Research
Case Report/Case Series
Autosomal Recessive Cerebellar Ataxia Type 3 Due to ANO10 Mutations Delineation and Genotype-Phenotype Correlation Study Mathilde Renaud, MD; Mathieu Anheim, MD, PhD; Erik-Jan Kamsteeg, PhD; Martial Mallaret, MD; Fanny Mochel, MD, PhD; Sascha Vermeer, MD, PhD; Nathalie Drouot, MSc; Jean Pouget, MD; Claire Redin, PhD; Emmanuelle Salort-Campana, MD; Hubertus P. H. Kremer, MD, PhD; Corien C. Verschuuren-Bemelmans, MD; Jean Muller, PhD; Hans Scheffer, PhD; Alexandra Durr, MD, PhD; Christine Tranchant, MD; Michel Koenig, MD, PhD
IMPORTANCE ANO10 mutations have been reported to cause a novel form of autosomal
recessive cerebellar ataxia (ARCA). Our objective was to report 9 ataxic patients carrying 8 novel ANO10 mutations to improve the delineation of this form of ARCA and provide genotype-phenotype correlation. OBSERVATIONS The ANO10 gene has been sequenced in 186 consecutive patients with ARCA. The detailed phenotype of patients with ANO10 mutations was investigated and compared with the 12 previously reported cases. The mean age at onset was 33 years (range, 17-43 years), and the disease progression was slow. Corticospinal tract signs were frequent, including extensor plantar reflexes and/or diffuse tendon reflexes and/or spasticity. No patient in our series had peripheral neuropathy. Magnetic resonance imaging of the brains of our patients revealed marked cerebellar atrophy. The most frequent mutation, a mononucleotide expansion from a polyA repeat tract (c.132dupA) that causes protein truncation, was never observed in homozygosity. Only 2 truncating mutations were reported in homozygosity, one of which (c.1150-1151del) was associated with juvenile or adolescent onset and mental retardation, whereas we show that the presence of at least 1 missense or in-frame mutation is associated with adult onset and slow progression. CONCLUSIONS AND RELEVANCE An ANO10 mutation is responsible for ARCA that is
mainly characterized by cerebellar atrophy and lack of peripheral neuropathy. We therefore suggest naming this entity autosomal recessive cerebellar ataxia type 3 (ARCA3). JAMA Neurol. 2014;71(10):1305-1310. doi:10.1001/jamaneurol.2014.193 Published online August 4, 2014.
A
utosomal recessive cerebellar ataxias (ARCAs) correspond to a heterogeneous group of inherited neurodegenerative disorders that affect the cerebellum and/or the peripheral nerves.1 A new classification of ARCA has been proposed including 3 groups: ARCA with pure sensory neuropathy, ARCA with sensorimotor axonal neuropathy, and ARCA without neuropathy. 2 Recently, a new form of ARCA due to mutations in the ANO10 gene has been reported. 3 To which group of the classification does this new entity belong and whether genotype-phenotype correlation may be proposed have not yet been elucidated. We report 9 unrelated patients carrying 8 novel ANO10 mutations, and we compared these patients with the 12 previously reported cases in order to provide the delineation of the disease and genotype-phenotype correlations. jamaneurology.com
Author Affiliations: Author affiliations are listed at the end of this article. Corresponding Author: Michel Koenig, MD, PhD, Laboratoire de Génétique des Maladies Rares, Institut National de la Santé et de la Recherche Médicale UMR_S 827, Institut Universitaire de Recherche Clinique, 641 Avenue du Doyen Gaston Giraud, 34093 Montpellier CEDEX 5, France ([email protected]).
Methods Between 2010 and 2013, a total of 186 unrelated index patients were consecutively recruited from 4 tertiary centers for inherited neurodegenerative disorders. Inclusion criteria were the combination of (1) sporadic or recessive progressive cerebellar ataxia; (2) age at onset of younger than 50 years; and (3) unknown etiology despite extensive investigations, including molecular analysis for Friedreich ataxia and obvious forms of recessive ataxias. Written informed consent was obtained from all participants. Participants were not financially compensated. The recessive ataxia research project was approved by the Strasbourg ethics committee. A detailed neurological examination, including scales of severity and magnetic resonance imaging of the brain, was performed for all patients. JAMA Neurology October 2014 Volume 71, Number 10
Copyright 2014 American Medical Association. All rights reserved.
Downloaded From: http://archneur.jamanetwork.com/ by a University of St. Andrews Library User on 05/14/2015
1305
Research Case Report/Case Series
ARCA3 Due to ANO10 Mutations
Table 1. Main Clinical, Molecular, Morphologic, and Electrophysiologic Features of 9 Patients Carrying 8 Novel ANO10 Mutations Feature
Case 1
Case 2
Case 3
Case 5
Case 6
Case 7
Case 8
Age at onset, y
17
33
40
Case 4 37
30
32
43
33
32
Age at examination, y
23
44
47
43
33
59
68
52
37
Sex
Female
Male
Male
Female
Female
Male
Female
Female
Race/Ethnicity
White (Turkish)
White (French)
White (French) West Indies
West Indies
White (French)
White (Dutch)
White (Dutch)
White (French)
First sign
Cerebellar dysarthria
Gait ataxia
Gait ataxia
Gait ataxia
Diplopia
Ataxia with vertigo
Ataxia
Ataxia, vertigo, cephalalgia
Ataxia
+
Female
Case 9
+
Gait ataxia
+
+
+
+
+
+
+
Tendon reflexes
Increased
Increased
Increased
Increased
Increased
Increased
Normal
Increased
Increased
Plantar reflexes
Flexor
Extensor
Flexor
Flexor
Flexor
Flexor
Flexor
Extensor
Extensor
Ocular motor findings
Gaze-evoked nystagmus, hypermetric saccades
Gaze-evoked nystagmus
Vertical nystagmus
Gaze-evoked nystagmus
Hypermetric Gaze-evoked saccades nystagmus
Downbeat nystagmus, slow saccades
Vertical and horizontal nystagmus
Saccadic pursuit
Other findings
Linguistic difficulties in French
Left hypoacousia since 20 y
Lower limb spasticity
Tongue fasciculations
Ankle clonus
Diplopia, dysphagia
Stridor, anxiety
None
Lower limb spasticity, paroxysmal limb weakness
Cognitive status
Normal
Normal
Normal
Normal
Normal
Normal
MMSE score of 19-29
Mild intellectual disabilities
Normal
SARA
11
14
NP
6.5
10
NP
NP
NP
10.5
ICARS
27
44
NP
NP
NP
NP
NP
NP
NP
SDFS
3
4
4
3
3
4
NP
NP
NP
NP
EMG results
Normal
Normal
Motor neuron Normal involvement in tibialis anterior
Results of ophthalmic examination with fundus
Normal
Normal
Normal
MRI findings
Diffuse cerebellar atrophy at 23 y
Diffuse cerebellar atrophy at 43 y
Cerebellar Diffuse atrophy at 43 y cerebellar atrophy at 47 t
Moderate cerebellar atrophy at 33 y
Cerebellar atrophy at 59 y
α1-Fetoprotein Normal levela
Normal
Increased 16.6 Normal ng/mL
Normal
Increased 11.8 ng/mL
Mutations
Compound heterozygous: missense (c.685G>T, p.Gly229Trp [exon 6]), nonsense (c .1291C>T, p.Gln431* [exon 8])
Compound heterozygous: missense (c.1009T>G, p.Phe337Val [exon 6]), frameshift (c.132dupA, p.Asp45Argfs*9 [exon 2])
Homozygous: donor splice site (c.1668+ 1G>A [exon 10])
NP
Compound heterozygous: frameshift (c.1214delT, p.Leu405* [exon 7]), donor splice site (c.1476+ 1G>T [exon 9])
NP
Normal
Normal
NP
3 Normal
Normal
Normal
Cerebellar atrophy at 40 y
Cerebellar Cerebellar atrophy at 34 y atrophy at 34 y
NP
Homozygous Homozygous Compound heterozygous: deletion deletion exon 12 missense exon 12 (c.1009T>G, p.Phe337Val [exon 6]), frameshift (c.132dupA, p.Asp45Argfs*9 [exon 2])
Normal
Normal
Normal
Compound heterozygous: deletion exon 12, frameshift (c.132dupA, p.Asp45Argfs*9 [exon 2])
Compound heterozygous: missense (c.512T>C, p.Phe171Ser [exon 5]), frameshift (c.132dupA, p.Asp45Argfs*9 [exon 2])
Abbreviations: EMG, electroneuromyography; ICARS, International Cooperative Ataxia Rating Scale; MMSE, Mini-Mental State Examination; MRI, magnetic resonance imaging; NP, not performed; SARA, Scale for the Assessment and Rating of Ataxia; SDFS, Spinocerebellar Degeneration Functional Score; +, tested positive. SI conversion factor: To convert α1-fetoprotein to micrograms per liter, multiply by 1.0. a
Normal range is less than 7 ng/mL.
