Intrafamilial phenotypic heterogeneity in siblings with pseudohypoparathyroidism 1B due to maternal STX16 deletion.

OBJECTIVES: Pseudohypoparathyroidism (PHP1B) is most commonly caused by epigenetic defects resulting in loss of methylation at the GNAS locus, although deletions of STX16 leading to GNAS methylation abnormalities have been previously reported. The phenotype of this disorder is variable and can include hormonal resistances and severe infantile obesity with hyperphagia. A possible time relationship between the onset of obesity and endocrinopathies has been previously reported but remains unclear. Understanding of the condition's natural history is limited, partly due to a scarcity of literature, especially in children. CASE PRESENTATION: We report three siblings with autosomal dominant PHP1B caused by a deletion in STX16 who presented with early childhood onset PTH-resistance with normocalcemia with a progressive nature, accompanied by TSH-resistance and severe infantile obesity with hyperphagia in some, not all of the affected individuals. CONCLUSIONS: PHP1B from a STX16 deletion displays intrafamilial phenotypic variation. It is a novel cause of severe infantile obesity, which is not typically included in commercially available gene panels but must be considered in the genetic work-up. Finally, it does not seem to have a clear time relationship between the onset of obesity and hormonal resistance.

Phosphorylation of the N-terminus of Syntaxin-16 controls interaction with mVps45 and GLUT4 trafficking in adipocytes.

The ability of insulin to stimulate glucose transport in muscle and fat cells is mediated by the regulated delivery of intracellular vesicles containing glucose transporter-4 (GLUT4) to the plasma membrane, a process known to be defective in disease such as Type 2 diabetes. In the absence of insulin, GLUT4 is sequestered in tubules and vesicles within the cytosol, collectively known as the GLUT4 storage compartment. A subset of these vesicles, known as the 'insulin responsive vesicles' are selectively delivered to the cell surface in response to insulin. We have previously identified Syntaxin16 (Sx16) and its cognate Sec1/Munc18 protein family member mVps45 as key regulatory proteins involved in the delivery of GLUT4 into insulin responsive vesicles. Here we show that mutation of a key residue within the Sx16 N-terminus involved in mVps45 binding, and the mutation of the Sx16 binding site in mVps45 both perturb GLUT4 sorting, consistent with an important role of the interaction of these two proteins in GLUT4 trafficking. We identify Threonine-7 (T7) as a site of phosphorylation of Sx16 in vitro. Mutation of T7 to D impairs Sx16 binding to mVps45 in vitro and overexpression of T7D significantly impaired insulin-stimulated glucose transport in adipocytes. We show that both AMP-activated protein kinase (AMPK) and its relative SIK2 phosphorylate this site. Our data suggest that Sx16 T7 is a potentially important regulatory site for GLUT4 trafficking in adipocytes.

Progression of PTH Resistance in Autosomal Dominant Pseudohypoparathyroidism Type Ib Due to Maternal STX16 Deletions.

CONTEXT: Maternally inherited STX16 deletions that cause loss of methylation at GNAS exon A/B and thereby reduce Gsα expression are the most frequent cause of autosomal dominant pseudohypoparathyroidism type Ib (AD-PHP1B). Early identification of these disease-causing variants in the children of affected and unaffected female carriers would prompt treatment with calcium and calcitriol once parathyroid hormone (PTH) levels increase, thereby preventing hypocalcemia and associated complications. OBJECTIVE: This study aimed to determine when PTH and calcium abnormalities develop after birth if a STX16 deletion is inherited maternally. METHODS: Forty-four children of affected (n = 7) or unaffected (n = 7) females with a STX16 deletion were investigated for the presence of these variants. If a deletion was identified, measurement of PTH, calcium, phosphate, and thyrotropin (TSH) was advised. RESULTS: The STX16 deletion that causes AD-PHP1B was identified in 25 children. Pretreatment laboratory results were available for 19 of those cases. Elevated PTH levels were detected by 2 years of age, and these were progressively higher if laboratory testing was first performed after establishing the genetic defect later in life. Total serum calcium levels remained within normal limits until about 5 years of age. TSH levels showed no consistent rise over time. CONCLUSION: Establishing whether a STX16 deletion is inherited from a female carrier of a disease-causing variant rapidly establishes the diagnosis of AD-PHP1B. Several years before overt hypocalcemia developed, PTH levels increased, thereby establishing the onset of PTH resistance. Our findings provide diagnostic guidance and when treatment with calcium and calcitriol should be considered in order to prevent hypocalcemia and associated sequelae.

