Identification of <em>ATP1A3</em> Mutations by Exome Sequencing as the Cause of Alternating Hemiplegia of Childhood in Japanese Patients

<div><h3>Background</h3><p>Alternating hemiplegia of childhood (AHC) is a rare disorder characterized by transient repeated attacks of paresis and cognitive impairment. Recent studies from the U.S. and Europe have described <em>ATP1A3</em> mutations in AHC. However, the genotype-phenotype relationship remains unclear. The purpose of this study was to identify the genetic abnormality in a Japanese cohort of AHC using exome analysis.</p> <h3>Principal Findings</h3><p>A total of 712,558 genetic single nucleotide variations in 8 patients with sporadic AHC were found. After a series of exclusions, mutations of three genes were regarded as candidate causes of AHC. Each patient harbored a heterozygous missense mutation of <em>ATP1A3</em>, which included G755C, E815K, C927Y and D801N. All mutations were at highly conserved amino acid residues and deduced to affect ATPase activity of the corresponding ATP pump, the product of <em>ATP1A3</em>. They were <em>de novo</em> mutations and not identified in 96 healthy volunteers. Using Sanger sequencing, E815K was found in two other sporadic cases of AHC. In this study, E815K was found in 5 of 10 patients (50%), a prevalence higher than that reported in two recent studies [19 of 82 (23%) and 7 of 24 (29%)]. Furthermore, the clinical data of the affected individuals indicated that E815K resulted in a severer phenotype compared with other <em>ATP1A3</em> mutations.</p> <h3>Interpretation</h3><p>Heterozygous <em>de novo</em> mutations of <em>ATP1A3</em> were identified in all Japanese patients with AHC examined in this study, confirming that <em>ATP1A3</em> mutation is the cause of AHC.</p> </div

Distribution of novel non-synonymous single nucleotide polymorphisms including brain-expressed genes in eight patients with AHC.

<p>NS: non-synonymous variants, SS: splice-site acceptor/donor variants.</p

Chromatograms of four <i>de novo</i> mutations identified in <i>ATP1A3</i>.

<p>Data were obtained by Sanger sequencing during the confirmation process. In trio of each pedigree, black shadow represents the proband. In the chromatograms, <i>Black letters</i> show exonic nucleotide sequences, <i>gray letters</i> show intronic nucleotide sequences. Amino acids are shown in a single letter notation. Nucleotides and amino acids in red indicate mutations. (A) G755C was identified only in Patient I-1. (B) E815K was identified in Patients II-1, III-1, IV-1, IX-1 and X-1. (C) C927Y was identified in Patient V-1 only. (D) D801N was identified in Patients VI-1, VII-1 and VIII-1. None of the mutations was detected in the father or mother except for Patient IX-1, whose parents refused to undergo genetic analysis.</p

Clinical data of 10 unrelated individuals with AHC.

<p>MDL: midazolam, CZP: clonazepam, L: left, R: right.</p

<i>ATP1A3</i> mutations and their protein domain structures.

<p><i>Black lined circle:</i> Mutations reported recently <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0056120#pone.0056120-Heinzen1" target="_blank">[10]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0056120#pone.0056120-Rosewich1" target="_blank">[11]</a>. <i>Red colored circle:</i> Mutations identified in the present study in a Japanese cohort with AHC. The <i>ATP1A3</i> gene consists of 23 exons that encode several domains in the ATP1A3 protein molecule, including 6 cytoplasmic, 10 helical and 5 extracellular domains. G755C and E815K were located in the cytoplasmic domains. Notably, E815K was resident of the transmembrane domain rather than the cytoplasmic domain. D801N and C927Y were located in the helical domains. C927Y was identified in this study only and hence considered novel.</p

Pipeline for detection of novel <i>de nov</i>o mutations.

<p>The pipeline was used to identify pathogenic mutations of alternating hemiplegia of childhood (AHC). All genetic variants detected by exome sequencing are sequentially filtered through the pipeline. First, variations are screened according to databases of registered single nucleotide polymorphisms (SNP) and only non-registered SNP undergo the next selection as “Novel variants”. In the next step, non-synonymous novel variants of genes expressed in the central nervous system are selected. When variations of the same gene are found in the patient, the impact of such variation is evaluated <i>in silico</i> using Grantham score and PolyPhen-2. Mutations identified at this stage are reconfirmed by Sanger sequence. <i>De novo</i> mutation is validated by analyzing samples from parents. Mutations considered pathogenic are sought in other patients with AHC if necessary.</p

Homologous comparison of altering-protein.

<p><i>Blue letters:</i> altering-protein by mutation, <i>red letters:</i> differential protein with human. (A) G755C changed by novel SNVs (c.2263G>T) of <i>ATP1A3</i> in Patient I-1. (B) E815K changed by novel SNVs (c.2443 G>A) of <i>ATP1A3</i> in Patients II-1, III-1, IV-1, IX-1 and X-1. (C) C927Y changed by novel SNVs (c.2780 G>A) of <i>ATP1A3</i> in Patient V-1. (D) D801N changed by novel SNVs (c.2401 G>A) of <i>ATP1A3</i> in Patient VI-1, VII-1 and VIII-1.</p

Pedigrees of six Japanese familial episodic pain syndrome in Japanese families.

<p>(A) Some ^a)Family 2 and ^b)Family 3 members have been reported previously [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0154827#pone.0154827.ref013" target="_blank">13</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0154827#pone.0154827.ref014" target="_blank">14</a>]. Black and white symbols indicate affected and unaffected individuals, respectively. Gray symbols indicate individuals with unknown phenotypic status. Squares and circles indicate males and females, respectively. Slashes indicate deceased individuals. “P” indicates probands. Blue arrows indicate exome sequenced individuals. * indicates linkage analysis performed individuals. The genotype of <i>SCN11A</i> p.R222H (Family 1, 2, 4, 5 and 6) or <i>SCN11A</i> p.R222S (Family 3) for each individual is illustrated. (B) Sequence chromatography of the identified <i>SCN11A</i> mutations.</p

Three subtypes of clinical manifestations.

<p>Three subtypes of clinical manifestations.</p

Genome-wide linkage analysis in two Japanese familial episodic pain syndrome families.

<p>Genome-wide linkage analysis was performed for eight affected and five unaffected members in Family 1, and four affected and four unaffected members in Family 2. Parametric linkage analysis was performed using 386 genetic markers (including 382 microsatellite genetic markers) that were 10 cM apart and covered 22 autosomes, as well as additional SNP markers. GeneHunter software was used.</p