Genetic polymorphisms in cell cycle regulatory genes MDM2 and TP53 are associated with susceptibility to lung cancer

The tumor suppressor TP53 pathway plays a crucial role in preventing carcinogenesis through its ability to impose cell cycle arrest and apoptosis following DNA damage and oncogene activation. MDM2 is a key negative regulator of the TP53 pathway and is overexpressed in many cancers as oncoprotein. We investigated the association between genetic variation in the promoter region of MDM2 (c.-5+309G>T, rs2279744:g.G>T) and the coding region of TP53 (c.215G>C, rs1042522:g.G>C, designated Arg72Pro) and the risk of developing lung cancer. The genotypes of 1,106 patients and 1,420 controls were determined by tetra-primer amplification refractory mutation system (ARMS)-PCR or PCR-based restriction fragment length polymorphism (RFLP). Associations with risk of lung cancer were estimated by logistic regression. We observed an increased lung cancer risk associated with the MDM2 GG (odds ratio [OR] = 1.83, 95% confidence interval [CI] = 1.45-2.32) and TG (OR = 1.33, 95% CI = 1.09-1.63) genotypes. An increased risk was also associated with the TP53 Pro/Pro genotype (OR = 1.47, 95% CI = 1.17-1.85, P = 0.003) compared to the Arg/Arg genotype. The gene-gene interaction of MDM2 and TP53 polymorphisms increased lung cancer risk in a supermultiplicative manner (OR for the presence of both MDM2 GG and TP53 Pro/Pro genotypes = 4.56, 95% CI = 2.76-7.54). Significant interactions were observed between these polymorphisms (respectively and jointly) and smoking (OR = 10.41, 95% CI = 5.26-20.58) for smokers with both the MDM2 GG and TP53 Pro/Pro genotypes. In conclusion, genetic polymorphisms in cell cycle regulatory genes MDM2 and TP53 contribute to the risk of developing lung cancer.     

Polymorphisms of the TP53 codon 72 and WRN codon 1367 in individuals from Northern Brazil with gastric adenocarcinoma

Abstract Gastric cancer is the second most frequent type of neoplasia and also the second most common cause of death in the world. TP53 codon 72, which produces variant proteins with an arginine (Arg) or proline (Pro), has been reported to be associated with cancers of the lung, oesophagus, stomach and cervix. Werner’s syndrome (WS) is a premature ageing disease caused by a mutation in the WRN gene. The WRN protein acts as a DNA helicase and as an exonuclease. WRN codon 1367 produces variant proteins with an Arg or cysteine (Cys). This polymorphism has been studied, in order to understand the clinical impact of the molecular variants in WS and in age-related disorders. In the present study, the TP53 codon 72 and the WRN codon 1367 polymorphisms were investigated in 54 gastric adenocarcinoma patients (31 diffuse-type and 25 intestinal-type) and 54 controls. DNA samples were extracted, and PCR-RFLP was utilised for genotyping TP53 codon 72 and WRN codon 1367. The allele frequencies of the TP53 polymorphism were: Arg=0.74 and Pro=0.26. The allele frequencies of the WRN polymorphism were: Cys=0.73 and Arg=0.27. The crude genotypic frequencies in gastric cancer patients were similar to those of the controls, but in the WRN codon 1367 polymorphisms the mean age tended to be higher in the Arg/Arg genotypes. There also was an association, although not statistically significant, between the presence of Helicobacter pylori and the genotypes Cys/Cys and Cys/Arg and a higher percentage of cardia cancer among the Arg/Arg genotypes, and of non-cardia cancer among genotypes Cys/Cys and Cys/Arg. These findings may be a reflection of differences in the interaction between WRN codon 1367 polymorphisms and local factors in the stomach. To our knowledge, this is the first study to examine a genetic polymorphism of the WRN gene in cancer. The precise mechanisms of action of the TP53 and WRN polymorphisms involved in the aetiopathogeny of this disease need further investigation.     

A TP53-truncating germline mutation (E287X) in a family with characteristics of both hereditary diffuse gastric cancer and Li-Fraumeni syndrome

Mutations in CDH1, which encodes E-cadherin, have been associated with hereditary diffuse gastric cancer (HDGC) in Western populations but have not been shown to play a major role in Asians. Recently, a patient with familial gastric cancer (FGC) was shown to harbor a germline mutation in the TP53 gene, which encodes p53 and has been previously associated with Li-Fraumeni Syndrome (LFS). To determine whether mutations in TP53 are associated with FGC in Asians, we screened the entire coding region of TP53 in probands from 23 Korean FGC families. We identified a nonsense (E287X) TP53 germline mutation in a family whose history is compatible with both HDGC and LFS. Two members of this family (SNU-G2) were afflicted with brain tumors, seven with gastric cancers, two with sarcomas, and one with both gastric cancer and a sarcoma. The E287X TP53 mutation segregated with the cancer phenotype in the family members from whom DNA samples were available. To our knowledge, this is the first report of a large family with both HDGC and LFS. Our results suggest that TP53 mutational screening in FGC families should be interpreted with caution because additional TP53 mutation-carrying HDGC families may also show LFS-related phenotypes.     

