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.

Chronic hypogammaglobulinemia after allogeneic stem cell transplantation and their treatment with subcutaneous immunoglobulin in pediatric patients.

INTRODUCTION: Hypogammaglobulinemia in the first months after allogeneic hematopoietic stem cell transplantation (HSCT) is common in paediatric patients. During this phase, replacement therapy with human immunoglobulin must be administered parenterally to prevent infections. In some cases, this hypogammaglobulinemia persists over time, which forces further treatment when the patient is usually no longer a carrier of a central line, making them ideal candidates for subcutaneous replacement therapy. There is little published literature describing the use of this method in paediatric patients undergoing HSCT, widely described in replacement treatment in children with primary immunodeficiencies with very good results. PATIENTS AND METHODS: An observational, descriptive, longitudinal and retrospective study is carried out. During the years 2008-2019, we evaluated all paediatric patients undergoing HSCT in our center with persistent chronic hypogammaglobulinemia (for over a year). The treatment phase with intravenous immunoglobulin (Privigen®) and the first four years of treatment with subcutaneous immunoglobulin (Hizentra®) are evaluated using a questionnaire. RESULTS: During the years 2008-2019, 175 patients underwent HSCT, 143 (82%) of whom exceeded three months after transplantation. Three (2%) of them had persistent hypogammaglobulinemia. All three share factors described in the literature involved in immune reconstitution. After analysing the questionnaire, it is observed that switching from intravenous to subcutaneous gammaglobulin has involved a great improvement in their quality of life. CONCLUSIONS: The origin of chronic hypogammaglobulinemia in our patients shows different factors and cannot be attributed to a single cause. Due to the limited number of patients no conclusions can be drawn at the population level. We have been able to observe that replacement treatment with Hizentra 20% has been as effective as the intravenous administration without evidence of an increase in bacterial infections. Furthermore, it has also led to an improvement in quality of life and increased comfort, as the patients themselves have stated.

Unexpected relevant role of gene mosaicism in patients with primary immunodeficiency diseases.

BACKGROUND: Postzygotic de novo mutations lead to the phenomenon of gene mosaicism. The 3 main types are called somatic, gonadal, and gonosomal mosaicism, which differ in terms of the body distribution of postzygotic mutations. Mosaicism has been reported occasionally in patients with primary immunodeficiency diseases (PIDs) since the early 1990s, but its real involvement has not been systematically addressed. OBJECTIVE: We sought to investigate the incidence of gene mosaicism in patients with PIDs. METHODS: The amplicon-based deep sequencing method was used in the 3 parts of the study that establish (1) the allele frequency of germline variants (n = 100), (2) the incidence of parental gonosomal mosaicism in families with PIDs with de novo mutations (n = 92), and (3) the incidence of mosaicism in families with PIDs with moderate-to-high suspicion of gene mosaicism (n = 36). Additional investigations evaluated body distribution of postzygotic mutations, their stability over time, and their characteristics. RESULTS: The range of allele frequency (44.1% to 55.6%) was established for germline variants. Those with minor allele frequencies of less than 44.1% were assumed to be postzygotic. Mosaicism was detected in 30 (23.4%) of 128 families with PIDs, with a variable minor allele frequency (0.8% to 40.5%). Parental gonosomal mosaicism was detected in 6 (6.5%) of 92 families with de novo mutations, and a high incidence of mosaicism (63.9%) was detected among families with moderate-to-high suspicion of gene mosaicism. In most analyzed cases mosaicism was found to be both uniformly distributed and stable over time. CONCLUSION: This study represents the largest performed to date to investigate mosaicism in patients with PIDs, revealing that it affects approximately 25% of enrolled families. Our results might have serious consequences regarding treatment and genetic counseling and reinforce the use of next-generation sequencing-based methods in the routine analyses of PIDs.

Anti-NF155 chronic inflammatory demyelinating polyradiculoneuropathy strongly associates to HLA-DRB15.

