Differential methylation as a cause of allele dropout at the imprinted GNAS locus.
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.
Tomaz
Rute A
RA
Centro de Investigação de Patobiologia Molecular (CIPM), Instituto Português de Oncologia de Lisboa Francisco Gentil, Lisboa, Portugal.
Cavaco
Branca M
BM
Leite
Valeriano
V
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
D002864:Chromogranins; C406732:GNAS protein, human; D019205:GTP-Binding Protein alpha Subunits, Gs