Clinical and molecular genetic assessment of a chorea-acanthocytosis pedigree

Journal of the Neurological Sciences 263 (2007) 124 – 132 www.elsevier.com/locate/jns

Clinical and molecular genetic assessment of a chorea-acanthocytosis pedigree Mio Ichiba a , Masayuki Nakamura a , Akira Kusumoto a , Emiko Mizuno a , Yutaka Kurano a , Mieko Matsuda a , Maiko Kato a , Asumi Agemura a , Yuko Tomemori a , Shinji Muroya a , Yoshiaki Nakabeppu b , Akira Sano a,⁎ a b

Department of Psychiatry, Kagoshima University Graduate School of Medical and Dental Sciences, Kagoshima University, Japan Department of Radiology, Kagoshima University Graduate School of Medical and Dental Sciences, Kagoshima University, Japan Received 8 September 2006; received in revised form 26 June 2007; accepted 3 July 2007 Available online 1 August 2007

Abstract Background: Chorea-acanthocytosis (ChAc) is an autosomal recessive hereditary disease characterized by neurodegeneration in the striatum and acanthocytosis that is caused by mutations in the VPS13A gene. There are only few reports that studied clinical status of the obligate carriers of ChAc. Clinical courses with follow-up neuroradiological and neuropsychological evaluations in individuals with ChAc have been rarely reported. Methods: We followed an index patient with ChAc and evaluated the clinical features of the pedigree members. Genetic analyses of VPS13A and genes responsible for other neuroacanthocytotic and neurodegenerative diseases were performed. Conclusions: The index patient was homozygous for a 3889C N T nonsense mutation in the VPS13A gene and presented with a typical ChAc phenotype. Neuropsychological evaluation with brain imaging in the patient over 3 years revealed atrophy and a decrease in blood flow at the basal ganglia and frontal lobe, and impairment in cognitive function reflecting frontal lobe dysfunction in progressive manners. Four out of five heterozygous mutation carriers in the pedigree showed signs or symptoms potentially attributable to a heterozygous VPS13A mutation. © 2007 Elsevier B.V. All rights reserved. Keywords: Chorea-acanthocytosis; VPS13A gene (ChAc); Signs or symptoms potentially attributable to a heterozygous VPS13A mutation; Heterozygous mutation carriers; Frontosubcortical dementia; Neurodegeneration

1. Introduction Chorea-acanthocytosis (ChAc) is a rare hereditary neurodegenerative disease characterized by adult-onset progressive involuntary choreic movements and erythrocyte acanthocytosis. The main neuropathological feature of ChAc is degeneration of the striatum [1]. Other clinical symptoms, including psychiatric features, epilepsy, peripheral neuropathy, myopathy, and oral self-mutilation, are often found. ⁎ Corresponding author. Department of Psychiatry, Kagoshima University Graduate School of Medical and Dental Sciences, Kagoshima University, 8-35-1 Sakuragaoka, Kagoshima 890-8520, Japan. Tel.: +81 99 275 5346; fax: +81 99 265 7089. E-mail address: [email protected] (A. Sano). 0022-510X/$ - see front matter © 2007 Elsevier B.V. All rights reserved. doi:10.1016/j.jns.2007.07.011

Several prior case studies on ChAc have reported neuroimaging results, clinical neuropsychiatric symptoms, and neuropathological findings at autopsy [1,2], but no study has followed the clinical course with neuroradiological and neuropsychological evaluations for more than 1 year. Serial neuroradiological and neuropsychological studies of a patient with ChAc who was homozygous for mutations of the causative gene are shown in the present report. VPS13A, the gene responsible for ChAc, is located on chromosome 9q21 [3] [4]. Although the inheritance of ChAc has been recognized as autosomal recessive, single heterozygous VPS13A mutations have been occasionally found in patients with ChAc. Dobson-Stone et al. reported 57 different VPS13A mutations in 43 patients with ChAc and 7 of the 43 patients possessed only a single heterozygous

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Fig. 1. Pedigree of a Japanese family. An arrow indicates the proband. Blackened symbols indicate individuals with symptoms of ChAc; blackened symbols at the upper left, lower left, upper right, and lower right indicate chorea, acanthocytosis, neuropsychiatric symptoms, and epilepsy, respectively. The combinations of plus and minus signs indicate the genotypes of VPS13A mutations; a plus sign (+) indicates the mutant allele of VPS13A point mutation, 3889C N T (Arg1297Ter), and a minus sign (−) indicates the normal VPS13A allele.

