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. 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. 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.

Pseudohypoparathyroidism type 1B (PHP1B) is a rare endocrine disorder caused by epigenetic defects at the GNAS locus, leading to isolated renal resistance to parathyroid hormone (PTH). Although typically identified in childhood, adult-onset cases are uncommon and easily overlooked. This case highlights the diagnostic challenge of late-onset PHP1B and the critical role of methylation-specific testing. A 33-year-old man was referred for evaluation of incidentally detected hypocalcemia (serum calcium 5.9 mg/dL) during a routine health examination. He complained of mild paresthesia of the hands and eyelid twitching but had no family history of endocrine disorders and exhibited no phenotypic features of Albright hereditary osteodystrophy. Laboratory evaluation revealed persistent hypocalcemia (serum calcium 7.7 mg/dL) and markedly elevated PTH levels (284.2 pg/mL) despite correction of magnesium and vitamin D insufficiency. Standard sequencing of the GNAS and STX16 genes showed no pathogenic variants. However, methylation-specific multiplex ligation-dependent probe amplification (MS-MLPA) identified gain of methylation in the NESP55 region and loss of methylation in the AS, XL, and A/B differentially methylated regions confirming a diagnosis of sporadic PHP1B. The patient received oral calcium carbonate and cholecalciferol supplementation. Magnesium deficiency was corrected with oral magnesium oxide. The patient remained asymptomatic during follow-up with adequate calcium supplementation. Adult-onset PHP1B should be considered in the differential diagnosis of unexplained hypocalcemia with elevated PTH, even in the absence of Albright hereditary osteodystrophy. Because conventional sequencing cannot detect imprinting defects, epigenetic testing such as MS-MLPA is essential for definitive diagnosis. Increased awareness of atypical, late-onset presentations can aid in timely diagnosis and appropriate management.

Pseudohypoparathyroidism type 1B (PHP1B) is a multihormone resistance disorder caused by aberrant GNAS methylation. Characteristic epigenetic changes at GNAS differentially methylated regions (DMRs), i.e., NESP, AS1, AS2, XL, and A/B, are associated with specific structural defects in different autosomal dominant PHP1B (AD-PHP1B) subtypes. However, mechanisms underlying abnormal GNAS methylation remain incompletely defined, largely because viable PHP1B mouse models are lacking. Using lymphoblastoid cells and induced pluripotent stem cells, we show that various GNAS methylation patterns in PHP1B reflect differential disruption of sense and antisense GNAS transcripts. In cases with broad GNAS methylation changes, loss of the maternal, sense-transcribed exon H/AS region impairs methylation of the AS1 DMR, which results in biallelic expression of an antisense transcript, GNAS-AS1, and NESP hypermethylation. In contrast, cases with normal AS1 methylation, including STX16 deletions, show monoallelic GNAS-AS1 expression and normal NESP methylation. The roles of these GNAS transcripts were confirmed by a retrotransposon in GNAS-AS1 intron 1, identified in an AD-PHP1B family. This insertion impaired exon H/AS transcription when located on the maternal allele, thus preventing the complete establishment of methylation at all maternal GNAS DMRs, leading to biallelic GNAS-AS1 transcription. However, maternal GNAS-AS1 transcription was profoundly attenuated, thus allowing only a small gain-of-methylation at NESP. Likewise, on the paternal allele, the retrotransposon attenuated GNAS-AS1 transcription, thus preventing complete NESP methylation. Our findings support a model of bidirectional transcription-mediated regulation of methylation at GNAS DMRs and will help to refine systematic approaches for establishing molecular defects underlying different PHP1B subtypes.

Genomic imprinting controls parental allele-specific gene expression via epigenetic mechanisms. Abnormal imprinting at the GNAS gene causes multiple phenotypes, including pseudohypoparathyroidism type-1B (PHP1B), a disorder of multihormone resistance. Microdeletions affecting the neighboring STX16 gene ablate an imprinting control region (STX16-ICR) of GNAS and lead to PHP1B upon maternal but not paternal inheritance. Mechanisms behind this imprinted inheritance mode remain unknown. Here, we show that the STX16-ICR forms different chromatin conformations with each GNAS parental allele and enhances two GNAS promoters in human embryonic stem cells. When these cells differentiate toward proximal renal tubule cells, STX16-ICR loses its effect, accompanied by a transition to a somatic cell-specific GNAS imprinting status. The activity of STX16-ICR depends on an OCT4 motif, whose disruption impacts transcript levels differentially on each allele. Therefore, a biallelically active embryonic enhancer dictates GNAS imprinting via different chromatin conformations, underlying the allele-specific pathogenicity of STX16-ICR microdeletions.

We present a rare familial case of two brothers presenting with late-onset symptoms of posturing, cramps, and occasional falls. The brothers, who were initially under the pediatric team being investigated for rare thiamine-transporter-related genetic disorders, had significant symptomatic hypocalcemia and cerebral calcifications identified on brain imaging. They were both diagnosed with pseudohypoparathyroidism type 1b in their late teenage years and achieved stable calcium levels and symptom control after treatment titration. This case highlights the importance of identifying key hypocalcemia symptoms such as fatigue, muscle cramps, and paresthesia, which may have led to earlier recognition of the condition.