The Mutation Screening Of LKB1Gene In Familial Peutz-jeghers Syndrome Patients

Background:Peutz-Jeghers syndrome （PJS） is an uncommon autosomal dominant inherited disease, characterized by the occurrence of gastrointestinal hamartomatous polyps and pigmentation of lips, buccal mucosa and digits. Patients with PJS have a significant risk for developing tumors in multiple organs. Germline mutation of LKB1gene, encoding a serine/threonine kinase as a tumor suppressor, has been identified responsible for developing of PJS. Up to now, more than200different kinds of mutations in LKB1gene have been shown at the HGMD website （http://www.hgmd.cf.ac.uk/ac/）. in which most are missense/nonsense mutation and small deletions/insertions.Objectives:PJS is a disease with apparente genetic predisposition. The detection of pathogenic gene mutation will help diagnosis earlier in high risk group. This study was to characterize the germline mutations in LKB1gene of familial Peutz-Jeghers syndrome （PJS） patients as well as their clinical manifestation which will help to diagnose PJS early.Methods:Eleven PJS families were collected. Typically mucosal pigmentation and hamartomatous polyps were all presence in the11probands. Genemic DNA was extracted from the peripheral blood of11probands and available family members. Nine exons and flanking introns of LKBl gene of11PJS probands were amplified using polymerase chain reaction （PCR） and then direct sequencd. Meanwhile, two hundred and fifth health adults were enrolled in this study for control, genomic DNA of peripheral blood was also extracted from these controls. Mutations identified in patients were checked in250normal controls by PCR and DHPLC （denaturing high performance liquid chromatography） to exclude polymorphisms. Total RNA was extracted from peripheral blood of the patient who was found to have dubious splice site mutation and his available family members. Reverse transcription was performed and cDNA of LKB1was amplified to identify the abnormal splicing caused by the dubious splice site mutation. MLPA （multiplex ligation-dependent probe amplification） was carried out to detect large fragment deletion of LKB1gene that was not found by the method of routine PCR.Results:Through the PCR and direct sequencing, nine kinds of germline mutations were found in eight PJS patients, most of which were point mutation （7/9）, two kinds were deletion or insertion of small base fragments. Of the nine kinds of mutations, four were considered to be pathogenic of which two were de novel, four were just polymorphisms, and one was pathogenic indefinitely. Four pathogenic mutations were c.465or469₄94or498de130, c.701T>A, c.924G>C, and c.734+5G>A, in which the mutation of c.465or469494or498de130and c.701T>A were first reported. Four gene polymorphisms were c.374+24G>T, c.464+47₄8insGGGGGCC, c.920+7G>C, and C,1062C>G The mutation pathogenic indefinitely was c.48G>A which was a samesense mutation. The result of MLPA shown that two PJS patients were detected to have lager fragment deletion in LKB1, exon1deletion and exon3to exon10deletion, respectively.Conclusion:LKB1gene germline mutation with pathogenic effect is still a common cause of familial PJS in Chinese patients. Most type of mutations is base substitution. The deletion of large fragment of LKB1is also common. We detected four new mutation type unreported previously, which expanded the spectrum of LKB1gene mutation.