PYCR2 Mutations Cause a Lethal Syndrome of Microcephaly and Failure to Thrive
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RESEARCH ARTICLE PYCR2 Mutations Cause a Lethal Syndrome of Microcephaly and Failure to Thrive Maha S. Zaki, MD, PhD,1 Gifty Bhat, MD,2,3 Tipu Sultan, MD,4 Mahmoud Issa, MD, PhD,1 Hea-Jin Jung, PhD,2 Esra Dikoglu, MD, PhD,2 Laila Selim, MD,5 Imam G. Mahmoud, MD, PhD,5 Mohamed S. Abdel-Hamid, MD,6 Ghada Abdel-Salam, MD, PhD,1 Isaac Marin-Valencia, MD, MS,2 and Joseph G. Gleeson, MD2 Objective: A study was undertaken to characterize the clinical features of the newly described hypomyelinating leu- kodystrophy type 10 with microcephaly. This is an autosomal recessive disorder mapped to chromosome 1q42.12 due to mutations in the PYCR2 gene, encoding an enzyme involved in proline synthesis in mitochondria. Methods: From several international clinics, 11 consanguineous families were identified with PYCR2 mutations by whole exome or targeted sequencing, with detailed clinical and radiological phenotyping. Selective mutations from patients were tested for effect on protein function. Results: The characteristic clinical presentation of patients with PYCR2 mutations included failure to thrive, microce- phaly, craniofacial dysmorphism, progressive psychomotor disability, hyperkinetic movements, and axial hypotonia with variable appendicular spasticity. Patients did not survive beyond the first decade of life. Brain magnetic reso- nance imaging showed global brain atrophy and white matter T2 hyperintensities. Routine serum metabolic profiles were unremarkable. Both nonsense and missense mutations were identified, which impaired protein multimerization. Interpretation: PYCR2-related syndrome represents a clinically recognizable condition in which PYCR2 mutations lead to protein dysfunction, not detectable on routine biochemical assessments. Mutations predict a poor outcome, probably as a result of impaired mitochondrial function. ANN NEUROL 2016;80:59–70 roline is a nonessential amino acid involved in pro- have major deleterious effects on health, leading to Ptein biosynthesis and antioxidant reactions.1,2 It is intellectual disability,2–4 skin and joint hyperelasticity, endogenously synthesized from glutamate via pyrroline- osteopenia,5,6 and cataracts.7 5-carboxylate synthase (P5CS), which converts glutamate The clinical and molecular phenotype of disorders to pyrroline-5-carboxylate (P5C). P5C can be converted of proline synthesis is currently under intensive investiga- to proline by 1 of 3 paralogues in mammals: PYCR1, tion. To date, mutations only in PYCR1 (Online Mende- PYCR2, and PYCRL, linked to the group of rare PYCR- lian Inheritance in Man [OMIM] 616406) and PYCR2 related disorders (Fig 1A). Defects of proline synthesis (OMIM 179035) have been associated with human View this article online at wileyonlinelibrary.com. DOI: 10.1002/ana.24678 Received Jan 12, 2016, and in revised form Mar 18, 2016. Accepted for publication Apr 17, 2016. Address correspondence to Dr Gleeson, Laboratory for Pediatric Brain Disease, Howard Hughes Medical Institute, Rockefeller University, New York, NY 10065. E-mail: [email protected] From the 1Human Genetics and Genome Research Division, Clinical Genetics Department, National Research Center, Cairo, Egypt; 2Laboratory for Pediatric Brain Disease, Howard Hughes Medical Institute, Rockefeller University, New York, NY; 3Division of Pediatric Genetics, Children’s Hospital at Montefiore, Bronx, NY; 4Pediatric Neurology, Institute of Child Health, Children Hospital, Lahore, Pakistan; 5Cairo University Children’s Hospital, Division of Neurology and Metabolic Disease, Cairo, Egypt; and 6Department of Medical Molecular Genetics, Human Genetics and Genome Research Division, National Research Center, Cairo, Egypt. Additional supporting information can be found in the online version of this article. VC 2016 American Neurological Association 59 ANNALS of Neurology FIGURE 1: Overview of proline metabolism and individuals with PCYR2 mutations. (A) Pyrroline-5-carboxylate reductase 2 (PYCR2) functions in the mitochondria with the paralogue PYCR1 for conversion of pyrroline-5-carboxylate (P5C) to proline. P5C can also be converted to proline in the cytoplasm, mediated by PYCR-like (PYCRL). P5C can alternatively be used as a sub- strate for ornithine synthesis, which can enter the urea cycle, or can be interconverted to glutamate, mediated by pyrroline-5- carboxylate dehydrogenase (P5CDH). Glutamate can then be used as a substrate for the citric acid cycle (CAC) or can be con- verted back to P5C by pyrroline-5-carboxylate synthetase (P5CS). (B) Bar diagram depicting cumulative survival of the 18 sub- jects, including 14 study patients, as well as 4 deceased siblings with similar clinical presentation at the time of ascertainment. The white