Gsα-dependent signaling is required for postnatal establishment of a functional ß-cell mass.

OBJECTIVE: Early postnatal life is a critical period for the establishment of the functional ß-cell mass that will sustain whole-body glucose homeostasis during the lifetime. ß cells are formed from progenitors during embryonic development but undergo significant expansion in quantity and attain functional maturity after birth. The signals and pathways involved in these processes are not fully elucidated. Cyclic adenosine monophosphate (cAMP) is an intracellular signaling molecule that is known to regulate insulin secretion, gene expression, proliferation, and survival of adult ß cells. The heterotrimeric G protein Gs stimulates the cAMP-dependent pathway by activating adenylyl cyclase. In this study, we sought to explore the role of Gs-dependent signaling in postnatal ß-cell development. METHODS: To study Gs-dependent signaling, we generated conditional knockout mice in which the α subunit of the Gs protein (Gsα) was ablated from ß-cells using the Cre deleter line Ins1Cre. Mice were characterized in terms of glucose homeostasis, including in vivo glucose tolerance, glucose-induced insulin secretion, and insulin sensitivity. ß-cell mass was studied using histomorphometric analysis and optical projection tomography. ß-cell proliferation was studied by ki67 and phospho-histone H3 immunostatining, and apoptosis was assessed by TUNEL assay. Gene expression was determined in isolated islets and sorted ß cells by qPCR. Intracellular cAMP was studied in isolated islets using HTRF-based technology. The activation status of the cAMP and insulin-signaling pathways was determined by immunoblot analysis of the relevant components of these pathways in isolated islets. In vitro proliferation of dissociated islet cells was assessed by BrdU incorporation. RESULTS: Elimination of Gsα in ß cells led to reduced ß-cell mass, deficient insulin secretion, and severe glucose intolerance. These defects were evident by weaning and were associated with decreased proliferation and inadequate expression of key ß-cell identity and maturation genes in postnatal ß-cells. Additionally, loss of Gsα caused a broad multilevel disruption of the insulin transduction pathway that resulted in the specific abrogation of the islet proliferative response to insulin. CONCLUSION: We conclude that Gsα is required for ß-cell growth and maturation in the early postnatal stage and propose that this is partly mediated via its crosstalk with insulin signaling. Our findings disclose a tight connection between these two pathways in postnatal ß cells, which may have implications for using cAMP-raising agents to promote ß-cell regeneration and maturation in diabetes.

DNA methylation changes during long-term in vitro cell culture are caused by epigenetic drift.

Culture expansion of primary cells evokes highly reproducible DNA methylation (DNAm) changes. We have identified CG dinucleotides (CpGs) that become continuously hyper- or hypomethylated during long-term culture of mesenchymal stem cells (MSCs) and other cell types. Bisulfite barcoded amplicon sequencing (BBA-seq) demonstrated that DNAm patterns of neighboring CpGs become more complex without evidence of continuous pattern development and without association to oligoclonal subpopulations. Circularized chromatin conformation capture (4C) revealed reproducible changes in nuclear organization between early and late passages, while there was no enriched interaction with other genomic regions that also harbor culture-associated DNAm changes. Chromatin immunoprecipitation of CTCF did not show significant differences during long-term culture of MSCs, however culture-associated hypermethylation was enriched at CTCF binding sites and hypomethylated CpGs were devoid of CTCF. Taken together, our results support the notion that DNAm changes during culture-expansion are not directly regulated by a targeted mechanism but rather resemble epigenetic drift.

Genetic barcoding reveals clonal dominance in iPSC-derived mesenchymal stromal cells.

