129个常染色体显性遗传性多囊肾病家系的致病基因检测及分析

引用本文

卜磊, 马熠熠, 梅长林. 129个常染色体显性遗传性多囊肾病家系的致病基因检测及分析[J]. 第二军医大学学报, 2019, 40(1): 7-13

BU Lei, MA Yi-yi, MEI Chang-lin. Detection and analysis of virulence genes for autosomal dominant polycystic kidney disease in 129 Chinese families[J]. Academic Journal of Second Military Medical University, 2019, 40(1): 7-13 (in Chinese with English abstract)

129个常染色体显性遗传性多囊肾病家系的致病基因检测及分析

卜磊, 马熠熠, 梅长林

海军军医大学(第二军医大学)长征医院肾内科, 解放军肾脏病研究所, 上海 200003

收稿日期: 2018-10-08 接受日期: 2018-12-14

基金项目: 国家自然科学基金（81670612），上海市加强公共卫生体系建设三年行动计划（SCREENING STUDY GWIV-18），山东省自然科学基金（ZR2014HL024）.

作者简介: 卜磊, 博士生, 主治医师.现在山东省临沂市人民医院工作.E-mail:bulei003@126.com

通信作者(Corresponding author): 梅长林, Tel:021-81885391, E-mail:chlmei1954@126.com

摘要: 目的探讨中国常染色体显性遗传性多囊肾病（ADPKD）患者多囊肾病1型致病基因（PKD1）和多囊肾病2型致病基因（PKD2）的突变类型。方法采用长链PCR和高通量测序方法对129个ADPKD家系的PKD1和PKD2基因进行突变分析，并用双脱氧链终止法测序技术对阳性突变进行验证。结果在129个ADPKD遗传家系中共检测到116个家系存在PKD1或PKD2基因的118个突变位点，检出率为89.9%（116/129）。PKD1和PKD2的突变率分别为92.2%（107/116）和8.6%（10/116）。在这118个突变位点中，80个（67.8%）为新突变，38个（32.2%）为已知突变；109个位于PKD1（33个已知突变和76个新突变），9个位于PKD2（5个已知突变和4个新突变）。结论新发现的PKD1和PKD2突变位点将有助于ADPKD患者的早期诊断和预后预测，并为临床干预提供基本的遗传信息。

关键词: 高通量测序常染色体显性多囊肾多囊肾病致病基因突变

Detection and analysis of virulence genes for autosomal dominant polycystic kidney disease in 129 Chinese families

BU Lei, MA Yi-yi, MEI Chang-lin

Department of Nephrology, Kidney Institute of PLA, Changzheng Hospital, Naval Medical University(Second Military Medical University), PLA Institute of Nephrology, Shanghai 200003, China

Supported by National Natural Science Foundation of China (81670612), Shanghai 3-Year Plan for Public Health System Construction (SCREENING STUDY GWIV-18), and Natural Science Foundation of Shandong Province (ZR2014HL024).

Abstract: Objective To investigate the mutation types of polycystic kidney disease 1 gene (PKD1) and polycystic kidney disease 2 gene (PKD2) in Chinese patients with autosomal dominant polycystic kidney disease (ADPKD). Methods The mutations of PKD1 and PKD2 in 129 inherited ADPKD families were analyzed by long PCR and highthroughput sequencing. The positive mutation was verified by Sanger sequencing method. Results A total of 118 mutation sites of PKD1 or PKD2 in 116 inherited ADPKD families were detected from 129 families, with the detection rate being 89.9% (116/129). The mutation rates of PKD1 and PKD2 were 92.2% (107/116) and 8.6% (10/116), respectively. Of the 118 mutation sites, 80 (67.8%) were new mutations and 38 (32.2%) were known mutations; and 109 mutation sites were located in PKD1 (33 known mutations and 76 new mutations) and 9 in PKD2 (5 known mutations and 4 new mutations). Conclusion The newly discovered PKD1 and PKD2 mutations may contribute to early diagnosis and prognosis prediction of ADPKD patients, and may provide basic genetic information for clinical intervention.

