Castellsagué Ester, González Sara, Nadal Marga, Campos Olga, Guinó Elisabet, Urioste Miguel, Blanco Ignacio, Frebourg Thierry, Capellá Gabriel
Translational Research Laboratory, IDIBELL-Institut Català d'Oncologia, Barcelona, Spain.
Clin Chem. 2008 Jul;54(7):1132-40. doi: 10.1373/clinchem.2007.101006. Epub 2008 May 16.
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.
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.
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.
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.
大约20%的经典家族性腺瘤性息肉病（FAP）病例以及70%至80%的轻度FAP（AFAP）病例的APC/MUTYH点突变为阴性。短荧光片段定量多重PCR（QMPSF）是一种检测拷贝数改变的技术，已成功应用于多个癌症综合征基因。我们使用针对APC基因的QMPSF来筛查FAP的APC/MUTYH突变阴性家族，以改善其诊断监测。
我们使用23个阴性对照和1个阳性对照建立并验证了APC基因QMPSF，并检测了45名（13名FAP和32名AFAP）APC/MUTYH突变阴性家族的无关成员的拷贝数改变。我们使用多重连接依赖探针扩增（MLPA）来确认结果。我们使用测序、定量实时PCR（QRT-PCR）和荧光原位杂交（FISH）等不同方法进一步表征所鉴定的缺失。
APC QMPSF能够以可接受的变异性检测缺失，从实验内和实验间重复获得的缺失对照的等位基因剂量平均值（标准差）表明了这一点[0.52（0.05）和0.45（0.10）]。在分析的13例（23%）经典FAP病例中，我们检测到3处大片段缺失（1处完整基因缺失，2处分别包含外显子9和10以及外显子11 - 15的部分缺失）。在32例AFAP病例中未检测到重排。
QMPSF能够检测APC基因的重排。我们的研究结果强调了在临床环境中对FAP家族进行常规基因检测时，将拷贝数改变方法作为第一步的重要性。