TP53 polymorphisms in gliomas from Indian patients: Study of codon 72 genotype, rs1642785, rs1800370 and 16 base pair insertion in intron-3

Several single nucleotide polymorphisms of the TP53 gene have been reported, amongst which polymorphism in codon 72 (rs1042522) has received significant attention and shown to be associated with disease susceptibility in different cancer types. However, there are variable reports on this polymorphism in gliomas from worldwide with inconsistent results. In addition, the implications of other polymorphic loci are not much explored in gliomas. Hence, in the present study the TP53 sequence was analyzed for all polymorphism and mutations in a total of 84 gliomas of different types and grades from patients of Indian origin. The complete sequence of all coding exons (2 to 11) and introns 2, 3, 5 and 8 of TP53 gene were studied while for introns 1, 4, 6, 7, 9 and 10, only exon flanking regions could be studied. The polymorphic loci were compared with control population. In addition to the well known codon 72 polymorphism (rs1042522), three other polymorphisms rs1642785, rs1800370 and a 16 base pair insertion in intron-3 were found. At codon 72, our study showed higher Arg/Arg genotype in gliomas compared to normal population (38% versus 13%). The Arg allele frequency in glioma patients was comparatively higher than controls (0.55 versus 0.45; P = 0.037). The Arg allele frequency was also high in adult glioblastomas compared to paediatric counterparts (0.55 versus 0.36). However, there was no significant association of TP53 mutations with any genotype of codon 72. At rs1642785, the G allele frequency was significantly higher in gliomas than in control population (0.55 versus 0.36, P = 0.005). The genotype at a 16 base pair insertion in intron-3 was almost similar in case and control. However, the polymorphism at rs1800370 was exclusive to gliomas. This is the first report of TP53 gene polymorphism in glioma patients from India. Our study also delineates the frequency of four polymorphisms in gliomas for the first time. The codon 72 variant (rs1042522) and rs1642785 polymorphisms possibly poses risk to glioma development in Indian population. However, the functional significance of these polymorphism needs further elucidation.     

Polymorphisms in apoptosis-related genes and TP53 mutations in non-small cell lung cancer

Apoptosis plays an essential role in the elimination of mutated or transformed cells from the body. Therefore, polymorphisms of apoptosis-related genes may lead to an alteration in apoptotic capacity, thereby affecting the occurrence of TP53 mutations in lung cancer. We investigated the relationship between potentially functional polymorphisms of apoptosis-related genes and TP53 mutations in non-small cell lung cancer (NSCLC). Twenty-seven single nucleotide polymorphisms in 20 apoptosis-related genes were genotyped by a sequenome mass spectrometry-based genotyping assay in 173 NSCLCs and the associations with TP53 mutations in the entire coding exons (exons 2-11), including splicing sites of the gene, were analyzed. None of the 27 polymorphisms was significantly associated with the occurrence of TP53 mutations. This suggests that apoptosis-related genes may not play an important role in the occurrence of TP53 mutations in lung cancer.     

Higher‐than‐expected population prevalence of potentially pathogenic germline TP53 variants in individuals unselected for cancer history

Li–Fraumeni syndrome (LFS) is an autosomal‐dominant cancer predisposition disorder associated with pathogenic germline variants in TP53, with a high penetrance over an individual's lifetime. The actual population prevalence of pathogenic germline TP53 mutations is still unclear, most likely due to biased selection of cancer affected families. The aim of this study was to estimate the population prevalence of potentially pathogenic TP53 exonic variants in three sequencing databases, totaling 63,983 unrelated individuals. Potential pathogenicity was defined using an original algorithm combining bioinformatic prediction tools, suggested clinical significance, and functional data. We identified 34 different potentially pathogenic TP53 variants in 131 out of 63,983 individuals (0.2%). Twenty‐eight (82%) of these variants fell within the DNA‐binding domain of TP53, with an enrichment for specific variants that were not previously identified as LFS mutation hotspots, such as the p.R290H and p.N235S variants. Our findings reveal that the population prevalence of potentially pathogenic TP53 variants may be up to 10 times higher than previously estimated from family‐based studies. These results point to the need for further studies aimed at evaluating cancer penetrance modifiers as well as the risk associated between cancer and rare TP53 variants.     