VPS13A mutation [5]. Heterogeneous phenotypes in terms of age of onset and symptoms were observed among patients with the same mutations [2]. We herein describe the clinical findings from a Japanese family harboring a 3889C N T (Arg1297Ter) nonsense mutation in the VPS13A gene and the genetic analyses of this pedigree, in which some of the family members with a heterozygous mutation showed signs and symptoms potentially attributable to a heterozygous VPS13A mutation. Then, we analyzed the polymorphism in the genes responsible for neurodegenerative choreiform diseases such as Huntington's disease, Huntington disease-like 2, McLeod syndrome, spinocerebellar ataxia 17, and ChAc.

PowerScript™ Reverse Transcriptase (BD Biosciences Clontech, Palo Alto, CA, USA) and random hexamers.

2. Materials and methods

Genotyping of triplet repeats was performed using fluorescent dye-labeled primer sets. Polymerase chain reaction (PCR) products were run on an ABI 3100 Avant Genetic Analyzer and analyzed using the fragment analysis software GeneMapper version 3.7 (Applied Biosystems). The following primers were used for identification of the triplet repeats in HD: 5'CCTTCGAGTCCCTCAAGTCCTTC-3' and 5'GGCTGAGGAAGCTGAGGAG-3' (for detecting CAG triplets), 5'-AGCAGCAGCAGCAACAGCC-3' and 5'GGCTGAGGAAGCTGAGGAG-3' (for detecting CTG triplets) [9–11]. Those for JPH3 were 5'-AGATGCCACCGCATTCGG-3' and 5'-GGTTCCCTGCACAGAAACCAT-3'. Those for TBP were 5'-GACCCCACAGCCTATTCAGA-3' and 5'-TTGACTGCTGAACGGCTGCA-3'. Those for genotyping the VPS13A triplet repeat polymorphism were 5'TACAGGGAGTGGATTATGA-3' and 5'-AATACAATTATTTTGCTTTATGA-3'.

2.1. Patients and family members We investigated a large family originating from Kagoshima Prefecture, Japan (Fig. 1). Nine family members including the proband with ChAc caused by a homozygous mutation in the VPS13A gene (V-2), heterozygous carriers of the mutation (III3, -5, -8, -9, and -11), and noncarriers (III-6, -10, and V-1) were examined in the present study. 2.2. Genetic analysis All patients and family members were referred by their primary psychiatrist or neurologist and provided written informed consent. The research protocol and consent form were approved by the Institutional Review Board of Kagoshima University. Genomic DNA (gDNA) was extracted from the leukocytes by standard methods and total RNA was extracted with a QIAamp® RNA blood mini kit (Qiagen, Hilden, Germany). Complementary DNA (cDNA) was prepared by reverse transcription of messenger RNA (mRNA) using BD

2.3. Mutation analysis Mutations in VPS13A were detected in cDNA and gDNA corresponding to regions encoding transcripts A and B using an ABI PRISM 3100 Avant Genetic Analyzer (Applied Biosystems, Foster, CA, USA) [3,4]. All translated regions in XK were analyzed from gDNA by cycle sequencing analysis [6–8]. 2.4. Genotyping using polymerase chain reaction for triplet repeats — polymorphisms or expansion

2.5. Clinical assessment Neuropsychiatric signs and symptoms were assessed by a group of psychiatrists. Psychiatric diagnosis was based on

