Association of gene polymorphisms and XPD expression on risk of nasopharyngeal carcinoma and survival among Malaysians

Nasopharyngeal carcinoma (NPC) is a rare form of cancer across the world except in certain areas such as Southern China, Hong Kong and Malaysia. NPC is the 4th most common cancer in Malaysia and the incidence rate for Malaysian Chinese is exceptionally high compared to other races. NPC is considered as a relatively radiosensitive tumor and patients diagnosed at early stages tend to survive longer compared to those who with advanced disease. Given that early symptoms of NPC are non-specific and that the nasopharynx is relatively inaccessible, less invasive screening methods such as biomarker screening might be the key to improve NPC survival and management. A matched case-control study was conducted to investigate the effect of hOGG1 Ser326Cys, ITGA2 C807T, TNF-α -308G>A and XPD Lys751Gln polymorphisms on the risk of nasopharyngeal carcinoma and all-cause survival. The association of XPD Lys751Gln polymorphism with XPD mRNA expression was investigated in order to substantiate the finding of significant association between XPD Lys751Gln polymorphism with NPC risk. A number of genes with their respective polymorphisms were shown in past studies to be associated with various cancers. hOGG1 and XPD genes encode for a DNA glycosylase and a DNA helicase respectively; both are proteins that are involved in DNA repair. ITGA2 is the alpha subunit of the transmembrane receptor integrin and is mainly responsible for cell-cell and cellextracellular matrix interaction. TNF-α is a cytokine that is released by immune cells during inflammation. In the present study, NPC cases and controls were matched by age, gender and ethnicity. Restriction fragment length polymorphism-polymerase chain reaction (RFLP-PCR) was used to process DNA genotyping studies involving all aforementioned gene polymorphisms. Conditional logistic regression was used for the analysis of NPC risk on gene polymorphisms, controlling for cigarette smoking, salted fish and alcohol consumption. Quantitative real-time PCR was utilized to process the XPD expression. 2-ΔΔCt relative expression fold-change method was chosen for the analysis of expression study. Linkage disequilibrium and haplotype analysis were conducted to explore the association of allele combinations arising from all aforementioned gene polymorphisms with NPC risk. Kaplan-Meier survival function, log-rank test, and Cox regression were used to investigate the effect of gene polymorphisms on the all-cause survival of NPC. XPD homozygous wildtype Lys/Lys genotype was associated with increased NPC risk (adjusted OR=1.65, 95% CI=1.09- 2.50). No association was found between ITGA2 and TNF-α polymorphism on NPC risk. Lys/Lys genotype of XPD polymorphism was associated with reduced XPD expression. Interaction between gene polymorphisms showed that Ser/Gln (hOGG1- Ser326/XPD-Gln751) (adjusted OR=2.18, 95% CI=1.00-4.75), Ser/T (hOGG1- Ser326/ITGA2-T807) (adjusted OR=1.48, 95% CI=1.02-2.16) and G/Gln (TNF-α- G308/XPD-Gln751) (adjusted OR=1.59, 95% CI=1.07-2.35) allelic combinations conferred higher risk of NPC. 5-year survival rates for ITGA2 807 C/C, C/T and T/T carriers were 55 %, 50 % and 43 %, respectively. The application of XPD Lys751Gln and ITGA2 C807T polymorphisms in early NPC detection and prognostic prediction for NPC survival is not warranted at the current moment and the finding should be subjected to further validation and testing involving studies with larger sample size

Homozygous Wildtype of XPD K751Q Polymorphism Is Associated with Increased Risk of Nasopharyngeal Carcinoma in Malaysian Population

<div><p>The xeroderma pigmentosum group D (XPD) gene encodes a DNA helicase, an important component in transcription factor IIH (TFIIH) complex. XPD helicase plays a pivotal role in unwinding DNA at the damaged region during nucleotide excision repair (NER) mechanism. Dysfunctional XPD helicase protein from polymorphic diversity may contribute to increased risk of developing cancers. This study aims to determine the association between XPD K751Q polymorphism (rs13181) and risk of nasopharyngeal carcinoma (NPC) in the Malaysian population. In this hospital-based matched case-control study, 356 controls were matched by age, gender and ethnicity to 356 cases. RFLP-PCR was used to genotype the XPD K751Q polymorphism. A significant association was observed between XPD K751Q polymorphism and the risk of NPC using conditional logistic regression. Subjects with homozygous Lys/Lys (wildtype) genotype have 1.58 times higher odds of developing NPC compared to subjects with recessive combination of heterozygous Lys/Gln and homozygous Gln/Gln genotypes (OR = 1.58, 95% CI = 1.05–2.38 p = 0.028) adjusted for cigarette smoking, alcohol and salted fish consumption. Our data suggests that Lys/Lys (wildtype) of XPD K751Q contributes to increased risk of NPC in the Malaysian population.</p></div

Partial sequence chromatograms of Lys751Gln polymorphism from study subjects.

