Wolman病臨床及LIPA基因突變1例

朱燕鳳 張 婷,4 陳 揚 林 凱 陸鑄今 王曉紅 俞 蕙 陸國平

·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2,3林 凱1陸鑄今2王曉紅1俞 蕙1陸國平2

目的 探討Wolman病的臨床特點及分子診斷的意義。方法 對1例符合Wolman病臨床表現的患兒進行白細胞溶酶體酸性脂酶LIPA基因測序，分析其突變的類型。結果 約5月齡女嬰，發(fā)現“皮膚黃染10余天，加重伴發(fā)熱3 d”入院。查體見肝脾顯著腫大，黃疸。實驗室檢查提示貧血，肝功能衰竭，高三酰甘油血癥；X線胸腹片和腹部增強CT均提示特征性雙側腎上腺增大和廣泛鈣化。骨髓涂片可見海藍色泡沫狀組織細胞，PAS染色提示脂質沉積。DNA測序顯示，LIPA基因編碼區(qū)第7外顯子上發(fā)生c.796G>T，p.G266*的純合無義突變，導致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。結論 Wolman病嬰兒期起病，以顯著肝脾腫大、特征性雙側腎上腺增大和廣泛鈣化、高三酰甘油血癥為特征，相應的酶學分析和LIPA基因檢測均可確診Wolman病。

Wolman病； 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)現皮膚黃染10余天，加重伴發(fā)熱3 d”收治復旦大學附屬兒科醫(yī)院(我院)PICU。入院前12 d被撿拾后發(fā)現其腹脹明顯送外院就診，發(fā)現肝脾腫大伴肝功能異常，遂轉入我院。

個人史、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，精神反應欠佳。前囟平軟，皮膚鞏膜重度黃染，無皮疹。頸軟，淺表淋巴結未觸及明顯腫大。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，質韌。脾臟肋下捫及10 cm，質韌。腸鳴音3～5·min-1。雙下肢脛前凹陷性水腫。四肢肌張力正常，活動可，神經系統(tǒng)檢查未見異常。

實驗室檢查：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天行血串聯質譜檢查示肉堿輕度升高，尿有機酸串聯質譜未見異常。

表1 本文患兒肝功能、血脂和凝血功能檢測數據

影像學檢查：入院第1天X線胸腹片示肺炎，雙側腎上腺區(qū)多發(fā)細小斑片狀鈣化 (圖1A)。入院第2天腹部增強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CT所見

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?。磕氿?。

診治經過：入院后完善上述相關輔助檢查，監(jiān)測生命體征。先后予保肝、止血、降血氨、促肝細胞生長等對癥治療肝功能衰竭。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充支持?；純喝朐寒斕靀線胸腹片及上腹部CT均提示腎上腺廣泛鈣化，血脂指標升高，肝脾腫大，肝功能衰竭，入院后第2和4天肝病科會診認為患兒可能存在少見或罕見的遺傳代謝性貯積性疾病，通過文獻檢索考慮Wolman病可能，行LIPA基因檢測，同時建議骨髓檢查。入院第7天行骨髓穿刺，骨髓涂片見海藍色泡沫狀組織細胞，胞質內多個脂質空泡(圖 2A, B)，PAS染色陰性(圖 2C)。入院第9天患兒出現少尿伴明顯氣急，繼而出現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、心肺復蘇等各種搶救措施，但患兒口鼻腔涌出較多鮮血，無自主心率及呼吸，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PAS染色陰性的泡沫狀細胞

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基因測序分析：以患兒入院第4天抗凝外周血提取基因組DNA，PCR擴增LIPA全基因并測序，測序結果經DNAMAN軟件序列對比分析顯示，在LIPA基因編碼區(qū)第7外顯子發(fā)現純合無義突變(c.796G>T)(圖3)，導致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[1]，屬罕見的常染色體隱性遺傳性溶酶體貯積病(Lysosomal storage disease, LSD)，檢索PubMed數據庫，至2012年11月報道的病例不足100例。本病是由LAL缺陷導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活產嬰兒，以猶太人多見[4]。中國香港和臺灣分別于1978[5]和1989年各報道1例[6]，中國大陸地區(qū)在2012年報道了首例Wolman病[7]，可能與該病罕見以及臨床對該病的認識不足有關。

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

在大多數報道的病例中，先證者往往來自一個有血緣關系的帶有純合突變的家系[10～12]。編碼該酶的基因為LIPA，由36 kb核苷酸系列和10個外顯子組成，位于10q23.2～23.3[10,11,13]。LIPA突變的類型包括錯義突變(L179P, G321W, G60V)[13～15]，無義突變(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、插入和無義突變都會導致LAL活性缺失或非常低下，致體內大量的膽固醇酯和三酰甘油不能被正常水解，從而沉積在體內各個組織和細胞內，繼而引起明顯的臨床表現，常在生后數周內起病，病情進展迅速，預后不良，通常1歲內死亡，即Wolman病。相比之下常見的局限于第10外顯子的錯義和缺失突變引起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。在兒童期常表現為肝腫大，有發(fā)展為肝纖維化可能，其他臨床表現多樣，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)性動脈粥樣硬化為特征。

