Mutations In STIM1 Research Articles

P.112Two murine models for tubular aggregate myopathy with mutations in Stim1 and Orai1

Calcium (Ca2+) is a key mediator in a wide range of cellular functions. Ca2+ released from the sarcoplasmic reticulum (SR) induces sarcomeric contraction of myofibrils, which is a primary mechanism mediating Ca2+ homeostasis in skeletal muscle. At the depletion of Ca2+ in the SR, store-operated Ca2+ entry (SOCE) is induced to obtain Ca2+ from the extracellular area. SR protein, STIM1 and Ca2+ channel protein on the plasma membrane, ORAI1 work on SOCE in skeletal myofibers. Tubular aggregate myopathy (TAM) has been known to be caused by constitutively activated SOCE owing to the dominant mutations in ORAI1 or STIM1 consequently.

CONGENITAL MYOPATHIES: CENTRONUCLEAR AND OTHERS: P.103STIM1 over-activation generates a multi-systemic phenotype affecting skeletal muscle, spleen, eye, skin, bones, and the immune system in mice

Tubular aggregate myopathy (TAM) and Stormorken syndrome (STRMK) are spectra of the same disease affecting muscle, platelets, spleen, and skin. TAM and STRMK arise from gain-of-function mutations in STIM1 or ORAI1, both encoding key regulators of Ca2+ homeostasis, and mutations in either gene results in excessive Ca2+ entry. To elucidate the physiological consequences of STIM1 over-activation, we generated a murine model harboring the most common TAM/STRMK mutation and characterized the phenotype at the histological, ultrastructural, metabolic, physiological, and functional level. In accordance with the clinical picture of TAM/STRMK, the Stim1R304W/+ mice manifested muscle weakness, thrombocytopenia, skin and eye anomalies, and spleen dysfunction, as well as additional features not yet observed in patients such as abnormal bone architecture and immune system dysregulation. In situ force measurements of the Stim1R304W/+ tibialis anterior revealed a reduction of the specific force, and we also measured sustained muscle contraction upon stimulation, demonstrating delayed muscle relaxation. Histological and ultrastructural analyses of Stim1R304W/+ tibialis anterior, soleus and gastrocnemius disclosed mild dystrophic features including internalized nuclei, regenerating fibers, fibrosis, and inflammation, fibers with high Ca2+ content, and swollen mitochondria. Tubular aggregates, the histopathological hallmark of TAM/STRMK were however not seen, demonstrating that they do no contribute to disease development. Ca2+ measurements on differentiated Stim1R304W/+ myotubes revealed elevated basal Ca2+ levels and an increased extracellular Ca2+ entry despite normal Ca2+ stores. Altogether, the Stim1R304W/+ mice recapitulate the main clinical features of TAM/STRMK, and provide insight into the pathophysiological effect STIM1 over-activation and aberrant Ca2+ homeostasis in different cells, tissues, and organs.

Tubular aggregate myopathy and Stormorken syndrome: Mutation spectrum and genotype/phenotype correlation.

Calcium (Ca2+ ) acts as a ubiquitous second messenger, and normal cell and tissue physiology strictly depends on the precise regulation of Ca2+ entry, storage, and release. Store-operated Ca2+ entry (SOCE) is a major mechanism controlling extracellular Ca2+ entry, and mainly relies on the accurate interplay between the Ca2+ sensor STIM1 and the Ca2+ channel ORAI1. Mutations in STIM1 or ORAI1 result in abnormal Ca2+ homeostasis and are associated with severe human disorders. Recessive loss-of-function mutations impair SOCE and cause combined immunodeficiency, while dominant gain-of-function mutations induce excessive extracellular Ca2+ entry and cause tubular aggregate myopathy (TAM) and Stormorken syndrome (STRMK). TAM and STRMK are spectra of the same multisystemic disease characterized by muscle weakness, miosis, thrombocytopenia, hyposplenism, ichthyosis, dyslexia, and short stature. To date, 42 TAM/STRMK families have been described, and here we report five additional families for which we provide clinical, histological, ultrastructural, and genetic data. In this study, we list and review all new and previously reported STIM1 and ORAI1 cases, discuss the pathomechanisms of the mutations based on the known functions and the protein structure of STIM1 and ORAI1, draw a genotype/phenotype correlation, and delineate an efficient screening strategy for the molecular diagnosis of TAM/STRMK.

