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Background: Homozygous loss of DIAPH1 results in seizures, cortical blindness, and microcephaly syndrome (SCBMS). We studied five Finnish and two Omani patients with loss of DIAPH1 presenting with SCBMS, mitochondrial dysfunction and immunodeficiency.

Objective: 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, CRISPR-Cas9 DIAPH1 knock-out in heathy donor PBMCs.

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 (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 three patients developed B cell lymphoma. Patients' immunophenotype was characterized by poor lymphocyte activation and proliferation, defective B cell maturation, and lack of naïve T cells. CRISPR-Cas9 knock-out 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 immunological 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.

Conclusion: Our data demonstrate that individuals with SCBMS can have combined immune deficiency and implicate defective cytoskeletal organization and mitochondrial dysfunction in SCBMS pathogenesis.