Adaptation of Candida albicans to specific host environments by gain-of-function mutations in transcription factors.

The yeast Candida albicans is usually a harmless member of the normal microbiota in healthy persons but is also a major fungal pathogen that can colonize and infect almost every human tissue. A successful adaptation to environmental changes encountered in different host niches requires an appropriate regulation of gene expression. The zinc cluster transcription factors are the largest family of transcriptional regulators in C. albicans and are involved in the control of virtually all aspects of its biology. Under certain circumstances, mutations in these transcription factors that alter their activity and the expression of their target genes confer a selective advantage, which results in the emergence of phenotypically altered variants that are better adapted to new environmental challenges. This review describes how gain-of-function mutations in different zinc cluster transcription factors enable C. albicans to overcome antifungal therapy and to successfully establish itself in specific host niches.

Three cases of autoinflammatory disease with novel NLRC4 mutations, and the first mutation reported in the CARD domain of NLRC4 associated with autoinflammatory infantile enterocolitis (AIFEC).

BACKGROUND: Gain of function (GOF) mutations in NOD-like receptor family CARD-containing 4 protein (NLRC4) gene induce a wide spectrum of autoinflammatory phenotypes. Currently, we categorize them into four groups: familial cold autoinflammatory syndrome (FCAS)4, autoinflammatory infantile enterocolitis (AIFEC), NLRC4-macrophage associated syndrome (MAS), and neonatal-onset multisystem inflammatory disease (NOMID). The rarity and complexity of the disease necessitate the description of new cases and a reexamination of our understanding of the condition. CASE PRESENTATIONS: We present three patients with NLRC4-GOF mutations and AIFEC phenotypes. The first patient is an infant girl with periodic fever, seizure, high inflammatory markers, and an episode of macrophage associated syndrome (MAS). History of recurrent fever episodes since childhood was reported in mother and maternal grandmother. A heterozygous mutation was found in CARD domain of NLRC4: c.A91C: p.Asn31His. The second patient is an adolescent boy with periodic fever, diarrhea, aphthous stomatitis, seizure, and central nervous system (CNS) vasculitis. A heterozygous mutation was found in NLRC4 gene: c.1202T > C. p. Val401Ala. The third patient is a child with chronic diarrhea and elevated inflammatory markers. We found a heterozygous mutation in NLRC4 gene: c.390delG: p.S132Afs*21. All mutations have been reported for the first time as NLRC4 mutations associated with autoinflammation. We introduced novel mutations in the CARD domain and between CARD and NBD domain in the first and third cases, respectively. All three children are under remission following treatment. CONCLUSIONS: NLRC4-GOF mutations can be associated with autoinflammation with diverse symptoms. Given the rarity of the disease and the possibility of new mutations being identified, the existence of a phenotype/genotype correlation has yet to be thoroughly investigated. The variety in manifestations and severity spectrum mandates a variety of treatments. Adalimumab has shown favorable outcomes in our AIFEC cases.

Exploring the molecular pathways of the activation process in PPARγ recurrent bladder cancer mutants.

The intricate involvement of Peroxisome Proliferator-Activated Receptor Gamma (PPARγ) in glucose homeostasis and adipogenesis is well-established. However, its role in cancer, particularly luminal bladder cancer, remains debated. The overexpression and activation of PPARγ are implicated in tumorigenesis. Specific gain-of-function mutations (M280I, I290M, and T475M) within the ligand-binding domain of PPARγ are associated with bladder cancer and receptor activation. The underlying molecular pathways prompted by these mutations remain unclear. We employed a dual-basin structure-based model (db-SBM) to explore the conformational dynamics between the inactive and active states of PPARγ and examined the effects of the M280I, I290M, and T475M mutations. Our findings, consistent with the existing literature, reveal heightened ligand-independent transcriptional activity in the I290M and T475M mutants. Both mutants showed enhanced stabilization of the active state compared to the wild-type receptor, with the I290M mutation promoting a specific transition route, making it a prime candidate for further study. Electrostatic analysis identified residues K303 and E488 as pivotal in the I290M activation cascade. Biophysical assays confirmed that disrupting the K303-E488 interaction reduced the thermal stabilization characteristic of the I290M mutation. Our study demonstrates the predictive capabilities of combining simulation and cheminformatics methods, validated by biochemical experiments, to gain insights into molecular activation mechanisms and identify target residues for protein modulation.