BACKGROUND: The use of mesenchymal stromal cells (MSCs) for research and clinical application is hampered by cellular heterogeneity and replicative senescence. Generation of MSC-like cells from induced pluripotent stem cells (iPSCs) may circumvent these limitations, and such iPSC-derived MSCs (iMSCs) are already tested in clinical trials. So far, a comparison of MSCs and iMSCs was particularly addressed in bulk culture. Despite the high hopes in cellular therapy, only little is known how the composition of different subclones changes in these cell preparations during culture expansion. METHODS: In this study, we used multicolor lentiviral genetic barcoding for the marking of individual cells within cell preparations. Based on this, we could track the clonal composition of syngenic MSCs, iPSCs, and iMSCs during culture expansion. Furthermore, we analyzed DNA methylation patterns at senescence-associated genomic regions by barcoded bisulfite amplicon sequencing. The proliferation and differentiation capacities of individual subclones within MSCs and iMSCs were investigated with limiting dilution assays. RESULTS: Overall, the clonal composition of primary MSCs and iPSCs gradually declined during expansion. In contrast, iMSCs became oligoclonal early during differentiation, indicating that they were derived from few individual iPSCs. This dominant clonal outgrowth of iMSCs was not associated with changes in chromosomal copy number variation. Furthermore, clonal dynamics were not clearly reflected by stochastically acquired DNA methylation patterns. Limiting dilution assays revealed that iMSCs are heterogeneous in colony formation and in vitro differentiation potential, while this was even more pronounced in primary MSCs. CONCLUSIONS: Our results indicate that the subclonal diversity of MSCs and iPSCs declines gradually during in vitro culture, whereas derivation of iMSCs may stem from few individual iPSCs. Differentiation regimen needs to be further optimized to achieve homogeneous differentiation of iPSCs towards iMSCs.

A MAFG-lncRNA axis links systemic nutrient abundance to hepatic glucose metabolism.

Obesity and type 2 diabetes mellitus are global emergencies and long noncoding RNAs (lncRNAs) are regulatory transcripts with elusive functions in metabolism. Here we show that a high fraction of lncRNAs, but not protein-coding mRNAs, are repressed during diet-induced obesity (DIO) and refeeding, whilst nutrient deprivation induced lncRNAs in mouse liver. Similarly, lncRNAs are lost in diabetic humans. LncRNA promoter analyses, global cistrome and gain-of-function analyses confirm that increased MAFG signaling during DIO curbs lncRNA expression. Silencing Mafg in mouse hepatocytes and obese mice elicits a fasting-like gene expression profile, improves glucose metabolism, de-represses lncRNAs and impairs mammalian target of rapamycin (mTOR) activation. We find that obesity-repressed LincIRS2 is controlled by MAFG and observe that genetic and RNAi-mediated LincIRS2 loss causes elevated blood glucose, insulin resistance and aberrant glucose output in lean mice. Taken together, we identify a MAFG-lncRNA axis controlling hepatic glucose metabolism in health and metabolic disease.

Senescence-Associated Metabolomic Phenotype in Primary and iPSC-Derived Mesenchymal Stromal Cells.

Long-term culture of primary cells is characterized by functional and secretory changes, which ultimately result in replicative senescence. It is largely unclear how the metabolome of cells changes during replicative senescence and if such changes are consistent across different cell types. We have directly compared culture expansion of primary mesenchymal stromal cells (MSCs) and induced pluripotent stem cell-derived MSCs (iMSCs) until they reached growth arrest. Both cell types acquired similar changes in morphology, in vitro differentiation potential, senescence-associated ß-galactosidase, and DNA methylation. Furthermore, MSCs and iMSCs revealed overlapping gene expression changes, particularly in functional categories related to metabolic processes. We subsequently compared the metabolomes of MSCs and iMSCs and observed overlapping senescence-associated changes in both cell types, including downregulation of nicotinamide ribonucleotide and upregulation of orotic acid. Taken together, replicative senescence is associated with a highly reproducible senescence-associated metabolomics phenotype, which may be used to monitor the state of cellular aging.

LincRNA H19 protects from dietary obesity by constraining expression of monoallelic genes in brown fat.