Genetic analysis of ANO10 was performed for 44 patients by conventional Sanger sequencing. A total of 142 additional patients were directly analyzed by a targeted exon-capture strategy coupled with multiplexing and high-throughput sequencing of 57 genes causing ataxia when mutated, including ANO10. The splice-site mutations were evaluated with the SplicePort program (http://spliceport.cbcb.umd.edu/) and were analyzed by reverse transcription of RNA from patients’ fibroblasts followed by polymerase chain reaction from exon 8 to 1306
exon 11 (520–base pair [bp] fragment) and from exon 6 to exon 12 (809-bp fragment). Polymerase chain reaction fragments were visualized on agarose gels and sequenced by use of the Sanger method. The patients from this and previous studies were divided into 2 groups according to the type of the ANO10 mutation: (1) at least 1 missense or in-frame mutation, or (2) 2 truncating mutations. The 2 groups were compared by use of the t test.
JAMA Neurology October 2014 Volume 71, Number 10
Copyright 2014 American Medical Association. All rights reserved.
Downloaded From: http://archneur.jamanetwork.com/ by a University of St. Andrews Library User on 05/14/2015
jamaneurology.com
ARCA3 Due to ANO10 Mutations
Case Report/Case Series Research
Results The clinical, radiological, and biological findings of the 9 patients are shown in Table 1 and Figure 1. Of the 186 patients, 9 (5%) had ANO10 mutations. All 9 of these patients carrying ANO10 mutations had obvious cerebellar atrophy detected on magnetic resonance imaging scans of the brain and a lack of peripheral neuropathy. There was a subgroup of 19 patients with recessive progressive cerebellar ataxia and 37 patients with sporadic progressive cerebellar ataxia for whom cerebellar atrophy and lack of peripheral neuropathy were documented. Of these 56 patients, 6 (11%) had ANO10 mutations. The 9 patients carried 1 previously reported ANO10 mutation and 8 novel ANO10 mutations (Table 1). The c.132dupA mutation was identified in 4 unrelated patients. The mutation is an insertion of an A (adenine nucleotide) following a homopolymer of 9 A’s and can be considered as a mononucleotide expansion of a mononucleotide repeat tract. Indeed, the c.132dupA mutation is a frequent mutation with a heterozy-
gous carrier frequency of 1/184 in different ethnic groups (Exome Variant Server [http://evs.gs.washington.edu/EVS]) and probably arose because of multiple independent homopolymer instability events. The c.132dupA mutation leads to early protein truncation (p.Asp45Argfs*9). Surprisingly, all 4 patients with this mutation were compound heterozygous, and no homozygous c.132dupA patient was identified. The 3 identified missense mutations, p.Phe171Ser, p.Gly229Trp, and p.Phe337Val, are likely pathogenic because they affect amino acid positions that are conserved in the orthologous sequences of almost all metazoan species (Figure 2). The p.Gly229Trp mutation is located at the C-terminal border of the first α-helical transmembrane segment, and the p.Phe337Val mutation is located at the C-terminal border of the third transmembrane segment. Reverse transcriptase–polymerase chain reaction analysis of the 2 donor splice-site mutations (Figure 3) revealed complete alteration of the splice sites. Surprisingly, skipping the mutated exon or using cryptic splice sites resulted in all cases in in-frame transcripts. The deletion mutation (deletion of exon 12), identified in 3 unrelated patients, is
Figure 1. Magnetic Resonance Imaging of the Brain of Patient 2 A
B
Marked cerebellar atrophy with normal supratentorial structures at 43 years of age. A sagittal T1-weighted sequence (A) and a coronal T1-weighted inversion recovery sequence (B) are shown.
Figure 2. Conservation of Amino Acids Altered by Missense Mutation (p.Phe171Ser, p.Gly229Trp, and p.Phe337Val, Respectively)
hs gg xl tr dr bf sp cg ct phc ag nv hm
S G I V I Q V F P L H D S E A L K K L E D T WY T R G I L V Q I F P L H D R E E L K K L R H T WY G R G I M I Q V F P L H D K E E L K R L R H QW Y L R G I L I Q I F P L H E Q E D L K R L S F T WY K R G V L V Q Y F P L H N K E D L K R L S F S WY K K H L L E K M Y P V H N P E Q L K R L Q H DWY H L G L I D G I Y P L H T P T E L V S L E N HW Y N H D I I T K M Y P L H E P E D L K K L G A DWY Q I G I I A Q L F P L H D K R S L K T L S F DWY Q S G Y V E K C F P L H E I E Y L K K F S Q T WY L T E L I T N L Y S L H D K R T L K E L R H RW I K P G V V S S I Y P L H E A Q K L K L L E K KW F L N H K I V Q I Y P L H H I E S L K S L E N QW Y L G
W E T I A L Y F G F L E Y F T F A L I P MA V I G L P Y Y L F E T I A L Y F G F L E Y F T F A L I P MA V I G I P Y Y V F D T L G L Y F A F L E Y F T MA L I P MA L I G I P Y Y F F E G Q A L Y F G F L E F F T F A L V P MA L V G V P Y Y L F E G L G L Y F G F L E Y F T F A L I P MA L I G I P Y Y L F E S V AMY F S F L G H Y T K A L L V P T V I G V L Y Y L F EK I G I YF S F LGF YTKAL F L PAA FGL L Y F L L EK I ALYFAFLGYYT I AL I PPAF I G I I YF I T G T V AM Y F A F L G F Y TMA L I P P A L I G V I S E Y G EN I A L Y F I F L G Y Y T C A L V P PM I L G L I E A L I E S V GMY F S F L G F Y T Y A L V V P T V F G F L Q L G L E TMAMY F A F L Q F Y S MT L I P P V L L I V V F A L S ES I AL YF T F LGF YTK F L L P TAV I G I LHY F F
Alignment of the surrounding sequence of ANO10 orthologues from 13 metazoan species is shown. The transmembrane sequences are underlined. Mutant amino acids (1-letter code) are indicated on top of the corresponding mutated amino acids. Conserved amino acids are indicated in bold: hs indicates Homo sapiens;
jamaneurology.com
V Y L V S L P F V C L C L Y F S L Y V MM I Y F D M E V W Y L V S V P F V C L C L Y F S L Y V MM I Y F D L E HW Y L V S V P F V C V C L Y L A S Y V MM I Y F D L E HW Y L V S L P F V L L C L Y L S L Y V MM I Y F Q M E GW Y L V S V P F V L L C L Y L S F Y V MM V Y F D M E F W Y C V S F P I V I L C M L V A V V V MM I Y F W A E N I Y C V S A P I I L V F L C L A F L MM L G F F W F E D Y Y C V T V P I V S V A L G I A F Y I M L G Y F V MQ E W Y G V S L P V V L L C L F G A F Y I ML V Y FW S E D L Y C V S L P I V F L C L T G A F F F M L F S F Y M E NM Y C V T A P I I M L CMG I A A F V T I F Q F Y V E A Y Y G I S Y P I V L L CMK V A T V VM L L Y F K L Q F Y Y F I S Y P L L L I S L L L V T V GM L F Y F H L N E K
gg, Gallus gallus; xl, Xenope laevis; tr, Tachifugu rubripes; dr, Danio rerio; bf, Branchiostoma floridae; sp, Strongylocentrotus purpuratus; cg; Crassostrea gigas; ct, Capitella teleta, phc, Pediculus humanus corporis; ag, Anopheles gambiae; nv, Nematostella vectensis; and hm, Hydra magnapapillata.
JAMA Neurology October 2014 Volume 71, Number 10
Copyright 2014 American Medical Association. All rights reserved.
