Functional non-synonymous variants of ABCG2 and gout risk.

Objectives: Common dysfunctional variants of ATP binding cassette subfamily G member 2 (Junior blood group) (ABCG2), a high-capacity urate transporter gene, that result in decreased urate excretion are major causes of hyperuricemia and gout. In the present study, our objective was to determine the frequency and effect on gout of common and rare non-synonymous and other functional allelic variants in the ABCG2 gene. Methods: The main cohort recruited from the Czech Republic consisted of 145 gout patients; 115 normouricaemic controls were used for comparison. We amplified, directly sequenced and analysed 15 ABCG2 exons. The associations between genetic variants and clinical phenotype were analysed using the t-test, Fisher's exact test and a logistic and linear regression approach. Data from a New Zealand Polynesian sample set and the UK Biobank were included for the p.V12M analysis. Results: In the ABCG2 gene, 18 intronic (one dysfunctional splicing) and 11 exonic variants were detected: 9 were non-synonymous (2 common, 7 rare including 1 novel), namely p.V12M, p.Q141K, p.R147W, p.T153M, p.F373C, p.T434M, p.S476P, p.D620N and p.K360del. The p.Q141K (rs2231142) variant had a significantly higher minor allele frequency (0.23) in the gout patients compared with the European-origin population (0.09) and was significantly more common among gout patients than among normouricaemic controls (odds ratio = 3.26, P < 0.0001). Patients with non-synonymous allelic variants had an earlier onset of gout (42 vs 48 years, P = 0.0143) and a greater likelihood of a familial history of gout (41% vs 27%, odds ratio = 1.96, P = 0.053). In a meta-analysis p.V12M exerted a protective effect from gout (P < 0.0001). Conclusion: Genetic variants of ABCG2, common and rare, increased the risk of gout. Non-synonymous allelic variants of ABCG2 had a significant effect on earlier onset of gout and the presence of a familial gout history. ABCG2 should thus be considered a common and significant risk factor for gout.

Prevalence of URAT1 allelic variants in the Roma population.

The Roma represents a transnational ethnic group, with a current European population of 8-10 million. The evolutionary process that had the greatest impact on the gene pool of the Roma population is called the founder effect. Renal hypouricemia (RHUC) is a rare heterogenous inherited disorder characterized by impaired renal urate reabsorption. The affected individuals are predisposed to recurrent episodes of exercise-induced nonmyoglobinuric acute kidney injury and nephrolithiasis. To date, more than 150 patients with a loss-of-function mutation for the SLC22A12 (URAT1) gene have been found, most of whom are Asians. However, RHUC 1 patients have been described in a variety of ethnic groups (e.g., Arab Israelis, Iraqi Jews, Caucasians, and Roma) and in geographically noncontiguous countries. This study confirms our previous findings regarding the high frequency of SLC22A12 variants observed. Frequencies of the c.1245_1253del and c.1400C>T variants were found to be 1.92% and 5.56%, respectively, in a subgroup of the Roma population from five regions in three countries: Slovakia, Czech Republic, and Spain. Our findings suggested that the common dysfunction allelic variants of URAT1 exist in the general Roma population and thus renal hypouricemia should be kept in differential diagnostic algorithm on Roma patients with defect in renal tubular urate transport. This leads to confirm that the genetic drift in the Roma have increased the prevalence of hereditary disorders caused by very rare variants in major population.

DNA methylation and mRNA expression of HLA-DQA1 alleles in type 1 diabetes mellitus.