Key words: high-throughput sequencing autosomal dominant polycystic kidney polycystic kidney disease gene mutation

常染色体显性遗传性多囊肾病（autosomal dominant polycystic kidney disease，ADPKD）是人类最常见的遗传性疾病，其特征是双侧、多发并逐渐增长的液性囊肿压迫及破坏周围正常肾组织、引起肾脏不可逆性功能丧失，约占终末期肾病（end-stage renal disease，ESRD）的10%^[1]。ADPKD表型由位于16p13.3的多囊肾病1型致病基因（polycystic kidney disease 1 gene，PKD1）和位于4q21的多囊肾病2型致病基因（polycystic kidney disease 2 gene，PKD2）突变引起^[2-3]，它们分别编码多囊蛋白1（polycystin 1，PC1）和多囊蛋白2（polycystin 2，PC2）。其中约85%~90%的患者为PKD1基因突变导致，约10%~15%为PKD2基因突变导致，PKD1突变的患者比PKD2突变者显现出更严重的临床表现和更差的预后，而且就致病突变的类型来说具有截短突变的患者比非截短突变的患者具有更严重的临床表现，这表明遗传因素在ADPKD患者的预后方面起着重要作用^[4-5]。目前，ADPKD的临床诊断主要基于使用年龄相关的囊肿数目的肾成像技术^[6-7]，对于没有阳性家族史的年轻患者或PKD2基因突变的患者，使用目前的标准很难提供确定的诊断。因此，全面了解PKD1和PKD2基因的突变信息是ADPKD早期诊断和早期干预的关键。

PKD1基因由46个外显子组成，编码序列为12 912个碱基；PKD2基因由15个外显子组成，编码序列为2 907个碱基^[8]。ADPKD的致病以点突变致病为主，并且没有明显的突变热点或者热区，整个基因外显子区及内含子剪切位点附近均可能发生突变而致病，PKD1和PKD2基因序列都是高度可变的^[9]。迄今为止，在ADPKD突变数据库（Autosomal Dominant Polycystic Kidney Disease Mutation Database，PKDB；http://pkdb.mayo.edu/）中已经报道了共1 273种致病性PKD1突变和202种致病性PKD2突变。然而，大多数突变只在单一的家系中出现1次，重复的突变位点仅占30%^[5]。因此，有必要调查更多的家系确认这些已知的突变，并扩展新发现的突变。此外，在PKDB中列出的突变是从西方人群的研究中获得的，来自亚洲人群的突变数据有助于确认和拓宽这个数据库。本研究从129个中国ADPKD家系中筛选突变位点，通过与PKDB对比，确定已知的突变，发现未知的突变，为进一步深入了解ADPKD的致病基因突变情况提供有价值的信息。

1 资料和方法 1.1 临床样本收集

血液样本来自129个家系（均为中国汉族人），每个家系至少包括1个患病个体。患病个体为2016年1月至2018年8月在海军军医大学（第二军医大学）长征医院肾内科确诊为ADPKD的患者。ADPKD诊断标准：腹部超声或计算机断层扫描（computed tomography，CT）检查发现双肾囊肿数≥5，有明确的多囊肾病家族史^{[6, 10]}。该研究通过海军军医大学（第二军医大学）长征医院伦理委员会审批，所有入选对象均签署知情同意书。每例入选对象取5 mL外周血，使用DNA提取试剂盒（德国QIAGEN公司）从淋巴细胞中提取基因组DNA。

1.2 长链PCR-高通量测序法检测突变位点^[11]

采用长链PCR扩增PKD1和PKD2外显子以及外显子和内含子交界的非翻译区（untranslated region，UTR）。使用长链PCR扩增的产物构建文库，上机进行高通量测序，对发现的突变位点采用一代测序（以基因组DNA为模板重新设计引物）进行验证。利用TMAP软件（Ion Torrent测序平台自带软件，美国Thermo Fisher公司）将所有测序读数映射到PKD1和PKD2参考基因组（PKD1：NM_001009944.2；PKD2：NM_000297.2）。采用人类基因组变异协会（Human Genome Variation Society，HGVS）推荐的标准命名法（http://www.hgvs.org/）命名本研究中鉴定的突变。使用dbSNP 138、1000 Genomes Project、NHLBI GO Exome Sequencing Project（ESP，http://esp.gs.washington.edu）、HapMap和PVFD（正常人群中的变异频率）数据库剔除非致病性突变。根据人类基因突变数据库（http://www.hgmd.cf.ac.uk）和PKDB，将剩余的突变分为已知突变和新突变。对未检测出PKD1和PKD2突变的家系采用同样的方法检测常染色体隐性遗传多囊肾病致病基因——多囊肾/多囊肝病变1基因（polycystic kidney and hepatic disease 1，PKHD1）。

