Maternal GNAS Contributes to the Extra-Large G Protein α-Subunit (XLαs) Expression in a Cell Type-Specific Manner

Abstract

GNAS encodes the stimulatory G protein alpha-subunit (Gsα) and its large variant XLαs. Studies have suggested that XLαs is expressed exclusively paternally. Thus, XLαs deficiency is considered to be responsible for certain findings in patients with paternal GNAS mutations, such as pseudo-pseudohypoparathyroidism, and the phenotypes associated with maternal uniparental disomy of chromosome 20, which comprises GNAS. However, a study of bone marrow stromal cells (BMSC) suggested that XLαs could be biallelically expressed. Aberrant BMSC differentiation due to constitutively activating GNAS mutations affecting both Gsα and XLαs is the underlying pathology in fibrous dysplasia of bone. To investigate allelic XLαs expression, we employed next-generation sequencing and a polymorphism common to XLαs and Gsα, as well as A/B, another paternally expressed GNAS transcript. In mouse BMSCs, Gsα transcripts were 48.4 ± 0.3% paternal, while A/B was 99.8 ± 0.2% paternal. In contrast, XLαs expression varied among different samples, paternal contribution ranging from 43.0 to 99.9%. Sample-to-sample variation in paternal XLαs expression was also detected in bone (83.7–99.6%) and cerebellum (83.8 to 100%) but not in cultured calvarial osteoblasts (99.1 ± 0.1%). Osteoblastic differentiation of BMSCs shifted the paternal XLαs expression from 83.9 ± 1.5% at baseline to 97.2 ± 1.1%. In two human BMSC samples grown under osteoinductive conditions, XLαs expression was also predominantly monoallelic (91.3 or 99.6%). Thus, the maternal GNAS contributes significantly to XLαs expression in BMSCs but not osteoblasts. Altered XLαs activity may thus occur in certain cell types irrespective of the parental origin of a GNAS defect.

The long-range interaction between two GNAS imprinting control regions delineates pseudohypoparathyroidism type 1B pathogenesis

“…Note that maternal XLαs transcription is not completely silenced in hESCs (see Supplementary Fig. 1E), which is consistent with previous studies showing maternal allele contribution to XLαs in bone marrow stromal cells (45,46).…”

“…Bisulfite PCR and amplicon sequencing were performed as we previously described ( 45 ). Genomic DNA was bisulfite converted using the EZ DNA Methylation-Gold Kit (ZYMO RESEARCH) following the manufacturer’s protocol, and each target region was amplified by a single-step PCR using KOD One.…”

GNAS gene mutations affecting XLαs and bone health: A long neglected relationship

“…39,41 It is now known that mouse-and humanderived BMSCs and adipose-derived stem cells may exhibit biallelic expression of Gsα equally without gene imprinting expression. 8,34 Thus, it is speculated that the key factor underlying the phenotypic differences in HO within offspring with paternal gene defects in GNAS exons 5-13 (ALEX without defect) may also be associated with another imprinted gene, XLαs. XLαs mRNA is expressed in bone marrow stromal cells and MC3T3-E1 preosteoblastic cells, and the expression of XLαs progressively increases during the differentiation of osteoblasts.…”

“…Coincidentally, Gsα is biallelic in most tissues but maternal in the vast majority of neuroendocrine tissues 8 . Notably, paternal expression of XLαs may not be absolute, as recent studies have demonstrated that XLαs can be biallelic in mouse‐ and human‐derived BMSCs, with paternal expression higher than maternal expression 34 . By Northern blots and RT‐PCR, XXLαs (extralarge XLαs ) mRNA was detected primarily in the mouse brain, cerebellum, and spleen 19 …”

“…Paternally inherited mutations in the same gene can lead to differences in the incidence and severity of ectopic ossification between PPHP and POH in the offspring 39,41 . It is now known that mouse‐ and human‐derived BMSCs and adipose‐derived stem cells may exhibit biallelic expression of Gsα equally without gene imprinting expression 8,34 . Thus, it is speculated that the key factor underlying the phenotypic differences in HO within offspring with paternal gene defects in GNAS exons 5‐13 ( ALEX without defect) may also be associated with another imprinted gene, XLαs .…”