The Research On The Mutation Feature Of Exon 13 Of RET Gene In Chinese Hirschsprung's Disease

BACKGROUND and AIMHirschsprung's disease (HD) is characterized by a congenital absence of enteric ganglia along a variable length of colon. The incidence of Hirschsprung's disease is 1/5000 births which is second morbidity in total congenital malformation disease in the world. Twenty percent of patients with Hirschsprung's disease is considered to be genetically caused, moreover , it's inheritability is 80%. Although patterns of inheritance in HD is different, the most of HD is classified to be multigened disease.HD's pathogenesis remains uncertain till now. Both RET and EDNRB gene mutations exist in HD, RET gene plays an important role in HD. RET gene encodes a 80 KD protein which is one of the receptor tyrosines of cell-surface that transmit signals for cell growth and differentiation. RET gene are required for ganglia migration toward the distal portion of the colon and neuron development. It was found homozygosity for mutation of RET gene as the cause of the most severe Hirschsprung phenotype, namely ,long segment colon aganglionosis. They have demonstrated that the orientation of the RET gene on 10qll.2 of chromosome.There is few report on RET gene mutations in Chinese cases with HD till now, and they didn't display the mutation location pattern and heredity. The purpose of the study is to examine mutation character of RET 13 exon in Chinese HD,which will set a basis for gene diagnosis in clinic. METHODSThe blood specimens of 20 HD patients and 7 normal controls was obtained by surgery in Children's Hospital between 1998 to 2000. Patients' age is range from 3 days to 13 years old . All of the 20 cases, who were diagnosed definitely by clinical history and pathological determine , came from diffuse families. Genomic DMA was extracted form peripheral blood leukocytes. Using the approach of PCR single strand conformation polymorphism analysis(SSCP),the thirteenth exon of the RET gene was studied, and further direct sequence of DNA was analyzed. RESULTSFour cases among 20 patients examined display abnormal shift of bands on SSCP gels. There are three abnormal SSCP band which suggest heterozygous and one abnormal SSCP band are homozygous. According the shifts of SSCP gels, four mutations were identified by direct sequencing , whose mutation of rate is 20%. Compared with foreign researches (25%),the mutation rate is lower in our experiment. There are two different patterns in all mutations, for example, point mutation: 18888 T to G and frameshift 18926 ins G.Combining the family members' data, the heterozygous mutation of RET gene in 5th specimen child derived from his father suggested autosomal dominant inheritance (AD).On the other hand, the homozygous mutation of RET gene in 17th specimen child suggested autosomal recessive inheritance (AR), because his father and mother's phenotype is normal. The result revealed genetic heterogeneity and incompletely penetrant. CONCLUSIONSOur study revealed that RET gene mutation plays an important role in HD and the point mutation is the dominant mutation pattern of RET gene in Chinese HD patient. Furthermore, the clinic symptoms appeared in the homozygous is more serious than heterozygous of RET 136exon.Moreover, point mutations of RET gene can occur in long segment or short segment of HD. HD has a genetic tendency.