1.3 突变的致病性评价

首先，采用人类基因突变数据库和PKDB筛选出已知的致病性突变。然后，对先前未报道的可能致病的突变，使用SIFT（https://sift.bii.a-star.edu.sg/）、MutationAssessor（http://mutationassessor.org/）、PolyPhen-2（http://genetics.bwh.harvard.edu/pph2/）等生物医学软件和美国医学遗传学与基因组学学会（American College of Medical Genetics，ACMG）序列变异的解释标准指南评估这些突变的致病潜力。最后，对于评估的致病突变，通过一代测序检测直系亲属的DNA序列进行家系共分离分析验证。

1.4 突变域分析

使用多个域数据库来分析PC1和PC2的突变域，包括简单模块体系结构研究工具（http://smart.embl-heidelberg.de）、Pfam（http://pfam.xfam.org/）和PROSITE（http://prospace.expac.org/）。

2 结果 2.1 总体突变情况

本研究共调查了129个家系的突变特征。其中116个家系共检测到107个家系PKD1突变和10个家系PKD2突变，总突变率为116/129（89.9%）。PKD1和PKD2的突变率分别为92.2%（107/116）和8.6%（10/116）。有4个家系发现PKD1等位基因纯合子突变（等位基因各有1个致病突变），为PKD1纯合子突变致病，有1个家系检测出PKD1和PKD2均发生突变，2个突变位点均有致病性。116个家系共检测到118个致病突变位点，其中80个（67.8%）为新突变，38个（32.2%）为已知突变；109个突变（33个已知突变和76个新突变）位于PKD1，9个突变（5个已知突变和4个新突变）位于PKD2。

2.2 明确的致病突变

将检测到的突变与PKDB比对确定了38个已知致病性突变，其中33个位于PKD1，5个位于PKD2（表 1）。在33个已知PKD1突变中，18个（54.5%）为无义突变，4个（12.1%）为移码突变，9个（27.3%）为错义突变，2个（6.1%）为剪接位点突变；5个已知PKD2突变均为无义突变。采用生物医学软件（SIFT、MutationAssessor、PolyPhen-2）对新发现的突变进行评估，根据ACMG原则筛选出可能的致病突变，并在患者家系中进行共分离分析验证，发现80个新发现突变（表 2）。其中76个PKD1新发现突变中有10个（13.2%）为无义突变，33个（43.4%）为移码突变，5个（6.6%）为整码突变，20个（26.3%）为错义突变，7个（9.2%）为剪接位点突变，1个（1.3%）为内含子变异；4个PKD2新发现突变分别为内含子变异、无义突变、错义突变和移码突变。

表 1 129个ADPKD家系中发现的38个PKD1和PKD2已知致病突变 Tab 1 Thirty-eight known pathogenic mutations of PKD1 and PKD2 from 129 ADPKD families

