A maternal exon H splice-site variant leading to pseudohypoparathyroidism type 1B with broad methylation defects in GNAS-differentially methylated regions

BACKGROUND: The GNAS locus produces multiple transcripts, including the maternally derived, GNAS-H, and GNAS-NESP55 and the paternally derived GNAS-XL, GNAS-A/B, and GNAS-AS, all of which are expressed in specific tissues. GNAS-Gsα is biparentally expressed in most tissues and imprinted in several tissues, specifically, the proximal tubules, thyroid, gonads, and pituitary. These imprinted transcripts are regulated by five differentially methylated regions (GNAS-DMRs), and hypomethylation in the GNAS-A/B:TSS-DMR causes resistance to hormones, including parathyroid hormone, leading to pseudohypoparathyroidism type 1B (PHP1B). Sporadic PHP1B shows broad methylation defects in the GNAS-DMRs, whereas most familial PHP1B cases show localized methylation defects at the GNAS-A/B:TSS-DMR and GNAS-AS2:TSS-DMR. We identified an inherited maternal exon H variant that causes a GNAS methylation pattern typically observed in sporadic PHP1B. RESULTS: To determine the underlying genetic cause in this family, we conducted long-read sequencing (LRS). LRS revealed a maternal exon H variant together with methylation defects of CpGs in the GNAS-DMRs. To evaluate the association of the variant with this familial PHP1B, we established patient-derived induced pluripotent stem cells (iPSCs). Reverse-transcription PCR (RT-PCR) and quantitative RT-PCR in patient-derived iPSCs showed no expression of GNAS-NESP55 or GNAS-H and increased expression of GNAS-AS, together with hypermethylation of the GNAS-NESP:TSS-DMR and hypomethylation of the GNAS-AS1:TSS-DMR and GNAS-XL:Ex1-DMR. RNA sequencing revealed no abnormal GNAS transcripts in iPSCs established from patients. To determine whether loss of GNAS-H expression itself causes PHP1B, or whether methylation defects of the GNAS-DMRs, followed by loss of GNAS-H expression, result in PHP1B in this family, we conducted RT-PCR after demethylation treatment in patient-derived iPSCs. We detected biallelic expressed GNAS-NESP55 transcript and only paternally expressed GNAS-H transcript. These findings indicated impaired maternal GNAS-H transcription in the patients, regardless of the methylation levels in the GNAS-DMRs. Furthermore, amplicon LRS spanning the region from exon H to GNAS-AS exon 1 in 40 sporadic PHP1B patients showed that genomic variants in this region are infrequent. CONCLUSION: Loss of maternal GNAS-H transcript precedes the onset of abnormal methylation imprinting at the GNAS-DMRs and contributes to its establishment. We also revealed that LRS is useful for diagnosing and researching imprinting disorders.