| Background: Hirschsprung's disease (HD) is a common congenital malformation affecting 1 in 5000 live births, which also named congenital aganglionsis and occupies the second in the congenital intestinal malformation. The proportion of cases between male and female is 4:1. Beyond 3.8-7.8% of all HD cases are of familial origin, the majority of HD is sporadic patients. As a complex disease, HD has been ascribed to the absence in the terminal hindgut of ganglion cells from the neural crest, which causes the abnormal contraction of involved intestines, and then the proximal end of the sick colon appears compensatory dilated thickness and forms megacolon.Although the pathogenesis of HD has not been fully understood, the familial occurrence with an increased risk for siblings as well as an uneven gender distribution indicates a genetic cause to the disease. Recently, RET pro-oncogene, the endothelin-3 gene and the endothelin receptor-B gene and so on have been considered as main genes which linked to HD in human. In 1993, Genetic mapping in multiplex families and the mutational analysis of candidate genes have led to the definitive identification of the defects that contribute to the HD risk. It has been found that the gene defects present in a major proportion of Hirschsprung's disease families are mutations in chromosome 10q11.2, which have now been found to be associated with RET gene. It's reported that RET mutations have been identified in approximately50% of the familial cases of HD and in 10%-20%of the sporadic cases abroad.The human RET proto-oncogene comprises 20 exons, with the length about 80kb. A receptor tyrosine kinase encoding by RET proto-oncogene consists of an intracellular tyrosine kinase domain, a transmembrane domain and an extracellular domain which includes a "cadherin-like" region. Receptor tyrosine kinases generally function as ligand dependent dimmers, which phosphorylate "second messenger" proteins in the cytoplasm and they are commonly associated with regulation of cell growth and differentiation, the development of normal nerves, and expressed in gangliogenic source cells (such as neurogenic ganglia and ganglia of peripheral nerve system, neuroendocrine cells, epidermic pigment cells, etc.). RET proto-oncogene is controlled by a promoter harboring four randomly repeated 5'-CG-3' sequences that are part of a 5'-CG-3' dense region in proximity to the transcriptional start site. In a region of about 900 base pairs, 95 unmethylated 5'-CG-3' dinucleotides are located of which the majority lies within the putative promoter segment. During embryogenesis, RET proto-oncogene expression is regulated in a temporally and spatially defined pattered. In adults, RET mRNA transcripts can be detected exclusively in the substantia nigra, the adrenal medulla, cerebellum and other parts of the brain. Through detailed examination, more than 60 missense mutations or deletions were found along the whole coding sequence of RET in HD.Objective: To investigate the relationship between mutations of RET proto-oncogene and Chinese patients with Hirschsprung's disease (HD), and to elucidate the genetic mechanism of familial HD patient at the molecular level. Methods: Genomic DNA was extracted from venous blood of 48 sporadic HD patients and 30 normal children. Polymerase chain reaction (PCR) products, which were amplified using specific primers (RET, exons 11, 13, 15, 17), were electrophoresised to analyze the single-strand conformational polymorphism (SSCP)patterns. The positive amplifying products were sequenced. Other probands and their relatives in two genealogies were screened for mutations of RET proto-oncogene simultaneously.Results: Mutation changes were detected in 8 of 48 sporadic HD patients in RET proto-oncogene. But in 30 normal individual no mutation changes were observed. This mutation rate in the gene was estimated at 16.7% (8/48). DNA sequence analysis showed that there were 3 patterns of nucleotide change, missense mutation (4 cases), frameshift mutation (2 cases) and silent mutat...
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