Characterisation of PLA2G6 as a locus for dystonia-parkinsonism

Abstract

Background:

Although many recessive loci causing parkinsonism dystonia have been identified, these do not explain all cases of the disorder

Methods:

We used homozygosity mapping and mutational analysis in three individuals from two unrelated families who presented with adult-onset levodopa-responsive dystonia-parkinsonism, pyramidal signs and cognitive/psychiatric features and cerebral and cerebellar atrophy on MRI but absent iron in the basal ganglia.

Results:

We identified areas of homozygosity on chromosome 22 and, subsequently PLA2G6 mutations.

Conclusions:

PLA2G6 mutations are associated with INAD and have previously been reported to have early cerebellar signs and the syndrome was classified as neurodegeneration with brain iron accumulation (type 2). Our cases have neither of these previously pathognomic features. Thus, mutations in PLA2G6 should additionally be considered in patients with adult-onset dystonia-parkinsonism even with absent iron on brain imaging.

Introduction

In patients with recessive dystonia-parkinsonism, the differential diagnosis is complex and includes pantothenate kinase-associated neurodegeneration (PKAN, also called Hallervorden Spatz disease or Neurodegeneration with Brain iron Accumulation (NBIA) type 1) caused by mutations in PANK2 on chromosome 20 (OMIM 234200)¹, Kufor-Rakeb syndrome caused by mutations in ATP13A2² on chromosome 1p (OMIM 606693) and DYT16 linked to mutations in PRKRA on chromosome 2.³ It may also include the parkinsonism loci PARK2⁴ (chromosome 6q, OMIM 602544), PARK6⁵ (chromosome 1p, OMIM 608309), PARK7⁶ (chromosome 1p, OMIM 602533) and the dopa-responsive dystonias. In addition, it is likely that there will be other loci for this syndrome.

In order to find the causes of parkinsonism dystonia in consanguineous families and to better group them for clinical characterization and to identify any families which do not have mutations in the known loci, we have perfomed systematic screening using whole genome genotyping methods. Families showing homozygosity at the different loci were then sequenced to find the homozygous gene mutations.

Here, we identified homozygosity on chromosome 22 and, subsequently PLA2G6 mutations in two families. These individuals did not fit the previously described phenotype of this syndrome, which is of Infantile Neuroaxonal Dystrophy (INAD) which is clinically characterized by mental retardation, early cerebellar degeneration pyramidal signs and visual disturbances.⁷ Our finding demonstrates that PLA2G6 mutations should be considered in patients with adult-onset dystonia parkinsonism, even in the absence of iron deposition on MRI.

Methods

Subjects

Ethical/IRB committee approval was obtained at our institutions. We started with ten individuals (8 affected and 2 unaffected) from five unrelated consanguineous families affected by an atypical akinetic rigid syndrome which were sampled. 10ml of venous blood was taken and DNA extracted by conventional methods.

Genotyping

Genotyping was carried out using either Infinium II HumanHap317 BeadChips, which assay 317,000 individual SNPs, or Infinium II HumanHap240 BeadChips and homozygosity mapping was performed as previously described.³

PLA2G6 mutation screening

In the three families which showed homozygosity all 17 coding exons and at least 50bp of flanking intronic sequences were analyzed by performing PCR analysis using 10pmol of both forward and reverse genomic primers (sequence available upon request) as previously described.³

Results

Mutation Analysis

Five affected members from three of the five families shared a homozygous region of chromosome 22q12.3–22q13.1 which contains the PLA2G6 gene. The restricted area compiling 320 consecutive SNPs is flanked by rs2076083 and rs135737 SNPs and was delimited by one affected member from family 1..

Sequencing the coding region of PLA2G6, revealed the presence of different novel mutations in affected members from families 1 and 2. In family 1 two affected members and 6 unaffected members were available for genetic testing. A homozygous mutation at position c.2222G>A resulting in p.R741Q was detected in both patients. Analysis of the p.R741Q mutation in the unaffected individual showed segregation with the disease (figure 1). In family 2, where one affected individual was available for genetic testing, homozygous c.2239C>T (p.R747W) mutation was found. No other family members were available for analysis. Neither variant was found in 186 Pakistani individuals of the Human Genome Diversity Project DNA panel (http://www.cephb.fr/HGDP-CEPH-Panel/) or 40 samples of ethnic Indian/Pakistani origin. Both mutations are conserved among species.

Figure 1:

(A) Pedigree of family 1 (p.R741Q mutation) showing the segregation. (B) Pedigree of family 2 (p.R747W mutation). Full symbols represent affected individuals, empty symbols unaffected family members. Diamond-shaped symbols indicate (unaffected) individuals of unspecified gender. #, further siblings. The index cases are highlighted by an arrow. Double lines indicate consanguineous marriages between first cousins. +/+, mutant; −/+, carrier; −/−, wild type; Neither mutation variant was found in 226 control individuals of ethnic Indian/Pakistani origin.