STIM1 holds a STING in its (N-terminal) tail

The Ca2+ sensor STIM1 is essential for adaptive immune responses, yet patients with hypomorphic STIM1 mutations develop both immunodeficiency and autoimmunity, implying that STIM1 also restrains immune responses. This study by Srikanth et al demonstrates that STIM1 tethers STING, a major inducer of the interferon (IFN) response, to the endoplasmic reticulum (ER) to prevent constitutive STING activation.

STIM1 over-activation generates a multi-systemic phenotype affecting the skeletal muscle, spleen, eye, skin, bones and immune system in mice.

Strict regulation of Ca2+ homeostasis is essential for normal cellular physiology. Store-operated Ca2+ entry (SOCE) is a major mechanism controlling basal Ca2+ levels and intracellular Ca2+ store refilling, and abnormal SOCE severely impacts on human health. Overactive SOCE results in excessive extracellular Ca2+ entry due to dominant STIM1 or ORAI1 mutations and has been associated with tubular aggregate myopathy (TAM) and Stormorken syndrome (STRMK). Both disorders are spectra of the same disease and involve muscle weakness, myalgia and cramps, and additional multi-systemic signs including miosis, bleeding diathesis, hyposplenism, dyslexia, short stature and ichthyosis. To elucidate the physiological consequences of STIM1 over-activation, we generated a murine model harboring the most common TAM/STRMK mutation and characterized the phenotype at the histological, ultrastructural, metabolic, physiological and functional level. In accordance with the clinical picture of TAM/STRMK, the Stim1R304W/+ mice manifested muscle weakness, thrombocytopenia, skin and eye anomalies and spleen dysfunction, as well as additional features not yet observed in patients such as abnormal bone architecture and immune system dysregulation. The murine muscles exhibited contraction and relaxation defects as well as dystrophic features, and functional investigations unraveled increased Ca2+ influx in myotubes. In conclusion, we provide insight into the pathophysiological effect of the STIM1 R304W mutation in different cells, tissues and organs and thereby significantly contribute to a deeper understanding of the pathomechanisms underlying TAM/STRMK and other human disorders involving aberrant Ca2+ homeostasis and affecting muscle, bones, platelets or the immune system.

The Rare Diseases Pilot for the 100,000 Genomes Project: Findings in Known and New Genes by Analysis of 3,549 Whole Genome Sequenced Samples from Patients and Relatives with Haematological, Haemostasis and Immune Disorders

