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Context: Sporadic pseudohypoparathyroidism type 1B (sporPHP1B) is an imprinting disease without a defined genetic cause, characterized by broad methylation changes in differentially methylated regions (DMRs) of the GNAS gene., Objective: This work aims to provide insights into the causative event leading to the GNAS methylation defects through comprehensive molecular genetic analyses of a pair of female monozygotic twins concordant for sporPHP1B who were conceived naturally, that is, without assisted reproductive techniques., Methods: Using the leukocyte genome of the twins and family members, we performed targeted bisulfite sequencing, methylation-sensitive restriction enzyme (MSRE)-quantitative polymerase chain reaction (qPCR), whole-genome sequencing (WGS), high-density single-nucleotide polymorphism (SNP) array, and Sanger sequencing., Results: Methylation analyses by targeted bisulfite sequencing and MSRE-qPCR revealed almost complete losses of methylation at the GNAS AS, XL, and A/B DMRs and a gain of methylation at the NESP55 DMR in the twins, but not in other family members. Except for the GNAS locus, we did not find apparent methylation defects at other imprinted genome loci of the twins. WGS, SNP array, and Sanger sequencing did not detect the previously described genetic defects associated with familial PHP1B. Sanger sequencing also ruled out any novel genetic alterations in the entire NESP55/AS region. However, the analysis of 28 consecutive SNPs could not exclude the possibility of paternal heterodisomy in a span of 22 kb comprising exon NESP55 and AS exon 5., Conclusion: Our comprehensive analysis of a pair of monozygotic twins with sporPHP1B ruled out all previously described genetic causes. Twin concordance indicates that the causative event was an imprinting error earlier than the timing of monozygotic twinning., (© The Author(s) 2021. Published by Oxford University Press on behalf of the Endocrine Society.)

Most patients with autosomal dominant pseudohypoparathyroidism type Ib (AD-PHP-Ib) carry maternally inherited microdeletions upstream of GNAS that are associated with loss of methylation restricted to GNAS exon A/B. Only few AD-PHP-Ib patients carry microdeletions within GNAS that are associated with loss of all maternal methylation imprints. These epigenetic changes are often indistinguishable from those observed in patients affected by an apparently sporadic PHP-Ib form that has not yet been defined genetically. We have now investigated six female patients affected by PHP-Ib (four unrelated and two sisters) with complete or almost complete loss of GNAS methylation, whose healthy children (11 in total) showed no epigenetic changes at this locus. Analysis of several microsatellite markers throughout the 20q13 region made it unlikely that PHP-Ib is caused in these patients by large deletions involving GNAS or by paternal uniparental isodisomy or heterodisomy of chromosome 20 (patUPD20). Microsatellite and single-nucleotide variation (SNV) data revealed that the two affected sisters share their maternally inherited GNAS alleles with unaffected relatives that lack evidence for abnormal GNAS methylation, thus excluding linkage to this locus. Consistent with these findings, healthy children of two unrelated sporadic PHP-Ib patients had inherited different maternal GNAS alleles, also arguing against linkage to this locus. Based on our data, it appears plausible that some forms of PHP-Ib are caused by homozygous or compound heterozygous mutation(s) in an unknown gene involved in establishing or maintaining GNAS methylation., (Copyright © 2011 American Society for Bone and Mineral Research.)

Context: GNAS encodes the alpha-subunit of the stimulatory G protein as well as additional imprinted transcripts including the maternally expressed NESP55 and the paternally expressed XLalphas, antisense, and A/B transcripts. Most patients with pseudohypoparathyroidism type Ib (PHP-Ib) exhibit imprinting defects affecting the maternal GNAS allele, which are thought to reduce/abolish Gsalpha expression in renal proximal tubules and thereby cause resistance to PTH., Objective: Our objective was to define the genetic defect in a previously unreported family with autosomal dominant PHP-Ib., Design and Setting: Analyses of serum and urine chemistries and of genomic DNA and lymphoblastoid-derived RNA were conducted at a tertiary hospital and research laboratory., Patients: Affected individuals presented with muscle weakness and/or paresthesia and showed hypocalcemia, hyperphosphatemia, and elevated serum PTH. Obligate carriers were healthy and revealed no obvious abnormality in mineral ion homeostasis., Results: A novel 4.2-kb microdeletion was discovered in the affected individuals and the obligate carriers, ablating two noncoding GNAS antisense exons while preserving the NESP55 exon. On maternal transmission, the deletion causes loss of all maternal GNAS imprints, partial gain of NESP55 methylation, and PTH resistance. Paternal transmission of the mutation leads to epigenetic alterations in cis, including a partial loss of NESP55 methylation and a partial gain of A/B methylation., Conclusions: The identified deletion points to a unique cis-acting element located telomeric of the NESP55 exon that is critical for imprinting both GNAS alleles. These findings provide novel insights into the molecular mechanisms underlying PHP and GNAS imprinting.