Increasing brown adipose tissue (BAT) thermogenesis in mice and humans improves metabolic health and understanding BAT function is of interest for novel approaches to counteract obesity. The role of long noncoding RNAs (lncRNAs) in these processes remains elusive. We observed maternally expressed, imprinted lncRNA H19 increased upon cold-activation and decreased in obesity in BAT. Inverse correlations of H19 with BMI were also observed in humans. H19 overexpression promoted, while silencing of H19 impaired adipogenesis, oxidative metabolism and mitochondrial respiration in brown but not white adipocytes. In vivo, H19 overexpression protected against DIO, improved insulin sensitivity and mitochondrial biogenesis, whereas fat H19 loss sensitized towards HFD weight gains. Strikingly, paternally expressed genes (PEG) were largely absent from BAT and we demonstrated that H19 recruits PEG-inactivating H19-MBD1 complexes and acts as BAT-selective PEG gatekeeper. This has implications for our understanding how monoallelic gene expression affects metabolism in rodents and, potentially, humans.

Effects of senolytic drugs on human mesenchymal stromal cells.

BACKGROUND: Senolytic drugs are thought to target senescent cells and might thereby rejuvenate tissues. In fact, such compounds were suggested to increase health and lifespan in various murine aging models. So far, effects of senolytic drugs have not been analysed during replicative senescence of human mesenchymal stromal cells (MSCs). METHODS: In this study, we tested four potentially senolytic drugs: ABT-263 (navitoclax), quercetin, nicotinamide riboside, and danazol. The effects of these compounds were analysed during long-term expansion of MSCs, until replicative senescence. Furthermore, we determined the effect on molecular markers for replicative senescence, such as senescence-associated beta-galactosidase staining (SA-ß-gal), telomere attrition, and senescence-associated DNA methylation changes. RESULTS: Co-culture experiments of fluorescently labelled early and late passages revealed that particularly ABT-263 had a significant but moderate senolytic effect. This was in line with reduced SA-ß-gal staining in senescent MSCs upon treatment with ABT-263. However, none of the drugs had significant effects on the maximum number of population doublings, telomere length, or epigenetic senescence predictions. CONCLUSIONS: Of the four tested drugs, only ABT-263 revealed a senolytic effect in human MSCs-and even treatment with this compound did not rejuvenate MSCs with regard to telomere length or epigenetic senescence signature. It will be important to identify more potent senolytic drugs to meet the high hopes for regenerative medicine.

Primary Osteoporosis Is Not Reflected by Disease-Specific DNA Methylation or Accelerated Epigenetic Age in Blood.

Osteoporosis is an age-related metabolic bone disease. Hence, osteoporotic patients might suffer from molecular features of accelerated aging, which is generally reflected by specific age-associated DNA methylation (DNAm) changes. In this study, we analyzed genomewide DNAm profiles of peripheral blood from patients with manifest primary osteoporosis and non-osteoporotic controls. Statistical analysis did not reveal any individual CG dinucleotides (CpG sites) with significant aberrant DNAm in osteoporosis. Subsequently, we analyzed if age-associated DNAm patterns are increased in primary osteoporosis (OP). Using three independent age-predictors we did not find any evidence for accelerated epigenetic age in blood of osteoporotic patients. Taken together, osteoporosis is not reflected by characteristic DNAm patterns of peripheral blood that might be used as biomarker for the disease. The prevalence of osteoporosis is age-associated-but it is not associated with premature epigenetic aging in peripheral blood. © 2017 American Society for Bone and Mineral Research.

Human Platelet Lysate versus Fetal Calf Serum: These Supplements Do Not Select for Different Mesenchymal Stromal Cells.

