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中华眼科医学杂志(电子版)

中华眼科医学杂志(电子版) ›› 2017, Vol. 07 ›› Issue (01) : 18 -24. doi: 10.3877/cma.j.issn.2095-2007.2017.01.004

所属专题： 文献；

论著

利用全外显子组测序法筛选Leber先天性黑矇一家系候选致病基因的实验研究
赵军1,†(), 黄晓生1, 彭诗茗1, 祝天辉1, 贺温玲1, 曾映虾1, 范先明1, 范宝剑2   
  1. 1. 518040　广东省深圳市眼科医院　暨南大学附属深圳眼科医院　深圳眼科学重点实验室
    2. 02114　美国，哈佛医学院附属麻省眼耳医院　眼科基因组学研究所
  • 收稿日期:2017-02-22 出版日期:2017-02-28
  • 通信作者: 赵军
  • 基金资助:
    广东省自然科学基金(S2013010013464); 广东省医学科研基金(A2014518); 深圳市科技计划项目(JCYJ20140415174819509); 深圳市科技计划项目(GJHZ20160229170608241)

Experimental study on using whole exome sequencing method for screening Leber congenital amaurosis in a family of candidate genes

Jun Zhao1,†(), Xiaosheng Huang1, Shiming Peng1, Tianhui Zhu1, Wenling He1, Yingxia Zeng1, Xianming Fan1, Baojian Fan2   

  1. 1. Shenzhen Eye Hospital Affiliated to Jinan University, Shenzhen Key Laboratory of Ophthalmology, Shenzhen, 518040, China
    2. Massachusetts Eye and Ear Infirmary, Harvard Medical School, Ocular Genomics Institute, Boston 02114, United States of America
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赵军, 黄晓生, 彭诗茗, 祝天辉, 贺温玲, 曾映虾, 范先明, 范宝剑. 利用全外显子组测序法筛选Leber先天性黑矇一家系候选致病基因的实验研究[J]. 中华眼科医学杂志(电子版), 2017, 07(01): 18-24.

Jun Zhao, Xiaosheng Huang, Shiming Peng, Tianhui Zhu, Wenling He, Yingxia Zeng, Xianming Fan, Baojian Fan. Experimental study on using whole exome sequencing method for screening Leber congenital amaurosis in a family of candidate genes[J]. Chinese Journal of Ophthalmologic Medicine(Electronic Edition), 2017, 07(01): 18-24.

目的

利用全外显子组测序法筛选Leber先天性黑矇(LCA)一个家系的候选致病基因，为补充或验证LCA致病基因的研究奠定基础。

方法

采用横断面研究方法。收集一个在深圳市眼科医院就诊的中国汉族LCA家系的临床资料。详细询问并记录全部家系成员的疾病史、家族史及婚育史，并对全部家系成员进行全面的身体检查。其中，眼部检查项目包括视力、眼位、眼压、验光、眼球运动情况、眼前段、眼底照相、光学相干断层扫描及视网膜电图检查等。提取先证者、另外1例患者及其父母的静脉血基因组DNA，采用磁珠提取法对DNA进行提纯。使用高通量测序平台对质量检测通过的DNA文库进行测序。在进行生物信息学分析时，对采集的数据行标准信息分析流程处理，同时对数据进行质控检测。采用GATK基因组分析网络工具库检索单核苷酸多态性位点和缺失标记位点的数量。测序结果经生物信息学分析工具SeattleSeq Annotation138注释后，与人类HAPMAP、dbSNP138、Exome Sequencing Project及Exome Aggregation Consortium数据库进行比对。过滤掉已报道的常见变异后，再过滤掉位于非编码区的变异和同义突变，并进一步筛选出该LCA家系的候选致病基因。

结果

该家系成员共有3代16人。其中，3名LCA患者均为第Ⅱ代成员，第Ⅰ代及第Ⅲ代成员均无发病者，符合常染色体隐性遗传规律。经测序及分析后，得到17个LCA的候选致病基因，分别为ACTN1、C1QTNF3、FAN1、MMP28、MYO9B、NAV1、NUP62、PHLDB3、PRDM12、SLC24A4、ST3GAL3、TCIRG1、TP53、USP54、YIF1B、ZDHHC17及ZNF107。这些基因均与目前已报道的24个LCA相关的致病基因不同。

结论

对该家系的DNA进行分析后，发现了17个新的LCA候选致病基因，可为进一步明确该家系LCA致病基因的研究奠定了基础。

关键词: Leber先天性黑矇, 家系, 全外显子组测序, 候选致病基因
Objective

To screen a Leber congenital amaurosis (LCA) pedigree for candidate genes and to provide the research foundation for identification of the pathogenic gene in this pedigree.

