The Mutation Detection Of Ret Proto-oncogene In A Family With Multiple Endocrine Neoplasia Type 2A Syndrome

Multiple endocrine neoplasia type 2(MEN2) is an autosomal dominant familial disorder characterized by occurrence of tumors or hyperplasias in cells of thyroid, adrenal medullary and hyperparathyroid. The genetic test is helpful for the early screening of this disease and the discovery of the carrier of gene mutation. Early diagnosis and treatment can greatly improve the clinical prognosis.The aims of this study were: (1) Clarifying the genotype of the MEN2A pedigree and exploring the clinical value of ret gene mutational analysis in this family. (2) Comparing the immunohistochemical expressions of ret proto-oncogene between MEN2A and single parathyroid adenoma.Methods:The subjects of this study are 19 family numbers of a MEN2A family. Methods were as bellow:1. Genome DNA was extracted from peripheral blood leucocytes and 2 pair of primers for PCR amplification of 10 and 11 exons of ret gene were used. Gel-purified PCR products were sequenced. Sequences were compared with genome DNA and cDNA sequences in the NCBI website. If discovering abnormality, we further inquired Human Gene Mutation Database (HGMD) to identify the abnormality. Sequencing was carried 4 times if the mutation was discovered.2. Expressions of ret proto-oncogene in one case with MEN2A and two cases of single parathyroid adenoma were immunohistochemically checked.3. The 19 family members were investigated by thyroid gland, parathyroid and adrenal gland ultrasound examination, bone density inspection as well as the serum calcium, the serum phosphorus, carcino-embryonic antigen and the parathyroid hormone examination.Results:1. No abnormality was found in exon 10 in this MEN2A pedigree. T to C replacement was found in nt.14994, G to A polymorphism was found in nt.15165 in the postpand (II-9). When the DNA sequences were compared with the cDNA, codon 634 TGC was changed to CGC, and the responding amino acid change was from Cys (C) to Arg (R). Codon 691 GGT was changed to AGT, and the responding amino acid change was from Gly (G) to Ser (S). The results of sequencing in the members(II-5,II-7 andâ…¢-6) were as same as the postpand. Only the polymorphism G691S in exon 11 of the ret proto-oncogene was detected in one member (â…¢-5).2. The results of immunohistochemical staining showed that there were more ret-positive cells and greater coloring extents in parathyroid adenoma tissues of MEN2A patients than those in single parathyroid adenoma patients.3. The results of the ultrasound examination were shown as follows: the solid lesions of bilateral thyroid, one side of parathyroid and one side of adrenal were seen in the member II-5; the solid space-occupying lesions of bilateral thyroid and one side of adrenal were seen in the member II-9; the solid space-occupying lesions of bilateral thyroid, bilateral adrenal and one side of parathyroid were seen in the member II-7; the solid space-occupying lesions of bilateral thyroid, one side of adrenal and one side of parathyroid were seen in the memberâ…¢-6; multiple thyroid small nodules were only seen in the memberâ…¢-5. Additionally, there were another three members whose ultrasound examinations were abnormal and no gene mutation.Conclusion1. The genotype of this MEN2A family was clarified: The coexistence of genotype C634R and the polymorphism G691S was found in 4 members(II-5,II-7,II-9,â…¢-6)of the family members. The members without this gene mutation were not patients. So the coexistence of genotype C634R and the polymorphism G691S was the virulence gene of the family.2. Only the polymorphism G691S in exon 11 of the ret proto-oncogene was detected in family memberâ…¢-5. This member may be the carrier of gene mutation. Close observations and follow-up to him should be done.3. The immunohistochemical expression of ret oncogene in parathyroid adenoma tissues of MEN2A patients was greater than that in single parathyroid adenoma.4. After the screening of clinical examination and virulence gene, two new MEN2A patients were diagnosed in this pedigree and their genotype was identified, which showed that identifying of the genotype was helpful in the discovery of new patients in this pedigree, and was also useful for the early diagnosis and treatment of the patients.