Homozygous mutation in the eukaryotic translation initiation factor 2alpha phosphatase gene, PPP1R15B, is associated with severe microcephaly, short stature and intellectual disability

Human Molecular Genetics, 2015, Vol. 24, No. 22 6293–6300 doi: 10.1093/hmg/ddv337 Advance Access Publication Date: 24 August 2015 Original Article ORIGINAL ARTICLE Homozygous mutation in the eukaryotic translation Kristin D. Kernohan1, Martine Tétreault4,5, Urszula Liwak-Muir1, Michael T. Geraghty1,2, Wen Qin1, Sunita Venkateswaran3, Jorge Davila6, Care4Rare Canada Consortium1, Martin Holcik1, Jacek Majewski4,5, Julie Richer7,† and Kym M. Boycott1,7,†,* 1 Children’s Hospital of Eastern Ontario Research Institute, 2Division of Metabolics and Newborn Screening, Department of Pediatrics, 3Division of Neurology, Department of Pediatrics, University of Ottawa, Ottawa, Ontario, Canada KIH 8L1, 4Department of Human Genetics, McGill University, Montreal, Quebec, Canada H3A 1B1, 5 McGill University and Genome Quebec Innovation Centre, Montreal, Quebec, Canada H3A 0G1, 6Department of Radiology and 7Department of Genetics, Children’s Hospital of Eastern Ontario, 401 Smyth Road, Ottawa, Ontario, Canada K1H 8L1 *To whom correspondence should be addressed at: Children’s Hospital of Eastern Ontario Research Institute, 401 Smyth Road, Ottawa, ON, Canada K1H 8L1. Tel: +1 6137377600; Email: Abstract Protein translation is an essential cellular process initiated by the association of a methionyl–tRNA with the translation initiation factor eIF2. The Met-tRNA/eIF2 complex then associates with the small ribosomal subunit, other translation factors and mRNA, which together comprise the translational initiation complex. This process is regulated by the phosphorylation status of the α subunit of eIF2 (eIF2α); phosphorylated eIF2α attenuates protein translation. Here, we report a consanguineous family with severe microcephaly, short stature, hypoplastic brainstem and cord, delayed myelination and intellectual disability in two siblings. Whole-exome sequencing identified a homozygous missense mutation, c.1972G>A; p.Arg658Cys, in protein phosphatase 1, regulatory subunit 15b (PPP1R15B), a protein which functions with the PPP1C phosphatase to maintain dephosphorylated eIF2α in unstressed cells. The p.R658C PPP1R15B mutation is located within the PPP1C binding site. We show that patient cells have greatly diminished levels of PPP1R15B–PPP1C interaction, which results in increased eIF2α phosphorylation and resistance to cellular stress. Finally, we find that patient cells have elevated levels of PPP1R15B mRNA and protein, suggesting activation of a compensatory program aimed at restoring cellular homeostasis which is ineffective due to PPP1R15B alteration. PPP1R15B now joins the expanding list of translation-associated proteins which when mutated cause rare genetic diseases. † J.R. and K.M.B. contributed equally as senior authors. Received: May 23, 2015. Revised and Accepted: August 11, 2015 © The Author 2015. Published by Oxford University Press. This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/ licenses/by-nc/4.0/), which permits non-commercial re-use, distribution, and reproduction in any medium, provided the original work is properly cited. For commercial re-use, please contact 6293 initiation factor 2alpha phosphatase gene, PPP1R15B, is associated with severe microcephaly, short stature and intellectual disability 6294 | Human Molecular Genetics, 2015, Vol. 24, No. 22 Introduction Results Patient description The female proband was born to second cousin parents following a pregnancy with exposure to maternal smoking and H1N1. Intrauterine growth retardation was detected prenatally at 5 months of gestation. She was born at 36 weeks 1 day of gestational age, weighing 1.64 kg (−3.1 SD) and measuring 38.1 cm (−6.1 SD) in length with a head circumference of 28.5 cm (−5.0 SD). There were no neonatal complications. She was assessed by the Medical Genetics Service at 12 months of chronological age and noted to have significant developmental delay, distinctive facial features and severe symmetric growth retardation (Fig. 1A): Protein translation is the final step in the genetic expression program, wherein mRNA transcripts are decoded to produce proteins. Translation begins with the association of a methionyltRNA with GTP-bound eukaryotic translation initiation factor 2 (a trimer consisting of eIF2α, eIF2β and eIF2γ subunits) (1,2). The methionyl-tRNA/eIF2-GTP complex (termed ternary complex) then binds a 40S ribosomal subunit which, with the help of a host of other translation initiation factors, assembles on the 5′ end of an mRNA which it scans to find the appropriate start codon and begin polypeptide synthesis (1,2). In stress conditions, eukaryotic cells conserve resources by attenuating protein translation. One such mechanism involves a number of stress-induced kinases that phosphorylate eIF2α at serine 51 ( p-eIF2α), blocking the production of eIF2-GTP (1,2). Once the stress is resolved, PPP1R15A/GADD34 is induced to restore translation by dephosphorylating eIF2α (3). Unstressed cells also contain basal levels of p-eIF2α, and this is countered by constitutively expressed PPP1R15B/CreP (4). PPP1R15A and PPP1R15B function by recruiting the phosphatase PPP1C to eIF2 (3–6). For years, it was assumed that translational dysfunction early in development was not compatible with life; however, a growing number of rare disease mutations have been identified in translational constituents, countering this assumption (7). Examples of translation machinery genes associated with disease include the translation factor eIF2B [leukoencephalopathy with vanishing white matter (VWM)] (8,9), 40S ribosomal subunits RPS19 and RPS24 (Diamond–Blackfan anemia) (10,11), ribosome biogenesis genes SBDS and RMPR (Shwachman–Diamond and cartilagehair hypoplasia, respectively) (12,13) and tRNA maturation and synthesis genes TRNT1, GARS, YARS, KARS and AARS (SIFD syndrome, Charcot–Marie–Tooth disease) (14–18). Given the large number of proteins involved in translation, and the relatively new advent of whole-exome and genome sequencing, it is clear we are just beginning to realize the spectrum of disease-causing mutations impacting this essential biological process. Here, we report two children from a consanguineous family with a novel autosomal recessive disorder characterized by microcephaly, short stature, hypoplastic brainstem and cord, delayed myelination and intellectual disability. Whole-exome sequencing revealed a homozygous missense mutation in the PPP1R15B gene, and studies in patient cells revealed greatly decreased PPP1R15B–PPP1C interactions, which resulted in increased basal levels of p-eIF2α and resistance to cellular stress, and elevation of PPP1R15B mRNA and protein, suggesting activation of an ineffective compensatory response. Our findings add PPP1R15B to the list of translation pathway components which when mutated cause rare genetic diseases. head circumference of 37.3 cm (−6 SD), length 58.6 cm (−5.0 (...truncated)