Inter-individual variation in nucleotide excision repair pathway is modulated by non-synonymous polymorphisms in ERCC4 and MBD4 genes.

Inter-individual differences in DNA repair capacity (DRC) may lead to genome instability and, consequently, modulate individual cancer risk. Among the different DNA repair pathways, nucleotide excision repair (NER) is one of the most versatile, as it can eliminate a wide range of helix-distorting DNA lesions caused by ultraviolet light irradiation and chemical mutagens. We performed a genotype-phenotype correlation study in 122 healthy subjects in order to assess if any associations exist between phenotypic profiles of NER and DNA repair gene single nucleotide polymorphisms (SNPs). Individuals were genotyped for 768 SNPs with a custom Illumina Golden Gate Assay, and peripheral blood mononuclear cells (PBMCs) of the same subjects were tested for a NER comet assay to measure DRC after challenging cells by benzo(a)pyrene diolepoxide (BPDE). We observed a large inter-individual variability of NER capacity, with women showing a statistically significant lower DRC (mean ± SD: 6.68 ± 4.76; p = 0.004) than men (mean ± SD: 8.89 ± 5.20). Moreover, DRC was significantly lower in individuals carrying a variant allele for the ERCC4 rs1800124 non-synonymous SNP (nsSNP) (p = 0.006) and significantly higher in subjects with the variant allele of MBD4 rs2005618 SNP (p = 0.008), in linkage disequilibrium (r(2) = 0.908) with rs10342 nsSNP. Traditional in silico docking approaches on protein-DNA and protein-protein interaction showed that Gly875 variant in ERCC4 (rs1800124) decreases the DNA-protein interaction and that Ser273 and Thr273 variants in MBD4 (rs10342) indicate complete loss of protein-DNA interactions. Our results showed that NER inter-individual capacity can be modulated by cross-talk activity involving nsSNPs in ERCC4 and MBD4 genes, and they suggested to better investigate SNP effect on cancer risk and response to chemo- and radiotherapies.

Genetic variants associated with increased risk of malignant pleural mesothelioma: a genome-wide association study.

Asbestos exposure is the main risk factor for malignant pleural mesothelioma (MPM), a rare aggressive tumor. Nevertheless, only 5-17% of those exposed to asbestos develop MPM, suggesting the involvement of other environmental and genetic risk factors. To identify the genetic risk factors that may contribute to the development of MPM, we conducted a genome-wide association study (GWAS; 370,000 genotyped SNPs, 5 million imputed SNPs) in Italy, among 407 MPM cases and 389 controls with a complete history of asbestos exposure. A replication study was also undertaken and included 428 MPM cases and 1269 controls from Australia. Although no single marker reached the genome-wide significance threshold, several associations were supported by haplotype-, chromosomal region-, gene- and gene-ontology process-based analyses. Most of these SNPs were located in regions reported to harbor aberrant alterations in mesothelioma (SLC7A14, THRB, CEBP350, ADAMTS2, ETV1, PVT1 and MMP14 genes), causing at most a 2-3-fold increase in MPM risk. The Australian replication study showed significant associations in five of these chromosomal regions (3q26.2, 4q32.1, 7p22.2, 14q11.2, 15q14). Multivariate analysis suggested an independent contribution of 10 genetic variants, with an Area Under the ROC Curve (AUC) of 0.76 when only exposure and covariates were included in the model, and of 0.86 when the genetic component was also included, with a substantial increase of asbestos exposure risk estimation (odds ratio, OR: 45.28, 95% confidence interval, CI: 21.52-95.28). These results showed that genetic risk factors may play an additional role in the development of MPM, and that these should be taken into account to better estimate individual MPM risk in individuals who have been exposed to asbestos.

Validation of the nucleotide excision repair comet assay on cryopreserved PBMCs to measure inter-individual variation in DNA repair capacity.