BACKGROUND: The aim of the research is to study the human leukocyte antigen (HLA) class II allele frequencies in chronic inflammatory demyelinating polyradiculoneuropathy (CIDP) associated with anti-neurofascin 155 (NF155) antibodies. METHODS: Thirteen anti-NF155+ and 35 anti-NF155 negative (anti-NF155neg) CIDP patients were included in a case-control study. The frequencies of the DRB1 HLA allele were analyzed in all patients while DQ frequencies were only studied in patients sharing the DRB1*15 allele. In silico HLA-peptide binding and NF155 antigenicity, predictions were performed to analyze overlap between presented peptides and antigenic regions. RESULTS: DRB1*15 alleles (DRB1*15:01 and DRB1*15:02) were present in 10 out of 13 anti-NF155+ CIDP patients and in only 5 out of 35 anti-NF155neg CIDP patients (77 vs 14%; OR = 20, CI = 4.035 to 99.13). DRB1*15 alleles appeared also in significantly higher proportions in anti-NF155+ CIDP than in normal population (77 vs 17%; OR = 16.9, CI = 4.434 to 57.30). Seven anti-NF155+ CIDP patients (53%) and 5 anti-NF155neg CIDP patients had the DRB1*15:01 allele (OR = 7, p = 0.009), while 3 anti-NF155+ CIDP patients and none of the anti-NF155neg CIDP patients had the DRB1*15:02 allele (OR = 23.6, p = 0.016). In silico analysis of the NF155 peptides binding to DRB1*15 alleles showed significant overlap in the peptides presented by the 15:01 and 15:02 alleles, suggesting functional homology. CONCLUSIONS: DRB1*15 alleles are the first strong risk factor associated to a CIDP subset, providing additional evidence that anti-NF155+ CIDP patients constitute a differentiated disease within the CIDP syndrome.

A novel gain-of-function STAT1 mutation resulting in basal phosphorylation of STAT1 and increased distal IFN-γ-mediated responses in chronic mucocutaneous candidiasis.

Gain-of-function STAT1 mutations have recently been associated with autosomal dominant chronic mucocutaneous candidiasis (CMC). The purpose of this study was to characterize the three members of a non-consanguineous family, the father and his two sons, who presented with recurrent oral thrush and ocular candidiasis since early childhood. The three patients had reduced levels of IL-17-producing T cells. This reduction affected specifically IL-17(+)IFN-γ(-) T cells, because the levels of IL-17(+)IFN-γ(+) T cells were similar to controls. We found that PBMC (peripheral blood mononuclear cells) from the patients did not respond to Candida albicans ex vivo. Moreover, after polyclonal activation, patients' PBMC produced lower levels of IL-17 and IL-6 and higher levels of IL-4 than healthy controls. Genetic analyses showed that the three patients were heterozygous for a new mutation in STAT1 (c.894A>C, p.K298N) that affects a highly conserved residue of the coiled-coil domain of STAT1. STAT1 phosphorylation levels were significantly higher in patients' cells than in healthy controls, both in basal conditions and after IFN-γ stimulation, suggesting a permanent activation of STAT1. Cells from the patients also presented increased IFN-γ-mediated responses measured as MIG and IP-10 production. In conclusion, we report a novel gain-of-function mutation in the coiled-coil domain of STAT1, which increases STAT1 phosphorylation and impairs IL-17-mediated immunity. The mutation is responsible for CMC in this family with autosomal dominant inheritance of the disease.

Polymorphisms of Pyrimidine Pathway Enzymes Encoding Genes and HLA-B*40∶01 Carriage in Stavudine-Associated Lipodystrophy in HIV-Infected Patients.

