Molecular methods in preimplantation genetic diagnosis with emphasis on the Fragile X syndrome

Preimplantation genetic diagnosis (PGD) is a form of prenatal diagnosis. It is performed on the third day after fertilisation, on embryos of couples at risk of transmitting an inherited disorder to their offspring. In this way, only embryos without the mutation under investigation are transferred in order to initiate a pregnancy. Embryos used for PGD are generated by routine IVF procedures. Fragile X syndrome is the most common form of inherited mental retardation. The causative mutation has been identified as an expansion of a triplet (CGG)n repeat in the 5' untranslated region of the FMR1 gene. The expansion is refractory to PCR due to preferential amplification of the smaller allele in heterozygous cells and the high GC content of the repeat and surrounding sequences. Currently, the only method of diagnosis available for this disease is PCR followed by Southern blotting in order to detect the expanded allele. This, however, is not suitable for preimplantation diagnosis as it is time consuming and requires a lot of DNA. Furthermore, the time at which the amplification occurs in the embryo is not yet determined so that a test relying on detecting the expansion may not be suitable for preimplantation diagnosis. For this reason polymorphic markers linked to the mutation are used to diagnose this disorder in preimplantation embryos. Meanwhile methods for the direct detection of the mutation in single cells have been tested in order to study the timing of the expansion in embryos diagnosed as affected after preimplantation genetic diagnosis. These were applied to spare oocytes and embryos from an IVF cycle. Finally, methods for PGD of sickle cell anaemia have been tested in order to determine the most efficient one. These include restriction enzyme digestion of PCR products, single strand conformation polymorphism (SSCP) followed by staining of the DNA with the silver staining technique, and fluorescent PCR followed by SSCP on an automated fluorescent sequencer (ALF, Pharmacia)

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 <it>BRCA1 </it>gene in a Greek cohort of families with suspected hereditary breast cancer

Abstract 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.</p

Mutation analysis of the BRCA1 and BRCA2 genes in Turkish patients with breast cancer.

Annual Meeting of the American-Society-of-Clinical-Oncology (ASCO) / Clinical Science Symposium on Predicting and Improving Adverse Outcomes in Older Adults with Cancer -- MAY 29-JUN 02, 2015 -- Chicago, ILWOS: 000358036902345Amer Soc Clin Onco