Gene	Position	cDNA change	Amino acid variant	Mutation type
PKD1	chr16:2160153_2160154	c.5014_5015del	p.Arg1672Glyfs^*98	Frameshift mutation
PKD1	chr16:2142493	c.11257C＞T	p.Arg3753Trp	Missense mutation
PKD1	chr16:2161446	c.3722T＞A	p.Ile1241Asn	Missense mutation
PKD1	chr16:2168821	c.385T＞C	p.Cys129Arg	Missense mutation
PKD1	chr16:2158883	c.6285C＞A	p.Asp2095Glu	Missense mutation
PKD1	chr16:2158773	c.6395T＞G	p.Phe2132Cys	Missense mutation
PKD1	chr16:2158336	c.6832G＞A	p.Gly2278Arg	Missense mutation
PKD1	chr16:2156405	c.7483T＞C	p.Cys2495Arg	Missense mutation
PKD1	chr16:2153747	c.8311G＞A	p.Glu2771Lys	Missense mutation
PKD1	chr16:2152079	c.9380G＞C	p.Gly3127Ala	Missense mutation
PKD1	chr16:2166120	c.1723-1G＞A	No change	Splice site mutation
PKD1	chr16:2143104	IVS37-10C＞A	p.Arg3672fs1X	Splice site mutation
PKD1	chr16:2167942	c.1051C＞T	p.Gln351Ter	Nonsense mutation
PKD1	chr16:2141004	c.11884C＞T	p.Gln3962Ter	Nonsense mutation
PKD1	chr16:2140953	c.11935C＞T	p.Gln3979Ter	Nonsense mutation
PKD1	chr16:2140803	c.12010C＞T	p.Gln4004Ter	Nonsense mutation
PKD1	chr16:2140777	c.12036G＞A	p.Trp4012Ter	Nonsense mutation
PKD1	chr16:2140689	c.12124C＞T	p.Gln4042^*	Nonsense mutation
PKD1	chr16:2140482	c.12248C＞A	p.Ser4083^*	Nonsense mutation
PKD1	chr16:2139967	c.12673C＞T	p.Gln4225Ter	Nonsense mutation
PKD1	chr16:2139949	c.12691C＞T	p.Gln4231^*	Nonsense mutation
PKD1	chr16:2165489	c.1987C＞T	p.Gln663Ter	Nonsense mutation
PKD1	chr16:2160862	c.4306C＞T	p.Arg1436Ter	Nonsense mutation
PKD1	chr16:2160371	c.4797C＞A	p.Tyr1599Ter	Nonsense mutation
PKD1	chr16:2168427	c.566C＞A	p.Ser189^*	Nonsense mutation
PKD1	chr16:2158696	c.6472C＞T	p.Gln2158^*	Nonsense mutation
PKD1	chr16:2156811	c.7204C＞T	p.Arg2402Ter	Nonsense mutation
PKD1	chr16:2155424	c.7915C＞T	p.Arg2639X	Nonsense mutation
PKD1	chr16:2152610	c.8972dupA	p.Tyr2991Ter	Nonsense mutation
PKD1	chr16:2152581	c.9002G＞A	p.Trp3001^*	Nonsense mutation
PKD1	chr16:2143663_2143666	c.10895_10898delAGAG	p.Glu3632Alafs^*4	Frameshift mutation
PKD1	chr16:2161098	c.4070delT	p.Leu1357ArgfsTer9	Frameshift mutation
PKD1	chr16:2158439	c.6727_6728delCA	p.Gln2243Glufs^*18	Frameshift mutation
PKD2	chr4:88986631	c.2224C＞T	p.Arg742Ter	Nonsense mutation
PKD2	chr4:88989098	c.2407C＞T	p.Arg803^*	Nonsense mutation
PKD2	chr4:88940695	c.681C＞A	p.Tyr227^*	Nonsense mutation
PKD2	chr4:88959475	c.916C＞T	p.Arg306^*	Nonsense mutation
PKD2	chr4:88967864	c.1390C＞T	p.Arg464Ter	Nonsense mutation
ADPKD: Autosomal dominant polycystic kidney disease; PKD1: Polycystic kidney disease 1 gene; PKD2: Polycystic kidney disease 2 gene

表 1 129个ADPKD家系中发现的38个PKD1和PKD2已知致病突变 Tab 1 Thirty-eight known pathogenic mutations of PKD1 and PKD2 from 129 ADPKD families

表 2 129个ADPKD家系中发现的80个PKD1和PKD2新发现致病突变 Tab 2 Eighty new pathogenic mutations of PKD1 and PKD2 from 129 ADPKD families

