The expanding clinical and genetic spectrum of ATP1A3-related disorders Hendrik Rosewich, Andreas Ohlenbusch, Peter Huppke, et al. Neurology 2014;82;945-955 Published Online before print February 12, 2014 DOI 10.1212/WNL.0000000000000212 This information is current as of February 12, 2014

The online version of this article, along with updated information and services, is located on the World Wide Web at: http://www.neurology.org/content/82/11/945.full.html

Neurology ® is the official journal of the American Academy of Neurology. Published continuously since 1951, it is now a weekly with 48 issues per year. Copyright © 2014 American Academy of Neurology. All rights reserved. Print ISSN: 0028-3878. Online ISSN: 1526-632X.

The expanding clinical and genetic spectrum of ATP1A3-related disorders

Hendrik Rosewich, MD Andreas Ohlenbusch, PhD Peter Huppke, MD Lars Schlotawa, MD Martina Baethmann, MD Inês Carrilho, MD Simona Fiori, MD Charles Marques Lourenço, MD Sarah Sawyer, MD, PhD Robert Steinfeld, MD, PhD Jutta Gärtner, MD* Knut Brockmann, MD*

Correspondence to Dr. Gärtner: [email protected]

ABSTRACT

Objective: We aimed to delineate the clinical and genetic spectrum of ATP1A3-related disorders and recognition of a potential genotype-phenotype correlation.

Methods: We identified 16 new patients with alternating hemiplegia of childhood (AHC) and 3 new patients with rapid-onset dystonia-parkinsonism (RDP) and included these as well as the clinical and molecular findings of all previously reported 164 patients with mutation-positive AHC and RDP in our analyses. Results: Major clinical characteristics shared in common by AHC and RDP comprise a strikingly asymmetric, predominantly dystonic movement disorder with rostrocaudal gradient of involvement and physical, emotional, or chemical stressors as triggers. The clinical courses include an early-onset polyphasic for AHC, a later-onset mono- or biphasic for RDP, as well as intermediate forms. Meta-analysis of the 8 novel and 38 published ATP1A3 mutations shows that the ones affecting transmembrane and functional domains tend to be associated with AHC as the more severe phenotype. The majority of mutations are located in exons 8, 14, 17, and 18.

Conclusion: AHC and RDP constitute clinical prototypes in a continuous phenotypic spectrum of ATP1A3-related disorders. Intermediate phenotypes combining criteria of both conditions are increasingly recognized. Efficient stepwise mutation analysis of the ATP1A3 gene may prioritize those exons where current state of knowledge indicates mutational clusters. Neurology® 2014;82:945–955 GLOSSARY AHC 5 alternating hemiplegia of childhood; ATP1A3 5 ATPase, Na1/K1 transporting, alpha 3 polypeptide; FD 5 functional domain; RDP 5 rapid-onset dystonia-parkinsonism; TM 5 transmembrane domain.

ATP1A3 is a neuron-specific p-type Na1/K1-ATPase with particular importance in sodiumcoupled transport of various molecules, osmoregulation, and excitability of nerves and muscles.1 The recent discovery that mutations in the ATP1A3 gene are not only the primary cause for rapid-onset dystonia-parkinsonism (RDP, DYT12)2,3 but also for alternating hemiplegia of childhood (AHC) has widened the spectrum of neurologic disorders associated with ATP1A3.4–6 RDP comprises abrupt onset of asymmetric dystonia and parkinsonism, frequently after a trigger, with bradykinesia and gait instability as well as prominent bulbar symptoms. Disease onset is often in the second and third decade of life, with reported age ranges of 9 months to 55 years.7,8 AHC is characterized by recurrent episodes of hemiplegia and dystonia alternating in laterality. All patients display first symptoms before the age of 18 months.9 The paroxysmal manifestations are typically triggered by physical or emotional stress and vanish with sleep. Nonparoxysmal manifestations evolve after months or years and include developmental delay, dysarthria, and ataxia. Supplemental data at Neurology.org *These authors contributed equally to this work. From the Department of Pediatrics and Adolescent Medicine (H.R., A.O., P.H., L.S., R.S., J.G., K.B.), Division of Pediatric Neurology, University Medical Center Göttingen, Georg August University; Department of Pediatrics (M.B.), Hospital Dritter Orden, Munich, Germany; Departments of Pediatric Neurology (I.C.), Hospital Maria Pia do Centro Hospitalar do Porto, Portugal; 4IRCCS Stella Maris (S.F.), Calambrone, Pisa; Department of Clinical and Experimental Medicine (S.F.), University of Pisa, Italy; Neurogenetics Unit (C.M.L.), Department of Neurology, School of Medicine of Ribeirao Preto, University of Sao Paulo, Brazil; and Children’s Hospital of Eastern Ontario (S.S.), Ottawa, Canada. Go to Neurology.org for full disclosures. Funding information and disclosures deemed relevant by the authors, if any, are provided at the end of the article. © 2014 American Academy of Neurology

