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Multiple system atrophy and CAG repeat length: A genetic screening of polyglutamine disease genes in Italian patients
Accepted Manuscript Title: Multiple system atrophy and CAG repeat length: a genetic screening of polyglutamine disease genes in Italian patients Authors: Alessia Mongelli, Lidia Sarro, Elena Rizzo, Lorenzo Nanetti, Nicoletta Meucci, Gianni Pezzoli, Stefano Goldwurm, Franco Taroni, Caterina Mariotti, Cinzia Gellera PII: DOI: Reference:
S0304-3940(18)30307-0 https://doi.org/10.1016/j.neulet.2018.04.044 NSL 33567
To appear in:
Neuroscience Letters
Received date: Revised date: Accepted date:
7-2-2018 30-3-2018 23-4-2018
Please cite this article as: Alessia Mongelli, Lidia Sarro, Elena Rizzo, Lorenzo Nanetti, Nicoletta Meucci, Gianni Pezzoli, Stefano Goldwurm, Franco Taroni, Caterina Mariotti, Cinzia Gellera, Multiple system atrophy and CAG repeat length: a genetic screening of polyglutamine disease genes in Italian patients, Neuroscience Letters https://doi.org/10.1016/j.neulet.2018.04.044 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Multiple system atrophy and CAG repeat length: a genetic screening of polyglutamine disease genes in Italian patients
Alessia Mongellia, Lidia Sarroa, Elena Rizzoa, Lorenzo Nanettia, Nicoletta Meuccib, Gianni Pezzolib, Stefano Goldwurmb,c, Franco Taronia, Caterina Mariottia,*, Cinzia Gelleraa.
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Affiliations
Unit of Genetics of Neurodegenerative and Metabolic Diseases, IRCCS-Foundation Neurological
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Parkinson Institute, ASST Gaetano Pini-CTO, Milan, Italy
Department of Neuroscience "Rita Levi Montalcini", University of Turin, Turin, Italy.
Caterina Mariotti, MD
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*Corresponding author:
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Institute Carlo Besta, Milan, Italy
Genetics of Neurodegenerative and Metabolic Diseases
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IRCCS-Foundation Neurological Institute Carlo Besta, via Celoria 11, 20133 Milan, Italy
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Tel.: +39 02 2394 2269; Fax +39 02 2394 2140
Highlights
Utility of polyQ genetic screening in Italian MSA population MSA-C had higher percentage of longer normal SCA1 alleles compared to other groups
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MSA phenotype shows a challenging overlap with dominant polyglutamine diseases.
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e-mail: [email protected]
One MSA-C patient carried an expanded SCA2 allele, suggesting possible misdiagnosis
Abstract Multiple system atrophy (MSA) is an adult onset, progressive, neurodegenerative disorder of unknown etiology characterized by autonomic dysfunction, parkinsonism (MSA-P) and cerebellar 1
ataxia (MSA-C). The phenotypic spectrum may present overlapping features with other neurodegenerative diseases, particularly the autosomal dominant inherited polyglutamine disorders. To investigate the possible contribution of CAG expansions in the MSA phenotype, we analyzed the triplet repeat length in the autosomal dominant causative genes for spinocerebellar ataxia (SCA) type 1, 2, 3, 6, 7, 17, dentatorubral-pallidoluysian atrophy (DRPLA) and Huntington disease (HD) in a
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cohort of 246 Italian MSA patients. As comparison, 223 controls were also analyzed. The alleles were classified on the basis of CAG repeat length as “normal”, “intermediate” or “expanded” according to
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literature.
The MSA patients (101 men/145 women) had a mean age at onset of 58 years and a mean age at genetic testing of 63 years. MSA-C patients had significantly younger age at onset and at examination
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in comparison to MSA-P (p<0.0001). We identified a SCA1 intermediate allele in a MSA-C subject
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(36 CAG), a SCA2 intermediate allele in a MSA-P patient (31 CAG), and a pathologically expanded
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SCA2 allele (36 CAG) in a patient initially misdiagnosed as MSA-C.
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No intermediate or expanded SCA alleles were detected in controls. The distribution of CAG repeat length was similar among groups except for SCA1 gene that showed a higher percentage of longer
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normal alleles repeats in MSA-C as compared to MSA-P and controls (p<0.0001).
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This study supports the utility of polyQ genetic testing in the differential diagnosis of MSA, and may suggest a possible role of SCA1 repeat length as risk factor for MSA-C. SCA1 and SCA2 genetic
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screening is recommended in MSA Italian patients.
