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Open Access Highly Accessed Research article

A novel SYBR-based duplex qPCR for the detection of gene dosage: detection of an APC large deletion in a familial adenomatous polyposis patient with an unusual phenotype

Giovana Tardin Torrezan1, Felipe Cavalcanti Carneiro da Silva1, Ana Cristina Victorino Krepischi12, Érika Maria Monteiro dos Santos1, Benedito Mauro Rossi3 and Dirce Maria Carraro12*

Author Affiliations

1 CIPE - International Center of Research and Training - A. C. Camargo Hospital, Rua Taguá, 440, CEP: 01508-010, São Paulo, SP, Brazil

2 National Institute of Science and Technology in Oncogenomics (INCITO), São Paulo, SP, Brazil

3 Barretos Cancer Hospital - Pio XII Foundation, Barretos, SP, Brazil

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BMC Medical Genetics 2012, 13:55  doi:10.1186/1471-2350-13-55

Published: 16 July 2012

Abstract

Background

Familial adenomatous polyposis (FAP) is a hereditary colorectal cancer syndrome caused by a loss of function of the APC gene. Large deletions in APC are a common cause of FAP; despite the existence of a variety of gene dosage detection methodologies, most are labor intensive and time and resource consuming.

Methods

We describe a new duplex qPCR method for gene dosage analysis based on the coamplification of a target and a reference gene in a SYBR Green reaction, followed by a comparison of the ratio between the target and the reference peaks of the melting curve for the test (patient) and control samples. The reliability of the described duplex qPCR was validated for several genes (APC, HPRT1, ATM, PTEN and BRCA1).

Results

Using this novel gene dosage method, we have identified an APC gene deletion in a FAP patient undergoing genetic testing. Comparative genomic hybridization based on microarrays (aCGH) was used to confirm and map the extent of the deletion, revealing a 5.2 MB rearrangement (5q21.3-q22.3) encompassing the entire APC and 19 additional genes.

Conclusion

The novel assay accurately detected losses and gains of one copy of the target sequences, representing a reliable and flexible alternative to other gene dosage techniques. In addition, we described a FAP patient harboring a gross deletion at 5q21.3-q22.3 with an unusual phenotype of the absence of mental impairment and dysmorphic features.

Keywords:
Gene dosage; Quantitative PCR; Familial adenomatous polyposis; APC whole gene deletion