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.

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.

Autosomal dominant pseudohypoparathyroidism type Ib: a novel inherited deletion ablating STX16 causes loss of imprinting at the A/B DMR.

CONTEXT: Pseudohypoparathyroidism type Ib (PHP-Ib) is a rare imprinting disorder characterized by end-organ resistance to PTH and, frequently, to thyroid-stimulating hormone. PHP-Ib familial form, with an autosomal dominant pattern of transmission (autosomal dominant pseudohypoparathyroidism type Ib [AD-PHP-Ib]), is typically characterized by an isolated loss of methylation at the guanine nucleotide-binding protein α-stimulating activity polypeptide 1 A/B differentially methylated region (DMR), secondary to genetic deletions disrupting the upstream imprinting control region in the syntaxin-16 (STX16) locus. However, deletions described up to now failed to account some cases of patients with a methylation defect limited to the A/B DMR; thus, it is expected the existence of other still unknown rearrangements, undetectable with conventional molecular diagnostic methods. OBJECTIVE: We investigated a PHP-Ib patient with a methylation defect limited to the A/B DMR and no known STX16 deletions to find the underlying primary genetic defect. PATIENT AND METHODS: A PHP-Ib patient (hypocalcaemia, hyperphosphataemia, raised serum PTH levels, no vitamin D deficiency) and his unaffected relatives were investigated by methylation specific-multiplex ligand-dependent probe amplification to search for novel pathogenetic defects affecting the guanine nucleotide-binding protein α-stimulating activity polypeptide 1 and STX16 loci. RESULTS: We report the clinical, biochemical, and molecular analysis of an AD-PHP-Ib patient with a novel STX16 deletion overlapping with previously identified STX16 deletions but that, unlike these genetic defects associated with AD-PHP-Ib, goes unnoticed with commonly used first-level diagnostic techniques. CONCLUSIONS: Our work highlights the importance of performing accurate investigations in PHP-Ib patients with methylation defects to allow precise genetic counseling because, in case of deletions, the segregation ratio is about 50% and the disease phenotype is transmitted in an autosomal dominant fashion via the mother.

VAMP4 is required to maintain the ribbon structure of the Golgi apparatus.

The Golgi apparatus forms a twisted ribbon-like network in the juxtanuclear region of vertebrate cells. Vesicle-associated membrane protein 4 (VAMP4), a v-SNARE protein expressed exclusively in the vertebrate trans-Golgi network (TGN), plays a role in retrograde trafficking from the early endosome to the TGN, although its precise function within the Golgi apparatus remains unclear. To determine whether VAMP4 plays a functional role in maintaining the structure of the Golgi apparatus, we depleted VAMP4 gene expression using RNA interference technology. Depletion of VAMP4 from HeLa cells led to fragmentation of the Golgi ribbon. These fragments were not uniformly distributed throughout the cytoplasm, but remained in the juxtanuclear area. Electron microscopy and immunohistochemistry showed that in the absence of VAMP4, the length of the Golgi stack was shortened, but Golgi stacking was normal. Anterograde trafficking was not impaired in VAMP4-depleted cells, which contained intact microtubule arrays. Depletion of the cognate SNARE partners of VAMP4, syntaxin 6, syntaxin 16, and Vti1a also disrupted the Golgi ribbon structure. Our findings suggested that the maintenance of Golgi ribbon structure requires normal retrograde trafficking from the early endosome to the TGN, which is likely to be mediated by the formation of VAMP4-containing SNARE complexes.

Syntaxin 16 regulates lumen formation during epithelial morphogenesis.

