OPEN Molecular Psychiatry (2018) 23, 1487–1495 www.nature.com/mp ORIGINAL ARTICLE The PNKD gene is associated with Tourette Disorder or Tic disorder in a multiplex family N Sun1,2, C Nasello1,2, L Deng1,2, N Wang1,2, Y Zhang1,2,ZXu3, Z Song4, K Kwan4, RA King5, ZP Pang3, J Xing1,2, GA Heiman1,2 and JA Tischfield1,2 Tourette Disorder (TD) is a childhood-onset neuropsychiatric and neurodevelopmental disorder characterized by the presence of both motor and vocal tics. The genetic architecture of TD is believed to be complex and heterogeneous. Nevertheless, DNA sequence variants co-segregating with TD phenotypes within multiplex families have been identified. This report examines whole exomes of affected and unaffected individuals in a multiplex TD family to discover genes involved in the TD etiology. We performed whole exome sequencing on six out of nine members in a three-generation TD multiplex family. Putative deleterious sequence variants co-segregating with TD patients were identified by our in-house bioinformatics pipeline. Induced pluripotent stem cells (iPSCs) were generated from one unaffected and two TD affected individuals. Neurons were derived from the iPSCs and biochemical assays were conducted to evaluate possible molecular differences between affected and unaffected. A rare heterozygous nonsense mutation in PNKD was co-segregated with TD in this multiplex family. Transcript and protein levels of the PNKD long isoform were reduced in neurons derived from the individuals with TD due to the nonsense mutation, indicating nonsense-mediated mRNA decay. We demonstrated that the PNKD long isoform monomer oligomerizes with itself as well as interacts with the synaptic active zone protein RIMS1α. We concluded that reduced PNKD long isoform levels are detected in all affected individuals and we provide evidence for a mechanism whereby this might contribute to the TD phenotype. Molecular Psychiatry (2018) 23, 1487–1495; doi:10.1038/mp.2017.179; published online 12 September 2017 INTRODUCTION the self-oligomerization of the PNKD (L) protein monomer and its Tourette Disorder (TD) is a heritable early-onset neurodevelop- interaction with synaptic protein RIMS1α in transfected human mental disorder characterized by the presence of both motor and cells and their co-localization in the human iPSC-derived neurons. vocal tics with unclear neuropathogenesis as well as complex 1 genetic architecture. The etiology of TD appears to be polygenic MATERIALS AND METHODS and heterogeneous, probably involving dozens of common variants with small effect size or rare variants with larger effect Human subjects size.2 An initial genome-wide association study did not identify A three-generation multiplex family was recruited through the Tourette fi 3 International Collaborative Genetics (TIC Genetics)6 study and New Jersey common variants meeting signi cance thresholds. However, a 7 linkage study on a multiplex family identified a rare dominant- Center for Tourette Syndrome (NJCTS) with informed consent by all participants. The study protocol has been approved by Rutgers University negative nonsense mutation (W317X) at the L-histidine decarbox- Institutional Review Board. ylase (HDC) gene.4 In addition, targeted sequencing of the region where chromosomal abnormalities were found in a TD patient identified the SLITRK1 gene, in which rare recurrent variants were The whole exome sequencing and sanger sequencing further identified.5 To identify potential TD-associated rare Genomic DNA was extracted from the bloods of subjects 3001, 3002, 4001, sequence variant(s) with large effect size, we focused on whole 4002, 5001, 5003 and 5005 and the saliva of subject 5004. whole exome exome sequencing (WES) of multiplex families. In one particular sequencing was performed on subjects 4001, 4002, 5001, 5003 (proband), 5004 and 5005 (Figure 1a). For the WES library preparation, the SureSelect family heavily affected by TD and Tic disorders, a novel nonsense Human All Exon V5+UTR kit (Agilent Technologies, Santa Clara, CA, USA) mutation within the paroxysmal nonkinesigenic dyskinesia (PNKD) was used to capture the targeted region for sequencing on the Illumina gene was identified, which appeared to be a strong TD candidate. HiSeq2000. Variants were called by Geospiza’s GeneSifter (PerkinElmer, To evaluate the impact(s) that the PNKD nonsense mutation Waltham, MA, USA). For Sanger sequencing, the genomic region harboring might have on gene expression and cellular functions, we the nonsense mutation was PCR-amplified with primers shown in generated induced pluripotent stem cells (iPSCs) and differen- Supplementary Table S1. tiated them into neuronal cells in vitro. With the iPSC-derived neurons from unaffected and affected family members, we were pVAAST analysis able to measure the endogenous transcript level and protein The pedigree Variant Annotation, Analysis and Search Tool (pVAAST) expression of the neuronal-specific PNKD long (L) isoform and we program prioritized candidate genes8 under both dominant and recessive examined its subcellular localization. In addition, we demonstrated modes of inheritance (see supplement). 