Retrograde and Anterograde Transport of Lat-Vesicles during the Immunological Synapse Formation: Defining the Finely-Tuned Mechanism.

LAT is an important player of the signaling cascade induced by TCR activation. This adapter molecule is present at the plasma membrane of T lymphocytes and more abundantly in intracellular compartments. Upon T cell activation the intracellular pool of LAT is recruited to the immune synapse (IS). We previously described two pathways controlling LAT trafficking: retrograde transport from endosomes to the TGN, and anterograde traffic from the Golgi to the IS. We address the specific role of four proteins, the GTPase Rab6, the t-SNARE syntaxin-16, the v-SNARE VAMP7 and the golgin GMAP210, in each pathway. Using different methods (endocytosis and Golgi trap assays, confocal and TIRF microscopy, TCR-signalosome pull down) we show that syntaxin-16 is regulating the retrograde transport of LAT whereas VAMP7 is regulating the anterograde transport. Moreover, GMAP210 and Rab6, known to contribute to both pathways, are in our cellular context, specifically and respectively, involved in anterograde and retrograde transport of LAT. Altogether, our data describe how retrograde and anterograde pathways coordinate LAT enrichment at the IS and point to the Golgi as a central hub for the polarized recruitment of LAT to the IS. The role that this finely-tuned transport of signaling molecules plays in T-cell activation is discussed.

Genetic and Epigenetic Characteristics of Autosomal Dominant Pseudohypoparathyroidism Type 1B: Case Reports and Literature Review.

Autosomal dominant pseudohypoparathyroidism 1B (AD-PHP1B) is a rare endocrine and imprinted disorder. The objective of this study is to clarify the imprinted regulation of the guanine nucleotide binding-protein α-stimulating activity polypeptide 1 (GNAS) cluster in the occurrence and development of AD-PHP1B based on animal and clinical patient studies. The methylation-specific multiples ligation-dependent probe amplification (MS-MLPA) was conducted to detect the copy number variation in syntaxin-16 (STX16) gene and methylation status of the GNAS differentially methylated regions (DMRs). Long-range PCR was used to confirm deletion at STX16 gene. In the first family, DNA analysis of the proband and proband's mother revealed an isolated loss of methylation (LOM) at exon A/B and a 3.0 kb STX16 deletion. The patient's healthy grandmother had the 3.0 kb STX16 deletion but no epigenetic abnormality. The patient's healthy maternal aunt showed no genetic or epigenetic abnormality. In the second family, the analysis of long-range PCR revealed the 3.0 kb STX16 deletion for the proband but not her children. In this study, 3.0 kb STX16 deletion causes isolated LOM at exon A/B in two families, which is the most common genetic mutation of AD-PHP1B. The deletion involving NESP55 or AS or genomic rearrangements of GNAS can also result in AD-PHP1B, but it's rare. LOM at exon A/B DMR is prerequisite methylation defect of AD-PHP1B. STX16 and NESP55 directly control the imprinting at exon A/B, while AS controls the imprinting at exon A/B by regulating the transcriptional level of NESP55.

Preferential Maternal Transmission of STX16-GNAS Mutations Responsible for Autosomal Dominant Pseudohypoparathyroidism Type Ib (PHP1B): Another Example of Transmission Ratio Distortion.