In family 3, we detected a region of homozygosity over 28,56Mb (table 1) at chromosome 22 as well as homozygous tracts in other areas of the genome. Analysis of PLA2G6 in affected individuals in family 3 and in the remaining two families (4 and 5), without homozygosity at chromosome 22q12.3–22q13.1, did not reveal variation in PLA2G6. We conclude that the disease in these other three families is caused by mutations at unknown loci.

Table 1:

Chromosome position of the SNPs flanking the homozygous chromosome 22 region found in the three ARS families.

ARS families	SNPs (rs number)		Localization (bp)
	Start	End	Start	End
Family 1 (2 patients)	rs2076083	rs135737	35,242,670	37,010,307
Family 2 (1 patient)	rs16996781	rs7288109	35,198,850	37,076,315
Family 3 (2 patients)	rs382013	rs3788684	16,656,101	45,215,256

Clinical features of PLA2G6 homozygotes

Family 1

This 34-year old Indian woman from a consanguineous background (parents were first cousins) had normal milestones. At age 26, over 6 months she developed a rapid cognitive decline, slow movements, clumsiness, imbalance, hand tremor and dysarthria. At 27 she could not walk without assistance. Neuropsychological assessment revealed severe, widespread cognitive dysfunction (verbal IQ 66, severe impairment of verbal and visual memory (scores <5%ile), word retrieval difficulties, frontal executive dysfunction) and depression. She had facial hypomimia, eye lid opening apraxia, square wave jerks in the primary position, supranuclear vertical gaze palsy and hypometric vertical saccades. Kayser-Fleischer rings and pigmentary retinopathy were absent. She was dysarthric with slow tongue movements. There was marked generalized rigidity and dystonia in all limbs, a left pill rolling rest tremor, mild postural arm tremor and bilateral bradykinesia. Power was normal with brisk reflexes, ankle clonus but flexor plantar responses. Assisted gait was narrow-based, stiff-legged and with extremely poor postural reflexes. She tended to walk on her toes.

Investigations revealed increased CK (215 IU/L; normal 24–173). CSF analysis revealed oligoclonal IgG and decreased homovanillic acid but was otherwise normal. Normal results inlcuded electrolytes, coagulation screen, serum copper, caeruloplasmin, ferritin, glucose, liver/renal function tests, serum amino acids, urinary organic acids, very long fatty acids, white cell enzymes, acanthocytes screen, vitamin B12 levels, ANA, TPHA, VDRL, neutrophil cytoplasmatic antibodies, thyroid function tests. Gene tests were normal for IT15, DRPLA, PARK2, SCA-1, −2, −3, −6, −7, ATP13A2. She had a normal phenylalanine loading test, EEG, EMG, nerve conduction studies, VEPs, skin biopsy and bone marrow aspiration.

Brain MRI (Figure 2) showed generalized cerebral atrophy and frontal white signal changes. Absence of iron deposition particularly in the basal ganglia was confirmed by T2* sequence. Spinal MRI was normal. A DaT SPECT scan showed markedly reduced uptake bilaterally in the basal ganglia.

Figure 2:

Bain MRI of patients 1 (A) and 2 (B). A) There is no evidence of iron accumulation in the basal ganglia (T2 *-weighted image, (left)). There are white matter increased signal changes, mainly around the frontal horn (T2 FLAIR (top right, long arrow). There is progressive generalized volume loss. Cerebellar volume is normal (T1-weighted image (bottom right, short arrow)). B) There is no iron deposition in the basal ganglia on T2*-weighted imaging (left). A normal MRI study, apart from mild cerebral atrophy (T1-weighted slides, (right)).

Trihexyphenidyl was not beneficial but levodopa commenced at age 28 had an unsustained but initially dramatic response resulting in almost independent walking. However, she developed prominent dyskinesias.

At 29 she had deteriorated with generalized bradykinesia and severe rigidity causing immobility. She had a staring expression and facial muscle twitches with a factitious smile. At age 32, a PEG had to be inserted.

A cousin was similarly affected with gradual leg onset with dragging and dystonia at age 10. At age 26 she developed arm and leg tremor, bradykinesia and became immobile. Similarly to her cousin, dopaminergic treatment was beneficial but caused prominent dyskinesias. However, little is known about this individual.

Family 2

This 21-year old man of Pakistani descent was a product of double consanguinity (Figure 1). There was no family history of neurological disease. Birth, early milestones and childhood were normal and he attended university. At 18, he developed a fairly rapid onset of dragging of his foot, cognitive decline and personality changes with aggression. He developed urinary frequency and nocturia. Swallowing was normal.

At age 19, neuropsychological assessment revealed impaired intellectual function (verbal IQ 88, performance IQ 74), visual memory, nominal functions and frontal executive dysfunction. He had blepharoclonus. Saccadic pursuit was jerky. There was no supranuclear gaze palsy or nystagmus or Kayser-Fleischer rings. There were asymmetric pyramidal features with spasticity, hyperreflexia, marked ankle clonus and extrapyramidal signs with cogwheeling, bradykinesia and foot dystonia. (see video) The gait was hemiparetic with right leg circumduction and extensor axial dystonia. Postural reflexes were markedly impaired.

