www.nature.com/gim ARTICLE Biallelic UBE4A loss-of-function variants cause intellectual disability and global developmental delay ✉ Uirá Souto Melo, PhD 1,15,16 , Devon Bonner, MD2,16, Kevin C. Kent Lloyd, DVM, PhD3,4, Ala Moshiri, MD, PhD5, Brandon Willis, BS3, Louise Lanoue, PhD3, Lynette Bower, BS3, Brian C. Leonard, DVM, PhD6, Davi Jardim Martins, MSc7, Fernando Gomes, PhD1, Felipe de Souza Leite, PhD1, Danyllo Oliveira, PhD1, João Paulo Kitajima, PhD8, Fabiola P. Monteiro, MD8, Mayana Zatz, PhD1, Carlos Frederico Martins Menck, PhD7, Matthew T. Wheeler, MD, PhD9, Jonathan A. Bernstein, MD, PhD2, Kevin Dumas, PhD10, Elizabeth Spiteri, PhD10, Nataliya Di Donato, MD, PhD11, Arne Jahn, MD, PhD11, Mais Hashem, BS12, Hessa S. Alsaif, BS12, ✉ Aziza Chedrawi, MD13, Fowzan S. Alkuraya, MD12,14, Fernando Kok, MD, PhD1,8 and Heather M. Byers, MD2 PURPOSE: To identify novel genes associated with intellectual disability (ID) in four unrelated families. METHODS: Here, through exome sequencing and international collaboration, we report eight individuals from four unrelated families of diverse geographic origin with biallelic loss-of-function variants in UBE4A. RESULTS: Eight evaluated individuals presented with syndromic intellectual disability and global developmental delay. Other clinical features included hypotonia, short stature, seizures, and behavior disorder. Characteristic features were appreciated in some individuals but not all; in some cases, features became more apparent with age. We demonstrated that UBE4A loss-of-function variants reduced RNA expression and protein levels in clinical samples. Mice generated to mimic patient-specific Ube4a loss-of- function variant exhibited muscular and neurological/behavioral abnormalities, some of which are suggestive of the clinical 1234567890():,; abnormalities seen in the affected individuals. CONCLUSION: These data indicate that biallelic loss-of-function variants in UBE4A cause a novel intellectual disability syndrome, suggesting that UBE4A enzyme activity is required for normal development and neurological function. Genetics in Medicine _#####################_ ; https://doi.org/10.1038/s41436-020-01047-z INTRODUCTION the ubiquitin–proteasome system (UPS).7,8 Overall, the Intellectual disability (ID) occurs in 1–3% of the general population ubiquitin–proteasome pathway has two critical steps: (1) covalent and is defined by limitations in intellectual functioning and attachment of ubiquitin to the targeted protein substrate followed adaptive behavior.1,2 ID is characterized by significant genetic by several ubiquitin molecules creating a chain assembly, and heterogeneity with over 1,000 genes implicated to date, while (2) degradation of the ubiquitylated protein by the proteasome thousands of genes likely remain to be identified.3–6 Next- complex. To do this, the UPS requires an ubiquitin-activating generation sequencing technologies and collaborative research enzyme (E1), an ubiquitin conjugating enzyme (E2), and a networks have greatly advanced the identification and character- substrate-specific ubiquitin-protein isopeptide ligase (E3) to ization of ID-related genes. In most families, ID is sporadic, covalently attach the 76–amino acid protein ubiquitin to proteins typically due to a de novo pathogenic variant. However, there are targeted for degradation. This multistep process plays an likely many rare Mendelian genetic causes of ID associated with important role in protein degradation including that of many autosomal recessive inheritance, most of which have not yet been short-lived regulatory proteins, such as those that contribute to described.3 Genes involved in protein ubiquitination play a the cell cycle and cellular signaling in response to DNA repair, significant role in ID and neurodevelopmental disorders (NDDs) environmental stress, secretion, and morphogenesis.9 U-box and remain an important pathway for discovery of ID- proteins, including UBE4A, were initially classified as a fourth related genes. class of ubiquitin molecule, ubiquitination protein ligase (E4) and A careful balance of protein synthesis and degradation is thought to promote polyubiquination and efficient ubiquitin chain required to maintain cellular protein steady-state and proper assembly.7,8 Additional studies demonstrated that U-box proteins function. Protein ubiquitination is a highly conserved, post- can uniquely function as both E3 and E4 ligases.10,11 translational modification that labels proteins for diverse functions Impairment of ubiquitination can cause ID and other neurolo- within the cell as well as misfolded proteins for degradation via gical conditions due to multiple mechanisms, including the 1Human Genome and Stem Cell Research Center, Department of Genetics and Evolutionary Biology, Biosciences Institute, University of São Paulo (USP), São Paulo, SP, Brazil. 