Background
Biallelic loss‐of‐function mutations in CARMIL2 cause combined immunodeficiency associated with dermatitis, inflammatory bowel disease (IBD), and EBV‐related smooth muscle tumors. Clinical ...and immunological characterizations of the disease with long‐term follow‐up and treatment options have not been previously reported in large cohorts. We sought to determine the clinical and immunological features of CARMIL2 deficiency and long‐term efficacy of treatment in controlling different disease manifestations.
Methods
The presenting phenotypes, long‐term outcomes, and treatment responses were evaluated prospectively in 15 CARMIL2‐deficient patients, including 13 novel cases. Lymphocyte subpopulations, protein expression, regulatory T (Treg), and circulating T follicular helper (cTFH) cells were analyzed. Three‐dimensional (3D) migration assay was performed to determine T‐cell shape.
Results
Mean age at disease onset was 38 ± 23 months. Main clinical features were skin manifestations (n = 14, 93%), failure to thrive (n = 10, 67%), recurrent infections (n = 10, 67%), allergic symptoms (n = 8, 53%), chronic diarrhea (n = 4, 27%), and EBV‐related leiomyoma (n = 2, 13%). Skin manifestations ranged from atopic and seborrheic dermatitis to psoriasiform rash. Patients had reduced proportions of memory CD4+ T cells, Treg, and cTFH cells. Memory B and NK cells were also decreased. CARMIL2‐deficient T cells exhibited reduced T‐cell proliferation and cytokine production following CD28 co‐stimulation and normal morphology when migrating in a high‐density 3D collagen gel matrix. IBD was the most severe clinical manifestation, leading to growth retardation, requiring multiple interventional treatments. All patients were alive with a median follow‐up of 10.8 years (range: 3–17 years).
Conclusion
This cohort provides clinical and immunological features and long‐term follow‐up of different manifestations of CARMIL2 deficiency.
CARMIL2 deficiency results in variable phenotypes encompassing skin disease, combined immune deficiency, early‐onset inflammatory bowel disease, and allergic manifestations. Patients have impaired differentiation of mainly CD4+ T cells, resulting in elevated naïve CD4+ T cells. Gastrointestinal involvement and EBV‐related smooth muscle tumors are major determinants of poor prognosis.
Abbreviations: CARMA, CARD‐Containing MAGUK Protein; CARMIL, capping protein regulator and myosin 1 linker 2; CBR, C‐terminal domain including a capping protein binding region; CP, capping protein; HD, homodimerization domain; EBV, Epstein‐Barr virus; LRR, leucine‐rich repeat; NF‐κB, nuclear factor kappa B; PH, pleckstrin homolog; PKCθ, protein kinase C theta; PRR, proline‐rich region; SMT, smooth muscle tumor
Background
Autosomal‐recessive mutations in SPEG (striated muscle preferentially expressed protein kinase) have been linked to centronuclear myopathy with or without dilated cardiomyopathy (CNM5). ...Loss of SPEG is associated with defective triad formation, abnormal excitation–contraction coupling, calcium mishandling and disruption of the focal adhesion complex in skeletal muscles. To elucidate the underlying molecular pathways, we have utilized multi‐omics tools and analysis to obtain a comprehensive view of the complex biological processes and molecular functions.
Methods
Skeletal muscles from 2‐month‐old SPEG‐deficient (Speg‐CKO) and wild‐type (WT) mice were used for RNA sequencing (n = 4 per genotype) to profile transcriptomics and mass spectrometry (n = 4 for WT; n = 3 for Speg‐CKO mice) to profile proteomics and phosphoproteomics. In addition, interactomics was performed using the SPEG antibody on pooled muscle lysates (quadriceps, gastrocnemius and triceps) from WT and Speg‐CKO mice. Based on the multi‐omics results, we performed quantitative real‐time PCR, co‐immunoprecipitation and immunoblot to verify the findings.
Results
We identified that SPEG interacts with myospryn complex proteins CMYA5, FSD2 and RyR1, which are critical for triad formation, and that SPEG deficiency results in myospryn complex abnormalities (protein levels decreased to 22 ± 3% for CMYA5 P < 0.05 and 18 ± 3% for FSD2 P < 0.01). Furthermore, SPEG phosphorylates RyR1 at S2902 (phosphorylation level decreased to 55 ± 15% at S2902 in Speg‐CKO mice; P < 0.05), and its loss affects JPH2 phosphorylation at multiple sites (increased phosphorylation at T161 1.90 ± 0.24‐fold, S162 1.61 ± 0.37‐fold and S165 1.66 ± 0.13‐fold; decreased phosphorylation at S228 and S231 39 ± 6%, S234 50 ± 12%, S593 48 ± 3% and S613 66 ± 10%; P < 0.05 for S162 and P < 0.01 for other sites). On analysing the transcriptome, the most dysregulated pathways affected by SPEG deficiency included extracellular matrix–receptor interaction (P < 1e−15) and peroxisome proliferator‐activated receptor signalling (P < 9e−14).