High-throughput amplicon scanning of the TP53 gene in breast cancer using high-resolution fluorescent melting curve analyses and automatic mutation calling

Identifying mutations in the TP53 gene is important for cancer prognosis, predicting response to therapy, and determining genetic risk. We have developed a high-throughput scanning assay with automatic calling to detect TP53 mutations in DNA from fresh frozen (FF) and formalin-fixed paraffin-embedded (FFPE) tissues. The coding region of the TP53 gene (exons 2-11) was PCR-amplified from breast cancer samples and scanned by high-resolution fluorescent melting curve analyses using a 384-well format in the LightCycler 480 instrument. Mutations were confirmed by direct sequencing. Sensitivity and specificity of scanning and automatic mutation calling was determined for FF tissue (whole genome amplified [WGA] and non-WGA) and FFPE tissue. Thresholds for automatic mutation calling were established for each preparation type. Overall, we confirmed 27 TP53 mutations in 68 primary breast cancers analyzed by high-resolution melting curve scanning and direct sequencing. Using scanning and automatic calling, there was high specificity (>95%) across all DNA preparation methods. Sensitivities ranged from 100% in non-WGA DNA from fresh tissue to 86% in WGA DNA and DNA from formalin-fixed, paraffin-embedded tissue. Scanning could detect mutated DNA at a dilution of 1:200 in a background of wild-type DNA. Mutation scanning by high-resolution fluorescent melting curve analyses can be done in a high-throughput and automated fashion. The TP53 scanning assay can be performed from a variety of specimen types with high sensitivity/specificity and could be used for clinical and research purposes.     

TP53 gene mutations of lung cancer patients in upper northern Thailand and environmental risk factors

TP53 mutations are observed in about 40–70% of lung cancer tissues, and the hot spot codon mutations are in exons 5 through 8. Previous studies revealed that the distinct TP53 mutational pattern between population groups may be due to different racial or exogenous factors. This research aims to identify risk factors that influence TP53 gene mutation in lung cancer patients residing areas with high lung cancer incidence, in the upper northern part of Thailand. Germline TP53 mutational analyses were also performed to determine the inherited cancer predisposition. Exons 5–8 of the TP53 gene were analyzed by sequencing DNA of cancerous tissue and peripheral blood leukocyte samples from 55 non–small lung cell cancer patients. The results showed that the TP53 germline mutation was not found in all patients, indicating that the TP53 germline mutations were not exclusively responsible for cancer predisposition in this group of lung cancer patients. A total of 19 somatic mutations were found in 18 patients. Mutations were predominantly found in exons, with only 10.53% observed at the splice sites of intron 7. No characteristic hot spot codons were observed. The data suggest that TP53 mutations in this study group are induced by exposure to substances other than tobacco smoke. Pesticide exposure or habitation in poorly ventilated houses may instead be related to the tumorigenesis of lung cancer via TP53 mutations.     

Polymorphisms in TP53 are associated with risk and survival of osteosarcoma in a Chinese population

Osteosarcoma (OS) is the most frequent histological form of primary bone cancer in adolescence. TP53 is a tumor suppressor gene which is essential for regulating cell division and preventing tumor formation. The purpose of this study is to examine whether genetic mutations in the TP53 gene are associated with OS risk and survival in a Chinese population. Five polymorphisms in the TP53 gene were selected in a case-control study, including 210 OS patients and 420 cancer-free controls. We found that subjects carrying rs12951053 CC genotype and rs1042522 GG genotype were significantly associated with risk of OS [odds ratio (OR) = 1.68, 95% confidence intervals (CI): 1.05-2.68; OR = 1.89, 95% CI: 1.16-3.07] compared with subjects carrying the common genotypes. Results of haplotype analysis also showed that A-G-G-A-C haplotype (rs12951053, rs1042522, rs8064946, rs9895829 and rs12602273) conferred significant decreased risk of OS (OR = 0.37, 95% CI: 0.19-0.72) compared with A-C-G-A-C haplotype. Besides, rs1042522 was an independent prognostic factor for OS with hazard radio (HR) = 1.94 (95% CI: 1.03-3.65) in GG genotype than in CC genotype. Our data suggest that genetic mutations in the TP53 gene are associated with risk and survival of OS in Chinese population.     