Culture medium of mesenchymal stromal cells (MSCs) is usually supplemented with either human platelet lysate (HPL) or fetal calf serum (FCS). Many studies have demonstrated that proliferation and cellular morphology are affected by these supplements - it is therefore important to determine if they favor outgrowth of different subpopulations and thereby impact on the heterogeneous composition of MSCs. We have isolated and expanded human bone marrow-derived MSCs in parallel with HPL or FCS and demonstrated that HPL significantly increases proliferation and leads to dramatic differences in cellular morphology. Remarkably, global DNA-methylation profiles did not reveal any significant differences. Even at the transcriptomic level, there were only moderate changes in pairwise comparison. Furthermore, the effects on proliferation, cytoskeletal organization, and focal adhesions were reversible by interchanging to opposite culture conditions. These results indicate that cultivation of MSCs with HPL or FCS has no systematic bias for specific cell types.

Effects of sex steroids on the pattern of methylation and expression of the promoter region of estrogen and androgen receptors in people with gender dysphoria under cross-sex hormone treatment.

Cross-sex hormone therapy (CHT) is critical for phenotypical and physiological transition in adults with gender dysphoria (GD). However, the impact of the CHT onto the molecular level/epigenetic regulation has not been comprehensively addressed. We postulate that CHT in GD could drive changes at the androgen receptor (AR), estrogen receptor alpha (ESR1) and estrogen receptor beta (ESR2), affecting their DNA methylation pattern and mRNA expression that may influence in the phenotypical changes associated to CHT. We carried out a prospective observational study on individuals with a diagnosis of GD. 18 subjects (no previous CHT): 12 female to male (FtoM) and 6 male to female (MtoF). An Epityper Mass array TM method was used to study the DNA methylation and Real-time PCR quantitative reverse transcription PCR (qRT-PCR) was used to quantify the gene expression. The analysis of AR, ESR1 and ESR2 receptor was performed at baseline, 6 and 12 months after CHT. No differences in DNA methylation of ESR were found in MtoF, while DNA methylation was increased in FtoM at 6 and 12 months of CHT. The AR showed a significant increase of methylation in MtoF group after 12 months of estrogenic treatment. Regarding the expression analysis, AR expression was significantly decreased in FtoM upon CHT treatment. AR, ESR1 and ESR2 methylation were correlated with anthropometric, metabolic and hormonal parameters in FtoM and MtoF. Our results support that CHT is associated to epigenetic changes that might affect the response to treatment with sex steroids.

The lncRNA HOTAIR impacts on mesenchymal stem cells via triple helix formation.

There is a growing perception that long non-coding RNAs (lncRNAs) modulate cellular function. In this study, we analyzed the role of the lncRNA HOTAIR in mesenchymal stem cells (MSCs) with particular focus on senescence-associated changes in gene expression and DNA-methylation (DNAm). HOTAIR binding sites were enriched at genomic regions that become hypermethylated with increasing cell culture passage. Overexpression and knockdown of HOTAIR inhibited or stimulated adipogenic differentiation of MSCs, respectively. Modification of HOTAIR expression evoked only very moderate effects on gene expression, particularly of polycomb group target genes. Furthermore, overexpression and knockdown of HOTAIR resulted in DNAm changes at HOTAIR binding sites. Five potential triple helix forming domains were predicted within the HOTAIR sequence based on reverse Hoogsteen hydrogen bonds. Notably, the predicted triple helix target sites for these HOTAIR domains were also enriched in differentially expressed genes and close to DNAm changes upon modulation of HOTAIR Electrophoretic mobility shift assays provided further evidence that HOTAIR domains form RNA-DNA-DNA triplexes with predicted target sites. Our results demonstrate that HOTAIR impacts on differentiation of MSCs and that it is associated with senescence-associated DNAm. Targeting of epigenetic modifiers to relevant loci in the genome may involve triple helix formation with HOTAIR.

Jansen Metaphyseal Chondrodysplasia due to Heterozygous H223R-PTH1R Mutations With or Without Overt Hypercalcemia.