Gene	Position	cDNA change	Amino acid variant	Mutation type
PKD1	chr16:2156251	c.7544G＞C	p.Arg2515Pro	Missense mutation
PKD1	chr16:2152447	c.9136C＞T	p.Arg3046Cys	Missense mutation
PKD1	chr16:2142955	c.11156G＞A	p.Arg3719Gln	Missense mutation
PKD1	chr16:2166025	c.1817G＞C	p.Arg606Pro	Missense mutation
PKD1	chr16:2166011	c.1831C＞T	p.Arg611Trp	Missense mutation
PKD1	chr16:2152906	c.8857A＞G	p.Asn2953Asp	Missense mutation
PKD1	chr16:2150268	c.9611A＞T	p.Asp3204Val	Missense mutation
PKD1	chr16:2168353	c.640T＞G	p.Cys214Gly	Missense mutation
PKD1	chr16:2156898	c.7117T＞G	p.Cys2373Gly	Missense mutation
PKD1	chr16:2166875	c.1565G＞C	p.Cys522Ser	Missense mutation
PKD1	chr16:2147950	c.10086G＞T	p.Gln3362His	Missense mutation
PKD1	chr16:2143912	c.10721G＞T	p.Gly3574Val	Missense mutation
PKD1	chr16:2156143	c.7652T＞C	p.Leu2551Pro	Missense mutation
PKD1	chr16:2155336	c.8003T＞C	p.Leu2668Pro	Missense mutation
PKD1	chr16:2150223	c.9656T＞C	p.Leu3219Pro	Missense mutation
PKD1	chr16:2164844	c.2180T＞C	p.Leu727Pro	Missense mutation
PKD1	chr16:2168826	c.380T＞C	p.Phe127Ser	Missense mutation
PKD1	chr16:2160131	c.5037C＞A	p.Ser1679Arg	Missense mutation
PKD1	chr16:2153600	c.8458A＞G	p.Ser2820Gly	Missense mutation
PKD1	chr16:2141595	c.11541C＞A	p.Ser3847Arg	Missense mutation
PKD1	chr16:2147987	c.10051-2A＞C	No change	Splice site mutation
PKD1	chr16:2167489	c.1385+1G＞A	No change	Splice site mutation
PKD1	chr16:2163296	c.2854-3C＞G	No change	Splice site mutation
PKD1	chr16:2167063	c.1386-9C＞A	No change	Splice site mutation
PKD1	chr16:2140284	IVS45+1delG	No change	Splice site mutation
PKD1	chr16:2140810	c.12004-1G＞C	No change	Splice site mutation
PKD1	chr16:2156805	c.7209+1	No change	Splice site mutation
PKD1	chr16:2143742	c.10822-3C＞G	No change	Intronic mutation
PKD1	chr16:2158936	c.6232C＞T	p.Gln2078Ter	Nonsense mutation
PKD1	chr16:2161654	c.3514C＞T	p.Gln1172^*	Nonsense mutation
PKD1	chr16:2161648	c.3520C＞T	p.Gln1174Ter	Nonsense mutation
PKD1	chr16:2168776	c.430C＞T	p.Gln144^*	Nonsense mutation
PKD1	chr16:2159295	c.5873G＞A	p.Trp1958^*	Nonsense mutation
PKD1	chr16:2143909	c.10724G＞A	p.Trp3575^*	Nonsense mutation
PKD1	chr16:2141123	c.11765G＞A	p.Trp3922Ter	Nonsense mutation
PKD1	chr16:2158824_2158827	c.6341_6344delACCT	p.Tyr2114Ter	Nonsense mutation
PKD1	chr16:2168000	c.993T＞A	p.Tyr331^*	Nonsense mutation
PKD1	chr16:2142548	c.11202C＞A	p.Tyr3734stop	Nonsense mutation
PKD1	chr16:2141113_2141116	c.11772_11775dupGGAA	p.3926fs	Frameshift mutation
PKD1	chr16:2161566	c.3597_3601delTGCGG	p.Ala1200Glyfs^*9	Frameshift mutation
PKD1	chr16:2156141	c.7654delG	p.Ala2552Profs^*68	Frameshift mutation
PKD1	chr16:2149883	c.9901dupG	p.Ala3301GlyfsTer89	Frameshift mutation
PKD1	chr16:2167593	c.1282delG	p.Ala428ProfsTer37	Frameshift mutation
PKD1	chr16:2185566	c.118_124dupGGCCCAG	p.Ala42Glyfs^*74	Frameshift mutation