The Rare Diseases Pilot study of the 100,000 Genomes Project had two objectives. Firstly, to identify the DNA variants underlying unresolved Mendelian disorders. Secondly, to develop an accredited framework for delivering whole genome sequencing (WGS) results across a national healthcare system. From February 2014 to June 2017, 13,037 individuals with a rare disease and their relatives were recruited at 57 National Health Service (NHS) hospitals in the UK and 26 non-UK hospitals using standardized eligibility criteria for 12 rare disease domains. This cohort includes cases with haematology (n=1021), immunology (n=1359) and haemostasis disorders (n=1169).With informed consent, clinical and laboratory data were collected and coded into a single research database using Human Phenotype Ontology (HPO) terms and 13,037 samples of DNA were Illumina WGS analysed to clinical standard at a mean depths >30X in all samples and 90% of the reference genome was covered at 19X minimum in all samples. The pilot resource contains over 165 million unique variants with 91.5%, 8,5% and 5.6% single nucleotide variants (SNVs), short insertions / deletions and large deletions of the 10,258 genetically independent samples with 47% of variants previously unobserved in large scale genome datasets (e.g. TopMED, gnomAD, UK10K). Across all domains 2,067 unique diagnostic-grade genes (DGGs) were curated to clinical standards to support pertinent finding reporting by 12 multi-disciplinary teams (MDTs) with domain-relevant clinical and genetic expertise. Over 1,300 MDT reports assigning pathogenic or likely pathogenic causal variants have been returned to referring clinicians, with the diagnostic yield ranging from 1.6 to 53.8%, depending on the extent of genetic screening pre-enrolment and the importance of the non-genetic component of the disorder (e.g. in immune disorders). About 30% of the causal variants identified have never been reported (absent from the Human Gene Mutation Database v.2018.1); interestingly, 51 variants have been reported in 11 DGGs linked to phenotypes belonging to different domains. A comparison with standard whole exome sequencing results revealed WGS to have at least 12.5% superiority in sensitivity for detecting known pathogenic variants.For the haematology, immunology and haemostasis domains 330 causal variants were reported in 83 DGGs, revealing novel modes of inheritance (Sivapalaratnam et al Blood 2016), and entire new clinical phenotypes linked to mutations in ABCC4, GNE, KDSR and STIM1 amongst other DGGs. The genotype and HPO-coded phenotypes of all pilot cases were analysed with BeviMed, a rapid and scalable Bayesian association test (Greene et al AJHG 2017) to identify causal variants in hitherto unknown genes. This identified more than 30 genes with posterior probabilities indicating a high likelihood of being implicated in underlying as yet unresolved Mendelian disorders. Results from co-segregation and cell biology studies have already corroborated this statistical inference and 15 novel genes acquired DGG status. Including a new method for the analysis of the ‘gene-regulatory’ elements we also identified an example of a causal variant in such an element controlling the function of both GATA1 and HDAC6 resulting in a severe syndromic pathology characterized by abnormal erythropoiesis and megakaryopoiesis.In conclusion, the pilot of the 100,000 Genomes Project has shown the feasibility of using WGS across a national health system, such as the NHS, to deliver a molecular diagnosis for patients with rare inherited diseases and how a national genotype/HPO-coded phenotype resource provides a powerful platform for the identification of novel diagnostic-grade genes. DisclosuresNo relevant conflicts of interest to declare.

ORAI1 channel gating and selectivity is differentially altered by natural mutations in the first or third transmembrane domain.

Key points Gain‐of‐function mutations in the highly selective Ca2+ channel ORAI1 cause tubular aggregate myopathy (TAM) characterized by muscular pain, weakness and cramping.TAM‐associated mutations in ORAI1 first and third transmembrane domain facilitate channel opening by STIM1, causing constitutive Ca2+ influx and increasing the currents evoked by Ca2+ store depletion.Mutation V107M additionally decreases the channel selectivity for Ca2+ ions and its inhibition by acidic pH, while mutation T184M does not alter the channel sensitivity to pH or to reactive oxygen species.The ORAI blocker GSK‐7975A prevents the constitutive activity of TAM‐associated channels and might be used in therapy for patients suffering from TAM. Skeletal muscle differentiation relies on store‐operated Ca2+ entry (SOCE) mediated by STIM proteins linking the depletion of endoplasmic/sarcoplasmic reticulum Ca2+ stores to the activation of membrane Ca2+‐permeable ORAI channels. Gain‐of‐function mutations in STIM1 or ORAI1 isoforms cause tubular aggregate myopathy (TAM), a skeletal muscle disorder with muscular pain, weakness and cramping. Here, we characterize two overactive ORAI1 mutants from patients with TAM: V107M and T184M, located in the first and third transmembrane domain of the channel. When ectopically expressed in HEK‐293T cells or human primary myoblasts, the mutated channels increased basal and store‐operated Ca2+ entry. The constitutive activity of V107M, L138F, T184M and P245L mutants was prevented by low concentrations of GSK‐7975A while the G98S mutant was resistant to inhibition. Electrophysiological recordings confirmed ORAI1‐V107M constitutive activity and revealed larger STIM1‐gated V107M‐ and T184M‐mediated currents with conserved fast and slow Ca2+‐dependent inactivation. Mutation V107M altered the channel selectivity for Ca2+ ions and conferred resistance to acidic inhibition. Ca2+ imaging and molecular dynamics simulations showed a preserved sensitivity of T184M to the negative regulation by reactive oxygen species. Both mutants were able to mediate SOCE in Stim1−/−/Stim2−/− mouse embryonic fibroblasts expressing the binding‐deficient STIM1‐F394H mutant, indicating a higher sensitivity for STIM1‐mediated gating, with ORAI1‐T184M gain‐of‐function being strictly dependent on STIM1. These findings provide new insights into the permeation and regulatory properties of ORAI1 mutants that might translate into therapies against diseases with gain‐of‐function mutations in ORAI1.