CONTEXT: Jansen's metaphyseal chondrodysplasia (JMC) is a rare skeletal dysplasia characterized by abnormal endochondral bone formation and typically severe hypercalcemia despite normal/low levels of PTH. Five different heterozygous activating PTH/PTHrP receptor (PTH1R) mutations that change one of three different amino acid residues are known to cause JMC. OBJECTIVES: Establishing the diagnosis of JMC during infancy or early childhood can be challenging, especially in the absence of family history and/or overt hypercalcemia. We therefore sought to provide radiographic findings supporting this diagnosis early in life. PATIENTS AND METHODS: Three patients, a mother and her two sons, had radiographic evidence for JMC. However, obvious hypercalcemia and suppressed PTH levels were encountered only in both affected children. Sanger sequencing and endonuclease (SphI) digestion of PCR-amplified genomic DNA were performed to search for the H223R-PTH1R mutation. RESULTS: The heterozygous H223R mutation was identified in all three affected individuals. Surprisingly, however, the now 38-year-old mother was never overtly hypercalcemic and was therefore not diagnosed until her sons were found to be affected by JMC at the ages of 28 months and 40 days, respectively. The presented radiographic findings at different ages will help diagnose other infants/toddlers suspected of having JMC. CONCLUSION: The H223R mutation is typically associated with profound hypercalcemia despite low/normal PTH levels. However, the findings presented herein show that overt hypercalcemia is not always encountered in JMC, even if caused by this relatively frequent mutation, which is similar to observations with other PTH1R mutations that show less constitutive activity.

New splice site acceptor mutation in AIRE gene in autoimmune polyendocrine syndrome type 1.

Autoimmune polyglandular syndrome type 1 (APS-1, OMIM 240300) is a rare autosomal recessive disorder, characterized by the presence of at least two of three major diseases: hypoparathyroidism, Addison's disease, and chronic mucocutaneous candidiasis. We aim to identify the molecular defects and investigate the clinical and mutational characteristics in an index case and other members of a consanguineous family. We identified a novel homozygous mutation in the splice site acceptor (SSA) of intron 5 (c.653-1G>A) in two siblings with different clinical outcomes of APS-1. Coding DNA sequencing revealed that this AIRE mutation potentially compromised the recognition of the constitutive SSA of intron 5, splicing upstream onto a nearby cryptic SSA in intron 5. Surprisingly, the use of an alternative SSA entails the uncovering of a cryptic donor splice site in exon 5. This new transcript generates a truncated protein (p.A214fs67X) containing the first 213 amino acids and followed by 68 aberrant amino acids. The mutation affects the proper splicing, not only at the acceptor but also at the donor splice site, highlighting the complexity of recognizing suitable splicing sites and the importance of sequencing the intron-exon junctions for a more precise molecular diagnosis and correct genetic counseling. As both siblings were carrying the same mutation but exhibited a different APS-1 onset, and one of the brothers was not clinically diagnosed, our finding highlights the possibility to suspect mutations in the AIRE gene in cases of childhood chronic candidiasis and/or hypoparathyroidism otherwise unexplained, especially when the phenotype is associated with other autoimmune diseases.

Differential methylation of TCF7L2 promoter in peripheral blood DNA in newly diagnosed, drug-naïve patients with type 2 diabetes.

TCF7L2 is the susceptibility gene for Type 2 diabetes (T2D) with the largest effect on disease risk that has been discovered to date. However, the mechanisms by which TCF7L2 contributes to the disease remain largely elusive. In addition, epigenetic mechanisms, such as changes in DNA methylation patterns, might have a role in the pathophysiology of T2D. This study aimed to investigate the differences in terms of DNA methylation profile of TCF7L2 promoter gene between type 2 diabetic patients and age- and Body Mass Index (BMI)- matched controls. We included 93 type 2 diabetic patients that were recently diagnosed for T2D and exclusively on diet (without any pharmacological treatment). DNA was extracted from whole blood and DNA methylation was assessed using the Sequenom EpiTYPER system. Type 2 diabetic patients were more insulin resistant than their matched controls (mean HOMA IR 2.6 vs 1.8 in controls, P<0.001) and had a poorer beta-cell function (mean HOMA B 75.7 vs. 113.6 in controls, P<0.001). Results showed that 59% of the CpGs analyzed in TCF7L2 promoter had significant differences between type 2 diabetic patients and matched controls. In addition, fasting glucose, HOMA-B, HOMA-IR, total cholesterol and LDL-cholesterol correlated with methylation in specific CpG sites of TCF7L2 promoter. After adjustment by age, BMI, gender, physical inactivity, waist circumference, smoking status and diabetes status uniquely fasting glucose, total cholesterol and LDL-cholesterol remained significant. Taken together, newly diagnosed, drug-naïve type 2 diabetic patients display specific epigenetic changes at the TCF7L2 promoter as compared to age- and BMI-matched controls. Methylation in TCF7L2 promoter is further correlated with fasting glucose in peripheral blood DNA, which sheds new light on the role of epigenetic regulation of TCF7L2 in T2D.