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Key words: Polyglutamine disease; MSA; Spinocerebellar ataxia; SCA; Huntington disease
1. Introduction
Multiple system atrophy (MSA) is an adult onset, progressive, neurodegenerative disorder of unknown etiology characterized by autonomic dysfunction, parkinsonism and cerebellar ataxia [1]. Clinical diagnosis of probable and possible MSA has been standardized by international criteria, 2
which also defined two categories based on the phenotype, MSA-P with predominant parkinsonism signs and MSA-C with predominant cerebellar features [2]. Definite diagnosis requires pathological identification of alpha-synuclein glial cytoplasmic inclusions in the brain. Genetic factors’ role in MSA etiology is still matter of debate, with inconsistent findings coming from genome wide association studies [3]. MSA diagnosis is often challenging especially during the early symptomatic
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phase due to a wide number of mimicking diseases [4,5]. Recently, MSA hereditability was estimated to range between 2.09 and 6.65%, which is still
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explainable by possible misdiagnosed cases included in the selected cohort [3]. Polyglutamine (polyQ) diseases such as spinocerebellar ataxias (SCA1, SCA2, SCA3, SCA6, SCA17, DRPLA) and also late-onset Huntington disease (HD) represent the most frequent genetic misdiagnosis due the
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considerable clinical overlap with MSA [5].
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The aim of this study was to search for possible associations between MSA phenotype (both MSA-C
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and MSA-P subtypes) and CAG repeat length in the dominant inherited polyQ disease genes in a large
2. Material and methods
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cohort of Italian MSA patients.
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2.1 Patient and control subjects
A total of 246 Italian MSA patients (118 MSA-P; 128 MSA-C) were consecutively recruited at two
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neurological centers specialized in movement disorders. Clinical diagnosis of possible or probable MSA was made according to the criteria proposed by Gilman et al. [6]. None of the included patients reported familiar history of movement disorders. As controls, we
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selected 223 age and sex-matched healthy Italian subjects who reported no neurological or major medical disease, and had no familiar history of polyQ diseases. Written informed consent was obtained from all study participants. 2.2 Genotyping
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An experienced biotechnologist blinded to the diagnosis performed genetic screening for CAG trinucleotide repeat size in the following polyQ disease-associated genes: Ataxin-1 (SCA1, MIM 164400), Ataxin-2 (SCA2, MIM 183090), Ataxin-3 (SCA3, MIM 109150), alpha-1A calcium channel Cav2.1 (SCA6, MIM 183086), Ataxin-7 (SCA7, MIM 164500), TATA binding protein (SCA17, MIM 607136), Atrophin-1 (dentatorubral-pallidoluysian atrophy, DRPLA, MIM 607462), and Huntingtin
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(HD, MIM 613004). The CAG repeat regions were amplified by polymerase chain reaction (PCR) in the presence of fluorescent (FAM-labelled) forward and reverse primers. Fluorescent PCR products
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were run on an Applied Biosystems 3130xl Genetic Analyser and CAG trinucleotide repeat size was determined using PeakScanner v1.0 software.
Alleles were classified on the basis of CAG length as “normal”, “intermediate” or “expanded”
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according to recent literature [7,8]. For SCA1 intermediate alleles, the presence of CAT interruption
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was tested using the SfaNI restriction enzyme. This enzyme cuts the repetitive CAG sequence when
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interrupted by at least one CAT trinucleotide [9]. In the control group we analyzed SCA1, SCA2, and
2.3 Statistical analysis
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HD genes in order to compare the frequency of the intermediate alleles identified in our MSA cohort.
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Statistical analyses were performed using JMP® 11.2.0 (SAS Institute Inc., United States). The
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Wilcoxon-Kruskal-Wallis test or Analysis of Variance (ANOVA) test with Tukey-Kramer HSD (Honest Significance Difference) post-hoc analyses were used as appropriate. For the MSA-C group
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we also performed a linear regression analysis with age-at-onset as dependent variable and SCA1 repeat length as predictor of the model. For the comparison of SCA1 allele distribution in the patient
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and in the control groups, the χ2 test was used. P-values ≤0.05 were considered significant.
3. Results
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The MSA patients were 101 men (41%) and 145 women (59%), and had a mean age at disease onset of 58 years and a mean age at examination of 63 years. Controls were 116 men (52%) and 107 women (48%) with a mean age at genetic screening of 63 years (Table 1). The subgroup of MSA-C subjects showed a younger age at disease onset (56 years) in comparison with MSA-P patients (60 years, p<0.0001), and also a younger age at examination (62 versus 64 years,
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p<0.0001). 3.1 Genetic findings
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The CAG repeat lengths in SCA3, SCA6, SCA7, SCA17 and DRPLA genes were within the normal range in three groups of subjects (Table 1).
In HD gene we found several intermediate length alleles (27-35 CAG repeats) with similar
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frequencies in all groups: 3% in MSA-C, 4% in MSA-P, and 2% in controls (Table 1).
