TaqMan PCR-based Gene Dosage Assay for Predictive Testing in Individuals from a Cancer Family with INK4 Locus Haploinsufficiency

Abstract

Background: A genetic syndrome of cutaneous malignant melanoma and nervous system tumors recently has been characterized and shown to be linked to the INK4 locus in the 9p21 region. Hemizygosity at adjacent physically mapped microsatellite markers indicated deletion of p16, p19, and p15 clustered tumor suppressors. Because individuals from this family could benefit from predictive testing in terms of cancer prevention, we developed a direct test without need to analyze parental DNAs to comply with the rules of individual consent and secrecy. Methods: We developed an assay using TaqManTM real-time quantitative PCR, with p15 as the test sequence and albumin (ALB) as the reference gene. The normalized ratio of p15/ALB is expected to yield a value of ∼1 in individuals without the deletion, whereas a ratio of ∼0.5, indicating p15 haploinsufficiency, is expected in predisposed individuals. Results: All patients harboring the previously defined at-risk haplotype were correctly identified using this approach. In six individuals with deletions, the p15/ALB ratios were 0.472–0.556 (SD, 0.013–0.078). In the five individuals without deletions, the ratios were 0.919–1.019 (SD, 0.006–0.075). Conclusions: This is the first report of a high-throughput, automatable gene dosage assay successfully applied to the identification of a germ-line deletion. This approach, not limited by marker informativeness or the need for harvesting live cells, can be applied to any condition with haploinsufficiency and extended to the characterization of most abnormalities of the ploidy.

Herpesvirus detection and cytokine levels (IL‐10, IL‐6, and IFN‐γ) in ocular fluid from Tunisian immunocompetent patients with uveitis

“…VZV is the most frequently detected virus in uveitis patients. It is often described as being an etiologic agent of uveitis occurring in adults over 60 years, and it is followed frequently by acute course [Laurendeau et al, 1999;Liesegang, 2004;Rothova et al, 2008]. In the current study, the majority of patients positive for VZV-DNA had acute anterior uveitis, which was consistent with data from previous literature [Jap and Chee, 2011].…”

“…Genomic DNA of HHVs and human albumin gene in aqueous humor samples were detected and quantified [Laurendeau et al, 1999;Gautheret-Dejean et al, 2000;Niesters et al, 2000;Oksenhendler et al, 2000;Fernandez et al, 2002;Bressollette-Bodin et al, 2009].…”

Development of duplex real-time PCR for detection of two DNA respiratory viruses

“…The albumin gene was quantified in DNA extracts from all nasal samples that were positive for HAdV or HBoV. The real-time PCR was performed using a Smart Cycler instrument according to a method described previously (Laurendeau et al, 1999). A dilution series of human genomic DNA was used as a standard (Human Genomic DNA, Roche, France).…”

Evaluation of the role of human herpes virus 6 and 8 in parapsoriasis

“…All samples were tested separately for albumin quantification allowing for estimation of the number of extracted cells. Real time PCR of albumin gene was adapted from previously published method (27).…”