Conclusions
We have elucidated the critical role of SPEG in the triad as it works closely with myospryn complex proteins (CMYA5, FSD2 and RyR1), it regulates phosphorylation levels of various residues in JPH2 and S2902 in RyR1, and its deficiency is associated with dysregulation of several pathways. The study identifies unique SPEG‐interacting proteins and their phosphorylation functions and emphasizes the importance of using a multi‐omics approach to comprehensively evaluate the molecular function of proteins involved in various genetic disorders.
Advances in bioinformatic tools paired with the ongoing accumulation of genetic knowledge and periodic reanalysis of genomic sequencing data have led to an improvement in genetic diagnostic rates. ...Candidate gene variants (CGVs) identified during sequencing or on reanalysis but not yet implicated in human disease or associated with a phenotypically distinct condition are often not revisited, leading to missed diagnostic opportunities. Here, we revisited 33 such CGVs from our previously published study and determined that 16 of them are indeed disease‐causing (novel or phenotype expansion) since their identification. These results emphasize the need to focus on previously identified CGVs during sequencing or reanalysis and the importance of sharing that information with researchers around the world, including relevant functional analysis to establish disease causality.
Septins play key roles in mammalian cell division and cytokinesis but have not previously been implicated in a germline human disorder. A male infant with severe neutropenia and progressive ...dysmyelopoiesis with tetraploid myeloid precursors was identified. No known genetic etiologies for neutropenia or bone marrow failure were found. However, next‐generation sequencing of germline samples from the patient revealed a novel, de novo germline stop‐loss mutation in the X‐linked gene SEPT6 that resulted in reduced SEPT6 staining in bone marrow granulocyte precursors and megakaryocytes. Patient skin fibroblast‐derived induced pluripotent stem cells (iPSCs) produced reduced myeloid colonies, particularly of the granulocyte lineage. CRISPR/Cas9 knock‐in of the patient's mutation or complete knock‐out of SEPT6 was not tolerated in non‐patient‐derived iPSCs or human myeloid cell lines, but SEPT6 knock‐out was successful in an erythroid cell line and resulting clones revealed a propensity to multinucleation. In silico analysis predicts that the mutated protein hinders the dimerization of SEPT6 coiled‐coils in both parallel and antiparallel arrangements, which could in turn impair filament formation. These data demonstrate a critical role for SEPT6 in chromosomal segregation in myeloid progenitors that can account for the unusual predisposition to aneuploidy and dysmyelopoiesis.
KIF26B is a member of the kinesin superfamily with evolutionarily conserved functions in controlling aspects of embryogenesis, including the development of the nervous system, though its function is ...incompletely understood. We describe an infant with progressive microcephaly, pontocerebellar hypoplasia, and arthrogryposis secondary to the involvement of anterior horn cells and ventral (motor) nerves. We performed whole exome sequencing on the trio and identified a de novo KIF26B missense variant, p.Gly546Ser, in the proband. This variant alters a highly conserved amino acid residue that is part of the phosphate-binding loop motif and motor-like domain and is deemed pathogenic by several in silico methods. Functional analysis of the variant protein in cultured cells revealed a reduction in the KIF26B protein's ability to promote cell adhesion, a defect that potentially contributes to its pathogenicity. Overall, KIF26B may play a critical role in the brain development and, when mutated, cause pontocerebellar hypoplasia with arthrogryposis.
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Inborn errors of immunity have been implicated in causing immune dysregulation, including allergic diseases. STAT6 is a key regulator of allergic responses.
This study sought to ...characterize a novel gain-of-function STAT6 mutation identified in a child with severe allergic manifestations.
Whole-exome and targeted gene sequencing, lymphocyte characterization, and molecular and functional analyses of mutated STAT6 were performed.
