Case Report: Aplastic anemia related to a novel CTLA4 variant.

A 20-year-old male patient with a history of celiac disease came to medical attention after developing profound fatigue and pancytopenia. Evaluation demonstrated pan-hypogammaglobulinemia. There was no history of significant clinical infections. Bone marrow biopsy confirmed hypocellular marrow consistent with aplastic anemia. Oncologic and hematologic evaluations were unremarkable for iron deficiency, paroxysmal nocturnal hemoglobinuria, myelodysplastic syndromes, T-cell clonality, and leukemia. A next generation genetic sequencing immunodeficiency panel revealed a heterozygous variant of uncertain significance in CTLA4 c.385T >A, p.Cys129Ser (C129S). Cytotoxic T-lymphocyte-associated protein 4 (CTLA-4) is an inhibitory receptor important in maintaining immunologic homeostasis. To determine the functional significance of the C129S variant, additional testing was pursued to assess for diminished protein expression, as described in other pathogenic CTLA4 variants. The results demonstrated severely impaired CTLA-4 expression and CD80 transendocytosis, consistent with other variants causing CTLA-4 haploinsufficiency. He was initially treated with IVIG and cyclosporine, and became transfusion independent for few months, but relapsed. Treatment with CTLA-4-Ig fusion protein (abatacept) was considered, however the patient opted for definitive therapy through reduced-intensity haploidentical hematopoietic stem cell transplant, which was curative.

Functional overlap of inborn errors of immunity and metabolism genes defines T cell metabolic vulnerabilities.

Inborn errors of metabolism (IEMs) and immunity (IEIs) are Mendelian diseases in which complex phenotypes and patient rarity have limited clinical understanding. Whereas few genes have been annotated as contributing to both IEMs and IEIs, immunometabolic demands suggested greater functional overlap. Here, CRISPR screens tested IEM genes for immunologic roles and IEI genes for metabolic effects and found considerable previously unappreciated crossover. Analysis of IEMs showed that N-linked glycosylation and the hexosamine pathway enzyme Gfpt1 are critical for T cell expansion and function. Further, T helper (TH1) cells synthesized uridine diphosphate N-acetylglucosamine more rapidly and were more impaired by Gfpt1 deficiency than TH17 cells. Screening IEI genes found that Bcl11b promotes the CD4 T cell mitochondrial activity and Mcl1 expression necessary to prevent metabolic stress. Thus, a high degree of functional overlap exists between IEM and IEI genes, and immunometabolic mechanisms may underlie a previously underappreciated intersection of these disorders.

Molecular and clinical characterization of a founder mutation causing G6PC3 deficiency.

G6PC3 deficiency is a monogenic immunometabolic disorder that causes syndromic congenital neutropenia. Patients display heterogeneous extra-hematological manifestations, contributing to delayed diagnosis. Here, we investigated the origin and functional consequence of the G6PC3 c.210delC variant found in patients of Mexican origin. Based on the shared haplotypes amongst carriers of the c.210delC mutation, we estimated that this variant originated from a founder effect in a common ancestor. Furthermore, by ancestry analysis, we concluded that it originated in the indigenous Mexican population. At the protein level, we showed that this frameshift mutation leads to an aberrant protein expression in overexpression and patient-derived cells. G6PC3 pathology is driven by the intracellular accumulation of the metabolite 1,5-anhydroglucitol-6-phosphate (1,5-AG6P) that inhibits glycolysis. We characterized how the variant c.210delC impacts glycolysis by performing extracellular flux assays on patient-derived cells. When treated with 1,5-anhydroglucitol (1,5-AG), the precursor to 1,5-AG6P, patient-derived cells exhibited markedly reduced engagement of glycolysis. Finally, we compared the clinical presentation of patients with the mutation c.210delC and all other G6PC3 deficient patients reported in the literature to date, and we found that c.210delC carriers display all prominent clinical features observed in prior G6PC3 deficient patients. In conclusion, G6PC3 c.210delC is a loss-of-function mutation that arose from a founder effect in the indigenous Mexican population. These findings may facilitate the diagnosis of additional patients in this geographical area. Moreover, the in vitro 1,5-AG-dependent functional assay used in our study could be employed to assess the pathogenicity of additional G6PC3 variants.

Immune dysregulation due to bi-allelic mutation of the actin remodeling protein DIAPH1.

Children with severe inflammatory diseases are challenging to diagnose and treat, and the etiology of disease often remains unexplained. Here we present DIAPH1 deficiency as an unexpected genetic finding in a child with fatal inflammatory bowel disease who also displayed complex neurological and developmental phenotypes. Bi-allelic mutations of DIAPH1 were first described in patients with a severe neurological phenotype including microcephaly, intellectual disability, seizures, and blindness. Recent findings have expanded the clinical phenotype of DIAPH1 deficiency to include severe susceptibility to infections, placing this monogenic disease amongst the etiologies of inborn errors of immunity. Immune phenotypes in DIAPH1 deficiency are largely driven aberrant lymphocyte activation, particularly the failure to form an effective immune synapse in T cells. We present the case of a child with a novel homozygous deletion in DIAPH1, leading to a premature truncation in the Lasso domain of the protein. Unlike other cases of DIAPH1 deficiency, this patient did not have seizures or lung infections. Her major immune-related clinical symptoms were inflammation and enteropathy, diarrhea and failure to thrive. This patient did not show T or B cell lymphopenia but did have dramatically reduced naïve CD4+ and CD8+ T cells, expanded CD4-CD8- T cells, and elevated IgE. Similar to other cases of DIAPH1 deficiency, this patient had non-hematological phenotypes including microcephaly, developmental delay, and impaired vision. This patient's symptSoms of immune dysregulation were not successfully controlled and were ultimately fatal. This case expands the clinical spectrum of DIAPH1 deficiency and reveals that autoimmune or inflammatory enteropathy may be the most prominent immunological manifestation of disease.