Heterozygous expression of a Kcnt1 gain-of-function variant has differential effects on somatostatin- and parvalbumin-expressing cortical GABAergic neurons.

More than 20 recurrent missense gain-of-function (GOF) mutations have been identified in the sodium-activated potassium (KNa) channel gene KCNT1 in patients with severe developmental and epileptic encephalopathies (DEEs), most of which are resistant to current therapies. Defining the neuron types most vulnerable to KCNT1 GOF will advance our understanding of disease mechanisms and provide refined targets for precision therapy efforts. Here, we assessed the effects of heterozygous expression of a Kcnt1 GOF variant (Kcnt1Y777H) on KNa currents and neuronal physiology among cortical glutamatergic and GABAergic neurons in mice, including those expressing vasoactive intestinal polypeptide (VIP), somatostatin (SST), and parvalbumin (PV), to identify and model the pathogenic mechanisms of autosomal dominant KCNT1 GOF variants in DEEs. Although the Kcnt1Y777H variant had no effects on glutamatergic or VIP neuron function, it increased subthreshold KNa currents in both SST and PV neurons but with opposite effects on neuronal output; SST neurons became hypoexcitable with a higher rheobase current and lower action potential (AP) firing frequency, whereas PV neurons became hyperexcitable with a lower rheobase current and higher AP firing frequency. Further neurophysiological and computational modeling experiments showed that the differential effects of the Kcnt1Y777H variant on SST and PV neurons are not likely due to inherent differences in these neuron types, but to an increased persistent sodium current in PV, but not SST, neurons. The Kcnt1Y777H variant also increased excitatory input onto, and chemical and electrical synaptic connectivity between, SST neurons. Together, these data suggest differential pathogenic mechanisms, both direct and compensatory, contribute to disease phenotypes, and provide a salient example of how a pathogenic ion channel variant can cause opposite functional effects in closely related neuron subtypes due to interactions with other ionic conductances.

Parvalbumin interneuron impairment causes synaptic transmission deficits and seizures in SCN8A developmental and epileptic encephalopathy.

SCN8A developmental and epileptic encephalopathy (DEE) is a severe epilepsy syndrome resulting from mutations in the voltage-gated sodium channel Nav1.6, encoded by the gene SCN8A. Nav1.6 is expressed in excitatory and inhibitory neurons, yet previous studies primarily focus on how SCN8A mutations affect excitatory neurons, with limited studies on the importance of inhibitory interneurons. Parvalbumin (PV) interneurons are a prominent inhibitory interneuron subtype that expresses Nav1.6. To assess PV interneuron function within SCN8A DEE, we used 2 mouse models harboring patient-derived SCN8A gain-of-function variants, Scn8aD/+, where the SCN8A variant N1768D is expressed globally, and Scn8aW/+-PV, where the SCN8A variant R1872W is selectively expressed in PV interneurons. Expression of the R1872W SCN8A variant selectively in PV interneurons led to development of spontaneous seizures and seizure-induced death. Electrophysiology studies showed that Scn8aD/+ and Scn8aW/+-PV interneurons were susceptible to depolarization block and exhibited increased persistent sodium current. Evaluation of synaptic connections between PV interneurons and pyramidal cells showed synaptic transmission deficits in Scn8aD/+ and Scn8aW/+-PV interneurons. Together, our findings indicate that PV interneuron failure via depolarization block along with inhibitory synaptic impairment likely elicits an overall inhibitory reduction in SCN8A DEE, leading to unchecked excitation and ultimately resulting in seizures and seizure-induced death.

STAT1 and STAT3 gain of function: clinically heterogenous immune regulatory disorders.