Tubular aggregate myopathy and Stormorken syndrome

Calcium (Ca2+) is an essential regulator for a large number of cellular functions in various tissues and organs, and small disturbances of Ca2+ homeostasis can severely compromise normal physiology. Intracellular Ca2+ balance is mainly controlled by the reticular Ca2+ sensor STIM1 and the plasma membrane Ca2+ channel ORAI1 through a mechanism known as store-operated Ca2+ entry (SOCE). Gain-of-function mutations in STIM1 or ORAI1 cause excessive extracellular Ca2+ influx, resulting in tubular aggregate myopathy (TAM) and Stormorken syndrome (STRMK). Both disorders are spectra of the same disease and involve muscle weakness, miosis, thrombocytopenia, hyposplenism, ichthyosis, dyslexia, and short stature. Here we summarize the clinical and histological characteristics of both disorders, provide an overview on the genetic causes, and recapitulate the current knowledge on the pathomechanisms leading to the multi-systemic phenotype of tubular aggregate myopathy and Stormorken syndrome.

Store-Operated Calcium Entry Mediated byORAIand STIM.

The calcium release-activated calcium (CRAC) channel, composed of ORAI and stromal interaction molecules (STIM), represents a prototypical example of store-operated calcium entry in mammals. The ORAI-STIM signaling occurs at membrane contact sites formed by close appositions between the endoplasmic reticulum (ER) and the plasma membrane. ORAI1 is a four-pass transmembrane protein that forms a highly calcium-selective ion channel in the plasma membrane. STIM1 is an ER-resident, a single-pass transmembrane protein that serves as a calcium sensor within the ER lumen and a potent activator of ORAI1 calcium channels. The intricate interplay between ORAI and STIM controls calcium entry into cells to regulate a myriad of physiological processes. We highlight herein the current knowledge on the structure-function relationship of CRAC channel, with a focus on key structural elements that mediate STIM1 conformational switch and the dynamic coupling between STIM1 and ORAI1. Furthermore, we discuss the physiological roles of STIM-ORAI signaling in various tissues and organs, as well as major pathological conditions arising from loss- or gain-of-function mutations in human ORAI1 and STIM1. © 2017 American Physiological Society. Compr Physiol 8:981-1002, 2018.

Increased susceptibility to hypertensive renal disease in spontaneously hypertensive rats due to a mutation in Stim1

