RESUMO
BACKGROUND: The patient with 10 or more adenomas in the colon poses a diagnostic challenge. Beside germline mutations in the APC and MUTYH genes, only four cases of mosaic APC mutations have been reported. AIM: Given the relatively high frequency of de novo APC mutations in familial adenomatous polyposis (FAP), an investigation was carried out into whether the proportion of somatic mosaic APC mutations is currently underestimated. METHODS: Between 1 January 1994 and 31 December 2005 germline mutation analysis was performed in 599 consecutive index patients with polyposis coli referred for diagnostic APC scanning using a combination of denaturing gradient gel electrophoresis (DGGE) and protein truncation test (PTT). Variants were analysed by direct sequencing with primers flanking those used for DGGE and PTT, and quantified using pyrosequencing. RESULTS: Scrutinizing the molecular genetic results and family data of 242 index patients with pathogenic APC mutations led to the identification of 10 mosaic cases (4%). C>T transitions were observed in CGA sites in four of the 10 cases with somatic mosaicism, which is significantly more than 26 of the 232 non-mosaic cases (p = 0.02). Phenotypes of patients with somatic mosaicism ranged from an attenuated form of polyposis coli to florid polyposis with major extracolonic manifestations. CONCLUSIONS: Mosaicism occurs in a significant number of APC mutations and it is estimated that one-fifth of the de novo cases of FAP are mosaic. Clinically, the severity of manifestations in offspring and the recurrence risk for siblings of apparently sporadic polyposis patients may be underestimated due to parental APC mosaicism.
Assuntos
Polipose Adenomatosa do Colo/genética , Genes APC , Mosaicismo , Adulto , Idoso , Estudos de Coortes , Feminino , Genótipo , Mutação em Linhagem Germinativa , Humanos , Masculino , Pessoa de Meia-Idade , Linhagem , FenótipoRESUMO
BACKGROUND: Protein Truncation Test (PTT) was used to detect mutations in exon 15 of the APC gene in patients with Familial Adenomatous Polyposis. This method is limited by its ability to detect polypetide chains up to a certain minimum length. The aim of this study was to increase the sensitivity of detection of mutations in this region by using the technique of Denaturing Gradient Gel Electrophoresis (DGGE). METHODS AND RESULTS: Study were performed on 122 patients without detected mutations in the APC gene. The patients were divided into two independent groups 15A and 15A+15B (with 51 and 71 patients respectively). All the patients were tested with the DGGE and the positive findings were confirmed with sequencing. No mutation was detected in the group 15A (0%). In group 15A+15B one (1.4%) polymorphism and four (5.63%) patients with nonsense mutations were detected. CONCLUSIONS: DGGE is an effective method for detecting mutations in the first part of exon 15 of APC gene. It allows detecting any change in DNA strand. DGGE complements PTT in scanning of the whole exon 15 of APC gene.
Assuntos
Polipose Adenomatosa do Colo/genética , Genes APC , Mutação , Eletroforese , Humanos , Reação em Cadeia da PolimeraseRESUMO
BACKGROUND: DNA sequences from chromosome Y can cause gonadoblastoma development in patients with Turner syndrome (TS). Estimated risk is about 30%. The aim of the study is detection of Y-sequences of DNA level, calculation of mosaicism and its cytogenetic location. Clinical result of the study is the recommendation to gonadectomy of proved positive patients. METHODS AND RESULTS: Samples from 110 patients were collected. The PCR method and analysis of products on agarose gel was compared with analysis of DNA fragments from quantitative fluorescent (QF) PCR on capillary electrophoresis. The loci DYZ3, AMGX/Y and SRY were used for detection. The method QF PCR was effected for DYZ3 and AMGX/Y loci. The positive cases were examined by FISH method. Five (4.5%) and 3 (2.7%) positive cases were detected in DYZ3 and SRY resp. loci by electrophoresis on agarose gel. Seventeen (15.5%) and 7 (6.4%) positive cases were detected in DYZ3 and AMGX/Y resp. by capillary electrophoresis. The estimated mosaicism ranged from 1:5 to 1:100,000. CONCLUSIONS: QG PCR is the most sensitive method for diagnostics of Y-sequences. Simultaneously the incidence of Y-positive cells can be estimated. The positive cases with marker in karyotype were confirmed by FISH.