PURPOSE OF REVIEW: The identification of STAT1 gain-of-function (GOF) in 2011 and STAT3 GOF in 2014 has advanced our understanding of the host immunity along the JAK/STAT pathway and allowed targeted treatment approaches. We review the clinical features and pathogenesis of STAT1 and STAT3 GOF and how this has shaped new approaches to therapy. RECENT FINDINGS: STAT1 GOF, initially described in patients with chronic mucocutaneous candidiasis (CMC) and autoimmune thyroid disease, is now recognized to cause early-onset multisystem autoimmunity and a range of infections. STAT3 GOF comprises mostly lymphoproliferation and autoimmunity but also with varying severity, including some with life threatening organ dysfunction. Treatment has evolved along with the understanding of the pathogenesis, with patients now receiving JAK inhibition to block upstream of the STAT defect with good response in autoimmunity and CMC in STAT1 GOF. Blockade of IL-6 signaling has also been used in STAT3 GOF. Hematopoietic cell transplantation had initial poor outcomes, but outcomes are now improving with focus on the control of inflammation pretransplant. SUMMARY: Understanding the pathogenesis of STAT1 and STAT3 GOF has allowed great recent advancements in therapy, but many questions remain as to the best approach to therapy for each patient's clinical presentation as well as the durability of these therapies.

Novel gain-of-function mutants identify a critical region within CFTR membrane-spanning domain 2 controlling cAMP-dependent and ATP-independent channel activation.

CFTR is an anion channel that has evolved from the mold of an ABC transporter. It possesses specific structural features, including a lateral portal between the cytoplasmic extensions of its transmembrane helices TM4 and TM6. This TM4-TM6 portal is lined by basic residues attracting anions from the cytosol towards the intracellular vestibule. Even though a symmetric, open portal is not observed at the level of the TM10/TM12 interface, basic amino acids are also present at this level, exposed to solvent in the vicinity of the regulatory R region, whose phosphorylation enables channel activation. Here, using all-atom molecular dynamics simulations in combination with functional and biochemical assays, we investigate the importance of these basic amino acids (R1158 and R1030), and of a neighboring aromatic amino acid (W846) in the regulation of CFTR activity. Results indicate that mutation of these amino acids globally increased channel activity and enabled channel opening by potentiators without the need to elevate cAMP levels. These effects (i) were observed even when the binding site of the potentiator VX-770 was mutated, revealing a probable independent mechanism, and (ii) were additive to one gain-of-function mutant within the selectivity filter. Taken together, our results indicate that the region of the membrane-spanning domain 2 (MSD2), symmetric to the lateral portal located between MSD1 TM4 and TM6, is a novel critical actor of CFTR regulation.

Somatic RAP1B gain-of-function variant underlies isolated thrombocytopenia and immunodeficiency.

The ubiquitously expressed small GTPase Ras-related protein 1B (RAP1B) acts as a molecular switch that regulates cell signaling, cytoskeletal remodeling, and cell trafficking and activates integrins in platelets and lymphocytes. The residue G12 in the P-loop is required for the RAP1B-GTPase conformational switch. Heterozygous germline RAP1B variants have been described in patients with syndromic thrombocytopenia. However, the causality and pathophysiological impact remained unexplored. We report a boy with neonatal thrombocytopenia, combined immunodeficiency, neutropenia, and monocytopenia caused by a heterozygous de novo single nucleotide substitution, c.35G>A (p.G12E) in RAP1B. We demonstrate that G12E and the previously described G12V and G60R were gain-of-function variants that increased RAP1B activation, talin recruitment, and integrin activation, thereby modifying late responses such as platelet activation, T cell proliferation, and migration. We show that in our patient, G12E was a somatic variant whose allele frequency decreased over time in the peripheral immune compartment, but remained stable in bone marrow cells, suggesting a differential effect in distinct cell populations. Allogeneic hematopoietic stem cell transplantation fully restored the patient's hemato-immunological phenotype. Our findings define monoallelic RAP1B gain-of-function variants as a cause for constitutive immunodeficiency and thrombocytopenia. The phenotypic spectrum ranged from isolated hematological manifestations in our patient with somatic mosaicism to complex syndromic features in patients with reported germline RAP1B variants.