Preferential transmission of a genetic mutation to the next generation, referred to as transmission ratio distortion (TRD), is well established for several dominant disorders, but underlying mechanisms remain undefined. Recently, TRD was reported for patients affected by pseudohypoparathyroidism type Ia or pseudopseudohypoparathyroidism. To determine whether TRD is observed also for autosomal dominant pseudohypoparathyroidism type Ib (AD-PHP1B), we analyzed kindreds with the frequent 3-kb STX16 deletion or other STX16/GNAS mutations. If inherited from a female, these genetic defects lead to loss-of-methylation at exon A/B alone or at all three differentially methylated regions (DMR), resulting in parathyroid hormone (PTH)-resistant hypocalcemia and hyperphosphatemia and possibly resistance to other hormones. In total, we investigated 212 children born to 80 females who are unaffected carriers of a STX16/GNAS mutation (n = 47) or affected by PHP1B (n = 33). Of these offspring, 134 (63.2%) had inherited the genetic defect (p = .00012). TRD was indistinguishable for mothers with a STX16/GNAS mutation on their paternal (unaffected carriers) or maternal allele (affected). The mechanisms favoring transmission of the mutant allele remain undefined but are likely to include abnormalities in oocyte maturation. Search for mutations in available descendants of males revealed marginally significant evidence for TRD (p = .038), but these analyses are less reliable because many more offspring of males than females with a STX16/GNAS mutation were lost to follow-up (31 of 98 versus 6 of 218). This difference in follow-up is probably related to the fact that inheritance of a mutation from a male does not have clinical implications, whereas inheritance from an affected or unaffected female results in PHP1B. Lastly, affected PHP1B females had fewer descendants than unaffected carriers, but it remains unclear whether abnormal oocyte development or impaired actions of reproductive hormones are responsible. Our findings highlight previously not recognized aspects of AD-PHP1B that are likely to have implications for genetic testing and counseling. © 2020 American Society for Bone and Mineral Research (ASBMR).

Vps54 regulates Drosophila neuromuscular junction development and interacts genetically with Rab7 to control composition of the postsynaptic density.

Vps54 is a subunit of the Golgi-associated retrograde protein (GARP) complex, which is involved in tethering endosome-derived vesicles to the trans-Golgi network (TGN). In the wobbler mouse, a model for human motor neuron (MN) disease, reduction in the levels of Vps54 causes neurodegeneration. However, it is unclear how disruption of the GARP complex leads to MN dysfunction. To better understand the role of Vps54 in MNs, we have disrupted expression of the Vps54 ortholog in Drosophila and examined the impact on the larval neuromuscular junction (NMJ). Surprisingly, we show that both null mutants and MN-specific knockdown of Vps54 leads to NMJ overgrowth. Reduction of Vps54 partially disrupts localization of the t-SNARE, Syntaxin-16, to the TGN but has no visible impact on endosomal pools. MN-specific knockdown of Vps54 in MNs combined with overexpression of the small GTPases Rab5, Rab7, or Rab11 suppresses the Vps54 NMJ phenotype. Conversely, knockdown of Vps54 combined with overexpression of dominant negative Rab7 causes NMJ and behavioral abnormalities including a decrease in postsynaptic Dlg and GluRIIB levels without any effect on GluRIIA. Taken together, these data suggest that Vps54 controls larval MN axon development and postsynaptic density composition through a mechanism that requires Rab7.

A novel long-range deletion spanning STX16 and NPEPL1 causing imprinting defects of the GNAS locus discovered in a patient with autosomal-dominant pseudohypoparathyroidism type 1B.

