Clinicopathologic Features of IDEDNIK (MEDNIK) Syndrome in a Term Infant: Histopathologic Features of the Gastrointestinal Tract and Report of a Novel AP1S1 Variant.

Inherited syndromes of congenital enteropathy are rare, with many genetic causes described. Mutations of the AP1S1 gene results in the syndrome of intellectual disability, enteropathy, deafness, peripheral neuropathy, ichthyosis, and keratoderma (IDEDNIK, formerly in the medical literature as MEDNIK). The clinicopathologic features of the enteropathy in IDEDNIK syndrome have not been fully explored. We describe a female infant who presented with metabolic acidosis, lethargy, and 14 watery stools per day. In the intensive care unit she required parenteral nutrition. She was found to have a novel homozygous pathogenic variant in the AP1S1 gene c.186T>G (p.Y62*). Esophagogastroduodenoscopy and colonoscopy at 6 months of age were grossly normal. However, histologic sections of the duodenum showed mild villous blunting and enterocytes with cytoplasmic vacuoles. CD10 immunostaining highlighted the disrupted brush border. MOC31 immunostaining was wild-type with a membranous pattern of expression. Electron microscopy of the duodenum showed scattered enterocytes cells with shortened and disrupted apical microvilli. Although there is a mixed gap diarrhea and disrupted brush border, there are no significant inclusions typical of microvillus inclusion disease, nor tufted enterocytes typical of tufting enteropathy, making the clinical and histopathologic features for this syndrome unique.

MiR-204-5p-targeted AP1S2 is necessary for papillary thyroid carcinoma.

MiR-204-5p, as a tumour suppressor, has been found in several cancers. However, whether miR-204-5p is involved in papillary thyroid carcinoma (PTC) has not yet been investigated. In this study, we identified miR-204-5p as a down-regulated miRNA in PTC tissues, unveiling that the levels of miR-204-5p in serum of patients with PTC were linked to PTC risk, and that the expression in patients concomitant with both PTC and benign lesions was much lower than that in patients only with PTC. Furthermore, we documented that miR-204-5p inhibited proliferation, migration, invasion, and cell cycle progression and triggered apoptosis of PTC cells via cell biology experiments. Finally, we identified that AP1S2 was a target of miR-204-5p using RNA-seq, iTRAQ, and bioinformatics prediction. Overall, miR-204-5p functions as a suppressor for PTC pathogenesis via the miR-204-5p/AP1S2 axis.

AP2S1 regulates APP degradation through late endosome-lysosome fusion in cells and APP/PS1 mice.

AP2S1 is the sigma 2 subunit of adaptor protein 2 (AP2) that is essential for endocytosis. In this study, we investigated the potential role of AP2S1 in intracellular processing of amyloid precursor protein (APP), which contributes to the pathogenesis of Alzheimer disease (AD) by generating the toxic ß-amyloid peptide (Aß). We found that knockdown or overexpression of AP2S1 decreased or increased the protein levels of APP and Aß in cells stably expressing human full-length APP695, respectively. This effect was unrelated to endocytosis but involved lysosomal degradation. Morphological studies revealed that silencing of AP2S1 promoted the translocalization of APP from RAB9-positive late endosomes (LE) to LAMP1-positive lysosomes, which was paralleled by the enhanced LE-lysosome fusion. In support, silencing of vacuolar protein sorting-associated protein 41 (VPS41) that is implicated in LE-lyso fusion prevented AP2S1-mediated regulation of APP degradation and translocalization. In APP/PS1 mice, an animal model of AD, AAV-mediated delivery of AP2S1 shRNA in the hippocampus significantly reduced the protein levels of APP and Aß, with the concomitant APP translocalization, LE-lyso fusion and the improved cognitive functions. Taken together, these data uncover a LE-lyso fusion mechanism in APP degradation and suggest a novel role for AP2S1 in the pathophysiology of AD.

Identification of a 5 bp duplicate in the AP1S2 gene of an individual with X-linked intellectual disability.