TP53基因多态与结直肠癌肝转移遗传易感性的相关性

目的 探讨TP53基因C-8343G、C-1863T及第72密码子(R72P)单核苷酸多态性与结直肠癌(colorectal cancer,CRC)肝转移风险的关系.方法 采用TaqMan和聚合酶链反应-限制性片段长度多态性方法,检测121例伴肝转移CRC与性别、年龄匹配的280例不伴肝转移CRC的各单核苷酸多态性的基因型分布及差异.结果 C-8343G和C-1863T基因型分布在伴和不伴肝转移的两组CRC人群间差异均无统计学意义.R72P增加CRC肝转移的发生风险:与PP基因型相比,RP基因型、RR基因型和R等位基因携带者(RP或RR基因型)的肝转移风险分别增加至2.21倍(95%CI:1.13～4.33)、2.26倍(95%CI:1.03～4.94)和2.22倍(95%CI:1.16～4.26).CRC组织中P53表达状态的分层分析结果显示:对于P53表达阳性者,72R携带者的肝转移风险与PP基因型相比进一步增加至3.28倍(95%CI:1.21～8.88);而对于P53表达阴性者,PP基因型与72R携带者的肝转移风险差异无统计学意义(比值比为1.37,95%CI:0.52～3.62).结论 TP53增加CRC,特别是P53表达阳性CRC的肝转移风险,可作为CRC肝转移高危人群的筛选指标;C-8343G和C-1863T可能均与CRC肝转移风险无关.     

TP53 mutations in astrocytic gliomas: an association with histological grade,TP53 codon 72 polymorphism and p53 expression

TP53 mutations and polymorphisms have been widely related to many cancers as long as these alterations may impair its capacity to induce cell cycle arrest, DNA repair mechanisms, and apoptosis. Although TP53 alterations have been studied in astrocytic tumors, there is a lack of analysis considering specific TP53 mutations and their associations with p53 immunostainning, polymorphisms and their significance among the histological grades. Thus, we analyzed TP53 alterations in exons 2–11, including the codon 72 polymorphism, using DNA sequencing in 96 astrocytic gliomas (18 grade I, 20 grade II, 14 grade III, and 44 grade IV). Also, immunohistochemistry was assessed to evaluate the p53 protein expression. In this study, we found that the higher histological grades were statistically associated with TP53 mutations. Some of these mutations, such as TP53 P98T and TP53 G244S, seemed to be a specific marker for the higher grades, and the TP53 E286K mutation appears to be a World Health Organization grade III–IV progression marker. Also, the TP53 P98T mutation, in exon 4, is very likely to be important on the stabilization of the p53 protein, leading to its immunopositivity and it is potentially associated with the TP53 72Pro/Pro genotype.     

Lack of association between the unique LMP2 gene polymorphism and the outcome of lung cancer in a population of Chinese Han nationality

Lung cancer is characterized by a widely ranging incidence variation; it is the most common cancer in China. In this study we will assess the association of low-molecular-mass protease 2 (LMP2) gene codon 60 polymorphism with the risk of lung cancer. Genomic DNA of peripheral blood mononuclear cells was isolated from 207 patients with lung cancer and 264 healthy controls. DNA direct sequencing and polymerase chain reaction-restriction fragment length polymorphism were performed to scrutinize LMP2 gene codon 60 polymorphism. The risk of LMP2 gene polymorphism in lung cancer was assessed using an unconditional logistic regression model adjusted by the confounding factors. As a result of DNA direct sequencing, the LMP2 codon 60 polymorphic substitution of the nucleotide was CGC → TGC in Chinese individuals, not CGC → CAC as reported in other ethnic populations. In histology-specific analysis and TNM stages, there was no apparent association between this LMP2 gene polymorphism and any of the histologic types or TNM stages of lung cancer using the Arg/Arg genotypes as the reference group (all p values > 0.05). These results suggest that the polymorphic site is unique in the Chinese population of Han nationality at the LMP2 codon 60 loci (Arg60Cys), but a lack of association with lung cancer exists.     

Genetic variants in <Emphasis Type="Italic">PCSK9</Emphasis> affect the cholesterol level in Japanese

Mutations in the proprotein convertase subtilisin/kexin 9 (PCSK9) gene have been reported in affected members of two families with autosomal dominant hypercholesterolemia. To investigate the effects of common variants in PCSK9 on the cholesterol level, we conducted an association study using a large cohort representing the general population in Japan (n=1,793). Direct sequencing in all of the exonic regions identified 21 polymorphisms. After consideration of linkage disequilibrium among these polymorphisms, we selected and genotyped nine polymorphisms by the TaqMan method. The intron 1/C(-161)T and exon 9/I474 V polymorphisms were associated with levels of total cholesterol (TC) [C(-161)T, P=0.0285; I474 V, P=0.0069] and low-density lipoprotein cholesterol (LDL-C) [C(-161)T, P=0.0257; I474 V, P=0.0007]. The distributions of these polymorphisms in subjects with miocardial infarction (MI) (n=649) were not different from those in the control population. These results provide the first evidence that common variants intron 1/C(-161)T and exon 9/I474 V in PCSK9 significantly affect TC and LDL-C levels in the general population in Japan.     