Emerging evidence implicates CD4+ T cells in the pathogenesis of hypertensive renal injury, though the underlying mechanisms that drive T cell dysfunction are not well understood. We identified a novel truncating mutation in the renal injury‐prone spontaneously hypertensive rat (SHR‐A3/SHRSP) line affecting the C‐terminus of STIM1, a protein involved in the store‐operated Ca2+ entry (SOCE) pathway. SOCE is required by T cells to activate the transcription factor NFAT and regulate T cell proliferation and cytokine production and regulatory T cell (Treg) development. In humans with loss‐of‐function mutations in Stim1, defective SOCE leads to a complex phenotype of immunodeficiency coupled with autoimmunity and uncontrolled lymphoproliferation. Our prior work identified defects in CD4+ T cell effector as well as regulatory function in SHR‐A3 rats as a result of impaired SOCE. We hypothesized that aberrant Stim1‐mediated SOCE leads to susceptibility to progressive renal injury in hypertension, potentially as a result of defective regulation of the immune response by T cells. Here, we test whether the emergence or renal injury in SHR‐A3 is affected by gene rescue of Stim1. A Stim1 congenic line (SHR‐A3(Stim1‐SHR‐B2)), in which the functional ‘wild type’ Stim1 allele was transferred from the SHR‐B2 line, which resists renal injury, into the injury‐susceptible SHR‐A3 genome. SOCE was restored to SHR‐B2 levels in the congenic SHR‐A3(Stim1‐SHR‐B2) line. This resulted in adequate NFAT activation (% cells with nuclear NFAT: 67±4.8 vs 8±3.8, p<0.001) in SHR‐A3(Stim1‐SHR‐B2) rats compared to SHR‐A3 rats. TCR‐induced lymphokine production was markedly higher in CD4+ T cells from SHR‐A3(Stim1‐SHR‐B2) compared with SHR‐A3 rats (IL‐2: 2074±367.1 vs 168±83.4 pg/mL, p<0.001; IFNγ: 2069±307.7 vs 235±69.5 pg/mL, p=0.002). Flow cytometric analysis of circulating T cell subsets revealed normalized CD4+CD25+FoxP3+ Treg frequencies in SHR‐A3(Stim1‐SHR‐B2) compared to SHR‐A3 (8.44±0.59 vs 4.34±0.59%, p=0.01). Restoration in SOCE in the congenic line was also associated with reduced renal T cell infiltration and decreased susceptibility to renal injury (uACR: 7.3±1.5 vs 13.4±2.9 p<0.05) at 40 weeks of age. Urinary renal injury biomarker levels were also lower in the congenic line than in SHR‐A3 rats at 30 weeks of age (KIM1: 0.7±0.07 vs 2.9±0.29 ng/mL, p<0.001; OPN: 6.9±1.4 vs 32.3±8.1 ng/mL, p<0.01; NGAL: 201.9±42.7 vs 1701±264.4 pg/mL, p<0.01). This correlated with lower renal injury scores assessed histologically. BP at 18 weeks, prior to the emergence of renal injury, was comparable between the SHR‐A3(Stim1‐SHR‐B2) and SHR‐A3 lines. Based on our findings, we conclude that defects in SOCE in SHR‐A3 attributable to the Stim1 mutation alter T cell function, reduce Treg numbers and may disturb regulatory interactions between T cells and other immune cells involved in hypertensive renal injury.Support or Funding InformationSupported by NIH‐NIDDK DK069632 and DK114235 to PAD and AHA 17POST33660779 to ISDThis abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Exercise Prevents Formation of Tubular Aggregates in Ageing Skeletal Muscle Fibers of Wild-Type Mice

Tubular aggregates (TAs) are ordered arrays of sarcoplasmic reticulum (SR) tubes found in muscle biopsies from patients affected by TA myopathy (TAM). TAM is a disorder linked to mutations in STIM1 and Orai1, the two main players in store-operated Ca2+ entry (SOCE), a mechanism that allows recovery of extracellular Ca2+ during repetitive muscle activity. TAs are also found in ageing 24 month old C57Bl6 mice in about 50% of fast-twitch EDL fibers (while they are not present in adult 4 month old mice) and, using immunofluorescence, we verified that such TAs are strongly positive for both STIM1 and Orai1. Measurements of contractile force during repetitive stimulation (30 consecutive tetanic pulses of 1 sec at 60 Hz applied every 5 seconds) revealed that 24 month old EDL muscles exhibit a faster decay of contractile force than adult (relative force at the 15th tetanus: 40.7±2.1% vs. 48.3.6±2.8% respectively). In addition, experiments performed in presence or absence of 2.5 mM extracellular Ca2+ suggest that the faster force decay of ageing EDLs is likely caused by their inability to recruit extracellular Ca2+. We finally analyzed EDL muscles from 24 month old mice that exercised voluntarily in wheel cages for 15 months (average of 396±29 km; n=3): formation of TAs was greatly reduced in trained vs. untrained age-matched mice (found in only 7% of fibers), while the capability of EDL muscles to maintain contractile force during repetitive stimulation was significantly rescued (relative force after 15th tetanus: 52.4±4.7%). Our findings suggest that: a) TAs accumulate dysfunctional STIM1 and Orai1; b) exercise limits formation of TAs and improves muscle function during repetitive stimulation, possibly improving the capability of aged muscle to use external Ca2+ via SOCE.