Type 1 diabetes (T1D) belongs among polygenic multifactorial autoimmune diseases. The highest risk is associated with human leucocyte antigen (HLA) class II genes, including HLA-DQA1 gene. Our aim was to investigate DNA methylation of HLA-DQA1 promoter alleles (QAP) and correlate methylation status with individual HLA-DQA1 allele expression of patients with T1D and healthy controls. DNA methylation is one of the epigenetic modifications that regulate gene expression and is known to be shaped by the environment.Sixty one patients with T1D and 39 healthy controls were involved in this study. Isolated DNA was treated with sodium bisulphite and HLA-DQA1 promoter sequence was amplified using nested PCR. After sequencing, DNA methylation of HLA-DQA1 promoter alleles was analysed. Individual mRNA HLA-DQA1 relative allele expression was assessed using two different endogenous controls (PPIA, DRA). We have found statistically significant differences in HLA-DQA1 allele 02:01 expression (PPIA normalization, Pcorr = 0·041; DRA normalization, Pcorr = 0·052) between healthy controls and patients with T1D. The complete methylation profile of the HLA-DQA1 promoter was gained with the most methylated allele DQA1*02:01 and the least methylated DQA1*05:01 in both studied groups. Methylation profile observed in patients with T1D and healthy controls was similar, and no correlation between HLA-DQA1 allele expression and DNA methylation was found. Although we have not proved significant methylation differences between the two groups, detailed DNA methylation status and its correlation with expression of each HLA-DQA1 allele in patients with T1D have been described for the first time.

Microarray analysis of circulating micro RNAs in the serum of patients with polymyositis and dermatomyositis reveals a distinct disease expression profile and is associated with disease activity.

OBJECTIVES: The aim of this study was a large scale investigation of myositis-associated circulating miRNA molecules and also determination of expression of these candidate molecules in relation to clinical activity of myositis. METHODS: RNA, containing also miRNAs, was isolated from sera of 28 patients suffering from idiopathic inflammatory myopathies (IIM) and 16 healthy controls. Expression of miRNAs was determined using a miRNA microarray method. Statistical analysis of miRNA expression was carried out using Arraystar software. RESULTS: Our results showed 23 significantly differentially expressed miRNAs. Six miRNAs were differentially expressed in IIM compared to healthy controls. In dermatomyositis (DM) we found 3 and in polymyositis (PM) 6 differentially expressed miRNAs compared to controls. Three miRNAs were up-regulated in patients with highly active disease compared to patients with low disease activity. Furthermore, we found 26 significantly differentially expressed miRNAs in SLE patients compared to IIM, DM and PM patients. CONCLUSIONS: This is the first study that comprehensively describes expression levels of circulating miRNAs in serum of patients suffering from IIM. It can be expected that some of these deregulated miRNA molecules are involved in aetiology of IIM and may potentially serve as molecular markers for IIM development or for monitoring of disease activity.

Differences in promoter DNA methylation and mRNA expression of individual alleles of the HLA class II DQA1 gene.

OBJECTIVES: Extensive polymorphism of HLA class II genes is not restricted to the coding region of the gene. It extends also to the linked promoter region, where it forms the basis for different levels of individual allele's expression. Differential expression of HLA class II alleles can shape an immune response and influence the risk of developing autoimmune disease. In addition to genetic variability, variation in epigenetic modifications, including DNA methylation, can be another cause of the uneven expression of individual alleles. We aimed to analyze the DNA methylation of promoter sequences and the levels of expression of individual DQA1 gene alleles, interallelic variation of these two characteristics and the relationship between them. METHODS: The 60 healthy donors included into study were HLA-DRB1, HLA-DQB1 and HLA-DQA1 genotyped using PCR-SSP. Genomic DNA was treated by sodium bisulfite and the target segment in the HLA-DQA1 gene promoter was PCR amplified. PCR product was cloned into Escherichia coli and individual clones were sequenced. Transcripts of individual DQA1 alleles in peripheral blood leukocytes were quantified by Real-Time PCR. RESULTS: In this study, we have described detailed DNA methylation profile of promoter area of DQA1 gene alleles. The overall promoter methylation is increased for DQA1*02:01 and DQA1*04:01 alleles, on the other side, DQA1*05:01 allele shows decreased methylation level. Our results suggest that there are only minor interindividual differences in DRA-normalized expression level of specific allele. Furthermore, expression levels of individual alleles followed DQA1*03>*01:03 (in DRB1*13-DQA1*01:03-DQB1*06:03 haplotype)>*01:01,*05:05, and DQA1*03>*02:01>*05:05 hierarchy. The statistically significantly most expressed allele, DQA1*03, comprises part of DQ8 molecule, which is commonly linked to autoimmune diseases. A clear relationship between promoter DNA methylation and mRNA expression level of the DQA1 gene could not be identified.