Downloaded From: http://archneur.jamanetwork.com/ by a University of St. Andrews Library User on 05/14/2015
1307
Research Case Report/Case Series
ARCA3 Due to ANO10 Mutations
Figure 3. RT-PCR Analysis of Exons 8 to 11 of ANO10 Transcripts of Patients With Splice-Site Mutation A
M
C
4
1
C
No RT
No cDNA
M
B
C TGGAAAAAGAAA TGGGAA C T T A T T TGGGC A C C T T TGA TGA T T A C T TGGAGT T A T T C C TGC AGT T TGGT T A TGTGAGC C T T T T C T C C TGTGT T T A C C C A T T AGC AGC TGC C T T TGC TGTGT T AAA T AA C T T C A C TGAAGT AAA T T C AGA TGC C T T AAAAA TGTGC AGGGT C T T C AAA CGT C C A T T C T C AGAA C C T T C AGC C AA T A T TGGTGT AAAGAGAGGGC TGAA T TGC AAGGT C A TGAGGAA T C T T T TGGGGGAAA T GGAAA TGT C C TGTGT C T TGT T TGTGGTGGTGGTGGT T T C A C AGT TGGC T T T TGAAA CGA TGAGTG T T A T A T C TGTGGT C A C T AA C TGTGCGC TGA T TGGAA TGT C A C C A C AAGTGAA TGC A C TGGAAAAAGAAA TGGGAA C T T A T T TGGGC A C C T T TGA TGA T T A C T TGGAGT T A T T C C TGC AGT T TGGT T A T T TGGC T T T TGAAA CGA TGAGTGT T A T A T C TGTGGT C A C T AAGTGTGCGC TGA T TGGAA TGT CACCACAAGTGAATGCA C TGGAAAAAGAAA TGGGAA C T T A T T TGT TGGC T T T TGAAA CGA TGAGTGT T A T A T C TGTGGT C A C T AA C TGTGCGC TGA T TGGAA TGT C A C C A C AAGTGAA TGC A
A, Reverse transcriptase–polymerase chain reaction (RT-PCR) fragments were run on agarose gel and visualized with ethidium bromide. The letter M indicates size markers; the letter C, control individual (520–base pair normal fragment); and the number 4, patient 4 (compound heterozygous c.1476+1G>T, exon 9 and p.Leu405*, exon 7). The sequence of the fragment for patient 4 revealed complete skipping of exon 9 (in-frame deletion from codon 432 to codon 492, which includes part of the coding sequence of the large third extracellular loop [Figure 4]); the absence of the normal fragment indicates degradation of the p.Leu405* allele by nonsense-mediated RNA decay. The number 1 indicates patient 1 (homozygous c.1668+1G>A, exon 10); the abnormal fragments are indicated by white arrowheads (sequence in panel B), and heteroduplexes are indicated by black arrowheads. No RT indicates negative control with no
reverse transcriptase; no cDNA, negative control with no complementary DNA. B, Sequence of the RT-PCR of the fragments of patient 1 (homozygous c.1668+1G>A, exon 10 [A]). Part of exon 9 and part of a cryptic exon in intron 10 have a single underline. Parts of exon 10 have a double underline. Part of exon 11 has no underline. Strikingly, all 3 identified fragments are in-frame. The 2 larger fragments result from the use of cryptic splice sites within exon 10. The largest fragment includes most of exon 10 and a cryptic exon, resulting in the replacement of tryptophane 555 by 29 novel amino acids. The smallest fragment (the most abundant) corresponds to complete skipping of exon 10, which results in in-frame deletion from codon 493 to codon 556, including the coding sequence of the sixth transmembrane segment.
also in frame. A phenotype vs genotype comparison, including also previously reported patients (Figure 4), revealed that the age at onset was higher for patients with at least 1 missense or in-frame exon skipping mutation than for patients with 2 truncating mutations (homozygous c.1050_1051del and homozygous p.Tyr203*) (P < .01).
to name this new entity autosomal recessive cerebellar ataxia type 3 (ARCA3) (Table 2). The mean age at onset is late in our series (including 8 patients with onset at older than 25 years of age), but, overall, age at onset for those with ARCA3 appears highly variable, ranging from 6 to 43 years. Disease progression is slow, with patients remaining ambulatory for up to 25 years after the onset of the disease. However, it may be more severe, as in one of our patients (case 4), who needed a stick for walking after 7 years of disease duration. Lower limb reflexes appeared to be always increased, but extensor plantar reflexes, mental retardation, or cognitive decline were variably present (Table 1).3,4,8 Two patients exhibited spasticity; therefore, ARCA3 should also be considered in case of spastic ataxia. One patient (case 9) reported paroxysmal episodes of ataxia and vertigo, which could be explained by the abnormal function of mutated anoctamin-
Discussion Although electrophysiological signs of lower motor neuron involvement in lower limbs were found in a few patients3,4 and in case 3, none of the 21 reported patients had polyneuropathy, which classifies ANO10-linked ataxia among the group of cerebellar ataxias without neuropathy.3 Because ARCA15,6 and ARCA27 are already representatives of this group, we propose 1308
JAMA Neurology October 2014 Volume 71, Number 10
Copyright 2014 American Medical Association. All rights reserved.
Downloaded From: http://archneur.jamanetwork.com/ by a University of St. Andrews Library User on 05/14/2015
jamaneurology.com
ARCA3 Due to ANO10 Mutations
Case Report/Case Series Research
Figure 4. Correlation Between Predicted Consequences of Mutations (Missense, In-Frame Exon Skipping, In-Frame Exon Deletion and Truncation) and Age at Onset of Disease Mutations p.Gly229Trp
Age at Onset, y p.Phe337Val
p.Leu510Arg
33, 40, 32 (20, 25, 32), 32
p.Phe171Ser
c.1476+1G>T
(25, 42), 37
c.1668+1G>A
17,
Del exon 12
30, 43, 33
c.1150-1151del
(6, 7, 7), (13, 15, 15),
p.Tyr203*
42
The ages at onset of patients from the same family are indicated in parentheses. The ages at onset of patients reported in our study are underlined. Previously reported patients were either homozygous for the p.Leu510Arg missense mutation (affecting the sixth transmembrane segment of ANO10), homozygous for the c.1050_1051del or p.Tyr203* truncating mutations, or compound heterozygous for a nonsense mutation and the same exon 9 splice-site mutation reported here for patient 4. The correlations imply that homozygous or compound heterozygous mutations that cause a partial loss of function (missense or in-frame mutation) will be associated with a milder phenotype, even if associated with a truncating mutation on the other allele.
Table 2. Clinical and Genetic Characteristics of Patients With ARCA1, ARCA2, or ARCA3 Characteristic
Patients With ARCA1
Age at onset, mean (range), y
32 (17-45)
Patients With ARCA2
Patients With ARCA3
4 (1-11)
21 (6-45)
Cerebellar ataxia
Always present
Always present
Always present
Other optional clinical features
Enhanced tendon reflexes
Mental retardation, myoclonus, epilepsy stroke like episodes, exercise intolerance
Corticospinal tract signs
Progression
Very slow
Variable but mostly very slow
Slow
EMG results
No polyneuropathy, motor neuron involvement in 1 patient6
No polyneuropathy
No polyneuropathy, occasional motor neuron involvement
Biomarker
None
Variable elevation of serum lactic acid levels, variable decrease of CoQ10
None (increased AFP level in 2 patients)
Brain MRI findings
Cerebellar atrophy
Cerebellar atrophy, variable cortical hyperintensities
Cerebellar atrophy
Gene, protein
SYNE1, nesprin-1 (nuclear envelope and spectrin-like repeats 1)
ADKC3, aarF domain containing kinase 3
ANO10, anoctamin-10
10, which is a putative calcium-activated chloride channel.9 No extraneurological signs were found in our series. The α1-fetoprotein serum level, which is known to be a reliable biomarker for ataxia with oculomotor apraxia type 210 and ataxia telangiectasia, was mildly increased in 2 cases. Whether α1-fetoprotein could be a biomarker for ARCA3 remains to be elucidated by further large studies. Overall, we report an ARCA3 phenotype in accordance with previous reports and, more precisely, define a core presentation that should be kept in mind when facing a patient with recessive or sporadic ataxia. Autosomal recessive cerebellar ataxia type 3 appears to be a common cause of recessive ataxias. While the frequency is accounted for, in part, by the high frequency of the major mutation, c.132dupA, which belongs to the group of unstable sequences, this mutation is underrepresented in patients with ARCA3 with respect to the jamaneurology.com
Abbreviations: AFP, α1-fetoprotein; ARCA1, autosomal recessive cerebellar ataxia type 1; CoQ10, coenzyme Q10; EMG, electromyography; MRI, magnetic resonance imaging.
other mutations that are at least 10-fold less frequent (http: //evs.gs.washington.edu/EVS). The fact that all patients with this mutation were compound heterozygotes and that no homozygous c.132dupA patient was identified suggests that c.132dupA homozygotes are either not viable or have a more severe phenotype, similar to the p.Arg141His mutation of phosphomannomutase 2 in the congenital disorder of glycosylation type Ia.11 In accordance with this correlation is the fact that all 6 previously reported patients with the homozygous truncating mutation c.1150-1151del3,4 have a significantly earlier age at onset (juvenile or adolescent onset associated with mental retardation) compared with the patients with at least 1 missense or in-frame mutation (Figure 4) who have adult onset, slow progression, and persistence of ambulation over a long disease duration. However, a recently reported patient,8 hoJAMA Neurology October 2014 Volume 71, Number 10
Copyright 2014 American Medical Association. All rights reserved.
