sybr-green-i and Prader-Willi-Syndrome

Prader-Willi syndrome and Angelman syndrome are distinct neurodevelopmental disorders that are associated with the deletion of the chromosomal 15q11-13 region or uniparental disomy of chromosome 15. In this article, we applied SYBR Green I-based real-time PCR and melting curve analysis assay for rapid genotyping of the small nuclear ribonucleoprotein polypeptide N (SNRPN) gene methylation status and for detecting aberrations in copy number in a single tube. A single pair of primers was designed to create a 357 bp fragment containing the cytosine phosphodiester guanine islands in the SNRPN promoter and to amplify both unmethylated and methylated sequences. Genotypes were identified based on the TC value for copy number changes and the characteristic melting temperature of methylated cytosine phosphodiester guanine. Genotyping of SNRPN was performed on blood samples of 20 individuals with Prader-Willi syndrome, 3 individuals with Angelman syndrome, and 20 unaffected individuals. The promoter methylation status and the copy number changes were successfully determined and compared with standard methylation-specific PCR, and were validated by multiplex ligation-dependent probe amplification. This single-tube, SYBR Green I, real-time PCR with melting curve assay is rapid, reliable, sensitive, and easy to perform. It is suitable for high-throughput analysis as an alternative technique for quantitative and qualitative analysis of target genes.

Topics: Angelman Syndrome; Benzothiazoles; Chromosomes, Human, Pair 15; Diamines; DNA Copy Number Variations; DNA Methylation; Genotype; Humans; Organic Chemicals; Prader-Willi Syndrome; Quinolines; Real-Time Polymerase Chain Reaction; snRNP Core Proteins

Prader-Willi syndrome (PWS) is a contiguous gene syndrome caused by the loss of function of genes situated within the 15q11-q13 region. The loss of function arises as a result of paternally derived mutations complemented by maternal imprinting. The molecular events underlying the disorder include interstitial deletions (70%), uniparental disomy (UPD) (25%), imprinting center defects (<5%), and rarely chromosomal translocations (<1%). The standard diagnosis of PWS is based on clinical observations and genetic investigations involving DNA methylation studies and fluorescence in situ hybridization (FISH) analysis. The absence of a paternal methylation pattern within 15q11 is sufficient for a diagnosis of PWS, and FISH analyses are used for the additional categorization of patients as either deletion or nondeletion. The main limitation of these investigations is that they neither determine the size of the molecular deletions nor permit detection of individuals with microdeletions in the PWS imprinting center that regulates imprinting in this region. We have designed and implemented a real-time PCR assay using genomic DNA and SYBR green I intercalating dye to determine the size of the chromosomal deletions in patients with PWS. This has been successfully performed on genomic DNA isolated from both peripheral blood leukocytes and buccal epithelial cells. Through this assay, the two common deletion classes in PWS were observed, and all results were 100% concordant with previous FISH assays performed on the same patients.

Topics: Benzothiazoles; Chromosome Deletion; Chromosomes, Human, Pair 15; Diamines; DNA; Epithelial Cells; Gene Dosage; Humans; In Situ Hybridization, Fluorescence; Leukocytes, Mononuclear; Mouth Mucosa; Organic Chemicals; Polymerase Chain Reaction; Prader-Willi Syndrome; Quinolines