Loss of DIAPH1 causes SCBMS, combined immunodeficiency, and mitochondrial dysfunction.

BACKGROUND: Homozygous loss of DIAPH1 results in seizures, cortical blindness, and microcephaly syndrome (SCBMS). We studied 5 Finnish and 2 Omani patients with loss of DIAPH1 presenting with SCBMS, mitochondrial dysfunction, and immunodeficiency. OBJECTIVE: We sought to further characterize phenotypes and disease mechanisms associated with loss of DIAPH1. METHODS: Exome sequencing, genotyping and haplotype analysis, B- and T-cell phenotyping, in vitro lymphocyte stimulation assays, analyses of mitochondrial function, immunofluorescence staining for cytoskeletal proteins and mitochondria, and CRISPR-Cas9 DIAPH1 knockout in heathy donor PBMCs were used. RESULTS: Genetic analyses found all Finnish patients homozygous for a rare DIAPH1 splice-variant (NM_005219:c.684+1G>A) enriched in the Finnish population, and Omani patients homozygous for a previously described pathogenic DIAPH1 frameshift-variant (NM_005219:c.2769delT;p.F923fs). In addition to microcephaly, epilepsy, and cortical blindness characteristic to SCBMS, the patients presented with infection susceptibility due to defective lymphocyte maturation and 3 patients developed B-cell lymphoma. Patients' immunophenotype was characterized by poor lymphocyte activation and proliferation, defective B-cell maturation, and lack of naive T cells. CRISPR-Cas9 knockout of DIAPH1 in PBMCs from healthy donors replicated the T-cell activation defect. Patient-derived peripheral blood T cells exhibited impaired adhesion and inefficient microtubule-organizing center repositioning to the immunologic synapse. The clinical symptoms and laboratory tests also suggested mitochondrial dysfunction. Experiments with immortalized, patient-derived fibroblasts indicated that DIAPH1 affects the amount of complex IV of the mitochondrial respiratory chain. CONCLUSIONS: Our data demonstrate that individuals with SCBMS can have combined immune deficiency and implicate defective cytoskeletal organization and mitochondrial dysfunction in SCBMS pathogenesis.

Image-based and machine learning-guided multiplexed serology test for SARS-CoV-2.

We present a miniaturized immunofluorescence assay (mini-IFA) for measuring antibody response in patient blood samples. The method utilizes machine learning-guided image analysis and enables simultaneous measurement of immunoglobulin M (IgM), IgA, and IgG responses against different viral antigens in an automated and high-throughput manner. The assay relies on antigens expressed through transfection, enabling use at a low biosafety level and fast adaptation to emerging pathogens. Using severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) as the model pathogen, we demonstrate that this method allows differentiation between vaccine-induced and infection-induced antibody responses. Additionally, we established a dedicated web page for quantitative visualization of sample-specific results and their distribution, comparing them with controls and other samples. Our results provide a proof of concept for the approach, demonstrating fast and accurate measurement of antibody responses in a research setup with prospects for clinical diagnostics.

ß-Catenin-mediated immune evasion pathway frequently operates in primary cutaneous melanomas.

Immunotherapy prolongs survival in only a subset of melanoma patients, highlighting the need to better understand the driver tumor microenvironment. We conducted bioinformatic analyses of 703 transcriptomes to probe the immune landscape of primary cutaneous melanomas in a population-ascertained cohort. We identified and validated 6 immunologically distinct subgroups, with the largest having the lowest immune scores and the poorest survival. This poor-prognosis subgroup exhibited expression profiles consistent with ß-catenin-mediated failure to recruit CD141+ DCs. A second subgroup displayed an equally bad prognosis when histopathological factors were adjusted for, while 4 others maintained comparable survival profiles. The 6 subgroups were replicated in The Cancer Genome Atlas (TCGA) melanomas, where ß-catenin signaling was also associated with low immune scores predominantly related to hypomethylation. The survival benefit of high immune scores was strongest in patients with double-WT tumors for BRAF and NRAS, less strong in BRAF-V600 mutants, and absent in NRAS (codons 12, 13, 61) mutants. In summary, we report evidence for a ß-catenin-mediated immune evasion in 42% of melanoma primaries overall and in 73% of those with the worst outcome. We further report evidence for an interaction between oncogenic mutations and host response to melanoma, suggesting that patient stratification will improve immunotherapeutic outcomes.