Polymorphisms Of Caspase-8, -10 Genes And Their Relationship With Pathogenesis Of Non-Hodgkin Lymphoma

Apoptosis is one of the most important regulatory mechanisms of tissue homeostasis in organisms. Many human pathologies are associated with aberrant apoptosis. It is now believed that clonal expansion and tumor growth are the results of the deregulation of intrinsic proliferation and apoptosis. Failure of apoptosis could allow the survival of transformed cells that are prone to undergo further genetic damage and play an important role in the pathogenesis of tumors. In recent years, studies of occurrence and regulation of apoptosis suggest that many genes are implicated in the process. A family of cysteine proteases that cleave the substrates at aspartate residues, known as caspases, plays a main role during the execution of apoptosis. Among fourteen members identified so far, caspase-10 and caspase-8 are most closely related. Their genes are located in chromosome 2q33-34 in sequence and they are both initiator caspases in death receptor-mediated apoptosis. Disruption of this pathway may result in illegitimate cell survival and contribute to neoplasia or autoimmune lymphoproliferative syndrome (ALPS). It was reported that germline mutations of either caspase-8 or caspase-10 were directly involved in ALPS II. Non-Hodgkin lymphoma (NHL) is a group of malignant tumors with very complicated cellular morphology, transformed from the process of development of lymphocytes. There are accumulating evidences that NHL cells are often resistant to the death receptor-mediated apoptosis by several mechanisms, including inactivating mutations of the genes involved in this pathway. Somatic mutations of CD95 and caspase-10 have been reportedin NHL. caspase-8 mutations have been detected in colorectal cancer tissues and some cancer cell lines. However, data on caspase-8 mutation in cancer tissues are few, whether caspase-8 mutation exists in NHL is not determined. In addition, the previous loss of heterozygosity (LOH) studies suggested that chromosome 2q33-34, where caspase-10 and -8 genes reside, was deleted in several tumors, such as NHL, neuroblastoma (NB), gastric cancer and so on. It is necessary to explore the possibility that alterations of the caspase-8 and -10 gene might play a role in NHL development.As a complex disease, the evolution of NHL is a multi-step, multi-phase and multi-factor process. Study on polymorphisms of caspase family genes is an important content of disease genomics in NHL. Single nucleotide polymorphisms (SNPs) in regulatory regions could affect expression of mRNA and proteins. SNPs in coding regions could change structures and functions of proteins. They greatly decide phenotype variation of human. A tremendous amount of SNPs exists in the human genome with high density, but their patterns of distribution differ considerably among populations. To date, there is no report on polymorphisms of caspase-8, -10 genes in healthy people of Han nationality or on association analysis between SNPs in caspase-8, -10 genes and complex diseases. Denaturing high performance liquid chromatography (DHPLC) is a novel high-throughput technique for screening DNA variations. It can be highly sensitive to detect mutations, SNPs, LOH and make genotype analysis. In this study, we performed PCR, DHPLC, DNA sequencing to analyse the entire coding region and their flanking sequences of caspase-8, -10 genes in healthy people and NHL patients of Han nationality and to determine whether SNPs and mutations of these genes could be involved in NHL development.Materials and Methods1. Materials(1) Healthy people group: 140 apparently healthy and unrelated individuals of Han nationality in Zhejiang province were collected, of which males accounted for 61.4% and females accounted for 38.6%. Their average age was 48 16 years old. Genomic DNA was preparedfrom 2ml peripheral blood by high salt method.(2) NHL patients group: 84 cases of NHL were obtained from affiliated hospitals of Zhejiang University and other local hospitals from the year of 1997 to 2003. Males accounted for 65.5% and females accounted for 34.