Blood tests were all normal. Brain MRI was unremarkable apart from mild generalized volume loss. Iron deposition was excluded by T2* imaging. (Figure 2) Spinal MRI was normal. A DaT SPECT scan shoed markedly reduced uptake in both striata. CSF examination was normal without oligoclonal bands. An EEG was normal.

Ropinirole introduced at age 20 resulted in marked improvement of gait, bradykinesia and dystonia.

Discussion

We studied five families with an atypical akinetic rigid syndrome and describe a yet unrecognized phenotype of PLA2G6 mutations that is adult-onset levodopa-responsive complicated parkinsonism without brain iron accumulation on MRI in two of these families. The remaining families showed more extensive homozygousity tracts (family 3) or no homozygosity of the area which contains the PLA2G6 gene (family 4 and 5).

Mutations in PLA2G6 also cause NBIA and iron was present in all PLA2G6-related NBIA cases recently described.⁸ In addition to marked cerebellar atrophy and progressive white matter changes, iron accumulation of the pallida (affecting medial and lateral portions) were also detected in further six PLA2G6-related INAD patients.⁹ In one, iron was not (yet) present on T2-weighted imaging (T2* scans not presented) 2 years after disease onset but prominent on both T2 and T2*-weighted MRI on 6-year follow up. Our finding of absent iron in the basal ganglia as confirmed by T2* weighted imaging, up to 12 years after disease duration, illustrates that a diagnosis of PLA2G6-related neurodegeneration should not only be considered in patients with dystonia-parksinsonism with brain iron accumulation but also those without.

VEPs and EMGs which are typically abnormal in classic young-onset INAD⁷ were normal in our patient.

Pathologically, INAD is characterized by axonal degeneration with distended axons (spheroid bodies), which stained ubiquitin-positive in PLA2G6-mouse models.¹⁰ However, there is evidence for pathological heterogeneity of INAD as cases with clinical and pathological features of INAD negative for PLA2G6 mutations and in contrast PLA2G6-positve patients without spheroid bodies have both been described.⁸ Brain or peripheral nerve pathological data for patients with adult-onset PLA2G6-related NBIA are not yet available. However, a skin biopsy was normal in our case without evidence of spheroid bodies.

Clinically, our patients showed striking resemblance to Kufor-Rakeb syndrome¹¹ where disease onset was at age 12 −15 years, within the age range of our cases. However, none of the additional clinical features of facial-faucial-finger mini-myoclonus, visual hallucinations, or oculogyric dystonic spasms found in further Kufor Rakeb cases¹² were present in our cases. There also was clinical overlap with PKAN. However, lack of the “eye of the tiger” sign on MRI, the absence of oromandibular dystonia which is often severe in PKAN patients¹³, as well as the absence of pigmentary retinopathy appears to distinguish the syndromes.¹⁴ Onset was in the legs in our cases, in contrast to DYT16.³ where the cranio-cervical region is prominently affected. Furthermore, reported DYT16 cases did not respond to levodopa.

Both mutations we report herein (p.R741Q and p.R747W) are novel: however, different mutations to the same amino acid (R741W) have been previously described in patients with INAD.^8,15 The clustering of mutations at this part of the protein suggests this domain is critical for its function. Little, however, is known about this function. The clinical and pathological similarity of the syndrome caused by PLA2G2 deficiency to those caused by PKAN and ATP13A2 deficiencies suggest that all three gene products may lie on a single biochemical pathway. The fact that we have identified gene mutations in only two of the five families with this syndrome and that the other three families do not all share areas of homozygosity, suggests there must be at least two other genes causing this syndrome. These other genes may map to this same pathway.

Supplementary Material

Video

Segment 1 shows facial hypomimia, dystonic posturing of the right arm, and bilateral slowness (R>L) of finger tapping. When walking the patient has pronounced dystonic axial retropulsion and loss of postural reflexes. Segment 2 shows blepharoclonus (excessive blinking), increased tone with rigidity and a spastic catch in both arms, more on the right, and bilateral ankle clonus. He has difficulty standing up from the chair, postural instability and an assisted stiff gait with dystonic inturning of the right foot.

Table 2:

Summary of demographic and clinical findings..

	Family 1 – pat 1	Family 1 – pat 2	Family 2
Current age	34	n.k.	21
Age of onset / first symptom	26 / cognitive deterioration (subacute)	10/ foot drag	18 / foot drag (subacute)
Cognitive decline	+	n.k.	+
Psychiatric features	+	n.k.	+
Extrapyramidal features:
Tremor	+#	+	Absent
Bradykinesia	++	++	++
Levodopa response	++	++	++##
Levodopa-induced dyskinesias	+	+	n.a.##
Dystonia	++	++	++
Eye movement abnormalities	+	n.k.	+
Imbalance / impaired postural reflexes	++	n.k.	++
Dysarthria	+	n.k.	Absent
Cerebellar signs	Absent	n.k.	Absent
Pyramidal signs	++	n.k.	++
Sensory abnormalities	Absent	n.k.	Absent
Autonomic involvement	Absent	n.k.	+

N.k. = not known; n.a. = not applicable; += mild; ++= severe;

^#=tremor with rest component (pill-rolling);

^{## =}levodopa-naïve, treated with a dopamine agonist