Heterogeneous molecular mechanisms underlie attenuated familial adenomatous polyposis.

PURPOSE: Familial adenomatous polyposis is a phenotypically heterogeneous disease predisposing to colorectal cancer. It is dominantly transmitted, when associated with the APC gene, and recessively inherited, when associated with MUTYH gene. We searched for APC and MUTYH germline alterations in Italian and Greek patients with attenuated polyposis, a phenotypic variant whose genetic cause remains unknown in many cases. METHODS: We studied 26 unrelated patients (and 16 relatives) with multiple colorectal adenomas (3-100, by endoscopic analysis) that had screened APC mutation-negative by protein truncation test. We searched for APC rearrangements by multiplex ligation-dependent probe amplification and for MUTYH mutations by sequencing. We performed a screening of five MUTYH recurrent pathogenic mutations in 501 Italian and 144 Greek controls. RESULTS: One patient proved to carry an APC whole-gene deletion; 4 of 25 (16%) patients showed biallelic and 3 of 25 (12%) monoallelic MUTYH mutations. In the three heterozygous subjects no pathogenetic variants were found in OGG1, MTH1, APE1, MSH2, and MSH6 genes. Frequency assessment of MUTYH mutations in healthy subjects showed that only Y165C and G382D reach a subpolymorphic frequency. CONCLUSION: Attenuated polyposis patients without "conventional" APC mutations are genetically heterogeneous, and the phenotype is not directly related to the germline defect. Therefore, the families' appropriate management requires an accurate genetic and clinical investigation.

Rare mutations predisposing to familial adenomatous polyposis in Greek FAP patients.

BACKGROUND: Familial Adenomatous Polyposis (FAP) is caused by germline mutations in the APC (Adenomatous Polyposis Coli) gene. The vast majority of APC mutations are point mutations or small insertions/deletions which lead to truncated protein products. Splicing mutations or gross genomic rearrangements are less common inactivating events of the APC gene. METHODS: In the current study genomic DNA or RNA from ten unrelated FAP suspected patients was examined for germline mutations in the APC gene. Family history and phenotype were used in order to select the patients. Methods used for testing were dHPLC (denaturing High Performance Liquid Chromatography), sequencing, MLPA (Multiplex Ligation - dependent Probe Amplification), Karyotyping, FISH (Fluorescence In Situ Hybridization) and RT-PCR (Reverse Transcription - Polymerase Chain Reaction). RESULTS: A 250 Kbp deletion in the APC gene starting from intron 5 and extending beyond exon 15 was identified in one patient. A substitution of the +5 conserved nucleotide at the splice donor site of intron 9 in the APC gene was shown to produce frameshift and inefficient exon skipping in a second patient. Four frameshift mutations (1577insT, 1973delAG, 3180delAAAA, 3212delA) and a nonsense mutation (C1690T) were identified in the rest of the patients. CONCLUSION: Screening for APC mutations in FAP patients should include testing for splicing defects and gross genomic alterations.

Characterization of a novel large deletion and single point mutations in the BRCA1 gene in a Greek cohort of families with suspected hereditary breast cancer.

BACKGROUND: Germline mutations in BRCA1 and BRCA2 predispose to breast and ovarian cancer. A multitude of mutations have been described and are found to be scattered throughout these two large genes. We describe analysis of BRCA1 in 25 individuals from 18 families from a Greek cohort. METHODS: The approach used is based on dHPLC mutation screening of the BRCA1 gene, followed by sequencing of fragments suspected to carry a mutation including intron--exon boundaries. In patients with a strong family history but for whom no mutations were detected, analysis was extended to exons 10 and 11 of the BRCA2 gene, followed by MLPA analysis for screening for large genomic rearrangements. RESULTS: A pathogenic mutation in BRCA1 was identified in 5/18 (27.7 %) families, where four distinct mutations have been observed. Single base putative pathogenic mutations were identified by dHPLC and confirmed by sequence analysis in 4 families: 5382insC (in two families), G1738R, and 5586G > A (in one family each). In addition, 18 unclassified variants and silent polymorphisms were detected including a novel silent polymorphism in exon 11 of the BRCA1 gene. Finally, MLPA revealed deletion of exon 20 of the BRCA1 gene in one family, a deletion that encompasses 3.2 kb of the gene starting 21 bases into exon 20 and extending 3.2 kb into intron 20 and leads to skipping of the entire exon 20. The 3' breakpoint lies within an AluSp repeat but there are no recognizable repeat motifs at the 5' breakpoint implicating a mechanism different to Alu-mediated recombination, responsible for the majority of rearrangements in the BRCA1 gene. CONCLUSIONS: We conclude that a combination of techniques capable of detecting both single base mutations and small insertions/deletions and large genomic rearrangements is necessary in order to accurately analyze the BRCA1 gene in patients at high risk of carrying a germline mutation as determined by their family history. Furthermore, our results suggest that in those families with strong evidence of linkage to the BRCA1 locus in whom no point mutation has been identified re-examination should be carried out searching specifically for genomic rearrangements.

