PCR-Based Analysis of Differentially Methylated Regions of GNAS Enables Convenient Diagnostic Testing of Pseudohypoparathyroidism Type Ib

Abstract

BACKGROUND: Pseudohypoparathyroidism type Ib (PHPIb) is characterized by parathyroid hormone (PTH) resistance, which can lead to hypocalcemia, hyperphosphatemia, and increased serum PTH. The disorder is caused by mutations in regulatory regions of the GNAS gene (GNAS complex locus) that lead to interferences in the methylation status of alternative GNAS promoters, such as exon A/B, NESP55, and XL␣-s. PHPIb comprises disorders that show distinctive changes in methylation status but share the same clinical phenotype: (a) loss of methylation only at exon A/B of the GNAS gene and involving no other obvious epigenetic abnormalities [e.g., those caused by heterozygous microdeletions in the STX16 (syntaxin 16) region and found in many patients with autosomal dominant (AD) PHPIb]; (b) methylation abnormalities at several differentially methylated regions (DMRs), which are observed in most patients with sporadic PHPIb and some families with AD PHPIb.

Establishment of diagnosis by bisulfite-treated methylation-specific PCR method and analysis of clinical characteristics of pseudohypoparathyroidism type 1b

“…They all had at least 3 symptoms of 6 AHO symptoms, discussion MSPCR was invented by Herman for rapid detection of DNA methylation pattern in CpG islands [8], and was clinically applied for rapid diagnosis of Prader-Willi and Angelman syndromes by Kosaki [14]. Weinhaeusel also reported about diagnostic testing of PHP-1b with MSPCR, but his method needs DNA digestion with restriction enzyme prior to PCR reaction [15]. Our method is more rapid and simple.…”

Inhibitors of DNA methylation support TGF-β1-induced IL11 expression in gingival fibroblasts

“…Positive amplification generated from methylated DNA upon restriction confirmed hypermethylation. DNA restriction digestion, control PCR testing the completion of digestion, and IL11-PCR were conducted under conditions described previously [37]. Amplicons were visualized on 2% agarose gels.…”

A Human Model of Epithelial to Mesenchymal Transition to Monitor Drug Efficacy in Hepatocellular Carcinoma Progression

“…Quantitative polymerase chain reaction (qPCR) was done on MSRE-digested DNA covering the CpG rich area at chr5:151,066,289-151,066,501 (human genome annotation: hg; ref. 19), including part of the first gene coding exon. This sequence region contains 9 MSRE sites when using the mixture of 3 enzymes mentioned earlier.…”

“…This sequence region contains 9 MSRE sites when using the mixture of 3 enzymes mentioned earlier. Completion of digestion was confirmed using a control PCR covering known differentially methylated regions (DMRs, H19, IGF2, ABL1, PITX2, XIST, and FMR1) as published recently (19). Ten nanograms of MSREdigested DNA was amplified in 10 mL PCR volume using 0.3 U HotStarTaq polymerase and buffer containing 1.5 mmol/L MgCl 2 (Qiagen) using a 384-well format and the LightCycler LC480 (Roche).…”