Elsevier

Neuroscience

Volume 384, 1 August 2018, Pages 152-164
Neuroscience

Research Article
Gain-of-function KCNJ6 Mutation in a Severe Hyperkinetic Movement Disorder Phenotype

https://doi.org/10.1016/j.neuroscience.2018.05.031Get rights and content

Highlights

  • Case study of human with de novo mutation in KCNJ6 (GIRK2)

  • Clinical findings of severe hyperkinetic movement disorder and developmental delay.

  • Mutation near pore of GIRK2 alters G protein activation and ion selectivity.

  • Inward depolarizing current blocked by QX-314, similar to GIRK2 weaver mutation.

  • Screen for possible KCNJ6 mutations in patients with severe movement disorders.

Abstract

Here, we describe a fourth case of a human with a de novo KCNJ6 (GIRK2) mutation, who presented with clinical findings of severe hyperkinetic movement disorder and developmental delay, similar to the Keppen–Lubinsky syndrome but without lipodystrophy. Whole-exome sequencing of the patient’s DNA revealed a heterozygous de novo variant in the KCNJ6 (c.512T>G, p.Leu171Arg). We conducted in vitro functional studies to determine if this Leu-to-Arg mutation alters the function of GIRK2 channels. Heterologous expression of the mutant GIRK2 channel alone produced an aberrant basal inward current that lacked G protein activation, lost K+ selectivity and gained Ca2+ permeability. Notably, the inward current was inhibited by the Na+ channel blocker QX-314, similar to the previously reported weaver mutation in murine GIRK2. Expression of a tandem dimer containing GIRK1 and GIRK2(p.Leu171Arg) did not lead to any currents, suggesting heterotetramers are not functional. In neurons expressing p.Leu171Arg GIRK2 channels, these changes in channel properties would be expected to generate a sustained depolarization, instead of the normal G protein-gated inhibitory response, which could be mitigated by expression of other GIRK subunits. The identification of the p.Leu171Arg GIRK2 mutation potentially expands the Keppen–Lubinsky syndrome phenotype to include severe dystonia and ballismus. Our study suggests screening for dominant KCNJ6 mutations in the evaluation of patients with severe movement disorders, which could provide evidence to support a causal role of KCNJ6 in neurological channelopathies.

Introduction

Genetic neurological channelopathies are a group of heterogeneous disorders, usually with dominant inheritance, and causing some form of paroxysmal neurological disturbances in function, which may become permanent in time (Spillane et al., 2016). For example, ion channelopathies have been implicated in diseases including epileptic encephalopathies, ataxia, paroxysmal dyskinesias, migraine, pain syndromes, skeletal muscle disorders, and periodic paralysis (Spillane et al., 2016). Under normal conditions, ion channels serve an important role of permitting rapid and selective movement of ions across plasma membranes, influencing the excitability of neurons and subsequent signaling in the brain. Channelopathies result from defects in the ion channel function or from changes in trafficking to the plasma membrane. A large number of channelopathies involve potassium channels, which can be divided into three families: voltage-gated K+ channels (KV1–18), two-pore K+ channels (K2P1–K2P18), and inwardly rectifying K+ channels (KIR1–KIR7) (Serratrice et al., 2010). KV channels contribute to the repolarization of the action potential. Defects in this family of channels commonly lead to some form of epilepsy. For example, mutations in KCNQ2 (KV7.2) and KCNA1 (KV1.2) cause benign familial neonatal seizures and myoclonic epilepsy, respectively (Villa and Combi, 2016). Thus, loss of these channels leads to dramatic increase in neuronal excitability. K2P channelopathies have been linked to familial migraine with aura, and have been attributed to have a role in pain sensation (Serratrice et al., 2010).