EC.O.2 - The blurred scenario of the new Calcium-related myopathies: clinical, radiological and molecular characterization of CASQ1, STIM1 and ORAI1 myopathies diagnosed in Padova neuromuscular center

The triad is the skeletal muscle substructure responsible for excitation–contraction coupling by regulation of intracellular calcium homeostasis. A rapidly growing list of skeletal myopathies are caused by gene mutations in components of the triad (CASQ1, STIM1 and ORAI1), but the clinical and molecular features of these diseases are still largely unknown. We collected the clinical, radiological, molecular and histopathological features of patients with mutations in CASQ1, STIM1 and ORAI1 genes diagnosed at Padova neuromuscular center. Thirty patients were identified: 18 CASQ1-mutated, all but one sharing the same mutation; 10 STIM1 from a single family; 2 unrelated ORAI1. CASQ1 patients presented in the 5th decade with exercise intolerance, myalgias, rhabdomyolysis and late moderate lower limb proximal weakness. STIM1 patients presented early onset, proximal greater than distal muscle weakness and progression over time. ORAI1 patients presented exercise intolerance and myalgias. Muscle MRI of CASQ1 patients (n = 10) showed a peculiar pattern of fibro-fatty substitution (asymmetric involvement of anterior > posterior thigh and posterior leg). STIM1 patients' MRI (n = 7) showed a homogeneous pattern of substitution with a posterior-to-anterior gradient. Muscle histopathology showed in all p.Asp244Gly CASQ1 patients large optically empty vacuoles, positive to sarcoplasmic reticulum protein. STIM1 and ORAI1 patients showed typical tubular aggregates (TAs) defined histopathological. The patient presenting the novel CASQ1 mutation presented histopathological features resembling more to TA than to vascular CASQ1 pattern (NADH-positive debris). We present a large series of patients with calcium-related myopathy, caused by mutation of CASQ1, STIM1 and ORAI1. The diseases differ for clinical features (exercise intolerance vs. weakness; early vs. late onset; slow vs. rapid evolution) and histopathological pattern (TAs vs. vascular myopathy), but overlaps are frequent.

EC.O.1 - Genetic and histological characterisation of excitation-contraction coupling related structural myopathy cohort

Tubular aggregates and cylindrical spirals appear to be related entities, both arising from the sarcoplasmic reticulum, and sometimes appearing in the same biopsy. Tubular aggregates are inclusions of regular arrays of membrane tubules, while cylindrical spirals are accumulations of spiral lamellae around a central core. Although these have been reported as secondary findings in a range of conditions, some pure genetic forms of myopathy are characterised by the presence of tubular aggregates or cylindrical spirals. Tubular aggregate myopathy typically presents in childhood, causing slowly progressive weakness and cramps. Cylindrical spiral myopathy variably causes weakness, myalgia and exertional cramps, with onset in adulthood. Both diseases are dominant. The scarcity of cases impedes genetic and functional investigation. We have a cohort of two families and 10 isolated probands with tubular aggregate myopathy or cylindrical spirals. They underwent histological investigation, electron microscopy and next-generation DNA sequencing. Tubular aggregate myopathy genetic findings include known ORAI1 mutation c.G325A, p.V109M, and a novel putative STIM1 mutation c.A356G:p.D119G, and other candidate genes. Compound heterozygous TTN variants c.28654C>T, p.Q9552* and c.A15925G, p.I5309V were found in a cylindrical spiral myopathy case. The TTN missense change is predicted to affect splicing. Functional characterisation is ongoing, including mutant cell lines, cDNA analysis and fluorescence-based calcium handling assays. Here, we describe the work to date on characterising this cohort, including the histological and genetic features.

