Mónica Martínez-Gallo

Objective: PIGM encodes a critical enzyme in the glycosylphosphatidylinositol (GPI)‐anchor biosynthesis pathway. While promoter‐region mutations in PIGM have been associated with a relatively mild phenotype characterized by portal vein thrombosis and absence seizures, recent evidence suggests that coding‐region mutations result in a more severe multisystemic disorder. Methods: Whole‐exome sequencing reanalysis was performed in a patient with early‐onset developmental and epileptic encephalopathy, and subsequent matchmaking identified another patient with a similar phenotype. The pathogenicity of the variant was evaluated through multiple functional assays. To further delineate the genotypic and phenotypic spectrum of PIGM, we reviewed all patients reported to date with PIGM variants. Results: We describe two unrelated patients, both carrying the same homozygous missense variant in PIGM (c.1001A > C, p.Gln334Pro), presenting with early‐onset developmental and epileptic encephalopathy, profound neurodevelopmental impairment, multi‐organ involvement, and distinctive neuroimaging findings including hypomyelination. Both patients died in infancy due to super‐refractory status epilepticus. Treatment with sodium phenylbutyrate was attempted in one patient without clinical benefit. Flow cytometry revealed partial GPI‐anchor deficiency. Comparative analysis with previously reported cases highlights a potential genotype–phenotype correlation between coding region variants and disease severity. Interpretation: Our findings establish PIGM as a causative gene of early‐onset developmental and epileptic encephalopathy and expand the clinical and radiological spectrum of PIGM deficiency to include hypomyelination and prenatal onset. This study underscores the importance of including PIGM in the differential diagnosis of developmental and epileptic encephalopathy and leukoencephalopathies and provides further insight into the molecular mechanisms underlying phenotypic variability of GPI‐anchor disorders.

Background: Studies investigating the long-term cellular immune response to SARS-CoV-2 mRNA vaccines in patients with inflammatory bowel disease (IBD) remain limited, particularly among those receiving immunosuppressive therapy. Methods: We prospectively evaluated humoral and cellular immune responses at short-term (4–6 weeks) and long-term (6–12 months) time points following SARS-CoV-2 mRNA vaccination in patients with IBD receiving anti-TNF agents, thiopurines, or combination therapy. We defined the short-term response as the measurement taken 4–6 weeks after the second vaccine dose and the long-term response as the measurement taken between 6 and 12 months after the first determination. A cohort of healthy controls was included for short-term comparative analysis. Results: At long-term follow-up, quantitative humoral responses were reduced in patients receiving anti-TNF monotherapy. In contrast, a reduced quantitative cellular response was found in the thiopurine (median 0.7 UI/mL, p < 0.05) and anti-TNF combo groups (median 0.4 UI/mL, p < 0.01) compared to anti-TNF monotherapy (median 2.2 UI/mL). Conclusions: There was a robust long-term humoral and cellular response to vaccination, but a diminished quantitative cellular response in patients treated with thiopurines or combo therapy compared to anti-TNF monotherapy.

Background: Rituximab is commonly used to treat patients with primary glomerular diseases; however, its pharmacokinetics in this population have not been fully described yet. Materials and methods: This single-center, open-label, uncontrolled clinical trial included adult patients with glomerular diseases who required rituximab treatment (NEFRTX; EudraCT: 2020-000484-23). Patients received 1 or 0.5 g of rituximab on day 1 (and d14 in some cases). Blood and urine samples were collected at days 1, 7, 14, 28, and 45 to measure biochemical parameters (proteinuria, albuminemia, plasma immunoglobulin, and urine immunoglobulin), rituximab, and antidrug antibody concentrations. The gene encoding the neonatal fragment-crystallizable receptor was also characterized. Linear regression and Win-Nonlin 1.1 were used for pharmacokinetic analysis. Results: Thirty-five cases (30 patients) were included in this study. Pharmacokinetic parameters were expressed as mean (SD): maximum plasma concentration, 179.4 (71.8) mcg/mL; volume of distribution, 78.9 (31.4) mL/kg; clearance, 0.30 (0.27) mL/h/kg; half-life (t _1/2 ), 11.6 (5.8) d; elimination rate constant, 0.0036 (0.0030) hour ⁻¹ ; and area under the curve, 117,756.1 (88,228.1) mcg·h/mL. Antidrug antibody was detected on d1 in 3 cases (8.6%) and was negative by d28. Rituximab t _1/2 was represented by the formula: t _1/2 = A-B·Log (Proteinuria)+C·Albuminemia, where A = 515.1 (128.8–901.3), B = 182.1 (−108.6 to −35.4), and C = 39.5 (−10.9 to 89.9). There were significant differences in rituximab t _1/2 based on diagnosis ( P = 0.025), early treatment ( P = 0.008), proteinuria >2.4 g /24h ( P < 0.001), plasma immunoglobulin 2.4 g /24h may require higher frequent dosing for adequate rituximab exposure. Establishing an optimal dosing regimen in this population remains warranted.

