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.
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.


CONCLUSIONS
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.
CIPE - International Center of Research and Training - A, C, Camargo Hospital, Rua Taguá, São Paulo, Brazil.
Giovana Tardin
GT
Torrezan
Felipe Cavalcanti Carneiro
FC
da Silva
Ana Cristina Victorino
AC
Krepischi
Erika Maria Monteiro
EM
dos Santos
Benedito Mauro
BM
Rossi
Dirce Maria
DM
Carraro
D011125:Adenomatous Polyposis Coli / Q000235:genetics*; D025601:Adenomatous Polyposis Coli Protein / Q000235:genetics*; D000328:Adult; D052160:Benzothiazoles; D002874:Chromosome Mapping; D003959:Diamines; D005091:Exons; D005260:Female; D018628:Gene Dosage; D015321:Gene Rearrangement; D006801:Humans; D007438:Introns; D008297:Male; D009930:Organic Chemicals / Q000737:chemistry*; D016133:Polymerase Chain Reaction / Q000379:methods*; D011804:Quinolines; D017384:Sequence Deletion
C555337:APC protein, human; D025601:Adenomatous Polyposis Coli Protein; D052160:Benzothiazoles; D003959:Diamines; D009930:Organic Chemicals; D011804:Quinolines; C098022:SYBR Green I