The formation and maintenance of cell-cell junctions, both under physiological and pathological conditions, requires the targeting and trafficking of junctional proteins. Proteins of the syntaxin (Stx)-family localize to a variety of subcellular membranes and contribute to intracellular transport of cargo by regulating vesicle fusion events at these sites. Unlike plasma membrane localized Stxs, the roles of endosome- and Golgi-localized stx proteins in epithelial morphogenesis are less understood. Here we show that Stx16- an endosome- and Golgi-localized target-membrane soluble N-ethylmaleimide attachment protein receptor (t-SNARE) that plays a role in membrane trafficking between these compartments - is essential for lumen development. In cultured Madin Darby Canine Kidney (MDCK) cells, Stx16 was selectively upregulated as sparsely plated cells attained confluency. Stx16-depleted confluent monolayers consistently showed lower transepithelial resistance than control monolayers, and failed to maintain endogenous and ectopically expressed E-cadherin at the adherens junctions due to decreased recycling. We further found that whereas cysts formed by MDCK cells cultured in Matrigel have a single hollow lumen, those formed by stx16-depleted counterparts had multiple lumens, due to abnormal orientiation of the mitotic spindle. Finally, a similar role for stx16 function in vivo is indicated by our analysis of pronephric-duct development in zebrafish expressing the claudinB:lynGFP transgene; lack of stx16 function in this structure (in stx16-morphant embryos) led to the development of enlarged, torturous pronephric ducts with more than one lumen. Taken together, our in vitro and in vivo studies establish a role for Stx16 in maintaining the integrity of cell-cell junctions, and thereby in morphogenesis of the kidney epithelial lumen.

Development and treatment of tertiary hyperparathyroidism in patients with pseudohypoparathyroidism type 1B.

CONTEXT: Pseudohypoparathyroidism type 1B (PHP1B) patients have PTH resistance at the renal proximal tubule and develop hypocalcemia and secondary hyperparathyroidism. Hyperparathyroid bone disease also develops in some patients. PHP1B patients are at theoretical risk of developing tertiary hyperparathyroidism. SETTING: Patients were studied in a clinical research center. PATIENTS: Five female PHP1B patients presented with hypercalcemia and elevated PTH. INTERVENTION: Patients either underwent parathyroidectomy (n = 4) or received cinacalcet (n = 1). MAIN OUTCOME MEASURES: Serum calcium and PTH were serially measured before and after intervention. RESULTS: Five PHP1B patients developed concomitantly elevated serum calcium and PTH levels (range, 235-864 ng/liter) requiring termination of calcium and vitamin D therapy (time after diagnosis, 21-42 yr; median, 34 yr), consistent with tertiary hyperparathyroidism. Four patients underwent parathyroidectomy with removal of one (n = 2) or two (n = 2) enlarged parathyroid glands. Calcium and vitamin D therapy was reinstituted postoperatively, and at 93-month median follow-up, PTH levels ranged between 56 and 182 (normal, <87) ng/liter. One patient was treated with cinacalcet, resulting in resolution of hypercalcemia. CONCLUSIONS: PHP1B patients are at risk of developing tertiary hyperparathyroidism and/or hyperparathyroid bone disease and should therefore be treated with sufficient doses of calcium and vitamin D to achieve serum calcium and PTH levels within or as close to the normal range as possible. Surgery is the treatment of choice in this setting. Cinacalcet may be a useful alternative in those who do not undergo surgery.

No evidence for GNAS copy number variants in patients with features of Albright's hereditary osteodystrophy and abnormal platelet Gs activity.

