利用全外显子测序在肥胖人群中筛查致病突变

Screening for pathogenic variants in obese cohort using whole-exome sequencing

目的·利用全外显子测序技术(whole-exome sequencing,WES)筛查瘦素-促黑素细胞激素(leptin-melanocyte stimulating hormone,LEP-MSH)通路中的关键基因在肥胖人群中的突变情况。方法·招募2011年1月—2019年7月在上海交通大学医学院附属第六人民医院接受袖状胃切除术的119名肥胖患者:年龄17~65岁,体质量指数(body mass index,BMI)≥34 kg/m2。采集研究对象的外周血,提取全基因组DNA并进行全外显子测序,应用生物信息学方法筛选LEP-MSH通路上16个基因(ADCY3、AGRP、BDNF、KSR2、LEP、LEPR、MC3R、MC4R、MCHR1、MRAP2、NTRK2、PCSK1、PHIP、POMC、SH2B1和SIM1)的突变。选取1000 Genomes(1000G)、Exome Aggregation Consortium(ExAC)和Genome Aggregation Database(gnomAD)数据库中总人群最小等位基因频率小于0.02且东亚人群频率小于0.01的罕见变异位点用于后续分析。使用6种突变有害性预测软件来评估变异的损害程度。最后,基于每位患者的临床信息,并根据美国医学遗传学和基因组学学院(America College of Medical Genetics and Genomics,ACMG)指南对所有变异进行致病性判断,仅保留“致病的”“可能致病的”“意义不明的”突变。结果·119名患者中有24名患者检测到了LEP-MSH通路中16个关键基因的26个22种突变,均为杂合突变;其中SH2B1基因突变7个(占总突变数的26.92%),MCHR1基因突变4个(占15.38%),PHIP基因突变3个(占11.53%),ADCY3、LEPR基因突变各2个(各占7.69%),LEP、NTRK2、AGRP、KSR2、MC3R、MC4R、BDNF、PCSK1基因突变各1个(各占3.85%)。3名患者携带SH2B1基因上的相同变异位点,LEPR基因和MCHR1基因上分别有2名患者携带相同变异位点。共有12种变异在3个数据库的东亚人群中都没有被收录,为东亚人群新发变异,分别位于SH2B1(p.V209I、p.R67C、p.L149F)、KSR2(p.P155T)、LEP(p.D106N)、LEPR(p.W132R)、PHIP(p.K1461R)、BDNF(p.N84S)、PCSK1(p.R282W)、NTRK2(p.T732M)、MC3R(p.S71P)和MC4R(p.W174X)。结论·共检测到LEP-MSH通路上22种肥胖可能相关的罕见变异,其中12种为东亚人群新发变异。

Objective·To screen mutations of key genes in the leptin-melanocyte stimulating hormone(LEP-MSH)pathway by whole-exome sequencing(WES)in the obese cohort.Methods·A total of 119 obese patients aged 17-65 years old with body mass index(BMI)≥34 kg/m2,who underwent laparoscopic sleeve gastrectomy from January 2011 to July 2019 at Shanghai Sixth People′s Hospital,Shanghai Jiao Tong University School of Medicine were collected.The peripheral blood samples of the research subjects were collected,and whole genome DNA was extracted to perform WES.Bioinformatic methods were applied to detect the mutations in 16 genes in the LEP-MSH pathway(ADCY3,AGRP,BDNF,KSR2,LEP,LEPR,MC3R,MC4R,MCHR1,MRAP2,NTRK2,PCSK1,PHIP,POMC,SH2B1,and SIM1).Rare variants with the minor allele frequency in the total population less than 0.02 and inthe East Asian population less than 0.01 in the 1000 Genome(1000G),Exome Aggregation Consortium(ExAC)and GenomeAggregation Database(gnomAD)were selected for subsequent analysis.Six pieces of prediction software were used to evaluate thedeleteriousness of the mutations.Finally,based on the clinical information of each patient,the pathogenicity of all variants wasdetermined according to the guidelines of America College of Medical Genetics and Genomics(ACMG),and only the"pathogenic","likely pathogenic",and"uncertain significance"variants were retained.Results·A total of 26 variants,22 kinds of variants weredetected in 24 patients from 119 subjects,all of which were heterozygous mutations.The detected variants included 7 in SH2B1gene(accounting for 26.92%of the total variants),4 in MCHR1 gene(accounting for 15.38%),3 in PHIP gene(accounting for11.53%),2 in ADCY3 and LEPR genes(accounting for 7.69%,respectively),and 1 in LEP,NTRK2,AGRP,KSR2,MC3R,MC4R,BDNF,and PCSK1 genes,respectively(accounting for 3.85%,respectively).There were 3 patients having the same mutation site inSH2B1 gene,and 2 patients having the same mutation sites in LEPR gene and MCHR1 gene,respectively.In addition,among thesemutations,there were 12 ones not included in the East Asian population in 3 public databases,which were novel mutations in theEast Asian population,located in SH2B1(p.V209I,p.R67C,and p.L149F),KSR2(p.P155T),LEP(p.D106N),LEPR(p.W132R),PHIP(p.K1461R),BDNF(p.N84S),PCSK1(p.R282W),NTRK2(p.T732M),MC3R(p.S71P),and MC4R(p.W174X).Conclusion·A total of 22 kinds of rare variations possibly associated with obesity in the LEP-MSH pathway are detected,12 of which are novelin the East Asian population.