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A complex rearrangement in the APC gene uncovered by multiplex ligation-dependent probe amplification.

ABSTRACT: Germline mutations in the tumor suppressor gene APC are the underlying cause of familial adenomatous polyposis, an autosomal-dominant cancer predisposition syndrome of the colorectum. Here, we describe a complex pathogenic rearrangement in the APC gene that was detected during deletion screening and transmitted throughout at least three generations. The rearrangement consists of a deletion of 604 bp in intron 4 that impairs the binding site of the reverse primer for exon 4 and of an insertion of 119 bp in exon 4 that interferes with the binding site of the multiplex ligation-dependent probe amplification (MLPA) probes for exon 4. The insertion is composed of three duplicated sequences derived from exon 4, intron 3, and intron 4, all in inverse direction. By transcript analysis, we found that the mutation results in complete skipping of exon 4 and that it leads to a frameshift. The rearrangement would not have been identified had it occurred outside the MLPA hybridization site. Our findings demonstrate that part of the pathogenic mutations remain undetected by routine methods. Moreover, MLPA and RNA analysis alone would have led to an incorrect interpretation of a genomic deletion of exon 4.

SUBMITTER: Pagenstecher C

PROVIDER: S-EPMC1867434 | biostudies-literature | 2007 Feb

REPOSITORIES: biostudies-literature

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A complex rearrangement in the APC gene uncovered by multiplex ligation-dependent probe amplification.

Pagenstecher Constanze C Gadzicki Dorothea D Stienen Dietlinde D Uhlhaas Siegfried S Mangold Elisabeth E Rahner Nils N Arslan-Kirchner Mine M Propping Peter P Friedl Waltraut W Aretz Stefan S

The Journal of molecular diagnostics : JMD 20070201 1

Germline mutations in the tumor suppressor gene APC are the underlying cause of familial adenomatous polyposis, an autosomal-dominant cancer predisposition syndrome of the colorectum. Here, we describe a complex pathogenic rearrangement in the APC gene that was detected during deletion screening and transmitted throughout at least three generations. The rearrangement consists of a deletion of 604 bp in intron 4 that impairs the binding site of the reverse primer for exon 4 and of an insertion of ...[more]

PMID: 17251345

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Project description:Connective tissue diseases characterized by aortic aneurysm, such as Marfan syndrome, Loeys-Dietz syndrome and Ehlers Danlos syndrome type IV are heterogeneous and despite overlapping phenotypes, the natural history, clinical manifestations and interventional course for each diagnosis can be quite unique. The majority of mutations involved in the etiology of these disorders are missense and nonsense mutations. However, large deletions and duplications undetected by sequencing may be implicated in their pathogenesis, and may explain the apparent lack of genotype-phenotype correlation in a subset of patients. The objective of this study was to search for large pathogenic deletions and/or duplications in the FBN1, TGF?R1, and TGF?R2 genes using multiplex-ligation dependent probe amplification (MLPA) in patients with aortopathy, in whom no mutations in the FBN1, TGF?R1, and TGF?R2 genes were identified by sequencing.The study included 14 patients from 11 unrelated families with aortic aneurysm. Of those, six patients (including 3 first-degree relatives), fulfilled the revised Ghent criteria for Marfan syndrome, and eight had predominantly aortic aneurysm/dilatation with variable skeletal and craniofacial involvement. MLPA for FBN1, TGF?R1, and TGF?R2 was carried out in all patients. A 385 K chromosome 15 specific array was used in two patients with a deletion of the entire FBN1 in order to define its size and boundaries.We identified two novel large deletions in the FBN1 gene in four patients of two unrelated families who met clinical diagnostic criteria for Marfan syndrome. One patient was found to have a FBN1 deletion encompassing exons 1-5. The other three patients had a 542 Kb deletion spanning the whole FBN1 gene and five additional genes (SLC24A5, MYEF2, CTXN2, SLC12A1, DUT) in the chromosome 15.Our findings expand the number of large FBN1 deletions, and emphasize the importance of screening for large genomic deletions in connective tissue disorders featuring aortopathies, especially for those with classic Marfan phenotype.

Project description:BackgroundWith the zebrafish increasingly being used in immunology and infectious disease research, there is a need for efficient molecular tools to evaluate immune gene expression in this model species. RT-MLPA (reverse transcription - multiplex ligation-dependent probe amplification) provides a sensitive and reproducible method, in which fluorescently labelled amplification products of unique lengths are produced for a defined set of target transcripts. The method employs oligonucleotide probes that anneal to adjacent sites on a target sequence and are then joined by a heat-stable ligase. Subsequently, multiplex PCR with universal primers gives rise to amplicons that can be analyzed with standard sequencing equipment and relative quantification software. Allowing the simultaneous quantification of around 40 selected markers in a one-tube assay, RT-MLPA is highly useful for high-throughput screening applications.FindingsWe employed a dual-colour RT-MLPA probe design for chemical synthesis of probe pairs for 34 genes involved in Toll-like receptor signalling, transcriptional activation of the immune response, cytokine and chemokine production, and antimicrobial defence. In addition, six probe pairs were included for reference genes unaffected by infections in zebrafish. First, we established assay conditions for adult zebrafish infected with different strains of Mycobacterium marinum causing acute and chronic disease. Addition of competitor oligonucleotides was required to achieve peak heights in a similar range for genes with different expression levels. For subsequent analysis of embryonic samples it was necessary to adjust the amounts of competitor oligonucleotides, as the expression levels of several genes differed to a large extent between adult and embryonic tissues. Assay conditions established for one-day-old Salmonella typhimurium-infected embryos could be transferred without further adjustment to five-day-old M. marinum-infected larvae. RT-MLPA results were compared with results of previous transcriptome analyses and with real-time PCR data, demonstrating a good correlation between all expression analysis methods.ConclusionsThe RT-MLPA assay developed in this study provides a rapid, cheap, and robust analysis tool for simultaneous quantification of a set of 34 innate immune response genes. With adjustment of conditions, the assay is suitable for infection studies in both adult and embryonic zebrafish. Application of RT-MLPA will facilitate high-throughput screening of immune responses in the zebrafish model.

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A complex rearrangement in the APC gene uncovered by multiplex ligation-dependent probe amplification.

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A complex rearrangement in the APC gene uncovered by multiplex ligation-dependent probe amplification.

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