PKD1	chr16:2185681	c.10delG	p.Ala4Profs^*69	Frameshift mutation
PKD1	chr16:215857	c.6597delG	p.Arg2200Alafs^*12	Frameshift mutation
PKD1	chr16:2167794	c.1198delC	p.Arg400Glufs^*65	Frameshift mutation
PKD1	chr16:2159987	c.5179_5180dupCC	p.Asn1728ArgfsTer32	Frameshift mutation
PKD1	chr16:2150203	c.9666_9675dup	p.Asn3226Aspfs^*30	Frameshift mutation
PKD1	chr16:2161607	c.3553_3558delinsTGCACCTA	p.Gly1185CysfsTer14	Frameshift mutation
PKD1	chr16:2185615	c.74_75delGCinsT	p.Gly25Valfs^*48	Frameshift mutation
PKD1	chr16:2160224	c.4943delA	p.His1648Profs^*74	Frameshift mutation
PKD1	chr16:2152607	c.8974delCA	p.His2992Serfs^*76	Frameshift mutation
PKD1	chr16:2139909	c.12730delC	p.His4244Thrfs^*114	Frameshift mutation
PKD1	chr16:2158431_2158434	c.6734_6737delTCCA	p.Ile2245Argfs^*4	Frameshift mutation
PKD1	chr16:2156921	c.7087_7093dupGTGCCCA	p.Ile2365Serfs^*57	Frameshift mutation
PKD1	chr16:2156812	c.7199_7202dupCCAA	p.Lys2401fs^*20	Frameshift mutation
PKD1	chr16:2152418	c.9164delT	p.Leu3055Profs^*19	Frameshift mutation
PKD1	chr16:2141440	c.11695dupC	p.Leu3899Profs^*62	Frameshift mutation
PKD1	chr16:2140566	c.12163delC	p.Leu4055SerfsTer143	Frameshift mutation
PKD1	chr16:2143071_2143072	c.11039_11040	p.Phe3680Serfs^*41	Frameshift mutation
PKD1	chr16:2159810	c.5357delC	p.Pro1786Argfs16X	Frameshift mutation
PKD1	chr16:2156925	c.7088_7089dup	p.Pro2364CysfsTer19	Frameshift mutation
PKD1	chr16:2141559	c.11576delC	p.Pro3859ArgfsTer86	Frameshift mutation
PKD1	chr16:2140531	c.12197_12198dupGC	p.Pro4132067AlafsTer	Frameshift mutation
PKD1	chr16:2147890	c.10145delC	p.r3382Serfs^*15	Frameshift mutation
PKD1	chr16:2156472	c.7415dupT	p.Ser2475LeufsTer26	Frameshift mutation
PKD1	chr16:2141442	c.11692_11693delTC	p.Ser3898Alafs^*62	Frameshift mutation
PKD1	chr16:2160372	c.4795dupT	p.Tyr1599Leufs^*16	Frameshift mutation
PKD1	chr16:2160630	c.4537dupG	p.Val1513Glyfs^*10	Frameshift mutation
PKD1	chr16:2163281	c.2865dupC	p.Val956Argfs^*145	Frameshift mutation
PKD1	chr16:2155902_2155904	c.7825_7827delATC	p.2608delIle	Codon mutation
PKD1	chr16:2140322	c.12399_12407del	p.Phe4133_Arg4136delinsLeu	Codon mutation
PKD1	chr16:2153731	c.8318_8326delCCCTGACGC	p.Pro2773_Thr2775del	Codon mutation
PKD1	chr16:2154287_2164295	c.2729_2723del	p.Asp910_Val912del	Codon mutation
PKD1	chr16:2162371_2162379	c.3254_3263delinsG	p.Val1085_Glu1088delinsGly	Codon mutation
PKD2	chr4:88989186	c.2495G＞C	p.Ser832Thr	Missense mutation
PKD2	chr4:88964368	c.1095_16_1095-8del9	No change	Intronic mutation
PKD2	chr4:88989203	c.2512G＞T	p.Glu838Ter	Nonsense mutation
PKD2	chr4:88959649	c.1092delC	p.A365fs^*10	Frameshift mutation
ADPKD: Autosomal dominant polycystic kidney disease; PKD1: Polycystic kidney disease 1 gene; PKD2: Polycystic kidney disease 2 gene

