A Drosophila genetic resource facilitates the identification of variants in ANKLE2 in a unique family with severe microcephaly. W.-L. Charng1,2, M. Jaiswal2,3, N. Link2, S. Yamamoto1,2,4, T. Gambin2,5, E. Karaca2, G. Mirzaa6,7, W. Wiszniewski2,8, B. Xiong1, V. Bayat1, T. Harel2,8, D. Pehlivan2, S. Penney2,8, L. E. Vissers9, J. de Ligt9, S. Jhangiani10, D. Muzny2,10, R. D. Clark11, C. J. Curry12, E. Boerwinkle10,13, W. B. Dobyns6,7,14, R. A. Gibbs2,10, R. Chen1,2,10, M. F. Wangler2,8, H. J. Bellen1,2,3,4, 9,15, J. R. Lupski2,8,10,16 1) Program in Developmental Biology, Baylor College of Medicine (BCM), Houston, TX, 77030; 2) Department of Molecular and Human Genetics, BCM, Houston, TX, 77030; 3) Howard Hughes Medical Institute, Houston, TX, 77030; 4) Jan and Dan Duncan Neurological Research Institute, Texas Childrens Hospital (TCH), Houston, TX, 77030; 5) Institute of Computer Science, Warsaw University of Technology, 00-661 Warsaw, Poland; 6) Department of Pediatrics, University of Washington, Seattle, WA, 98195; 7) Center for Integrative Brain Research, Seattle Childrens Research Institute, Seattle, WA, 98101; 8) Texas Childrens Hospital, Houston, TX, 77030; 9) 9Department of Human Genetics, Radboudumc, PO Box 9101, 6500 HB, Nijmegen, The Netherlands; 10) Human Genome Sequencing Center, BCM, Houston, TX, 77030; 11) Division of Medical Genetics, Department of Pediatrics, Loma Linda University Medical Center, Loma Linda, CA, 92354; 12) Department of Pediatrics, University of California San Francisco, San Francisco, CA, 94143, and Genetic Medicine Central California, Fresno, CA, 93701; 13) Human Genetics Center, University of Texas, Health Science Center at Houston, Houston, TX, 77030; 14) Department of Neurology, University of Washington, 98195; 15) Department of Neuroscience, BCM, Houston, TX, 77030; 16) Department of Pediatrics, Baylor College of Medicine, Houston, TX, 77030.

   To create a resource for the study of human disease genes, we conducted a large scale forward genetic screen on fly X-chromosome and isolated lethal mutations in 165 genes involved in neuronal development, function, or maintenance. We then explored the corresponding 250 human homologs/orthologs in 1,929 human exomes from the Baylor Hopkins Center for Mendelian Genomics (see Wangler et al.). Many study subjects have neurological disease phenotypes. We identified disease associated mutations in ANKLE2, which is the only homolog of fly dAnkle2, and has not been associated with human disorders. In a single family, two children exhibit severe microcephaly, and hyper and hypo-pigmented macules of the skin. The head size of the proband was over 9 standard deviations below the mean and MRI revealed a polymicrogyria-like cortical malformation. ANKLE2 is one of the 10 genes that segregate in a pattern consistent with Mendelian recessive expectations and one of four genes with high expression in the central nervous system (CNS). In our screen, dAnkle2 mutant flies exhibit bristles loss in the peripheral nervous system due to an underdevelopment of sensory organs. In addition, the brain of dAnkle2 mutant larva is much smaller than controls, indicating defective CNS development. Indeed, the number of dividing dAnkle2 mutant neuroblasts in the developing brain is largely reduced and these neuroblasts undergo apoptosis. All the fly phenotypes can be rescued by human ANKLE2, indicating a functional conservation across species. Interestingly, ANKLE2 is an interactor of VRK1, a gene implicated in pontocerebellar hypoplasia and reported by us in association with microcephaly and sensorimotor axonal distal symmetric polyneuropathy. We are currently culturing fibroblasts of proband and studies are underway to determine the defects observed in fly. Overall, our approach represents a unique combination of fly forward genetic screens and subsequent disease gene identification in human genomic data sets with additional functional studies in the fly and human cell lines. Recently, a mutation in CLP1 has been reported by us to cause apoptosis of neuronal progenitors and lead to microcephaly. In summary, the finding of neuroblast susceptibility to apoptosis in dAnkle2 mutant flies supports an emerging theme in neurodevelopmental disorders associated with both VRK1 and CLP1 variant alleles.

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