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. 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.

Association of the p53 codon 72 polymorphism with clinicopathological characteristics of colorectal cancer through mRNA analysis.

TP53 represents a suitable candidate for a colorectal cancer susceptibility locus. The polymorphism in the p53 72nd codon involves a proline to arginine substitution, leading to changes in gene transcription activity, interaction with other proteins and modulation of apoptosis. Studies evaluating the association between this polymorphism and colorectal cancer (CRC) have shown inconsistent results, and none have evaluated the mRNA status of TP53. The aim of the present study was to evaluate the association between this SNP expression at the mRNA level in CRC samples and patient clinicopathological variables and prognosis, p53 protein expression and TP53 mutation. This is the first report to describe the mRNA expression of p53 codon 72 alleles in CRC. We evaluated 101 non-related patients with CRC treated at the A.C. Camargo Cancer Center in Brazil. RNA was isolated from frozen tumor tissues using a trizol-based protocol. The polymorphism was detected using RT-PCR followed by Sanger sequencing. Associations were analyzed using Pearson's Chi-square or Fisher's exact tests, logistic regression and Cox. This polymorphism was significantly associated with clinicopathological variables related to increased tumor aggressiveness. The expression of Arg72 (OR, 3.83; CI 1.02-14.35; P=0.046) and the TNM stage (OR, 7.15; CI 1.45-35.29; P=0.016) were found to be independent predictors for recurrence. These data suggest that the mRNA expression of the Pro72 allele is associated with less favorable tumor features. The allele frequency of the p53 Pro72 was 0.26. The analysis of mRNA is important to determine the specific contribution of the allele expressed. These results suggest that this polymorphism may play a role in CRC.

Characterization of germline mutations of MLH1 and MSH2 in unrelated south American suspected Lynch syndrome individuals.

Lynch syndrome (LS) is an autosomal dominant syndrome that predisposes individuals to development of cancers early in life. These cancers are mainly the following: colorectal, endometrial, ovarian, small intestine, stomach and urinary tract cancers. LS is caused by germline mutations in DNA mismatch repair genes (MMR), mostly MLH1 and MSH2, which are responsible for more than 85% of known germline mutations. To search for germline mutations in MLH1 and MSH2 genes in 123 unrelated South American suspected LS patients (Bethesda or Amsterdam Criteria) DNA was obtained from peripheral blood, and PCR was performed followed by direct sequencing in both directions of all exons and intron-exon junctions regions of the MLH1 and MSH2 genes. MLH1 or MSH2 pathogenic mutations were found in 28.45% (34/123) of the individuals, where 25/57 (43.85%) fulfilled Amsterdam I, II and 9/66 (13.63%) the Bethesda criteria. The mutations found in both genes were as follows: nonsense (35.3%), frameshift (26.47%), splicing (23.52%), and missense (9%). Thirteen alterations (35.14%) were described for the first time. The data reported in this study add new information about MLH1 and MSH2 gene mutations and contribute to better characterize LS in Brazil, Uruguay and Argentina. The high rate of novel mutations demonstrates the importance of defining MLH1 and MSH2 mutations in distinct LS populations.