Zhenming Cai

Association of Base Excision Repair Gene Polymorphisms with ESRD Risk in a Chinese Population

The base excision repair (BER) pathway, containing OGG1, MTH1 and MUTYH, is a major protector from oxidative DNA damage in humans, while 8-oxoguanine (8-OHdG), an index of DNA oxidation, is increased in maintenance hemodialysis (HD) patients. Four polymorphisms of BER genes, OGG1 c.977C > G (rs1052133), MTH1 c.247G > A (rs4866), MUTYH c.972G > C (rs3219489), and AluYb8MUTYH (rs10527342), were examined in 337 HD patients and 404 healthy controls. And the 8-OHdG levels in leukocyte DNA were examined in 116 HD patients. The distribution of MUTYH c.972 GG or AluYb8MUTYH differed between the two groups and was associated with a moderately increased risk for end-stage renal disease (ESRD) (P = 0.013 and 0.034, resp.). The average 8-OHdG/106 dG value was significantly higher in patients with the OGG1 c.977G, MUTYH c.972G or AluYb8MUTYH alleles (P < 0.001 via ANOVA). Further analysis showed that combination of MUTYH c.972GG with OGG1 c.977GG or AluYb8MUTYH increased both the risk for ESRD and leukocyte DNA 8-OHdG levels in HD patients. Our study showed that MUTYH c.972GG, AluYb8MUTYH, and combination of OGG1 c.977GG increased the risk for ESRD development in China and suggested that DNA oxidative damage might be involved in such process.

The Polymorphic AluYb8 Insertion in the MUTYH Gene is Associated with Reduced Type 1 Protein Expression and Reduced Mitochondrial DNA Content

The human mutY homolog (MUTYH) participates in base excision repair (BER), which is critical for repairing oxidized DNA bases and maintaining DNA replication fidelity. The polymorphic AluYb8 insertion in the 15th intron of the MUTYH gene (AluYb8MUTYH) has been shown to associate with an aggregated 8-hydroxy-2′-deoxyguanosine (8-OH-dG) lesion in genomic DNA and to serve as a risk factor for age-related diseases. In this work, we demonstrate that this variant is associated with a significant reduction of the type 1 MUTYH protein that localizes to mitochondria. Notably, this variant affects mitochondrial DNA (mtDNA) maintenance and functional mitochondrial mass in individuals homozygous for the AluYb8MUTYH variant. These findings provide evidence for an association between the AluYb8MUTYH variant and decreased mitochondrial homeostasis and, consequently, contribute to elucidating the roles of the AluYb8MUTYH variant in impairing the mitochondrial base excision repair (mtBER) system and increasing the risk of acquiring an age-related disease.

Base excision repair gene polymorphisms are associated with inflammation in patients undergoing chronic hemodialysis.

Chronic inflammation may increase the risk of mortality for patients undergoing hemodialysis, while enhanced oxidative stress and DNA oxidative damage are involved in the inflammatory response. The purpose of this study was to examine the associations between inflammation and polymorphisms in the base excision repair (BER) system, which protects against oxidative DNA damage, among hemodialysis patients. Data were analyzed from 167 hemodialysis patients and 66 healthy controls. All subjects were evaluated for the expression of inflammatory cytokines (IL-1β and IL-6) and genotyped for two BER genes, including hOGG1 c.977C>G, MUTYH c.972G>C and AluYb8MUTYH. The results showed that the hemodialysis patients had significantly higher levels of IL-1β and IL-6 than the healthy controls. In the healthy controls, no patterns of association were observed between the hOGG1 c.977C>G or MUTYH c.972G>C genotypes and IL-1β or IL-6 levels; however, patients with the MUTYH c.972G/G genotype presented higher levels of IL-1β than those with the C/C genotype. The AluYb8MUTYH genotype was strongly associated with increased IL-1β levels among controls and increased IL-1β and IL-6 levels among hemodialysis patients. Additionally, the synergetic effect of these variations of the BER genes on the levels of IL-1β and IL-6 was investigated. The combinations of the AluYb8MUTYH genotype with the hOGG1 c.977 C>G or MUTYH c.972 G>C genotypes were associated with the IL-1β and IL-6 levels in hemodialysis patients. This is the first report showing an association between BER genetic polymorphisms and the inflammatory state during hemodialysis; this association might be mediated by impaired anti-oxidant defense mechanisms.