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Fig. 2. Data of the proband. (A, B, C) Axial T1-weighted MRI studies, which were taken at 36 year 9 month (A), 38 year 0 month (B), and 39 year 11 month (C), showed progressive bilateral atrophy of the caudate heads and the cavity of the septum pellucidum. (D) Axial T2-weighted MRI taken at the same time as (C) showed increased signal intensity bilaterally in the striatum, caudate heads, and a large area of the cortex. (E, F, G) Cerebral blood flow assessed using 123I-IMP SPECT images taken at 34 year 2 month (E), 36 year 8 month (F) and 37 year 5 month (G). 3D-SSP Z-score views demonstrate areas of blood flow reduction. They showed a progressive decrease in cerebral blood flow. (H) Peripheral blood smear stained with May–Grünwald–Giemsa solution showed many irregular shapes including acanthocytes. The scale bar corresponds to 10 μm.

International Statistical Classification of Diseases and Related Health Problems, Tenth Revision, ICD-10 [12]. Severities of the psychiatric and neurological symptoms were determined based on the Neuropsychiatric Inventory [13], Hamilton Depression Rating Scale [14], Yale–Brown Obsessive Compulsive Scale [15], Huntington's Disease Rating Scale [16], and Abnormal Involuntary Movement Scale [17].

required to read all the words was recorded in seconds. In Part II, the patient is required to name the colors of the ink in which three words for colors (‘red’, ‘blue’, and ‘white’ in Japanese kanji) were randomly printed in 50 letters, and in which the color of the ink differed from the color expressed by the word itself. The time required to name every ink color without reading the words was recorded. 2.7. Clinical laboratory test

2.6. Neuropsychological examination The subjects were examined using the Mini-Mental State Examination (MMSE) [18], Wechsler Adult Intelligence Scale-Revised (WAIS-R) [19], Trail Making Test, Modified Stroop Test, and Raven's Colored Progressive Matrices (RCPM) test [20]. The Modified Stroop Test consists of two parts. In Part I, the patient is required to read three words (‘red’, ‘blue’, and ‘white’ in Japanese kanji) printed in colored ink and randomly placed within 50 letters. The time

Dry blood smears from the family members of the proband with ChAc were stained with May–Grünwald– Giemsa solution. The normal value of acanthocytes was defined as b 1.2% [21]. 2.8. Brain imaging test Cerebral blood flow was assessed by single photon emission computed tomography using N-isopropyl-p-[123I]

M. Ichiba et al. / Journal of the Neurological Sciences 263 (2007) 124–132 Table 1 Neuropsycological examinations Age

37 year 7 month

MMSE 25/30 Items on which Calculation—3 she lost points Memory recall—1 Cube copying—1 WAIS-R VIQ 70 PIQ 65 FIQ 64 Stroop test Read around 35 s incorrect Color naming 45 s incorrect Trail making test Part A 50 s incorrect Part B 175 s incorrect RCPM

38 year 4 month

39 year 11 month

26/30 26/30 Calculation—3 Calculation—2 Cube copying—1 Command—1 Cube copying—1 71 65 65

68 61 61

45 s incorrect 75 s incorrect

82 s correct 90 s incorrect

75 s correct 61 s incorrect 170 s incomplete 95 s incomplete 22/36 310 s

MMSE: Mini-Mental State Examination. WAIS-R: Wechsler Adult Intelligence Scale-Revised. RCPM: Raven's Colored Progressive Matrices.

iodoamphetamine (123I-IMP SPECT). The data were compared with the age matched normal control databases using the three-dimensional stereotactic surface projections (3DSSP) [22,23]. 3. Results

