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The apical Par complex, which contains atypical protein kinase C (aPKC), Bazooka (Par-3), and Par-6, is required for establishing polarity during asymmetric division of neuroblasts in Drosophila, and its activity depends on L(2)gl. We show that loss of Ankle2, a protein associated with microcephaly in humans and known to interact with Zika protein NS4A, reduces brain volume in flies and impacts the function of the Par complex. Reducing Ankle2 levels disrupts endoplasmic reticulum (ER) and nuclear envelope morphology, releasing the kinase Ballchen-VRK1 into the cytosol. These defects are associated with reduced phosphorylation of aPKC, disruption of Par-complex localization, and spindle alignment defects. Importantly, removal of one copy of ballchen or l(2)gl suppresses Ankle2 mutant phenotypes and restores viability and brain size. Human mutational studies implicate the above-mentioned genes in microcephaly and motor neuron disease. We suggest that NS4A, ANKLE2, VRK1, and LLGL1 define a pathway impinging on asymmetric determinants of neural stem cell division., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Arthrogryposis is a clinical finding that is present either as a feature of a neuromuscular condition or as part of a systemic disease in over 400 Mendelian conditions. The underlying molecular etiology remains largely unknown because of genetic and phenotypic heterogeneity. We applied exome sequencing (ES) in a cohort of 89 families with the clinical sign of arthrogryposis. Additional molecular techniques including array comparative genomic hybridization (aCGH) and Droplet Digital PCR (ddPCR) were performed on individuals who were found to have pathogenic copy number variants (CNVs) and mosaicism, respectively. A molecular diagnosis was established in 65.2% (58/89) of families. Eleven out of 58 families (19.0%) showed evidence for potential involvement of pathogenic variation at more than one locus, probably driven by absence of heterozygosity (AOH) burden due to identity-by-descent (IBD). RYR3, MYOM2, ERGIC1, SPTBN4, and ABCA7 represent genes, identified in two or more families, for which mutations are probably causative for arthrogryposis. We also provide evidence for the involvement of CNVs in the etiology of arthrogryposis and for the idea that both mono-allelic and bi-allelic variants in the same gene cause either similar or distinct syndromes. We were able to identify the molecular etiology in nine out of 20 families who underwent reanalysis. In summary, our data from family-based ES further delineate the molecular etiology of arthrogryposis, yielded several candidate disease-associated genes, and provide evidence for mutational burden in a biological pathway or network. Our study also highlights the importance of reanalysis of individuals with unsolved diagnoses in conjunction with sequencing extended family members., (Copyright © 2019 American Society of Human Genetics. Published by Elsevier Inc. All rights reserved.)

Neurodegenerative diseases can occur so early as to affect neurodevelopment. From a cohort of more than 2,000 consanguineous families with childhood neurological disease, we identified a founder mutation in four independent pedigrees in cleavage and polyadenylation factor I subunit 1 (CLP1). CLP1 is a multifunctional kinase implicated in tRNA, mRNA, and siRNA maturation. Kinase activity of the CLP1 mutant protein was defective, and the tRNA endonuclease complex (TSEN) was destabilized, resulting in impaired pre-tRNA cleavage. Germline clp1 null zebrafish showed cerebellar neurodegeneration that was rescued by wild-type, but not mutant, human CLP1 expression. Patient-derived induced neurons displayed both depletion of mature tRNAs and accumulation of unspliced pre-tRNAs. Transfection of partially processed tRNA fragments into patient cells exacerbated an oxidative stress-induced reduction in cell survival. Our data link tRNA maturation to neuronal development and neurodegeneration through defective CLP1 function in humans., (Copyright © 2014 Elsevier Inc. All rights reserved.)