Inwardly rectifying potassium (KIR) channels play a key role in the maintenance of the resting potential and regulation of cell excitability (Hibino et al., 2010, Lüscher and Slesinger, 2010). These channels lack the voltage-sensitivity of their Kv counterparts and preferentially limit the outward flow of K+ to a voltage range near the resting membrane potential. KIR channels can have high basal activity (constitutively open), such as KIR2 channels, or low basal activity that can be enhanced by ligands such as G proteins and alcohol, such as KIR3 channels. There are seven different KIR families (KIR1–KIR7) that form either homotetramers or heterotetramers. However, KIR channels typically assemble with only members from the same subfamily, for example, KIR3.1 does not coassemble with KIR2.1 but it does co-assemble with KIR3.2 (Hibino et al., 2010, Lüscher and Slesinger, 2010). The constitutively active KIR2 (KCNJ2) has been associated with the Andersen syndrome, a human disease that is characterized by periodic paralysis, cardiac arrhythmia, and dysmorphism (Plaster et al., 2001). Point mutations in KIR6.2 (KCNJ11) have been described for the human disease developmental delay, epilepsy and neonatal diabetes mellitus (DEND) syndrome, a treatable channelopathy of the brain and pancreas (Hibino et al., 2010, Lüscher and Slesinger, 2010). The G protein-gated inwardly-rectifying K+ (GIRK) channel belongs to the KIR3 subfamily and is a regulator of cardiac and neuronal excitability (Hibino et al., 2010, Lüscher and Slesinger, 2010). There are four GIRK channel subunits in humans, referred to as KIR3.1 (KCNJ3), KIR3.2 (KCNJ6, Girk2), KIR3.3 (KCNJ9), and KIR3.4 (KCNJ5) (for review, see Hibino et al., 2010, Lüscher and Slesinger, 2010). Mice lacking GIRK channels display altered responses to addictive drugs and in the case of the Girk2 knockout, result in epileptic seizures (for review, see Rifkin et al., 2017). Heretofore, human neurological diseases caused by mutations in GIRK channels have been uncommon.

Recently, dominant mutations in KCNJ6 (GIRK2) have been linked to the Keppen–Lubinsky Syndrome (MIM# 614098) (Gorlin and Hennekam, 2001), a disorder characterized by lipodystrophy, severe developmental delay, intellectual disability, hypertonia, hyperreflexia and growth retardation (Gorlin and Hennekam, 2001, Masotti et al., 2015). Three patients were reported to have KCNJ6 mutations near the ion selective pore; one patient with a mutation (p. Gly154Ser) corresponding to the developmentally impaired weaver mouse (Rakic and Sidman, 1973, Rakic and Sidman, 1973, Goldowitz and Mullen, 1982, Hatten et al., 1986, Goldowitz, 1989, Patil et al., 1995, Hess, 1996) and two patients with deletions in the pore (p.Thr152del). These three patients all show lipodystrophy, which ranges from a generalized loss of adipose tissue in one case, to a restricted loss of facial adipose tissue in two cases (Masotti et al., 2015). (De Brasi et al., 2003, Basel-Vanagaite et al., 2009). Thus, lipodystrophy is a hallmark of the Keppen–Lubinsky Syndrome.

Here we report a case study with a de novo KCNJ6 missense mutation [NM_002240: c.512T>G; NP_002231: p. Leu171Arg], without recognizable lipodystrophy, i.e., no obvious absence of facial adipose tissue. Instead, the patient presented with developmental delay, hypotonia, and a severe hyperkinetic movement disorder. Epilepsy was also not present. The p. Leu171Arg mutation was determined to be a gain-of-function, with dramatic changes in G protein activation and ion selectivity. Though, p. Leu171Arg resides in a different region of the protein, the electrophysiological phenotype resembles that described in the murine weaver (wv) mouse, which has extensive developmental defects and also carries a mutation in KCNJ6 (Patil et al., 1995, Hess, 1996).

Section snippets

Experimental procedures

Whole-exome sequencing. Patient and family were enrolled into the TIDEX gene discovery study (UBC IRB approval H12-00067) and provided informed and written consent for data and sample collection, WES, as well as publication of the current case report. WES was performed for the index and her unaffected parents using the Agilent SureSelect kit and Illumina HiSeq 2000 (Perkin-Elmer, Waltham, MA, USA). Data were analyzed using our semi-automated bioinformatics pipeline (Tarailo-Graovac et al., 2016

Clinical findings

The patient is a 4-year old girl from non-consanguineous parents, with an unremarkable pregnancy and term delivery. Her birth weight was 3210 g (30th percentile), and there were no neonatal complications. The first concerns arose when she did not achieve head control by the age of 6 months. Her mother also noticed posturing of her legs, abnormal movements of her arms, and arching of her back. The patient had frequent episodes in which her eyes rolled up. These movements at times were quite

Discussion

Here, we report a gain-of-function mutation in the KCNJ6 (Girk2) gene contributes to a human neurological disease that has many similarities to the Keppen–Lubinsky syndrome. Whole-exome sequencing of the patient and her parents revealed a de novo variant, p.Leu171Arg, located on chromosome 21 in KCNJ6 of the patient. Expression of the GIRK2 mouse homolog with the mutation, GIRK2(L173R), alone or in the presence of the GIRK1 subunit, revealed several significant changes in channel properties.

