University of Southern Denmark Biallelic VARS variants cause developmental encephalopathy with microcephaly that is recapitulated in vars knockout zebrafish Siekierska, Aleksandra; Stamberger, Hannah; Deconinck, Tine; Oprescu, Stephanie N.; Partoens, Michèle; Zhang, Yifan; Sourbron, Jo; Adriaenssens, Elias; Mullen, Patrick; Wiencek, Patrick; Hardies, Katia; Lee, Jeong Soo; Giong, Hoi Khoanh; Distelmaier, Felix; Elpeleg, Orly; Helbig, Katherine L.; Hersh, Joseph; Isikay, Sedat; Jordan, Elizabeth; Karaca, Ender; Kecskes, Angela; Lupski, James R.; Kovacs-Nagy, Reka; May, Patrick; Narayanan, Vinodh; Pendziwiat, Manuela; Ramsey, Keri; Rangasamy, Sampathkumar; Shinde, Deepali N.; Spiegel, Ronen; Timmerman, Vincent; von Spiczak, Sarah; Helbig, Ingo; C4RCD Research Group; Møller, Rikke S.; Hjalgrim, Helle; AR working group of the EuroEPINOMICS RES Consortium; Weckhuysen, Sarah; Francklyn, Christopher; Antonellis, Anthony; de Witte, Peter A M; De Jonghe, Peter Published in: Nature Communications DOI: 10.1038/s41467-018-07953-w Publication date: 2019 Document version: Final published version Document license: CC BY Citation for pulished version (APA): Siekierska, A., Stamberger, H., Deconinck, T., Oprescu, S. N., Partoens, M., Zhang, Y., Sourbron, J., Adriaenssens, E., Mullen, P., Wiencek, P., Hardies, K., Lee, J. S., Giong, H. K., Distelmaier, F., Elpeleg, O., Helbig, K. L., Hersh, J., Isikay, S., Jordan, E., ... De Jonghe, P. (2019). Biallelic VARS variants cause developmental encephalopathy with microcephaly that is recapitulated in vars knockout zebrafish. Nature Communications, 10, [708]. https://doi.org/10.1038/s41467-018-07953-w Go to publication entry in University of Southern Denmark's Research Portal ARTICLE https://doi.org/10.1038/s41467-018-07953-w OPEN Biallelic VARS variants cause developmental encephalopathy with microcephaly that is recapitulated in vars knockout zebrafish Aleksandra Siekierska, Hannah Stamberger et al.# Aminoacyl tRNA synthetases (ARSs) link specific amino acids with their cognate transfer RNAs in a critical early step of protein translation. Mutations in ARSs have emerged as 1234567890():,; a cause of recessive, often complex neurological disease traits. Here we report an allelic series consisting of seven novel and two previously reported biallelic variants in valyl-tRNA synthetase (VARS) in ten patients with a developmental encephalopathy with microcephaly, often associated with early-onset epilepsy. In silico, in vitro, and yeast complementation assays demonstrate that the underlying pathomechanism of these mutations is most likely a loss of protein function. Zebrafish modeling accurately recapitulated some of the key neurological disease traits. These results provide both genetic and biological insights into neurodevelopmental disease and pave the way for further in-depth research on ARS related recessive disorders and precision therapies. Correspondence and requests for materials should be addressed to P.d.W. (email: [email protected]) or to P.D.J. (email: [email protected]). #A full list of authors and their affiliations appears at the end of the paper. NATURE COMMUNICATIONS | (2019) 10:708 | https://doi.org/10.1038/s41467-018-07953-w | www.nature.com/naturecommunications 1 ARTICLE NATURE COMMUNICATIONS | https://doi.org/10.1038/s41467-018-07953-w minoacyl tRNA synthetases (ARSs) play a key role in mo, median 4.3 mo). Seizure types included generalized or protein translation as they catalyze the attachment of bilateral tonic-clonic seizures (seven patients), myoclonia (four A fi speci c amino acids to their cognate transfer RNA patients), tonic seizures (one patient), focal seizures (two (tRNA) molecules1,2. The nuclear encoded ARS gene loci are patients), and atypical absences (one patient). In patient 2, subdivided into 17 cytoplasmic, 17 mitochondrial, and three bi- migrating focal seizures were documented on EEG. In four functional ARSs3,4. The canonical aminoacylation and proof- patients more than two anti-epileptic drug regimens failed reading functions of ARSs are highly conserved across species. In meeting the definition of drug resistance25. No seizures were addition, during evolution many ARSs acquired additional observed in patients 4 and 5 (family III), and repeated EEGs were domains with unique structural characteristics that are not normal. Both siblings were reported to have a notably happy essential for tRNA charging but account for non-canonical demeanor resembling Angelman syndrome, but genetic testing functions5,6. These alternative functions are critical for cellular for this syndrome was negative. homeostasis and include among others: regulation of signal Other clinical neurological features included (axial) hypotonia transduction and cell migration, angiogenesis