Downloaded From: http://archneur.jamanetwork.com/ by a University of St. Andrews Library User on 05/14/2015
1309
Research Case Report/Case Series
ARCA3 Due to ANO10 Mutations
mozygous for the truncating nonsense mutation p.Tyr203*, does not fit with this correlation because he had onset at 42 to 46 years of age. It is nevertheless interesting to note that the only 2 reported homozygous truncating mutations (p.Tyr203* and c.1150-1151del) are both located in exon 6, which is in frame and differentially spliced in vivo (absent from isoform 2 of ANO10 [http://www.uniprot.org/uniprot/]). It is possible that these 2 mutations variably affect differential splicing (or cryptic splice-site activation, as in case 1 of this study) of exon 6 and result in production of in-frame transcripts, because the 2 mutations are located close to the acceptor and donor splice sites, respectively, of exon 6. Such a mechanism would explain the differential consequence of homozygous truncating mutations p.Tyr203* and c.1150-1151del
ARTICLE INFORMATION Published Online: August 4, 2014. doi:10.1001/jamaneurol.2014.193. Author Affiliations: Département de neurologie, hôpitaux universitaires de Strasbourg, hôpital de Hautepierre, Strasbourg, France (Renaud, Anheim, Mallaret, Tranchant); Institut de Génétique et de Biologie Moléculaire et Cellulaire, Institut National de la Santé et de la Recherche Médicale, Centre National de la Recherche Scientifique, Université de Strasbourg, Strasbourg, France (Renaud, Anheim, Mallaret, Drouot, Redin, Muller, Tranchant, Koenig); Fédération de Médecine Translationnelle de Strasbourg, Université de Strasbourg, Strasbourg, France (Renaud, Anheim, Mallaret, Tranchant); Department of Human Genetics, Radboud University Medical Centre, Nijmegen, the Netherlands (Kamsteeg, Vermeer, Scheffer); Université Pierre et Marie Curie Paris 6, Centre de recherche de l’institut du cerveau et de la moelle épinière, Paris, France (Mochel, Durr); AP-HP, Hôpital de la Salpêtrière, Département de Génétique et Cytogénétique, Paris, France (Mochel, Durr); Département de neurologie, hôpitaux universitaires de Marseille, hôpital de la Timone, Marseille, France (Pouget, Salort-Campana); Department of Neurology, Radboud University Medical Centre, Donders Institute for Brain, Cognition and Behaviour, Nijmegen, the Netherlands (Kremer, Verschuuren-Bemelmans); Laboratoire de Génétique des Maladies Rares, Institut National de la Santé et de la Recherche Médicale UMR_S 827, Institut Universitaire de Recherche Clinique, Montpellier, France (Koenig). Author Contributions: Drs Renaud and Koenig had full access to all of the data in the study and take responsibility for the integrity of the data and the accuracy of the data analysis. Drs Anheim and Renaud contributed equally to this article as first authors. Drs Tranchant and Koenig contributed equally to this article as last authors. Study concept and design: Renaud, Anheim, Redin, Muller, Scheffer, Tranchant, Koenig. Acquisition, analysis, or interpretation of data: Renaud, Anheim, Kamsteeg, Mallaret, Mochel, Vermeer, Drouot, Pouget, Salort-Campana, Kremer,
1310
and the absence of homozygous truncating mutations c.132dupA, despite the high frequency of heterozygote carriers (1/184). RNA studies of the p.Tyr203* and c.1150-1151del homozygous patients are warranted to solve this issue.
Conclusions In conclusion, ARCA3, which has a major mutation (c.132dupA) that is a mononucleotide expansion from a mononucleotide repeat tract, is not a rare form of recessive cerebellar ataxia. It should be considered in cases of progressive cerebellar ataxia with cerebellar atrophy but without peripheral neuropathy, even in sporadic cases and whatever the age at onset.
Verschuuren-Bemelmans, Muller, Durr, Tranchant, Koenig. Drafting of the manuscript: Renaud, Anheim, Drouot, Muller, Durr, Tranchant, Koenig. Critical revision of the manuscript for important intellectual content: Anheim, Kamsteeg, Mallaret, Mochel, Vermeer, Pouget, Redin, Salort-Campana, Kremer, Verschuuren-Bemelmans, Scheffer, Tranchant, Koenig. Statistical analysis: Renaud, Muller. Obtained funding: Muller, Scheffer, Koenig. Administrative, technical, or material support: Mochel, Vermeer, Drouot, Pouget, Redin, Durr, Koenig. Study supervision: Anheim, Tranchant, Koenig. Conflict of Interest Disclosures: None reported. Funding/Support: Dr Renaud was supported by a fellowship from the Journées de Neurologie de Langue Française. This study was supported by funds from the Institut National de la Santé et de la Recherche Médicale, the Centre National de la Recherche Scientifique, the Agence Nationale pour la Recherche-Maladies Rares and Maladies Neurologiques et Psychiatriques (grant ANR-09MNPS-001-01 to Dr Koenig), and the ANR/E-rare JTC 2011 “Euro-SCAR” (grant 2011-RARE-004-01 to Dr Koenig). Role of the Sponsor: The funding agencies had no role in the design and conduct of the study; collection, management, analysis, or interpretation of the data; preparation, review, or approval of the manuscript; and decision to submit the manuscript for publication. REFERENCES 1. Anheim M, Fleury M, Monga B, et al. Epidemiological, clinical, paraclinical and molecular study of a cohort of 102 patients affected with autosomal recessive progressive cerebellar ataxia from Alsace, Eastern France: implications for clinical management. Neurogenetics. 2010;11(1):1-12.
3. Vermeer S, Hoischen A, Meijer RP, et al. Targeted next-generation sequencing of a 12.5 Mb homozygous region reveals ANO10 mutations in patients with autosomal-recessive cerebellar ataxia. Am J Hum Genet. 2010;87(6):813-819. 4. Chamova T, Florez L, Guergueltcheva V, et al. ANO10 c.1150_1151del is a founder mutation causing autosomal recessive cerebellar ataxia in Roma/Gypsies. J Neurol. 2012;259(5):906-911. 5. Gros-Louis F, Dupré N, Dion P, et al. Mutations in SYNE1 lead to a newly discovered form of autosomal recessive cerebellar ataxia. Nat Genet. 2007;39(1):80-85. 6. Izumi Y, Miyamoto R, Morino H, et al. Cerebellar ataxia with SYNE1 mutation accompanying motor neuron disease. Neurology. 2013;80(6):600-601. 7. Lagier-Tourenne C, Tazir M, López LC, et al. ADCK3, an ancestral kinase, is mutated in a form of recessive ataxia associated with coenzyme Q10 deficiency. Am J Hum Genet. 2008;82(3):661-672. 8. Maruyama H, Morino H, Miyamoto R, Murakami N, Hamano T, Kawakami H. Exome sequencing reveals a novel ANO10 mutation in a Japanese patient with autosomal recessive spinocerebellar ataxia. Clin Genet. 2014;85(3):296-297. 9. Duran C, Hartzell HC. Physiological roles and diseases of Tmem16/Anoctamin proteins: are they all chloride channels? Acta Pharmacol Sin. 2011;32 (6):685-692. 10. Anheim M, Monga B, Fleury M, et al. Ataxia with oculomotor apraxia type 2: clinical, biological and genotype/phenotype correlation study of a cohort of 90 patients. Brain. 2009;132(pt 10): 2688-2698. 11. Matthijs G, Schollen E, Van Schaftingen E, Cassiman JJ, Jaeken J. Lack of homozygotes for the most frequent disease allele in carbohydrate-deficient glycoprotein syndrome type 1A. Am J Hum Genet. 1998;62(3):542-550.
2. Anheim M, Tranchant C, Koenig M. The autosomal recessive cerebellar ataxias. N Engl J Med. 2012;366(7):636-646.
JAMA Neurology October 2014 Volume 71, Number 10
Copyright 2014 American Medical Association. All rights reserved.
