李國民 方曉燕 徐 虹 沈 茜孫 利翟亦暉郭慕依 安 宇 吳冰冰

·論著·

兒童2型Dent病1例并文獻復習

李國民1,4方曉燕1,4徐 虹1沈 茜1孫 利1翟亦暉1郭慕依2安 宇3吳冰冰3

目的 總結(jié)1例2型Dent病患兒臨床資料，提高對該病的認識。方法 報道1例2型Dent病患兒的臨床發(fā)現(xiàn)、相關實驗室檢查指標和腎活檢病理改變。對該家系相關成員行CLCN5和OCRL基因外顯子及附近調(diào)控區(qū)域直接測序，分析突變位點，并文獻復習。結(jié)果 患兒，男，6歲起病，首發(fā)癥狀為單純性蛋白尿，未見先天性白內(nèi)障、智力低下、認知障礙及發(fā)育遲緩。實驗室檢查提示低分子蛋白尿，高鈣尿癥，鏡下血尿，血清乳酸脫氫酶和磷酸肌酸激酶增高，腎活檢病理提示輕微病變。患兒CLCN5和OCRL基因測序分析發(fā)現(xiàn)，CLCN5基因未見致病性突變，ORCL基因c.260delA(p.Q87fs105X)純合突變，確診為2型Dent病。家系OCRL基因突變分析顯示，患兒父親c.260delA未檢出突變，母親攜帶c.260delA雜合突變。c.260delA突變?yōu)橐拼a突變，可導致OCRL基因編碼蛋白截短。結(jié)論 2型Dent病以低分子蛋白尿、高鈣尿癥和鏡下血尿為特征，血清乳酸脫氫酶、磷酸肌酸激酶增高和OCRL基因純合移碼突變支持2型Dent病診斷。

2型Dent??；CLCN5基因；OCRL基因

1 病例資料

男，漢族，2004年6月出生，2010年10月因“體檢發(fā)現(xiàn)蛋白尿3個月”就診復旦大學附屬兒科醫(yī)院(我院)，以蛋白尿待查收入我院。

患兒3個月前體檢時發(fā)現(xiàn)尿蛋白2+，不伴有發(fā)熱、腹痛，無尿頻、尿急、尿痛，未見水腫及少尿，未見肉眼血尿。當?shù)蒯t(yī)院多次復查尿常規(guī)，尿蛋白均陽性(+～3+)，24 h尿蛋白定量在1.40～1.45 g·L-1，予貝那普利、黃芪顆粒治療，尿蛋白未轉(zhuǎn)陰。

患兒系G1P1，足月剖宮產(chǎn)，出生無產(chǎn)傷及窒息，出生體重4 100 g。既往體健，無腎臟病病史。父母均體健，非近親結(jié)婚，家族成員中無類似疾病史。

入院查體：身高120 cm，體重21 kg。血壓90/60 mmHg，神志清楚，精神尚可，全身未見水腫，兩肺及心臟聽診正常，腹平坦，腎區(qū)無叩擊痛，肋下未捫及肝、脾腫大。神經(jīng)系統(tǒng)檢查無認知障礙，智力測試正常，眼科檢查未發(fā)現(xiàn)角膜色素沉著和白內(nèi)障。

血電解質(zhì)、肝腎功能、血膽固醇正常。血清乳酸脫氫酶278 U·L-1，磷酸肌酸激酶42 U·L-1。尿常規(guī)蛋白2+，鏡下血尿，24 h尿蛋白定量1.02 g·L-1，α1微球蛋白79 mg·L-1。24 h尿Ca 5.31 mg·kg-1，尿Ca/Cr 0.43。泌尿系統(tǒng)B超示腎臟結(jié)構(gòu)和大小未見異常，未見結(jié)石和鈣鹽沉著。腎活檢示：光鏡下可見30個腎小球，除1個腎小球硬化外，余腎小球形態(tài)結(jié)構(gòu)大致正常，腎小管無異常(圖1A,B)。免疫熒光提示IgG、IgA、IgM、C3、C4、C1q和Fb均陰性，Ⅳ型膠原α1、α3和α5鏈均陽性。電鏡下可見5個腎小球，未有其他異常發(fā)現(xiàn)(圖1C)。

圖1 本文患兒腎臟病理改變

Fig 1 Renal pathological changes in the proband

Notes Globally sclerosed glomerulus shown in A (HE stain original magnification 200×), minute lesion shown in B ( PAS stain original magnification 200×) under light microscope; Normal glomerular structure shown in C under electron microscopy ( original magnification 11 500×)