根據本文病例和黃永蘭等[7]報道，證實國內人群中存在LIPA基因突變的現象，推測國內人群中同一基因突變的輕型表現CESD應有一定的發(fā)病，但至今國內尚未有報道，可能是由于尚未被認識和重視而被漏診，提示以肝纖維化、高脂血癥以及早發(fā)動脈粥樣硬化的病例，特別是成人需警惕CESD可能。就目前中國的人口規(guī)模，運用基因測序技術檢測符合Wolman病和CESD臨床表現的病例，會識別出更多的病例。結合目前分子檢測在國內的應用現狀，基因測序特異性好，但相對于酶功能的檢測費用高，有一定技術難度，檢測時間較長，尚不能普遍開展。

Wolman病應與其他以脂質代謝障礙的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胞即尼曼-匹克細胞，與Wolman病骨髓象中的泡沫細胞相似很難鑒別，但尼曼-匹克氏病骨髓涂片PAS染色呈弱陽性[22]，而Wolman病PAS染色陰性。結合相應的臨床癥狀，檢測相應的酶活性對臨床診斷非常重要。

Wolman病以嬰兒期起病，伴生長遲緩，喂養(yǎng)不耐受，難治性腹瀉，肝脾腫大等臨床表現，腎上腺廣泛鈣化是該病的特征性表現。本文病例入院后因肝脾腫大，黃疸、肝功能衰竭，請肝病科會診發(fā)現其X線胸腹片及腹部CT均提示腎上腺廣泛鈣化，這種表現與臨床常見的嬰兒肝病綜合征不同，極為少見，結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，考慮到患兒可能存在少見或罕見的遺傳代謝性儲積性疾病，通過文獻檢索和會診討論考慮Wolman??；為了除外其他的先天代謝性疾病和感染因素，還進行了血、尿串聯質譜，病原學方面的檢測。骨髓涂片可見海藍色泡沫狀組織細胞(PAS染色陰性提示脂質沉積可能)，之后LIPA基因檢測結果均支持最初的Wolman病臨床診斷；而血、尿串聯質譜和病原學方面的檢測結果則排除了其他常見的可引起類似臨床表現的先天代謝性疾病和感染因素。本文病例血標本LIPA基因測序結果顯示，該基因編碼區(qū)第7外顯子上發(fā)生c.796G>T，p.G266*的純合無義突變，使LIPA基因編碼的LAL蛋白質合成提前終止，這是Wolman病一個常見的熱點突變位點[15]。

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

致謝 由衷的感謝為該病例提供LIPA全基因檢測分析的我院分子轉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。

[1]Abramov AS, Schorr S, Wolman M. Generalized xanthomatosis with calcified adrenals. AMA J Dis Child, 1956,91(3):282-286

[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

[4]Valles-Ayoub Y, Esfandiarifard S, No D, et al. Wolman disease (LIPA p.G87V) genotype frequency in people of Iranian-Jewish ancestry. Genet Test Mol Biomarkers, 2011,15(6): 395-398

[5]Ho FC, Lin HJ, Chan WC. Wolman′s disease: the first reported Chinese patient. Mod Med Asia, 1978,14(11): 23-26

[6]Wang TR, Chuang SM. A Chinese case of Wolman disease. J Inherit Metab Dis, 1989, 12(3): 328

[7]Huang YL(黃永蘭), Sheng HY, Zhao XY, et al. Wolman disease with novel mutation of LIPA gene in a Chinese infant. Chin J Pediatr(中華兒科雜志), 2012, 50(8): 601-605

[8]Sando GN, Rosenbaum LM. Human lysosomal acid lipase/cholesteryl ester hydrolase. Purification and properties of the form secreted by fibroblasts in microcarrier culture. J Biol Chem, 1985, 260(28): 15186-15193

[9]Patrick AD, Lake BD. Deficiency of an acid lipase in Wolman′s disease. Nature, 1969, 222(5198): 1067-1068

[10]Pagani F, Pariyarath R, Garcia R, et al, New lysosomal acid lipase gene mutants explain the phenotype of Wolman disease and cholesteryl ester storage disease. J Lipid Res, 1998,39(7): 1382-1388

[11]Ries S, Aslanidis C, Fehringer P, et al, A new mutation in the gene for lysosomal acid lipase leads to Wolman disease in an African kindred. J Lipid Res, 1996.,37(8): 1761-1765