945

In this study, we analyzed the clinical and genetic features of 16 new patients with AHC, 3 new patients with RDP, and of all AHC and RDP cases with proven ATP1A3 mutation reported in the literature. We addressed 3 questions: (1) Are AHC and RDP allelic conditions or do both diseases rather represent a phenotypic continuum of ATP1A3related disorders? (2) Do the genotypic spectra of AHC and RDP differ and is there any genotype-phenotype correlation? (3) Can we delineate mutation clusters for a more reasonable and economic diagnostic workup? METHODS Patient cohort. For this study, we recruited 19 patients referred to the Department of Pediatric Neurology, University Medicine Göttingen, from neurology departments in Germany, and neurologists from different European Union countries, Canada, and Brazil between February 2009 and April 2013. All patients met the published clinical diagnostic criteria for AHC10,11 or RDP,2,12 and whole blood samples were provided.

Standard protocol approvals, registrations, and patient consents. The institutional review board of the Faculty of Medicine, University of Göttingen, Germany approved this study (file reference 15/6/11). Written informed consent was obtained from adult participants, parents of participants younger than 18 years, and guardians of adult participants with intellectual disability.

Clinical analysis. Tables 1, 2, and 3 display detailed clinical information for all new patients. Furthermore, the available clinical data of all AHC and RDP patients with ATP1A3 mutations described in the literature are included.3–8,13–26 Assessment of clinical data from the literature did not rely on established dystonia rating scales because they do not allow for adequate description of the particular phenotypic characteristics of AHC and RDP. Especially the paroxysmal manifestations of AHC and, to a much lesser extent of RDP, are not reflected by conventional rating scales. Moreover, there is no agreed on a rating scale for AHC. Instead, to provide the most robust phenotypic information, we collected all signs and symptoms implying an outstanding phenotypic significance for ATP1A3-associated disorders, as they have evolved in clinical and genetic studies over the past years. Mutation analysis. Mutation analysis was performed according to standard protocols (for details see appendix e-1 on the Neurology® Web site at Neurology.org). RESULTS Clinical spectrum of ATP1A3-related disorders.

Tables 1 and 2 summarize the paroxysmal and nonparoxysmal manifestations of 16 new patients meeting clinical criteria for AHC. Meta-analysis of clinical features in AHC. Meta-analysis

of the available clinical data of AHC patients with ATP1A3 mutations published previously and the ones first described in this study allows the assessment of neurologic symptoms and disease course by frequency (see table e-1). Hemiplegic episodes, disappearance of symptoms on falling asleep, and a rostrocaudal gradient (face to arm to leg) of involvement were reported in all 946

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March 18, 2014

patients. Other paroxysmal manifestations included, in decreasing frequency, episodes of abnormal ocular movements, dystonic episodes, bulbar symptoms, episodes of autonomic dysfunction, seizures, and paroxysmal respiratory disturbances. In nearly all patients with AHC, paroxysmal manifestations were triggered at least once by a provoking event. Developmental impairment of varying severity was the most common nonparoxysmal clinical feature followed by dysarthria, ataxia, muscular hypotonia, and choreoathetosis. A progression of nonparoxysmal symptoms was seen in one-fourth of all patients. In almost all patients, the disease course was polyphasic with an onset in the first 18 months of life and a rapid recovery of paroxysmal symptoms (see table e-2). Meta-analysis of clinical features in RDP and AHC/RDP intermediate cases. Meta-analysis of the available pub-

lished clinical data on patients with RDP with ATP1A3 mutations and those first described in this study allows the assessment of neurologic symptoms and disease course by frequency, as displayed in detail in table e-2. While characteristic neurologic symptoms such as abrupt onset of dystonia/hemiplegia, a rostrocaudal gradient of involvement, prominent bulbar findings, and trigger factors are seen in both AHC and RDP, age at onset and clinical course are different in the majority of cases. Meta-analysis of these clinical hallmarks allows for identification of intermediate AHC/RDP phenotypes (see table e-2 and appendix e-2). Two patients with atypical AHC and age at onset of 3 and 2 years, respectively,21 and one patient with RDP phenotype and age at onset of 11 months8 were reported previously. Patients ATP1A3-31 and ATP1A3-42 in this study (described in detail in appendix e-2) show a sequence of symptoms combining a polyphasic and monophasic course and thus merge AHC and RDP criteria. Recurrent paroxysmal neurologic symptoms were previously observed in patients with predominant RDP phenotype.8,13,19 Genetic spectrum of ATP1A3-related disorders (AHC, RDP, and AHC/RDP). Table 2 and the figure summa-

rize the molecular genetic findings in our cohort of 16 patients with newly diagnosed AHC. We identified ATP1A3 mutations in 15 cases. Five heterozygous missense mutations were new (see tables 2 and 3). The mutation T335P in exon 9 is located only 2 amino acids downstream of the already reported mutation C333F. Both mutations are located close to the transmembrane domain 4 (TM4) and within the functional domain (FD) E1-E2 ATPase of the Na1/K1-ATPase a3-subunit. The second new missense mutation, L757P, is located only 2 amino acids downstream of the 2 known missense mutations

