J Neurol (2014) 261:983–991 DOI 10.1007/s00415-014-7310-2
ORIGINAL COMMUNICATION
Episodic ataxia type 2: phenotype characteristics of a novel CACNA1A mutation and review of the literature Wolfgang Nachbauer • Michael Nocker • Elfriede Karner • Iva Stankovic Iris Unterberger • Andreas Eigentler • Rainer Schneider • Werner Poewe Margarete Delazer • Sylvia Boesch
• •
Received: 8 January 2014 / Revised: 5 March 2014 / Accepted: 5 March 2014 / Published online: 22 March 2014 Ó Springer-Verlag Berlin Heidelberg 2014
Abstract Episodic ataxia type 2 (EA2) is an autosomal dominant inherited neurological disorder that is characterized by paroxysmal episodes of ataxia. The causative gene for EA2 is CACNA1A which codes for the alpha 1A subunit of the voltage-gated P/Q-type calcium channel (Cav2.1). We detected a novel point mutation in the CACNA1A gene in a large Austrian family. All ten affected family members harbored a heterozygous c.3089?2T[C nucleotide exchange in intron 19. In silico modeling demonstrated a loss of the splice site of exon 19 by the mutation, which most likely results in exon skipping without frameshifting or use of an alternative splice site. Clinically, the family exhibited frequent ataxic episodes accompanied by headache in some individuals, which showed a good treatment response to acetazolamide or aminopyridine. Interictal phenotype variability was high ranging from an unremarkable clinical examination to a progressive cerebellar syndrome. Detailed cognitive testing with standardized neuropsychological tests revealed specific deficits in various domains including memory, Electronic supplementary material The online version of this article (doi:10.1007/s00415-014-7310-2) contains supplementary material, which is available to authorized users. W. Nachbauer M. Nocker E. Karner I. Unterberger A. Eigentler W. Poewe M. Delazer S. Boesch (&) Department of Neurology, Medical University Innsbruck, Anichstrasse 35, 6020 Innsbruck, Austria e-mail: [email protected] I. Stankovic Department of Neurology, University of Belgrade, Belgrade, Serbia R. Schneider Department of Biochemistry, Leopold-Franzens-University Innsbruck, Innsbruck, Austria
executive functions and visual abilities. Moreover, a striking coincidence of socio-phobic behavior and anxiety disorders was detected within this family, which interfered with activities of daily living and has to be taken in consideration in EA2 patient management. We here characterize the phenotype of this novel CACNA1A mutation, review the respective literature and discuss implications on diagnosis and patient management. Keywords Episodic ataxia CACNA1A Clinical phenotype Cognition
Introduction Episodic ataxias (EAs) are autosomal dominant inherited neurological disorders characterized by paroxysmal attacks of ataxia and eventually progressive cerebellar dysfunction in later disease stages. The most common subtype of EAs is EA type 2 (EA2), caused by mutations in the CACNA1A gene coding for the pore forming alpha 1A subunit of the voltage gated P/Q-type calcium channel (Cav2.1) [1, 2]. Typically, EA2 is characterized by episodes of ataxia, vertigo and nausea lasting for minutes to hours. Overlapping clinical features to the allelic disorders familial hemiplegic migraine (FHM1) and spinocerebellar ataxia type 6 (SCA6) have been described [3–5]. Therefore, attacks can be accompanied by dysarthria, diplopia, tinnitus, hemiplegia and headache [6, 7]. Interictal symptoms such as nystagmus and ataxia occur in EA2, although individuals may be asymptomatic between single episodes. Attacks are triggered by emotional stress, exertion, caffeine or alcohol. Acetazolamide [8] and the potassium channel blocker 4-aminopyridine [9, 10] have been shown to be effective in EA2, particularly in reducing frequency and
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severity of attacks. We herein give a detailed clinical description of an Austrian family with affected individuals in four generations harbouring a novel CACNA1A mutation and review phenotype variability in previously published EA2 families.
Patients and methods Clinical evaluation Probands were identified within a family exhibiting episodic ataxic features in combination with a mild progressive cerebellar syndrome. The index patient (III-1) was initially referred to the ataxia outpatient clinic of the Department of Neurology, Medical University Innsbruck in 2009. Further family members were consecutively seen and examined on a routine basis. Ten individuals out of four generations were clinically affected (compare Fig. 1). Clinical information on deceased individuals (I-1 and II-1) was obtained by interview of family members and review of available medical records. Clinical work-up comprised detailed medical history including duration, frequency and severity of attacks, as well as neurological assessment. For measuring and monitoring the severity of ataxia the Scale for the Assessment and Rating of Ataxia (SARA) [11] was implemented in routine examination. Additionally, standardized neuropsychological testing was carried out. Cerebral magnetic resonance imaging (MRI) was performed on 1.5 T whole body scanner (Magnetom Avanto, Siemens Erlangen, Germany). Investigations and assessments were conducted in accordance with the Declaration of Helsinki on ethical principles for medical research involving human subjects.