Methods

A cross-sectional study was designed. Clinical data of a Chinese family with LCA collected from Chinese Han population in Shenzhen Eye Hospital. A detailed inquiry and record all the family members with disease history, family history and obstetrical history, and conduct a comprehensive physical examination of all family members. Among them, the eye examination including visual acuity, eye position, eye movement, anterior segment examination, fundus examination, non-contact intraocular pressure, refraction, fundus photography, optical coherence tomography and electroretinogram. Venous blood genomic DNA was extracted from proband, one patients and their parents. The DNA was purified by magnetic beads extraction method. Sequencing of DNA libraries for quality detection using high-throughput sequencing platforms. Carries on the bioinformatics analysis, collects the data to carry on the standard information analysis processing, concurrently carries on the quality control examination to the data. The number of single nucleotide polymorphism and the number of deletion markers were retrieved using GATK genomic analysis network tool library. The sequencing results were annotated with SeattleSeq Annotation138, and compared with public databases of human HAPMAP, dbSNP138, Exome Sequencing Project and Exome Aggregation Consortium. The candidate genes were identified after common variants, non-coding variants and synonymous mutations were filtered out.

Results

The LCA pedigree had 3 generations and 16 family members. All patients with LCA were only in the second generation, in accordance with an autosomal recessive inheritance pattern. Seventeen candidate genes were identified, including ACTN1, C1QTNF3, FAN1, MMP28, MYO9B, NAV1, NUP62, PHLDB3, PRDM12, SLC24A4, ST3GAL3, TCIRG1, TP53, USP54, YIF1B, ZDHHC17 and ZNF107. None of these candidate genes were previously related to LCA.

Conclusions

After analyzing the DNA of the family, seventeen novel candidate genes were identified for this LCA pedigree. Our findings provided a basis for identification of the pathogenic gene in this LCA family.