Table 1

Paroxysmal manifestations of 16 patients with alternating hemiplegia of childhood identified in this study

Patients

Onset

Paroxysmal features

Neurology 82

Patient code

Sex

Present age, y, mo

Age at onset, y, mo

First symptoms

Hemiplegic episodes

Trigger

Seizures

Dystonic episodes

Episodes of abnormal ocular movements

Episodes of Bulbar autonomic symptoms dysfunction

Disappearance on falling asleep

ATP1A3-25

F

9, 1

1, 0

Nystagmus

s 1/f 5–10/d up to 1 h

1

2

s 1, 0

s 1, 0

1

2

1

ATP1A3-26

M

12, 9

0, 3

Nystagmus, dystonic episode, apathia

s 0, 4/f 10–15/d 1 min up to 2 days

1

s 4, 0

s 0, 3

s 0, 3

1

1

1

ATP1A3-27

F

7, 2

0, 2

Monocular exotropia and oculogyria

s 1, 8

1

2

s 0, 6

s 0, 2

2

?

1

ATP1A3-28

F

13, 7

0, 1

Monocular exotropia and nystagmus

s 0, 3

1

s 11, 0

s 0, 3

s 0, 1

1

?

1

ATP1A3-29

M

2, 6

0, 2

Conjugate eye deviation, monocular nystagmus

s 0, 6

1

2

s 0, 5

s 0, 2

1

?

1

ATP1A3-30

M

2, 3

0, 7

Dystonic episodes

s 0, 9/f/d 1 h–2 days

1

2

s 0, 7

2

1

?

1

ATP1A3-31

M

18, 9

1, 6

Hemiplegic episode

s 1, 6/f 1–25/d 10 min–4 wk

1

s 4, 3

s 0, 1

s 4, 3

1

2

2/1

ATP1A3-32

F

12, 4

0, 2

Up and down eye movement

s?/f 20–30/d hours to days

2

s 1, 1

s 0, 10

s 0, 2

1

1

1

ATP1A3-33

F

5, 4

0, 1

Seizures

s?/f 1–12/d 1–14 days

1

s 0, 1

s?

2

2

?

1

ATP1A3-34

M

4, 7

0, 11

Tonic-clonic seizures

s 1, 6/f 2–4/d 3 h–3 days

2

s 0, 11

s 1, 1

s 1, 1

1

1

1

ATP1A3-35

F

4, 9

0, 4

Dystonic episodes and intermittent strabismus

s 0, 9/f 2–28/d 1–8 h

2

?

s 0, 4

s 0, 4

1

1

1

March 18, 2014

ATP1A3-36

M

18, 10

1, 0

Hemiplegic episodes

s 1, 0/f 2–4/d 1 h–3 days

2

2

s 5, 0

2

1

2

1

ATP1A3-37

M

0, 8

0, 1

Abnormal ocular movement, hemiplegic episodes

s 0, 1/f 1–30/d 2 h–4 days

1

2

s 0, 9

s 0, 1

2

1

1

ATP1A3-38

M

1, 0

0, 5

Hemiplegic episodes

s 0, 5/f 4–30/d 20 min–3 days 2

2

2

s?

2

2

1

ATP1A3-39

F

4, 3

0, 1

Nystagmus, dystonic episodes

s 1, 9/f 3–10/d minutes up to 3 days

1

?

s 0, 1

s 0, 1

1

2

1

ATP1A3-40

F

5, 7

0, 10

Abnormal ocular movement, hemiplegic episodes

s 0, 10/f 4/d 4–5 h

1

s 1, 2

s 2, 0

s 0, 10

1

?

1

Abbreviations: s 5 start (year, month); f 5 frequency (range of no. of episodes/month); d 5 duration.

947

948 Table 2

ATP1A3 mutations and nonparoxysmal clinical features of 16 patients with alternating hemiplegia of childhood identified in this study

Neurology 82 March 18, 2014

Mutation

Nonparoxysmal features

Patient code

Exon

c.DNA

Protein

Protein

Cognitive development

Best motor function, y, mo

Rostrocaudal gradient

Muscular hypotonia

Choreoathetosis Ataxia

Dysarthria

Progression of nonparoxysmal features

ATP1A3-25

21

c.2839G.A

p.Gly947Arg

G947R

Intellectual disability

Unaided walking at 3, 0

1

1

2

1

1

2

ATP1A3-26

16

c.2263G.A

p.Gly755Ser

G755S

Intellectual disability

Unaided walking at 2, 6

1

2

2

1

1

2

ATP1A3-27

17

c.2401G.A

p.Asp801Asn

D801N

Intellectual disability

Unaided walking at 7, 0

1

1

2

1

?

?

ATP1A3-28

17

c.2401G.A

p.Asp801Asn

D801N

Intellectual disability

Unaided walking at 1, 6

1

1

1

1

?

