Article

Dosage analysis of cancer predisposition genes by multiplex ligation-dependent probe amplification

National Genetics Reference Laboratory (Wessex), Salisbury District Hospital, Salisbury SP2 8BJ, UK.
British Journal of Cancer (Impact Factor: 4.82). 10/2004; 91(6):1155-9. DOI: 10.1038/sj.bjc.6602121
Source: PubMed

ABSTRACT Multiplex ligation-dependent probe amplification (MLPA) is a recently described method for detecting gross deletions or duplications of DNA sequences, aberrations which are commonly overlooked by standard diagnostic analysis. To determine the incidence of copy number variants in cancer predisposition genes from families in the Wessex region, we have analysed the hMLH1 and hMSH2 genes in patients with hereditary nonpolyposis colorectal cancer (HNPCC), BRCA1 and BRCA2 in families with hereditary breast/ovarian cancer (BRCA) and APC in patients with familial adenomatous polyposis coli (FAP). Hereditary nonpolyposis colorectal cancer (n=162) and FAP (n=74) probands were fully screened for small mutations, and cases for which no causative abnormality were found (HNPCC, n=122; FAP, n=24) were screened by MLPA. Complete or partial gene deletions were identified in seven cases for hMSH2 (5.7% of mutation-negative HNPCC; 4.3% of all HNPCC), no cases for hMLH1 and six cases for APC (25% of mutation negative FAP; 8% of all FAP). For BRCA1 and BRCA2, a partial mutation screen was performed and 136 mutation-negative cases were selected for MLPA. Five deletions and one duplication were found for BRCA1 (4.4% of mutation-negative BRCA cases) and one deletion for BRCA2 (0.7% of mutation-negative BRCA cases). Cost analysis indicates it is marginally more cost effective to perform MLPA prior to point mutation screening, but the main advantage gained by prescreening is a greatly reduced reporting time for the patients who are positive. These data demonstrate that dosage analysis is an essential component of genetic screening for cancer predisposition genes.

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    • "A contrasting approach that screens candidate genes for small microdeletions/duplications complements the genome-wide approach of array-CGH. Multiplex ligationdependent probe amplification (MLPA) was developed to identify copy number variation of targeted DNA sequences such as candidate genes (Koolen et al. 2004; Bunyan et al. 2004; Schouten et al. 2002; Zhi 2010). MLPA has evolved into a popular method to identify microdeletions/duplications from known candidate genes in a clinical and research environment (Koolen et al. 2004). "
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    ABSTRACT: Congenital heart defects are the most common malformation, and are the foremost causes of mortality in the first year of life. Among congenital heart defects, conotruncal defects represent about 20% and are severe malformations with significant morbidity. Insulin gene enhancer protein 1 (ISL1) has been considered a candidate gene for conotruncal heart defects based on its embryonic expression pattern and heart defects induced in Isl1 knockout mice. Nevertheless no mutation of ISL1 has been reported from any human subject with a heart defect. From a population base of 974,579 births during 1999–2004, we used multiplex ligation-dependent probe amplification to screen for microdeletions/duplications of ISL1 among 389 infants with tetralogy of Fallot or d-transposition of the great arteries (d-TGA). We also sequenced all exons of ISL1. We identified a novel 20-kb microdeletion encompassing the entire coding region of ISL1, but not including either flanking gene, from an infant with d-TGA. We confirmed that the deletion was caused by nonhomologous end joining mechanism. Sequencing of exons of ISL1 did not reveal any subject with a novel nonsynonymous mutation. This is the first report of an ISL1 mutation of a child with a congenital heart defect.
    07/2014; 2(4). DOI:10.1002/mgg3.75
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    • "Germline mutations in the tumor suppressor gene TP53 account for over 50% of the families matching LFS criteria [3] but for only 20-40% of the LFL families [4]; lack of TP53 mutation in a significant proportion of LFS/LFL families, suggests that other types of inherited alterations must contribute to their cancer susceptibility. Although point mutations have been commonly described, DNA copy number variations (CNVs) have been reported as an alternative mechanism for cancer predisposition for at least 30% of known Mendelian cancer genes [5,6], including TP53[7,8], APC[9], BRCA1[10] and the mismatch repair gene MSH2[9]. "
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    ABSTRACT: The Li-Fraumeni syndrome (LFS) is an inherited rare cancer predisposition syndrome characterized by a variety of early-onset tumors. Although germline mutations in the tumor suppressor gene TP53 account for over 50% of the families matching LFS criteria, the lack of TP53 mutation in a significant proportion of LFS families, suggests that other types of inherited alterations must contribute to their cancer susceptibility. Recently, increases in copy number variation (CNV) have been reported in LFS individuals, and are also postulated to contribute to LFS phenotypic variability. Seventy probands from families fulfilling clinical criteria for either Li-Fraumeni or Li-Fraumeni-like (LFS/LFL) syndromes and negative for TP53 mutations were screened for germline CNVs. We found a significantly increased number of rare CNVs, which were smaller in size and presented higher gene density compared to the control group. These data were similar to the findings we reported previously on a cohort of patients with germline TP53 mutations, showing that LFS/LFL patients, regardless of their TP53 status, also share similar CNV profiles. These results, in conjunction with our previous analyses, suggest that both TP53-negative and positive LFS/LFL patients present a broad spectrum of germline genetic alterations affecting multiple loci, and that the genetic basis of LFS/LFL predisposition or penetrance in many cases might reside in germline transmission of CNVs.
    Orphanet Journal of Rare Diseases 04/2014; 9(1):63. DOI:10.1186/1750-1172-9-63 · 3.96 Impact Factor
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    • "PTEN intron/exon copy number was determined by exporting peak heights into an Excel spreadsheet, specifically designed to assess the ratio of each test peak relative to all other peaks for the given individual. Further, ratios of test:control peaks and control:control peaks for each sample were compared with normal individuals included in each run (Bunyan et al, 2004). "
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    ABSTRACT: Background: A cohort of 629 patients with suspected Bannayan–Riley–Ruvalcaba syndrome or Cowden syndrome was tested for mutations in the PTEN gene. Methods: Dosage analysis of PTEN was carried out using a PTEN-specific multiplex ligation-dependent probe amplification (MLPA) kit, whereas point mutation analysis was performed using direct sequencing. Results: Approximately 4% of the patients from the testing cohort were heterozygously deleted for the two MLPA probe-binding sites situated in intron 1. The same deletion was subsequently seen in ∼3% of 220 normal controls, and in patients from the testing cohort with a causative mutation elsewhere in the PTEN gene. Sequencing of the variant revealed an 899 bp deletion, the 3′ breakpoint of which is only 58 bp from the start of exon 2. Conclusion: Although all evidence suggests that the 899 bp deletion is a polymorphism with no clinical effect, it removes the binding sites of almost all published PTEN exon 2 forward primers, resulting in allelic loss during PCR.
    British Journal of Cancer 01/2013; 108(2). DOI:10.1038/bjc.2012.562 · 4.82 Impact Factor
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