1Department of Genetics, Rutgers University, Piscataway, NJ, USA; 2Human Genetics Institute of New Jersey, Piscataway, NJ, USA; 3Child Health Institute of New Jersey, New Brunswick, NJ, USA; 4Department of Cell Biology and Neuroscience, Piscataway, NJ, USA and 5Child Study Center, Yale School of Medicine, New Haven, CT, USA. Correspondence: Dr JA Tischfield, Department of Genetics, Rutgers University, 145 Bevier Road, Piscataway, NJ 08854, USA. E-mail: [email protected] Received 13 December 2016; revised 24 May 2017; accepted 7 June 2017; published online 12 September 2017 PNKD gene is associated with Tourette or Tic Disorders N Sun et al 1488 Figure 1. A rare heterozygous nonsense mutation (C to T transition) was identified in a TD multiplex family by whole exome sequencing. (a) We performed whole exome sequencing on 4001, 4002, 5001, 5003 (proband), 5004 and 5005. (b) The heterozygous nonsense mutation at the PNKD gene was present in subjects 3001, 4001, 5001, 5003, 5004 and 5005. TD, Tourette disorder. Generation and maintenance of iPSCs Real-time quantitative PCR Induced pluripotent stem cells were generated from the CD4+ T lymphocytes Total RNA was extracted using the RNeasy Mini kit (QIAGEN, Hilden, using CytoTune-iPS reprogramming kit.9 Induced pluripotent stem cells were Germany). cDNA was made from 1ug total RNA using TaqMan Reverse cultured on Matrigel-coated plates in mTeSR (see supplement). Transcription Reagents (Thermo Fisher Scientific, Waltham, MA, USA). Real- time quantitative PCR (RT-qPCR) was performed on ABI PRISM 7900HT Sequence Detection System (Applied Biosystems, Foster City, CA, USA). Generation and maintenance of neural progenitor cells and Primer information is shown in Supplementary Table S1. neurons neural progenitor cells were generated from iPSCs using a neural rosette- Plasmids construction and transfection based protocol. Neurons were derived from NPCs by culturing the NPCs in Human PNKD (L) cDNA was cloned from cDNA ORF Clone (RC206179) neural differentiation medium (see supplement). (Origene, Rockville, MD, USA) and was inserted into the pcDNA4/TO vector. Molecular Psychiatry (2018), 1487 – 1495 PNKD gene is associated with Tourette or Tic Disorders N Sun et al 1489 CLASS III β TUBULIN / MAP2 CLASS III β TUBULIN / MAP2 CLASS III β TUBULIN / MAP2 CLASS III β TUBULIN / MAP2 Unrelated Unaffected 20 µm TD1 TD2 Unaffected Spontaneous MAP2 / Synapsin Whole cell currents action potentials n=5/11 n=6/11 µ 50µm Unaffected 20 m 200 pA 25 mV MAP2 / Synapsin 5 ms 5 s Sodium currents n=5/12 n=9/12 TD1 MAP2 / Synapsin n=4/14 n=9/14 TD2 n=5/11 MAP2 / Synapsin n=8/11 Unrelated Unaffected 500 pA 200 ms 40 Ω 1600 60 e 30 1200 ran b 40 m 20 800 e ntial (-mV) e ng M t i t 20 o 10 400 P Res 11 12 14 11 11 12 14 11 11 12 14 11 Membrane Capacitance (pF) 0 Memberane Resistance (M ) 0 0 TD1 TD2 TD1 TD2 TD1 TD2 Unrelated Unrelated Unrelated Unaffected Unaffected Unaffected Unaffected Unaffected Unaffected Figure 2. Functional characterizations of neurons derived from iPSCs. (a) Neurons derived from unaffected as wells as TD subjects express mature neuronal marker MAP2 and Class III β-tublin. (b) Whole cell currents responses were recorded from human neurons derived from subjects with and without TD. (c) Spontaneous action potentials were recorded from human neurons derived from subjects with and without TD. (d) Synapsin, a synaptic vesicle marker, was detected in human neurons with and without TD. (e) Membrane capacitance, (f) Membrane resistance and (g) resting membrane potentials of human neurons derived from subjects with and without TD were quantified. iPSC, induced pluripotent stem cells; TD, Tourette disorder. By PCR, a FLAG tag or a Myc tag was added to the PNKD (L) cDNA sequence transfection reagent (Thermo Fisher Scientific). To transfect 293FT (Thermo as shown: 5′-FLAG-PNKD-3′ or 5′-PNKD-Myc-3′. To express human RIMS1α, Fisher Scientific) cells plated in 10 cm dishes, we used 1 mg ml − 1 PEI RIMS1 transcript variant 1, cDNA ORF Clone (RC213013) (Origene) was used. solution (Sigma-Aldrich, St. Louis, MO, USA). A room temperature mixture of The expression of GFP cloned into the pcDNA4/TO vector monitored plasmids, transfection reagents and Opti-MEM reduced-serum medium were transfection efficiency. To transfect neurons plated on poly-L-ornithine/ added to the cultures. Two days later, cells were collected for RNA laminin/fibronectin-coated 12 mm coverslips, we used Lipofectamine 2000 extraction, or lysed in protein lysis buffer or fixed for immunocytochemistry. Molecular Psychiatry (2018), 1487 – 1495 PNKD gene is associated with Tourette or Tic Disorders N Sun et al 1490 Western blot and co-immunoprecipitation Neuronal cells were generated from the human subjects through Cells were lysed in RIPA buffer for western blot analysis or in RIPA IP buffer iPSC intermediates for co-immunoprecipitation. Anti-FLAG M2 Magnetic beads (Sigma-Aldrich) We generated NPCs and subsequently neurons from iPSCs of eluted FLAG-tagged proteins.