JAK inhibitors to treat STAT3 gain-of-function: a single-center report and literature review.

Objective: The signal transducer and activator of transcription 3 (STAT3) gain-of-function (GOF) syndrome (STAT3-GOF) is an inborn error of immunity (IEI) characterized by diverse manifestations of immune dysregulation that necessitate systemic immunomodulatory treatment. The blockade of the interleukin-6 receptor and/or the inhibition of the Janus kinases has been commonly employed to treat diverse STAT3-GOF-associated manifestations. However, evidence on long-term treatment outcome, especially in the case of adult patients, is scarce. Methods: Clinical data, including laboratory findings and medical imaging, were collected from all seven patients, diagnosed with STAT3-GOF, who have been treated at the Hannover University School, focusing on those who received a Janus kinase (JAK) inhibitor (JAKi). Previously published cases of STAT3-GOF patients who received a JAKi were evaluated, focusing on reported treatment efficacy with respect to diverse STAT3-GOF-associated manifestations of immune dysregulation and safety. Results: Five out of seven patients diagnosed with STAT3-GOF were treated with a JAKi, each for a different indication. Including these patients, outcomes of JAKi treatment have been reported for a total of 41 patients. Treatment with a JAKi led to improvement of diverse autoimmune, inflammatory, or lymphoproliferative manifestations of STAT3-GOF and a therapeutic benefit could be documented for all except two patients. Considering all reported manifestations of immune dysregulation in each patient, complete remission was achieved in 10/41 (24.4%) treated patients. Conclusions: JAKi treatment improved diverse manifestations of immune dysregulation in the majority of STAT3-GOF patients, representing a promising therapeutic approach. Long-term follow-up data are needed to evaluate possible risks of prolonged treatment with a JAKi.

Targeted protein relocalization via protein transport coupling.

Subcellular protein localization regulates protein function and can be corrupted in cancers1 and neurodegenerative diseases2,3. The rewiring of localization to address disease-driving phenotypes would be an attractive targeted therapeutic approach. Molecules that harness the trafficking of a shuttle protein to control the subcellular localization of a target protein could enforce targeted protein relocalization and rewire the interactome. Here we identify a collection of shuttle proteins with potent ligands amenable to incorporation into targeted relocalization-activating molecules (TRAMs), and use these to relocalize endogenous proteins. Using a custom imaging analysis pipeline, we show that protein steady-state localization can be modulated through molecular coupling to shuttle proteins containing sufficiently strong localization sequences and expressed in the necessary abundance. We analyse the TRAM-induced relocalization of different proteins and then use nuclear hormone receptors as shuttles to redistribute disease-driving mutant proteins such as SMARCB1Q318X, TDP43ΔNLS and FUSR495X. TRAM-mediated relocalization of FUSR495X to the nucleus from the cytoplasm correlated with a reduction in the number of stress granules in a model of cellular stress. With methionyl aminopeptidase 2 and poly(ADP-ribose) polymerase 1 as endogenous cytoplasmic and nuclear shuttles, respectively, we demonstrate relocalization of endogenous PRMT9, SOS1 and FKBP12. Small-molecule-mediated redistribution of nicotinamide nucleotide adenylyltransferase 1 from nuclei to axons in primary neurons was able to slow axonal degeneration and pharmacologically mimic the genetic WldS gain-of-function phenotype in mice resistant to certain types of neurodegeneration4. The concept of targeted protein relocalization could therefore inspire approaches for treating disease through interactome rewiring.

An Alternatively Spliced Gain-of-Function NT5C2 Isoform Contributes to Chemoresistance in Acute Lymphoblastic Leukemia.