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In SCA1, we identified an intermediate allele in a MSA-C subject (36 CAG repeats, allele frequency
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0.4%). The digestion of the PCR product with SfaNI enzyme showed the presence of CAT interruption
patient (allele frequency 0.4%).
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in the SCA1-36 CAG allele. An intermediate SCA2 allele with 31 CAG repeats was found in a MSA-P
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In addition, we found a pathological expanded SCA2 allele with 36 CAG repeats (allelic
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frequency=0.4%) in a patient who was initially misdiagnosed as MSA-C (Table 1 and Figure 1). No SCA1 and SCA2 intermediate or expanded alleles were observed in controls.
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3.2 Allelic distribution of CAG repeat length The distribution of CAG repeat length was similar in MSA-C, MSA-P and controls in all the tested genes except for the SCA1 gene (Figure 1, Table 1).
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In fact, MSA-C patients had a higher mean of CAG repeat length in SCA1 gene (28.5 ±1.6) compared to MSA-P (27.8 ±1.7) and to controls (27.6 ± 1.3) (p<0.0001). This finding was also confirmed after excluding the MSA-C subject carrying the SCA1 intermediate allele with 36 CAG (p<0.0001) (Table 2).
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MSA-C patients carried a higher percentage of the SCA1 large normal allele with 30 repeats (χ2 test <0.0001) in comparison with both MSA-P patients and controls. Large normal SCA1 alleles (31-to-34 repeats) were also more represented in MSA-C and MSA-P subjects in comparison with controls, without reaching statistical significance (Table 2). To test the hypothesis that the large SCA1 alleles (30-34 CAG) in MSA-C patients could have an influence on the earlier age at onset observed in this
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group, we performed a linear regression analysis including the two variables, but no significant
3.3 Clinical features of MSA-C patient with SCA1 36 CAG
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correlation was found (ρ=-0.020, p=0.89).
The MSA-C patient with an intermediate SCA1 repeat length allele (36 CAG with CAT interruption)
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presented gait and postural instability at the age of 64. Familiar history was unremarkable, and brain
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MRI showed cerebellar atrophy. During the course of the disease the patient also presented autonomic
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failure with erectile dysfunction and orthostatic decrease of systolic blood pressure of 30 mm Hg
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within 3 minutes of standing, confirmed at the Tilt-test examination after 2 minutes of orthostatism. The patient did not complain of overt urinary dysfunctions. The clinical record reported the presence
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of sleep disorders characterized by noisy sleep and limb movements.
3.4 Clinical features of MSA-P patient with SCA2 31 CAG
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The MSA-P patient carrying an intermediate SCA2 allele (31 CAG) was a 77 year old woman presenting with bradykinesia, rigidity and postural instability. These symptoms were poorly responsive to levodopa therapy. She also had constipation and urinary urgency. Episodic REM Sleep
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Behavior Disorder (RBD) was referred at baseline visit and also during follow-up. Brain MRI showed moderate diffuse supratentorial cortical atrophy, cerebellar atrophy and bilateral atrophy of the putamen. Five years after the onset of symptoms, she had moderate dysarthria, dysphagia for both liquid and solid food, and urinary incontinence. She died after 8 years of disease duration.
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3.5 Clinical features of patient with SCA2 (36 CAG) The patient carrying an expanded SCA2 allele (36 CAG) presented gait unsteadiness at the age of 68. Later, he developed mild dysarthria, motor incoordination, and mild parkinsonian signs, such as camptocormia, mild bradykinesia, and impaired postural response. He also complained urinary urgency, constipation, and restless leg syndrome. MRI showed the "hot cross bun" sign in the pons, as
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well as cerebellar and brainstem volume reduction. A 99mTc SPET showed hypometabolism at the cerebellar cortex. When the genetic screening revealed the correct diagnosis of SCA2 disease, the
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family history was further inquired and we found that the patient's mother, deceased for cancer at age 72, was suffering from an undefined gait disorder.