[12]Gramatges MM, Dvorak CC, Regula DP, et al. Pathological evidence of Wolman′s disease following hematopoietic stem cell transplantation despite correction of lysosomal acid lipase activity. Bone Marrow Transplant, 2009, 44(7): 449-450

[13]Fujiyama J, Sakuraba H, Kuriyama M, et al. A new mutation (LIPA Tyr22X) of lysosomal acid lipase gene in a Japanese patient with Wolman disease. Hum Mutat, 1996,8(4): 377-380

[14]Fasano T, Pisciotta L, Bocchi L, et al. Lysosomal lipase deficiency: molecular characterization of eleven patients with Wolman or cholesteryl ester storage disease. Mol Genet Metab, 2012, 105(3): 450-456

[15]Lohse P, Maas S, Sewell AC, et al. Molecular defects underlying Wolman disease appear to be more heterogeneous than those resulting in cholesteryl ester storage disease. J Lipid Res, 1999, 40(2): 221-228

[16]Seedorf U, Muntoni S, Mayatepek E. Wolman disease due to homozygosity for a novel truncated variant of lysosomal acid lipase (351insA) associated with complete in situ acid lipase deficiency. Circulation, 1996,94(S1): 35

[17]Krivit W, Freese D, Chan KW, et al. Wolman′s disease: a review of treatment with bone marrow transplantation and considerations for the future. Bone Marrow Transplant, 1992, 10(S1): 97-101

[19]Aslanidis C, Ries S, Fehringer P, et al. Genetic and biochemical evidence that CESD and Wolman disease are distinguished by residual lysosomal acid lipase activity. Genomics, 1996, 33(1): 85-93

[19]Zschenker O, Jung N, Rethmeier J, et al. Characterization of lysosomal acid lipase mutations in the signal peptide and mature polypeptide region causing Wolman disease. J Lipid Res, 2001,42(7): 1033-1040

[20]Mayatepek E, Seedorf U, Wiebusch H, et al. Fatal genetic defect causing Wolman disease. J Inherit Metab Dis, 1999,22(1): 93-94

[21]Shi HP(施惠平). 溶酶體貯積癥病例診斷及產前診斷. Chin J Pediatr(中華兒科雜志), 1999, 37(1): 2

[22]胡亞美，江載芳，主編. 褚福棠實用兒科學. 第7版.北京:人民衛(wèi)生出版社, 2002

[23]Krivit W, Peters C, Dusenbery K, et al. Wolman disease successfully treated by bone marrow transplantation. Bone Marrow Transplant, 2000,26(5): 567-570

[24]Stein J, Garty BZ, Dror Y, et al. Successful treatment of Wolman disease by unrelated umbilical cord blood transplantation. Eur J Pediatr, 2007,166(7): 663-666

[25]Tolar J, Petryk A, Khan K, et al. Long-term metabolic, endocrine, and neuropsychological outcome of hematopoietic cell transplantation for Wolman disease. Bone Marrow Transplant, 2009,43(1): 21-27

[26]Anderson RA, Byrum RS, Coates PM, et al. Mutations at the lysosomal acid cholesteryl ester hydrolase gene locus in Wolman disease. Proc Natl Acad Sci U S A, 1994, 91(7): 2718-2722

[27]Kyriakides EC, Paul B, Balint JA. Lipid accumulation and acid lipase deficiency in fibroblasts from a family with Wolman′s disease, and their apparent correction in vitro. J Lab Clin Med, 1972,80(6): 810-816

[28]Brown MS, Sobhani MK, Brunschede GY, et al. Restoration of a regulatory response to low density lipoprotein in acid lipase-deficient human fibroblasts. J Biol Chem, 1976,251(11): 3277-3286

[29]Lee TM, Welsh M, Benhamed S, et al. Intragenic deletion as a novel type of mutation in Wolman disease. Mol Genet Metab, 2011,104(4): 703-705

[30]Du H, Cameron TL, Garger SJ, et al. Wolman disease/cholesteryl ester storage disease: efficacy of plant-produced human lysosomal acid lipase in mice. J Lipid Res, 2008,49(8): 1646-1657

[31]Du H, Levine M, Ganesa C, et al. The role of mannosylated enzyme and the mannose receptor in enzyme replacement therapy. Am J Hum Genet, 2005,77(6): 1061-1074

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

上海市自然科學基金:12ZR1403500;上海市浦江人才計劃:12PJ1401500

復旦大學附屬兒科醫(yī)院 1 感染科，2 重癥醫(yī)學科；上海，201102；3 共同第一作者;4 現在上海市兒童醫(yī)院工作

俞蕙，E-mail:yuhui20@yahoo.com；陸國平，E-mail:luxy2005@yahoo.com.cn

10.3969/j.issn.1673-5501.2013.01.012

2012-10-17

2012-12-17)

丁俊杰)