The gut microbiota enhances systemic immunoglobulin G (IgG) responses to vaccines, but it is unknown whether this effect involves IgA, which coats intestinal microbes. That IgA may amplify postimmune IgG production is suggested by the impaired IgG response to pneumococcal vaccines in some IgA-deficient patients. Here, we found that antipneumococcal but not total IgG production was impaired in mice with IgA deficiency. The positive effect of gut IgA on antipneumococcal IgG responses started very early in life and could implicate gut bacteria, as these responses were attenuated in germ-free mice recolonized with gut microbes from IgA-deficient donors. IgA could exert this effect by constraining the systemic translocation of gut antigens, which was associated with chronic immune activation, including T cell overexpression of programmed cell death protein 1 (PD-1). This inhibitory receptor may attenuate antipneumococcal IgG production by causing B cell hyporesponsiveness, which improved upon anti–PD-1 treatment. Thus, gut IgA functionally interacts with systemic IgG to enhance antipneumococcal vaccine responses.

TRAF3, a versatile adaptor protein within the TRAF family, participates in various signaling pathways involving the tumor necrosis factor receptor, toll-like receptor, and retinoic acid-inducible gene I-like receptor families. In 2010, autosomal dominant TRAF3 deficiency was reported in a patient with herpes simplex virus-1 encephalitis, consistent with the role of TRAF3 in type I interferon production. Recently, a novel, completely different clinical phenotype was described in patients with TRAF3 haploinsufficiency (TRAF3^Hl), characterized by recurrent bacterial infections, autoimmune features, systemic inflammation, and hypergammaglobulinemia. In this study, we conducted a TRAF3-targeted reanalysis of next-generation sequencing data from 800 patients with inborn errors of immunity. Through this reassessment and additional familial investigations, we identified three previously unidentified cases of TRAF3^Hl within two different families. These individuals harbored stop-gain variants (p.Arg163* and p.Gln407*) and experienced recurrent bacterial infections with hypogammaglobulinemia. Previously, the patients had been diagnosed with common variable immunodeficiency (CVID) and were receiving immunoglobulin replacement therapy. In addition, a TRAF3 start-loss variant (c.3G > A) was identified in a fourth patient, but after familial and molecular studies, it was not considered disease-causing, excluding TRAF3^Hl in this patient. This study illustrates the usefulness of targeted reanalysis of genes with reported novel phenotypes. We rescued three patients with TRAF3^Hl, presenting similarities and differences with the previously reported patients. The most significant differences were hypogammaglobulinemia and a CVID-like presentation. These data expand the clinical phenotype of TRAF3^Hl and pave the way for further investigation into loss-of-function variants in patients with CVID.

The human gut includes plasma cells (PCs) expressing immunoglobulin A1 (IgA1) or IgA2, two structurally distinct IgA subclasses with elusive regulation, function, and reactivity. We show here that intestinal IgA1+ and IgA2+ PCs co-emerged early in life, comparably accumulated somatic mutations, and were enriched within short-lived CD19+ and long-lived CD19− PC subsets, respectively. IgA2+ PCs were extensively clonally related to IgA1+ PCs and a subset of them presumably emerged from IgA1+ precursors. Of note, secretory IgA1 (SIgA1) and SIgA2 dually coated a large fraction of mucus-embedded bacteria, including Akkermansia muciniphila. Disruption of homeostasis by inflammatory bowel disease (IBD) was associated with an increase in actively proliferating IgA1+ plasmablasts, a depletion in long-lived IgA2+ PCs, and increased SIgA1+SIgA2+ gut microbiota. Such increase featured enhanced IgA1 reactivity to pathobionts, including Escherichia coli, combined with depletion of beneficial A. muciniphila. Thus, gut IgA1 and IgA2 emerge from clonally related PCs and show unique changes in both frequency and reactivity in IBD.