Downloaded From: http://archneur.jamanetwork.com/ by a University of St. Andrews Library User on 05/14/2015
jamaneurology.com
Case Report/Case Series
Autosomal Recessive Cerebellar Ataxia Type 3 Due to ANO10 Mutations Delineation and Genotype-Phenotype Correlation Study Mathilde Renaud, MD; Mathieu Anheim, MD, PhD; Erik-Jan Kamsteeg, PhD; Martial Mallaret, MD; Fanny Mochel, MD, PhD; Sascha Vermeer, MD, PhD; Nathalie Drouot, MSc; Jean Pouget, MD; Claire Redin, PhD; Emmanuelle Salort-Campana, MD; Hubertus P. H. Kremer, MD, PhD; Corien C. Verschuuren-Bemelmans, MD; Jean Muller, PhD; Hans Scheffer, PhD; Alexandra Durr, MD, PhD; Christine Tranchant, MD; Michel Koenig, MD, PhD
IMPORTANCE ANO10 mutations have been reported to cause a novel form of autosomal
recessive cerebellar ataxia (ARCA). Our objective was to report 9 ataxic patients carrying 8 novel ANO10 mutations to improve the delineation of this form of ARCA and provide genotype-phenotype correlation. OBSERVATIONS The ANO10 gene has been sequenced in 186 consecutive patients with ARCA. The detailed phenotype of patients with ANO10 mutations was investigated and compared with the 12 previously reported cases. The mean age at onset was 33 years (range, 17-43 years), and the disease progression was slow. Corticospinal tract signs were frequent, including extensor plantar reflexes and/or diffuse tendon reflexes and/or spasticity. No patient in our series had peripheral neuropathy. Magnetic resonance imaging of the brains of our patients revealed marked cerebellar atrophy. The most frequent mutation, a mononucleotide expansion from a polyA repeat tract (c.132dupA) that causes protein truncation, was never observed in homozygosity. Only 2 truncating mutations were reported in homozygosity, one of which (c.1150-1151del) was associated with juvenile or adolescent onset and mental retardation, whereas we show that the presence of at least 1 missense or in-frame mutation is associated with adult onset and slow progression. CONCLUSIONS AND RELEVANCE An ANO10 mutation is responsible for ARCA that is
mainly characterized by cerebellar atrophy and lack of peripheral neuropathy. We therefore suggest naming this entity autosomal recessive cerebellar ataxia type 3 (ARCA3). JAMA Neurol. 2014;71(10):1305-1310. doi:10.1001/jamaneurol.2014.193 Published online August 4, 2014.
A
utosomal recessive cerebellar ataxias (ARCAs) correspond to a heterogeneous group of inherited neurodegenerative disorders that affect the cerebellum and/or the peripheral nerves.1 A new classification of ARCA has been proposed including 3 groups: ARCA with pure sensory neuropathy, ARCA with sensorimotor axonal neuropathy, and ARCA without neuropathy. 2 Recently, a new form of ARCA due to mutations in the ANO10 gene has been reported. 3 To which group of the classification does this new entity belong and whether genotype-phenotype correlation may be proposed have not yet been elucidated. We report 9 unrelated patients carrying 8 novel ANO10 mutations, and we compared these patients with the 12 previously reported cases in order to provide the delineation of the disease and genotype-phenotype correlations. jamaneurology.com
Author Affiliations: Author affiliations are listed at the end of this article. Corresponding Author: Michel Koenig, MD, PhD, Laboratoire de Génétique des Maladies Rares, Institut National de la Santé et de la Recherche Médicale UMR_S 827, Institut Universitaire de Recherche Clinique, 641 Avenue du Doyen Gaston Giraud, 34093 Montpellier CEDEX 5, France ([email protected]).
Methods Between 2010 and 2013, a total of 186 unrelated index patients were consecutively recruited from 4 tertiary centers for inherited neurodegenerative disorders. Inclusion criteria were the combination of (1) sporadic or recessive progressive cerebellar ataxia; (2) age at onset of younger than 50 years; and (3) unknown etiology despite extensive investigations, including molecular analysis for Friedreich ataxia and obvious forms of recessive ataxias. Written informed consent was obtained from all participants. Participants were not financially compensated. The recessive ataxia research project was approved by the Strasbourg ethics committee. A detailed neurological examination, including scales of severity and magnetic resonance imaging of the brain, was performed for all patients. JAMA Neurology October 2014 Volume 71, Number 10
Copyright 2014 American Medical Association. All rights reserved.
Downloaded From: http://archneur.jamanetwork.com/ by a University of St. Andrews Library User on 05/14/2015
1305
Research Case Report/Case Series
ARCA3 Due to ANO10 Mutations
Table 1. Main Clinical, Molecular, Morphologic, and Electrophysiologic Features of 9 Patients Carrying 8 Novel ANO10 Mutations Feature
Case 1
Case 2
Case 3
Case 5
Case 6
Case 7
Case 8
Age at onset, y
17
33
40
Case 4 37
30
32
43
33
32
Age at examination, y
23
44
47
43
33
59
68
52
37
Sex
Female
Male
Male
Female
Female
Male
Female
Female
Race/Ethnicity
White (Turkish)
White (French)
White (French) West Indies
West Indies
White (French)
White (Dutch)
White (Dutch)
White (French)
First sign
Cerebellar dysarthria
Gait ataxia
Gait ataxia
Gait ataxia
Diplopia
Ataxia with vertigo
Ataxia
Ataxia, vertigo, cephalalgia
Ataxia
+
Female
Case 9
+
Gait ataxia
+
+
+
+
+
+
+
Tendon reflexes
Increased
Increased
Increased
Increased
Increased
Increased
Normal
Increased
Increased
Plantar reflexes
Flexor
Extensor
Flexor
Flexor
Flexor
Flexor
Flexor
Extensor
Extensor
Ocular motor findings
Gaze-evoked nystagmus, hypermetric saccades
Gaze-evoked nystagmus
Vertical nystagmus
Gaze-evoked nystagmus
Hypermetric Gaze-evoked saccades nystagmus
Downbeat nystagmus, slow saccades
Vertical and horizontal nystagmus
Saccadic pursuit
Other findings
Linguistic difficulties in French
Left hypoacousia since 20 y
Lower limb spasticity
Tongue fasciculations
Ankle clonus
Diplopia, dysphagia
Stridor, anxiety
None
Lower limb spasticity, paroxysmal limb weakness
Cognitive status
Normal
Normal
Normal
Normal
Normal
Normal
MMSE score of 19-29
Mild intellectual disabilities
Normal
SARA
11
14
NP
6.5
10
NP
NP
NP
10.5
ICARS
27
44
NP
NP
NP
NP
NP
NP
NP
SDFS
3
4
4
3
3
4
NP
NP
NP
NP
EMG results
Normal
Normal
Motor neuron Normal involvement in tibialis anterior
Results of ophthalmic examination with fundus
Normal
Normal
Normal
MRI findings
Diffuse cerebellar atrophy at 23 y
Diffuse cerebellar atrophy at 43 y
Cerebellar Diffuse atrophy at 43 y cerebellar atrophy at 47 t
Moderate cerebellar atrophy at 33 y
Cerebellar atrophy at 59 y
α1-Fetoprotein Normal levela
Normal
Increased 16.6 Normal ng/mL
Normal
Increased 11.8 ng/mL
Mutations
Compound heterozygous: missense (c.685G>T, p.Gly229Trp [exon 6]), nonsense (c .1291C>T, p.Gln431* [exon 8])
Compound heterozygous: missense (c.1009T>G, p.Phe337Val [exon 6]), frameshift (c.132dupA, p.Asp45Argfs*9 [exon 2])
Homozygous: donor splice site (c.1668+ 1G>A [exon 10])
NP
Compound heterozygous: frameshift (c.1214delT, p.Leu405* [exon 7]), donor splice site (c.1476+ 1G>T [exon 9])
NP
Normal
Normal
NP
3 Normal
Normal
Normal
Cerebellar atrophy at 40 y
Cerebellar Cerebellar atrophy at 34 y atrophy at 34 y
NP
Homozygous Homozygous Compound heterozygous: deletion deletion exon 12 missense exon 12 (c.1009T>G, p.Phe337Val [exon 6]), frameshift (c.132dupA, p.Asp45Argfs*9 [exon 2])
Normal
Normal
Normal
Compound heterozygous: deletion exon 12, frameshift (c.132dupA, p.Asp45Argfs*9 [exon 2])
Compound heterozygous: missense (c.512T>C, p.Phe171Ser [exon 5]), frameshift (c.132dupA, p.Asp45Argfs*9 [exon 2])
Abbreviations: EMG, electroneuromyography; ICARS, International Cooperative Ataxia Rating Scale; MMSE, Mini-Mental State Examination; MRI, magnetic resonance imaging; NP, not performed; SARA, Scale for the Assessment and Rating of Ataxia; SDFS, Spinocerebellar Degeneration Functional Score; +, tested positive. SI conversion factor: To convert α1-fetoprotein to micrograms per liter, multiply by 1.0. a
Normal range is less than 7 ng/mL.
