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

Abstract

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.

Copy Number Assessment by Competitive PCR with Limiting Deoxynucleotide Triphosphates and High-Resolution Melting

“…Limiting cycle number for relative quantification of targets with different primer pairs is well established (15)(16)(17). Presumably, PCR retains the relative amounts of multiple targets throughout the exponential phase.…”

“…Although prior methods of relative quantification estimate copy number ratios by relative peak heights (16,17 ), it is not easy to estimate the baseline on derivative melting curve plots. In our hands, better precision and clarity were obtained by normalizing the reference peaks of all derivative plots both vertically (fluorescence) and horizontally (temperature).…”

“…For example, preamplification methods maintain relative quantification by restricting the number of cycles to 10 -15 (15 ). Quantitative multiplex PCR of short fluorescent fragments uses low cycle number to maintain relative quantification, followed by separation on sequencing gels (16 ). Comparative high-resolution melting identifies copy number changes by melting at a low cycle number, followed by mutation detection by secondary melting at a high cycle number (17 ).…”

Genomic era diagnosis and management of hereditary and sporadic colon cancer

“…Approximately 90% of APC alterations in FAP introduce a stop codon causing truncation of the resulting protein at the C-terminus, with over 900 different germline APC alterations having been discovered in FAP individuals to date [26][27][28][29] . Though clinical genetic testing of the APC gene has a 90% mutation detection rate [30] , approximately 10% of classic FAP cases do not have an identifiable mutation in APC, requiring greater reliance on clinical presentation and empiric surveillance screening in all at risk individuals in these families [21,31] .…”

A Customized Simulated Annealing Suitable for Primer Design in Polymerase Chain Reaction Processes

“…Studies of gene expression level (Isenbarger et al, 2008;Dixon et al, 2007), loss of allelic heterozygosity (LOH) (Vladušić et al, 2010;Chih-Ming et al, 2009;Franko et al, 2008;Saelee et al, 2008), microsatellite instability (MSI) (Eveno et al, 2010;Bertagnolli et al, 2009), microdeletions (Kolb et al, 2010;Pasmant et al, 2009), quantification of small ncRNAs (Ro et al, 2006;Berezikov et al, 2006) and detection of lowlevel mutations (Milbury et al, 2009) are a few examples of the popularity, success and diversity of uses of PCR. Some of the examples can be characterized as quantitative PCR or Real Time PCR (Lan et al, 2009;Roux, 2009;VanGuilder et al, 2008;Pattyn et al, 2003;Vandesompele et al, 2002) or quantitative multiplex PCR (Sasaki et al, 2010;Wang et al, 2009;Castellsagué et al, 2008), Inter-Alu PCR (Bonafè et al, 2001;Srivastava et al, 2005), or COLD-PCR (Milbury et al, 2009) and miniprimer PCR (Isenbarger et al, 2008).…”