<p>Arrow indicates the location of the nucleotide changes. Partial sequence chromatogram (A) represents Lys/Lys genotype, partial sequence chromatogram (B) represents Lys/Gln genotype and partial sequence chromatogram (C) represents Gln/Gln genotype.</p

Gel electrophoresis of PCR-RFLP products for representative blood samples for the XPD Lys751Gln polymorphism.

<p>Lane M represents low range DNA ladder marker (Fermentas), lanes 1 and 4 represent Lys/Lys genotype (102 bp and 82 bp), lanes 2 and 5 represent Lys/Gln genotype (184bp, 102 bp and 82 bp), lanes 3 and 6 represent Gln/Gln genotype (184 bp), lane 7 represents negative control (RFLP reaction without PCR product) and lane 8 represents negative control (RFLP reaction without restriction enzyme, <i>MboII</i>).</p

PCR-RFLP assay of XPD K751Q polymorphism.

<p>PCR-RFLP assay of XPD K751Q polymorphism.</p

Characteristics of the nasopharyngeal carcinoma cases and controls.

<p>Characteristics of the nasopharyngeal carcinoma cases and controls.</p

Frequency of XPD K751Q recessive model genotypes and association with NPC.

<p>^aOR: odds ratio</p><p>^bCI: confidence interval</p><p>^cAdjusted: age, gender, ethnicity, salted fish consumption, cigarette smoking and alcohol consumption</p><p>Frequency of XPD K751Q recessive model genotypes and association with NPC.</p

Allelic and genotype frequencies of XPD K751Q polymorphism (Hardy-Weinberg equilibrium test).

<p>Allelic and genotype frequencies of XPD K751Q polymorphism (Hardy-Weinberg equilibrium test).</p

Haplotype CGC from XPD, hOGG1 and ITGA2 polymorphisms increases the risk of nasopharyngeal carcinoma in Malaysia

<div><p>Background</p><p>8-oxoG, a common DNA lesion resulting from reactive oxygen species (ROS), has been shown to be associated with cancer initiation. hOGG1 DNA glycosylase is the primary enzyme responsible for excision of 8-oxoG through base excision repair (BER). Integrins are members of a family of cell surface receptors that mediate the cell-cell and extracellular matrix (ECM) interactions. Integrins are involved in almost every aspect of carcinogenesis, from cell differentiation, cell proliferation, metastasis to angiogenesis. Loss of ITGA2 expression was associated with enhanced tumor intravasation and metastasis of breast and colon cancer. XPD gene encodes DNA helicase enzyme that is involved in nucleotide excision repair (NER). It is shown in previous research that XPD homozygous wildtype Lys/Lys genotype was associated with higher odds of NPC.</p><p>Methods</p><p>We conducted a 1 to N case-control study involving 300 nasopharyngeal carcinoma (NPC) cases and 533 controls matched by age, gender and ethnicity to investigate the effect of hOGG1 Ser326Cys, ITGA2 C807T and XPD Lys751Gln polymorphisms on NPC risk. Linkage disequilibrium and haplotype analysis were conducted to explore the association of allele combinations with NPC risk. Restriction fragment length polymorphism (RFLP-PCR) was used for DNA genotyping.</p><p>Results</p><p>No significant association was observed between hOGG1 Ser326Cys and ITGA2 C807T polymorphisms with NPC risk after adjustment for age, gender, ethnicity, cigarette smoking, alcohol and salted fish consumption. Lys/Lys genotype of XPD Lys751Gln polymorphism was associated with increased NPC risk (OR = 1.60, 95% CI = 1.06–2.43). Subjects with history of smoking (OR = 1.81, 95% CI = 1.26–2.60), and salted fish consumption before age of 10 (OR = 1.77, 95% CI = 1.30–2.42) were observed to have increased odds of NPC. The odds of developing NPC of CGC haplotype was significantly higher compared to reference AGC haplotype (OR = 2.20, 95% CI = 1.06–4.58).</p><p>Conclusion</p><p>The allele combination of CGC from hOGG1, ITGA2 and XPD polymorphisms was significantly associated with increased odds of NPC.</p></div

Linkage disequilibrium between hOGG1 Ser326Cys, ITGA2 C807T and XPD Lys751Glu polymorphisms (D’).

<p>Linkage disequilibrium between hOGG1 Ser326Cys, ITGA2 C807T and XPD Lys751Glu polymorphisms (D’).</p