bars represent the living and gray bars represent the deceased individuals at any given age interval. By age 8 years, 9 individuals (5 study patients and 4 siblings) had died; there are no living survivors beyond 10 years of age. (C) Pedigree dia- grams showing 11 families, with a total of 18 individuals who displayed features of neurodevelopmental delay; 14 children were recruited for the study and found to have homozygous mutations in PYCR2. All pedigrees but 1240 had documented parental consanguinity (double bar), either first-cousin once removed (2566) or first cousin (all others). Generation number and individual family IDs are labeled. Arrows 5 probands in each family with PYCR2 mutation, filled symbols 5 affected subjects, squares 5 males, circles 5 females, hashed 5 deceased, triangles 5 miscarriage. P 5 proband. 60 Volume 80, No. 1 Zaki et al: PYCR2-Related Syndrome disease. Homozygous mutations in PYCR1 were reported The study included 11 families, (family ID 1232, 1240, in families presenting with cutis laxa and progeroid fea- 2206, 2404, 2566, 2664, 2682, 2706, 3109, 3740, and 4592) tures.8,9 Fibroblasts from patients showed defective mito- with 14 affected members with pediatric onset neurological fea- chondrial morphology, reduced mitochondrial membrane tures and autosomal recessive inheritance. Two families (2206 potential, and a 5-fold increase in apoptosis upon oxida- and 2404) had 2 diseased siblings each who presented identi- tive stress.9 Proline levels in plasma were normal. Muta- cally to the ascertained probands, but were deceased prior to ascertainment. None of the families has been previously tions in PYCR2 were recently reported in 2 reported. All patients were examined by at least 1 of the consanguineous families with microcephaly and cerebral 10 authors, and clinical details including videos were recorded at hypomyelination. Similar to patients with PYCR1 the time of each visit. The study was approved by the ethical 8 mutations, no evidence for proline depletion was found committees of participating institutions, and all ascertainment in patient serum, suggesting functional redundancy of was performed with informed consent. multiple PYCR paralogues in proline production. PYCR2-related disorder has been classified as hypomyeli- Genotyping nating leukodystrophy-10 (OMIM 616420) based on the Genomic DNA was extracted from blood leukocytes or saliva report of these 2 families. samples by standard methods from all genetically informative Here, we expand the clinical account of PYCR2- members wherever possible, to include parents, and affected and unaffected offspring. The extracted DNA samples were tested for related disorders by presenting 14 patients from 11 con- quality and family integrity with a panel of fluorescent genotyp- sanguineous families with homozygous PYCR2 muta- ing markers using polymerase chain reaction and analyzed using tions. The clinical phenotype was remarkably consistent an ABI 3130xl automatic DNA Genetic Analyzer and ABI Peak between patients, characterized by microcephaly (occipi- Scanner Software v1.0 (Applied Biosystems, Foster City, CA). tofrontal circumference of 3–6 standard deviations [SD] below the mean), severe psychomotor delay, failure to Whole Exome Sequencing thrive, facial dysmorphism, and white matter hyperinten- Five hundred nanograms of genomic DNA were subjected to whole exome sequencing (WES), using Agilent (Santa Clara, sities with global brain atrophy on magnetic resonance CA) Sure Select Human All Exome 50Mb reagents and the imaging (MRI). Intellectual and motor functions wors- Illumina (San Diego, CA) HiSeq 2000 platform 150bp paired- ened over time, and none of the patients survived beyond end sequencing, resulting in >1003 average exon coverage. the first decade of life. Mutations clustered in the dimeri- Exomes were aligned to hg19 reference genome, and variants zation domain of the protein, and several tested muta- were identified using the GATK Toolkit,11 annotated and pri- tions impaired multimerization. We propose that oritized using custom Python scripts.12 PYCR2-related disorder is a clinically recognizable lethal genetic leukoencephalopathy syndrome without biochem- Sanger Sequencing Potential disease-causing variants identified from WES were ical alterations on standard clinical assays. validated and tested for segregation using fluorescent Sanger sequencing. Each member of the family for whom DNA was Subjects and Methods available was tested for zygosity. Allele frequencies were deter- Patient Material mined from the ExAC data set and from an in-house 5000 We recruited subjects to this study as part of an assessment of WES library of geographically similar individuals. Sequences children with neurodevelopmental disorders presenting to clin- were analyzed with Sequencher 5.1 software (Gene