Adaptor-related protein complex 1 subunit sigma 2 (AP1S2) is a subunit of AP1 that is crucial for the reformation of the synaptic vesicle. Variants in AP1S2 have been reported to cause a rare neurodevelopmental disorder, Pettigrew syndrome (PGS) (OMIM: 304,340), which is characterized by walking delay, abnormal speech, mild to profound X-linked intellectual disability (XLID), and abnormal brain, and behaviors. Here, we describe a 2-year- and 5-month-old male patient who presented with global developmental delay (GDD). Trio whole exome sequencing (WES) revealed a 5 bp duplicate in the AP1S2 gene (NM_003916.5: exon 2: c.96_100dup, p. Leu34Glnfs*8) predicted to cause early termination of translation, which was inherited from the unaffected mother. The clinical features of our patient were consistent with previous reports. This is the second case in the Chinese family and the eleventh variant found in AP1S2-related XLID. Our findings expand the AP1S2 variant spectrum in neurodevelopmental disorders and provide evidence for the application of WES in PGS diagnosis.

The EMT activator ZEB1 accelerates endosomal trafficking to establish a polarity axis in lung adenocarcinoma cells.

Epithelial-to-mesenchymal transition (EMT) is a transcriptionally governed process by which cancer cells establish a front-rear polarity axis that facilitates motility and invasion. Dynamic assembly of focal adhesions and other actin-based cytoskeletal structures on the leading edge of motile cells requires precise spatial and temporal control of protein trafficking. Yet, the way in which EMT-activating transcriptional programs interface with vesicular trafficking networks that effect cell polarity change remains unclear. Here, by utilizing multiple approaches to assess vesicular transport dynamics through endocytic recycling and retrograde trafficking pathways in lung adenocarcinoma cells at distinct positions on the EMT spectrum, we find that the EMT-activating transcription factor ZEB1 accelerates endocytosis and intracellular trafficking of plasma membrane-bound proteins. ZEB1 drives turnover of the MET receptor tyrosine kinase by hastening receptor endocytosis and transport to the lysosomal compartment for degradation. ZEB1 relieves a plus-end-directed microtubule-dependent kinesin motor protein (KIF13A) and a clathrin-associated adaptor protein complex subunit (AP1S2) from microRNA-dependent silencing, thereby accelerating cargo transport through the endocytic recycling and retrograde vesicular pathways, respectively. Depletion of KIF13A or AP1S2 mitigates ZEB1-dependent focal adhesion dynamics, front-rear axis polarization, and cancer cell motility. Thus, ZEB1-dependent transcriptional networks govern vesicular trafficking dynamics to effect cell polarity change.

Ap2s1 mutation causes hypercalcaemia in mice and impairs interaction between calcium-sensing receptor and adaptor protein-2.

Adaptor protein 2 (AP2), a heterotetrameric complex comprising AP2α, AP2ß2, AP2µ2 and AP2σ2 subunits, is ubiquitously expressed and involved in endocytosis and trafficking of membrane proteins, such as the calcium-sensing receptor (CaSR), a G-protein coupled receptor that signals via Gα11. Mutations of CaSR, Gα11 and AP2σ2, encoded by AP2S1, cause familial hypocalciuric hypercalcaemia types 1-3 (FHH1-3), respectively. FHH3 patients have heterozygous AP2S1 missense Arg15 mutations (p.Arg15Cys, p.Arg15His or p.Arg15Leu) with hypercalcaemia, which may be marked and symptomatic, and occasional hypophosphataemia and osteomalacia. To further characterize the phenotypic spectrum and calcitropic pathophysiology of FHH3, we used CRISPR/Cas9 genome editing to generate mice harboring the AP2S1 p.Arg15Leu mutation, which causes the most severe FHH3 phenotype. Heterozygous (Ap2s1+/L15) mice were viable, and had marked hypercalcaemia, hypermagnesaemia, hypophosphataemia, and increases in alkaline phosphatase activity and fibroblast growth factor-23. Plasma 1,25-dihydroxyvitamin D was normal, and no alterations in bone mineral density or bone turnover were noted. Homozygous (Ap2s1L15/L15) mice invariably died perinatally. Co-immunoprecipitation studies showed that the AP2S1 p.Arg15Leu mutation impaired protein-protein interactions between AP2σ2 and the other AP2 subunits, and also with the CaSR. Cinacalcet, a CaSR positive allosteric modulator, decreased plasma calcium and parathyroid hormone concentrations in Ap2s1+/L15 mice, but had no effect on the diminished AP2σ2-CaSR interaction in vitro. Thus, our studies have established a mouse model that is representative for FHH3 in humans, and demonstrated that the AP2S1 p.Arg15Leu mutation causes a predominantly calcitropic phenotype, which can be ameliorated by treatment with cinacalcet.