?

ATP1A3-29

17

c.2401G.A

p.Asp801Asn

D801N

Learning disability

Unaided walking at 1, 9

1

1

2

2

?

?

ATP1A3-30

No ATP1A3 mutation detected

Learning disability

Unaided walking at 1, 1

1

1

2

?

1

2

ATP1A3-31

18

c.2428A.T

p.ILle810Phe

I810F

Intellectual disability

Unaided walking at 1, 6

1

1

2

1

1

ATP1A3-32

17

c.2411C.T

p.Thr804Ile

T804I

Intellectual disability

Unaided walking at age ?

?

2

?

?

?

?

ATP1A3-33

18

c.2443G.A

p.Glu815Lys

E815K

Intellectual disability

Unaided sitting at 4, 1

?

1

?

1

?

?

ATP1A3-34

20

c.2780 G.A

p.Cys927Tyr

C927Y

Intellectual disability

Unaided sitting at 2, 0

1

1

2

2

1

1

ATP1A3-35

8

c.965T.A

p.Val322Asp

V322D

Intellectual disability

Unaided walking at 2, 6

1

2

2

1

1

2

ATP1A3-36

17

c.2270T.C

p.Leu757Pro

L757P

Intellectual disability

Unaided walking at 1, 0

1

2

1

2

1

2

ATP1A3-37

IVS18

c.254211G.A

Learning disability

Unaided walking at 1, 6

2

1

2

1

1

1 slow

ATP1A3-38

17

c.2401G.A

p.Asp801Asn

D801N

Intellectual disability

Unaided sitting at age ?

2

1

2

2

1

2

ATP1A3-39

9

c.1003A.C

p.Thr335Pro

T335P

Learning disability

Unaided walking at 2, 2

1

1

2

1

1

2

ATP1A3-40

17

c.2415C.G

p.Asp805Glu

D805E

Intellectual disability

Unaided walking at 3, 0

1

1

1

1

1

1

Table 3

Clinical features and mutations of patients with rapid-onset dystonia-parkinsonism reported in the literature and in this study

Patients

Mutation

Phenotype

Families or single cases

No.

Exon

c.DNA

Protein

Protein

Age at onset, y

Triggers

Rostrocaudal gradient

Bulbar symptoms

Improvement

Second event

References: Patients, mutations, and functional studies

1

1

8

c.821T.C

p.Ile274AThr

I274T

37

2

1

1

?

2

3, 7, 26

3

3

8

c.829G.A

p.Glu277Lys

E277K

20–26

2(1)/1(1 fever, head trauma; 1 respiratory tract infection)

1

1

2(2)/1(1)

2(1)/1(1; 1 with 3, 7, 23 third event)

1

1

8

c.979_981delCTG

p.327Leudel

327Ldel

12

1 physical overexertion

1

1

1

2

16

ATP1A3-41

1

9

c.1109C.A

p.Thr.370Asn

T370N

9

1 respiratory tract infection

1

1

1

1

This study

ATP1A3-42

1

9

c.1144T.C

p.Trp382Arg

W382R

17

1 physical activity

1

1

1

2

This study

ATP1A3-43

1

10

c.1250T.C

p.Leu417Pro

L417P

16

1 alcohol consumption

1

1

2

2

This study

5

18

14

c.1838C.T

p.Thr613Met

T613M

4–59

2(8)/1(5 head trauma and/or emotional trauma, 1 heat, 1 drill on a warm day, 2 alcohol); 1?

1(17)/2(1)

2(4)/1(14)

2(10)/1(4)/?(4) 2(6)/1(6)/?(6)

3, 7, 17–19, 25

Neurology 82 March 18, 2014

1

1

15

c.2051C.T

p.Ser684Phe

S684F

30

1 after childbirth

1

1

?

?

22

1

1

17

?

p.Arg756His

R756H

11 mo

1 fever

1

1

1

1 (and third event)

3

1

12

17

c.2273T.G

p.Ile758Ser

I758S

14–45

2(2)/1(1 after childbirth, 1(8); only arms 1 after running/?(8) and legs (4)

2(1)/1(5)/? (6)

2(4)/1(2)/?(6)

2(4)/1(1)/ repetitive writers cramp (1)/?(6)

2, 3, 7

1

2

17

c.2338T.C

p.Phe780Leu

F780L

16 and 35

2(1)/1(1 after running)

1

1

1

2

3, 7

1

4

17

c.2401G.T

p.Asp801Tyr

D801Y

17–22

2(1)/1(1 walking on a warm day)/?(2)

1

1

2

2(1)/1(3)

3, 7

2

2

20

c.2767G.A

p.Asp923Asn

D923N

4 and 20

1(1 long exhausting 1 walk, 1 mild head trauma and fever)

1

2(1)/1(1)

2(1)/1(1)

8, 13, 24

1

1

23

c.3191_3193dupTAC

p.1013Tyrdup

1013Ydup

16

?

1

1

?

?