2Division of Medical Genetics, Department of Pediatric, Stanford University School of Medicine, Stanford, CA, USA. 3Mouse Biology Program, University of California–Davis, Davis, CA, USA. 4Department of Surgery, School of Medicine, University of California–Davis, Sacramento, CA, USA. 5Department of Ophthalmology & Vision Science, School of Medicine, University of California Davis, Sacramento, CA, USA. 6Department of Surgical and Radiological Sciences, School of Veterinary Medicine, University of California–Davis, Davis, CA, USA. 7Department of Microbiology, Institute of Biomedical Sciences, University of São Paulo (USP), São Paulo, SP, Brazil. 8Mendelics, São Paulo, SP, Brazil. 9Division of Cardiovascular Medicine, Department of Medicine, Stanford University School of Medicine, Stanford, CA, USA. 10Clinical Genomics Program, Department of Pathology, School of Medicine, Stanford University, Stanford, CA, USA. 11Institute for Clinical Genetics, Technische Universität Dresden, Dresden, Germany. 12Department of Genetics, King Faisal Specialist Hospital and Research Center, Riyadh, Saudi Arabia. 13Department of Neuroscience, King Faisal Specialist Hospital and Research Center, Riyadh, Saudi Arabia. 14College of Medicine, Alfaisal University, Riyadh, Saudi Arabia. 15Present address: Max Planck Institute for Molecular Genetics, RG Development & Disease, Berlin, Germany. 16These authors ✉ contributed equally: Uirá Souto Melo, Devon Bonner. email: [email protected]; [email protected] © American College of Medical Genetics and Genomics 2020 U.S. Melo et al. 2 decline of the cellular proteolytic capacity, accumulation of mL, Stemcell Technologies), then plated in a dish with DMEM high-glucose misfolded protein, and reduction of effective protein produc- fibroblast media (Thermo Fisher, Waltham, MA, USA) supplemented with tion.12–14 Several genes involved in the UPS-mediated degrada- 10% fetal bovine serum (Thermo Fisher), 1% nonessential amino acids tion pathway are known to cause syndromic NDD including UBE3A (NEAA; Merck, Darmstadt, Germany), and 1% penicillin–streptomycin (Angelman syndrome, [MIM 105830]),12 and more recently (Sigma-Aldrich, St. Louis, MO, USA). described UBE2A (MIM 300860)13 and UBE3B (MIM 244450).14 An Peripheral blood mononuclear cells (PBMCs) were obtained from extensive list of many other E2, E3 ligases, and deubiquitinating sodium-heparin blood of patient D-1 by dilution with complete medium enzymes have been described in ID and NDD (e.g., HUWE1, HERC2, consisting of RPMI 1640 Glutamax supplemented with 15% FCS, 1% – HECW2, UBR1, USP7), suggesting that variants in ubiquitin-related penicillin streptomycin (Gibco, Thermo Fisher) and gradient centrifugation genes could explain some unsolved ID cases.15 However, our with Ficoll (Merck). PBMCs were washed twice with complete medium and resuspended with EBV (B95-8) supernatant (50%) supplemented with understanding of UBE4A function in humans and its implication in complete medium (48.5%) and PHL-A buffer (1.5%, Biochrom, Merck). After ID is sparse. Biallelic UBE4A loss-of-function (LoF) variants were incubation with transformation medium for 2–3 days, immortalized cells previously proposed as a candidate ID gene in a single family, also were cultured in complete medium. reported here (family C), but have not otherwise been described.5 Through the GeneMatcher platform16 and professional con- versations with colleagues, we collaboratively assembled a cohort Expression analysis of eight individuals in four families, all with syndromic ID and RNA was extracted from fibroblasts from families A (A-1, A-2) and D (D-1) global developmental delay. All affected individuals had homo- using RNeasy mini kit (Qiagen, Hilden, Germany). Total RNA (2 µg/µL) was ™ zygous LoF UBE4A variants. Functional studies presented further reverse-transcribed using oligo(dT) primers and SuperScript III First-strand evidence for pathogenicity, showing degradation of UBE4A Synthesis System (Thermo Fisher). Primers for reverse transcription in vitro. Mice engineered with a pathogenic variant of Ube4a quantitative PCR (RT-qPCR) are described in Table S2. RT-qPCR was performed using the Applied Biosystems® 7500 Fast Real-time PCR System exhibited ocular abnormalities, musculoskeletal weakness, and −ΔΔ and gene expression was calculated using the 2 CT method.17 behavioral hypersensitivity, which were also, to some degree, Protein extraction and western blot analysis were performed using observed in our cases. Herein, we report eight individuals with standard protocols using A-1 and A-2 samples. Whole-cell lysates were homozygous UBE4A LoF variants, with a consistent phenotype and obtained by lysing fibroblasts