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with oral involuntary movement and pseudobulbar palsy. The severity of dysphagia was assessed by barium contrast X-ray, which demonstrated that a small amount of barium had obviously flowed into the trachea. In spite of these problems, she ate excessively and became obese. After diet therapy, including caloric restriction and administration of a semi-liquid diet to prevent aspiration, her body mass index decreased from 33.0 to 21.8. Neurological examination revealed poor muscle tone and absent deep tendon reflexes in all limbs. Brain magnetic resonance imaging (MRI) showed progressive, symmetrical, mild atrophy of the caudate heads (Fig. 2A, B, and C) and increased signal intensity bilaterally in the striatum and in vast areas of cortex in T2-weighted images (Fig. 2D) over the course of time. Blood flow of striatum had decreased in the early phase, and the reduction of blood flow extended to the frontal lobe gradually (Fig. 2E, F, and G). Table 1 shows the results of the neuropsychological examinations . The IQ score measured using WAIS-R decreased slightly from 64 to 61 during the ages from 37 year 7 month to 39 year 11 month. On the MMSE, attention, calculation, and cube copying task performance were impaired, although memory and orientation were maintained. Assessments of frontal lobe function by Modified Stroop Test, Trail Making Test, and RCPM revealed progressive impairment of frontal lobe functions. The genetic test revealed homozygous 3889C N T (Arg1297Ter) nonsense mutations in the VPS13A gene, which was the same mutation as the one reported previously [5] (Table 2).

3.1. Case report 3.1.1. Proband (V-2) The proband is a 39-year-old right-handed woman. She was born to consanguineous parents. Since the age of 28 years, an oral involuntary movement, associated with oral self-mutilation, dysphagia, and dysarthria, appeared and gradually worsened. Subsequently, involuntary choreic movements in her limbs spread to her whole body. When the patient was 34 years old, a diagnosis of ChAc was suspected because acanthocytes were observed on blood smears. Serum beta-lipoprotein was normal. The diagnosis of McLeod syndrome was excluded since no mutation was found in sequencing of the XK gene. When the patient was in her mid-30s, she experienced epileptic seizures, which were treated with antiepileptic drugs. Also in her mid-30s, she began showing emotional lability, infantile behavior, and the existence of delusional mood, and she made repeated suicide attempts. She compulsively hid kitchenware in the closets of her home and became obese in a short time because of compulsive overeating. These problematic behaviors led to her hospitalization in a psychiatric hospital. When the patient was 36 years old, her peripheral blood smear revealed 20% acanthocytes. A slight elevation of creatine kinase was also found. Progressive involuntary tongue protrusion, which forced foods out of her mouth, caused self-mutilation and difficulty in eating. She also showed progressive dysphagia and dysarthria, associated

3.1.2. The eldest brother of the proband's mother (III-3) This subject, an 80-year-old man, showed left side hemichoreic movements in his extremities that had been diagnosed as senile chorea when the man was 72 years old. The involuntary movements disappeared without treatment within 1 year. He showed neither respiratory nor thyroid disturbances. Diabetes mellitus was not found either. Neuroleptics had been never administered. Although transient hemichorea is known to be associated with lacunar infarct in the basal ganglia [24], brain MRI showed a slight degree of atrophy consistent with his age without any lacunar infarctions in the basal ganglia (Fig. 3A and B). Evaluation by SPECT showed slightly decreased perfusion in the left thalamus (Fig. 3C). Peripheral blood smears showed 9% acanthocytes (Fig. 3D) and normal creatine kinase levels were observed in the serum. Genetic tests showed the subject to be heterozygous for mutation of VPS13A. The results of the WAIS-R suggested that his intelligence had been slightly impaired (standard scores: information 4, digit span 8, vocabulary 10, arithmetic 4, comprehension 8, similarities 4, picture completion 6, picture arrangement 4, block design 12, object assembly 4, and digit symbol 18). Because the Japanese version of WAIS-R is standardized for persons 16 to 74 years of age, accurate assessment of the subject's mental ability was not available. Calculating his age as 74 years, his verbal intelligence quotient (IQ) would be 74, performance IQ would be 75, and full IQ would be 73.