PURPOSE: To assess in a cohort of Caucasian patients exposed to stavudine (d4T) the association of polymorphisms in pyrimidine pathway enzymes and HLA-B*40∶01 carriage with HIV/Highly active antiretroviral therapy (HAART)-associated lipodystrophy syndrome (HALS). METHODS: Three-hundred and thirty-six patients, 187 with HALS and 149 without HALS, and 72 uninfected subjects were recruited. The diagnosis of HALS was performed following the criteria of the Lipodystrophy Severity Grading Scale. Polymorphisms in the thymidylate synthase (TS) and methylene-tetrahydrofolate reductase (MTHFR) genes were determined by direct sequencing, HLA-B genotyping by PCR-SSOr Luminex Technology, and intracellular levels of stavudine triphosphate (d4T-TP) by a LC-MS/MS assay method. RESULTS: HALS was associated with the presence of a low expression TS genotype polymorphism (64.7% vs. 42.9%, OR = 2.43; 95%CI: 1.53-3.88, P<0.0001). MTHFR gene polymorphisms and HLA-B*40∶01 carriage were not associated with HALS or d4T-TP intracellular levels. Low and high expression TS polymorphisms had different d4T-TP intracellular levels (25.60 vs. 13.60 fmol/10(6) cells, P<0.0001). Independent factors associated with HALS were(OR [95%CI]: (a) Combined TS and MTHFR genotypes (p = 0.006, reference category (ref.): 'A+A'; OR for 'A+B' vs. ref.: 1.39 [0.69-2.80]; OR for 'B+A' vs. ref.: 2.16 [1.22-3.83]; OR for 'B+B' vs. ref.: 3.13, 95%CI: 1.54-6.35), (b) maximum viral load ≥5 log10 (OR: 2.55, 95%CI: 1.56-4.14, P = 0.001), (c) use of EFV (1.10 [1.00-1.21], P = 0.008, per year of use). CONCLUSION: HALS is associated with combined low-expression TS and MTHFR associated with high activity polymorphisms but not with HLA-B*40∶01 carriage in Caucasian patients with long-term exposure to stavudine.

From Severe Combined Immunodeficiency to Omenn syndrome after hematopoietic stem cell transplantation in a RAG1 deficient family.

BACKGROUND: Mutations in RAG genes cause a spectrum of severe immunodeficiencies ranging from Severe Combined Immunodeficiency (SCID) T-B-NK+ to Omenn syndrome (OS) through intermediate phenotypes, even for the same alteration. Nowadays, hematopoietic stem cell transplantation (HSCT) is the unique curative treatment available. METHODS: We describe three related patients from a Moroccan consanguineous family. Patient 1 developed at 1 month of age moderate eczematous dermatitis with eosinophilia, followed by infections and enteritis. He was transplanted and received reduced intensity conditioning regimen previous to HSCT. His brother, patient 2, was born preterm with a severe neonatal erythroderma, hepatosplenomegaly and lymphadenopathy. Patient 3, cousin of the two siblings, was also born preterm and fulfilled all criteria for classical OS. Immunological evaluation was performed and RAG genes were sequenced. RESULTS: Immunological data from all three patients were very diversed, from T lymphopenia to marked lymphocytosis, and different degrees of eosinophilia and IgE levels. Non-responder T cells and absent B cells were constant. All patients presented the same homozygous mutation in RAG1 gene (c.631delT). Patient 1 fully recovered both clinically and immunologically after HSCT. Two years later, he lost the accomplished lymphoid chimera and the disease relapsed as a classical OS, leading to patient's death. CONCLUSIONS: This is the first report of a RAG1 deficient patient with a changed clinical and immunological phenotype from SCID to OS after HSCT. The use of a myeloablative conditioning regimen that eliminates reminiscent T cells might have improved patient's outcome and it should be considered in similar cases.

The polymorphism p.G219R of CD40L does not cause immunological alterations in vivo: conclusions from a X-linked hyper IgM syndrome kindred.