BACKGROUND: Pseudohypoparathyroidism (PHP) is a rare disorder characterized by hypocalcemia, hyperphosphatemia, and resistance to parathyroid hormone (PTH). According to different GNAS mutations, PHP is divided into several subtypes, among which autosomal-dominant PHP1B (AD-PHP1B) is caused by STX16 deletion and epigenetic alteration of GNAS. Although the deletion of STX16 exons 2-6 is commonly observed, other mutations involving STX16 can also result in AD-PHP1B. MATERIALS AND METHODS: The clinical information of a 38-year-old male PHP patient was collected. The genomic DNA from peripheral blood cells was extracted for genetic analysis of GNAS and upstream STX16 by methylation-specific multiplex ligation-dependent probe amplification (MS-MLPA) and whole-exome sequencing (WES). Sanger sequencing was performed to verify the break point of the novel long-range deletion. RESULTS: The patient's medical history of tetany and seizure as well as laboratory examination showing hypocalcemia and elevated PTH levels indicated the diagnosis of PHP. The results of MS-MLPA showed loss of methylation of GNAS A/B:TSS-DMR and half-reduced copy number of STX16 exon 1-9, which revealed the subtype of AD-PHP1B. Furthermore, the WES study displayed a 87.5 kb missing upstream of GNAS. A 87.5 kb deletion spanning STX16 and NPEPL1 together with an insertion of 28 bp of unknown origin was verified by PCR along with Sanger sequencing. CONCLUSIONS: A novel deletion of 87.5 kb spanning STX16 and NPEPL1 was discovered in an AD-PHP1B patient, which provides new information on molecular defects leading to AD-PHP1B.

Syntaxin 16's Newly Deciphered Roles in Autophagy.

Syntaxin 16, a Qa-SNARE (soluble N-ethylmaleimide-sensitive factor activating protein receptor), is involved in a number of membrane-trafficking activities, particularly transport processes at the trans-Golgi network (TGN). Recent works have now implicated syntaxin 16 in the autophagy process. In fact, syntaxin 16 appears to have dual roles, firstly in facilitating the transport of ATG9a-containing vesicles to growing autophagosomes, and secondly in autolysosome formation. The former involves a putative SNARE complex between syntaxin 16, VAMP7 and SNAP-47. The latter occurs via syntaxin 16's recruitment by Atg8/LC3/GABARAP family proteins to autophagosomes and endo-lysosomes, where syntaxin 16 may act in a manner that bears functional redundancy with the canonical autophagosome Qa-SNARE syntaxin 17. Here, I discuss these recent findings and speculate on the mechanistic aspects of syntaxin 16's newly found role in autophagy.

Mammalian Atg8 proteins regulate lysosome and autolysosome biogenesis through SNAREs.

Mammalian homologs of yeast Atg8 protein (mAtg8s) are important in autophagy, but their exact mode of action remains ill-defined. Syntaxin 17 (Stx17), a SNARE with major roles in autophagy, was recently shown to bind mAtg8s. Here, we identified LC3-interacting regions (LIRs) in several SNAREs that broaden the landscape of the mAtg8-SNARE interactions. We found that Syntaxin 16 (Stx16) and its cognate SNARE partners all have LIR motifs and bind mAtg8s. Knockout of Stx16 caused defects in lysosome biogenesis, whereas a Stx16 and Stx17 double knockout completely blocked autophagic flux and decreased mitophagy, pexophagy, xenophagy, and ribophagy. Mechanistic analyses revealed that mAtg8s and Stx16 control several properties of lysosomal compartments including their function as platforms for active mTOR. These findings reveal a broad direct interaction of mAtg8s with SNAREs with impact on membrane remodeling in eukaryotic cells and expand the roles of mAtg8s to lysosome biogenesis.

VAMP7 Regulates Autophagosome Formation by Supporting Atg9a Functions in Pancreatic ß-Cells From Male Mice.

Dysfunctional mitochondria are observed in ß-cells of diabetic patients, which are eventually removed by autophagy. Vesicle-associated membrane protein (VAMP)7, a vesicular SNARE protein, regulates autophagosome formation to maintain mitochondrial homeostasis and control insulin secretion in pancreatic ß-cells. However, its molecular mechanism is largely unknown. In this study, we investigated the molecular mechanism of VAMP7-dependent autophagosome formation using VAMP7-deficient ß-cells and ß-cell-derived Min6 cells. VAMP7 localized in autophagy-related (Atg)9a-resident vesicles of recycling endosomes (REs), which contributed to autophagosome formation, and it interacted with Hrb, Syntaxin16, and SNAP-47. Hrb recruited VAMP7 and Atg9a from the plasma membrane to REs. Syntaxin16 and SNAP-47 mediated autophagosome formation at a step later than the proper localization of VAMP7 to Atg9a-resident vesicles. Knockdown of Hrb, Syntaxin16, and SNAP-47 resulted in defective autophagosome formation, accumulation of dysfunctional mitochondria, and impairment of glucose-stimulated insulin secretion. Our data indicate that VAMP7 and Atg9a are initially recruited to REs to organize VAMP7 and Atg9a-resident vesicles in an Hrb-dependent manner. Additionally, VAMP7 forms a SNARE complex with Syntaxin16 and SNAP-47, which may cause fusions of Atg9a-resident vesicles during autophagosome formation. Thus, VAMP7 participates in autophagosome formation by supporting Atg9a functions that contribute to maintenance of mitochondrial quality.