對患兒(先證者)家系采用“2次試紙法”行尿液篩查。該家系共有3代成員26人，本例患兒尿液篩查尿蛋白陽性，其母親尿蛋白可疑陽性(±)，余24位家系成員2次尿液篩查均陰性。利用Sanger法首先對先證者及其父母行CLCN5基因外顯子和其附近區(qū)域進行直接測序，未發(fā)現(xiàn)致病性突變。 對OCRL基因外顯子和其附近區(qū)域進行直接測序，測序結(jié)果與美國國家生物技術(shù)信息中心(NCBI)GenBank (http://www.ncbi.nlm.nih.gov/Genbank/) 中的序列進行比對，其中核苷酸序列參照 NG_008638.1OCRL，cDNA序列參照 NM_0000276.3OCRL。氨基酸序列參照Uni-ProtKB (http://www.uniprot.org/uniprot/Q01968, ID: Q01968) 和 the Ensembl Genome Browser (http://www.ensembl.org, ID: ENSP00000360154) 提供的序列進行比對。測序結(jié)果顯示(圖2)，患兒OCRL基因5號外顯子c.260delA (p.Q87fs105X)純合缺失變異，確診為2型Dent病，患兒母親攜帶雜合c.260delA變異，其父親未發(fā)現(xiàn)c.260delA變異。在100例正常人群中未發(fā)現(xiàn)有c.260delA變異。c.260delA變異是單個堿基的缺失，引起移碼突變，造成OCRL基因編碼蛋白截短。其余家系成員尿蛋白陰性未行基因突變分析。

圖2 患兒及其父母OCRL基因檢測結(jié)果

Fig 2 Renal pathological changes in proband

Notes Mutational analysis ofOCRLin the family. Arrows indicate mutation. A: control, B: proband, C: mother, D: father

予患兒依那普利、氫氯噻嗪口服,并囑多飲水，并定期復查腎功能。患兒出院后3和4年來我院隨訪，血電解質(zhì)和血氣分析正常；血清乳酸脫氫酶300～324 U·L-1，磷酸肌酸激酶32～302 U·L-1；仍有鏡下血尿，24 h尿蛋白定量1.31～1.54 g·L-1，α1微球蛋白275～528.3 mg·L-1，24 h尿Ca 5.31～9.13 mg·kg-1，尿Ca/Cr 0.41～0.44；腎臟B超未見結(jié)石和鈣鹽沉積。隨訪患兒母親24 h尿蛋白定量0.18 g·L-1，磷酸肌酸激酶202 U·L-1,乳酸脫氫酶265 U·L-1;肝、腎功能、電解質(zhì)和血氣分析均正常。

2 討論

Dent病是一種罕見的X連鎖隱性遺傳性腎小管疾病，以近端小管功能異常為表現(xiàn)，其主要特征為低分子量蛋白尿(LMWP)、高鈣尿癥、腎結(jié)石、腎鈣質(zhì)沉積癥和腎功能異常[1, 2]。盡管Dent病累及近端腎小管功能，但極少患者有糖尿、氨基酸尿、高磷酸鹽尿和尿液酸化功能障礙等范可尼綜合征的表現(xiàn)[3]。典型Dent病癥狀僅見于男性，一般在兒童早期就可出現(xiàn)[4]。<10歲的兒童可表現(xiàn)為LMWP和(或)鏡下血尿，癥狀隱匿。30%～80%的Dent病患者于30～50歲進展為終末期腎病(ESRD)，然而也有部分患者至60歲以上也未進展為ESRD[1, 4, 5]。女性攜帶者癥狀輕微，可僅有鏡下血尿，罕有進展為ESRD的報道[1, 6]。佝僂病或軟骨病是Dent病常見的并發(fā)癥。典型Dent病的診斷需同時符合Hoopes標準[7]中的3條：①LMWP：尿中低分子蛋白至少升高5倍，用于監(jiān)測的低分子蛋白主要有視黃醇結(jié)合蛋白、α1微球蛋白和β2微球蛋白；②高鈣尿癥：24 h尿Ca定量>4.0 mg·kg-1或隨機尿Ca/Cr>0.25 mg·mg-1；③有下列情況之一：鏡下血尿、腎結(jié)石、腎鈣鹽沉著癥、低磷血癥和腎功能異常。本文患兒臨床特征為LMWP，α1微球蛋白定量異常增高；高鈣尿癥，24 h尿Ca定量和尿Ca/Cr均高于同年齡兒童的正常值；持續(xù)性鏡下血尿。因此，本文患兒符合Dent病的臨床診斷。

約65%的Dent病由CLCN5基因突變引起(1型Dent病, OMIM #300009)[1, 2]。另有15%的Dent病由OCRL基因突變引起(2型Dent病, MIM #300555)[2, 8, 9]，余下的20% Dent病尚未發(fā)現(xiàn)致病基因[5, 8]。Dent病發(fā)病率目前尚不清楚，以“Dent disease”為檢索詞檢索PubMed數(shù)據(jù)庫，1型Dent病病例約250例，2型Dent病有57例病例報道[2]。國內(nèi)曾有1型Dent病的報道[6, 10, 11]，尚未見2型Dent病報道。

