Genetic Studies On Abnormal Serum Uric Acid Metabolism And Related Disorders

Serum uric acid（SUA）is a complex trait with high heritability,which is regulated by multiple SUA-raising and SUA-decreasing alleles.Unlike the rare disease,hypouricemia,which is caused by single gene mutation,hyperuricemia（HUA）and gout are common.Most of them belong to complex disease with polygenic inheritance and their genetic susceptibility is composed of a series of susceptible genes.The prevalence of HUA and gout in China is close to those in developed countries such as Europe and the United States（1-4%）,and has become a kind of common metabolic disease,which has attracted wide attention from domestic and overseas scholars.In contrast,hypouricemia is a rare abnormal phenotype on SUA level.Hypouricemia has not obtained enough attention because of its low awareness,low prevalence and unclear long-term prognosis,only scattered cases have been reported in China.Our understanding of the etiology of abnormal SUA metabolism and related disorders is still limited.In order to understand the genetic risk variations more completely,this dissertation will apply a variety of genetic research methods to analyze the genetic susceptibility of hypouricemia,HUA and gout comprehensively and to identify alleles that raise or decrease SUA level and gout associated susceptibility genes.PART Ⅰ-Genetic analysis of the rare phenotype,hypouricemia.In the chapter 2of this dissertation,whole-exome sequencing（WES）was used to discover causal genes and mutations in a patient with renal hypouricemia and her healthy parents.We found two compound heterozygous mutations of c.269G>A/p.R90H and c.1289₁290insGG/p.M430fsX466,which are both located in the SLC22A12 gene,in the patient.Sanger sequencing further confirmed that the patient’s heterozygous c.269G>A/p.R90H mutation,which has been reported previously,derived from her mother,and the heterozygous c.1289₁290insGG/p.M430fsX466 mutation,which was found for the first time,derived from her father.This patient included,a total of nine cases of hypouricemia for Chinese descent were reported,all of whom were SLC22A12 and/or SLC2A9 gene mutation carriers by genetic analysis.Thus,it’s speculated that phenotype of hypouricemia in China is mainly caused by mutations in SLC22A12 and SLC2A9 genes.In the chapter 3 of this dissertation,amplicon multiplexed targeted resequencing of whole exon regions of SLC2A9 and SLC22A12were performed in three cohorts of additional 31 hypouricemia,288 HUA and 280normal controls and statistical analysis was conducted to verify whether the mutations confer real hypouricemia or HUA susceptibility.A total of 84 high-quality variants were identified in these three cohorts.18 variants were functional（non-synonymous or in splicing region）,and then included in the following association analysis.Common variants were not associated with hypouricemia or HUA.For rare variants,6 single nucleotide variations（SNVs）p.T21I and p.G13D in SLC2A9,p.W50fs,p.Q382L,p.V547L and p.E458K in SLC22A12,occurred in totally six hypouricemia subjects and were absent in HUA and normal controls.Among them,p.G13D in SLC2A9 and p.V547L in SLC22A12 were newly reported.Allelic and genotypic frequency distributions of the 6 SNVs differed significantly between the hypouricemia and normal controls（even after multiple testing correction）and the gene-based analysis substantiated the significant effects of rare SNVs on hypouricemia.These rare mutations had no significant effects on HUA susceptibility.In conclusion,we first present a comprehensive mutation spectrum of hypouricemia in Han Chinese population.In-silicon analysis suggests that mutation-induced structural instability or malfunction of the urate transporter（complex）may be the main mechanisms for hypouricemia.PART Ⅱ-Study on the genetic susceptibility of gout and HUA.Gout is a common arthritis,and HUA is the most important biochemical basis.Previous genome-wide association studies（GWAS）have identified dozens of susceptibility loci for SUA/gout,but few studies systematically validate them in Chinese population.In the chapter 4 of this dissertation,we attempt to extensively investigate whether these previously identified SUA/gout GWAS loci affect susceptibility to gout in Han Chinese using our previous gout GWAS dataset（1,255 gout patients and 1,848normal controls）.A total of 2,255 variants showed linkage disequilibrium（LD）with GWAS identified SUA/gout associated variants and further pruning resulted in 56 LD independent variants for subsequent association analysis.Next,for variants reaching nominal association with gout,cumulative genetic risk score analysis was performed to assess the cumulative effect of multiple risk alleles on gout incidence.23 variants（41%）showed nominal association with gout in our dataset（P<0.05）.The previously reported gout associated loci,including ABCG2,SLC2A9,GCKR,ALDH2 and CNIH2（except ALDH16A1）,were replicated.Cumulative genetic risk score analysis showed that the risk of gout increased for individuals with the growing number（≥8）of the risk alleles on gout associated loci.The tendency of increasing risk for gout escalated when additional risk alleles on SUA associated loci were considered.Our study shows that most of the SUA/gout susceptibility genes are identical in varied ethnic groups,which lays an important theoretical foundation for future cross-ancestral meta-analysis.In the chapter 5 of this dissertation,we used candidate gene strategy and case-control association study to determine whether SLC28A2 gene confers HUA and gout susceptibility in Han Chinese population.Three sample sets of 1,376 gout patients,1,290 HUA subjects（no gout attack）and 1,349 normal controls were recruited for this study.Eight single nucleotide polymorphisms（SNPs）in the SLC28A2gene were genotyped using ligase detection reaction-polymerase chain reaction（LDR-PCR）technology.rs16941238 showed the most significant association with HUA(OR=0.773,P=3.30E-03,P_perm=2.00E-03),but not with gout(OR=0.880,P=0.1315,P_perm=0.1491).rs2271437（p.L163W）was significantly associated with gout(OR=1.387,P=0.0277,P_perm=0.0288),and was further confirmed in the meta-analysis with our previous gout GWAS dataset（OR=1.322,P=8.90E-03）.Each SNP basically conferred consistent effect direction on gout and HUA,compared with the normal control.Our findings support the associations of the SLC28A2 gene with HUA and gout susceptibility in Han Chinese.In conclusion,genetic analysis on varied phenotypes of abnormal SUA can effectively detect the related risk variations and further enrich our understanding of genetic mechanism of SUA metabolism and gout,which may not only promote the establishment of molecular diagnosis and genetic counseling for hypouricemia,but also provide important biological basis for precise and personalized medical treatment of gout in future.