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4. Discussion
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MSA clinical diagnosis may be challenging due to the lack of definite biomarkers and the wide
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clinical overlap with other neurodegenerative disorders [4]. In particular, several diseases caused by
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trinucleotide expansions in coding regions of various genes, such as spinocerebellar ataxias subtypes and Huntington disease, may share common phenotypic characteristics with MSA patients. For this
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reason, genetic testing for SCAs, including SCA1, 2, 3, 6, 17 and DRPLA could be important in the
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differential diagnosis of MSA patients without a family history. In this study we analyzed the CAG repeat length in the dominant polyglutamine disease genes in a large cohort of Italian MSA patients
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with the aim of determining the prevalence of CAG expansions and the possible role of the repeat length on MSA phenotype. With the exception of SCA1 alleles, we found that the CAG repeat size distributions for most of the
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autosomal dominant polyglutamine diseases were similar among MSA patients and controls. Our findings were consistent with those previously reported in other cohort of patients with ataxia [10] or Parkinson disease [11] and in healthy controls. Interestingly, we found that SCA1 large normal alleles (30-35 CAG) were more frequent in the MSA-C group compared to both MSA-P and controls. This finding has not been previously reported in MSA-C. 7
Differences in the CAG repeat length of SCAs normal alleles have been reported in Caucasians and Japanese ethnic groups. In fact, Caucasian showed higher SCA1 and SCA2 CAG repeats length in large normal alleles (SCA1>30 repeats, SCA2>22 repeats) as compared to Japanese. This could contribute to the generation of fully expanded alleles and reflect the variable prevalence of the two SCA diseases in different populations [12].
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It is known that the larger size of normal SCA1 alleles can lower the age at onset of symptoms in SCA1 and SCA6 affected patients [13]. In our study, the group of MSA-C patients had a significantly
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younger age at onset a compared to MSA-P group. A similar observation was also described in a recent prospective study in American MSA patients, showing different ages at examination but similar natural histories among MSA subgroups [14]. In other studies no age differences were observed in
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large cohorts of European [15] and American [2] patients.
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In our work no significant correlation was found between the younger age at onset of MSA-C patients
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and longer SCA1 alleles. However, an enriched distribution of SCA1 large normal alleles (30-35
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CAG) was found in MSA-C patients, suggesting that large normal SCA1 alleles might have a role as a risk factor or potential phenotype modifier in MSA.
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Further investigation in larger population-based MSA-C cohorts is needed to confirm this hypothesis.
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In our study we also identified a SCA1 intermediate length allele in a patient with a clinical diagnosis of possible MSA-C. This subject carried an interrupted SCA1 allele with 36 CAT/CAG repeats. A few
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ataxic patients with this type of intermediate allele have been previously identified [9]. In the series described by Zühlke et al., subjects with 35-38 triplet (interrupted by CAT) showed a clinical phenotype consisting of cerebellar ataxia, but lacking of the additional characteristic features of SCA1
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[9]. Up to now, literature supports a pathogenic role only for the SCA1 alleles containing a pure CAG sequence of at least 39 repeats. Our patient presented some clinical and neuroimaging features in overlap with both SCA1 and MSA-C diseases, however the presence of CAT interruptions in a SCA1 intermediate allele did not support the diagnosis of SCA1.
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A clinical overlap was also encountered in the case of the MSA-P subjects carrying intermediate SCA2 allele. Parkinsonism with autonomic disturbances has been previously recognized as part of the phenotypic spectrum in SCA2 not only in patients carrying fully expanded alleles (≥ 34 CAG) [16], but also in a subject showing a SCA2 intermediate allele with 32 CAG [17]. Intermediate SCA2 alleles with ≥31 CAG were associated with an increase risk of amyotrophic lateral sclerosis (ALS) [18],
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however their specific pathogenic role in other neurodegenerative disorders is not yet fully determined [19].
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In our sample we found a pathologic trinucleotide expansions in SCA2. The patient carrying this allele (CAG 22/36) was initially misdiagnosed as possible MSA-C. At recruitment for this study, the patient seemed to meet the diagnostic criteria for possible MSA-C, supporting the observation of a potential
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clinical overlap between the two neurodegenerative disorders, particularly in the early phase of the
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diseases [3, 5]. Only a posteriori anamnestic evaluation identified the presence of analogous
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history in the differential diagnosis of MSA.
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neurological symptoms in his mother, emphasizing the importance of a correct and complete family
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We did not identify expanded alleles in other SCA genes. In a previous study on Korean MSA patients, high prevalence of expanded alleles (7.3%) was observed in SCA1, SCA2, SCA3, SCA6,
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SCA17 and DRPLA genes, with more than half of the mutations occurring in SCA17 gene [4]. Our findings in the SCA17 gene are in contrast with Kim et al., probably because of the different cutoff
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size used in the two studies. Kim et al., considered abnormal alleles with >40 repeats, whereas in our study we considered intermediate alleles with 43-48 repeats and expanded alleles ≥49 repeats. In the
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cohort described by Kim and colleagues [4] 7 MSA patients carried 41 CAG repeats (2.7%), 3 patients had 42 repeats (1.2%), and 3 MSA subjects had respectively 43, 44, and 46 CAG. None had allele size above 49 repeats [4]. In our study, we identified 5 patients carrying 41 CAG (2%), 1 patient carrying 42 CAG (0.4%), and no SCA17 alleles with ≥43 CAG were found. If we consider that in Italy SCA1 and SCA2 are the most frequent SCA genotypes and that SCA17 mutation accounts for less than 1% of the cases [20], the lack of SCA17 intermediate alleles could be likely due to the limited sample of our 9
cohort. In addition a possible bias of our study is represented by the fact that we did not screened for other trinucleotide disorders mimicking MSA phenotype such as late onset Friedreich ataxia or Xlinked tremor and ataxia syndrome [1]. In conclusion, our study confirms the value of genetic testing for the dominant polyQ disorders in the differential diagnosis of MSA, and in particular of SCA1 and SCA2 genes in the Italian population.