Association of the <Emphasis Type="Italic">HTRA1</Emphasis> gene variant with age-related macular degeneration in the Japanese population

The purpose of this investigation was to determine whether the high-temperature requirement A-1 (HTRA1) gene polymorphism is associated with age-related macular degeneration (AMD) in native, unrelated Japanese patients. A total of 123 patients with AMD and 133 control subjects without AMD were recruited for this study. The single-nucleotide polymorphism (SNP) rs11200638 in the HTRA1 gene was assessed using a TaqMan assay. The risk A allele frequencies in the AMD cases and control patients were 0.577 and 0.380, respectively, and were associated with a significant risk of developing AMD (p=7.75x10(-6)). The results were more significant in subtype analyses with wet AMD (p=5.96x10(-7)). We conclude that the rs11200638 variant in the HTRA1 gene is strongly associated with AMD in the Japanese population. This result supports the hypothesis that the HTRA1 gene may increase susceptibility to AMD development and can participate in a potential new molecular pathway for AMD pathogenesis by extending this association across diverse ethnicities.     

Genetic polymorphisms of TP53-binding protein 1 (TP53BP1) gene and association with breast cancer risk

In the present study, we evaluated the association between the TP53BP1 Glu353Asp and T-885G polymorphisms and breast cancer risk as well as with the clinicopathological characteristics of the patients. Genotyping of these polymorphisms was performed on 387 breast cancer patients and 252 normal and healthy women who had no history of any malignancy using PCR-RFLP method in a hospital-based Malaysian population. Breast cancer risk was not observed among women who were heterozygous (OR(adj) = 0.887; 95% CI, 0.632-1.245) or homozygous (OR(adj) = 1.083; 95% CI, 0.595-1.969) for Asp allele, and those carriers of Asp allele (OR(adj) = 0.979; 95% CI, 0.771-1.243). Similarly, women who were TG heterozygotes (OR(adj) = 1.181; 95% CI, 0.842-1.658) or GG homozygotes (OR(adj) = 1.362; 95% CI, 0.746-2.486) and carriers of G allele (OR(adj) = 1.147; 95% CI, 0.903-1.458) were not associated with increased risk of breast cancer. Asp allele genotype was significantly associated with ER negativity (p = 0.0015) and poorly differentiated tumours (p = 0.008), but G allele genotype was not associated with the clinicopathological characteristics. In conclusion, Glu353Asp and T-885G polymorphic variants might not have an influence on breast cancer risk, thus might not be potential candidates for cancer susceptibility. Glu353Asp variant might be associated with tumour aggressiveness as defined by its association with ER negativity and poorly differentiated tumours.     

The association study between CYP24A1 gene polymorphisms and risk of liver,lung and gastric cancer in a Chinese population

Recently, four single nucleotide polymorphisms (rs2585428, rs4809960, rs6022999 and rs6068816) in CYP24A1 gene were extensively studied for their associations with cancer risk. However, these studies included only a few types of cancer, which calls for further investigations. In view of this, we here conducted a case-control study to explore the associations between these four CYP24A1 gene polymorphisms and risk of liver, lung and gastric cancer in a Chinese population. A total of 480 liver cancer patients, 550 lung cancer patients, 460 gastric cancer patients and 800 normal controls were recruited in this study. The genotyping of CYP24A1 gene polymorphisms was applied with Sanger sequencing assay. Single-locus analysis demonstrated that rs6022999 was significantly associated with risk of liver and lung cancer, while rs6068816 was significantly associated with the risk of gastric cancer. Haplotype analysis revealed that haplotype GTAT was associated with an increased risk of liver cancer and a decreased risk of lung cancer, and haplotype ATGC was associated with a decreased risk of lung cancer. The further meta-analysis of rs6068816 and lung cancer risk showed that rs6068816 was not associated with lung cancer risk in Chinese population, which confirmed our present finding. Conclusively, rs6022999 may be a genetic biomarker for liver and lung cancer susceptibility in Chinese population, and rs6068816 may be used to predict gastric cancer risk in Chinese population.