Albright's hereditary osteodystrophy (AHO) is characterized by short stature, round face, calcifications, obesity, brachydactyly and intellectual disability. AHO without hormone resistance is called pseudopseudohypoparathyroidism (PPHP), a rare clinical condition difficult to diagnose with highly variable features. PPHP is caused by paternally inherited loss-of-function mutations in the GNAS. Patients with 2q37 microdeletions or HDAC4 mutations are also defined as having an AHO-like phenotype with normal stimulatory G (Gs) function. We have studied 256 patients with AHO features but no other diagnosis. Their platelet Gs activity was determined via the aggregation-inhibition test showing Gs hypo- or hyperfuncton in 24% and 15% of the patients, respectively. Before initiating with detailed (epi)genetic GNAS studies, we here wanted to excluded copy number variants (CNVs) in GNAS as cause of AHO with a novel large-scale screening technique. Multiplex amplicon quantification (MAQ) for CNVs screening was developed for the 20q13.3 region including GNAS and potential long-range imprinting control elements such as STX16. This is the first large-scale GNAS CNV study in patients with common AHO features but no CNVs were detected. In conclusion, CNVs in the GNAS region are not likely to cause an AHO-like phenotype with or without abnormal platelet Gs activity. Future studies will be undertaken to find out whether these AHO patients with abnormal Gs function are characterized by GNAS coding or methylation defects.

Genome-wide RNAi screens identify genes required for Ricin and PE intoxications.

Protein toxins such as Ricin and Pseudomonas exotoxin (PE) pose major public health challenges. Both toxins depend on host cell machinery for internalization, retrograde trafficking from endosomes to the ER, and translocation to cytosol. Although both toxins follow a similar intracellular route, it is unknown how much they rely on the same genes. Here we conducted two genome-wide RNAi screens identifying genes required for intoxication and demonstrating that requirements are strikingly different between PE and Ricin, with only 13% overlap. Yet factors required by both toxins are present from the endosomes to the ER, and, at the morphological level, the toxins colocalize in multiple structures. Interestingly, Ricin, but not PE, depends on Golgi complex integrity and colocalizes significantly with a medial Golgi marker. Our data are consistent with two intertwined pathways converging and diverging at multiple points and reveal the complexity of retrograde membrane trafficking in mammalian cells.

Syntaxin 16 binds to cystic fibrosis transmembrane conductance regulator and regulates its membrane trafficking in epithelial cells.

The cystic fibrosis transmembrane conductance regulator (CFTR) is a key membrane protein in the complex network of epithelial ion transporters regulating epithelial permeability. Syntaxins are one of the major determinants in the intracellular trafficking and membrane targeting of secretory proteins. In the present study we demonstrate the biochemical and functional association between CFTR and syntaxin 16 (STX16) that mediates vesicle transport within the early/late endosomes and trans-Golgi network. Immunoprecipitation experiments in rat colon and T84 human colonic epithelial cells indicate that STX16 associates with CFTR. Further analyses using the domain-specific pulldown assay reveal that the helix domain of STX16 directly interacts with the N-terminal region of CFTR. Immunostainings in rat colon and T84 cells show that CFTR and STX16 highly co-localize at the apical and subapical regions of epithelial cells. Interestingly, CFTR-associated chloride current was reduced by the knockdown of STX16 expression in T84 cells. Surface biotinylation and recycling assays indicate that the reduction in CFTR chloride current is due to decreased CFTR expression on the plasma membrane. These results suggest that STX16 mediates recycling of CFTR and constitutes an important component of CFTR trafficking machinery in intestinal epithelial cells.

Pseudohypoparathyroidism and GNAS epigenetic defects: clinical evaluation of albright hereditary osteodystrophy and molecular analysis in 40 patients.

CONTEXT: The two main subtypes of pseudohypoparathyroidism (PHP), PHP-Ia and -Ib, are caused by mutations in GNAS exons 1-13 and methylation defects in the imprinted GNAS cluster, respectively. PHP-Ia patients show Albright hereditary osteodystrophy (AHO) and resistance toward PTH and additional hormones, whereas PHP-Ib patients do not have AHO, and hormone resistance appears to be limited to PTH and TSH. Recently, methylation defects have been detected in few patients with PHP and mild AHO, indicating a molecular overlap between the two forms. OBJECTIVES: The aim of the study was to screen patients with clinically diagnosed PHP-Ia for methylation defects and to investigate the presence of correlations between the molecular findings and AHO severity. PATIENTS AND METHODS: We investigated differential methylation of GNAS regions and STX16 microdeletions in genomic DNA from 40 patients with sporadic AHO and multihormone resistance, with no mutations in Gsalpha-coding GNAS exons. RESULTS: Molecular analysis showed GNAS cluster imprinting defects in 24 of the 40 patients analyzed. No STX16 deletion was detected. The presence of imprinting defects was not associated with the severity of AHO or with specific AHO signs. CONCLUSIONS: We report the largest series of the literature of patients with clinical AHO and multihormone resistance and no mutation in the Gsalpha gene. Our findings of frequent GNAS imprinting defects further confirm the existence of an overlap between molecular and clinical features of PHP-Ia and PHP-Ib and highlight the necessity of a new clinical classification of the disease that takes into account the recent knowledge on the molecular basis underlying these defects.

