Detection of APC gene deletions using quantitative multiplex PCR of short fluorescent fragments

Abstract

BACKGROUND: Approximately 20% of classic familial adenomatous polyposis (FAP)casesand 70%to80%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 detectedinthe32 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. © 2008 American Association for Clinical Chemistry.