Mutations in PRRT2 Gene Cause Episodic Diseases: From Paroxysmal Dyskinesia to Episodic Ataxia and Hemiplegic Migraine

1MRC

Alice Gardiner1, Fatima Jaffer1,2, Dimitri Kullmann1,2, Nicholas Wood2, Louis Ptacek2, Henry Houlden1,2 and Michael Hanna1,2

Centre for Neuromuscular Diseases and the 2 Paroxysmal/episodic ataxia collaborative group, UCL Institute of Neurology

Background
Paroxysmal Kinesigenic Dyskinesia (PKD) Paroxysmal kinesigenic dyskinesia is the commonest of the paroxysmal dyskinesias, a group of episodic movement disorders. Patients with PKD experience up to 20 attacks of involuntary muscle movements a day, usually triggered by sudden voluntary movements such as standing after a period of sitting. It is an autosomal dominant disorder, and can be present with or without infantile convulsions (PKC/IC). The episodic nature of the disease as well as the connection with seizures meant that it was historically thought be part of the spectrum of neurological conditions caused my mutations in voltage gated ion channels, but for many years the exact genetic cause was unknown. However, in the past year several groups have used exome sequencing to identify the causative of PKD gene as not a channel gene, but PRRT2, a gene, coding for a proline-rich transmembrane protein (PRRT2)1,2,3,4 PRRT2 PRRT2 is a protein of unknown function. It has four exons, although all of exon and one and most of exon four are non-coding. It is known to have two transmembrane regions. Mutations in patients with PKD or PKD/IC have been found across the gene by several groups1,2,3,4 (Figure 1), although one mutation, p.R217Pfs*8, an insertion of a cysteine in a string of 8 cysteines is by far the most common. The mechanism of disease for PKD is unknown, although Lee et al recently showed that it is expressed in the brain and spinal cord, and that it interacts with the synaptic protein SNAP25, hypothesising .that it is caused by a disruption in the vesicle release pathway.
S172R*3 S124Vfs*10 Q163X ATG (1) PRRT2 Exon 1 218bp Exon 2 944bp Exon 3 133bp R217Pfs*8

Results
Mutations in PRRT2 were seen in 27 families/sporadic patients with PKD or PKD/IC, of which 11 also had migraine, hemiplegic migraine or seizures. In addition, a mutation was found in one family with EA and HM and one with just HM. As seen before, the majority of the mutations were the reported p.R217Pfs*8, but 5 other mutations were also found, and 4 were novel (Table 1). All mutations were heterozygous.

Mutation p.R217Pfs*8

Frequency Novel? Chromatogram 26 no yes yes yes no yes

p.311InsGAC 1 P215R P216H p.L171Lfs*3 G305W 1 1 1 1

Table 1 Details and chromatograms of the mutations found in this study

R266W A287T Splice W281R site

R308C V322Wfs*15 V325Sfs*12

Discussion
The cohort of patients with PKD and PKD/IC
Exon 4 1254bp

TGA (340)

As expected, we found that mutations in PRRT2 were a common cause of PKD and PKD/IC in our patients. Mutations were found in 47% of patients, which is similar to the previously reported figure of 50.4%3. The cohort of patients with episodic ataxia and hemiplegic migraine Although we found mutations in PRRT2 in both of these groups, the frequency was very low, at less than 1% each. Therefore, we have identified a new but rare genetic cause for both episodic ataxia and hemiplegic migraine and extended the clinical spectrum of this gene. Figure 2 shows how we think PRRT2 can be put together with our existing knowledge of these disorders to create an overlapping spectrum of phenotypes and causative genes. Dystonia

Figure 1 The PRRT2 gene has four exons, exon 1 is non-coding, the gene length is 340 amino acids, 1020bp. The start ATG (codon 1) is indicated in the figure and all mutations are labeled from this codon up to the stop codon TGA at 340. Reference sequence NM_145239, Ensemble ENST00000358758. Adapted from Gardiner et al5.

Objectives
There are two main objectives to this study; 1.To screen our large cohort of patients with PKD or PKD/IC for mutations in the PRRT2 gene. 2.To screen a cohort of patients with the similar episodic disorders, episodic ataxia and hemiplegic migraine, for mutations in the PRRT2 gene to extend the phenotypic spectrum of this gene, as well as try to understand the mechanism further.

Ataxia
KCNA1 PATX CACNB4 CACNA1A SLC1A3

DYT9 SLC2A1

PRRT2

Headache

Figure 2 A Venn diagram showing the three related disorders and the genes which are known to cause them.

Methods
We used Sanger sequencing to screen a cohort of 58 patients with PKD or PKD/IC, 182 with episodic ataxia (EA) and 128 hemiplegic migraine (HM) for mutations in the 3 coding exons of PRRT2. Mutations found were confirmed in both the forward and reverse directions

SCNA1 KCNK18 KCNG4 KCNAB3

Mechanism There is still much work to be done before the mechanism of this disease is fully understood, however this work indicates there could be an overlap with the pathways of the other channel disorders

References
1Chen

W. J., et al. (2011) Nat Genet 43, 1252-5 2Li J., et al. (2012) J Med Genet 49, 76-8 3Lee et al. (2012) Cell Reports 1, 2-12

4Wang

J. L., et al (2011) Brain 134, 3490-3498 5Gardiner et al (2012) Neurology in press