Heritability And Genome-wide Association Study Of Serum Alanine Aminotransferase In Twins

Objectives:Alanine aminotransferase（ALT）is an important liver enzyme,and its abnormal serum level often indicates liver injury.Genetic factors play an important role in the variation of serum ALT levels.Many studies worldwide have evaluated the heritability of serum ALT levels and conducted genome-wide association study（GWAS）for it,but there is a lack of relevant research on the Chinese general population.Therefore,this study aims to（1）construct a structural equation model using twin samples to evaluate the heritability of serum ALT levels,and（2）conduct a GWAS to investigate single nucleotide polymorphisms（SNPs）,genes,and pathways related to the variation of serum ALT levels.Methods:The study subjects were selected from theQingdao Twin Registry,and twin pairs that met the inclusion and exclusion criteria were selected.The zygosity of twins was identified by gender,blood type,and microsatellite DNA scanning and genotyping.All included twin samples underwent standardized questionnaire surveys,physical examinations,and laboratory tests.The serum ALT levels of the subjects were measured by an automated biochemical analyzer according to standard procedures.Mx was used to construct a structural equation model to evaluate the effects of genetic and environmental factors on the phenotype.The Infinium Omni2Exome-8v1.2Bead Chip genotyping array（Illumina,San Diego,California,USA）was used to genotype the dizygotic twin samples.The IMPUTE2 was used for imputation of ungenotyped SNPs.The Genome-Wide efficient mixed model analysis（GEMMA）was used for SNP-based analysis.Haploreg v4.1 was used for enhancer enrichment analysis of top SNPs.Locuszoom was used to draw the regional association plot of top SNPs associated with serum ALT levels.Regulome DB and3DSNP were used for functional annotation and scoring of top SNPs and SNPs associated with high linkage disequilibrium（LD）with top SNPs in the region.For gene-based analysis and pathway enrichment analysis,we utilized the Versatile Gene-based Association Study-2（VEGAS2）software and Pathway scoring algorithm（PASCAL）software,respectively.The GTEx database was used to query the number of expression quantitative trait loci（eQTLs）and gene expression levels of the target SNPs and genes in different tissues.Results:A total of 369 pairs of twins were included in this study,including 136 pairs of dizygotic twins.The median（interquartile rang,IQR）age of the study participants was 50（46,57）years.The median（IQR）serum ALT level was 18（12,27）U/L.In the heritability analysis,the heritability of serum ALT level estimated by the AE model was 65%,with the remaining 35%of the variance attributable to environmental factors.Among the 136 pairs of dizygotic twins,GWAS analysis was performed,yielding the following results:（1）In the SNP-based analysis,no SNPs reached significance level(P<5×10^-8)with serum ALT level,but 65 SNPs showed suggestive associations with this phenotype(P<1×10^-5).The most strongly associated SNP was rs2829565,located on chromosome 21 near RNA5SP489,while other SNPs such as rs372235626 and rs10793147 also reached suggestive association levels.Enhancer enrichment analysis indicated that genetic variations related to serum ALT level were significantly enriched in seven tissues and cells,including Colonic Mucosa,Mesenchymal Stem Cell Derived Chondrocyte Cultured Cells,H1 Derived Neuronal Progenitor Cultured Cells,h ESC Derived CD56+Ectoderm Cultured Cells,Monocytes-CD14+RO01746 Primary Cells,Primary hematopoietic stem cells G-CSF-mobilized Male,and Primary hematopoietic stem cells short term culture（P<0.05）.Furthermore,a large number of SNPs in the 5p15.31 region were found having high LD coefficient with SNP rs372235626 which was showed suggestive associations with serum ALT level.Several SNPs including rs372235626,rs545693502,rs3776464,rs59809395,and rs3822442 were predicted to have regulatory effects based on annotation and scoring.（2）In the gene-based analysis,no genes reached significance level(P<2.36×10^-6),but 1,073genes showed suggestive associations with serum ALT level（P<0.05）,including CHIC2,ZNF587B,MTRR,ZNF776,ZNF586,LOC101928436,BLOC1S5,and NLRP13.（3）In the pathway-based analysis,782 pathways were found to be associated with serum ALT level（empirical P<0.05）,including the 3 UTR MEDIATED TRANSLATIONAL REGULATION,ARACHIDONIC ACID METABOLISM,NUCLEOTIDE BINDING DOMAIN LEUCINE RICH REPEAT CONTAINING RECEPTOR NLR SIGNALING PATHWAYS,and others.（4）eQTL and gene expression analysis:Among the top 20 genes,at least 14 genes have cis-eQTL in one or more tissues related to serum ALT levels.The top 20 genes are expressed to varying degrees in serum ALT-related tissues,with the LHFP gene showing relatively high expression levels in these tissues.Conclusion:Based on the data of twins in Qingdao,the heritability analysis showed that serum ALT levels have a high heritability.Additionally,multiple SNPs,genes,and pathways that may be associated with serum ALT levels were identified.These findings provide clues for further exploration of the genetic mechanisms underlying serum ALT levels.