Functional Polymorphisms of the hOGG1 Gene Confer Risk to Type 2 Epithelial Ovarian Cancer in Chinese

Objective: 8-Hydroxydeoxyguanosine (8-OHdG) is an oxidized nucleoside that can lead to misincorporation of bases. Human 8-oxoguanine DNA glycosylase (hOGG1) is the key defense enzyme against mutation by the cellular 8-OHdG in duplex DNA. The present study was aimed to explore whether the hOGG1 gene variants play an important role in the carcinogenesis of epithelial ovarian cancer (EOC). Methods: Germ line variants in 5′-untranslated region (c.-18G>T, c.-23A>G, c.-45G>A, and c.-53G>C) and c.977C>G (Ser326Cys) polymorphism in exon7 of the hOGG1 gene in 420 sporadic EOCs and 840 controls were detected. Immunohistochemical and promoter luciferase activity assays were used to explore the effect of c.-18G>T variant on hOGG1 expression. Results: In contrast to type I EOC cases, patients with type II EOC were usually older, already in the advanced stage, and exhibited a common protein 53 (p53) overexpression. The frequencies of genotypes c.-18G/T and c.977G/G in hOGG1 were significantly high in the patients with type II EOC (odds ratio, 2.83; 95% confidence interval, 1.45-5.52; odds ratio, 1.66; 95% confidence interval, 1.26-2.17) but not in the patients with type I EOC. The average level of hOGG1 protein in the normal tissues adjacent to the type II EOC-carried c.-18G/T was lower than that with c.-18G/G (P = 0.01). The luciferase activity in the c.-18T allele was lower than that in the c.-18G allele (P = 0.001). Conclusion: The genotypes of c.-18G/T in 5′-untranslated region and c.977G/G in exon7 of the hOGG1 gene would confer risk to type II EOC.

A Splice Mutation and mRNA Decay of EXT2 Provoke Hereditary Multiple Exostoses

Background Hereditary multiple exostoses (HME) is an autosomal dominant disease. The classical paradigm of mutation screening seeks to relate alterations in the exostosin glycosyltransferase genes, EXT1 and EXT2, which are responsible for over 70% of HME cases. However, the pathological significance of the majority of these mutations is often unclear. Methods In a Chinese family with HME, EXT1 and EXT2 genes were screened by direct sequencing. The consequence of a detected mutant was predicted by in silico analysis and confirmed by mRNA analysis. The EXT1 and EXT2 mRNA and protein levels and the HS patterns in the HME patients were compared with those in healthy controls. Results A heterozygous transition (c.743+1G>A) in the EXT2 gene, which co-segregated with the HME phenotype in this family, was identified. The G residue at position +1 in intron 4 of EXT2 was predicted to be a 5′ donor splice site. The mRNA analysis revealed an alternative transcript with a cryptic splice site 5 bp downstream of the wild-type site, which harbored a premature stop codon. However, the predicted truncated protein was not detected by western blot analysis. Decay of the mutant mRNA was shown by clone sequencing and quantification analysis. The corresponding downregulation of the EXT2 mRNA will contribute to the abnormal EXT1/EXT2 ratio and HS pattern that were detected in the patients with HME. Conclusion The heterozygous mutation c.743+1G>A in the EXT2 gene causes HME as a result of abnormal splicing, mRNA decay, and the resulting haploinsufficiency of EXT2.