Fig. 1 Pedigree of the family. The family comprised ten affected individuals out of four generations. Roman numerals (I–IV) give the generation, Arabic numerals state the affected individuals of the respective generation ordered according to their age. Squares males; circles females; filled symbols affected subjects; blank symbols unaffected subjects; diagonal line deceased subjects
Neuropsychological assessment comprised a wide range of cognitive functions. Verbal memory (learning, short- and long-delay free recall, recognition) was tested with the Verbal Learning and Memory Test (VLMT), a German version of the Rey Auditory Verbal Learning Test [12]. Figural memory (short- and long-delay free recall and recognition) was examined with the Rey Complex Figure Test and Recognition Trial (RCFT) [13]. Further tasks assessed visuospatial processing [subtests 7,8,9 of the Leistungspru¨fsystem (LPS)] [14], visuo-constructional ability (Rey Complex Figure Copy Trial) and mental arithmetic (GDAE) [15]. Language tasks measured comprehension, confrontation naming by simple nouns and confrontation naming by compound nouns (AAT) [16]. Speed of articulation was tested by rapid syllable repetition [17]. Assessment of attention included the verbal memory span (WMS) [18], tonic alertness (reaction on a visual stimulus) and divided attention (TAP) [19]. Executive function tests assessed verbal working memory (WMS) [18], verbal and figural fluency (RWT, HAMASCH) [20, 21], mental flexibility and ability to maintain rules (OMO, TMT B) [22, 23], planning [24] and inhibition (TAP Go/No Go) [19]. Verbal intelligence was estimated by a multiple choice vocabulary test (MWT) [25]. Motor functions of the right and the left hand were assessed by the grooved pegboard (Lafayette Instruments). To avoid stress which could possibly trigger attacks, tests were distributed over several assessments. Subjects III-1, III-2 and IV-2 performed all neuropsychological tests. Patient IV-3 performed most of the tests. Patient IV-1 was available for parts of the tests. Patients’ scores were compared with standard norms. Performance was
I-1
II - 2
II - 1
III - 1
II - 3
III - 3
III - 2
IV- 1
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Neuropsychological tests
IV- 2
IV- 3
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considered as below average when the score was between the 15th and third percentile of standardized norms, and as impaired when it was at or below the second percentile. Genetic testing Genetic testing was performed in all affected individuals, except for the deceased individuals I-1 and II-1, after written informed consent. Analyses were carried out in a certified laboratory by standard procedures (Centogene, Rostock, Germany). All affected individuals showed a novel c.3089?2T[C mutation lying in the donor splice site of intron 19, which affects the splicing process of exon 19. In silico evaluation of the identified sequence mutation affecting the canonical donor splice site of exon 19/intron 19 was performed using as input exon 19 and 150 bp of intron 19 sequence as given in the NCBI genomic and mRNA reference sequences NC_000019.10 (genomic) and NM_001127222.1 (mRNA) corresponding to consensus cDNA CCDS45998.1 and protein isoform NP_001120694.1. The Berkeley Drosophila Genome Project server (http://www.fruitfly.org/seq_tools/splice.html) and the MaxEntScan server (http://genes.mit.edu/burgelab/ maxent/Xmaxentscan_scoreseq.html) [26] were employed by using default parameters to assess likely changes in the recognition of the exon19/intron 19 splice site caused by the c.3089?2T[C mutation. Both in silico tools predicted a loss of the exon 19/intron 19 splice-site by the mutation which affects an invariantly conserved donor splice site nucleotide.
Results Clinical findings Clinical description of the affected family member of generation I (I-1) was drawn from collateral sources. This person had spells of dizziness in her later adulthood. Accompanying ataxic symptoms and headache were not documented. No overt causes regarding blood pressure, cardiac function or the vestibular system were found. In the second generation, three (II-1, II-2, II-3) out of 11 children were affected. The clinical phenotype in this generation consisted of episodes of vertigo with ataxia and dysarthria. Additional interictal neurological symptoms were absent despite gaze-evoked nystagmus. An 85-year old woman (II-2) complained about short episodes of vertigo lasting for minutes, where she would not be able to stand or walk. Attacks occurred clustered up to several times per week and were markedly reduced in frequency and intensity after initiation of aminopyridine treatment.
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Her sister (II-3) has had vertigo attacks since her youth. The attacks were quite rare and did not interfere with activities of daily life. The clinical examination was unremarkable. The third affected sibling (II-1) died several years ago without being examined by a neurologist. In the third generation of this EA2 family, at least one affected family member revealed progressive ataxia with falls and dysarthria. Attacks appeared to result in phobic behavior and interfered with social activities. A 58-year old male patient (III-1) presented with a more than 10-year history of episodes with instability of gait and tendency to fall, as well as slurred speech and paresthetic sensations of the extremities. Initially, frequency of these episodes was low, but increased with disease duration and resulted in considerable socio-phobic behavior. During the attacks, the patient presented with a severe cerebellar syndrome including dysarthria, limb and gait ataxia. Brain MRI was performed at age 55 years and showed no signs of cerebellar atrophy. His 56-year old brother (III-2) has had disturbances of equilibrium associated with dysarthria since the age of 10 years. Contrary to his brother, attacks did not worsen in frequency or intensity over time. Attacks could be avoided by treatment with aminopyridine. A female cousin (II-3) has had vertigo attacks since her youth. In generation IV, the clinical phenotype worsened considerably, interfering with performance in school and profession, social activities and even activities of daily life. Especially, vegetative symptoms such as nausea accompanied by headache appeared in this generation. An affected 36-year old male patient (IV-3) has had attacks of vertigo since his early childhood. Initially, attacks were solely accompanied by nausea but additionally comprised dysarthria and limb ataxia in the later disease course. Moreover, attacks were accompanied with frontal headache and sensations of heat as preceding symptoms. Interictal neurological assessment revealed bilateral gaze evoked nystagmus without any further evidence of ataxia. His female cousin (IV-1) showed the most pronounced clinical symptoms. She reported severe ataxic episodes accompanied by dizziness, nausea and vomiting lasting up to 1 day. Attacks started in her early childhood and have occurred 3–5 times per month. In line with other members of this EA2 family, magnetic resonance imaging of the brain revealed no significant atrophy or structural lesions. Conditions of clinical state could be improved by treatment with acetazolamide. Because of pregnancy, acetazolamide treatment was stopped which led to a severe worsening of frequency and intensity of attacks. Her 18-year old halfsister (IV-2) has had episodes of ataxia since she was 10 years old. Additionally, scintillating scotoma with subsequent frontal headache and nausea may have accompanied these attacks.