Molecular and clinical characterization of a founder mutation causing G6PC3 deficiency.

Background: G6PC3 deficiency is a rare genetic disorder that causes syndromic congenital neutropenia. It is driven by the intracellular accumulation of a metabolite named 1,5-anhydroglucitol-6-phosphate (1,5-AG6P) that inhibits glycolysis. Patients display heterogeneous extra-hematological manifestations, contributing to delayed diagnosis. Objective: The G6PC3 c.210delC variant has been identified in patients of Mexican origin. We set out to study the origin and functional consequence of this mutation. Furthermore, we sought to characterize the clinical phenotypes caused by it. Methods: Using whole-genome sequencing data, we conducted haplotype analysis to estimate the age of this allele and traced its ancestral origin. We examined how this mutation affected G6PC3 protein expression and performed extracellular flux assays on patient-derived cells to characterize how this mutation impacts glycolysis. Finally, we compared the clinical presentations of patients with the c.210delC mutation relative to other G6PC3 deficient patients published to date. Results: Based on the length of haplotypes shared amongst ten carriers of the G6PC3 c.210delC mutation, we estimated that this variant originated in a common ancestor of indigenous American origin. The mutation causes a frameshift that introduces a premature stop codon, leading to a complete loss of G6PC3 protein expression. When treated with 1,5-anhydroglucitol (1,5-AG), the precursor to 1,5-AG6P, patient-derived cells exhibited markedly reduced engagement of glycolysis. Clinically, c.210delC carriers display all the clinical features of syndromic severe congenital neutropenia type 4 observed in prior reports of G6PC3 deficiency. Conclusion: The G6PC3 c.210delC is a loss-of-function mutation that arose from a founder effect in the indigenous Mexican population. These findings may facilitate the diagnosis of additional patients in this geographical area. Moreover, the in vitro 1,5-AG-dependent functional assay used in our study could be employed to assess the pathogenicity of additional G6PC3 variants.

Clinical and cellular phenotypes resulting from a founder mutation in IL10RB.

Inborn errors of immunity are a group of rare genetically determined diseases that impair immune system development or function. Many of these diseases include immune dysregulation, autoimmunity or autoinflammation as prominent clinical features. In some children diagnosed with very early onset inflammatory bowel disease (VEOIBD), monogenic inborn errors of immune dysregulation underlie disease. We report a case of VEOIBD caused by a novel homozygous loss of function mutation in IL10RB. We use CyTOF with a broad panel of antibodies to interrogate the immunophenotype of this patient and detect reduced frequencies of CD4 and CD8 T cells with additional defects in some populations of T helper cells, innate-like T cells and memory B cells. Finally, we identify the patient's mutation as a founder allele in an isolated indigenous population and estimate the age of this variant by studying the shared ancestral haplotype.

Inherited human ezrin deficiency impairs adaptive immunity.

BACKGROUND: Inborn errors of immunity (IEI) are a group of monogenic diseases that confer susceptibility to infection, autoimmunity, and cancer. Despite the life-threatening consequences of some IEI, their genetic cause remains unknown in many patients. OBJECTIVE: We investigated a patient with an IEI of unknown genetic etiology. METHODS: Whole-exome sequencing identified a homozygous missense mutation of the gene encoding ezrin (EZR), substituting a threonine for an alanine at position 129. RESULTS: Ezrin is one of the subunits of the ezrin, radixin, and moesin (ERM) complex. The ERM complex links the plasma membrane to the cytoskeleton and is crucial for the assembly of an efficient immune response. The A129T mutation abolishes basal phosphorylation and decreases calcium signaling, leading to complete loss of function. Consistent with the pleiotropic function of ezrin in myriad immune cells, multidimensional immunophenotyping by mass and flow cytometry revealed that in addition to hypogammaglobulinemia, the patient had low frequencies of switched memory B cells, CD4+ and CD8+ T cells, MAIT, γδ T cells, and centralnaive CD4+ cells. CONCLUSIONS: Autosomal-recessive human ezrin deficiency is a newly recognized genetic cause of B-cell deficiency affecting cellular and humoral immunity.

Hermansky-Pudlak syndrome with early onset inflammatory bowel disease due to loss of dysbindin expression.

Hermansky-Pudlak syndrome (HPS) is a heterogeneous group of autosomal recessive genetic disorders characterized by oculocutaneous albinism, bleeding diathesis, and variable presentation of immune deficiency and dysregulation. The pathogenesis of HPS involves mutations in genes responsible for biogenesis and trafficking of lysosome-related organelles, essential for the function of melanosomes, platelet granules, and immune cell granules. Eleven genes coding for proteins in the BLOC-1, BLOC-2, BLOC-3 and AP-3 complexes have been implicated in the pathogenesis of HPS. To date, the rare subtype HPS-7 associated with bi-allelic mutations in DTNBP1 (dysbindin) has only been reported in 9 patients. We report a novel DTNBP1 splicing mutation in a 15-month-old patient with HPS-7 phenotype and severe inflammatory bowel disease (IBD). This patient's leukocytes have undetectable dysbindin protein. We also identify dysregulated expression of several genes involved in activation of the adaptive immune response. This case underscores the emerging immunological consequences of dysbindin deficiency and suggests that DTNBP1 mutations may underlie some rare cases of very early onset IBD.