Relapsed or refractory B-cell acute lymphoblastic leukemia (B-ALL) is a major cause of pediatric cancer-related deaths. Relapse-specific mutations do not account for all chemotherapy failures in B-ALL patients, suggesting additional mechanisms of resistance. By mining RNA sequencing datasets of paired diagnostic/relapse pediatric B-ALL samples, we discovered pervasive alternative splicing (AS) patterns linked to relapse and affecting drivers of resistance to glucocorticoids, antifolates, and thiopurines. Most splicing variations represented cassette exon skipping, "poison" exon inclusion, and intron retention, phenocopying well-documented loss-of-function mutations. In contrast, relapse-associated AS of NT5C2 mRNA yielded an isoform with the functionally uncharacterized in-frame exon 6a. Incorporation of the 8-amino acid sequence SQVAVQKR into this enzyme created a putative phosphorylation site and resulted in elevated nucleosidase activity, which is a known consequence of gain-of-function mutations in NT5C2 and a common determinant of 6-mercaptopurine resistance. Consistent with this finding, NT5C2ex6a and the R238W hotspot variant conferred comparable levels of resistance to 6-mercaptopurine in B-ALL cells both in vitro and in vivo. Furthermore, both NT5C2ex6a and the R238W variant induced collateral sensitivity to the inosine monophosphate dehydrogenase inhibitor mizoribine. These results ascribe to splicing perturbations an important role in chemotherapy resistance in relapsed B-ALL and suggest that inosine monophosphate dehydrogenase inhibitors, including the commonly used immunosuppressive agent mycophenolate mofetil, could be a valuable therapeutic option for treating thiopurine-resistant leukemias. Significance: Alternative splicing is a potent mechanism of acquired drug resistance in relapsed/refractory acute lymphoblastic leukemias that has diagnostic and therapeutic implications for patients who lack mutations in known chemoresistance genes.

RAC2 gain-of-function variants causing inborn error of immunity drive NLRP3 inflammasome activation.

A growing number of patients presenting severe combined immunodeficiencies attributed to monoallelic RAC2 variants have been identified. The expression of the RHO GTPase RAC2 is restricted to the hematopoietic lineage. RAC2 variants have been described to cause immunodeficiencies associated with high frequency of infection, leukopenia, and autoinflammatory features. Here, we show that specific RAC2 activating mutations induce the NLRP3 inflammasome activation leading to the secretion of IL-1ß and IL-18 from macrophages. This activation depends on the activation state of the RAC2 variant and is mediated by the downstream kinase PAK1. Inhibiting the RAC2-PAK1-NLRP3 inflammasome pathway might be considered as a potential treatment for these patients.

De novo mutations promote inflammation in children with STAT3 gain-of-function syndrome by affecting IL-1ß expression.

STAT3 gain-of-function syndrome, characterized by early-onset autoimmunity and primary immune regulatory disorder, remains poorly understood in terms of its immunological mechanisms. We employed whole-genome sequencing of familial trios to elucidate the pivotal role of de novo mutations in genetic diseases. We identified 37 high-risk pathogenic loci affecting 23 genes, including a novel STAT3 c.508G>A mutation. We also observed significant down-regulation of pathogenic genes in affected individuals, potentially associated with inflammatory responses regulated by PTPN14 via miR378c. These findings enhance our understanding of the pathogenesis of STAT3 gain-of-function syndrome and suggest potential therapeutic strategies. Notably, combined JAK inhibitors and IL-6R antagonists may offer promising treatment avenues for mitigating the severity of STAT3 gain-of-function syndrome.

Mitochondrial Ca2+-coupled generation of reactive oxygen species, peroxynitrite formation, and endothelial dysfunction in Cantú syndrome.

Cantú syndrome is a multisystem disorder caused by gain-of-function (GOF) mutations in KCNJ8 and ABCC9, the genes encoding the pore-forming inward rectifier Kir6.1 and regulatory sulfonylurea receptor SUR2B subunits, respectively, of vascular ATP-sensitive K+ (KATP) channels. In this study, we investigated changes in the vascular endothelium in mice in which Cantú syndrome-associated Kcnj8 or Abcc9 mutations were knocked in to the endogenous loci. We found that endothelium-dependent dilation was impaired in small mesenteric arteries from Cantú mice. Loss of endothelium-dependent vasodilation led to increased vasoconstriction in response to intraluminal pressure or treatment with the adrenergic receptor agonist phenylephrine. We also found that either KATP GOF or acute activation of KATP channels with pinacidil increased the amplitude and frequency of wave-like Ca2+ events generated in the endothelium in response to the vasodilator agonist carbachol. Increased cytosolic Ca2+ signaling activity in arterial endothelial cells from Cantú mice was associated with elevated mitochondrial [Ca2+] and enhanced reactive oxygen species (ROS) and peroxynitrite levels. Scavenging intracellular or mitochondrial ROS restored endothelium-dependent vasodilation in the arteries of mice with KATP GOF mutations. We conclude that mitochondrial Ca2+ overload and ROS generation, which subsequently leads to nitric oxide consumption and peroxynitrite formation, cause endothelial dysfunction in mice with Cantú syndrome.