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Table 1 Clinical characteristics of patients II–2 $ 85 years
II–3 $ 82 years
III–1 # 58 years
III–2 # 56 years
III–3 # 51 years
IV–1 $ 35 years
IV–2 $ 18 years
IV–3 # 36 years
Age of onset
30 years
youth
45 years
10 years
youth
2 years
10 years
early childhood
Frequency of attacks
2–3/week
rare
daily
triggered
rare
3–5/month
1–2/week
2–3/month
Duration of attacks Trigger
min none
min none
30 min–3 h emotion
15–30 min exertion, alcohol
min none
h–days stress, exertion
min–h exertion, emotion
2–3 h exertion, coffee, emotion
Ictal symptoms Dysarthria
–
–
H
H
–
H
H
H
Diplopia
–
–
H
–
–
H
–
–
Gait ataxia
H
–
H
H
–
H
H
H
Vertigo
H
H
H
H
H
H
H
H
Cephalea Nausea
– –
– –
– –
– –
– –
H H
H H
H H
Interictal symptoms Nystagmus
H
–
H
–
–
H
H
H
Gait ataxia
–
–
H
–
–
H
H
–
Dysarthria
–
–
H
–
–
H
–
–
SARA score
3
2
6
0
0
4
2
0
Aminopyridine
no therapy
Acetazolamide
Aminopyridine
no therapy
Acetazolamide
no therapy
Acetazolamide
Therapy Frequency
H
H
H
H
H
Duration
–
H
–
H
H
Intensity
H
H
H
H
H
Point mutation (T[C) intron 19, CACNA1A
H
H
H
– H
H
H
H
$ female, # male, min minutes, h hours, SARA scale for the assessment and rating of ataxia
Detailed clinical characteristics of the affected patients are given in table 1. Neuropsychological findings Detailed cognitive testing revealed specific deficits in various cognitive domains including memory, executive functions and visual abilities despite the absence of cerebellar atrophy on MRI. Table 2 summarizes neuropsychological results. All patients scored in the average range in language tasks (comprehension and confrontation-naming of simple nouns). All but one patient scored in the average range on tasks assessing verbal intelligence (IV-3), verbal memory recognition (III-1), confrontation-naming of compound nouns (IV-3) and visual spatial abilities (IV-1, IV-2). However, various impairments were found in other cognitive domains. All patients scored below average in at least three executive tasks (verbal working-memory, phonemic verbal fluency, mental flexibility), even in the impaired range in the set-shifting category of phonemic
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verbal fluency. Rapid syllable was impaired in the youngest patient (IV-2) and below average in all others. Fine motor manipulations with the dominant right hand were impaired in three out of four patients (III-1, III-2, IV-3) and below average in the remaining fourth patient (IV-2). All patients scored below average or even in the impaired range in at least one subtest assessing attention (psychomotor speed, tonic alertness, divided attention). Three out of five patients scored below average or in the impaired range in verbal learning (III-1, IV-1, IV-3), in the short-delay and longdelay free recall of the Rey-Figure (IV-1, IV-2, IV-3) and in visuo-constructional abilities. Two out of five patients (III-1, IV-3) showed deficits in the short-delay and longdelay free recall of a word list. Apart from overt neuropsychological deficits, patients with severe or frequent attacks appear to exert socio-phobic behavior or anxiety disorder. Although psychotic symptoms were denied by all our patients, anxiety and bad performance in school, especially when the disease started early in life, was striking. This phenomenon was
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Table 2 Patients’ performance in neuropsychological tests Tasks
Patients III-1
III-2
IV-1
IV-2
IV-3
Learning sum (Trial 1-5) (maximum 75)
31*
45
42*
54
28**
Short-delay free recall (maximum 15)
7*
10
9
12
7*
Long-delay free recall (maximum 15)
7*
10
11
10
4**
Recognition hits minus false positives (maximum 15)
8*
13
12
13
11
18.5
23
7**
6.5**
10**
Memory Verbal Learning and Memory Test (VLMT)
Rey Complex Figure Test and Recognition Trial (RCFT) Short-delay free recall (maximum 36) Long-delay free recall (maximum 36)
20
24
3**
8.5**
10**
Recognition hits minus false positives (maximum 24)
20
23
n.a.
15**
14**
Visual Abilities Visuoperception Leistungspru¨fsystem (Achievement Test System (L-P-S) subtest 7 (maximum 40)
9
17
8*
8*
n.a.
Spatial processing (L-P-S) subtest 8 (maximum 40)
9
23
0**
15
n.a.
Spatial processing (L-P-S) subtest 9 (maximum 40)
9
8
15
13*
n.a.
Visuoconstruction (RCFT) Copy (maximum 36)
33
33
32*
26**
25**
17
15
n.a.
1**
0**
Token Test (Trial 5): Errors
1
0
2
1
2
Confrontation naming simple nouns (maximum 30)
30
30
29
28
30
Confrontation naming compound nouns (maximum 30) Rapid syllable repetition (‘‘badaga’’/5 s)
26 30*
29 30*
26 27*
29 20**
25* 21*
Trail Making Test, Part A (TMT-A) (s)
69*
29
n.a.
27
70**
Wechsler Memory Scale (WMS): digit span forward (max. 12)
4**
5*
4**
4*
6
Tonic alertness Median (ms)
418**
255
n.a.
316*
290*
Phasic alertness Median (ms)
355*
245
n.a.
253
306*
Mental Arithmetic Graded Difficulty Arithmetic Examination (GDAE) (maximum: 24) Language/Speed of Articulation Aachener Aphasie Test (AAT)
Attention
Test of Attentional Performance (TAP)
Divided attention: Auditive stimuli Median (ms)
493
489
n.a.
594
501
Visual stimuli Median (ms)
544
517
n.a.
641**
569*
Omissions
2
1
n.a.
10**
0
Errors
2
2
n.a.
0
10*
5*
5*
4*
4*
3*
Executive Functions Wechsler Memory Scale (WMS): digit span backward (max. 12) Regensburger Wortflu¨ssigkeits-Test (RWT) Animals/min
9*
23
n.a.
11**
12**
‘‘S’’ words/min Sports/fruits/min
6* 8*
6* 15
n.a. n.a.
3** 10*
3** n.a.
‘‘H’’/’’T’’ words/min
4**
6**
n.a.
4**
3**
Sum
24
38
n.a.
17*
43
Correct
12*
34
n.a.
17*
34
Trail Making Test, Part B (TMT-B) (s)
143*
121*
n.a.
76*
156**
Planungstest (Tower of London): trials to complete (min. = 33)
36
37
n.a.
39
n.a.
452
446
n.a.
532*
n.a.
HAMASCH 5-Point Test
Test of Attentional Performance (TAP) Go/No Go Test: median (ms)
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Table 2 continued Tasks
Patients III-1
III-2
IV-1
IV-2
IV-3
Omissions
11**
0
n.a.
0
n.a.
Errors
4*
1
n.a.
3*
n.a.
5
3
n.a.
1
14*
Right hand
97**
95**
n.a.
78*
111**
Left hand
101**
78
n.a.
77
102**
95
95
n.a.
