| BackgroundsWith the rapid development of social economy and the dramatic changes in the lifestyle of the residents,the prevalence of obesity in Chinese children are rapidly increasing.Obesity is not only a disease in itself,it also affects children's cardiovascular system,endocrine system,respiratory system,and growth of children.The vast majority of obesity is a complex disease that occurs in multiple genes and multiple factors.Although too much food intake and little energy consumption are important factors of obesity,genetic factors play a fatal role for the occurrence of obesity in a given environment.Association studies have an important role in finding alleles related to obesity susceptibility.In 2007,Frayling et al.first discovered the FTO associated with body mass index?BMI?,which triggered the upsurge of the Genome wide association study?GWAS?.However,most of these loci that discovered by GWAS are located in the noncoding region of genes or introns,and the frequency is high,but the effect is very small,so the contribution to obesity is also minute.A large number of genetic loci that have not been found may be more common variants and some low frequency or rare variants with strong effects,which need to be studied.In recent years,the next-generation sequencing technology has developed rapidly,it is an effective method to study the genetic variation of individuals and to find the rare functional gene loci.At present,studies on the genetic susceptibility to obesity in Chinese children are mostly repetitive and confirmatory studies,that is,to verify whether the genetic susceptibility sites found by foreign populations have genetic effects in Chinese children.It has not been found that the genetic susceptibility of Chinese children's obesity is new.To the best of our knowledge,there is no original large-scale,systematically genetic susceptibility study in Chinese population,no matter children or adults.In this study,a two-stage design was used.Firstly,we sequenced the whole-exome of children and screen out the candidate variants associated with obesity.Secondly,the candidate loci were genotyped in a large sample to identify functional variants associated with obesity in Chinese children.ObjectiveTo investigate novel functional genetic variants associated with obesity in Chinese children and adolescents.MethodsDesign The study comprised two stages.We performed whole exome sequencing in96 obese children and 96 controls?stage 1?.We extracted 92,249 variants from annotation?nonsynonymous,stop gain,stop loss,insertion/deletion,splice site?,and selected 48 variants probably relevant to obesity with case-control status.These variants were genotyped in 5,383 children to search for association with obesity?stage 2?.Exon capture and Illumina sequencing From peripheral blood leukocytes,genomic DNA was isolated using the QIAamp DNA blood kit?Qiagen?.The Roche/NimbleGen's SeqCap EZ Human Exome Kit v3.0 capture kit was used for the selection of genomic coding regions,followed by sequencing on the Illumina HiseqTM 2500.Sequence results were compared with GRCh37?hg19?as the reference genome.For every subject,data were returned in excel-readable format as a file.SNV selection and genotyping 48 variants probably relevant to obesity with case-control status.Genotyping was performed using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry.Phenotypes General obesity was defined by the age-and sex-specfic BMI cutoff points recommended by the International Obesity Task Force?IOTF?.Adiposity was defined as body fat mass percentage?FMP?greater than or equal to 20%for males,and 25%for females aged?14 years,30%for females aged>14 years.Those whose waist circumferences were equal to or greater than the 90th percentile for age and sex,a newly developed waist circumference references for Chinese school-age children,were defined as abdominal obesity,otherwise were normal.Statistical analyses Data analyses were performed with PLINK 1.90,SPSS 22.0 and R 3.4.3.Subjects were characterized by BMI,and values are reported as meanąSD for obese and controls separately.Hardy-Weinberg equilibrium?HWE?of the genotype frequencies among controls were checked prior to association analysis.Allelic association analysis were performed by additive genetic model,and odds ratio?OR?and 95%confidence interval?CI?were used to estimate the association.Age and sex were adjusted as covariates.Bonferroni correction and false discovery rate?FDR?were used to adjust the multiple tests.For single-variant association,we set the significance threshold to<5.420×10-7,corresponding to a Bonferroni correction for92,249 SNVs.ResultsA total of 48 genetic variants were found by screening the whole exome of 192subjects?126,344 variants?.After data quality control,there are 37 variants left.Among them,17 were low frequency variants?1%?MAF<5%?,and 20 was common mutations?MAF?5%?.According to the function annotation,34non-synonymous variation,3 splicing.Association analysis was performed with BMI and general obesity as the phenotypes,respectively.The new low frequency nonsynonymous MAP3K21 rs189326455-C was associated with BMI and obesity,and achieved genome-wide significance((?=-2.22,P=8.916×10-8),(OR=0.26,95%CI:0.17-0.42,P=9.605×10-9)).A novel nonsynonymous mutations ATP1A4 rs79938119-G were also associated with obesity?OR=0.71,95%CI:0.58-0.87,P=0.001?.The novel association was also demonstrated between the low frequency nonsynonymous EMILIN2 rs56288451-T and obesity and BMI in boys??OR=0.78,95%CI,0.66-0.91,P=0.002?,??=-0.79,P=0.001??.In analyses of TG,TC,HDL-C and LDL-C as phenotype,we discovered one low frequency nonsynonymous rs189326455-C in MAP3K21 gene that showed consistent association with TG and achieved genome-wide significance(TG:?=-0.25,P=5.990×10-11).ConclusionsWe found that the low frequency nonsynonymous rs189326455 in MAP3K21 was associated with adiposity.It provides important clues to the progress of obesity genetic susceptibility. |
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