Immunophenotyping and Therapeutic Insights from Chronic Mucocutaneous Candidiasis Cases with STAT1 Gain-of-Function Mutations.

PURPOSE: Heterozygous STAT1 Gain-of-Function (GOF) mutations are the most common cause of chronic mucocutaneous candidiasis (CMC) among Inborn Errors of Immunity. Clinically, these mutations manifest as a broad spectrum of immune dysregulation, including autoimmune diseases, vascular disorders, and malignancies. The pathogenic mechanisms of immune dysregulation and its impact on immune cells are not yet fully understood. In treatment, JAK inhibitors have shown therapeutic effectiveness in some patients. METHODS: We analyzed clinical presentations, cellular phenotypes, and functional impacts in five Taiwanese patients with STAT1 GOF. RESULTS: We identified two novel GOF mutations in 5 patients from 2 Taiwanese families, presenting with symptoms of CMC, late-onset rosacea, and autoimmunity. The enhanced phosphorylation and delayed dephosphorylation were displayed by the patients' cells. There are alterations in both innate and adaptive immune cells, including expansion of CD38+HLADR +CD8+ T cells, a skewed activated Tfh cells toward Th1, reduction of memory, marginal zone and anergic B cells, all main functional dendritic cell lineages, and a reduction in classical monocyte. Baricitinib showed therapeutic effectiveness without side effects. CONCLUSION: Our study provides the first comprehensive clinical and molecular characteristics in STAT1 GOF patient in Taiwan and highlights the dysregulated T and B cells subsets which may hinge the autoimmunity in STAT1 GOF patients. It also demonstrated the therapeutic safety and efficacy of baricitinib in pediatric patient. Further research is needed to delineate how the aberrant STAT1 signaling lead to the changes in cellular populations as well as to better link to the clinical manifestations of the disease.

Proteome-scale prediction of molecular mechanisms underlying dominant genetic diseases.

Many dominant genetic disorders result from protein-altering mutations, acting primarily through dominant-negative (DN), gain-of-function (GOF), and loss-of-function (LOF) mechanisms. Deciphering the mechanisms by which dominant diseases exert their effects is often experimentally challenging and resource intensive, but is essential for developing appropriate therapeutic approaches. Diseases that arise via a LOF mechanism are more amenable to be treated by conventional gene therapy, whereas DN and GOF mechanisms may require gene editing or targeting by small molecules. Moreover, pathogenic missense mutations that act via DN and GOF mechanisms are more difficult to identify than those that act via LOF using nearly all currently available variant effect predictors. Here, we introduce a tripartite statistical model made up of support vector machine binary classifiers trained to predict whether human protein coding genes are likely to be associated with DN, GOF, or LOF molecular disease mechanisms. We test the utility of the predictions by examining biologically and clinically meaningful properties known to be associated with the mechanisms. Our results strongly support that the models are able to generalise on unseen data and offer insight into the functional attributes of proteins associated with different mechanisms. We hope that our predictions will serve as a springboard for researchers studying novel variants and those of uncertain clinical significance, guiding variant interpretation strategies and experimental characterisation. Predictions for the human UniProt reference proteome are available at https://osf.io/z4dcp/.

Tumor-derived RHOA mutants interact with effectors in the GDP-bound state.