Genetic analysis of ANO10 was performed for 44 patients by conventional Sanger sequencing. A total of 142 additional patients were directly analyzed by a targeted exon-capture strategy coupled with multiplexing and high-throughput sequencing of 57 genes causing ataxia when mutated, including ANO10. The splice-site mutations were evaluated with the SplicePort program (http://spliceport.cbcb.umd.edu/) and were analyzed by reverse transcription of RNA from patients’ fibroblasts followed by polymerase chain reaction from exon 8 to 1306
exon 11 (520–base pair [bp] fragment) and from exon 6 to exon 12 (809-bp fragment). Polymerase chain reaction fragments were visualized on agarose gels and sequenced by use of the Sanger method. The patients from this and previous studies were divided into 2 groups according to the type of the ANO10 mutation: (1) at least 1 missense or in-frame mutation, or (2) 2 truncating mutations. The 2 groups were compared by use of the t test.
JAMA Neurology October 2014 Volume 71, Number 10
Copyright 2014 American Medical Association. All rights reserved.
Downloaded From: http://archneur.jamanetwork.com/ by a University of St. Andrews Library User on 05/14/2015
jamaneurology.com
ARCA3 Due to ANO10 Mutations
Case Report/Case Series Research
Results The clinical, radiological, and biological findings of the 9 patients are shown in Table 1 and Figure 1. Of the 186 patients, 9 (5%) had ANO10 mutations. All 9 of these patients carrying ANO10 mutations had obvious cerebellar atrophy detected on magnetic resonance imaging scans of the brain and a lack of peripheral neuropathy. There was a subgroup of 19 patients with recessive progressive cerebellar ataxia and 37 patients with sporadic progressive cerebellar ataxia for whom cerebellar atrophy and lack of peripheral neuropathy were documented. Of these 56 patients, 6 (11%) had ANO10 mutations. The 9 patients carried 1 previously reported ANO10 mutation and 8 novel ANO10 mutations (Table 1). The c.132dupA mutation was identified in 4 unrelated patients. The mutation is an insertion of an A (adenine nucleotide) following a homopolymer of 9 A’s and can be considered as a mononucleotide expansion of a mononucleotide repeat tract. Indeed, the c.132dupA mutation is a frequent mutation with a heterozy-
gous carrier frequency of 1/184 in different ethnic groups (Exome Variant Server [http://evs.gs.washington.edu/EVS]) and probably arose because of multiple independent homopolymer instability events. The c.132dupA mutation leads to early protein truncation (p.Asp45Argfs*9). Surprisingly, all 4 patients with this mutation were compound heterozygous, and no homozygous c.132dupA patient was identified. The 3 identified missense mutations, p.Phe171Ser, p.Gly229Trp, and p.Phe337Val, are likely pathogenic because they affect amino acid positions that are conserved in the orthologous sequences of almost all metazoan species (Figure 2). The p.Gly229Trp mutation is located at the C-terminal border of the first α-helical transmembrane segment, and the p.Phe337Val mutation is located at the C-terminal border of the third transmembrane segment. Reverse transcriptase–polymerase chain reaction analysis of the 2 donor splice-site mutations (Figure 3) revealed complete alteration of the splice sites. Surprisingly, skipping the mutated exon or using cryptic splice sites resulted in all cases in in-frame transcripts. The deletion mutation (deletion of exon 12), identified in 3 unrelated patients, is
Figure 1. Magnetic Resonance Imaging of the Brain of Patient 2 A
B
Marked cerebellar atrophy with normal supratentorial structures at 43 years of age. A sagittal T1-weighted sequence (A) and a coronal T1-weighted inversion recovery sequence (B) are shown.
Figure 2. Conservation of Amino Acids Altered by Missense Mutation (p.Phe171Ser, p.Gly229Trp, and p.Phe337Val, Respectively)
hs gg xl tr dr bf sp cg ct phc ag nv hm
S G I V I Q V F P L H D S E A L K K L E D T WY T R G I L V Q I F P L H D R E E L K K L R H T WY G R G I M I Q V F P L H D K E E L K R L R H QW Y L R G I L I Q I F P L H E Q E D L K R L S F T WY K R G V L V Q Y F P L H N K E D L K R L S F S WY K K H L L E K M Y P V H N P E Q L K R L Q H DWY H L G L I D G I Y P L H T P T E L V S L E N HW Y N H D I I T K M Y P L H E P E D L K K L G A DWY Q I G I I A Q L F P L H D K R S L K T L S F DWY Q S G Y V E K C F P L H E I E Y L K K F S Q T WY L T E L I T N L Y S L H D K R T L K E L R H RW I K P G V V S S I Y P L H E A Q K L K L L E K KW F L N H K I V Q I Y P L H H I E S L K S L E N QW Y L G
W E T I A L Y F G F L E Y F T F A L I P MA V I G L P Y Y L F E T I A L Y F G F L E Y F T F A L I P MA V I G I P Y Y V F D T L G L Y F A F L E Y F T MA L I P MA L I G I P Y Y F F E G Q A L Y F G F L E F F T F A L V P MA L V G V P Y Y L F E G L G L Y F G F L E Y F T F A L I P MA L I G I P Y Y L F E S V AMY F S F L G H Y T K A L L V P T V I G V L Y Y L F EK I G I YF S F LGF YTKAL F L PAA FGL L Y F L L EK I ALYFAFLGYYT I AL I PPAF I G I I YF I T G T V AM Y F A F L G F Y TMA L I P P A L I G V I S E Y G EN I A L Y F I F L G Y Y T C A L V P PM I L G L I E A L I E S V GMY F S F L G F Y T Y A L V V P T V F G F L Q L G L E TMAMY F A F L Q F Y S MT L I P P V L L I V V F A L S ES I AL YF T F LGF YTK F L L P TAV I G I LHY F F
Alignment of the surrounding sequence of ANO10 orthologues from 13 metazoan species is shown. The transmembrane sequences are underlined. Mutant amino acids (1-letter code) are indicated on top of the corresponding mutated amino acids. Conserved amino acids are indicated in bold: hs indicates Homo sapiens;
jamaneurology.com
V Y L V S L P F V C L C L Y F S L Y V MM I Y F D M E V W Y L V S V P F V C L C L Y F S L Y V MM I Y F D L E HW Y L V S V P F V C V C L Y L A S Y V MM I Y F D L E HW Y L V S L P F V L L C L Y L S L Y V MM I Y F Q M E GW Y L V S V P F V L L C L Y L S F Y V MM V Y F D M E F W Y C V S F P I V I L C M L V A V V V MM I Y F W A E N I Y C V S A P I I L V F L C L A F L MM L G F F W F E D Y Y C V T V P I V S V A L G I A F Y I M L G Y F V MQ E W Y G V S L P V V L L C L F G A F Y I ML V Y FW S E D L Y C V S L P I V F L C L T G A F F F M L F S F Y M E NM Y C V T A P I I M L CMG I A A F V T I F Q F Y V E A Y Y G I S Y P I V L L CMK V A T V VM L L Y F K L Q F Y Y F I S Y P L L L I S L L L V T V GM L F Y F H L N E K
gg, Gallus gallus; xl, Xenope laevis; tr, Tachifugu rubripes; dr, Danio rerio; bf, Branchiostoma floridae; sp, Strongylocentrotus purpuratus; cg; Crassostrea gigas; ct, Capitella teleta, phc, Pediculus humanus corporis; ag, Anopheles gambiae; nv, Nematostella vectensis; and hm, Hydra magnapapillata.
JAMA Neurology October 2014 Volume 71, Number 10
Copyright 2014 American Medical Association. All rights reserved.