86
70*
Odd-Man-Out Test (OMO): errors Fine-Motor Manipulations Grooved Pegboard
Intelligence Estimated Verbal IQ: Mehrfachwahl-Wortschatz Test (MWT-A) n.a. Not administered, min. minimum, max. maximum * 3rd \16th percentile = below average ** B2nd percentile = impaired
anticipated in patient III-2 who scored high for anxiety and depression. Moreover, socio-phobic behavior appears to ameliorate gradually after treatment or even disappears (as observed in patient IV-3 after treatment initiation; patient III-1 formally tested only when treated).
Discussion This paper provides a detailed clinical description of a family affected by episodic ataxia with a novel point mutation of the CACNA1A gene and reviews current literature in EA2 families comprising more than three affected subjects in at least two generations. The majority of CACNA1A mutations causing EA2 are nonsense mutations resulting in a truncated protein. The novel mutation in our family occurs in a crucial nucleotide (T[C) on intron 19. As demonstrated by in silico modelling the exon 19/intron 19 donor splice site is no longer recognized, which may, as a further consequence, lead to exon skipping. Alternatively, it is likely that part of the message will be processed with an abnormal and weaker splice-site down-stream, which would lead to incorporation of this part of the intron sequence, frameshift of the open reading frame and premature termination of the transcript. Intronic CACNA1A mutations leading to exon skipping and abnormal splicing have been described previously in EA2 [53, 54] and appear to result in high phenotype variability as it was the case in the present family [33, 42, 54]. EA2 attacks are typically characterized by episodes of ataxia and can be associated with dysarthria, diplopia, tinnitus, nausea, hemiplegia and headache. In our family, patients showed a high phenotype variability ranging from rare vertiginous attacks (II-2, II-3) to severe ataxic spells accompanied by dysarthria, nausea and vomiting with
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onset in early childhood (IV-1, IV-3). In some affected individuals (IV-1, IV-2, IV-3) attacks were accompanied by headache and scintillating scotoma (compare Table 1). Headache, if occurring in EA2, is usually associated with photophonophobia, nausea and vomiting but may additionally be accompanied by paresthesias, hemiplegia [27– 29], muscular weakness [5, 30], confusion or even coma (compare Table 3) [29, 31, 32]. Headache, however, may also appear without accompanying symptoms [5, 33–35]. Similarly to other EA2 families, frequency and duration of attacks was highly variable in our family. The index patient exhibited increasing frequency of attacks with progressing disease, while patient IV-3 experienced worsening of attacks in his third decade. Both increasing [36, 37] and decreasing frequency with age [38, 39], as well as high frequency during childhood with rebound in the fourth decade [39] have been reported. Between attacks, individuals may be free of symptoms but eventually develop nystagmus and ataxia. Interictal phenotypes in the present family were rather typical, manifesting as mild ataxia and gaze-evoked nystagmus or isolated gaze-evoked nystagmus in the majority of cases (compare Table 1). Broad heterogeneity of interictal symptoms may occasionally comprise mental retardation [39, 40], dystonia [37, 41], strabismus [41] and seizures [5, 33, 42]. Subjects with normal interictal phenotype have also been observed [33, 38, 43]. Different phenotypes, encompassing EA2, SCA6 or FHM features have been described in members of the same family [4, 5, 29, 44] and even within single individuals [5, 45]. In addition, up to 33 % of SCA6 patients may have episodic symptoms compatible with the diagnosis of EA2. In one SCA6 family some members had episodic ataxia and others progressive ataxia, whereas on a genetic basis all affected members showed 23 CAG repeats [4]. In
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another family with a CACNA1A missense mutation, phenotypes of both SCA6 and FHM were observed [29]. On the other hand, a progressive cerebellar deficit, typical for SCA6, often develops later in the disease course of EA2 [29, 39, 44], which is the case in our index patient (III-1) who presented with a progressive cerebellar syndrome accompanied by episodic worsening of dysarthria and gait ataxia. Disease onset was early in the majority of affected subjects, whereas the index patient (III-1) and his mother (II-2) developed first symptoms in their adulthood. Families comprising individuals with both early and late disease onset show tendency to decrease in age of onset in succeeding generations [27, 42, 43]. DNA repeat sequences influencing gene expression, CACNA1A gene modifiers or loss of protective susceptibility alleles from parents may explain this phenomenon [42, 46]. Alternatively, earlier disease onset may also be attributed to an observational bias in affected families. Recent reports suggest neuronal channelopathies to be associated with neuropsychiatric features [47]. In EA2, cognitive impairment and mental retardation have been described in some few mutations [48–50]. Mantuano et al. [51] suggested an increased incidence of psychiatric disorders in their EA2 cohorts. To the best of our knowledge, however, no study so far has systematically assessed cognitive functions and mood in EA2. Neuropsychological testing in our family showed impairments in various cognitive functions. All patients exhibited deficits in executive functions including verbal working-memory, phonematic verbal fluency, set-shifting and cognitive flexibility. Moreover, deficits were evident in verbal and figural memory, in attention and in visuo-constructional abilities. These results are in line with previous findings of executive dysfunction in genetically related FHM [52], suggesting that cerebro-cerebellar loops are not fully functional in patients with EA2. Apart from neuropsychological findings, patients appear to be prone to develop socio-phobic behavior and anxiety disorder [51], which has to be taken into consideration in patient management and treatment decision especially in young onset EA2 patients. More detailed neuropsychological assessment in a broader spectrum of EA2 mutations will be necessary to emphasize these findings. A wide range of clinical presentations emphasizes the absence of clear genotypic-phenotypic correlation both between and within families. In accordance with the current literature, EA2 mutations that are affecting splice site regions appear to result in higher phenotype variability and overlapping symptoms to allelic disease as FHM1 and SCA6. The underlying mechanisms for this phenomenon are currently unknown, but may be related to alternative splicing of the CACNA1A gene and/or epigenetic
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mechanisms. On clinical grounds, neuropsychological evaluation suggested some involvement of the cerebrocerebellar loops in the absence of overt cerebellar atrophy and, even more importantly, a considerable impact on social behaviour and mood. A precise diagnostic work-up of affected individuals, an accurate evaluation of relatives at risk and an early initiation of treatment are therefore of major significance in EA2 patient management. Acknowledgments The authors would like to acknowledge Andreas Janecke for his advice and support in genetic counselling. Conflicts of interest
The authors declare no conflict of interest.