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The observation of an enriched distribution of SCA1 large normal alleles (30-35 CAG) in MSA-C needs to be further investigated in a large series of cases. The identification of intermediate or
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expanded alleles in polyQ genes is crucial for a correct diagnosis and an appropriate genetic
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counseling for patients and families.
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Declarations of interest: none.
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Aknowledgement
The DNA samples of MSA patients of the Parkinson Institute of Milan were obtained from the
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“Parkinson Institute Biobank” member of the Telethon Network of Genetic Biobank (http://biobanknetwork.telethon.it, project n. GTB12001) funded by TELETHON Italy, and supported
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by the ‘Fondazione Grigioni per il Morbo di Parkinson’. Genetic analyses of MSA and control
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subjects, performed at the Foundation IRCCS Neurological Institute Carlo Besta, were partially supported by the Italian Ministry of Health (Grants GR-2013-02357821 to LN; and RF-2011-
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02347420 to CM).
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Orrell, R. Rademakers, W. Robberecht, G. Rouleau, O.A. Ross, F. Salachas, K. Sidle, B.N. Smith, B.W. Soong, G. Sorarù, G. Stevanin, E. Kabashi, C. Troakes, C. van Broeckhoven, J.H. Veldink, L.H. van den Berg, C.E. Shaw, J.F. Powell, A. Al-Chalabi, ATXN2 trinucleotide repeat length correlates with risk of ALS, Neurobiol. Aging. 51 (2017) 178.e1-178.e9.
[19] O.A. Ross, N.J. Rutherford, M. Baker, A.I. Soto-Ortolaza, M.M. Carrasquillo, M. DeJesus13
Hernandez, J. Adamson, M. Li, K. Volkening, E. Finger, W.W. Seeley, K.J. Hatanpaa, C. Lomen-Hoerth, A. Kertesz, E.H. Bigio, C. Lippa, B.K. Woodruff, D.S. Knopman, C.L. White, J.A. Van Gerpen, J.F. Meschia, I.R. Mackenzie, K. Boylan, B.F. Boeve, B.L. Miller, M.J. Strong, R.J. Uitti, S.G. Younkin, N.R. Graff-Radford, R.C. Petersen, Z.K. Wszolek, D.W. Dickson, R. Rademakers, Ataxin-2 repeat-length variation and neurodegeneration, Hum. Mol.
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Genet. 20 (2011) 3207–3212. [20] A. Brusco, C. Gellera, C. Cagnoli, A. Saluto, A. Castucci, C. Michielotto, V. Fetoni, C.
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Mariotti, N. Migone, S. Di Donato, F. Taroni, Molecular genetics of hereditary spinocerebellar ataxia: mutation analysis of spinocerebellar ataxia genes and CAG/CTG repeat expansion
A
CC E
PT
ED
M
A
N
U
detection in 225 Italian families. Arch. Neurol. 61 (2004) 727–33.
14
Figure Legend Figure 1. Allelic distribution of CAG repeat within SCA1, 2, 3, 6, 7, 17, DRPLA and HD genes in MSA-C (blue bars) and MSA-P (red bars) patients. Vertical axes represent allelic frequencies (%) while horizontal axes represent number of CAG repeat units. Arrows indicates: (a) the intermediate SCA1 allele (CAGn=36), (b) the intermediate SCA2 allele (CAGn=31), and (c) the expanded SCA2 (CAGn=36)
in
MSA-C
patients.
SCA
indicates
spinocerebellar
ataxia;
A
CC E
PT
ED
M
A
N
U
SC R
dentatorubropallidoluysian atrophy; and HD, Huntington disease.
15
DRPLA,
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allele
Table 1: Demographic and genetic characteristics of MSA patients and controls. MSA-C
MSA-P
Controls
Subjects N.
128
118
223
Total alleles N.
