title: Differential methylation as a cause of allele dropout at the imprinted GNAS locus.
abstract: BACKGROUND
  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.
  
  
  METHODS
  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).
  
  
  RESULTS
  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.
  
  
  CONCLUSIONS
  For the first time, we report that differential methylation of imprinted regions can lead to preferential amplification of unmethylated alleles. Addition of coadjuvants to the PCR may facilitate amplification of both alleles, providing an accurate genotyping in cases with methylation-related diseases.
authors.affiliation: Centro de Investigação de Patobiologia Molecular (CIPM), Instituto Português de Oncologia de Lisboa Francisco Gentil, Lisboa, Portugal.
authors.forename: Rute A
authors.initials: RA
authors.lastname: Tomaz
authors.forename: Branca M
authors.initials: BM
authors.lastname: Cavaco
authors.forename: Valeriano
authors.initials: V
authors.lastname: Leite
mesh_terms: D000483:Alleles; D022981:Allelic Imbalance / Q000235:genetics / Q000502:physiology; D001483:Base Sequence; D016022:Case-Control Studies; D002864:Chromogranins; D019175:DNA Methylation / Q000502:physiology*; D019205:GTP-Binding Protein alpha Subunits, Gs / Q000235:genetics*; D056426:Genetic Loci; D018392:Genomic Imprinting / Q000502:physiology*; D005838:Genotype; D006801:Humans; D020641:Polymorphism, Single Nucleotide; D011547:Pseudohypoparathyroidism / Q000235:genetics; D017422:Sequence Analysis, DNA
chemical_list: D002864:Chromogranins; C406732:GNAS protein, human; D019205:GTP-Binding Protein alpha Subunits, Gs