Background: The antibody response to SARS-CoV-2 does not follow the immunoglobulin isotype pattern expected in a primary response and is inconsistent with the current interpretation of COVID-19 immunopathology as the result of a primary infection. To better understand the immune response to SARS-CoV-2, it is essential to determine whether it is primary or secondary (or recall). The analysis of highly granular immunological variable trajectories of a homogeneous cohort of patients receiving standardised medical care should discern between primary and secondary responses. Methods: This is a prospective cohort study of 191 SARS-CoV-2 infection cases and 44 healthy controls from the second wave of COVID-19 in the Barcelona area. The study stratified patients by severity and analysed the trajectories of SARS-CoV-2 antibodies and multiple immune variables for features associated with primary and recall immune responses. Findings: Isotype-specific antibody trajectories to SARS-CoV-2 proteins revealed a pattern of recall response in 94·2% of cases. In these cases, the detailed trajectories of plasmablasts, B cells, cTfh high-resolution subsets, and cytokines were consistent with a secondary response. The transcriptomic data indicated that this cohort is strictly comparable to contemporary cohorts. Conclusions: In most cases, the immune response to SARS-CoV-2 is a recall response. This opens the possibility that most COVID-19 cases are subjected to immune imprinting by endemic coronavirus, which, in turn, can contribute to severity by interfering with the immune response to SARS-CoV-2 and by antibody-dependent enhancement. Considering the immune responses to SARS-CoV-2 secondary provides a better perspective to interpret COVID-19 pathology. Funding: Grants COV20/00416, Cov20/00654, and COV20/00388 from Instituto de Salud Carlos III (ISCIII), Madrid, Spain, co-financed by the European Regional Development Fund (ERDF).

The term common variable immunodeficiency (CVID) encompasses a clinically diverse group of disorders, mainly characterized by hypogammaglobulinemia, insufficient specific antibody production, and recurrent infections. The genetics of CVID is complex, and monogenic defects account for only a portion of cases, typically A), and patient 4 had a hypomorphic variant in IKBKG (p.Glu57Lys) and a heterozygous splice variant in TNFRSF13B (TACI) (c.61+2T>A). Overall, the analysis of our cohort suggests that CVID in a small proportion of females (1.6% in our cohort) is caused by skewed XCI and highly penetrant gene variants on the X-chromosome. Additionally, skewed XCI may contribute to polygenic effects (3.3% in our cohort). These results indicate that skewed XCI may represent another piece in the complex puzzle of CVID genetics.

Chronic granulomatous disease (CGD) is a prototypical inborn error of immunity affecting phagocytes, in which these cells are unable to produce reactive oxygen species. CGD is caused by defects in genes encoding subunits of the NADPH oxidase enzyme complex (CYBA, CYBB, CYBC1, NCF1, NCF2, NCF4); inflammatory responses are dysregulated, and patients are highly susceptible to recurrent severe bacterial and fungal infections. X-linked CGD (XL-CGD), caused by mutations in the CYBB gene, is the most common and severe form of CGD. In this study, we describe the analytical processes undertaken in 3 families affected with XL-CGD to illustrate several molecular challenges in the genetic diagnosis of this condition: in family 1, a girl with a heterozygous deletion of CYBB exon 13 and skewed X-chromosome inactivation (XCI); in family 2, a boy with a hemizygous deletion of CYBB exon 7, defining its consequences at the mRNA level; and in family 3, 2 boys with the same novel intronic variant in CYBB (c.1151 + 6 T > A). The variant affected the splicing process, although a small fraction of wild-type mRNA was produced. Their mother was a heterozygous carrier, while their maternal grandmother was a carrier in form of gonosomal mosaicism. In summary, using a variety of techniques, including an NGS-based targeted gene panel and deep amplicon sequencing, copy number variation calling strategies, microarray-based comparative genomic hybridization, and cDNA analysis to define splicing defects and skewed XCI, we show how to face and solve some uncommon genetic mechanisms in the diagnosis of XL-CGD.