256
236
446
Sex
52 M /76 F
49 M/69 F
116 M/107 F
Age at onset (years)
56.4 ± 7.7
60.3 ± 7.6
64.4 ± 7.3
62.5 ± 7.9
28.5 ± 1.6 [2536]
27.8 ± 1.7 [15-34]
27.6 ± 1.3 [19-33]
255 (99.6)
236 (100)
446 (100)
1 (0.4)
0
0
0
22.1 ± 1.1 [2136]
22.1 ± 0.8 [18-31]
22.1 ± 1.1 [12-30]
252 (99.6)
233 (99.6)
439 (98.4)
1 (0.4)
0
1 (0.4)
0
0
20.2 ± 5.1 [936]
20.2 ± 4.9 [10-34]
256 (100)
236 (100)
0
0
0
0
11.5 ±1.6 [7-14]
11.2 ± 1.9 [7-14]
256 (100) 0 0
236 (100) 0 0
9.8 ± 0.9 [9-13]
9.8 ± 0.9 [7-13]
254 (100)
236 (100)
0
0
0
0
37.1 ± 1.9 [2942]
36.9 ± 1.9 [29-41]
256 (100) 0
236 (100) 0
ED
SCA3
0
CC E
PT
Normal [≤44] Intermediate [4559] Expanded [≥61] SCA6
Normal [≤18] Intermediate [19] Expanded [≥20]
A
SCA7
Normal [≤27] Intermediate [2833] Expanded [≥34]
SCA17 Normal [≤42 ] Intermediate [43-
U N
0
A
Normal [≤26] Intermediate [3134] Expanded [≥35]
<0.0001 MSA-C vs. MSAP <0.0001 MSA-C vs. Controls ns MSA-P vs. Controls <0.0001 MSA-C vs. MSAP <0.0001 MSA-C vs. Controls ns MSA-P vs. Controls
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61.6 ± 8.2
Normal [≤35] Intermediate [3639§] Expanded [≥pure 39] SCA2
<0.0001
M
SCA1 (N. CAG repeats)
ns
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Age at examination (years)
P Value*
16
0
ns
ns
ns
ns
ns
0
17.4 ± 3.5 [928]
17.9 ± 3.3 [11-30]
254 (100)
236 (100)
0
0
0
0
18.5 ± 3.1 [928]
18.5 ± 3.3 [9-32]
18.2 ± 3.2 [8-33]
254 (96.8)
227 (96.2)
438 (98.2)
8 (3.2)
9 (3.8)
8 (1.8)
0
0
Normal [≤35] Intermediate [3648] Expanded [≥49] HD Normal [≤26] Intermediate [2735] Expanded [≥36]
0
Values show mean ± SD, [range] or
(%).§=
ns
ns
interrupt 39 CAG. *P values were assessed using: t-test
SC R
DRPLA
0
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48] Expanded [≥49]
(age at onset MSA-C vs MSA-P); ANOVA (age at examination in the three groups), WilcoxonKruskal-Wallis test (allelic distribution). In bold statistically significant p values.
Abbreviations: N, number; MSA-C, multiple system atrophy - cerebellar type; MSA-P, multiple
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system atrophy - parkinsonian type; vs, versus; M, male; F, female; ns, not significant (p≥0.05);
A
CC E
PT
ED
M
A
N
SCA, spinocerebellar ataxia; DRPLA, dentatorubropallidoluysian atrophy; HD, Huntington disease.
17
Table 2: SCA1 repeat length distribution in alleles from MSA- C, MSA-P and control subjects. Number of CAG repeats Tot. 15
19
20
21
24
25
26
27
28
29
30
31
32
33
34
N. allel es
0
2
0
2
3
6
24
182
158
45
20
2
Allelic frequency
0
0.
0
0.
0.
1.
5.
40.
35.
10.
4.5
0.
(%)
0.
5
0.
5
7
4
4
8
4
1
8.6
5
χ2 (z score)
48
1.
48
1.
0.
0.
1.
4.0
0.1
0.3
1
3.
15
16
77
92
6
1
4
15
MSA-C
1
1
0
0.
0.
0
2
2
0.95
2.
1.
71
97
63
SC R
N° alleles
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Controls
0
0
0
0
5
8
62
74
42
44
8
5
5
1
N° alleles
0
0
0
0
0
2.
3.
24.
29.
16.
17.
3.
2.
2.
0.4
Allelic frequency
0.
0.
0.
0.
1.
0
2
4
1
5
3
2
0
0
0.38
(%)
27
54
27
54
36
0.
0.
8.3
2.1
7.0
22.
3.
1.
5.
01
52
4
6
89
79
93
06
0
1
0
2
N° alleles
0.
0
0.
0
0.
Allelic frequency
4
0.
(%)
2.
50
χ2 (z score)
22
N
7
6
85
91
16
16
5
4
1
1
3.
2.
36.
35.
6.8
6.8
2.
1.
0.
0.4 0.49
ED
1
4
0.
9
0
5
0
6
3.8
0.8
1
7
4
2.
50
0.
1.
1.
0.0
1.1
3
6
0.