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ORIGINAL COMMUNICATION
Episodic ataxia type 2: phenotype characteristics of a novel CACNA1A mutation and review of the literature Wolfgang Nachbauer • Michael Nocker • Elfriede Karner • Iva Stankovic Iris Unterberger • Andreas Eigentler • Rainer Schneider • Werner Poewe Margarete Delazer • Sylvia Boesch
• •
Received: 8 January 2014 / Revised: 5 March 2014 / Accepted: 5 March 2014 / Published online: 22 March 2014 Ó Springer-Verlag Berlin Heidelberg 2014
Abstract Episodic ataxia type 2 (EA2) is an autosomal dominant inherited neurological disorder that is characterized by paroxysmal episodes of ataxia. The causative gene for EA2 is CACNA1A which codes for the alpha 1A subunit of the voltage-gated P/Q-type calcium channel (Cav2.1). We detected a novel point mutation in the CACNA1A gene in a large Austrian family. All ten affected family members harbored a heterozygous c.3089?2T[C nucleotide exchange in intron 19. In silico modeling demonstrated a loss of the splice site of exon 19 by the mutation, which most likely results in exon skipping without frameshifting or use of an alternative splice site. Clinically, the family exhibited frequent ataxic episodes accompanied by headache in some individuals, which showed a good treatment response to acetazolamide or aminopyridine. Interictal phenotype variability was high ranging from an unremarkable clinical examination to a progressive cerebellar syndrome. Detailed cognitive testing with standardized neuropsychological tests revealed specific deficits in various domains including memory, Electronic supplementary material The online version of this article (doi:10.1007/s00415-014-7310-2) contains supplementary material, which is available to authorized users. W. Nachbauer M. Nocker E. Karner I. Unterberger A. Eigentler W. Poewe M. Delazer S. Boesch (&) Department of Neurology, Medical University Innsbruck, Anichstrasse 35, 6020 Innsbruck, Austria e-mail: [email protected] I. Stankovic Department of Neurology, University of Belgrade, Belgrade, Serbia R. Schneider Department of Biochemistry, Leopold-Franzens-University Innsbruck, Innsbruck, Austria
executive functions and visual abilities. Moreover, a striking coincidence of socio-phobic behavior and anxiety disorders was detected within this family, which interfered with activities of daily living and has to be taken in consideration in EA2 patient management. We here characterize the phenotype of this novel CACNA1A mutation, review the respective literature and discuss implications on diagnosis and patient management. Keywords Episodic ataxia CACNA1A Clinical phenotype Cognition
Introduction Episodic ataxias (EAs) are autosomal dominant inherited neurological disorders characterized by paroxysmal attacks of ataxia and eventually progressive cerebellar dysfunction in later disease stages. The most common subtype of EAs is EA type 2 (EA2), caused by mutations in the CACNA1A gene coding for the pore forming alpha 1A subunit of the voltage gated P/Q-type calcium channel (Cav2.1) [1, 2]. Typically, EA2 is characterized by episodes of ataxia, vertigo and nausea lasting for minutes to hours. Overlapping clinical features to the allelic disorders familial hemiplegic migraine (FHM1) and spinocerebellar ataxia type 6 (SCA6) have been described [3–5]. Therefore, attacks can be accompanied by dysarthria, diplopia, tinnitus, hemiplegia and headache [6, 7]. Interictal symptoms such as nystagmus and ataxia occur in EA2, although individuals may be asymptomatic between single episodes. Attacks are triggered by emotional stress, exertion, caffeine or alcohol. Acetazolamide [8] and the potassium channel blocker 4-aminopyridine [9, 10] have been shown to be effective in EA2, particularly in reducing frequency and
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severity of attacks. We herein give a detailed clinical description of an Austrian family with affected individuals in four generations harbouring a novel CACNA1A mutation and review phenotype variability in previously published EA2 families.
Patients and methods Clinical evaluation Probands were identified within a family exhibiting episodic ataxic features in combination with a mild progressive cerebellar syndrome. The index patient (III-1) was initially referred to the ataxia outpatient clinic of the Department of Neurology, Medical University Innsbruck in 2009. Further family members were consecutively seen and examined on a routine basis. Ten individuals out of four generations were clinically affected (compare Fig. 1). Clinical information on deceased individuals (I-1 and II-1) was obtained by interview of family members and review of available medical records. Clinical work-up comprised detailed medical history including duration, frequency and severity of attacks, as well as neurological assessment. For measuring and monitoring the severity of ataxia the Scale for the Assessment and Rating of Ataxia (SARA) [11] was implemented in routine examination. Additionally, standardized neuropsychological testing was carried out. Cerebral magnetic resonance imaging (MRI) was performed on 1.5 T whole body scanner (Magnetom Avanto, Siemens Erlangen, Germany). Investigations and assessments were conducted in accordance with the Declaration of Helsinki on ethical principles for medical research involving human subjects.