15

Patients are sorted by mutation sequence. 2(#) 5 no. of patients not affected; 1(#) 5 no. of patients affected; ?(#) 5 no. of patients without available information.

949

Figure

Predicted locations of the ATP1A3 mutations in the Na1/K1-ATPase a3-subunit

(A) ATPase subunit a3 isoform 1 is the predominant splice variant of the a3-subunit. Mutations reported previously in patients with alternating hemiplegia of childhood (AHC) (mutations displayed above the protein) and rapid-onset dystonia-parkinsonism (RDP) (mutations displayed below the protein) are shown in black. Mutations identified in this study are shown in red (AHC) and blue (RDP). For patients with AHC, clustering of mutations at the first and the last third, especially at the transmembrane regions of the ATP1A3 protein, becomes evident (T1–10 5 transmembrane regions 1–10). (B) Various conserved functional ATP1A3 domains and transmembrane regions are predicted to be affected by amino acid changes. Cation ATPase N 5 cation transporter/ATPase, N-terminus; HAD-like 5 haloacid dehalogenase-like hydrolases; cation ATPase C 5 cation-transporting ATPase, C-terminus.

L755S and L755C. All 3 mutations are located close to the TM5 (figure). The remaining newly identified missense mutations T804I, D805E, and I810F lie within a cluster of known pathogenic sequence variations surrounding TM6 (figure). Table 3 and the figure display the molecular genetic findings in our 3 patients with RDP who had new missense mutations affecting exon 9 and exon 10, respectively. The mutation T370N is located in close proximity to the missense mutation L371P, reported to be pathogenic and associated with an AHC phenotype. The tryptophan of mutation W382R is located in a highly conserved region of the protein. The missense mutation L417P is the only mutation reported in RDP and AHC that is located in the hydrolase-like 2 FD of the Na1/K1-ATPase a3-subunit (figure). Genotype-phenotype aspects in ATP1A3-related disorders.

Evaluation of clinical and genetic data of all 118 AHC patients and 46 RDP patients with proven ATP1A3 mutations reported in the literature3–8,13–26 as well as of the 15 AHC and 3 RDP patients with ATP1A3 mutations described in this study provides clues for a genotype-phenotype correlation. 950

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March 18, 2014

Although mutations in patients with AHC and RDP are distributed over almost all ATP1A3 protein domains, there are characteristic features for each phenotype. Mutations in sporadic or familial AHC cases affect the TMs of the protein in 20 of 30 (67%), whereas mutations in sporadic or familial RDP cases affect TMs in 5 of 14 (36%) (p 5 0.1, odds ratio 5 3.6, Fisher exact test). The designated FDs of the Na1/ K1-ATPase a3-subunit (cation transporter/ATPase at the N-terminus, E1-E2 ATPase, hydrolase-like 2, haloacid dehalogenase-like hydrolases, and cationtransporting ATPase at the C-terminus) are affected in 22 of 30 mutations (73%) associated with an AHC phenotype vs 7 of 14 changes (50%) for RDP (p 5 0.18, odds ratio 5 2.75, Fisher exact test). No patient with RDP reported so far shows a mutation over a long stretch in the N-terminal part of the ATP1A3 protein and 50% of all mutations associated with RDP affect the loop between TM4 and TM5. Clustering of mutations at TMs 2, 4, 5, 6, 8, and 9 of the ATP1A3 protein is evident for AHC. Furthermore, approximately one-fourth of all mutations associated with AHC phenotypes are in the region around the TM6 of the Na1/K1-ATPase a3-subunit.