0.
0.
34
34
5
2
39
87
33
22
1
2
1
2
5
18
38
329
323
103
80
15
10
7
2
%
0.
0.
0.
0.
0.
1.
4.
35.
34.
11.
8.6
1.
1.
0.
0.2
1
2
1
2
5
9
1
2
5
0
6
1
8
CC E
43
Tot. N. alleles
PT
patients
A
MSA-P
3
M
χ2 (z score)
U
0
patients
446
254
236
936
In bold statistically significant p value obtained with χ2 test. The MSA-C patient carrying a SCA1
A
intermediate allele (CAGn=27/36) has been excluded from the statistical analysis. Abbreviations: N, number; MSA-C, multiple system atrophy - cerebellar type; MSA-P, multiple system atrophy parkinsonian type.
18
S0304-3940(18)30307-0 https://doi.org/10.1016/j.neulet.2018.04.044 NSL 33567
To appear in:
Neuroscience Letters
Received date: Revised date: Accepted date:
7-2-2018 30-3-2018 23-4-2018
Please cite this article as: Alessia Mongelli, Lidia Sarro, Elena Rizzo, Lorenzo Nanetti, Nicoletta Meucci, Gianni Pezzoli, Stefano Goldwurm, Franco Taroni, Caterina Mariotti, Cinzia Gellera, Multiple system atrophy and CAG repeat length: a genetic screening of polyglutamine disease genes in Italian patients, Neuroscience Letters https://doi.org/10.1016/j.neulet.2018.04.044 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Multiple system atrophy and CAG repeat length: a genetic screening of polyglutamine disease genes in Italian patients
Alessia Mongellia, Lidia Sarroa, Elena Rizzoa, Lorenzo Nanettia, Nicoletta Meuccib, Gianni Pezzolib, Stefano Goldwurmb,c, Franco Taronia, Caterina Mariottia,*, Cinzia Gelleraa.
a
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Affiliations
Unit of Genetics of Neurodegenerative and Metabolic Diseases, IRCCS-Foundation Neurological
b
Parkinson Institute, ASST Gaetano Pini-CTO, Milan, Italy
Department of Neuroscience "Rita Levi Montalcini", University of Turin, Turin, Italy.
Caterina Mariotti, MD
A
*Corresponding author:
N
U
c
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Institute Carlo Besta, Milan, Italy
Genetics of Neurodegenerative and Metabolic Diseases
M
IRCCS-Foundation Neurological Institute Carlo Besta, via Celoria 11, 20133 Milan, Italy
ED
Tel.: +39 02 2394 2269; Fax +39 02 2394 2140
Highlights
Utility of polyQ genetic screening in Italian MSA population MSA-C had higher percentage of longer normal SCA1 alleles compared to other groups
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MSA phenotype shows a challenging overlap with dominant polyglutamine diseases.
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e-mail: [email protected]
One MSA-C patient carried an expanded SCA2 allele, suggesting possible misdiagnosis
Abstract Multiple system atrophy (MSA) is an adult onset, progressive, neurodegenerative disorder of unknown etiology characterized by autonomic dysfunction, parkinsonism (MSA-P) and cerebellar 1
ataxia (MSA-C). The phenotypic spectrum may present overlapping features with other neurodegenerative diseases, particularly the autosomal dominant inherited polyglutamine disorders. To investigate the possible contribution of CAG expansions in the MSA phenotype, we analyzed the triplet repeat length in the autosomal dominant causative genes for spinocerebellar ataxia (SCA) type 1, 2, 3, 6, 7, 17, dentatorubral-pallidoluysian atrophy (DRPLA) and Huntington disease (HD) in a
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cohort of 246 Italian MSA patients. As comparison, 223 controls were also analyzed. The alleles were classified on the basis of CAG repeat length as “normal”, “intermediate” or “expanded” according to
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literature.
The MSA patients (101 men/145 women) had a mean age at onset of 58 years and a mean age at genetic testing of 63 years. MSA-C patients had significantly younger age at onset and at examination
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in comparison to MSA-P (p<0.0001). We identified a SCA1 intermediate allele in a MSA-C subject
N
(36 CAG), a SCA2 intermediate allele in a MSA-P patient (31 CAG), and a pathologically expanded
A
SCA2 allele (36 CAG) in a patient initially misdiagnosed as MSA-C.
M
No intermediate or expanded SCA alleles were detected in controls. The distribution of CAG repeat length was similar among groups except for SCA1 gene that showed a higher percentage of longer
ED
normal alleles repeats in MSA-C as compared to MSA-P and controls (p<0.0001).
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This study supports the utility of polyQ genetic testing in the differential diagnosis of MSA, and may suggest a possible role of SCA1 repeat length as risk factor for MSA-C. SCA1 and SCA2 genetic
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screening is recommended in MSA Italian patients.