and tumorigenesis, (four patients), spasticity (five patients), and an ataxic gait (two inflammatory responses, and control of cell death5. This func- patients). Three patients were reported to have significant sleep tional diversity may in part account for the association between problems. Brain imaging showed cerebral atrophy in eight mutations in ARS genes and a broad range of human disorders, patients and atrophy or partial agenesis of the corpus callosum including neurological disorders, cancer, and auto-immune in four patients. Furthermore, hypomyelination or delayed diseases2. myelination was reported in four patients. All patients had a Both monoallelic and biallelic pathogenic variants in ARS severe, progressive microcephaly on the background of a more genes, encoding dominant and recessive disease traits, respec- general failure-to-thrive. Patients 9 and 10 (carrying the same tively, have been increasingly reported in patients with various VARS variant) had additional systemic features including post- disorders that often have predominantly neurological features. natal anemia and hepatosplenomegaly in patient 9. Patient 10 Dominant heterozygous mutations in ARS genes have been died at the age of 3 years as a consequence of septic shock with no identified in patients with Charcot-Marie-Tooth disease and history of immunodeficiency. A detailed summary of the clinical related peripheral neuropathies, including AARS7, GARS8, findings in all patients is provided in Supplementary Data 1. HARS9,10, MARS11,12, WARS13, and YARS12,14. Recessive muta- All patients were referred for genetic testing with WES or whole- tions have been identified in complex disorders often involving genome sequencing (WGS) after standard diagnostic work-up did the central nervous system such as hypomyelination with brain- not reveal a cause for their neurodevelopmental disorder (details stem and spinal cord involvement (DARS)15, leukodystrophy available in Supplementary Notes 1 to 6). Three compound (RARS)16, congenital visual impairment and progressive micro- heterozygous VARS variants were identified in the affected cephaly (KARS)17, developmental delay with progressive micro- members of families I (p.Leu434Val/p.Gly822Ser), II (p.Gln400Pro/ cephaly and intractable seizures (QARS)18,19 and early onset p.Arg442Gln), and III (p.Leu78Argfs*35/p.Arg942Gln). Of note, in epileptic encephalopathy with myelination defect (AARS)20. addition to the biallelic VARS variant, patient 3 also carried a rare de Interestingly, some ARS genes have been associated with both novo variant of uncertain significance in the brain expressed gene dominant and recessive disease traits including mutations in UBE2O which has been reported 1 time in the Exome Aggregation AARS7,20, KARS21, and YARS14,22. Constortium (ExAC)26 (OMIM 617649, Supplementary Note 2). In this study, we report five newly diagnosed families with Two homozygous missense variants identified in families IV biallelic variants in valyl-tRNA synthetase (VARS), including (p.Leu885Phe) and V (p.Arg1058Gln) were previously reported in seven novel VARS variants. In addition, we present an in-depth a large study on brain malformations in mainly consanguineous description of two families previously reported in a large study on families23 and in families VI (Jordan ancestry) and VII (Israel/Arabic brain malformations in mainly consanguineous families wherein ancestry), the same, homozygous, p.Arg404Trp variant was identi- VARS was reported as a candidate disease gene23. In vitro studies fied. All VARS variants were deemed pathogenic based on their with patient-derived cell lines, including enzymatic assays, and absence or presence in very low frequency in ExAC and the Genome yeast complementation assays show that recessive VARS muta- Aggregation Database (gnomAD)26, impact on protein level, different tions most likely lead to a loss-of-protein function, i.e. loss of prediction tools, and segregation (Supplementary Table 1). All function (LoF) alleles. A vars knockout (KO) zebrafish model identified variants were located in the catalytic or the anticodon- further demonstrates that deficiency of vars results in micro- binding domain of the protein with the exception of the frameshift cephaly and epileptiform activity, replicating key characteristics variant, which was not located in a specificdomain(Fig.1b). of the human disease. In silico modeling in T. thermophilus ValRS. To gain insights Results into the potential structural and functional consequences of the Biallelic VARS variants cause developmental encephalopathy. human missense variants, the sequence of the human enzyme was In total, ten patients from seven families with biallelic VARS aligned to ValRS sequences from various taxa (Supplementary