Approximately 100 genes undergo genomic imprinting. Mutations in fewer than 10 imprinted genetic loci, including GNAS, are associated with complex human diseases that differ phenotypically based on the parent transmitting the mutation. Besides the ubiquitously expressed Gsalpha, which is of broad biological importance, GNAS gives rise to an antisense transcript and to several Gsalpha variants that are transcribed from the nonmethylated parental allele. We previously identified two almost identical GNAS microdeletions extending from exon NESP55 to antisense (AS) exon 3 (delNESP55/delAS3-4). When inherited maternally, both deletions are associated with erasure of all maternal GNAS methylation imprints and autosomal-dominant pseudohypoparathyroidism type Ib, a disorder characterized by parathyroid hormone-resistant hypocalcemia and hyperphosphatemia. As for other imprinting disorders, the mechanisms resulting in abnormal GNAS methylation are largely unknown, in part because of a paucity of suitable animal models. We now showed in mice that deletion of the region equivalent to delNESP55/delAS3-4 on the paternal allele (DeltaNesp55(p)) leads to healthy animals without Gnas methylation changes. In contrast, mice carrying the deletion on the maternal allele (DeltaNesp55(m)) showed loss of all maternal Gnas methylation imprints, leading in kidney to increased 1A transcription and decreased Gsalpha mRNA levels, and to associated hypocalcemia, hyperphosphatemia, and secondary hyperparathyroidism. Besides representing a murine autosomal-dominant pseudohypoparathyroidism type Ib model and one of only few animal models for imprinted human disorders, our findings suggest that the Nesp55 differentially methylated region is an additional principal imprinting control region, which directs Gnas methylation and thereby affects expression of all maternal Gnas-derived transcripts.

Context: Several endocrine diseases that share resistance to PTH are grouped under the term pseudohypoparathyroidism (PHP). Patients with PHP type Ia show additional hormone resistance, defective erythrocyte G(s)alpha activity, and dysmorphic features termed Albright's hereditary osteodystrophy (AHO). Patients with PHP-Ib show less diverse hormone resistance and normal G(s)alpha activity; AHO features are typically absent in PHP-Ib. Mutations affecting G(s)alpha coding exons of GNAS and epigenetic alterations in the same gene are associated with PHP-Ia and -Ib, respectively. The epigenetic GNAS changes in familial PHP-Ib are caused by microdeletions near or within GNAS but without involving G(s)alpha coding exons., Objective: We sought to identify the molecular defect in a patient who was diagnosed with PHP-Ia based on clinical presentation (hormone resistance and AHO) but displayed the molecular features typically associated with PHP-Ib (loss of methylation at exon A/B) without previously described genetic mutations., Methods: Microsatellite typing, comparative genome hybridization, and allelic dosage were performed for proband and her parents., Results: Comparative genome hybridization revealed a deletion of 30,431 bp extending from the intronic region between exons XL and A/B to intron 5. The same mutation was also demonstrated, by PCR, in the patient's mother, but polymorphism and allele dosage analyses indicated that she had this mutation in a mosaic manner., Conclusion: We discovered a novel multiexonic GNAS deletion transmitted to our patient from her mother who is mosaic for this mutation. The deletion led to different phenotypic manifestations in the two generation and appeared, in the patient, as loss of GNAS imprinting.

Epigenetic defects in the imprinted GNAS cluster are associated with pseudohypoparathyroidism type Ib. In two kindreds with this disorder, we now report deletions that remove the differentially methylated region encompassing exon NESP55 and exons 3 and 4 of the antisense transcript. When inherited from a female, either deletion abolishes all maternal GNAS imprints and derepresses maternally silenced transcripts, suggesting that the deleted region contains a cis-acting element that controls imprinting of the maternal GNAS allele.

Patients with pseudohypoparathyroidism type Ib (PHP-Ib) have hypocalcemia and hyperphosphatemia due to renal parathyroid hormone (PTH) resistance, but lack physical features of Albright hereditary osteodystrophy. PHP-Ib is thus distinct from PHP-Ia, which is caused by mutations in the GNAS exons encoding the G protein alpha subunit. However, an imprinted autosomal dominant form of PHP-Ib (AD-PHP-Ib) has been mapped to a region of chromosome 20q13.3 containing GNAS. Furthermore, loss of methylation at a differentially methylated region (DMR) of this locus, exon A/B, has been observed thus far in all investigated sporadic PHP-Ib cases and the affected members of multiple AD-PHP-Ib kindreds. We now report that affected members and obligate gene carriers of 12 unrelated AD-PHP-Ib kindreds and four apparently sporadic PHP-Ib patients, but not healthy controls, have a heterozygous approximately 3-kb microdeletion located approximately 220 kb centromeric of GNAS exon A/B. The deleted region, which is flanked by two direct repeats, includes three exons of STX16, the gene encoding syntaxin-16, for which no evidence of imprinting could be found. Affected individuals carrying the microdeletion show loss of exon A/B methylation but no epigenetic abnormalities at other GNAS DMRs. We therefore postulate that this microdeletion disrupts a putative cis-acting element required for methylation at exon A/B, and that this genetic defect underlies the renal PTH resistance in AD-PHP-Ib.

Heterozygous inactivating mutations in the GNAS1 exons (20q13.3) that encode the alpha-subunit of the stimulatory G protein (Gsalpha) are found in patients with pseudohypoparathyroidism type Ia (PHP-Ia) and in patients with pseudo-pseudohypoparathyroidism (pPHP). However, because of paternal imprinting, resistance to parathyroid hormone (PTH)-and, sometimes, to other hormones that require Gsalpha signaling-develops only if the defect is inherited from a female carrier of the disease gene. An identical mode of inheritance is observed in kindreds with pseudohypoparathyroidism type Ib (PHP-Ib), which is most likely caused by mutations in regulatory regions of the maternal GNAS1 gene that are predicted to interfere with the parent-specific methylation of this gene. We report a patient with PTH-resistant hypocalcemia and hyperphosphatemia but without evidence for Albright hereditary osteodystrophy who has paternal uniparental isodisomy of chromosome 20q and lacks the maternal-specific methylation pattern within GNAS1. Since studies in the patient's fibroblasts did not reveal any evidence of impaired Gsalpha protein or activity, it appears that the loss of the maternal GNAS1 gene and the resulting epigenetic changes alone can lead to PTH resistance in the proximal renal tubules and thus lead to impaired regulation of mineral-ion homeostasis.