Complex phenotypes associated with STIM1 mutations in both coiled coil and EF-hand domains

Dominant mutations in STIM1 are a cause of three allelic conditions: tubular aggregate myopathy, Stormorken syndrome (a complex phenotype including myopathy, hyposplenism, hypocalcaemia and bleeding diathesis), and a platelet dysfunction disorder, York platelet syndrome. Previous reports have suggested a genotype–phenotype correlation with mutations in the N-terminal EF-hand domain associated with tubular aggregate myopathy, and a common mutation at p.R304W in a coiled coil domain associated with Stormorken syndrome. In this study individuals with STIM1 variants were identified by exome sequencing or STIM1 direct sequencing, and assessed for neuromuscular, haematological and biochemical evidence of the allelic disorders of STIM1. STIM1 mutations were investigated by fibroblast calcium imaging and 3D modelling. Six individuals with STIM1 mutations, including two novel mutations (c.262A>G (p.S88G) and c.911G>A (p.R304Q)), were identified. Extra-neuromuscular symptoms including thrombocytopenia, platelet dysfunction, hypocalcaemia or hyposplenism were present in 5/6 patients with mutations in both the EF-hand and CC domains. 3/6 patients had psychiatric disorders, not previously reported in STIM1 disease. Review of published STIM1 patients (n = 49) confirmed that neuromuscular symptoms are present in most patients. We conclude that the phenotype associated with activating STIM1 mutations frequently includes extra-neuromuscular features such as hypocalcaemia, hypo-/asplenia and platelet dysfunction regardless of mutation domain.

ORAI1 Mutations with Distinct Channel Gating Defects in Tubular Aggregate Myopathy.

Calcium (Ca2+ ) is a physiological key factor, and the precise modulation of free cytosolic Ca2+ levels regulates multiple cellular functions. Store-operated Ca2+ entry (SOCE) is a major mechanism controlling Ca2+ homeostasis, and is mediated by the concerted activity of the Ca2+ sensor STIM1 and the Ca2+ channel ORAI1. Dominant gain-of-function mutations in STIM1 or ORAI1 cause tubular aggregate myopathy (TAM) or Stormorken syndrome, whereas recessive loss-of-function mutations are associated with immunodeficiency. Here, we report the identification and functional characterization of novel ORAI1 mutations in TAM patients. We assess basal activity and SOCE of the mutant ORAI1 channels, and we demonstrate that the G98S and V107M mutations generate constitutively permeable ORAI1 channels, whereas T184M alters the channel permeability only in the presence of STIM1. These data indicate a mutation-dependent pathomechanism and a genotype/phenotype correlation, as the ORAI1 mutations associated with the most severe symptoms induce the strongest functional cellular effect. Examination of the non-muscle features of our patients strongly suggests that TAM and Stormorken syndrome are spectra of the same disease. Overall, our results emphasize the importance of SOCE in skeletal muscle physiology, and provide new insights in the pathomechanisms involving aberrant Ca2+ homeostasis and leading to muscle dysfunction.

Role of STIM1 And Orai1 in the Formation of Tubular Aggregates in Ageing Skeletal Muscle Fibers

Tubular aggregates (TAs), ordered arrays of sarcoplasmic reticulum (SR) tubes, form in ageing fast-twitch fibers of mice, preferentially in males. TAs are also found in biopsies from patients affected by TA Myopathy (TAM), a muscle disorder linked to mutations in STIM1 and Orai1 (proteins involved in store-operated Ca2+ entry, SOCE) We have previously shown using Electron Microscopy (EM) that tubes of TAs appear linked by small bridges, resembling aggregated STIM1 molecules. Here, we compared extensor digitorum longus (EDL) muscles from 2 groups of mice (∼4 months, adults and ≥24 months, aged) to determine: a) presence of STIM1 (and Orai1) in TAs; b) the relative contribution of Ca2+ entry to muscle function during repetitive stimulation in ageing muscle. Immunofluorescence indicates that ageing causes STIM1 (a SR protein), but not Orai (that resides in TTs) to accumulate in TAs. This finding is consistent with: a) EM data showing that TTs are rarely seen in TAs; and b) western-bot analysis showing increased expression levels of both STIM1 splicing variants (arbitrary units: STIM1-short=0.87±0.09 vs. 1.18±0.04; STIM1-long=0.81±0.1 vs. 1.11±0.05, respectively adult vs. aged). During a repetitive stimulation protocol (30 x 1s-60Hz pulses every 5 seconds): i) in 2.5 mM Ca2+ external solution, EDL muscles from aged mice exhibit a decreased capability to maintain contractile (relative force after 10 tetani in adult vs. aged: 61.0±3.2%, and 49.9±2.4%); ii) in Ca2+-free external solution, aged EDL muscles display a lower decay in contractile force (relative force after 10 tetani in adult vs. aged: 41.0±3.1%, and 53.4±1.5%). Our results indicate that TAs a) accumulate STIM1, which is likely visible as electron-dense strands between tubes; b) while contain STIM1, do not functionally contribute to Ca2+ entry during repetitive stimulation.

