Targeted deletion of the Nesp55 DMR defines another Gnas imprinting control region and provides a mouse model of autosomal dominant PHP-Ib

Abstract

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 Gsα, which is of broad biological importance, GNAS gives rise to an antisense transcript and to several Gsα 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 (ΔNesp55 p ) leads to healthy animals without Gnas methylation changes. In contrast, mice carrying the deletion on the maternal allele (ΔNesp55 m ) showed loss of all maternal Gnas methylation imprints, leading in kidney to increased 1A transcription and decreased Gsα 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.genomic imprinting | Gsα | pseudohypoparathyroidism | parathyroid hormone | hormonal resistance

Loss of XLαs (extra-large αs) imprinting results in early postnatal hypoglycemia and lethality in a mouse model of pseudohypoparathyroidism Ib

“…This mouse strain, ΔNesp55 m , phenocopies AD-PHP-Ib with respect to the GNAS imprinting defects-i.e., loss of all of the maternal Gnas methylation imprints combined with increased (biallelic) 1A transcription-and with respect to the abnormal regulation of mineral ion homeostasis-i.e., hypocalcemia, hyperphosphatemia, and secondary hyperparathyroidism (30). However, unlike the findings in patients with deletions involving NESP55 and antisense exons 3 and 4 (AD-PHP-Ib delNASm ), there is 100% early postnatal lethality in ΔNesp55 m mice, whereas mice in which the paternal Nesp55 DMR is deleted (ΔNesp55 p mice) show no epigenetic and biochemical abnormalities and have an apparently normal phenotype and life span.…”

“…We have previously generated mice in which the Gnas Nesp55 DMR was ablated (30). Maternal deletion of this DMR led to a loss of all maternal Gnas imprint marks and biochemical abnormalities consistent with PTH resistance, similar to that observed in AD-PHP-Ib delNASm patients, who carry the equivalent deletion in the same locus.…”

“…The ΔNesp55 m mice die within 5 d of postnatal life in 129/S2 and C57BL/6J backgrounds (30). As an attempt to improve survival of these mice, we crossed them into the outbred CD1 strain, but the early postnatal lethality did not change significantly, with no pups surviving until postnatal day 10 (P10).…”

“…As an attempt to improve survival of these mice, we crossed them into the outbred CD1 strain, but the early postnatal lethality did not change significantly, with no pups surviving until postnatal day 10 (P10). We have previously shown that ΔNesp55 m mice are hypocalcemic at P2 and attributed the lethality to the possible worsening of the low calcium levels by day 5 (30). To determine whether other factors could underlie or contribute to the early postnatal lethality of the ΔNesp55 m mice, we measured blood glucose levels at P2.…”

“…Alternatively, the feeding difficulty in ΔNesp55 m mice can result from a generalized neurological or motor defect that prevents the pups from getting access to the mother. In fact, some of the ΔNesp55 m pups appear hyperactive (30), which may reflect a neurological defect.…”