Meri Kaustio1, Naemeh Nayebzadeh2, Reetta Hinttala2, Terhi Tapiainen3, Pirjo Åström4, Katariina Mamia5, Nora Pernaa4, Johanna Lehtonen6, Virpi Glumoff4, Elisa Rahikkala7, Minna Honkila8, Päivi Olsén8, Antti Hassinen1, Minttu Polso1, Nashat Al Sukaiti9, Jalila Al Shekaili10, Mahmood Al Kindi10, Nadia Al Hashmi11, Henrikki Almusa1, Daria Bulanova12, Emma Haapaniemi13, Pu Chen1, Maria Suo-Palosaari14, Päivi Vieira8, Hannu Tuominen15, Hannaleena Kokkonen16, Nabil Al Macki17, Huda Al Habsi18, Tuija Löppönen19, Heikki Rantala20, Vilja Pietiäinen1, Shen-Ying Zhang21, Marjo Renko22, Timo Hautala23, Tariq Al Farsi9, Johanna Uusimaa8, Janna Saarela24. 1. Institute for Molecular Medicine Finland (FIMM), HiLIFE, University of Helsinki, Helsinki, Finland. 2. PEDEGO Research Unit, University of Oulu, Oulu, Finland; Medical Research Center Oulu, University of Oulu, Oulu, Finland; Biocenter Oulu, Oulu, Finland. 3. PEDEGO Research Unit, University of Oulu, Oulu, Finland; Medical Research Center Oulu, University of Oulu, Oulu, Finland; Biocenter Oulu, Oulu, Finland; Department of Pediatrics and Adolescent Medicine, Oulu University Hospital, Oulu, Finland. 4. Research Unit of Biomedicine, University of Oulu, Oulu, Finland. 5. Centre for Molecular Medicine Norway (NCMM), University of Oslo, Oslo, Norway. 6. Institute for Molecular Medicine Finland (FIMM), HiLIFE, University of Helsinki, Helsinki, Finland; Centre for Molecular Medicine Norway (NCMM), University of Oslo, Oslo, Norway; Folkhälsan Research Center, Helsinki, Finland. 7. PEDEGO Research Unit, University of Oulu, Oulu, Finland; Medical Research Center Oulu, University of Oulu, Oulu, Finland; Department of Clinical Genetics, Oulu University Hospital, Oulu, Finland. 8. PEDEGO Research Unit, University of Oulu, Oulu, Finland; Medical Research Center Oulu, University of Oulu, Oulu, Finland; Department of Pediatrics and Adolescent Medicine, Oulu University Hospital, Oulu, Finland. 9. Department of Pediatric Allergy and Clinical Immunology, The Royal Hospital, Muscat, Oman. 10. Department of Microbiology and Immunology, Sultan Qaboos University Hospital, Muscat, Oman. 11. Department of Clinical and Biochemical Genetics, The Royal Hospital, Muscat, Oman. 12. Institute for Molecular Medicine Finland (FIMM), HiLIFE, University of Helsinki, Helsinki, Finland; Biotech Research and Innovation Centre (BRIC), University of Copenhagen, Copenhagen, Denmark. 13. Centre for Molecular Medicine Norway (NCMM), University of Oslo, Oslo, Norway; Department of Pediatric Research, Oslo University Hospital, Oslo, Norway; Research Programs Unit, Molecular Neurology and Biomedicum Stem Cell Centre, Faculty of Medicine, University of Helsinki, Helsinki, Finland. 14. Medical Research Center Oulu, University of Oulu, Oulu, Finland; Department of Diagnostic Radiology, Oulu University Hospital and University of Oulu, Oulu, Finland; Research Unit of Medical Imaging, Physics and Technology, Faculty of Medicine, University of Oulu, Oulu, Finland. 15. Department of Pathology, Oulu University Hospital, Oulu, Finland. 16. Medical Research Center Oulu, University of Oulu, Oulu, Finland; Department of Clinical Genetics, Northern Finland Laboratory Centre, Oulu University Hospital, Oulu, Finland. 17. Department of Pediatric Neurology, The Royal Hospital, Muscat, Oman. 18. Department of General Pediatrics, The Royal Hospital, Muscat, Oman. 19. Department of Pediatrics, University of Eastern Finland and Kuopio University Hospital, Kuopio, Finland. 20. PEDEGO Research Unit, University of Oulu, Oulu, Finland. 21. St. Giles Laboratory of Human Genetics of Infectious Diseases, Rockefeller Branch, The Rockefeller University, New York, NY; Paris Descartes University, Imagine Institute, Paris, France; Laboratory of Human Genetics of Infectious Diseases, Necker Branch, INSERM UMR 1163, Necker Hospital for Sick Children, Paris, France. 22. PEDEGO Research Unit, University of Oulu, Oulu, Finland; Department of Pediatrics, University of Eastern Finland and Kuopio University Hospital, Kuopio, Finland. 23. Research Unit of Biomedicine, University of Oulu, Oulu, Finland; Department of Internal Medicine, Oulu University Hospital, Oulu, Finland. 24. Institute for Molecular Medicine Finland (FIMM), HiLIFE, University of Helsinki, Helsinki, Finland; Centre for Molecular Medicine Norway (NCMM), University of Oslo, Oslo, Norway; Department of Medical Genetics, Oslo University Hospital, Oslo, Norway; Department of Clinical Genetics, Helsinki University Hospital, Helsinki, Finland. Electronic address: janna.saarela@helsinki.fi.
Abstract
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.
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.
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