Hyper-IgM syndromes (HIGM) are characterized by low levels of IgG, IgA and IgE and normal to high levels of IgM. Patients with these syndromes present recurrent infections due to an impaired immunoglobulin maturation. The most prevalent form of HIGM, X-linked hyper IgM syndrome (XHIM), is caused by mutations in the gene encoding the CD40 ligand (CD40LG). We present two siblings with XHIM caused by a large CD40LG deletion affecting more than half of the gene, and extended from the end of intron 3 to far upstream of the promoter regions. Genetic analysis in the maternal family discovered the CD40L(G219R) polymorphism in several members. Segregation of this polymorphism in the kindred indicated that the deletion of CD40LG was a de novo mutation in the mother. Although half of her CD4+ T cells did not express CD40L and the other half expressed the CD40L(G219R) variant, the mother was healthy. This suggests that this polymorphism is not pathogenic by itself although it has been recently related to X-linked lymphoproliferative syndrome.

Non-myeloablative hematopoietic stem cell transplantation in the treatment of severe idiopathic CD4+ lymphocytopenia.

A 40-year-old man with severe chronic idiopathic CD4+ lymphocytopenia complicated with opportunistic infections was successfully treated with non-myeloablative allogeneic hematopoietic stem cell transplantation. After conditioning with fludarabine plus low dose of total-body irradiation, CD34+ peripheral blood stem cells obtained by leukapheresis from his HLA-identical sister were infused. T cell and myeloid complete chimerism was achieved at day +28 and remained stable during the follow-up period. The patient did not develop infectious complications during the procedure. At 35 months of follow-up, his CD4+ T cell count was 1019 cells per microliter. Non-myeloablative allogeneic hematopoietic stem cell transplantation should be considered a treatment option for patients with severe forms of idiopathic CD4+ lymphocytopenia.

Gly111Ser mutation in CD8A gene causing CD8 immunodeficiency is found in Spanish Gypsies.

We describe the second case of CD8 immunodeficiency. It confirms the pathogenic effect of p.Gly111Ser, leading to complete deficit of CD8+ lymphocytes, although the clinical manifestations may vary in severity. Similarly to the first case reported, our patient is also from Spanish Gypsy origin and homozygous for the p.Gly111Ser mutation in CD8alpha chain. The patient has suffered repeated respiratory infections from childhood but with conservation of her pulmonary parenchyma, on the contrary to the first patient, who died because of his respiratory injury. We developed an AluI-PCR-RFLP assay to screen a total of 1127 unrelated control individuals: 734 subjects of Gypsy ancestry from different sub-isolates and geographic locations in Europe, and 393 of Spanish (non-Gypsy) ethnicity. The results indicate that p.Gly111Ser is confined to the Spanish Gypsy population, where it occurs at a carrier rate of 0.4%. Analysis of microsatellite markers flanking the CD8A mutated gene revealed a shared polymorphic haplotype suggesting a common founder for p.Gly111Ser mutation that causes CD8 deficiency in the Spanish Gypsy population. CD8 immunodeficiency should be given diagnostic consideration in Spanish Gypsies with recurrent infections. Our findings may also have implications for these patients in terms of specific recommendations in vaccination and healthy habits and for genetic counseling of affected families.

Gains of glycosylation comprise an unexpectedly large group of pathogenic mutations.

Mutations involving gains of glycosylation have been considered rare, and the pathogenic role of the new carbohydrate chains has never been formally established. We identified three children with mendelian susceptibility to mycobacterial disease who were homozygous with respect to a missense mutation in IFNGR2 creating a new N-glycosylation site in the IFNgammaR2 chain. The resulting additional carbohydrate moiety was both necessary and sufficient to abolish the cellular response to IFNgamma. We then searched the Human Gene Mutation Database for potential gain-of-N-glycosylation missense mutations; of 10,047 mutations in 577 genes encoding proteins trafficked through the secretory pathway, we identified 142 candidate mutations ( approximately 1.4%) in 77 genes ( approximately 13.3%). Six mutant proteins bore new N-linked carbohydrate moieties. Thus, an unexpectedly high proportion of mutations that cause human genetic disease might lead to the creation of new N-glycosylation sites. Their pathogenic effects may be a direct consequence of the addition of N-linked carbohydrate.