The PNKD gene is associated with Tourette Disorder or Tic disorder in a multiplex family

Abstract

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.

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.

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.

Figure 3. PNKD expression in LCL, NPC, iPSC and iPSC-derived D30 neurons by RT-qPCR

A. PNKD (L) transcript expression increased from iPSC to NPC and from NPC to D30 neurons in the Uaf subject, indicating that PNKD (L) was enriched in neuronal cells (n = 3). B. PNKD (S) transcript is expressed in all the cell types of all three subjects. PNKD (S) transcript level increased during neural differentiation (n = 3). C. PNKD (M) transcript is expressed at low levels in iPSCs, NPCs and iPSC-derived neurons (n = 3). D. PNKD (L) transcript was significantly reduced in iPSC-derived D30 neurons of TD1 and TD2 subjects compared to the Uaf subject (n = 3). E. PNKD (L) protein was detected in D30 neurons but not in LCL or iPSC. PNKD (L) protein was reduced in iPSC-derived D30 neurons of subjects TD1 and TD2 compared to the Uaf subject (n = 3).

Figure 4. The human PNKD (L) protein oligomerizes with itself and interacts with the RIMS1 α protein

A. 239FT cells overexpressing GFP, FLAG-PNKD(L), PNKD(L)-Myc or FLAG-PNKD(L) + PNKD(L)-Myc were lysed for Co-IP. The PNKD(L)-Myc was pulled down by the anti-FLAG magnetic beads only in the presence of the FLAG-PNKD(L). B. 239FT cells overexpressing GFP, RIMS1α-FLAG, PNKD(L)-Myc or RIMS1α-FLAG+PNKD(L)-Myc were lysed for Co-IP. The PNKD(L)-Myc was eluted by the anti-FLAG magnetic beads only in the presence of the RIMS1α-FLAG.

Figure 5. Endogenous RIMS1a expression in Uaf and TD cells

A. RIMS1α transcript was not expressed in LCL, was expressed at low levels in iPSC and NPC and was enriched in iPSC-derived D30 neurons (n = 3). B. RT-qPCR measured the RIMS1α transcript in iPSC-derived D30 neurons of Uaf, TD1 and TD2 subjects. D30 neurons from three independent differentiations were collected. There was no significant difference between the expression of the RIMS1α transcript in Uaf and TD neurons (n = 3). C. RIMS1 protein was examined by western blot in D30 neurons from three independent differentiations. RIMS1 protein was reduced in TD neurons.