Rab6-dependent retrograde traffic of LAT controls immune synapse formation and T cell activation.

The adapter molecule linker for activation of T cells (LAT) orchestrates the formation of signalosomes upon T cell receptor (TCR) stimulation. LAT is present in different intracellular pools and is dynamically recruited to the immune synapse upon stimulation. However, the intracellular traffic of LAT and its function in T lymphocyte activation are ill defined. We show herein that LAT, once internalized, transits through the Golgi-trans-Golgi network (TGN), where it is repolarized to the immune synapse. This retrograde transport of LAT depends on the small GTPase Rab6 and the target soluble N-ethylmaleimide-sensitive factor attachment protein receptor (t-SNARE) Syntaxin-16, two regulators of the endosome-to-Golgi/TGN retrograde transport. We also show in vitro in Syntaxin-16- or Rab6-silenced human cells and in vivo in CD4+ T lymphocytes of the Rab6 knockout mouse that this retrograde traffic controls TCR stimulation. These results establish that the retrograde traffic of LAT from the plasma membrane to the Golgi-TGN controls the polarized delivery of LAT at the immune synapse and T lymphocyte activation.

Early-Onset Obesity: Unrecognized First Evidence for GNAS Mutations and Methylation Changes.

Context: Early-onset obesity, characteristic for disorders affecting the leptin-melanocortin pathway, is also observed in pseudohypoparathyroidism type 1A (PHP1A), a disorder caused by maternal GNAS mutations that disrupt expression or function of the stimulatory G protein α-subunit (Gsα). Mutations and/or epigenetic abnormalities at the same genetic locus are also the cause of pseudohypoparathyroidism type 1B (PHP1B). However, although equivalent biochemical and radiographic findings can be encountered in these related disorders caused by GNAS abnormalities, they are considered distinct clinical entities. Objectives: To further emphasize the overlapping features between both disorders, we report the cases of several children, initially brought to medical attention because of unexplained early-onset obesity, in whom PHP1B or PHP1A was eventually diagnosed. Patients and Methods: Search for GNAS methylation changes or mutations in cohorts of patients with early-onset obesity. Results: Severe obesity had been noted in five infants, with a later diagnosis of PHP1B due to STX16 deletions and/or abnormal GNAS methylation. These findings prompted analysis of 24 unselected obese patients, leading to the discovery of inherited STX16 deletions in 2 individuals. Similarly, impressive early weight gains were noted in five patients, who initially lacked additional Albright hereditary osteodystrophy features but in whom PHP1A due to GNAS mutations involving exons encoding Gsα was diagnosed. Conclusions: Obesity during the first year of life can be the first clinical evidence for PHP1B, expanding the spectrum of phenotypic overlap between PHP1A and PHP1B. Importantly, GNAS methylation abnormalities escape detection by targeted or genome-wide sequencing strategies, raising the question of whether epigenetic GNAS analyses should be considered for unexplained obesity.

Excess of a Rassf1-targeting microRNA, miR-193a-3p, perturbs cell division fidelity.