Table 4

Mutational spectrum of ATP1A3-related disorders

Alternating hemiplegia of childhood Mutation

Frequency

Exon

c.DNA

Protein

Protein

Allele frequency

Percent

Described in reference

5

c.410C.A

p.Ser137Phe

S137F

2

1.50

4

5

c.410C.T

p.Ser137Tyr

S137Y

1

0.75

4

5

c.419A.T

p.Gln140Leu

Q140L

1

0.75

4

7

c.658G.A

p.Asp220Asn

D220N

1

0.75

4

8

c.821T.A

p.Ile274Asn

I274N

2

1.50

4, 6

8

c.965T.A

p.Val322Asp

V322D

2

1.50

This study, patient ATP1A3-35, 6

9

c.998G.T

p.Cys333Phe

C333F

2

1.50

4

9

c.1003A.C

p.Thr335Pro

T335P

1

0.75

This study, patient ATP1A3-39

9

c.1112T.C

p.Leu371Pro

L371P

1

0.75

6

16

c.2263G.A

p.Gly755Ser

G755S

2

1.50

This study, patient ATP1A3-26, 4

16

c.2263G.T

p.Gly755Cys

G755C

2

1.50

5, 6

MC 17

c.2270T.C

p.Leu757Pro

L757P

1

0.75

This study, patient ATP1A3-36

MC 17

c.2316C.A

p.Ser772Arg

S772R

1

0.75

6

MC 17

c.2318A.T

p.Asn773Ile

N773I

1

0.75

6

MC 17

c.2318A.G

p.Asn773Ser

N773S

1

0.75

4

MC 17

c.2401G.A

p.Asp801Asn

D801N

52

39.10

MC 17

c.2411C.T

p.Thr804Ile

T804I

1

0.75

This study, patient ATP1A3-32

MC 17

c.2415C.G

p.Asp805Glu

D805E

1

0.75

This study, patient ATP1A3-40

MC 18

c.2417T.G

p.Met806Arg

M806R

1

0.75

4

MC 18

c.2428A.T

p.Ile810Phe

I810F

1

0.75

This study, patient ATP1A3-31, see appendix e-1

MC 18

c.2429T.G

p.Ile810Ser

I810S

1

0.75

4

This study, patients ATP1A3-27–29/38, 4–6

MC 18

c.2431T.C

p.Ser811Pro

S811P

4

3.01

MC 18

c.2443G.A

p.Glu815Lys

E815K

30

22.56

This study, patient ATP1A3-33, 4–6

MC intron 18

c.25 4211G.A

3

2.26

This study, patient ATP1A3-37, 4, 6

Splice site

4

20

c.2755_2757delGTC p.Val919del

V919del

1

0.75

4

20

c.2767G.T

p.Asp923Tyr

D923Y

1

0.75

6

20

c.2767G.A

pAsp923Asn

D923N

4

3.01

21

20

c.2780G.A

p.Cys927Tyr

C927Y

2

1.50

This study, patient ATP1A3-34, 5

21

c.2839G.A

p.Gly947Arg

G947R

6

4.51

This study, patient ATP1A3-25, 4

21

c.2839G.C

p.Gly947Arg

G947R

2

1.50

4

21

c.2864C.A

p.Ala955Asp

A955D

1

0.75

4

22

c.2974G.T

p.Asp992Tyr

1

0.75

4

133

100.00

D992Y P

Rapid-onset dystonia-parkinsonism Mutation

Frequency

Exon

c.DNA

Protein

Protein

Allele frequency

Families

MC 8

c.821T.C

p.Ile274AThr

I274T

1

1

4.76

3, 7, 26

MC 8

c.829G.A

p.Glu277Lys

E277K

3

3

14.29

3, 7, 23

MC 8

c.979_981delCTG

p.327Leu

327Ldel

1

1

4.76

Percent

Described in reference

16

Continued

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Table 4

Continued

Rapid-onset dystonia-parkinsonism Mutation

Frequency Allele frequency

Exon

c.DNA

Protein

Protein

9

c.1109C.A

p.Thr370Asn

T370N

1

1

4.76

This study, patient ATP1A3-41, see appendix e-1

9

c.1144T.C

p.Trp382Arg

W382R

1

1

4.76

This study, patient ATP1A3-42, see appendix e-1

10

c.1250T.C

p.Leu417Pro

L417P

1

1

4.76

This study, patient ATP1A3-43, see appendix e-1

MC 14

c.1838C.T

p.Thr613Met

T613M

18

5

23.81

15

c.2051C.T

p.Ser684Phe

S684F

1

1

4.76

22

MC 17

?

p.Arg756His

R756H

1

1

4.76

8

MC 17

c.2273T.G

p.Ile758Ser

I758S

12

1

4.76

3

MC 17

c.2338T.C

p.Phe780Leu

F780L

2

1

4.76

3, 7

MC 17

c.2401G.T

p.Asp801Tyr

D801Y

4

1

4.76

3, 7

20

c.2767G.A

p.Asp923Asn

D923N

2

2

9.52

8, 13, 21

23

c.3191_3193dupTAC p.1013Tyrdup

15

1013Ydup P

Families

Percent

1

1

4.76

49

21

100.00

Described in reference

3, 7, 14, 17–20, 25

Abbreviation: MC 5 mutation cluster. Gene ID: 478; gene NG_008015.1 genomic; reference NM_152296.4 transcript; sequences NP_689509.1 protein; variation locations are based on these accessions.

Mutation clusters for ATP1A3-related disorders. Table 4

summarizes the new and already reported AHC and RDP patient mutations. Approximately 39% of all AHC cases have the missense mutation D801N in exon 17 and approximately 23% of all AHC patients carry the missense mutation E815K in exon 18. An additional 5% of mutations were found in exon 17 and another 8% were detected in exon 18. The remaining mutations in patients with AHC are distributed over exons 5, 7, 8, 9, 16, 20, 21, and 22. For RDP phenotypes, mutational clusters are located in exons 8, 14, and 17. Counting each RDP family or single case as once-only occurrence of the respective mutation, approximately 24% of all RDP families or single patients have a mutation in exon 8, approximately 24% carry the mutation T613M in exon 14, and about 19% display a mutation in exon 17. The other mutations reported so far in patients with RDP are scattered over exons 9, 10, 15, 20, and 23. Taken together, mutational hotspots in both AHC and RDP are located in exons 8 (AHC 3%, RDP 24%), 14 (AHC 0%, RDP 24%), 17 (AHC 44%, RDP 19%), and 18 (AHC 31%, RDP 0%). Thus, in clinically suspected AHC and RDP, a rational mutation analysis approach should include in the first step exons 17 and 18 for patients with AHC phenotype, and exons 8, 14, and 17 for patients with RDP phenotype. 952