[Epigenetics and pseudohypoparathyroidism]. / Épigénétique et pseudohypoparathyroïdies.

Parental imprinting and the type of the genetic alteration play a determinant role in the phenotype expression of GNAS locus associated to pseudohypoparathyroidism (PHP). This imprint is tissue-specific, mainly localized in the kidney and the thyroid. Only the maternal allele is expressed at this level. An alteration in the coding sequence of the gene leads to an haplo-insufficiency and a dysmorphic phenotype (Albright's syndrome). If the alteration is on the maternal allele, there is a hormonal resistance to the PTH at the kidney level and to the TSH at the thyroid level. The phenotype is known as a PHP1a. If the alteration is on the paternal allele, there are few clinical signs with no hormonal resistance and the phenotype is known as pseudo-pseudo-hypoparathyroidism (PPHP). Methylation anomalies of GNAS locus, in particular of exon 1A, are responsible for a lack of expression of Gαs at kidney and thyroid levels only. If these anomalies concern the maternal allele (the only one expressed) with a paternal pattern, there is no haplo-insufficiency and no dysmorphic syndrome. The hormonal resistance is yet again limited to PTH and TSH. The phenotype is known as PHP1b. In the familial forms, these methylation anomalies are associated with a deletion of the syntaxine 16 gene in the maternal allele. This gene contains probably the imprinting center of the locus.

High resolution analysis of DNA copy-number aberrations of chromosomes 8, 13, and 20 in gastric cancers.

DNA copy-number gains of chromosomes 8q, 13q, and 20q are frequently observed in gastric cancers. Moreover gain of chromosome 20q has been associated with lymph node metastasis. The aim of this study was to correlate DNA copy-number changes of individual genes on chromosomes 8q, 13q, and 20q in gastric adenocarcinomas to clinicopathological data. DNA isolated from 63 formalin-fixed and paraffin-embedded gastric adenocarcinoma tissue samples was analyzed by whole-genome microarray comparative genomic hybridization and by multiplex ligation-dependent probe amplification (MLPA), targeting 58 individual genes on chromosomes 8, 13, and 20. Using array comparative genomic hybridization, gains on 8q, 13q, and 20q were observed in 49 (77.8%), 25 (39.7%), and 49 (77.8%) gastric adenocarcinomas, respectively. Gain of chromosome 20q was significantly correlated with lymph node metastases (p = 0.05) and histological type (p = 0.02). MLPA revealed several genes to be frequently gained in DNA copy number. The oncogene c-myc on 8q was gained in 73% of the cancers, while FOXO1A and ATP7B on 13q were both gained in 28.6% of the cases. Multiple genes on chromosome 20q showed gains in more than 60% of the cancers. DNA copy-number gains of TNFRSF6B (20q13.3) and ZNF217 (20q13.2) were significantly associated with lymph node metastasis (p = 0.02) and histological type (p = 0.02), respectively. In summary, gains of chromosomes 8q, 13q, and 20q in gastric adenocarcinomas harbor DNA copy-number gains of known and putative oncogenes. ZNF217 and TNFRSF6B are associated with important clinicopathological variables, including lymph node status.