Cardiovascular and Hemostatic Disorders: SOCE and Ca2+ Handling in Platelet Dysfunction.

Among the Ca2+ entry mechanisms in platelets, store-operated Ca2+ entry (SOCE) plays a prominent role as it is necessary to achieve full activation of platelet functions and replenish intracellular Ca2+ stores. In platelets, as in other non-excitable cells, SOCE has been reported to involve the activation of plasma membrane channels by the ER Ca2+ sensor STIM1. Despite electrophysiological studies are not possible in human platelets, indirect analyses have revealed that the Ca2+-permeable channels involve Orai1 and, most likely, TRPC1 subunits. A relevant role for the latter has not been found in mouse platelets. There is a body of evidence revealing a number of abnormalities in SOCE or in its molecular regulators that result in qualitative platelet disorders and, as a consequence, altered platelet responsiveness upon stimulation with multiple physiological agonists. Platelet SOCE abnormalities include STIM1 and Orai1 mutations. This chapter summarizes the current knowledge in this field, as well as the disorders associated to platelet SOCE dysfunction.

P.357 - The novel STIM1 mutation with tubular aggregate myopathy and its pathogenicity

P.357 - The novel STIM1 mutation with tubular aggregate myopathy and its pathogenicity

Abstract 004: Aberrant CD4 + T Cell Function in Stroke-prone Hypertensive Rats Due to a Mutation in Stim1

The genetic mechanism of end organ injury in hypertension may involve gene variation in genes participating in inflammation and its regulation. We identified a novel truncating mutation in the hypertensive end organ injury-prone spontaneously hypertensive rat (SHR-A3/SHRSP) line affecting the C-terminus of STIM1, a protein involved in the store-operated Ca 2+ entry (SOCE) pathway. The genomes of injury-resistant SHR lines, including SHR-B2 used here, encode the ‘wild-type’ STIM1. SOCE is required by T cells to activate the transcription factor NFAT and regulate T cell proliferation and cytokine production. T cell receptor (TCR) stimulation depletes intracellular Ca 2+ stores, activates the ER Ca 2+ -sensor STIM1 and results in SOCE. We tested the effect of STIM1 mutation on lymphocyte SOCE and found it was dramatically reduced in SHR-A3, but not in SHR-B2 (maximal [Ca 2+ ] i : 150.5±34.9 vs 521.5±42.6 nM, p<0.0001). Flow cytometric analysis of circulating T cell subsets revealed comparable levels of CD4 + and CD8 + T cells in both lines, however circulating CD4 + CD25 + FoxP3 + T regs were reduced in SHR-A3 compared to SHR-B2 (4.34±0.59 vs 7.12±0.33%, p=0.01). TCR-induced lymphocyte proliferation was similar in both SHR-A3 and SHR-B2. T cell cytokine production in response to TCR stimulation was markedly impaired CD4 + T cells from SHR-A3 compared with SHR-B2 (IL-2: 168 ±83.4 vs 1385±377.0 pg/mL, p=0.01; IFNγ: 235±69.5 vs 2119±434.7 pg/mL, p=0.002). IL-2 and IFNγ production was completely inhibited by Pyr6, an inhibitor of Stim1-dependent SOCE. However, circulating levels of IL-2 and IFNγ were not different between the two lines. Based on our findings, we conclude that TCR-mediated effector signaling is impaired due to defective SOCE in SHR-A3 rats. Defects in SOCE in SHR-A3 attributable to STIM1 mutation alter T cell function, reduce T reg numbers and may disturb regulatory interactions between T cells and other immune cells involved in end organ injury.

A need for a systematic genetic evaluation of hereditary polyuric patients.

A need for a systematic genetic evaluation of hereditary polyuric patients.