Fig. 1 Pedigree of the family. The family comprised ten affected individuals out of four generations. Roman numerals (I–IV) give the generation, Arabic numerals state the affected individuals of the respective generation ordered according to their age. Squares males; circles females; filled symbols affected subjects; blank symbols unaffected subjects; diagonal line deceased subjects
Neuropsychological assessment comprised a wide range of cognitive functions. Verbal memory (learning, short- and long-delay free recall, recognition) was tested with the Verbal Learning and Memory Test (VLMT), a German version of the Rey Auditory Verbal Learning Test [12]. Figural memory (short- and long-delay free recall and recognition) was examined with the Rey Complex Figure Test and Recognition Trial (RCFT) [13]. Further tasks assessed visuospatial processing [subtests 7,8,9 of the Leistungspru¨fsystem (LPS)] [14], visuo-constructional ability (Rey Complex Figure Copy Trial) and mental arithmetic (GDAE) [15]. Language tasks measured comprehension, confrontation naming by simple nouns and confrontation naming by compound nouns (AAT) [16]. Speed of articulation was tested by rapid syllable repetition [17]. Assessment of attention included the verbal memory span (WMS) [18], tonic alertness (reaction on a visual stimulus) and divided attention (TAP) [19]. Executive function tests assessed verbal working memory (WMS) [18], verbal and figural fluency (RWT, HAMASCH) [20, 21], mental flexibility and ability to maintain rules (OMO, TMT B) [22, 23], planning [24] and inhibition (TAP Go/No Go) [19]. Verbal intelligence was estimated by a multiple choice vocabulary test (MWT) [25]. Motor functions of the right and the left hand were assessed by the grooved pegboard (Lafayette Instruments). To avoid stress which could possibly trigger attacks, tests were distributed over several assessments. Subjects III-1, III-2 and IV-2 performed all neuropsychological tests. Patient IV-3 performed most of the tests. Patient IV-1 was available for parts of the tests. Patients’ scores were compared with standard norms. Performance was
I-1
II - 2
II - 1
III - 1
II - 3
III - 3
III - 2
IV- 1
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Neuropsychological tests
IV- 2
IV- 3
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considered as below average when the score was between the 15th and third percentile of standardized norms, and as impaired when it was at or below the second percentile. Genetic testing Genetic testing was performed in all affected individuals, except for the deceased individuals I-1 and II-1, after written informed consent. Analyses were carried out in a certified laboratory by standard procedures (Centogene, Rostock, Germany). All affected individuals showed a novel c.3089?2T[C mutation lying in the donor splice site of intron 19, which affects the splicing process of exon 19. In silico evaluation of the identified sequence mutation affecting the canonical donor splice site of exon 19/intron 19 was performed using as input exon 19 and 150 bp of intron 19 sequence as given in the NCBI genomic and mRNA reference sequences NC_000019.10 (genomic) and NM_001127222.1 (mRNA) corresponding to consensus cDNA CCDS45998.1 and protein isoform NP_001120694.1. The Berkeley Drosophila Genome Project server (http://www.fruitfly.org/seq_tools/splice.html) and the MaxEntScan server (http://genes.mit.edu/burgelab/ maxent/Xmaxentscan_scoreseq.html) [26] were employed by using default parameters to assess likely changes in the recognition of the exon19/intron 19 splice site caused by the c.3089?2T[C mutation. Both in silico tools predicted a loss of the exon 19/intron 19 splice-site by the mutation which affects an invariantly conserved donor splice site nucleotide.
Results Clinical findings Clinical description of the affected family member of generation I (I-1) was drawn from collateral sources. This person had spells of dizziness in her later adulthood. Accompanying ataxic symptoms and headache were not documented. No overt causes regarding blood pressure, cardiac function or the vestibular system were found. In the second generation, three (II-1, II-2, II-3) out of 11 children were affected. The clinical phenotype in this generation consisted of episodes of vertigo with ataxia and dysarthria. Additional interictal neurological symptoms were absent despite gaze-evoked nystagmus. An 85-year old woman (II-2) complained about short episodes of vertigo lasting for minutes, where she would not be able to stand or walk. Attacks occurred clustered up to several times per week and were markedly reduced in frequency and intensity after initiation of aminopyridine treatment.
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Her sister (II-3) has had vertigo attacks since her youth. The attacks were quite rare and did not interfere with activities of daily life. The clinical examination was unremarkable. The third affected sibling (II-1) died several years ago without being examined by a neurologist. In the third generation of this EA2 family, at least one affected family member revealed progressive ataxia with falls and dysarthria. Attacks appeared to result in phobic behavior and interfered with social activities. A 58-year old male patient (III-1) presented with a more than 10-year history of episodes with instability of gait and tendency to fall, as well as slurred speech and paresthetic sensations of the extremities. Initially, frequency of these episodes was low, but increased with disease duration and resulted in considerable socio-phobic behavior. During the attacks, the patient presented with a severe cerebellar syndrome including dysarthria, limb and gait ataxia. Brain MRI was performed at age 55 years and showed no signs of cerebellar atrophy. His 56-year old brother (III-2) has had disturbances of equilibrium associated with dysarthria since the age of 10 years. Contrary to his brother, attacks did not worsen in frequency or intensity over time. Attacks could be avoided by treatment with aminopyridine. A female cousin (II-3) has had vertigo attacks since her youth. In generation IV, the clinical phenotype worsened considerably, interfering with performance in school and profession, social activities and even activities of daily life. Especially, vegetative symptoms such as nausea accompanied by headache appeared in this generation. An affected 36-year old male patient (IV-3) has had attacks of vertigo since his early childhood. Initially, attacks were solely accompanied by nausea but additionally comprised dysarthria and limb ataxia in the later disease course. Moreover, attacks were accompanied with frontal headache and sensations of heat as preceding symptoms. Interictal neurological assessment revealed bilateral gaze evoked nystagmus without any further evidence of ataxia. His female cousin (IV-1) showed the most pronounced clinical symptoms. She reported severe ataxic episodes accompanied by dizziness, nausea and vomiting lasting up to 1 day. Attacks started in her early childhood and have occurred 3–5 times per month. In line with other members of this EA2 family, magnetic resonance imaging of the brain revealed no significant atrophy or structural lesions. Conditions of clinical state could be improved by treatment with acetazolamide. Because of pregnancy, acetazolamide treatment was stopped which led to a severe worsening of frequency and intensity of attacks. Her 18-year old halfsister (IV-2) has had episodes of ataxia since she was 10 years old. Additionally, scintillating scotoma with subsequent frontal headache and nausea may have accompanied these attacks.