BACKGROUND: Several microRNA (miRNA) molecules have emerged as important post-transcriptional regulators of tumour suppressor and oncogene expression. Ras association domain family member 1 (RASSF1) is a critical tumour suppressor that controls multiple aspects of cell proliferation such as cell cycle, cell division and apoptosis. The expression of RASSF1 is lost in a variety of cancers due to the promoter hypermethylation. METHODS: miR-193a-3p was identified as a RASSF1-targeting miRNA by a dual screening approach. In cultured human cancer cells, immunoblotting, qRT-PCR, luciferase reporter assays, time-lapse microscopy and immunofluorescence methods were used to study the effects of excess miR-193a-3p on RASSF1 expression and cell division. RESULTS: Here, we report a new miRNA-mediated mechanism that regulates RASSF1 expression: miR-193a-3p binds directly to RASSF1-3'UTR and represses the mRNA and protein expression. In human cancer cells, excess of miR-193a-3p causes polyploidy through impairment of the Rassf1-Syntaxin 16 signalling pathway that is needed for completion of cytokinesis. In the next cell cycle the miR-193a-3p-overexpressing cells exhibit multipolar mitotic spindles, mitotic delay and elevated frequency of cell death. CONCLUSIONS: Our results suggest that besides epigenetic regulation, altered expression of specific miRNAs may contribute to the loss of Rassf1 in cancer cells and cause cell division errors.

A Large Inversion Involving GNAS Exon A/B and All Exons Encoding Gsα Is Associated With Autosomal Dominant Pseudohypoparathyroidism Type Ib (PHP1B).

Pseudohypoparathyroidism type Ib (PHP1B) is characterized primarily by resistance to parathyroid hormone (PTH) and thus hypocalcemia and hyperphosphatemia, in most cases without evidence for Albright hereditary osteodystrophy (AHO). PHP1B is associated with epigenetic changes at one or several differentially-methylated regions (DMRs) within GNAS, which encodes the α-subunit of the stimulatory G protein (Gsα) and splice variants thereof. Heterozygous, maternally inherited STX16 or GNAS deletions leading to isolated loss-of-methylation (LOM) at exon A/B alone or at all maternal DMRs are the cause of autosomal dominant PHP1B (AD-PHP1B). In this study, we analyzed three affected individuals, the female proband and her two sons. All three revealed isolated LOM at GNAS exon A/B, whereas the proband's healthy maternal grandmother and uncle showed normal methylation at this locus. Haplotype analysis was consistent with linkage to the STX16/GNAS region, yet no deletion could be identified. Whole-genome sequencing of one of the patients revealed a large heterozygous inversion (1,882,433 bp). The centromeric breakpoint of the inversion is located 7,225 bp downstream of GNAS exon XL, but its DMR showed no methylation abnormality, raising the possibility that the inversion disrupts a regulatory element required only for establishing or maintaining exon A/B methylation. Because our three patients presented phenotypes consistent with PHP1B, and not with PHP1A, the Gsα promoter is probably unaffected by the inversion. Our findings expand the spectrum of genetic mutations that lead to LOM at exon A/B alone and thus biallelic expression of the transcript derived from this alternative first GNAS exon. © 2017 American Society for Bone and Mineral Research.

Genetic and epigenetic methylation defects and implication of the ERMN gene in autism spectrum disorders.

Autism spectrum disorders (ASD) are highly heritable and genetically complex conditions. Although highly penetrant mutations in multiple genes have been identified, they account for the etiology of <1/3 of cases. There is also strong evidence for environmental contribution to ASD, which can be mediated by still poorly explored epigenetic modifications. We searched for methylation changes on blood DNA of 53 male ASD patients and 757 healthy controls using a methylomic array (450K Illumina), correlated the variants with transcriptional alterations in blood RNAseq data, and performed a case-control association study of the relevant findings in a larger cohort (394 cases and 500 controls). We found 700 differentially methylated CpGs, most of them hypomethylated in the ASD group (83.9%), with cis-acting expression changes at 7.6% of locations. Relevant findings included: (1) hypomethylation caused by rare genetic variants (meSNVs) at six loci (ERMN, USP24, METTL21C, PDE10A, STX16 and DBT) significantly associated with ASD (q-value <0.05); and (2) clustered epimutations associated to transcriptional changes in single-ASD patients (n=4). All meSNVs and clustered epimutations were inherited from unaffected parents. Resequencing of the top candidate genes also revealed a significant load of deleterious mutations affecting ERMN in ASD compared with controls. Our data indicate that inherited methylation alterations detectable in blood DNA, due to either genetic or epigenetic defects, can affect gene expression and contribute to ASD susceptibility most likely in an additive manner, and implicate ERMN as a novel ASD gene.

