Pseudohypoparathyroidism type Ib (PHP1B), a rare imprinting disorder, is caused by loss-of- methylation at one or several differentially methylation regions (DMR) on the maternal GNAS allele thereby reducing expression of the simulatory G protein (G?s). Besides PTH-resistant hypocalcemia and hyperphosphatemia, additional clinical and laboratory abnormalities are being recognized with increasing frequency. Thus, considerable overlap has become apparent between PHP1B and PHP1A. In fact, we encountered several PHP1B patients, who were evaluated for hyperphagia leading to early- onset obesity many years before a GNAS-related disorder was considered and before PTH-resistance had developed. Autosomal dominant PHP1B (AD-PHP1B) is caused by deletions in GNAS or STX16, but the majority of these patients are sporadic and with the exception of few cases caused by paternal uniparental isodisomy/heterodisomy, the genetic defect(s) leading to this disease variant remains unknown. A novel maternally inherited GNAS deletion that is associated with broad methylation changes at this locus was recently discovered, althought the patient had no family history of the disease; this raises the possibility that other ?sporadic? PHP1B cases are also caused by as-yet undefined GNAS/STX16 mutations. Through whole genome sequencing (WGS), we furthermore identified a large GNAS inversion in a new AD-PHP1B kindred, which causes loss-of-methylation at GNAS exon A/B alone. When this inversion is located on the paternal allele, no functional XL?s (extra- large variants of G?s) can be generated, which is likely to lead to failure-to-thrive and short stature, i.e. findings similar to those observed in humans or rodents with XL?s-deficiency. We now plan to determine whether genetic and epigenetic GNAS changes are a frequent cause of early-onset obesity, congenital hypothyroidism, and potentially short stature, and whether these abnormalities can be encountered even in the absence of PTH-resistant hypocalcemia (Aim 1). To determine whether disease-causing mutations in GNAS/STX16 lead to epigenetic GNAS changes when introduced in vitro, we developed cell lines from the stromal vascular fraction (SVF) of brown adipose tissue in which deletions were introduced through CRISPR/Cas9. We will determine whether these cellular manipulations change allele-specific GNAS methylation and/or parent-specific expression of G?s and its splice variants, and thus the response to hormonal stimulation (Aim 2). We will furthermore search in new AD-PHP1B families with GNAS methylation changes, but without mutations at known disease- causing sites, for novel GNAS/STX16 deletions/inversions, and we will determine through WGS, whether adult females with ?sporadic? PHP1B, who have healthy children without epigenetic GNAS changes, could be affected an autosomal recessive disease variant (Aim 3).
PTH-resistant hypocalcemia is the hallmark of pseudohypoparathyroidism type Ib (PHP1B), a rare imprinting disorder caused by deletions/inversions within GNAS or STX16. In addition to well-establised disease characteristics, previously not recognized clinical and laboratory abnormalities have emerged, including early-onset obesity and congenital hypothyroidism, which may occur even in the absence of mineral ion changes. By identifying novel mutations in GNAS/STX16 or elsewhere in the genome, and by introducing human mutations into clonal cell lines through CRISPR/Cas9, we will explore the mechanisms responsible for parent-specific GNAS methylation and G?s expression.
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