Video consent

The authors received a signed release form for the patient videotaped, authorizing the offline and/or online distribution of this video material.

Acknowledgments

We gratefully acknowledge the patient and family for their participation in this study; Dr. G. Sinclair and Dr. H. Vallance for interpretation of biochemical data; Ms. E. Aisenberg for creating the mouse L173R mutation in Girk2, Ms. X. Han for Sanger sequencing; Ms. A. Ghani for consenting and data management; Mrs. M. Higginson for DNA extraction, sample handling and technical data; Mr. D. Arenillas and Mr. M. Hatas for systems support, Ms E. Lomba and Mrs. D. Pak for research management

Author contributions

Gabriella Horvath worked on conception and design, acquisition of data and drafted a significant portion of the manuscript. Yulin Zhao collected all of the electrophysiology data, analyzed data and edited the manuscript. Maja Tarailo-Graovac worked on conception and design and edited a significant portion of the manuscript, worked on acquisition and analysis of the data, identifying the KCNJ6 variant. Cyrus Boelman worked on acquisition and analysis of the data and edited a significant portion

Conflict of interest

The authors have no conflict(s) of interest to declare.

References (49)

  • K. Arnold et al.

    The SWISS-MODEL workspace: a web-based environment for protein structure homology modelling

    Bioinformatics

    (2006)
  • L. Basel-Vanagaite et al.

    Keppen-Lubinsky syndrome: expanding the phenotype

    Am J Med Genet A

    (2009)
  • M. Biasini et al.

    SWISS-MODEL: modelling protein tertiary and quaternary structure using evolutionary information

    Nucleic Acids Res

    (2014)
  • K. Bodhinathan et al.

    Molecular mechanism underlying ethanol activation of G-protein-gated inwardly rectifying potassium channels

    Proc Natl Acad Sci USA

    (2013)
  • D. De Brasi et al.

    New syndrome with generalized lipodystrophy and a distinctive facial appearance: confirmation of Keppen-Lubinski syndrome?

    Am J Med Genet A

    (2003)
  • D. Goldowitz et al.

    Granule cell as a site of gene action in the weaver mouse cerebellum: evidence from heterozygous mutant chimeras

    J Neurosci

    (1982)
  • R.J.C.M. Gorlin et al.

    Keppen-Lubinski syndrome

    (2001)
  • N. Guex et al.

    Automated comparative protein structure modeling with SWISS-MODEL and Swiss-PdbViewer: a historical perspective

    Electrophoresis

    (2009)
  • M.E. Hatten et al.

    Weaver mouse cerebellar granule neurons fail to migrate on wild-type astroglial processes in vitro

    J Neurosci

    (1986)
  • H. Hibino et al.

    Inwardly rectifying potassium channels: their structure, function, and physiological roles

    Physiol Rev

    (2010)
  • A. Inanobe et al.

    Characterization of G-protein-gated K+ channels composed of Kir3.2 subunits in dopaminergic neurons of the substantia nigra

    J Neurosci

    (1999)
  • C. Karschin et al.

    IRK(1–3) and GIRK(1–4) inwardly rectifying K+ channel mRNAs are differentially expressed in the adult rat brain

    J Neurosci

    (1996)
  • F. Kiefer et al.

    The SWISS-MODEL Repository and associated resources

    Nucleic Acids Res

    (2009)
  • M. Kircher et al.

    A general framework for estimating the relative pathogenicity of human genetic variants

    Nat Genet

    (2014)
  • Cited by (0)

    This work was supported by grants from the National Institutes of Health (DA037170 to PAS; AA018734 to PAS); the B.C. Children’s Hospital Foundation (“1st Collaborative Area of Innovation” www.tidebc.org); Genome British Columbia (SOF-195); the Canadian Institutes of Health Research (#301221); NeuroDevNet (Strategic Opportunity Grant); and informatics infrastructure supported by Genome British Columbia and Genome Canada (ABC4DE Project). Dr. van Karnebeek is a recipient of the Michael Smith Foundation for Health Research Scholar Award.

    Denotes equal contribution.

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