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Table 2 Clinical data of the family Age Age at the Sex VPS13A Acanthocyte Chorea mutation (%) (year) first signs or symptoms appeared (year)

Orofacial Epilepsy Psychiatric involuntary symptoms movement

Creatine kinase (IU/l)

Mouscle tonus

Deep tendon reflexes

MRI

SPECT

III-3

80

72

M

+/−

9

133

Normal

Slightly diminished

Normal for his age

III-5 III-6 III-8 III-9

75 74 69 66

– – – 50s

F M M F

+/− −/− +/− +/−

ND ND ND 122

Normal Normal Hypotonic Normal

Normal Normal Diminished Normal

Slightly decreased perfusion in the left thalamus ND ND ND Decreased perfusion in frontal lobe and basal ganglia

III-10 III-11

62 60

– 59

F F

IV-1

40 – (age at death) 41 – 39 28

V-1 V-2

ND: not determined.

–

–

0 0 3 11

Transient hemichoreic movements – – – –

– – – –

– – – –

−/− +/−

0 1

– Extremities

– –

– –

– – Recurrent depressive 89 disorder

Normal Normal

M

ND

ND

–

–

–

–

ND

ND

M F

−/− +/+

0 20

– – Whole body +

– +

– Frontosubcortical dementia

ND Normal Normal 182–1348 Hypotonic absent

Intelligence had been slightly impaired – – – Mild depressive episode

ND ND ND Hyperintensity signal bilaterally in the striatum in T2-weighted images Diminished ND Left N right in Hyperintensity lower limbs signal bilaterally in the striatum in T2-weighted images ND ND

ND Mild atrophy of the caudate heads and hyperintensity signal in the striatumand the cortex in T2-weighted images

ND No obvious abnormalities

ND

ND Decrease in blood flow at striatum extending to the frontal lobe over time

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Fig. 3. Data of the proband's uncle and mother. (A, B, C, D) Data of the uncle, III-3. (A) Axial T1-weighted MRI study showed a slight degree of basal ganglia atrophy. (B) Axial T2-weighted MRI showed no lacunar infarctions. (C) 3D-SSP Z-score views demonstrate areas of blood flow reduction. They showed slightly decreased perfusion in left thalamus. (D) Peripheral blood smear stained with May–Grünwald–Giemsa solution showed many irregular shapes including acanthocytes. The scale bar corresponds to 10 μm. (E, F, G, H) Data of the mother, III-9. (E) Axial T1-weighted MRI study showed no obvious abnormalities. (F) Axial T2-weighted MRI showed increased signal intensity bilaterally in the striatum. (G) The SPECT result showed decreased perfusion in frontal lobe and basal ganglia. (H) Peripheral blood smear showed many irregular shapes including acanthocytes.

3.1.3. The eldest sister of the proband's mother (III-5) The subject, a 75-year-old woman, showed neither neuropsychiatric symptoms nor acanthocytosis. Heterozygous mutation of VPS13A was found in the genetic test. 3.1.4. Fifth brother of the proband's mother (III-8) This subject, a 69-year-old man, showed 3% acanthocytes on peripheral blood smear without any neuropsychiatric signs. Deep tendon reflexes were normal. Genetic tests showed the subject to be heterozygous for mutation of VPS13A. 3.1.5. Proband's mother (III-9) This 66-year-old woman did not show any neurological abnormalities. Deep tendon reflexes were normal. She had experienced appetite loss and insomnia since she was in her

50s. She had previously been given a diagnosis of having a depressive state and had been taking tranquilizer clotiazepam although such state is common among the elderly people and is not a specific symptom of ChAc. Brain MRI showed increased signal intensity bilaterally in the striatum on T2-weighted images (Fig. 3F) and SPECT showed decreased perfusion in frontal lobe and basal ganglia (Fig. 3G). Peripheral blood smears showed 11% acanthocytes (Fig. 3H) and the serum levels of creatine kinase were normal. The subject's genetic test revealed a heterozygous mutation of VPS13A. Her intelligence level was within the normal range (IQ = 85). 3.1.6. Youngest sister of the proband's mother (III-11) The subject, a 60-year-old woman, had previously been given a diagnosis of depression. Since the age of 59 years,