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Table 1 Clinical characteristics of patients II–2 $ 85 years
II–3 $ 82 years
III–1 # 58 years
III–2 # 56 years
III–3 # 51 years
IV–1 $ 35 years
IV–2 $ 18 years
IV–3 # 36 years
Age of onset
30 years
youth
45 years
10 years
youth
2 years
10 years
early childhood
Frequency of attacks
2–3/week
rare
daily
triggered
rare
3–5/month
1–2/week
2–3/month
Duration of attacks Trigger
min none
min none
30 min–3 h emotion
15–30 min exertion, alcohol
min none
h–days stress, exertion
min–h exertion, emotion
2–3 h exertion, coffee, emotion
Ictal symptoms Dysarthria
–
–
H
H
–
H
H
H
Diplopia
–
–
H
–
–
H
–
–
Gait ataxia
H
–
H
H
–
H
H
H
Vertigo
H
H
H
H
H
H
H
H
Cephalea Nausea
– –
– –
– –
– –
– –
H H
H H
H H
Interictal symptoms Nystagmus
H
–
H
–
–
H
H
H
Gait ataxia
–
–
H
–
–
H
H
–
Dysarthria
–
–
H
–
–
H
–
–
SARA score
3
2
6
0
0
4
2
0
Aminopyridine
no therapy
Acetazolamide
Aminopyridine
no therapy
Acetazolamide
no therapy
Acetazolamide
Therapy Frequency
H
H
H
H
H
Duration
–
H
–
H
H
Intensity
H
H
H
H
H
Point mutation (T[C) intron 19, CACNA1A
H
H
H
– H
H
H
H
$ female, # male, min minutes, h hours, SARA scale for the assessment and rating of ataxia
Detailed clinical characteristics of the affected patients are given in table 1. Neuropsychological findings Detailed cognitive testing revealed specific deficits in various cognitive domains including memory, executive functions and visual abilities despite the absence of cerebellar atrophy on MRI. Table 2 summarizes neuropsychological results. All patients scored in the average range in language tasks (comprehension and confrontation-naming of simple nouns). All but one patient scored in the average range on tasks assessing verbal intelligence (IV-3), verbal memory recognition (III-1), confrontation-naming of compound nouns (IV-3) and visual spatial abilities (IV-1, IV-2). However, various impairments were found in other cognitive domains. All patients scored below average in at least three executive tasks (verbal working-memory, phonemic verbal fluency, mental flexibility), even in the impaired range in the set-shifting category of phonemic
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verbal fluency. Rapid syllable was impaired in the youngest patient (IV-2) and below average in all others. Fine motor manipulations with the dominant right hand were impaired in three out of four patients (III-1, III-2, IV-3) and below average in the remaining fourth patient (IV-2). All patients scored below average or even in the impaired range in at least one subtest assessing attention (psychomotor speed, tonic alertness, divided attention). Three out of five patients scored below average or in the impaired range in verbal learning (III-1, IV-1, IV-3), in the short-delay and longdelay free recall of the Rey-Figure (IV-1, IV-2, IV-3) and in visuo-constructional abilities. Two out of five patients (III-1, IV-3) showed deficits in the short-delay and longdelay free recall of a word list. Apart from overt neuropsychological deficits, patients with severe or frequent attacks appear to exert socio-phobic behavior or anxiety disorder. Although psychotic symptoms were denied by all our patients, anxiety and bad performance in school, especially when the disease started early in life, was striking. This phenomenon was
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Table 2 Patients’ performance in neuropsychological tests Tasks
Patients III-1
III-2
IV-1
IV-2
IV-3
Learning sum (Trial 1-5) (maximum 75)
31*
45
42*
54
28**
Short-delay free recall (maximum 15)
7*
10
9
12
7*
Long-delay free recall (maximum 15)
7*
10
11
10
4**
Recognition hits minus false positives (maximum 15)
8*
13
12
13
11
18.5
23
7**
6.5**
10**
Memory Verbal Learning and Memory Test (VLMT)
Rey Complex Figure Test and Recognition Trial (RCFT) Short-delay free recall (maximum 36) Long-delay free recall (maximum 36)
20
24
3**
8.5**
10**
Recognition hits minus false positives (maximum 24)
20
23
n.a.
15**
14**
Visual Abilities Visuoperception Leistungspru¨fsystem (Achievement Test System (L-P-S) subtest 7 (maximum 40)
9
17
8*
8*
n.a.
Spatial processing (L-P-S) subtest 8 (maximum 40)
9
23
0**
15
n.a.
Spatial processing (L-P-S) subtest 9 (maximum 40)
9
8
15
13*
n.a.
Visuoconstruction (RCFT) Copy (maximum 36)
33
33
32*
26**
25**
17
15
n.a.
1**
0**
Token Test (Trial 5): Errors
1
0
2
1
2
Confrontation naming simple nouns (maximum 30)
30
30
29
28
30
Confrontation naming compound nouns (maximum 30) Rapid syllable repetition (‘‘badaga’’/5 s)
26 30*
29 30*
26 27*
29 20**
25* 21*
Trail Making Test, Part A (TMT-A) (s)
69*
29
n.a.
27
70**
Wechsler Memory Scale (WMS): digit span forward (max. 12)
4**
5*
4**
4*
6
Tonic alertness Median (ms)
418**
255
n.a.
316*
290*
Phasic alertness Median (ms)
355*
245
n.a.
253
306*
Mental Arithmetic Graded Difficulty Arithmetic Examination (GDAE) (maximum: 24) Language/Speed of Articulation Aachener Aphasie Test (AAT)
Attention
Test of Attentional Performance (TAP)
Divided attention: Auditive stimuli Median (ms)
493
489
n.a.
594
501
Visual stimuli Median (ms)
544
517
n.a.
641**
569*
Omissions
2
1
n.a.
10**
0
Errors
2
2
n.a.
0
10*
5*
5*
4*
4*
3*
Executive Functions Wechsler Memory Scale (WMS): digit span backward (max. 12) Regensburger Wortflu¨ssigkeits-Test (RWT) Animals/min
9*
23
n.a.
11**
12**
‘‘S’’ words/min Sports/fruits/min
6* 8*
6* 15
n.a. n.a.
3** 10*
3** n.a.
‘‘H’’/’’T’’ words/min
4**
6**
n.a.
4**
3**
Sum
24
38
n.a.
17*
43
Correct
12*
34
n.a.
17*
34
Trail Making Test, Part B (TMT-B) (s)
143*
121*
n.a.
76*
156**
Planungstest (Tower of London): trials to complete (min. = 33)
36
37
n.a.
39
n.a.
452
446
n.a.
532*
n.a.
HAMASCH 5-Point Test
Test of Attentional Performance (TAP) Go/No Go Test: median (ms)
123
988
J Neurol (2014) 261:983–991
Table 2 continued Tasks
Patients III-1
III-2
IV-1
IV-2
IV-3
Omissions
11**
0
n.a.
0
n.a.
Errors
4*
1
n.a.
3*
n.a.
5
3
n.a.
1
14*
Right hand
97**
95**
n.a.
78*
111**
Left hand
101**
78
n.a.
77
102**
95
95
n.a.