Misleading localization by 18F-fluorocholine PET/CT in familial hypocalciuric hypercalcemia type-3: a case report.

BACKGROUND: Familial hypocalciuric hypercalcemia (FHH) is a heterogeneous autosomal-dominant disorder of calcium hemostasis that may be difficult to distinguish clinically from mild primary hyperparathyroidism. Loss-of-function mutations mainly involving Arg15 residue of the adaptor-related protein complex 2, sigma subunit 1 (AP2S1) cause a rarer, more recently recognized form of FHH, FFH type-3. Recently, 18F-fluorocholine positron emission tomography/computed tomography (FCH-PET/CT) showed superior sensitivity to conventional imaging in localizing parathyroid adenomas. We report a new FFH type-3 patient who underwent unnecessary parathyroidectomy in association with misleading FCH-PET/CT imaging. CASE PRESENTATION: A 29-year old woman was initially evaluated for parathyroid hormone (PTH)-dependent hypercalcemia in 2013. Medical history was positive only for chronic constipation and malaise with no personal or family history of hypercalcemia, kidney stones, or neck surgery. Over seven years, serum calcium level was 2.51-2.89 mmol/L with concomitant PTH level of 58.7-94.8 mmol/L. Serum phosphate levels were in the low/low normal range. Serum creatinine and magnesium levels were normal. 25-hydroxy vitamin D level was 13 nmol/L. 24-hour urine calcium level was 1.92 mmol/day but increased to 6.99 mmol/day after treatment with cholecalciferol 1000 IU daily. Bone mineral density and renal ultrasound were normal. Parathyroid ultrasound showed two hypoechoic nodules inferior to the left and right thyroid lobes; however, 99mtechnitium-sestamibi scans (2013, 2016, 2018) were negative. FCH-PET/CT (2019) showed focal uptake co-localizing with the nodule inferior to the left thyroid lobe. The patient underwent left inferior parathyroidectomy and pathology was consistent with parathyroid hyperplasia. However, postoperatively, serum calcium and PTH levels remained elevated and FCH-PET/CT and ultrasound showed persistence of the uptake/nodule. Whole exome sequencing showed Arg15Cys mutation in the AP2S1 gene characteristic of FHH type-3. CONCLUSIONS: In this new case of FHH type-3, FCH-PET/CT failed to localize to the hyperplastic parathyroid glands and localized instead to apparently a lymph node. This, together with increased urinary calcium after vitamin D supplementation, led to unnecessary parathyroidectomy. Given the increasingly lower cost of genetic testing and the cost of follow up and unnecessary surgery, it may prudent to include genetic testing for FHH early on in patients with mild PTH-dependent hypercalcemia.

Familial hypocalciuric hypercalcaemia type 3: AP2S1 missense mutation.

A 45-year-old man was referred to endocrine for the evaluation of hypercalcaemia. The calcium was elevated, vitamin D was low with a normal parathyroid hormone. Dual-energy X-ray absorptiometry scan revealed osteoporosis at the lumbar spine and femoral neck. A 24-hour urine collection revealed low urinary calcium, which was believed to be secondary to vitamin D deficiency. A diagnosis of primary hyperparathyroidism was made. The patient underwent a four-gland parathyroid exploration surgery in which three of his parathyroid glands were removed. The pathology was consistent with benign parathyroid tissue. Post surgery, the patient had persistently elevated calcium levels. He was then started on bisphosphonate and cinacalcet for osteoporosis and hypercalcaemia, respectively. Genetic analysis of familial hypocalciuric hypercalcaemia (FHH) showed a p.arg15cys mutation in the AP2S1 gene, confirming the diagnosis of FHH type 3.

