斷非常重要。

Wolman病以嬰兒期起病，伴生長(zhǎng)遲緩，喂養(yǎng)不耐受，難治性腹瀉，肝脾腫大等臨床表現(xiàn)，腎上腺?gòu)V泛鈣化是該病的特征性表現(xiàn)。本文病例入院后因肝脾腫大，黃疸、肝功能衰竭，請(qǐng)肝病科會(huì)診發(fā)現(xiàn)其X線胸腹片及腹部CT均提示腎上腺?gòu)V泛鈣化，這種表現(xiàn)與臨床常見(jiàn)的嬰兒肝病綜合征不同，極為少見(jiàn)，結(jié)合患兒明顯的肝脾腫大、凝血功能障礙、高三酰甘油血癥和外周血三系下降的表現(xiàn)，考慮到患兒可能存在少見(jiàn)或罕見(jiàn)的遺傳代謝性儲(chǔ)積性疾病，通過(guò)文獻(xiàn)檢索和會(huì)診討論考慮Wolman?。粸榱顺馄渌南忍齑x性疾病和感染因素，還進(jìn)行了血、尿串聯(lián)質(zhì)譜，病原學(xué)方面的檢測(cè)。骨髓涂片可見(jiàn)海藍(lán)色泡沫狀組織細(xì)胞(PAS染色陰性提示脂質(zhì)沉積可能)，之后LIPA基因檢測(cè)結(jié)果均支持最初的Wolman病臨床診斷；而血、尿串聯(lián)質(zhì)譜和病原學(xué)方面的檢測(cè)結(jié)果則排除了其他常見(jiàn)的可引起類似臨床表現(xiàn)的先天代謝性疾病和感染因素。本文病例血標(biāo)本LIPA基因測(cè)序結(jié)果顯示，該基因編碼區(qū)第7外顯子上發(fā)生c.796G>T，p.G266*的純合無(wú)義突變，使LIPA基因編碼的LAL蛋白質(zhì)合成提前終止，這是Wolman病一個(gè)常見(jiàn)的熱點(diǎn)突變位點(diǎn)[15]。

Wolman病臨床以支持對(duì)癥姑息治療為主，如予低脂肪或無(wú)脂肪配方奶喂養(yǎng)，靜脈營(yíng)養(yǎng)、補(bǔ)充脂溶性維生素等，預(yù)后較差。目前造血干細(xì)胞移植是唯一被報(bào)道的能防止肝功能衰竭和死亡的治療措施[23～27]，來(lái)源于供者的細(xì)胞提供正常LAL，使機(jī)體可正常水解膽固醇酯和三酰甘油[2, 9, 28, 29]。近年來(lái)已經(jīng)有成功以骨髓移植治療Wolman病的報(bào)道，有報(bào)道個(gè)別患兒生存年齡至11歲，但目前移植成功率不高，治療相關(guān)死亡也很顯著，缺少長(zhǎng)期隨訪的資料等[23, 27]，仍處于探索階段。此外，酶替代療法還處在動(dòng)物實(shí)驗(yàn)階段[30, 31]。

致謝 由衷的感謝為該病例提供LIPA全基因檢測(cè)分析的我院分子轉(zhuǎn)化中心的王慧君博士；感謝所有參與該病例臨床處理及診斷分析的所有部門，尤其感謝我院徐虹教授、張靈恩教授、高怡瑾主任給予的支持。

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[2]Wolman M, Sterk W, Gatt S, et al. Primary familial xanthomatosis with involvement and calcification of the adrenals. Report of two more cases in siblings of a previously described infant. Pediatrics, 1961,28: 742-757

[3]Fitoussi G, Nègre-Salvayre A, Pieraggi MT, et al. New pathogenetic hypothesis for Wolman disease: possible role of oxidized low-density lipoproteins in adrenal necrosis and calcification. Biochem J, 1994, 301 (Pt 1): 267-273

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One case with Wolman disease: clinical features and LIPA gene mutation

ZHUYan-feng1,3,ZHANGTing1,3,4,CHENYang2,3,LINKai1,LUZhu-jin2,WANGXiao-hong1,YUHui1,LUGuo-ping2

(Children′sHospitalofFudanUniversity,Shanghai201102,China; 1DepartmentofInfectiousDiseases, 2PediatricIntensiveCareUnit, 3Hasequalcontributiontothestudy; 4NowatShanghaiChildren′sHospital)

YU Hui, E-mail:yuhui20@yahoo.com; LU Guo-ping, E-mail:luxy2005@yahoo.com.cn

ObjectiveWolman disease (WD) is a rare autosomal recessive lysosomal storage disease, caused by deficiency of lysosomal acid lipase (LAL). In China, WD, the same type disorder as cholesteryl ester storage disease (CESD), but with severe clinical manifestations, is still poorly recognized. The purpose of this study was to investigate the clinical features, genetic abnormality of WD.MethodsClinical manifestations, laboratory examinations, and genetic testing of one case of WD were presented, analyzed, and discussed. The related literatures were reviewed.ResultsA approximately 5-month-old female infant presented typical WD clinical features, including massive hepatosplenomegaly, remarkable jaundice, liver failure, severe coagulopathy, depressed bone marrow function, bilateral calcified adrenal glands, and in bone marrow aspirate, multiple foamy, vacuolated cytoplasm, sea-blue histiocytes with negative periodic acid schiff staining were observed. Sequencing of theLIPAgene revealed that she was a homozygote with the nonsense mutation (c.796G>T) in exon 7, resulting in the change of amino acid 266 from Gly to termination codon (GGA-TGA). This mutation finally result in a synthesis of the truncated protein (p.G266*).Conclusions The case presented here is the second WD case reported in mainland China. Based on the size of our population, WD and CESD are both underestimated. With the progress in DNA sequencing technique, and recognition the nature of WD and CESD, hopefully more cases will be diagnosed and recognized.

Wolman disease; Lysosomal acid lipase;LIPAgene mutation; Hepatosplenomegaly; Calcification of adrenal glands

上海市自然科學(xué)基金:12ZR1403500;上海市浦江人才計(jì)劃:12PJ1401500

復(fù)旦大學(xué)附屬兒科醫(yī)院 1 感染科，2 重癥醫(yī)學(xué)科；上海，201102；3 共同第一作者;4 現(xiàn)在上海市兒童醫(yī)院工作

俞蕙，E-mail:yuhui20@yahoo.com；陸國(guó)平，E-mail:luxy2005@yahoo.com.cn

10.3969/j.issn.1673-5501.2013.01.012

2012-10-17

2012-12-17)

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