Key words: Leber congenital amaurosis, Pedigree, Whole exome sequencing, Candidate pathogenic gene
图1 Leber先天性黑矇的一家系图谱　■表示LCA患者，□○均表示正常者，↗表示先证者
图2 Leber先天性黑矇患者的眼底彩照图像　A、B图分别表示Ⅱ－3的右眼、左眼，C、D图分别表示Ⅱ－6的右眼、左眼，E、F图分别表示Ⅱ－9的右眼、左眼；图中全部患者眼底呈现视网膜血管变细及视网膜散在骨细胞样及椒盐样色素沉着，C、D图还存在脉络膜萎缩性改变
图3 Leber先天性黑矇患者的视网膜骨细胞样色素沉着区光学相干断层扫描图像　A图表示视网膜扫描部位；B图表示光学相干断层扫描图像，箭头表示移行增殖的色素上皮
图4 Leber先天性黑矇患者Ⅱ－9的视网膜电图检测图像　A图显示右眼视杆细胞反应，B图显示左眼视杆细胞反应，C图显示右眼标准混合视锥杆细胞反应，D图显示左眼标准混合视锥杆细胞反应，E图显示右眼暗适应震荡电位，F图显示左眼暗适应震荡电位，G图显示右眼视锥细胞反应，H图显示左眼视锥细胞反应，I图显示右眼明适应30闪烁光反应，J图显示左眼明适应30闪烁光反应
图5 4个样本的DNA电泳图　S泳道表示标准品，M泳道表示参照标记物(Marker)，1～4泳道分别对应Ⅰ－1、Ⅰ－2、Ⅱ－6及Ⅱ－9的DNA样品，bp表示碱基对数目
[1]
Koenekoop RK. Mutations in NMNAT1 cause Leber congenital amaurosis and identify a new disease pathway for retinal degeneration[J]. Nat Genet, 2012, 44(9)：1035-1039.
[2]
Koenekoop RK. An overview of Leber congenital amaurosis: a model to understand human retinal development[J]. Surv Ophthalmol, 2004, 49(4)：379-398.
[3]
单海冬,赵培泉. Leber先天性黑矇基因研究进展[J]. 国际眼科纵览, 2005, 29(2)：113-116.
[4]
罗东凤,王少元. 全基因组外显子测序及其在遗传病研究中的应用[J]. 国际遗传学杂志, 2012, 35(3)：173-177.
[5]
Stone EM. Leber congenital amaurosis－a model for efficient genetic testing of heterogeneous disorders: LXIV Edward Jackson Memorial Lecture[J]. Am J Ophthalmol, 2007, 144(6)：791-811.
[6]
Schappert－Kimmijser J,Henkes HE,Van DBJ. Amaurosis congenita (Leber)[J]. AMA Arch Ophthalmol, 1959, 137(2)：211-218.
[7]
Lotery AJ,Namperumalsamy P,Jacobson SG, et al. Mutation analysis of 3 genes in patients with Leber congenital amaurosis[J]. Arch Ophthalmol, 2000, 118(4)：537-543.
[8]
Vaizey MJ,Sanders MD,Wybar KC, et al. Neurological abnormalities in congenital amaurosis of Leber. Review of 30 cases[J]. Arch Dis Child, 1977, 52(5)：399-402.
[9]
Dekaban AS. Mental Retardation and Neurologic Involvement in Patients with Congenital Retinal Blindness[J]. Dev Med Child Neurol, 1972, 14(4)：436-444.
[10]
Fazzi E,Rossi M,Signorini S, el al. Leber′s congenital amaurosis: is there an autistic component? [J]. Dev Med Child Neurol, 2007, 49(7)：503-507.
[11]
Rogers SJ,Newhartlarson S. Characteristics of infantile autism in five children with Leber′s congenital amaurosis[J]. Dev Med Child Neurol, 1989, 31(5)：598-608.
[12]
Zuhlke C,Stell A,Kasmann－Kellner B. Genetics of oculocutaneous albinism[J]. Ophthalmologe, 2007, 104(8)：674-680.
[13]
Cibis GW,Fitzgerald KM. Optic nerve hypoplasia in association with brain anomalies and an abnormal electrorentinogram[J]. Doc Ophthalmol, 1994, 86(1)：11-22.
[14]
Varsanyi B,Wissinger B,Kohl S, el al. Clinical and genetic features of Hungarian achromatopsia patients[J]. Mol Vis, 2005, 11(11)：996-1001.
[15]
Wissinger B,Gamer D,Jagle H, el al. CNGA3 mutations in hereditary cone photoreceptor disorders[J]. Am J Hum Genet, 2001, 69(4)：722-737.
[16]
Weleber RG,Tongue AC. Congenital stationary night blindness presenting as Leber′s congenital amaurosis[J]. Arch Ophthalmol, 1987, 105(3)：360-365.
[17]
Collin GB,Marshall JD,Ikeda A, et al. Mutations in ALMS1 cause obesity, type 2 diabetes and neurosensory degeneration in Alstorm syndrome[J]. Nat Genet, 2002, 31(1)：74-78.
[18]
Vesa J,Hellsten E,Verkruyse LA, el al. Mutations in the palmitoyl protein thiosesterase gene causing infantile neuronal ceroid lipofuscinosis[J]. Nature, 1995, 376：584-587.
[19]
Choi M,Scholl UI,Ji W, et al. Genetic diagnosis by whole exome capture and massively parallel DNA sequencing[J]. Proc Natl Acad Sci, 2009, 106(45)：19096-19101.
[20]
Maguire AM,Simonelli F,Pierce EA, et al. Safety and efficacy of gene transfer for Leber′s congenital amaurosis[J]. N Engl J Med, 2008, 358(21)：2240-2248.
[21]