AP1S1 missense mutations cause a congenital enteropathy via an epithelial barrier defect.

Congenital diarrheal disorders (CDD) comprise > 50 monogenic entities featuring chronic diarrhea of early-onset, including defects in nutrient and electrolyte absorption, enterocyte polarization, enteroendocrine cell differentiation, and epithelial integrity. Diarrhea is also a predominant symptom in many immunodeficiencies, congenital disorders of glycosylation, and in some defects of the vesicular sorting and transporting machinery. We set out to identify the etiology of an intractable diarrhea in 2 consanguineous families by whole-exome sequencing, and identified two novel AP1S1 mutations, c.269T>C (p.Leu90Pro) and c.346G>A (p.Glu116Lys). AP1S1 encodes the small subunit of the adaptor protein 1 complex (AP-1), which plays roles in clathrin coat-assembly and trafficking between trans-Golgi network, endosomes and the plasma membrane. An AP1S1 knock-out (KO) of a CaCo2 intestinal cell line was generated to characterize intestinal AP1S1 deficiency as well as identified mutations by stable expression in KO background. Morphology and prototype transporter protein distribution were comparable between parental and KO cells. We observed altered localization of tight-junction proteins ZO-1 and claudin 3, decreased transepithelial electrical resistance and an increased dextran permeability of the CaCo2-AP1S1-KO monolayer. In addition, lumen formation in 3D cultures of these cells was abnormal. Re-expression of wild-type AP1S1 in CaCo2-AP1S1-KO cells reverted these abnormalities, while expression of AP1S1 containing either missense mutation did not. Our data indicate that loss of AP1S1 function causes an intestinal epithelial barrier defect, and that AP1S1 mutations can cause a non-syndromic form of congenital diarrhea, whereas 2 reported truncating AP1S1 mutations caused MEDNIK syndrome, characterized by mental retardation, enteropathy, deafness, neuropathy, ichthyosis, and keratodermia.

A Hong Kong Chinese kindred with familial hypocalciuric hypercalcaemia caused by AP2S1 mutation.

Familial hypocalciuric hypercalcaemia (FHH) is a genetic disorder of altered calcium homeostasis. Mutations in the CASR, GNA11 and AP2S1 genes have been reported to cause FHH. We report a Hong Kong Chinese kindred with FHH type 3 (FHH3) caused by mutations in AP2S1. The proband, a 51-year-old woman with hypercalcaemia, was initially diagnosed to have primary hyperparathyroidism but repeated parathyroidectomy failed to normalize her plasma calcium concentrations. Later, FHH was suspected and yet no mutations were identified in the CASR gene which causes FHH type 1 (FHH1), the most common form of FHH. Genetic testing of AP2S1 revealed a heterozygous c.43C>T (p.Arg15Cys) mutation, confirming the diagnosis of FHH3. The elder brother and niece of the proband, who both have hypercalcaemia, were found to harbour the same mutation. To our knowledge, this is the first Chinese kindred of FHH3 reported in the English literature.

Long noncoding RNA LINC00518 acts as a competing endogenous RNA to promote the metastasis of malignant melanoma via miR-204-5p/AP1S2 axis.

Long intergenic nonprotein coding RNA 518 (LINC00518) has been shown to promote cancer cell growth and metastasis in some human tumors. Although it has been reported that LINC00518 is dysregulated in melanoma, its exact role and molecular mechanism in melanoma remain unclear. RNA-seq analysis and qRT-PCR was used to detect the expression of LINC00518 in melanoma tissues. Melanoma cases from The Cancer Genome Atlas (TCGA), GEO#GSE15605 and GEO#GSE24469 were included in this study. 3D migration, transwell and scratch wound assay were used to explore the role of LINC00518 in melanoma cells. Bioinformatics, luciferase reporter assays, MS2-RIP assay, RNA pull-down assay and RNA-ChIP assay were used to demonstrate the mechanism of LINC00518 in melanoma. We found that LICN00518 was significantly upregulated in melanoma tissue, and high LICN00518 level was an independent risk factor for melanoma patients. LICN00518 promoted the invasion and migration of melanoma cells. LICN00518 exerted its role by decoying miR-204-5p to upregulate Adaptor Related Protein Complex 1 Sigma 2 Subunit (AP1S2) expression. We also demonstrated that LICN00518 promoted melanoma metastasis in vivo through pulmonary metastasis assay. This result elucidates a new mechanism for LICN00518 in the metastasis of melanoma. LICN00518 may serve as a survival indicator and potential therapeutic target in melanoma patients.