86
70*
Odd-Man-Out Test (OMO): errors Fine-Motor Manipulations Grooved Pegboard
Intelligence Estimated Verbal IQ: Mehrfachwahl-Wortschatz Test (MWT-A) n.a. Not administered, min. minimum, max. maximum * 3rd \16th percentile = below average ** B2nd percentile = impaired
anticipated in patient III-2 who scored high for anxiety and depression. Moreover, socio-phobic behavior appears to ameliorate gradually after treatment or even disappears (as observed in patient IV-3 after treatment initiation; patient III-1 formally tested only when treated).
Discussion This paper provides a detailed clinical description of a family affected by episodic ataxia with a novel point mutation of the CACNA1A gene and reviews current literature in EA2 families comprising more than three affected subjects in at least two generations. The majority of CACNA1A mutations causing EA2 are nonsense mutations resulting in a truncated protein. The novel mutation in our family occurs in a crucial nucleotide (T[C) on intron 19. As demonstrated by in silico modelling the exon 19/intron 19 donor splice site is no longer recognized, which may, as a further consequence, lead to exon skipping. Alternatively, it is likely that part of the message will be processed with an abnormal and weaker splice-site down-stream, which would lead to incorporation of this part of the intron sequence, frameshift of the open reading frame and premature termination of the transcript. Intronic CACNA1A mutations leading to exon skipping and abnormal splicing have been described previously in EA2 [53, 54] and appear to result in high phenotype variability as it was the case in the present family [33, 42, 54]. EA2 attacks are typically characterized by episodes of ataxia and can be associated with dysarthria, diplopia, tinnitus, nausea, hemiplegia and headache. In our family, patients showed a high phenotype variability ranging from rare vertiginous attacks (II-2, II-3) to severe ataxic spells accompanied by dysarthria, nausea and vomiting with
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onset in early childhood (IV-1, IV-3). In some affected individuals (IV-1, IV-2, IV-3) attacks were accompanied by headache and scintillating scotoma (compare Table 1). Headache, if occurring in EA2, is usually associated with photophonophobia, nausea and vomiting but may additionally be accompanied by paresthesias, hemiplegia [27– 29], muscular weakness [5, 30], confusion or even coma (compare Table 3) [29, 31, 32]. Headache, however, may also appear without accompanying symptoms [5, 33–35]. Similarly to other EA2 families, frequency and duration of attacks was highly variable in our family. The index patient exhibited increasing frequency of attacks with progressing disease, while patient IV-3 experienced worsening of attacks in his third decade. Both increasing [36, 37] and decreasing frequency with age [38, 39], as well as high frequency during childhood with rebound in the fourth decade [39] have been reported. Between attacks, individuals may be free of symptoms but eventually develop nystagmus and ataxia. Interictal phenotypes in the present family were rather typical, manifesting as mild ataxia and gaze-evoked nystagmus or isolated gaze-evoked nystagmus in the majority of cases (compare Table 1). Broad heterogeneity of interictal symptoms may occasionally comprise mental retardation [39, 40], dystonia [37, 41], strabismus [41] and seizures [5, 33, 42]. Subjects with normal interictal phenotype have also been observed [33, 38, 43]. Different phenotypes, encompassing EA2, SCA6 or FHM features have been described in members of the same family [4, 5, 29, 44] and even within single individuals [5, 45]. In addition, up to 33 % of SCA6 patients may have episodic symptoms compatible with the diagnosis of EA2. In one SCA6 family some members had episodic ataxia and others progressive ataxia, whereas on a genetic basis all affected members showed 23 CAG repeats [4]. In
J Neurol (2014) 261:983–991
another family with a CACNA1A missense mutation, phenotypes of both SCA6 and FHM were observed [29]. On the other hand, a progressive cerebellar deficit, typical for SCA6, often develops later in the disease course of EA2 [29, 39, 44], which is the case in our index patient (III-1) who presented with a progressive cerebellar syndrome accompanied by episodic worsening of dysarthria and gait ataxia. Disease onset was early in the majority of affected subjects, whereas the index patient (III-1) and his mother (II-2) developed first symptoms in their adulthood. Families comprising individuals with both early and late disease onset show tendency to decrease in age of onset in succeeding generations [27, 42, 43]. DNA repeat sequences influencing gene expression, CACNA1A gene modifiers or loss of protective susceptibility alleles from parents may explain this phenomenon [42, 46]. Alternatively, earlier disease onset may also be attributed to an observational bias in affected families. Recent reports suggest neuronal channelopathies to be associated with neuropsychiatric features [47]. In EA2, cognitive impairment and mental retardation have been described in some few mutations [48–50]. Mantuano et al. [51] suggested an increased incidence of psychiatric disorders in their EA2 cohorts. To the best of our knowledge, however, no study so far has systematically assessed cognitive functions and mood in EA2. Neuropsychological testing in our family showed impairments in various cognitive functions. All patients exhibited deficits in executive functions including verbal working-memory, phonematic verbal fluency, set-shifting and cognitive flexibility. Moreover, deficits were evident in verbal and figural memory, in attention and in visuo-constructional abilities. These results are in line with previous findings of executive dysfunction in genetically related FHM [52], suggesting that cerebro-cerebellar loops are not fully functional in patients with EA2. Apart from neuropsychological findings, patients appear to be prone to develop socio-phobic behavior and anxiety disorder [51], which has to be taken into consideration in patient management and treatment decision especially in young onset EA2 patients. More detailed neuropsychological assessment in a broader spectrum of EA2 mutations will be necessary to emphasize these findings. A wide range of clinical presentations emphasizes the absence of clear genotypic-phenotypic correlation both between and within families. In accordance with the current literature, EA2 mutations that are affecting splice site regions appear to result in higher phenotype variability and overlapping symptoms to allelic disease as FHM1 and SCA6. The underlying mechanisms for this phenomenon are currently unknown, but may be related to alternative splicing of the CACNA1A gene and/or epigenetic
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mechanisms. On clinical grounds, neuropsychological evaluation suggested some involvement of the cerebrocerebellar loops in the absence of overt cerebellar atrophy and, even more importantly, a considerable impact on social behaviour and mood. A precise diagnostic work-up of affected individuals, an accurate evaluation of relatives at risk and an early initiation of treatment are therefore of major significance in EA2 patient management. Acknowledgments The authors would like to acknowledge Andreas Janecke for his advice and support in genetic counselling. Conflicts of interest
The authors declare no conflict of interest.
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