Inter-individual susceptibility to mutagens/carcinogens can be assessed by either genotyping DNA repair genes in different pathways or phenotyping DNA repair capacity (DRC) at the molecular or cellular level. Due to the large number of known DNA repair genes, and the interactions between repair pathways, phenotyping is becoming the preferred approach to measure DRC, and reliable assays are therefore increasingly needed. The use of a cellular phenotype comet assay for the nucleotide excision repair (NER) pathway using benzo[a]pyrene diol epoxide (BPDE) has been described in previous papers, but no thorough evaluation of its applicability in large genotype-phenotype studies has been presented. Our aim was to evaluate the possibility of using cryopreserved instead of fresh peripheral blood mononuclear cells (PBMCs) to evaluate intra- and inter-assay variation, and inter-individual variation, for the aphidicolin (APC)-block NER comet assay. Moreover, we measured the variation for the designated internal standard (K562 erythroleukaemia cell line) and we evaluated the feasibility to use lymphoblastoid cell lines (LCLs) as surrogate of PBMCs. Our results showed a low intra-assay [coefficient of variation (CV) 19.9%] and inter-assay (CV 32.3%) variation, with a good inter-individual variation (122 subjects, mean ± standard deviation 7.38 ± 4.99; range 0.66-26.14; CV 67.63%). A significant correlation between results derived from cryopreserved and fresh PBMCs from the same individuals was found (10 subjects, r = 0.62, P = 0.05). Results from LCLs and cryopreserved PBMCs from the same subjects showed an inverse significant correlation (10 subjects, r = -0.712, P = 0.02). K562 cells as internal standard showed low intra-assay variation. In the present study the APC-block NER comet assay on cryopreserved PBMCs seemed to be a reliable method to measure DRC variation in epidemiological studies; LCLs were not a good surrogate in this assay.

An overview of the genetic structure within the Italian population from genome-wide data.

In spite of the common belief of Europe as reasonably homogeneous at genetic level, advances in high-throughput genotyping technology have resolved several gradients which define different geographical areas with good precision. When Northern and Southern European groups were considered separately, there were clear genetic distinctions. Intra-country genetic differences were also evident, especially in Finland and, to a lesser extent, within other European populations. Here, we present the first analysis using the 125,799 genome-wide Single Nucleotide Polymorphisms (SNPs) data of 1,014 Italians with wide geographical coverage. We showed by using Principal Component analysis and model-based individual ancestry analysis, that the current population of Sardinia can be clearly differentiated genetically from mainland Italy and Sicily, and that a certain degree of genetic differentiation is detectable within the current Italian peninsula population. Pair-wise F(ST) statistics Northern and Southern Italy amounts approximately to 0.001 between, and around 0.002 between Northern Italy and Utah residents with Northern and Western European ancestry (CEU). The Italian population also revealed a fine genetic substructure underscoring by the genomic inflation (Sardinia vs. Northern Italy = 3.040 and Northern Italy vs. CEU = 1.427), warning against confounding effects of hidden relatedness and population substructure in association studies.

Association between total number of deaths, diabetes mellitus, incident cancers, and haplotypes in chromosomal region 8q24 in a prospective study.

The 8q24 region is a gene desert, although chromosomal aberrations and somatic amplification involving this region, including translocations involving the protooncogene c-MYC, have been frequently reported in people with cancer. To investigate the role of variants in 8q24 region, the authors analyzed data from a prospective study (n = 10,372 participants who were followed for 11 years) in which a large number of health events (>1,500) occurred (1993-1998). They genotyped all subjects for 5 candidate single nucleotide polymorphisms (rs672888, rs1447295, rs9642880, rs16901979, and rs6983267) that were identified in previous genome-wide scans. Although significant associations with individual single nucleotide polymorphisms were small in magnitude, the authors observed higher increases in the risks of different types of cancer with specific haplotypes, particularly when subjects were homozygous for the haplotype: for breast cancer and homozygotes for haplotype CAGCT, hazard ratio = 3.40, 95% confidence interval: 1.24, 9.21; for prostate cancer and grouped rare haplotypes, hazard ratio = 7.43, 95% confidence interval: 3.00, 18.37; and for brain cancer and homozygotes for haplotype CGGCT, hazard ratio = 13.48, 95% confidence interval: 3.00, 59.53. Significant associations were also observed between haplotypes and deaths from cardiovascular diseases and cerebrovascular diseases; the most stable association was between homozygotes for haplotypes CGTCG and CAGCT and total deaths in men (hazard ratio = 3.5, 95% confidence interval: 1.8, 6.9, and hazard ratio = 2.8, 95% confidence interval: 1.3, 6.4, respectively). In conclusion, the authors have observed a strong pleiotropic effect of the 8q24 region in a large prospective study. This observation can shed light on the mechanisms underlying reported associations between 8q24 variants and disparate chronic diseases.

DNA repair gene expression level in peripheral blood and tumour tissue from non-small cell lung cancer and head and neck squamous cell cancer patients.

