Differential methylation as a cause of allele dropout at the imprinted GNAS locus

Genetic Testing and Molecular Biomarkers
Rute A TomazValeriano Leite

Abstract

We detected false homozygosity at the NESP55 differentially methylated region of the imprinted GNAS locus while analyzing the segregation of single-nucleotide polymorphisms (SNPs) in families with pseudohypoparathyroidism type Ib (PHP-Ib). We hypothesized that differential methylation of NESP55 could affect polymerase chain reaction (PCR) amplification, resulting in allele dropout. We genotyped 10 normal controls for four SNPs in NESP55 differentially methylated region. SNPs were amplified by standard PCR conditions and with the addition of dimethyl sulfoxide. The methylated allele was identified by HpaII analysis, and haplotypes were confirmed using subcloning strategies. All SNPs were also genotyped in a PHP-Ib patient (P1), carrying methylation at both NESP55 alleles, and in an in vitro methylated control DNA (SSSI-N4). In the control samples, we identified allele dropout of the methylated allele in 85% of the amplifications, using standard PCR conditions. Addition of dimethyl sulfoxide to the PCR successfully prevented dropout in all cases. No amplification bias was observed for P1 and SSSI-N4 samples. For the first time, we report that differential methylation of imprinted regions can lead to preferential amplification of ...Continue Reading

References

May 1, 1992·PCR Methods and Applications·P S WalshR Higuchi
Mar 17, 2001·Nature Reviews. Genetics·W Reik, J Walter
Nov 9, 2002·Bioinformatics·Long-Cheng Li, Rajvir Dahiya
Aug 28, 2004·Endocrinology·Lee S WeinsteinMin Chen
Oct 6, 2007·The Journal of Molecular Diagnostics : JMD·Natalia KholodPhilippe Delvenne
Jan 6, 2009·BMC Bioinformatics·Chumpol NgamphiwSissades Tongsima

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Citations

Apr 13, 2012·Travel Medicine and Infectious Disease·Remington L Nevin
Oct 7, 2016·Journal of Pediatric Endocrinology & Metabolism : JPEM·Maria Francisca CoutinhoSandra Alves
Jan 10, 2021·Scientific Reports·Jonas M den HeijerGeert Jan Groeneveld

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Datasets Mentioned

BETA
AL132655

Methods Mentioned

BETA
genotyping
PCR
electrophoresis
PCRs

Software Mentioned

Sequencing Analysis
MethPrimer
VarDetect

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