Bainbridge JW,Smith AJ,Barker SS, et al. Effect of gene therapy on visual function in Leber′s congenital amaurosis[J]. N Engl J Med, 2008, 358(21)：2231-2239.
[22]
Cideciyan AV,Hauswirth WW,Aleman TS, et al. Vision 1 year after gene therapy for Leber′s congenital amaurosis[J]. N Engl J Med, 2009, 361(7)：725-727.
[23]
Simonelli F,Maguire AM,Testa F, et al. Gene therapy for Leber′s congenital amaurosis is safe and effective through 1.5 years after vector administration[J]. Mol Ther, 2010, 18(3)：643-650.
[24]
Wang S,Zhang Q,Zhang X, et al. Clinical and genetic characteristics of Leber congenital amaurosis with novel mutations in known genes based on a Chinese eastern coast Han population[J]. Graefes Arch Clin Exp Ophthalmol, 2016, 254(11)：2227-2238.
[25]
Chen Y,Zhang Q,Shen T, et al. Comprehensive mutation analysis by whole－exome sequencing in 41 Chinese families with Leber congenital amaurosis[J]. Invest Ophthalmol Vis Sci, 2013, 54(6)：4351-4357.
[26]
Lin Li,Shiqiang Li,Xiaoyun Jia, et al. Detection of variants in 15 genes in 87 unrelated Chinese patients with Leber congenital amaurosis[J]. PLoS One, 2011, 6(5)：e19458.
[27]
Wang H,Wang X,Zou X, et al. Comprehensive molecular diagnosis of a large chinese Leber congenital amaurosis cohort[J]. Invest Ophthalmol Vis Sci, 2015, 56(6)：3642-3655.
[28]
Chung DC,Traboulsi EI. Leber congenital amaurosis: clinical correlations with genotypes, gene therapy trials update, and future directions[J]. JAAPOS, 2009, 13(6)：587-592.
[29]
Acland GM,Aguirre GD,Ray J, et al. Gene therapy restores vision in a canine model of childhood blindness[J]. Nat Genet, 2001, 28(1)：92-95.
[30]
Chacon－Camacho OF,Zenteno JC. Review and update on the molecular basis of Leber congenital amaurosis[J]. World J Clin Cases, 2015, 3(2)：112-124.
[31]
Perrault I,Rozet JM,Calvas P, et al. Retinal－specific guanylate cyclase gene mutations in Leber′s congenital amaurosis[J]. Nat Genet, 1996, 14(4)：461-464.
[32]
Minegishi Y,Sheng X,Yoshitake K, et al. CCT2 mutations evoke Leber congenital amaurosis due to chaperone complex instability[J]. Sci Rep, 2016, 6(2016)：33742.
[33]
Hollander AID,Roepman R,Koenekoop RK, et al. Leber congenital amaurosis: genes, proteins and disease mechanisms [J]. Prog Retin Eye Res, 2008, 27(4)：391-419.
[34]
Hollander AID,Black A,Bennett J, et al. Lighting a candle in the dark: advances in genetics and gene therapy of recessive retinal dystrophies[J]. J Clin Inves, 2012, 120(3)：3042-3053.
[35]
Wang SY,Zhang Q,Zhang X, et al. Comprehensive analysis of genetic variations in strictly－defined Leber congenital amaurosis with whole－exome sequencing in Chinese[J]. Int J Ophthalmol.2016, 9(9)：1260-1264.
[36]
Clellan JM,King MC. Genetic heterogeneity in human disease[J]. Cell, 2010, 141(2)：210-217.
[37]
Mcclellan JM,Susser E,King MC. Schizophrenia: a common disease caused by multiple rare alleles[J]. Br J Psychiatry, 2007, 190(8)：194-199.
[38]
Wang H,Chen X,Dudinsky L, et al. Exome capture sequencing identifies a novel mutations in BBS4[J]. Mol Vis, 2011, 17(379-82)：3529-3540.
[39]
王乐今. Leber眼病与先天性白内障的临床与基础研究[D]. 武汉：华中科技大学，2008.
[40]
Chiang PW,Wang J,Chen Y, et al. Exome sequencing identifies NMNAT1 mutations as a cause of Leber congenital amaurosis[J]. Nat Genet, 2012, 44(9)：972-974.
[41]
Wang X,Wang H,Sun V, et al. Comprehensive molecular diagnosis of 179 Leber congenital amaurosis and juvenile retinitis pigmentosa patients by targeted next generation sequencing[J]. J Med Genet, 2013, 50(10)：674-688.
[42]
Wang X,Wang H,Cao M, et al. Whole－exome sequencing identifies ALMS1, IQCB1, CNGA3, and MYO7A mutations in patients with Leber congenital amaurosis[J]. Hum Mutat, 2011, 32(12)：1450-1459.
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