This study reports a child with a missense mutation in the DNA binding domain of STAT6 (c.1114G>A, p.E372K) who presented with severe atopic dermatitis, eosinophilia, and elevated IgE. Naive lymphocytes from the affected patient displayed increased TH2- and suppressed TH1- and TH17-cell responses. The mutation augmented both basal and cytokine-induced STAT6 phosphorylation without affecting dephosphorylation kinetics. Treatment with the Janus kinase 1/2 inhibitor ruxolitinib reversed STAT6 hyperresponsiveness to IL-4, normalized TH1 and TH17 cells, suppressed the eosinophilia, and improved the patient’s atopic dermatitis.
This study identified a novel inborn error of immunity due to a STAT6 gain-of-function mutation that gave rise to severe allergic dysregulation. Janus kinase inhibitor therapy could represent an effective targeted treatment for this disorder.
OBJECTIVE:To identify causative genes for centronuclear myopathies (CNM), a heterogeneous group of rare inherited muscle disorders that often present in infancy or early life with weakness and ...hypotonia, using next-generation sequencing of whole exomes and genomes.
METHODS:Whole-exome or -genome sequencing was performed in a cohort of 29 unrelated patients with clinicopathologic diagnoses of CNM or related myopathy depleted for cases with mutations of MTM1, DNM2, and BIN1. Immunofluorescence analyses on muscle biopsies, splicing assays, and gel electrophoresis of patient muscle proteins were performed to determine the molecular consequences of mutations of interest.
RESULTS:Autosomal recessive compound heterozygous truncating mutations of the titin gene, TTN, were identified in 5 individuals. Biochemical analyses demonstrated increased titin degradation and truncated titin proteins in patient muscles, establishing the impact of the mutations.
CONCLUSIONS:Our study identifies truncating TTN mutations as a cause of congenital myopathy that is reported as CNM. Unlike the classic CNM genes that are all involved in excitation-contraction coupling at the triad, TTN encodes the giant sarcomeric protein titin, which forms a myofibrillar backbone for the components of the contractile machinery. This study expands the phenotypic spectrum associated with TTN mutations and indicates that TTN mutation analysis should be considered in cases of possible CNM without mutations in the classic CNM genes.
Clinical exome sequencing (CES) is increasingly being utilized; however, a large proportion of patients remain undiagnosed, creating a need for a systematic approach to increase the diagnostic yield. ...We have reanalyzed CES data for a clinically heterogeneous cohort of 102 probands with likely Mendelian conditions, including 74 negative cases and 28 cases with candidate variants, but reanalysis requested by clinicians. Reanalysis was performed by an interdisciplinary team using a validated custom-built pipeline, "Variant Explorer Pipeline" (VExP). This reanalysis approach and results were compared with existing literature. Reanalysis of candidate variants from CES in 28 cases revealed 1 interpretation that needed to be reclassified. A confirmed or potential genetic diagnosis was identified in 24 of 75 CES-negative/reclassified cases (32.0%), including variants in known disease-causing genes (n = 6) or candidate genes (n = 18). This yield was higher compared with similar studies demonstrating the utility of this approach. In summary, reanalysis of negative CES in a research setting enhances diagnostic yield by about a third. This study suggests the need for comprehensive, continued reanalysis of exome data when molecular diagnosis is elusive.
Current vaccine development disregards human immune ontogeny, relying on animal models to select vaccine candidates targeting human infants, who are at greatest risk of infection worldwide, and ...receive the largest number of vaccines. To help accelerate and de-risk development of early-life effective immunization, we engineered a human age-specific microphysiologic vascular-interstitial interphase, suitable for pre-clinical modeling of distinct age-targeted immunity
. Our Tissue Constructs (TCs) enable autonomous extravasation of monocytes that undergo rapid self-directed differentiation into migratory Dendritic Cells (DCs) in response to adjuvants and licensed vaccines such as Bacille Calmette-Guérin (BCG) or Hepatitis B virus Vaccine (HBV). TCs contain a confluent human endothelium grown atop a tri-dimensional human extracellular matrix substrate, employ human age-specific monocytes and autologous non heat-treated plasma, and avoid the use of xenogenic materials and exogenous cytokines. Vaccine-pulsed TCs autonomously generated DCs that induced single-antigen recall responses from autologous naïve and memory CD4+ T lymphocytes, matching study participant immune-status, including BCG responses paralleling donor PPD status, BCG-induced adenosine deaminase (ADA) activity paralleling infant cohorts
, and multi-dose HBV antigen-specific responses as demonstrated by lymphoproliferation and TCR sequencing. Overall, our microphysiologic culture method reproduced age- and antigen-specific recall responses to BCG and HBV immunization, closely resembling those observed after a birth immunization of human cohorts
, offering for the first time a new approach to early pre-clinical selection of effective age-targeted vaccine candidates.
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