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This Article Full Text Full Text (PDF) Supplemental Data All Versions of this Article: clinchem.2007.101006v1 54/7/1132    most recent Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in Web of Science Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via Google Scholar Google Scholar Articles by Castellsagué, E. Articles by Capellá, G. Search for Related Content PubMed PubMed Citation Articles by Castellsagué, E. Articles by Capellá, G. Related Collections Molecular Diagnostics and Genetics

Detection of APC Gene Deletions Using Quantitative Multiplex PCR of Short Fluorescent Fragments

¹ Translational Research Laboratory, IDIBELL-Institut Català d’Oncologia, Barcelona, Spain; ² Bioinformatics and Biostatistics Unit, Department of Epidemiology, IDIBELL-Institut Català d’Oncologia, Barcelona, Spain; ³ Human Genetics Group, Spanish National Cancer Centre (CNIO), and Centre for Biomedical Research on Rare Diseases, Instituto de Salud Carlos III, CIBERER; ⁴ Genetic Counselling Unit, IDIBELL-Institut Català d’Oncologia, Barcelona, Spain; ⁵ Department of Genetics, INSERM U614, Faculty of Medicine, Rouen, France.

^aAddress correspondence to this author at: Laboratori de Recerca Translacional (LRT1), Institut Català d’Oncologia, Hospital Duran i Reynals, Gran via s/n Km 2.7, 08907 L’Hospitalet de Llobregat, Spain. Fax +34 93-260-74-66; e-mail gcapella{at}iconcologia.net.

Background: Approximately 20% of classic familial adenomatous polyposis (FAP) cases and 70% to 80% of attenuated FAP (AFAP) cases are negative for the APC/MUTYH point mutation. Quantitative multiplex PCR of short fluorescent fragments (QMPSF), a technique for detecting copy number alterations, has been successfully applied to several cancer syndrome genes. We used QMPSF for the APC gene to screen FAP APC/MUTYH mutation-negative families to improve their diagnostic surveillance.

Methods: We set up and validated APC-gene QMPSF using 23 negative and 1 positive control and examined 45 (13 FAP and 32 AFAP) unrelated members of APC/MUTYH mutation-negative families for copy number alterations. We confirmed the results using multiplex ligation-dependent probe amplification (MLPA). We used different approaches such as sequencing, quantitative real time-PCR (QRT-PCR), and fluorescence in situ hybridization (FISH) to further characterize the identified deletions.

Results: APC QMPSF was capable of detecting deletions with an acceptable variability, as shown by mean values (SD) of allele dosage for the deleted control obtained from intra- and interexperimental replicates [0.52 (0.05) and 0.45 (0.10)]. We detected 3 gross deletions in 13 (23%) of the classic FAP cases analyzed (1 complete gene deletion and 2 partial deletions encompassing exons 9 and 10 and exons 11–15, respectively). No rearrangements were detected in the 32 AFAP cases.

Conclusions: QMPSF is able to detect rearrangements of the APC gene. Our findings highlight the importance of using a copy number alteration methodology as a first step in the routine genetic testing of FAP families in the clinical setting.