Intragenic Deletions of GNAS in Pseudohypoparathyroidism Type 1A Identify a New Region Affecting Methylation of Exon A/B

Context: Pseudohypoparathyroidism type 1A (PHP1A) and pseudopseudohypoparathyroidism (PPHP) are caused by inactivating mutations in the exons of GNAS that encode the alpha-subunit of the stimulatory G protein (G sa). In some cases abnormal methylation of exon A/B of GNAS, a hallmark of PHP1B, has been reported. Objective: To identify the underlying genetic basis for PHP1A/PPHP in patients in whom molecular defects were not detected by GNAS sequencing and microarray-based analysis of copy number variations. Methods: Whole genome sequencing (WGS) and pyrosequencing of differentially methylated regions (DMRs) of GNAS using genomic deoxyribonucleic acid from affected patients. Results: We identified 2 novel heterozygous GNAS deletions: a 6.4 kb deletion that includes exon 2 of GNAS in the first proband that was associated with normal methylation (57%) of exon A/B DMR, and a 1438 bp deletion in a second PHP1A patient that encompasses the promoter region and 5' untranslated region of G sa transcripts, which was inherited from his mother with PPHP. This deletion was associated with reduced methylation (32%) of exon A/B DMR. Conclusions: WGS can detect exonic and intronic mutations, including deletions that are too small to be identified by microarray analysis, and therefore is more sensitive than other techniques for molecular analysis of PHP1A/PPHP. One of the deletions we identified led to reduced methylation of exon A/B DMR, further refining a region needed for normal imprinting of this DMR. We propose that deletion of this region can explain why some PHP1A patients have reduced of methylation of the exon A/B DMR.