Postnatal establishment of allelic Gαs silencing as a plausible explanation for delayed onset of parathyroid hormone resistance owing to heterozygous Gαs disruption.

Pseudohypoparathyroidism type-Ia (PHP-Ia), characterized by renal proximal tubular resistance to parathyroid hormone (PTH), results from maternal mutations of GNAS that lead to loss of α-subunit of the stimulatory G protein (Gαs) activity. Gαs expression is paternally silenced in the renal proximal tubule, and this genomic event is critical for the development of PTH resistance, as patients display impaired hormone action only if the mutation is inherited maternally. The primary clinical finding of PHP-Ia is hypocalcemia, which can lead to various neuromuscular defects including seizures. PHP-Ia patients frequently do not present with hypocalcemia until after infancy, but it has remained uncertain whether PTH resistance occurs in a delayed fashion. Analyzing reported cases of PHP-Ia with documented GNAS mutations and mice heterozygous for disruption of Gnas, we herein determined that the manifestation of PTH resistance caused by the maternal loss of Gαs, ie, hypocalcemia and elevated serum PTH, occurs after early postnatal life. To investigate whether this delay could reflect gradual development of paternal Gαs silencing, we then analyzed renal proximal tubules isolated by laser capture microdissection from mice with either maternal or paternal disruption of Gnas. Our results revealed that, whereas expression of Gαs mRNA in this tissue is predominantly from the maternal Gnas allele at weaning (3 weeks postnatal) and in adulthood, the contributions of the maternal and paternal Gnas alleles to Gαs mRNA expression are equal at postnatal day 3. In contrast, we found that paternal Gαs expression is already markedly repressed in brown adipose tissue at birth. Thus, the mechanisms silencing the paternal Gαs allele in renal proximal tubules are not operational during early postnatal development, and this finding correlates well with the latency of PTH resistance in patients with PHP-Ia.

Gastric inhibitory polypeptide receptor methylation in newly diagnosed, drug-naïve patients with type 2 diabetes: a case-control study.

GIP action in type 2 diabetic (T2D) patients is altered. We hypothesized that methylation changes could be present in GIP receptor of T2D patients. This study aimed to assess the differences in DNA methylation profile of GIPR promoter between T2D patients and age- and Body Mass Index (BMI)-matched controls. We included 93 T2D patients (cases) that were uniquely on diet (without any anti-diabetic pharmacological treatment). We matched one control (with oral glucose tolerance test negative, non diabetic), by age and BMI, for every case. Cytokines and hormones were determined by ELISA. DNA was extracted from whole blood and DNA methylation was assessed using the Sequenom EpiTYPER system. Our results showed that T2D patients were more insulin resistant and had a poorer ß cell function than their controls. Fasting adiponectin was lower in T2D patients as compared to controls (7.0±3.8 µgr/mL vs. 10.0±4.2 µgr/mL). Levels of IL 12 in serum were almost double in T2D patients (52.8±58.3 pg/mL vs. 29.7±37.4 pg/mL). We found that GIPR promoter was hypomethylated in T2D patients as compared to controls. In addition, HOMA-IR and fasting glucose correlated negatively with mean methylation of GIPR promoter, especially in T2D patients. This case-control study confirms that newly diagnosed, drug-naïve T2D patients are more insulin resistant and have worse ß cell function than age- and BMI-matched controls, which is partly related to changes in the insulin-sensitizing metabolites (adiponectin), in the proinflammatory profile (IL12) and we suggest in the methylation pattern of GIPR. Our study provides novel findings on GIPR promoter methylation profile which may improve our ability to understand type 2 diabetes pathogenesis.