CLCN5基因位于染色體Xp11.22-11.23，編碼746個氨基酸長度的CLC-5蛋白[12]。CLC-5蛋白屬于電壓門控性氯離子通道蛋白家族成員，具有高度保守的結(jié)構(gòu)，在體內(nèi)發(fā)揮著重要作用，包括調(diào)節(jié)膜的興奮性、維持細胞內(nèi)外離子的穩(wěn)態(tài)和調(diào)節(jié)酸化功能[13～16]。OCRL基因位于染色體Xq25，編碼磷脂酰肌醇4，5-二磷酸5-磷酸酶。該酶可以水解磷脂酰肌醇4，5-二磷酸(PIP2)[7, 17]。參與調(diào)節(jié)肌動蛋白聚合過程，進而影響細胞遷移和細胞間接觸，在腎小管、眼晶狀體和腦組織等發(fā)育過程中起重要作用[2, 18]。本文患兒CLCN5基因突變分析未發(fā)現(xiàn)致病性突變，OCRL基因突變分析發(fā)現(xiàn)5號外顯子c.260delA (p.Q87fs105X)純合缺失變異。在100例正常人群中未發(fā)現(xiàn)有c.260delA變異。c.260delA變異是單個堿基的缺失，引起移碼突變，造成OCRL基因編碼蛋白截短，為致病性突變。故患兒發(fā)病與致病性c.260delA純合突變有關。人類基因突變資料庫未發(fā)現(xiàn)該突變報道(http://www.hgmd.cf.ac.uk/)， 應為新的突變。家系OCRL基因突變分析提示，患兒母親有c.260delA雜合突變，為攜帶者，除α1微球蛋白、24 h尿蛋白定量、磷酸肌酸激酶和乳酸脫氫酶偏高外，無其他臨床表現(xiàn)。

OCRL基因最早被發(fā)現(xiàn)是Lowe 綜合征(眼-腦-腎綜合征, OMIM #309000)的致病基因[18]。Lowe 綜合征典型的臨床特征為雙側(cè)先天性白內(nèi)障、近端小管功能異常(常表現(xiàn)為范可尼綜合征)、精神和運動發(fā)育落后[19, 20]。PubMed數(shù)據(jù)庫檢索到的57例2型Dent病[5,7,17,21～29]和本文報道的1例匯總分析結(jié)果如表1所示，18/58例2型Dent病患者有輕度智力障礙，發(fā)育延遲等腎外癥狀，尚未見嚴重的精神和運動發(fā)育落后報道，58例均無腎小管酸中毒、糖尿、氨基酸等范可尼綜合征的表現(xiàn),4例有外周性白內(nèi)障，但無先天性白內(nèi)障。27/54例伴血清磷酸肌酸激酶和乳酸脫氫酶的增高。本文患兒無眼部和腦部異常發(fā)現(xiàn)，近端腎小管障礙表現(xiàn)為LMWP和高鈣尿癥，未見糖尿、氨基酸尿等范可尼綜合征的表現(xiàn)，血氣分析提示也無腎小管酸中毒表現(xiàn)，提示符合2型Dent病臨床特點。多次血清磷酸肌酸激酶和乳酸脫氫酶均升高，提示肌肉受累。引起2型Dent病的突變位點通常發(fā)生在OCRL基因5′端，位于外顯子1～7，為磷脂酰肌醇4，5-二磷酸5-磷酸酶結(jié)構(gòu)域編碼區(qū)，而Lowe 綜合征突變位點一般位于OCRL基因的外顯子8～24。本文患兒OCRL基因突變位點位于外顯子5上，符合2型Dent病遺傳學特點[18, 30]。

表1 已報道的2型Dent病患者的腎外表現(xiàn)(n)

Notes MR: Mental retardation; ND:not done. 1) mild; 2) normal vision; 3) peripheral early cataract detected by slit-lamp

1型Dent病患者通常表現(xiàn)為LMWP，24 h尿蛋白定量為輕度或中度蛋白尿。近年來有研究發(fā)現(xiàn)，少部分低齡1型Dent病可有腎病水平蛋白尿，但這部分患兒無低蛋白血癥，尿蛋白電泳表現(xiàn)，LMWP和白蛋白均增高[9]。2型Dent病患者不僅有腎臟表現(xiàn)，還可有腎外表現(xiàn)。1型和2型Dent病的癥狀相似，主要靠CLCN5和OCRL基因突變分析來鑒別。2型Dent病患者均有磷酸肌酸激酶和乳酸脫氫酶升高，部分患者還伴腎外癥狀。因此，臨床上對肌酶升高或有腎外癥狀的病例可先行OCRL基因突變分析，而對無腎外癥狀或肌酶升高的病例應先行CLCN5突變分析。