Downloaded From: http://archneur.jamanetwork.com/ by a University of St. Andrews Library User on 05/14/2015
1307
Research Case Report/Case Series
ARCA3 Due to ANO10 Mutations
Figure 3. RT-PCR Analysis of Exons 8 to 11 of ANO10 Transcripts of Patients With Splice-Site Mutation A
M
C
4
1
C
No RT
No cDNA
M
B
C TGGAAAAAGAAA TGGGAA C T T A T T TGGGC A C C T T TGA TGA T T A C T TGGAGT T A T T C C TGC AGT T TGGT T A TGTGAGC C T T T T C T C C TGTGT T T A C C C A T T AGC AGC TGC C T T TGC TGTGT T AAA T AA C T T C A C TGAAGT AAA T T C AGA TGC C T T AAAAA TGTGC AGGGT C T T C AAA CGT C C A T T C T C AGAA C C T T C AGC C AA T A T TGGTGT AAAGAGAGGGC TGAA T TGC AAGGT C A TGAGGAA T C T T T TGGGGGAAA T GGAAA TGT C C TGTGT C T TGT T TGTGGTGGTGGTGGT T T C A C AGT TGGC T T T TGAAA CGA TGAGTG T T A T A T C TGTGGT C A C T AA C TGTGCGC TGA T TGGAA TGT C A C C A C AAGTGAA TGC A C TGGAAAAAGAAA TGGGAA C T T A T T TGGGC A C C T T TGA TGA T T A C T TGGAGT T A T T C C TGC AGT T TGGT T A T T TGGC T T T TGAAA CGA TGAGTGT T A T A T C TGTGGT C A C T AAGTGTGCGC TGA T TGGAA TGT CACCACAAGTGAATGCA C TGGAAAAAGAAA TGGGAA C T T A T T TGT TGGC T T T TGAAA CGA TGAGTGT T A T A T C TGTGGT C A C T AA C TGTGCGC TGA T TGGAA TGT C A C C A C AAGTGAA TGC A
A, Reverse transcriptase–polymerase chain reaction (RT-PCR) fragments were run on agarose gel and visualized with ethidium bromide. The letter M indicates size markers; the letter C, control individual (520–base pair normal fragment); and the number 4, patient 4 (compound heterozygous c.1476+1G>T, exon 9 and p.Leu405*, exon 7). The sequence of the fragment for patient 4 revealed complete skipping of exon 9 (in-frame deletion from codon 432 to codon 492, which includes part of the coding sequence of the large third extracellular loop [Figure 4]); the absence of the normal fragment indicates degradation of the p.Leu405* allele by nonsense-mediated RNA decay. The number 1 indicates patient 1 (homozygous c.1668+1G>A, exon 10); the abnormal fragments are indicated by white arrowheads (sequence in panel B), and heteroduplexes are indicated by black arrowheads. No RT indicates negative control with no
reverse transcriptase; no cDNA, negative control with no complementary DNA. B, Sequence of the RT-PCR of the fragments of patient 1 (homozygous c.1668+1G>A, exon 10 [A]). Part of exon 9 and part of a cryptic exon in intron 10 have a single underline. Parts of exon 10 have a double underline. Part of exon 11 has no underline. Strikingly, all 3 identified fragments are in-frame. The 2 larger fragments result from the use of cryptic splice sites within exon 10. The largest fragment includes most of exon 10 and a cryptic exon, resulting in the replacement of tryptophane 555 by 29 novel amino acids. The smallest fragment (the most abundant) corresponds to complete skipping of exon 10, which results in in-frame deletion from codon 493 to codon 556, including the coding sequence of the sixth transmembrane segment.
also in frame. A phenotype vs genotype comparison, including also previously reported patients (Figure 4), revealed that the age at onset was higher for patients with at least 1 missense or in-frame exon skipping mutation than for patients with 2 truncating mutations (homozygous c.1050_1051del and homozygous p.Tyr203*) (P < .01).
to name this new entity autosomal recessive cerebellar ataxia type 3 (ARCA3) (Table 2). The mean age at onset is late in our series (including 8 patients with onset at older than 25 years of age), but, overall, age at onset for those with ARCA3 appears highly variable, ranging from 6 to 43 years. Disease progression is slow, with patients remaining ambulatory for up to 25 years after the onset of the disease. However, it may be more severe, as in one of our patients (case 4), who needed a stick for walking after 7 years of disease duration. Lower limb reflexes appeared to be always increased, but extensor plantar reflexes, mental retardation, or cognitive decline were variably present (Table 1).3,4,8 Two patients exhibited spasticity; therefore, ARCA3 should also be considered in case of spastic ataxia. One patient (case 9) reported paroxysmal episodes of ataxia and vertigo, which could be explained by the abnormal function of mutated anoctamin-
Discussion Although electrophysiological signs of lower motor neuron involvement in lower limbs were found in a few patients3,4 and in case 3, none of the 21 reported patients had polyneuropathy, which classifies ANO10-linked ataxia among the group of cerebellar ataxias without neuropathy.3 Because ARCA15,6 and ARCA27 are already representatives of this group, we propose 1308
JAMA Neurology October 2014 Volume 71, Number 10
Copyright 2014 American Medical Association. All rights reserved.
Downloaded From: http://archneur.jamanetwork.com/ by a University of St. Andrews Library User on 05/14/2015
jamaneurology.com
ARCA3 Due to ANO10 Mutations
Case Report/Case Series Research
Figure 4. Correlation Between Predicted Consequences of Mutations (Missense, In-Frame Exon Skipping, In-Frame Exon Deletion and Truncation) and Age at Onset of Disease Mutations p.Gly229Trp
Age at Onset, y p.Phe337Val
p.Leu510Arg
33, 40, 32 (20, 25, 32), 32
p.Phe171Ser
c.1476+1G>T
(25, 42), 37
c.1668+1G>A
17,
Del exon 12
30, 43, 33
c.1150-1151del
(6, 7, 7), (13, 15, 15),
p.Tyr203*
42
The ages at onset of patients from the same family are indicated in parentheses. The ages at onset of patients reported in our study are underlined. Previously reported patients were either homozygous for the p.Leu510Arg missense mutation (affecting the sixth transmembrane segment of ANO10), homozygous for the c.1050_1051del or p.Tyr203* truncating mutations, or compound heterozygous for a nonsense mutation and the same exon 9 splice-site mutation reported here for patient 4. The correlations imply that homozygous or compound heterozygous mutations that cause a partial loss of function (missense or in-frame mutation) will be associated with a milder phenotype, even if associated with a truncating mutation on the other allele.
Table 2. Clinical and Genetic Characteristics of Patients With ARCA1, ARCA2, or ARCA3 Characteristic
Patients With ARCA1
Age at onset, mean (range), y
32 (17-45)
Patients With ARCA2
Patients With ARCA3
4 (1-11)
21 (6-45)
Cerebellar ataxia
Always present
Always present
Always present
Other optional clinical features
Enhanced tendon reflexes
Mental retardation, myoclonus, epilepsy stroke like episodes, exercise intolerance
Corticospinal tract signs
Progression
Very slow
Variable but mostly very slow
Slow
EMG results
No polyneuropathy, motor neuron involvement in 1 patient6
No polyneuropathy
No polyneuropathy, occasional motor neuron involvement
Biomarker
None
Variable elevation of serum lactic acid levels, variable decrease of CoQ10
None (increased AFP level in 2 patients)
Brain MRI findings
Cerebellar atrophy
Cerebellar atrophy, variable cortical hyperintensities
Cerebellar atrophy
Gene, protein
SYNE1, nesprin-1 (nuclear envelope and spectrin-like repeats 1)
ADKC3, aarF domain containing kinase 3
ANO10, anoctamin-10
10, which is a putative calcium-activated chloride channel.9 No extraneurological signs were found in our series. The α1-fetoprotein serum level, which is known to be a reliable biomarker for ataxia with oculomotor apraxia type 210 and ataxia telangiectasia, was mildly increased in 2 cases. Whether α1-fetoprotein could be a biomarker for ARCA3 remains to be elucidated by further large studies. Overall, we report an ARCA3 phenotype in accordance with previous reports and, more precisely, define a core presentation that should be kept in mind when facing a patient with recessive or sporadic ataxia. Autosomal recessive cerebellar ataxia type 3 appears to be a common cause of recessive ataxias. While the frequency is accounted for, in part, by the high frequency of the major mutation, c.132dupA, which belongs to the group of unstable sequences, this mutation is underrepresented in patients with ARCA3 with respect to the jamaneurology.com
Abbreviations: AFP, α1-fetoprotein; ARCA1, autosomal recessive cerebellar ataxia type 1; CoQ10, coenzyme Q10; EMG, electromyography; MRI, magnetic resonance imaging.
other mutations that are at least 10-fold less frequent (http: //evs.gs.washington.edu/EVS). The fact that all patients with this mutation were compound heterozygotes and that no homozygous c.132dupA patient was identified suggests that c.132dupA homozygotes are either not viable or have a more severe phenotype, similar to the p.Arg141His mutation of phosphomannomutase 2 in the congenital disorder of glycosylation type Ia.11 In accordance with this correlation is the fact that all 6 previously reported patients with the homozygous truncating mutation c.1150-1151del3,4 have a significantly earlier age at onset (juvenile or adolescent onset associated with mental retardation) compared with the patients with at least 1 missense or in-frame mutation (Figure 4) who have adult onset, slow progression, and persistence of ambulation over a long disease duration. However, a recently reported patient,8 hoJAMA Neurology October 2014 Volume 71, Number 10
Copyright 2014 American Medical Association. All rights reserved.