Endocrine profile and phenotype-(epi)genotype correlation in Spanish patients with pseudohypoparathyroidism.

CONTEXT: Recent advances in genetics and epigenetics have revealed an overlap between molecular and clinical features of pseudohypoparathyroidism (PHP) subtypes, broadening the previous spectrum of PHP genotype-phenotype correlations and indicating limitations of the current classification of the disease. OBJECTIVES: The aim of the study was to screen patients with clinical diagnoses of PHP type I or pseudo-PHP for underlying molecular defects and explore possible correlations between molecular findings and clinical features. PATIENTS AND METHODS: We investigated the GNAS locus at the molecular level in 72 affected patients (46 women and 26 men) from 56 nonrelated families. Clinical data were obtained for 63 of these patients (38 women and 25 men). RESULTS: The molecular analysis showed that 35 patients carried structural mutations, 32 had loss of methylation, and 2 had a 2q37 deletion but did not reveal any (epi)mutation for 3 patients. Comparing these results and the clinical data, we observed that a younger age at diagnosis was associated with structural defects at the GNAS gene and epigenetic defects with a diagnosis later in life (9.19 ± 1.64 vs 24.57 ± 2.28 years, P < .0001). CONCLUSIONS: This first global review of PHP in Spain highlights the importance of a detailed clinical and genetic study of each patient and the integrated analysis of the findings from the two approaches. It may also help geneticists and clinicians to raise the suspicion of PHP earlier, reach more accurate diagnoses, and provide patients with PHP and their families with useful genetic information and counseling, thereby improving outcomes and quality of life.

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

Maternal deletion of the NESP55 differentially methylated region (DMR) (delNESP55/ASdel3-4(m), delNAS(m)) from the GNAS locus in humans causes autosomal dominant pseudohypoparathyroidism type Ib (AD-PHP-Ib(delNASm)), a disorder of proximal tubular parathyroid hormone (PTH) resistance associated with loss of maternal GNAS methylation imprints. Mice carrying a similar, maternally inherited deletion of the Nesp55 DMR (ΔNesp55(m)) replicate these Gnas epigenetic abnormalities and show evidence for PTH resistance, yet these mice demonstrate 100% mortality during the early postnatal period. We investigated whether the loss of extralarge αs (XLαs) imprinting and the resultant biallelic expression of XLαs are responsible for the early postnatal lethality in ΔNesp55(m) mice. First, we found that ΔNesp55(m) mice are hypoglycemic and have reduced stomach-to-body weight ratio. We then generated mice having the same epigenetic abnormalities as the ΔNesp55(m) mice but with normalized XLαs expression due to the paternal disruption of the exon giving rise to this Gnas product. These mice (ΔNesp55(m)/Gnasxl(m+/p-)) showed nearly 100% survival up to postnatal day 10, and a substantial number of them lived to adulthood. The hypoglycemia and reduced stomach-to-body weight ratio observed in 2-d-old ΔNesp55(m) mice were rescued in the ΔNesp55(m)/Gnasxl(m+/p-) mice. Surviving double-mutant animals had significantly reduced Gαs mRNA levels and showed hypocalcemia, hyperphosphatemia, and elevated PTH levels, thus providing a viable model of human AD-PHP-Ib. Our findings show that the hypoglycemia and early postnatal lethality caused by the maternal deletion of the Nesp55 DMR result from biallelic XLαs expression. The double-mutant mice will help elucidate the pathophysiological mechanisms underlying AD-PHP-Ib.