由于局灶節(jié)段腎小球硬化腎病綜合征(FSGS-NS)患兒的近端腎小管功能異常比微小病變腎病綜合征(MCD-NS)更常見。 據(jù)此， 有學者推斷FSGS可繼發(fā)于原發(fā)性近端腎小管功能異?；驌p害[31]。Kaneko等[28]研究也發(fā)現(xiàn)，2型Dent病可引起FSGS。本文患兒腎活檢結(jié)果顯示，電鏡下5個腎小球結(jié)構(gòu)無明顯異常，免疫熒光正常，光鏡下30個腎小球，僅1個腎小球硬化，其他腎小球結(jié)構(gòu)大致正常，腎小管無異常改變。1個硬化腎小球周圍的腎小球結(jié)構(gòu)大致正常，該硬化腎小球較孤立，推測其是生理狀態(tài)下的自然改變，與疾病關系不大。本文患兒腎活檢并未發(fā)現(xiàn)FSGS改變，可能與患兒病程較短有關，不排除進展為FSGS的可能。盡管Dent病患者腎臟病理光鏡下有FSGS改變，但電鏡下僅表現(xiàn)足突部分融合，而不是廣泛融合[9]。因此，F(xiàn)SGS可能不是Dent病特征性的病理改變，而僅僅可能是疾病病程的反映。

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(本文編輯：丁俊杰)

Dent-2 disease in one child and literature review

LIGuo-min1,4,FANGXiao-yan1,4,XUHong1,SHENQian1,SUNLi1,ZHAIYi-hui1,GUOMu-yi2,ANYu3,WUBing-bing3

(1DepartmentofKidneyandRheumatology,Children'sHospitalofFudanUniversity,Shanghai201102; 2DepartmentofPathology,ShanghaiMedicalCollegeofFudanUniversity,Shanghai200023; 3MedicalTranslationalCenterofChildren'sHospitalofFudanUniversity,Shanghai201102,China; 4hasequalcontribution)

Corresponding Author：XU Hong，E-mail：hxu@shmu.edu.cn

ObjectiveTo summarize and review the clinical data of a child with Dent-2 disease so as to improve our understanding to the disease.Methods Clinical data of the case with Dent-2 disease were summarized, including clinical manifestations, laboratory findings, renal pathological changes and family investigation. Mutation analysis inCLCN5 andOCRLgenes was performed by direct sequencing in his family. Mutations ofCLCN5 andOCRLgenes were examined in normal control and related literatures from PubMed were reviewed also.ResultsAge at onset was 6 years old. Proteinuria was the only symptom at onset. The patient had no extrarenal symptoms of Lowe syndrome, such as peripheral cataracts, mental impairment and stunted growth. Laboratory findings showed low-molecular weight proteinuria, hypercalciuria, microscopic hematuria and elevation of creatine kinase/lactate dehydrogenase. Renal pathological change detection showed a minute lesion under light microscope. A novel homozygous c.260delA (p.Q87fs105X) mutation was found in exon 5 ofOCRLgene. No mutations ofCLCN5 gene were detected. Mutation analysis showed his mother carried a c.260delA mutation, but his father did not. This deletion caused a frameshift of amino acids and resulted in a truncated protein (p.Q87fs105X), and was not identified in the 100 healthy, unrelated controls.ConclusionDent-2 disease is an X-linked tubulopathy characterized by low-molecular-weight proteinuria (LMWP), hypercalciuria, nephrocalcinosis, nephrolithiasis, and the development of renal insufficiency. Dent-2 disease can be diagnosed by these characteristics. Lowe syndrome was ruled out in the patient with no peripheral cataracts, mental impairment and stunted growth. Elevation of creatine kinase/lactate dehydrogenase and carrying the homozygous frameshift mutation inOCRLgene support the diagnosis of the patient in the current study. This is the first report of Dent-2 disease caused byOCRLgene mutation in Chinese family.

Dent-2 disease;CLCN5 gene;OCRLgene

復旦大學附屬兒科醫(yī)院人才工程-學科帶頭人(1125)培育計劃項目

1 復旦大學附屬兒科醫(yī)院腎臟風濕科 上海，201102; 2 復旦大學上海醫(yī)學院病理教研室 上海，200032；3 復旦大學附屬兒科醫(yī)院醫(yī)學轉(zhuǎn)化中心 上海，201102；4 共同第一作者

徐虹，E-mail:hxu@shmu.edu.cn

10.3969/j.issn.1673-5501.2014.06.011

2014-10-03

2014-11-20)