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she had experienced depression characterized by insomnia, appetite loss, agitation, and suicidal compulsions. She then became a heavy drinker and injured herself more than once, for example by stabbing a chopstick into her hand. When the woman was 60 years old, choreic movements appeared in her upper and lower limbs. Slight gait disturbance had appeared before she took notice of her choreic movements. Restlessness with mild intermittent exaggeration of gesture in the lower extremities was observed on the physical examination. She showed neither respiratory nor thyroid disturbances. Diabetes mellitus was not found either. Neuroleptics had never been administered. Patellar and Achilles' tendon reflexes were exaggerated in the left side, while Babinski and Chaddock reflexes were negative on both sides. Deep tendon reflexes in the upper extremities were normal. Thoracic ossification of the ligamentum flavum was detected when the woman was 60 years old and then a laminectomy for Th 10–12 was carried out. Brain MRI showed increased signal intensity bilaterally in the striatum on T2-weighted images. The SPECT analysis showed no obvious abnormalities. Peripheral blood smears showed 1% acanthocytes. Genetic tests showed that she had a heterozygous mutation of VPS13A. 3.1.7. Proband's father (IV-1) The proband's father had obesity, diabetes mellitus, and hypertension since his late 30s and died from a stroke at the age of 40 years. He did not have any neuropsychiatric symptoms. 3.1.8. Other family members Three family members carried no mutations of VPS13A: III-6, -10, and V-1. They were 74, 62, and 41 years old and showed neither neuropsychiatric symptoms nor acanthocytosis. Unfortunately, data on III-1, -2, and II-3 were unavailable. 3.2. Polymorphism analysis in the genes associated with choreiform neurodegenerative disorder The clinical symptoms of the heterozygous carriers of VPS13A mutations varied considerably, supporting the existence of modifier genes and/or some environmental factors. To identify these genes, polymorphisms were analyzed, including triplet repeats and mutations in genes related to striatal degeneration. We analyzed CAG and CCG triplet repeats in the HD gene. The CAG repeat expansion is responsible for Huntington disease (HD). Normal copy numbers (16 or 17 copies) of CAG repeats and CCG repeats (7 to 10) were found in all cases. No linkage between these HD polymorphisms and ChAc-related symptoms was found. The CTG triplet repeats in the JPH3 gene, which is responsible for Huntington disease-like 2, were analyzed. All family members had 14 copies of the repeat, which is within the normal range. The XK gene is responsible for McLeod syndrome and although all of the translated regions

in the XK gene were analyzed, no mutations were detected. Triplet repeats in the TBP gene, which codes for the TATA binding protein and the expansion of which is responsible for the pathogenesis of spinocerebellar ataxia 17, were also analyzed. Normal copy numbers (34 or 35 copies) of the repeat were observed in all family members. No linkage between the numbers of triplet repeats and symptoms associated with ChAc was found. The GAT triplet repeats coding for poly-aspartic acid on VPS13A exon 69 were analyzed. All family members had 10 copies of the triplet. 4. Discussion Studies have reported a wide range of neuropsychiatric symptoms among many patients with ChAc and their family members [1,2]. Although most of the neuropsychiatric symptoms are characteristic of frontosubcortical dementia, 60% of patients with ChAc show psychiatric problems such as schizophrenia, paranoia, obsessive–compulsive disorder, depression, and marked emotional lability [1]. The proband in the present study also showed diverse psychiatric problems. The patient's obesity caused by compulsive overeating contrasts with the nutritional deficiencies is usually seen in patients with ChAc due to their pseudobulbar symptoms and their orofaciolingual dystonias [25]. The results of neuropsychological tests, especially in the Modified Stroop and Trail Making tests, showed progressive frontal lobe dysfunction. Huntington's disease is the most common genetic disease associated with striatal degeneration and resultant chorea. Previous neuropsychological studies have demonstrated that patients with HD show greater impairment of memory function than of frontal lobe function [26]. In contrast, the proband maintained better memory function than frontal lobe function, suggesting that frontal lobe dysfunction is more severe in cases of ChAc than in HD, although memory function is better retained in this study. Recently, Henkel et al. conducted a study using MRI with voxel-based morphometry and found atrophy in the head of the caudate nucleus without significant global brain atrophy in patients with ChAc [27]. Arzberger et al., however, reported a moderate loss of neurons in the cerebral cortex [28]. Brain perfusion SPECT showed a decrease in blood flow in the basal ganglia prior to the atrophy of the caudate heads shown by MRI. Previous autopsy-based studies have found no abnormalities in the cortices of patients with ChAc [1], but the brain imaging in the present study showed decreased blood flow and a mild degree of atrophy of the cerebral cortices, especially of the frontal lobe. Weight gain due to carbohydrate craving is one of the prominent early symptoms in frontal lobe dementia [29]. This might mean that the excessive eating and obesity observed in the proband of the present study are the results of frontal lobe dysfunction associated with frontosubcortical dementia. Although ChAc is an autosomal recessive disease, in the present study, 4 out of 5 family members showed signs or