A novel type 2 diabetes risk allele increases the promoter activity of the muscle-specific small ankyrin 1 gene.

Genome-wide association studies have identified Ankyrin-1 (ANK1) as a common type 2 diabetes (T2D) susceptibility locus. However, the underlying causal variants and functional mechanisms remain unknown. We screened for 8 tag single nucleotide polymorphisms (SNPs) in ANK1 between 2 case-control studies. Genotype analysis revealed significant associations of 3 SNPs, rs508419 (first identified here), rs515071, and rs516946 with T2D (P < 0.001). These SNPs were in linkage disequilibrium (r2 > 0.80); subsequent analysis indicated that the CCC haplotype associated with increased T2D susceptibility (OR 1.447, P < 0.001). Further mapping showed that rs508419 resides in the muscle-specific ANK1 gene promoter. Allele-specific mRNA and protein level measurements confirmed association of the C allele with increased small ANK1 (sAnk1) expression in human skeletal muscle (P = 0.018 and P < 0.001, respectively). Luciferase assays showed increased rs508419-C allele transcriptional activity in murine skeletal muscle C2C12 myoblasts, and electrophoretic mobility-shift assays demonstrated altered rs508419 DNA-protein complex formation. Glucose uptake was decreased with excess sAnk1 expression upon insulin stimulation. Thus, the ANK1 rs508419-C T2D-risk allele alters DNA-protein complex binding leading to increased promoter activity and sAnk1 expression; thus, increased sAnk1 expression in skeletal muscle might contribute to T2D susceptibility.

Association of AluYb8 insertion/deletion polymorphism in the MUTYH gene with mtDNA maintain in the type 2 diabetes mellitus patients.

A common AluYb8-element insertion/deletion polymorphism of the MUTYH gene (AluYb8MUTYH) is a novel genetic risk factor for type 2 diabetes mellitus (T2DM). In the present study, mtDNA sequencing analysis indicated that the mtDNA sequence heteroplasmy was not associated with AluYb8MUTYH polymorphism. To better understand the genetic risk for T2DM, we investigated the association of this polymorphism with mtDNA content, mtDNA breakage and mtDNA transcription in the leukocytes of T2DM patients. The mtDNA content and unbroken mtDNA were significantly increased in the mutant patients than in the wild-type patients (P <0.05, respectively). However, no association between mtDNA transcription and AluYb8MUTYH variant was observed. The results suggested that the AluYb8MUTYH variant was associated with an altered mtDNA maintain in T2DM patients. The high level of mtDNA content observed in the mutant patients may have resulted from inefficient base excision repair of mitochondrial MUTYH and a compensatory mechanism that is triggered by elevated oxidative stress.

Detection of mosaicism for the polymorphic variants in the 5'-UTR of hOGG1 by cloning and sequence analysis and pyrosequencing.

Mosaicism refers to the presence of genetically distinct cell lines within an organism or a tissue. Somatic mosaicism exists in distinct populations of somatic cells and commonly arises as a result of somatic mutations, mainly in early embryonic development. SNPs are important markers that distinguish between different individuals in heterogeneous biological samples and contribute greatly to disease risk association studies. In this work, we investigated the relationship between the functional variants in the 5′‐UTR of the hOGG1 gene and the risk of type 2 diabetes. Upon detection of the polymorphisms c.‐53G>C, c.‐23A>G, and c.‐18G>T in the hOGG1 gene, we found that mosaicism was present in 3/28 (10.71%), 7/51 (13.73%), and 1/44 (2.27%) patients respectively, who were carriers of these single nucleotide variations, by cloning and sequence analysis and pyrosequencing. Statistical analysis showed that the frequency of the variation c.‐23A>G in the hOGG1 5′‐UTR in type 2 diabetic patients was significantly higher than that in healthy controls. However, sequencing of the mutant alleles in mosaic individuals showed weak peaks that may affect detection of the SNPs and impair association‐based investigations.