Desbuquois dysplasia (DBQD) is a severe condition characterized by short stature, joint laxity, and advanced carpal ossification. Based on the presence of additional hand anomalies, we have previously distinguished DBQD type 1 and identified CANT1 (calcium activated nucleotidase 1) mutations as responsible for DBQD type 1. We report here the identification of five distinct homozygous xylosyltransferase 1 (XYLT1) mutations in seven DBQD type 2 subjects from six consanguineous families. Among the five mutations, four were expected to result in loss of function and a drastic reduction of XYLT1 cDNA level was demonstrated in two cultured individual fibroblasts. Because xylosyltransferase 1 (XT-I) catalyzes the very first step in proteoglycan (PG) biosynthesis, we further demonstrated in the two individual fibroblasts a significant reduction of cellular PG content. Our findings of XYLT1 mutations in DBQD type 2 further support a common physiological basis involving PG synthesis in the multiple dislocation group of disorders. This observation sheds light on the key role of the XT-I during the ossification process., (Copyright © 2014 The American Society of Human Genetics. Published by Elsevier Inc. All rights reserved.)

Intraflagellar transport (IFT) depends on two evolutionarily conserved modules, subcomplexes A (IFT-A) and B (IFT-B), to drive ciliary assembly and maintenance. All six IFT-A components and their motor protein, DYNC2H1, have been linked to human skeletal ciliopathies, including asphyxiating thoracic dystrophy (ATD; also known as Jeune syndrome), Sensenbrenner syndrome, and Mainzer-Saldino syndrome (MZSDS). Conversely, the 14 subunits in the IFT-B module, with the exception of IFT80, have unknown roles in human disease. To identify additional IFT-B components defective in ciliopathies, we independently performed different mutation analyses: candidate-based sequencing of all IFT-B-encoding genes in 1,467 individuals with a nephronophthisis-related ciliopathy or whole-exome resequencing in 63 individuals with ATD. We thereby detected biallelic mutations in the IFT-B-encoding gene IFT172 in 12 families. All affected individuals displayed abnormalities of the thorax and/or long bones, as well as renal, hepatic, or retinal involvement, consistent with the diagnosis of ATD or MZSDS. Additionally, cerebellar aplasia or hypoplasia characteristic of Joubert syndrome was present in 2 out of 12 families. Fibroblasts from affected individuals showed disturbed ciliary composition, suggesting alteration of ciliary transport and signaling. Knockdown of ift172 in zebrafish recapitulated the human phenotype and demonstrated a genetic interaction between ift172 and ift80. In summary, we have identified defects in IFT172 as a cause of complex ATD and MZSDS. Our findings link the group of skeletal ciliopathies to an additional IFT-B component, IFT172, similar to what has been shown for IFT-A., (Copyright © 2013 The Authors. Published by Elsevier Inc. All rights reserved.)

We investigated three families whose offspring had extreme microcephaly at birth and profound mental retardation. Brain scans and postmortem data showed that affected individuals had brains less than 10% of expected size (≤10 standard deviation) and that in addition to a massive reduction in neuron production they displayed partially deficient cortical lamination (microlissencephaly). Other body systems were apparently unaffected and overall growth was normal. We found two distinct homozygous mutations of NDE1, c.83+1G>T (p.Ala29GlnfsX114) in a Turkish family and c.684_685del (p.Pro229TrpfsX85) in two families of Pakistani origin. Using patient cells, we found that c.83+1G>T led to the use of a novel splice site and to a frameshift after NDE1 exon 2. Transfection of tagged NDE1 constructs showed that the c.684_685del mutation resulted in a NDE1 that was unable to localize to the centrosome. By staining a patient-derived cell line that carried the c.83+1G>T mutation, we found that this endogeneously expressed mutated protein equally failed to localize to the centrosome. By examining human and mouse embryonic brains, we determined that NDE1 is highly expressed in neuroepithelial cells of the developing cerebral cortex, particularly at the centrosome. We show that NDE1 accumulates on the mitotic spindle of apical neural precursors in early neurogenesis. Thus, NDE1 deficiency causes both a severe failure of neurogenesis and a deficiency in cortical lamination. Our data further highlight the importance of the centrosome in multiple aspects of neurodevelopment., (Copyright © 2011 The American Society of Human Genetics. Published by Elsevier Inc. All rights reserved.)