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symptoms potentially attributable to a heterozygous VPS13A mutation. We also genotyped the mutation, 3889C N T, microsatellite markers and SNPs inside or around VPS13A gene. Many of them were detected heterozygously in the carrier family members presenting partial signs or symptoms potentially attributable to a heterozygous VPS13A mutation suggesting that the possibility of large deletion is eliminated. There was a discrepancy between acanthocytosis and neuropsychiatric symptoms in the heterozygous mutation carriers: III-3 showed transient hemichorea, acanthocytosis, slight degree of brain atrophy, and decrease in blood flow at basal ganglia; III-5 showed neither acanthocytosis nor neuropsychiatric symptoms; III-8 showed only acanthocytosis; III-9 showed acanthocytosis, mild depressive episode, and decrease in blood flow at basal ganglia; and III-11 showed chorea and neuropsychiatric symptoms without acanthocytosis. For clinical evaluation of acanthocytosis, Storch et al. reported that diluted blood combined with wet blood preparation showed high specificity (0.98) and the highest sensitivity (1.0) [21]. In contrast to the results by the above wet preparation, dry smear showed the highest specificity (0.99) and low sensitivity (0.4). In this study, dry blood preparations using undiluted samples were analyzed. Even under such a low sensitive condition, heterozygous carriers showed 9% (III-3), 3% (III-8), and 11% (III-9). Storch et al. showed that the percentage of acanthocytes in wet preparations was greater than that in dry preparations [21]. III-11 showed 1% acanthocytes in dry preparation which might be increased in wet preparation. We investigated whether polymorphisms in other genes that cause choreiform neurodegenerative diseases would have contributed to the development of the signs or symptoms of carriers. The association between the polymorphisms in the genes and partial signs and/or symptoms potentially attributable to a heterozygous VPS13A mutation was not found. Carriers of disease mutations of the responsible genes frequently show signs or symptoms potentially attributable to a heterozygous gene mutation in recessive diseases such as Fabry disease, adrenoleukodystrophy, galactosemia, phenylketonuria, cystinuria, spinal muscular atrophy, and Duchenne muscular dystrophy [30–33]. In some cases, possible modifier genes or gene loci have been already identified [34,35]. Some modifiers might take part similarly in ChAc. Our results support the existence of signs or symptoms that were potentially attributed to a heterozygous VPS13A mutation; however, case studies and molecular, specifically, linkage studies of additional heterozygous mutation carriers in families with ChAc are needed. Acknowledgements The authors thank all patients and their family members for their willingness to participate in this study. We also thank Ms. K. Tanabe and Ms. M. Ishigami for their excellent technical assistance. This work was supported in part by a

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