Cinacalcet sustainedly prevents pancreatitis in a child with a compound heterozygous SPINK1/AP2S1 mutation.

Familial hypocalciuric hypercalcemia is an autosomal dominant genetic disorder characterized by hypercalcemia associated with inappropriate hypocalciuria and normal parathyroid hormone levels. Acute recurrent pancreatitis (ARP) is rare in children. Predisposing factors include hypercalcemia and mutations in the serine protease inhibitor Kazal-type 1 (SPINK1) gene. The disease carries a heavy morbidity and preventive treatment options are scant. Here, we report a child with a novel genetic/metabolic form of ARP associated with compound heterozygous SPINK1/AP2S1 (adaptor protein-2 σ1-subunit) mutations, recurrence of which was completely abrogated for 6 years by cinacalcet treatment.

Expanding the spectrum of genetic variants in the calcium-sensing receptor (CASR) gene in hypercalcemic individuals.

OBJECTIVE: Familial hypocalciuric hypercalcemia (FHH) is an autosomal dominantly inherited disorder with overlapping biochemistry profile with primary hyperparathyroidism (PHPT), making the correct diagnosis a challenge. The objective of the study was to evaluate the results of the clinical work-up of a large group of hypercalcemic individuals. DESIGN: Cross-sectional study. PATIENTS: Patients undergoing clinical work-up of hypercalcemia. MEASUREMENTS: Molecular genetic analysis of the CASR gene and exon 2 of the AP2S1 gene. Plasma levels of ionized calcium and PTH as well as calcium creatinine clearance ratio (CCCR). RESULTS: A rare CASR variant was identified in 38 of 624 index patients (6.1%). A total of 18 CASR variants identified in this study were novel. No variants were identified in exon 2 of the AP2S1 gene. The majority of the variants (N = 16) were classified as likely pathogenic. The level of plasma calcium, plasma PTH and the CCCR was not affected by the type of variant (ie nonsense vs missense) (all P-values >.05). The CCCR was found to be significantly lower for variants in the transmembrane domain compared with variants located in the extracellular domain (P < .05). Plasma levels of calcium and PTH were not associated with the location of the variant (P > .05). CONCLUSIONS: We expanded the spectrum of CASR variants in hypercalcemia with 18 novel variants, and suggest that the location of the CASR variant may affect calcium excretion as determined by the CCCR.

Pluripotent stem cell-derived bile canaliculi-forming hepatocytes to study genetic liver diseases involving hepatocyte polarity.

BACKGROUND & AIMS: Hepatocyte polarity is essential for the development of bile canaliculi and for safely transporting bile and waste products from the liver. Functional studies of autologous mutated proteins in the context of the polarized hepatocyte have been challenging because of the lack of appropriate cell models. The aims of this study were to obtain a patient-specific hepatocyte model that recapitulated hepatocyte polarity and to employ this model to study endogenous mutant proteins in liver diseases that involve hepatocyte polarity. METHODS: Urine cell-derived pluripotent stem cells, taken from a patient with a homozygous mutation in ATP7B and a patient with a heterozygous mutation, were differentiated towards hepatocyte-like cells (hiHeps). HiHeps were also derived from a patient with MEDNIK syndrome. RESULTS: Polarized hiHeps that formed in vivo-like bile canaliculi could be generated from embryonic and patient urine cell-derived pluripotent stem cells. HiHeps recapitulated polarized protein trafficking processes, exemplified by the Cu2+-induced redistribution of the copper transporter protein ATP7B to the bile canalicular domain. We demonstrated that, in contrast to the current dogma, the most frequent yet enigmatic Wilson disease-causing ATP7B-H1069Q mutation per se did not preclude trafficking of ATP7B to the trans-Golgi Network. Instead, it prevented its Cu2+-induced polarized redistribution to the bile canalicular domain, which could not be reversed by pharmacological folding chaperones. Finally, we demonstrate that hiHeps from a patient with MEDNIK syndrome, suffering from liver copper overload of unclear etiology, showed no defect in the Cu2+-induced redistribution of ATP7B to the bile canaliculi. CONCLUSIONS: Functional cell polarity can be achieved in patient pluripotent stem cell-derived hiHeps, enabling, for the first time, the study of the endogenous mutant proteins, patient-specific pathogenesis and drug responses for diseases where hepatocyte polarity is a key factor. LAY SUMMARY: This study demonstrates that cells that are isolated from urine can be reprogrammed in a dish towards hepatocytes that display architectural characteristics similar to those seen in the intact liver. The application of this methodology to cells from patients diagnosed with inherited copper metabolism-related liver diseases (that is, Wilson disease and MEDNIK syndrome) revealed unexpected and novel insights into patient mutation-specific disease mechanisms and drug responses.