Evaluation of dHPLC in mutation screening of the APC gene in a Greek FAP cohort.

BACKGROUND: Germline mutations in the APC gene predispose to colorectal adenomas leading to cancer in over 80% of patients. A multitude of mutations, dispersed throughout the gene, have been described. We wanted to evaluate the usefulness of denaturing high performance liquid chromatography (dHPLC) for mutation screening. MATERIALS AND METHODS: Ten amplicons containing 14 mutations in the APC, previously identified by sequencing in 22 FAP patients, were analysed by dHPLC. dHPLC was also used to screen members of a family for a mutation identified in the proband. RESULTS: We analysed 10 amplicons under a total of 59 temperatures. Successful results were obtained from 51 out of 59 tested temperatures (86.4%). In all cases a different heteroduplex-homoduplex pattern was obtained from mutant DNA in at least two of the temperatures. All 14 mutations identified by sequencing were also detected using dHPLC. Sequence analysis of a large family confirmed the dHPLC results. CONCLUSION: Using dHPLC we detected all mutations previously identified by sequencing.

X-linked adrenoleukodystrophy: are signs of hypogonadism always due to testicular failure?

We present the clinical and hormonal findings of a young male with X-linked adrenoleukodystrophy (X-ALD), with special emphasis on the biochemical and clinical pattern of hypogonadism. A patient, with primary adrenal insufficiency since the age of 5 years, developed progressive neurological symptoms at the age of 29. Diagnosis of X-ALD was established by elevated serum very long chain fatty acids (VLCFAs) and genetic testing. His sexual body hair was sparse. Hormonal investigations revealed normal testosterone and inappropriately elevated LH levels. Androgen receptor gene analysis was negative for mutations or polymorphic variants associated with decreased receptor activity. Signs of hypogonadism in patients with confirmed X-ALD are not exclusively due to primary testicular failure. Tissue specific androgen resistance represents an alternative possibility. Since no loss-of-function mutations were detected in the androgen receptor, it is speculated that the patient's androgen resistance could be part of a functional defect mediated through VLCFA accumulation at the testosterone receptor and/or post-receptor levels.

Complex chromosome rearrangement in a child with microcephaly, dysmorphic facial features and mosaicism for a terminal deletion del(18)(q21.32-qter) investigated by FISH and array-CGH: Case report.

We report on a 7 years and 4 months old Greek boy with mild microcephaly and dysmorphic facial features. He was a sociable child with maxillary hypoplasia, epicanthal folds, upslanting palpebral fissures with long eyelashes, and hypertelorism. His ears were prominent and dysmorphic, he had a long philtrum and a high arched palate. His weight was 17 kg (25th percentile) and his height 120 cm (50th percentile). High resolution chromosome analysis identified in 50% of the cells a normal male karyotype, and in 50% of the cells one chromosome 18 showed a terminal deletion from 18q21.32. Molecular cytogenetic investigation confirmed a del(18)(q21.32-qter) in the one chromosome 18, but furthermore revealed the presence of a duplication in q21.2 in the other chromosome 18. The case is discussed concerning comparable previously reported cases and the possible mechanisms of formation.