Downloaded From: http://archneur.jamanetwork.com/ by a University of St. Andrews Library User on 05/14/2015
1309
Research Case Report/Case Series
ARCA3 Due to ANO10 Mutations
mozygous for the truncating nonsense mutation p.Tyr203*, does not fit with this correlation because he had onset at 42 to 46 years of age. It is nevertheless interesting to note that the only 2 reported homozygous truncating mutations (p.Tyr203* and c.1150-1151del) are both located in exon 6, which is in frame and differentially spliced in vivo (absent from isoform 2 of ANO10 [http://www.uniprot.org/uniprot/]). It is possible that these 2 mutations variably affect differential splicing (or cryptic splice-site activation, as in case 1 of this study) of exon 6 and result in production of in-frame transcripts, because the 2 mutations are located close to the acceptor and donor splice sites, respectively, of exon 6. Such a mechanism would explain the differential consequence of homozygous truncating mutations p.Tyr203* and c.1150-1151del
ARTICLE INFORMATION Published Online: August 4, 2014. doi:10.1001/jamaneurol.2014.193. Author Affiliations: Département de neurologie, hôpitaux universitaires de Strasbourg, hôpital de Hautepierre, Strasbourg, France (Renaud, Anheim, Mallaret, Tranchant); Institut de Génétique et de Biologie Moléculaire et Cellulaire, Institut National de la Santé et de la Recherche Médicale, Centre National de la Recherche Scientifique, Université de Strasbourg, Strasbourg, France (Renaud, Anheim, Mallaret, Drouot, Redin, Muller, Tranchant, Koenig); Fédération de Médecine Translationnelle de Strasbourg, Université de Strasbourg, Strasbourg, France (Renaud, Anheim, Mallaret, Tranchant); Department of Human Genetics, Radboud University Medical Centre, Nijmegen, the Netherlands (Kamsteeg, Vermeer, Scheffer); Université Pierre et Marie Curie Paris 6, Centre de recherche de l’institut du cerveau et de la moelle épinière, Paris, France (Mochel, Durr); AP-HP, Hôpital de la Salpêtrière, Département de Génétique et Cytogénétique, Paris, France (Mochel, Durr); Département de neurologie, hôpitaux universitaires de Marseille, hôpital de la Timone, Marseille, France (Pouget, Salort-Campana); Department of Neurology, Radboud University Medical Centre, Donders Institute for Brain, Cognition and Behaviour, Nijmegen, the Netherlands (Kremer, Verschuuren-Bemelmans); Laboratoire de Génétique des Maladies Rares, Institut National de la Santé et de la Recherche Médicale UMR_S 827, Institut Universitaire de Recherche Clinique, Montpellier, France (Koenig). Author Contributions: Drs Renaud and Koenig had full access to all of the data in the study and take responsibility for the integrity of the data and the accuracy of the data analysis. Drs Anheim and Renaud contributed equally to this article as first authors. Drs Tranchant and Koenig contributed equally to this article as last authors. Study concept and design: Renaud, Anheim, Redin, Muller, Scheffer, Tranchant, Koenig. Acquisition, analysis, or interpretation of data: Renaud, Anheim, Kamsteeg, Mallaret, Mochel, Vermeer, Drouot, Pouget, Salort-Campana, Kremer,
1310
and the absence of homozygous truncating mutations c.132dupA, despite the high frequency of heterozygote carriers (1/184). RNA studies of the p.Tyr203* and c.1150-1151del homozygous patients are warranted to solve this issue.
Conclusions In conclusion, ARCA3, which has a major mutation (c.132dupA) that is a mononucleotide expansion from a mononucleotide repeat tract, is not a rare form of recessive cerebellar ataxia. It should be considered in cases of progressive cerebellar ataxia with cerebellar atrophy but without peripheral neuropathy, even in sporadic cases and whatever the age at onset.
Verschuuren-Bemelmans, Muller, Durr, Tranchant, Koenig. Drafting of the manuscript: Renaud, Anheim, Drouot, Muller, Durr, Tranchant, Koenig. Critical revision of the manuscript for important intellectual content: Anheim, Kamsteeg, Mallaret, Mochel, Vermeer, Pouget, Redin, Salort-Campana, Kremer, Verschuuren-Bemelmans, Scheffer, Tranchant, Koenig. Statistical analysis: Renaud, Muller. Obtained funding: Muller, Scheffer, Koenig. Administrative, technical, or material support: Mochel, Vermeer, Drouot, Pouget, Redin, Durr, Koenig. Study supervision: Anheim, Tranchant, Koenig. Conflict of Interest Disclosures: None reported. Funding/Support: Dr Renaud was supported by a fellowship from the Journées de Neurologie de Langue Française. This study was supported by funds from the Institut National de la Santé et de la Recherche Médicale, the Centre National de la Recherche Scientifique, the Agence Nationale pour la Recherche-Maladies Rares and Maladies Neurologiques et Psychiatriques (grant ANR-09MNPS-001-01 to Dr Koenig), and the ANR/E-rare JTC 2011 “Euro-SCAR” (grant 2011-RARE-004-01 to Dr Koenig). Role of the Sponsor: The funding agencies had no role in the design and conduct of the study; collection, management, analysis, or interpretation of the data; preparation, review, or approval of the manuscript; and decision to submit the manuscript for publication. REFERENCES 1. Anheim M, Fleury M, Monga B, et al. Epidemiological, clinical, paraclinical and molecular study of a cohort of 102 patients affected with autosomal recessive progressive cerebellar ataxia from Alsace, Eastern France: implications for clinical management. Neurogenetics. 2010;11(1):1-12.
3. Vermeer S, Hoischen A, Meijer RP, et al. Targeted next-generation sequencing of a 12.5 Mb homozygous region reveals ANO10 mutations in patients with autosomal-recessive cerebellar ataxia. Am J Hum Genet. 2010;87(6):813-819. 4. Chamova T, Florez L, Guergueltcheva V, et al. ANO10 c.1150_1151del is a founder mutation causing autosomal recessive cerebellar ataxia in Roma/Gypsies. J Neurol. 2012;259(5):906-911. 5. Gros-Louis F, Dupré N, Dion P, et al. Mutations in SYNE1 lead to a newly discovered form of autosomal recessive cerebellar ataxia. Nat Genet. 2007;39(1):80-85. 6. Izumi Y, Miyamoto R, Morino H, et al. Cerebellar ataxia with SYNE1 mutation accompanying motor neuron disease. Neurology. 2013;80(6):600-601. 7. Lagier-Tourenne C, Tazir M, López LC, et al. ADCK3, an ancestral kinase, is mutated in a form of recessive ataxia associated with coenzyme Q10 deficiency. Am J Hum Genet. 2008;82(3):661-672. 8. Maruyama H, Morino H, Miyamoto R, Murakami N, Hamano T, Kawakami H. Exome sequencing reveals a novel ANO10 mutation in a Japanese patient with autosomal recessive spinocerebellar ataxia. Clin Genet. 2014;85(3):296-297. 9. Duran C, Hartzell HC. Physiological roles and diseases of Tmem16/Anoctamin proteins: are they all chloride channels? Acta Pharmacol Sin. 2011;32 (6):685-692. 10. Anheim M, Monga B, Fleury M, et al. Ataxia with oculomotor apraxia type 2: clinical, biological and genotype/phenotype correlation study of a cohort of 90 patients. Brain. 2009;132(pt 10): 2688-2698. 11. Matthijs G, Schollen E, Van Schaftingen E, Cassiman JJ, Jaeken J. Lack of homozygotes for the most frequent disease allele in carbohydrate-deficient glycoprotein syndrome type 1A. Am J Hum Genet. 1998;62(3):542-550.
2. Anheim M, Tranchant C, Koenig M. The autosomal recessive cerebellar ataxias. N Engl J Med. 2012;366(7):636-646.
JAMA Neurology October 2014 Volume 71, Number 10
Copyright 2014 American Medical Association. All rights reserved.
Downloaded From: http://archneur.jamanetwork.com/ by a University of St. Andrews Library User on 05/14/2015
jamaneurology.com