RHOA mutations are found at diverse residues in various cancer types, implying mutation- and cell-specific mechanisms of tumorigenesis. Here, we focus on the underlying mechanisms of two gain-of-function RHOA mutations, A161P and A161V, identified in adult T-cell leukemia/lymphoma. We find that RHOAA161P and RHOAA161V are both fast-cycling mutants with increased guanine nucleotide dissociation/association rates compared with RHOAWT and show reduced GTP-hydrolysis activity. Crystal structures reveal an altered nucleotide association in RHOAA161P and an open nucleotide pocket in RHOAA161V. Both mutations perturb the dynamic properties of RHOA switch regions and shift the conformational landscape important for RHOA activity, as shown by 31P NMR and molecular dynamics simulations. Interestingly, RHOAA161P and RHOAA161V can interact with effectors in the GDP-bound state. 1H-15N HSQC NMR spectra support the existence of an active population in RHOAA161V-GDP. The distinct interaction mechanisms resulting from the mutations likely favor an RHOAWT-like "ON" conformation, endowing GDP-bound state effector binding activity.

Distinct neurodevelopmental and epileptic phenotypes associated with gain- and loss-of-function GABRB2 variants.

BACKGROUND: Variants in GABRB2, encoding the ß2 subunit of the γ-aminobutyric acid type A (GABAA) receptor, can result in a diverse range of conditions, ranging from febrile seizures to severe developmental and epileptic encephalopathies. However, the mechanisms underlying the risk of developing milder vs more severe forms of disorder remain unclear. In this study, we conducted a comprehensive genotype-phenotype correlation analysis in a cohort of individuals with GABRB2 variants. METHODS: Genetic and electroclinical data of 42 individuals harbouring 26 different GABRB2 variants were collected and accompanied by electrophysiological analysis of the effects of the variants on receptor function. FINDINGS: Electrophysiological assessments of α1ß2γ2 receptors revealed that 25/26 variants caused dysfunction to core receptor properties such as GABA sensitivity. Of these, 17 resulted in gain-of-function (GOF) while eight yielded loss-of-function traits (LOF). Genotype-phenotype correlation analysis revealed that individuals harbouring GOF variants suffered from severe developmental delay/intellectual disability (DD/ID, 74%), movement disorders such as dystonia or dyskinesia (59%), microcephaly (50%) and high risk of early mortality (26%). Conversely, LOF variants were associated with milder disease manifestations. Individuals with these variants typically exhibited fever-triggered seizures (92%), milder degrees of DD/ID (85%), and maintained ambulatory function (85%). Notably, severe movement disorders or microcephaly were not reported in individuals with loss-of-function variants. INTERPRETATION: The data reveals that genetic variants in GABRB2 can lead to both gain and loss-of-function, and this divergence is correlated with distinct disease manifestations. Utilising this information, we constructed a diagnostic flowchart that aids in predicting the pathogenicity of recently identified variants by considering clinical phenotypes. FUNDING: This work was funded by the Australian National Health & Medical Research Council, the Novo Nordisk Foundation and The Lundbeck Foundation.

A patient-based murine model recapitulates human STAT3 gain-of-function syndrome.

STAT3 gain-of-function (GOF) variants results in a heterogeneous clinical syndrome characterized by early onset immunodeficiency, multi-organ autoimmunity, and lymphoproliferation. While 191 documented cases with STAT3 GOF variants have been reported, the impact of individual variants on immune regulation and the broad clinical spectrum remains unclear. We developed a Stat3p.L387R mouse model, mirroring a variant identified in a family exhibiting common STAT3 GOF symptoms, and rare phenotypes including pulmonary hypertension and retinal vasculitis. In vitro experiments revealed increased STAT3 phosphorylation, nuclear migration, and DNA binding of the variant. Our Stat3p.L387R model displayed similar traits from previous Stat3GOF strains, such as splenomegaly and lymphadenopathy. Notably, Stat3p.L387R/+ mice exhibited heightened embryonic lethality compared to prior Stat3GOF/+ models and ocular abnormalities were observed. This research underscores the variant-specific pathology in Stat3p.L387R/+ mice, highlighting the ability to recapitulate human STAT3 GOF syndrome in patient-specific transgenic murine models. Additionally, such models could facilitate tailored treatment development.