Detection Of Apc Gene Germline Mutations In Chinese Familial Adenomatous Polyposis Families

BACKGROUND AND OBJECTIVE Familial adenomatous polyposis(FAP)is a rare, autosomal dominant inherited disease. The prevalence of FAP is estimated at 1 in 5000-10,000. and is characterized by the development of multiple adenomatous polyps(more than hundred) in colorectal at a young age.If left untreated, cancer 100% inevitably develops before 50 years. Because of the large number of polyps, they almost inevitably develop colorectal carcinoma by the age of 50 years. It is caused by germline mutations in the adenomatous polyposis coli (APC) gene located in 5q21-q22., The APC gene is a tumour suppressor gene,The coding region of the gene consists of 15 exons, encoding a protein consisting of 2843 amino acids . The APC gene product (APC) is protein with a molecular mass of approximately 310 kd. APC is a multidomain protein that consists of an oligomerization domain and an armadillo domain in the N-terminus, a number of 15- and 20-amino-acid repeats in the central portion, and a basic domain and binding sites for EB1 and the mammalian homologue of discs large (DLG) in the C-terminus Through these multiple domains, APC binds numerous proteins, allowing APC to perform functions in not only tumor suppression but also cell differentiation, adhesion,polarity formation, migration, development, apoptosis, and neuronal functions. APC is highly expressed in the intestinal and colorectal epithelia and may be involved in homeostasis of the enterocyte renewal phenomena, in which proliferation, migration, differentiation, and apoptosis are highly regulated both temporally and spatially. the preponderance of these were nonsense mutations and frameshift mutations, resulting in a truncated protein with a broad range of molecular masses less than the wild-type APC protein. Micro Mutations in the APC gene are present in 60% to 80% of patients with FAP ,and large fragment deletions were detected in 10-15% of mutation-negative patients, Since the identification of the APC gene in 1991 , more than 900 germline mutations have been reported ,but the reports are fewer about the APC gene germline mutation in Chinese FAP. Therefore, we design the experiment to study the characteristics of APC gene germline mutation in Chinese patients with familial adenomatous polyposis (FAP)METHODS1. Patients To collect 14 unrelated familial adenomatous polyposis patients who were Diagnosis and treatment in Beijing Military General Hospital from 2002 to 2008 and all patients signed informed consent.2. The detection of micro mutation Peripheral blood was drawn from 14 FAP probands. Genomic DNA and RNA were purified from peripheral blood leukocytes, All 15 exons including splice junctions of APC were amplified by PCR (Polymerase Chain Reaction), and the amplifiled pruducts were detected by direct sequencing3. large fragment deletion Large fragment deletion was detected by multiplex ligation dependent probe amplification (MLPA) in APC mutation negative patients.RESULTS(1)9 micro mutations were identified in the 14 unrelated FAP families by direct sequencing,the mutation rate is 64.3% (9/14), including 6 frameshift mutations (66.7%), 1 nonsense mutations(11.1%),2 splicing mutation (22.2%).and 4 are novel, they are c.532-2A>T, c.2336-2337insT,c.3923-3929delAAGAAAAå'Œc.4179-4180 GadelinsT. (2) 2 large fragment deletions were detected by MLPA in 5 APC mutation negative families by direct sequencing. one is the deletions of exon 10A and exon 11. the other is the deletion of exon15 start.Both of them are novel.The large fragment deletion rate is 14.3 % in FAP and is 40 in APC mutation negative patient.(3) The total mutation rate of small mutation and large fragment deletion is 78.6%.(4) 7 SNP site are detected.(5) missense mutation was detected respectively in Family 3,11,14CONCLUSION(1) The type of mutation is diverse , include frameshift mutation, nonsense mutations, splicing mutation and large fragment deletion.(2) The germline mutation rate of APC gene can be improved by direct squencing combined with large fragment deletetion MLPA. Therefore ,it's nessesary to add large fragment deletetion in routine molecule genetics detection.