Received on November 29, 2007
Accepted on April 4, 2008

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

^* To whom correspondence should be addressed. 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 and found 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.