Macrosomia, obesity, and macrocephaly as first clinical presentation of PHP1b caused by STX16 deletion.

Pseudohypoparathyroidism (PHP) is a genetic disorder with resistance to parathyroid hormone (PTH) as most important feature. Main subtypes of the disease are pseudohypoparathyroidism 1b (PHP1b) and pseudohypoparathyroidism 1a (PHP1a). PHP1b is characterized by PTH resistance of the renal cortex due to reduced activity of the stimulatory G protein α subunit (Gsα) of the PTH receptor. In addition to resistance to PTH, PHP1a patients also lack sensitivity for other hormones that signal their actions through G protein-coupled receptors and display physical features of Albright hereditary osteodystrophy (AHO), which is not classically seen in PHP1b patients. PHP1a is caused by heterozygous loss-of-function mutations in maternally inherited GNAS exons 1-13, which encode Gsα. PHP1b is often caused by deletion of the STX16 gene, which is thought to have an important role in controlling the methylation and thus imprinting at part of the GNAS locus. Here we present a patient with PHP1b caused by the previously described recurrent 3-kb STX16 deletion. The patient's first symptoms were macrosomia, early onset obesity, and macrocephaly. Since this is an atypical but previously described rare presentation of PHP1b, we reemphasize STX16 deletions and PHP1b as a rare cause for early onset obesity and macrosomia. © 2016 Wiley Periodicals, Inc.

Analysis of Multiple Families With Single Individuals Affected by Pseudohypoparathyroidism Type Ib (PHP1B) Reveals Only One Novel Maternally Inherited GNAS Deletion.

Proximal tubular resistance to parathyroid hormone (PTH) resulting in hypocalcemia and hyperphosphatemia are preeminent abnormalities in pseudohypoparathyroidism type Ib (PHP1B), but resistance toward other hormones as well as variable features of Albright's Hereditary Osteodystrophy (AHO) can occur also. Genomic DNA from PHP1B patients shows epigenetic changes at one or multiple differentially methylated regions (DMRs) within GNAS, the gene encoding Gαs and splice variants thereof. In the autosomal dominant disease variant, these methylation abnormalities are caused by deletions in STX16 or GNAS on the maternal allele. The molecular defect(s) leading to sporadic PHP1B (sporPHP1B) remains in most cases unknown and we therefore analyzed 60 sporPHP1B patients and available family members by microsatellite markers, single nucleotide polymorphisms (SNPs), multiplex ligation-dependent probe amplification (MLPA), and methylation-specific MLPA (MS-MLPA). All investigated cases revealed broad GNAS methylation changes, but no evidence for inheritance of two paternal chromosome 20q alleles. Some patients with partial epigenetic modifications in DNA from peripheral blood cells showed more complete GNAS methylation changes when testing their immortalized lymphoblastoid cells. Analysis of siblings and children of sporPHP1B patients provided no evidence for an abnormal mineral ion regulation and no changes in GNAS methylation. Only one patient revealed, based on MLPA and microsatellite analyses, evidence for an allelic loss, which resulted in the discovery of two adjacent, maternally inherited deletions (37,597 and 1427 bp, respectively) that remove the area between GNAS antisense exons 3 and 5, including exon NESP. Our findings thus emphasize that the region comprising antisense exons 3 and 4 is required for establishing all maternal GNAS methylation imprints. The genetic defect(s) leading in sporPHP1B to epigenetic GNAS changes and thus PTH-resistance remains unknown, but it seems unlikely that this disease variant is caused by heterozygous inherited or de novo mutations involving GNAS.