表 2 129个ADPKD家系中发现的80个PKD1和PKD2新发现致病突变 Tab 2 Eighty new pathogenic mutations of PKD1 and PKD2 from 129 ADPKD families

2.3 新生突变

对个体而言，基因突变既可以由其父母遗传而来，也可以后天获得，后天获得的突变称为新生突变。本研究发现了2个PKD1的新生突变（c.11257C＞T和c.1987C＞T），分别为错义突变和无义突变，2个突变均为已知致病性突变。

2.4 PKHD1的检测

在13个未检测出PKD1和PKD2突变的家系中，检测出3个个体存在PKHD1等位基因的纯合子突变，确定此3个个体为常染色体隐性遗传多囊肾病患者。

3 讨论

既往已有关于中国ADPKD患者中PKD1和PKD2突变分析的报道^[12-16]，但ADPKD家系的数量有限，而且在这些研究中缺乏家系分析，限制了对可能致病突变的验证。本研究共纳入129个ADPKD家系，共检测到118个PKD1和PKD2突变，并对所有发现的突变进行家系分析及验证，确定了它们的致病性。

在本研究中，PKD1和PKD2突变的总检出率为89.9%（116/129），这比中国人群既往研究报道的52.3%~85.8%略高^[12-16]。13例多囊肾患者未发现PKD1和PKD2突变，而临床表现符合ADPKD的诊断标准，在这13例患者中检测PKHD1，发现3例患者为常染色体隐性遗传多囊肾病。然而仍有10例患者没有发现PKD1、PKD2及PKHD1的突变。类似现象既往也有报道^{[13, 16]}。虽然在这些患者中没有发现PKD1、PKD2突变，但是PC1和PC2的蛋白水平可能因其他机制而下调。如微RNA-17和微RNA-92可以直接与PKD1 mRNA的3′UTR结合，转录后下调其表达水平^[17]；DNA远端上游元件结合蛋白1（far upstream element binding protein 1，FUBP1）也能结合PKD2的3′UTR抑制其翻译^[18]。这些机制可以解释PKD1、PKD2突变在一些ADPKD患者中的缺失。

PKD1和PKD2在本研究中的突变率分别为92.2%（107/116）和8.6%（10/116，其中1个家系PKD1和PKD2均发生致病性突变），而大多数白人人群中PKD1和PKD2突变的比例分别约为85%和15%^[5]。本研究发现的118个致病突变中，新突变80个，其余38个是已知突变。新发现的突变所占比例（67.8%）高于之前报道的16.7%~30%^{[5, 19-20]}。

无义突变、移码突变、典型剪接位点突变和较大的缺失突变为明确的致病突变；而错义突变、非典型剪接位点突变和框内缺失突变的致病性是不确定的，需要在家系的其他成员上验证^[18]。然而在家系信息不完整的情况下评价错义突变的致病性是一个挑战。一些预测工具（PolyPhen-2、SIFT和MutationAssessor）已经被开发用来帮助评估错义突变的致病性。虽然本研究中使用预测工具评估的大多数错义突变的致病性与家系分析结果一致，但经预测工具证实为良性突变的1个突变（PKD1 c.7544G＞C）在家系验证中检测到了遗传共分离，为致病突变。在其他相关研究中也发现了类似的现象^[21-22]。因此，推荐将家系共分离分析作为评估错义突变致病性的金标准，而不是预测工具。此外，在PKDB中，错义突变（PKD1 c.8572G＞A）被报道为高度可能的致病性突变^[23]，而本研究通过家系共分离分析证明是非致病性突变。同时，由于PKD1和PKD2的等位基因异质性高，ADPKD存在高频率的新突变。在本研究中，在116个家系中共发现2个新生突变（1.7%），和之前的报道（0.9%~3.1%）^{[19, 24-25]}相符。

本研究也存在一些局限性。首先，大多数新突变只在单个家系中发现，其致病性需要通过不同的家系资料进一步验证，特别是对于软件评估的可疑致病性突变。其次，虽然我们收集了尽可能多的家系信息，但是一些新的错义突变和框内缺失突变仍然没有完整的家系信息，因此，这些突变的致病性还有待确定。最后，本研究虽然通过家系分析评价了非典型剪接位点突变的致病性，但是最好使用小基因剪接实验（minigene splicing assay）^[26]进一步分析它们的致病性。

综上所述，本研究通过对129个ADPKD家系的PKD1和PKD2突变的研究，在116个ADPKD家系中共发现118个致病突变（109个位于PKD1、9个位于PKD2）。在118个突变中，首次报道了80个新发现的突变，另有38个是已知致病突变。新致病突变的发现将有助于ADPKD患者尤其是汉族人群患者的早期诊断和预后预测。

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第二军医大学学报 2019, Vol. 40 Issue (1): 7-13	PDF