A novel splice site mutation in AP1S2 gene for X-linked mental retardation in a Chinese pedigree and literature review.

BACKGROUND: Pettigrew syndrome (PGS) is a rare X-linked mental retardation that caused by AP1S2 mutation. The pathogenesis of AP1S2 deficiency has remained elusive. The purpose of this study is to give a comprehensive overview of the phenotypic and genetic spectrum of AP1S2 mutations. METHODS: This study systematically analyzed clinical features and genetic information of a Chinese family with AP1S2 variation, and reviewed previously reported literatures with the same gene variation. RESULTS: We identified a new c.1-1 G>C mutation in AP1S2 gene from a four generation family with seven affected individuals and found the elevated neuron-specific enolase (NSE) in a patient. We summarized the clinical manifestation of 59 patients with AP1S2 mutation. We found that pathogenic point mutations affecting AP1S2 are associated with dysmorphic features and neurodevelopmental problems, which included highly variable mental retardation (MR), delayed in walking, abnormal speech, hypotonia, abnormal brain, abnormal behavior including aggressive behavior, ASD, self-abusive, and abnormal gait. Patients with splice site mutation were more likely to lead to seizures. By contrast, patients with nonsense mutations are more susceptible to microcephaly. CONCLUSION: Our findings suggest AP1S2 mutations contribute to a broad spectrum of neurodevelopmental disorders and are important in the etiological spectrum of PGS.

MEDNIK syndrome with a frame shift causing mutation in AP1S1 gene and literature review of the clinical features.

MEDNIK syndrome is an autosomal recessive rare disease as one of the most recently described copper metabolism disorder characterized by intellectual disability, ichthyosis, hearing loss, peripheral neuropathy, enteropathy and keratodermia. Here in, we reported a case presented with ichthyosis and intellectual disability with MEDNIK syndrome that confirmed by mutation analysis in a Turkish child. She was finally diagnosed with MEDNIK syndrome by clinical findings, which were confirmed by molecular genetic testing. Sequencing of AP1S1 gene showed a homozygous insertion c.364dupG (NM_001283.4), which is predicted to cause a frameshift of the reading frame (p.D122Gfs*18). To our knowledge, this is the first case of MEDNIK syndrome from Turkey. Diagnosis of MEDNIK syndrome is still challenging and we hope that this case will contribute to further understanding.

High-Confidence Interactome for RNF41 Built on Multiple Orthogonal Assays.

Ring finger protein 41 (RNF41) is an E3 ubiquitin ligase involved in the ubiquitination and degradation of many proteins including ErbB3 receptors, BIRC6, and parkin. Next to this, RNF41 regulates the intracellular trafficking of certain JAK2-associated cytokine receptors by ubiquitinating and suppressing USP8, which, in turn, destabilizes the ESCRT-0 complex. To further elucidate the function of RNF41 we used different orthogonal approaches to reveal the RNF41 protein complex: affinity purification-mass spectrometry, BioID, and Virotrap. We combined these results with known data sets for RNF41 obtained with microarray MAPPIT and Y2H screens. This way, we establish a comprehensive high-resolution interactome network comprising 175 candidate protein partners. To remove potential methodological artifacts from this network, we distilled the data into a high-confidence interactome map by retaining a total of 19 protein hits identified in two or more of the orthogonal methods. AP2S1, a novel RNF41 interaction partner, was selected from this high-confidence interactome for further functional validation. We reveal a role for AP2S1 in leptin and LIF receptor signaling and show that RNF41 stabilizes and relocates AP2S1.