BACKGROUND: The nucleotide excision repair pathway is crucial for cellular DNA integrity and the ERCC1 helicase is also potentially involved in resistance to platinum-based chemotherapy, and high levels of ERCC1 mRNA in tumours have been associated with cisplatin resistance in different human cancers. The aim of this work was to investigate the correlation between DNA repair gene expression levels in tumour tissue, normal tissue and peripheral blood samples from patients with two common human cancers, non-small cell lung cancer (NSCLC) and squamous cell carcinoma of the head and neck (HNSCC), to test if blood gene expression could be a proxy for tumour tissue gene expression to predict response to platinum-based chemotherapy. METHODS: Using RT-qPCR we determined ERCC1, ERCC2, ERCC4, XPA, XPC, XRCC1, XRCC3, APEX, OGG1, MGMT mRNA levels in fresh NSCLC, normal lung and HNSCC tissue, as well as blood, from NSCLC and HNSCC patients who were treated surgically. RESULTS: Target gene expression in NSCLC and HNSCC tissue was higher than in blood. A statistically significant correlation (p<0.05) was found between target gene mRNA expression in tumour tissue and blood, in particular ERCC1, MGMT, XPC, XRCC1 and XRCC3 in NSCLC and APEX, ERCC1, ERCC2, ERCC4, XRCC1 and XRCC3 in HNSCC. CONCLUSIONS: The existence of a significant correlation between blood and tumour tissue expression of some genes of clinical interest, such as ERCC1 in NSCLC and HNSCC, could allow the introduction in clinical practice of a simple test that would measure mRNA levels of DNA repair genes in peripheral blood samples instead of tissue samples to determine prognostic and predictive factors in NSCLC and HNSCC patients.

Role of the 12q24.12 locus in the onset of preeclampsia: an Italian case-control study.

OBJECTIVE: The 12q24.12 locus has been reported to be involved in the control of many traits and also in severe diseases such as cardiovascular disease, hypertension and some immune-related disease. To our knowledge, no study has been published so far investigating the role of this locus in the pathogenesis of preeclampsia (PE). METHODS: We genotyped four single nucleotide polymorphisms (SNPs) in 12q24.12 locus in 198 preeclamptic, 224 chronic hypertensive and 265 normotensive women from Italy, to test the contribution polymorphisms/haplotypes on the onset of preeclampsia and their association with chronic hypertension. RESULTS: No association was observed for any single SNP, while a common haplotype CGTG (21% in normotensive women) revealed a possible protective effect (OR 0.64, 95% CI 0.42-0.97) against preeclampsia. CONCLUSIONS: Our data suggest that a common haplotype within 12q24.12 locus may be associated with a protective effect against preeclampsia. This observation may be linked with the potential role of this region in the control of microcirculation. To the best of our knowledge, our study is the first one that links the 12q24.12 locus with this life-threatening perinatal complication of unknown etiology. Further physiological and functional studies are needed to clarify the molecular mechanisms and pathways of preeclampsia.

Involvement of MRE11A and XPA gene polymorphisms in the modulation of DNA double-strand break repair activity: a genotype-phenotype correlation study.

DNA double-strand breaks (DSB) are the most lethal form of ionizing radiation-induced DNA damage, and failure to repair them results in cell death. In order to see if any associations exist between DNA repair gene polymorphisms and phenotypic profiles of DSB repair (DSBR) we performed a genotype-phenotype correlation study in 118 young healthy subjects (mean age 25.8±6.7years). Subjects were genotyped for 768 single nucleotide polymorphisms (SNPs) with a custom Illumina Golden Gate Assay, and an H2AX histone phosphorylation assay was done to test DSBR capacity. We found that H2AX phosphorylation at 1h was significantly lower in subjects heterozygous (no variant homozygotes were observed) for the XPA gene SNP rs3176683 (p-value=0.005), while dephosphorylation was significantly higher in subjects carrying the variant allele in three MRE11A gene SNPs: rs1014666, rs476137 and rs2508784 (p-value=0.003, 0.003 and 0.008, respectively). An additive effect of low-activity DNA repair alleles was associated with altered DSBR activity, as demonstrated by both H2AX phosphorylation at 1 h (p-trend <0.0001) and γH2AX dephosphorylation at 3h (p-trend <0.0001). Our study revealed that in addition to SNPs of genes that are well-established players in DSBR, non-DSBR genes, such as the XPA gene that is mainly involved in the nucleotide excision repair pathway, can also influence DSBR in healthy subjects. This suggests that successful DSBR may require both DSBR and non-DSBR mechanisms.