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Meta-analysis of clinical and genetic findings in 18 new patients with ATP1A3-related disorders investigated in this study together with the data on 118 AHC and 46 RDP cases described in previous reports3–8,13,15,17–20,22–27 indicates that (1) AHC and RDP constitute prototypic disorders within a continuous phenotypic spectrum, (2) there is evidence for a genotype-phenotype correlation, and (3) focusing mutation analysis on mutation clusters in 4 exons of the ATP1A3 gene allows for efficient diagnostic workup with 67% probability of mutation detection. Now that heterozygous ATP1A3 mutations are recognized as the genetic cause not only of RDP but of AHC as well, new light is shed on the nosologic relationship of these 2 conditions. Albeit considered to be 2 distinct disorders, accurate phenotyping shows that major clinical characteristics are almost identical in AHC and RDP (table e-2). These comprise a strikingly asymmetric movement disorder with predominantly dystonic and, to a lesser extent, atactic features as well as rostrocaudal (face to arm to leg) gradient of involvement with prominent bulbar symptoms including dysarthria or even anarthria, dysphagia, hypomimia, and abnormal eye movements. Bradykinesia and postural instability, known parkinsonian manifestations in RDP, are observed in AHC as well. Triggering of onset of symptoms by different stressors is also a highly typical feature in both conditions. DISCUSSION

The striking difference between AHC and RDP is the clinical course. Onset of the AHC phenotype is by definition within the first 18 months and thus earlier than in RDP. While in AHC frequently recurring paroxysms with abrupt onset and rapid recovery characterize the course of the disease, the RDP phenotype is considered to show an abrupt primary onset and sometimes a secondary abrupt worsening of features, but unchanged neurologic pattern thereafter. However, these differences dissolve with thorough analyses of the clinical course observed in several patients with RDP and of patients with AHC/RDP intermediate phenotypes. Previous reports of patients with RDP mentioned superimposing paroxysmal manifestations including oculogyric crises,2 paroxysmal dystonia,3 and other recurrent symptoms triggered by stressors and dissolving with sleep.8,13,19 Patients ATP1A3-42 and ATP1A3-31 in this study (for details see appendix e-2) represent further examples for intermediate phenotypes combining clinical features of RDP and AHC. A long-standing diagnostic criterion for AHC is onset of symptoms before 18 months of age.10,11 In contrast, the age range for onset of RDP was considered to be 4 to 55 years.7,13,19 However, recent reports describe “atypical AHC” cases with onset later than 18 months21 as well as a patient with RDP and onset as early as 9 months.8 Thus, while most patients with recognized ATP1A3 mutations still show clinical phenotypes in line with the classic criteria for either AHC or RDP, numerous cases with intermediate features provide enlarging evidence that both conditions are “different manifestations along a clinical spectrum” rather than allelic disorders.28 In this study, we identified 8 novel missense ATP1A3 mutations. Thus, the current mutational spectrum extends to 30 different missense mutations, 1 deletion, and 1 insertion for AHC as well as to 12 different missense mutations, 1 deletion, and 1 duplication for RDP. These mutations are distributed over the whole gene in both conditions (figure) with mutational clusters and clues for a genotype-phenotype correlation. The majority of mutations in patients with AHC are located in exons 17 and 18 of the ATP1A3 gene (74%). The mutation D801N in exon 17 accounts for approximately 39% and the mutation E815K in exon 18 for approximately 23% of all mutations. Mutational clusters in patients with RDP are exons 8, 14, and 17. The mutation T613M in exon 14 accounts for approximately 37% and I758S in exon 17 for approximately 24% of all mutations. This allows for a more efficient and economic molecular genetic testing in a patient showing clinical symptoms consistent with an ATP1A3-related disorder.