Large-scale exome datasets reveal a new class of adaptor-related protein complex 2 sigma subunit (AP2σ) mutations, located at the interface with the AP2 alpha subunit, that impair calcium-sensing receptor signalling.

Mutations of the sigma subunit of the heterotetrameric adaptor-related protein complex 2 (AP2σ) impair signalling of the calcium-sensing receptor (CaSR), and cause familial hypocalciuric hypercalcaemia type 3 (FHH3). To date, FHH3-associated AP2σ mutations have only been identified at one residue, Arg15. We hypothesized that additional rare AP2σ variants may also be associated with altered CaSR function and hypercalcaemia, and sought for these by analysing >111 995 exomes (>60 706 from ExAc and dbSNP, and 51 289 from the Geisinger Health System-Regeneron DiscovEHR dataset, which also contains clinical data). This identified 11 individuals to have 9 non-synonymous AP2σ variants (Arg3His, Arg15His (x3), Ala44Thr, Phe52Tyr, Arg61His, Thr112Met, Met117Ile, Glu122Gly and Glu142Lys) with 3 of the 4 individuals who had Arg15His and Met117Ile AP2σ variants having mild hypercalcaemia, thereby indicating a prevalence of FHH3-associated AP2σ mutations of ∼7.8 per 100 000 individuals. Structural modelling of the novel eight AP2σ variants (Arg3His, Ala44Thr, Phe52Tyr, Arg61His, Thr112Met, Met117Ile, Glu122Gly and Glu142Lys) predicted that the Arg3His, Thr112Met, Glu122Gly and Glu142Lys AP2σ variants would disrupt polar contacts within the AP2σ subunit or affect the interface between the AP2σ and AP2α subunits. Functional analyses of all eight AP2σ variants in CaSR-expressing cells demonstrated that the Thr112Met, Met117Ile and Glu142Lys variants, located in the AP2σ α4-α5 helical region that forms an interface with AP2α, impaired CaSR-mediated intracellular calcium (Cai2+) signalling, consistent with a loss of function, and this was rectified by treatment with the CaSR positive allosteric modulator cinacalcet. Thus, our studies demonstrate another potential class of FHH3-causing AP2σ mutations located at the AP2σ-AP2α interface.

Copper Regulates Maturation and Expression of an MITF:Tryptase Axis in Mast Cells.

Copper has previously been implicated in the regulation of immune responses, but the impact of this metal on mast cells is poorly understood. In this article, we address this issue and show that copper starvation of mast cells causes increased granule maturation, as indicated by higher proteoglycan content, stronger metachromatic staining, and altered ultrastructure in comparison with nontreated cells, whereas copper overload has the opposite effects. In contrast, copper status did not impact storage of histamine in mast cells, nor did alterations in copper levels affect the ability of mast cells to degranulate in response to IgER cross-linking. A striking finding was decreased tryptase content in mast cells with copper overload, whereas copper starvation increased tryptase content. These effects were associated with corresponding shifts in tryptase mRNA levels, suggesting that copper affects tryptase gene regulation. Mechanistically, we found that alterations in copper status affected the expression of microphthalmia-associated transcription factor, a transcription factor critical for driving tryptase expression. We also found evidence supporting the concept that the effects on microphthalmia-associated transcription factor are dependent on copper-mediated modulation of MAPK signaling. Finally, we show that, in MEDNIK syndrome, a condition associated with low copper levels and a hyperallergenic skin phenotype, including pruritis and dermatitis, the number of tryptase-positive mast cells is increased. Taken together, our findings reveal a hitherto unrecognized role for copper in the regulation of mast cell gene expression and maturation.