We found some evidence for a genotype-phenotype correlation. ATP1A3 mutations of patients with classic AHC more often affect the TMs and FDs of the Na1/ K1-ATPase a3-subunit than mutations of patients with the classic RDP phenotype (TM 67% vs 36%; FD 73% vs 50%). Although it seems reasonable that alterations in TM or FD contribute to a more severe phenotype, the position of the mutation alone does not sufficiently explain the phenotypic variability. Mutations affecting the same amino acid position were observed to be associated with different phenotypes, e.g., I274N in AHC and I274T in RDP or D801N in AHC and D801Y in RDP (table 4) and functional studies demonstrated that nonsynonymous amino acid substitutions resulted in remarkably different alterations of activities of the a3-subunit of the Na1/K1ATPase.20 Both patients with intermediate phenotypes carry novel ATP1A3 mutations. Functional consequences of these mutations have not been investigated. The mutation I810F of patient ATP1A3-31 affects TM6 as do most ATP1A3 mutations in patients with AHC. The nonsynonymous amino acid substitution I810S was described in a patient with a mild AHC phenotype without quadriparesis, epilepsy, dystonia, ataxia, or chorea.4 We speculate that mutations at this position may compromise ATPase activities and protein expression levels to a lesser extent and thus may result in intermediate phenotypes. The mutation W382R of the second patient (ATP1A3-42) with an intermediate phenotype is located between the fourth and fifth TM. Approximately 50% of all ATP1A3 mutations associated with RDP are located between these 2 TMs. Because this mutation affects a highly conserved region of the protein, ATPase activity may possibly be more severely impaired, which may explain the intermediate phenotype. To date, there is only one mutation of the ATP1A3 gene reported that is related to both phenotypes, classic RDP and classic AHC. The mutation D923N in exon 20 associated with RDP8,13,24 was also present in 4 affected members of a family with a clinical pattern essentially in line with AHC.21 The mutation first occurred de novo and then segregated with the disease over 3 generations. That observation provides preliminary evidence for an AHC/RDP overlap at the genotype level. One possible explanation for diversity of phenotypes in patients carrying the D923N mutation might be that the protein expression level is close to the one of AHC mutations.4 Thus, because the RDP mutations D801Y and L327del also show comparable expression levels to AHC mutations when expressed in COS-7 or HeLa cells,4 there might also be patients with a classic AHC, classic RDP, or intermediate AHC/RDP phenotype associated with these 2 mutations. Neurology 82

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The fact that nonsynonymous amino acid substitutions at the same position may result in different phenotypes and that one and the same mutation results in divergent features clearly indicates that the position of the mutation alone is not responsible for the phenotypic variability. Protein structural abnormalities and/or protein function including subcellular protein localization, residual ATPase activity, residual ion-binding and ion-transport capacity, as well as other genetic, epigenetic, and environmental factors may contribute to the variation of clinical phenotypes in ATP1A3-related disorders. In one of our 16 patients with clear clinical symptoms of AHC, we could not identify an ATP1A3 mutation. Mutation rates described in previous reports ranged from 74%4 to 100%5,6 in patients meeting diagnostic criteria of AHC. Possible explanations for negative mutation analysis in patients with AHC comprise misinterpretation of clinical phenotypes that overlap AHC such as Glut1 deficiency syndrome,29 presence of microdeletions or duplications affecting single exons not detectable by Sanger sequencing, and presence of further disease genes for the AHC phenotype. As we could show in this study, there is increasing evidence that RDP and AHC rather constitute a clinical continuum of ATP1A3-related disorders, with AHC at the severe end of the spectrum and RDP as a milder variant. Future studies should compile revised criteria for the clinical diagnosis of ATP1A3associated disorders. These criteria should include classic AHC and RDP cases as well as AHC/RDP intermediate phenotypes that are not captured in the existing diagnostic criteria.

3.

4.

5.

6.

7.

8.

9.

10. 11.

12.

13.

AUTHOR CONTRIBUTIONS Hendrik Rosewich: drafting/revising the manuscript for content, including medical writing for content, study concept or design, analysis and interpretation of data, acquisition of data, statistical analysis, study supervision or coordination. Andreas Ohlenbusch, Peter Huppke, Lars Schlotawa, Martina Baethmann, Inês Carrilho, Simona Fiori, Charles Marques Lourenço, Sarah Sawyer, and Robert Steinfeld: analysis or interpretation of data, acquisition of data. Jutta Gärtner and Knut Brockmann: drafting/ revising the manuscript for content, including medical writing for content, study concept or design, analysis and interpretation of data, acquisition of data, study supervision or coordination.

14.

STUDY FUNDING

17.

15.

16.

No targeted funding reported.

DISCLOSURE

18.

The authors report no disclosures relevant to the manuscript. Go to Neurology.org for full disclosures.

19. Received July 8, 2013. Accepted in final form December 3, 2013. REFERENCES 1. Dobretsov M, Stimers JR. Neuronal function and alpha3 isoform of the Na/K-ATPase. Front Biosci 2005;10:2373–2396. 2. Dobyns WB, Ozelius LJ, Kramer PL, et al. Rapid-onset dystonia-parkinsonism. Neurology 1993;43:2596–2602. 954

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The expanding clinical and genetic spectrum of ATP1A3-related disorders Hendrik Rosewich, Andreas Ohlenbusch, Peter Huppke, et al. Neurology 2014;82;945-955 Published Online before print February 12, 2014 DOI 10.1212/WNL.0000000000000212 This information is current as of February 12, 2014 Updated Information & Services

including high resolution figures, can be found at: http://www.neurology.org/content/82/11/945.full.html

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Supplementary material can be found at: http://www.neurology.org/content/suppl/2014/02/12/WNL.00000 00000000212.DC1.html http://www.neurology.org/content/suppl/2014/04/21/WNL.00000 00000000212.DC2.html

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