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Key words: Polyglutamine disease; MSA; Spinocerebellar ataxia; SCA; Huntington disease
1. Introduction
Multiple system atrophy (MSA) is an adult onset, progressive, neurodegenerative disorder of unknown etiology characterized by autonomic dysfunction, parkinsonism and cerebellar ataxia [1]. Clinical diagnosis of probable and possible MSA has been standardized by international criteria, 2
which also defined two categories based on the phenotype, MSA-P with predominant parkinsonism signs and MSA-C with predominant cerebellar features [2]. Definite diagnosis requires pathological identification of alpha-synuclein glial cytoplasmic inclusions in the brain. Genetic factors’ role in MSA etiology is still matter of debate, with inconsistent findings coming from genome wide association studies [3]. MSA diagnosis is often challenging especially during the early symptomatic
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phase due to a wide number of mimicking diseases [4,5]. Recently, MSA hereditability was estimated to range between 2.09 and 6.65%, which is still
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explainable by possible misdiagnosed cases included in the selected cohort [3]. Polyglutamine (polyQ) diseases such as spinocerebellar ataxias (SCA1, SCA2, SCA3, SCA6, SCA17, DRPLA) and also late-onset Huntington disease (HD) represent the most frequent genetic misdiagnosis due the
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considerable clinical overlap with MSA [5].
N
The aim of this study was to search for possible associations between MSA phenotype (both MSA-C
A
and MSA-P subtypes) and CAG repeat length in the dominant inherited polyQ disease genes in a large
2. Material and methods
ED
M
cohort of Italian MSA patients.
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2.1 Patient and control subjects
A total of 246 Italian MSA patients (118 MSA-P; 128 MSA-C) were consecutively recruited at two
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neurological centers specialized in movement disorders. Clinical diagnosis of possible or probable MSA was made according to the criteria proposed by Gilman et al. [6]. None of the included patients reported familiar history of movement disorders. As controls, we
A
selected 223 age and sex-matched healthy Italian subjects who reported no neurological or major medical disease, and had no familiar history of polyQ diseases. Written informed consent was obtained from all study participants. 2.2 Genotyping
3
An experienced biotechnologist blinded to the diagnosis performed genetic screening for CAG trinucleotide repeat size in the following polyQ disease-associated genes: Ataxin-1 (SCA1, MIM 164400), Ataxin-2 (SCA2, MIM 183090), Ataxin-3 (SCA3, MIM 109150), alpha-1A calcium channel Cav2.1 (SCA6, MIM 183086), Ataxin-7 (SCA7, MIM 164500), TATA binding protein (SCA17, MIM 607136), Atrophin-1 (dentatorubral-pallidoluysian atrophy, DRPLA, MIM 607462), and Huntingtin
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(HD, MIM 613004). The CAG repeat regions were amplified by polymerase chain reaction (PCR) in the presence of fluorescent (FAM-labelled) forward and reverse primers. Fluorescent PCR products
SC R
were run on an Applied Biosystems 3130xl Genetic Analyser and CAG trinucleotide repeat size was determined using PeakScanner v1.0 software.
Alleles were classified on the basis of CAG length as “normal”, “intermediate” or “expanded”
U
according to recent literature [7,8]. For SCA1 intermediate alleles, the presence of CAT interruption
N
was tested using the SfaNI restriction enzyme. This enzyme cuts the repetitive CAG sequence when
A
interrupted by at least one CAT trinucleotide [9]. In the control group we analyzed SCA1, SCA2, and
2.3 Statistical analysis
M
HD genes in order to compare the frequency of the intermediate alleles identified in our MSA cohort.
ED
Statistical analyses were performed using JMP® 11.2.0 (SAS Institute Inc., United States). The
PT
Wilcoxon-Kruskal-Wallis test or Analysis of Variance (ANOVA) test with Tukey-Kramer HSD (Honest Significance Difference) post-hoc analyses were used as appropriate. For the MSA-C group
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we also performed a linear regression analysis with age-at-onset as dependent variable and SCA1 repeat length as predictor of the model. For the comparison of SCA1 allele distribution in the patient
A
and in the control groups, the χ2 test was used. P-values ≤0.05 were considered significant.
3. Results
4
The MSA patients were 101 men (41%) and 145 women (59%), and had a mean age at disease onset of 58 years and a mean age at examination of 63 years. Controls were 116 men (52%) and 107 women (48%) with a mean age at genetic screening of 63 years (Table 1). The subgroup of MSA-C subjects showed a younger age at disease onset (56 years) in comparison with MSA-P patients (60 years, p<0.0001), and also a younger age at examination (62 versus 64 years,
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p<0.0001). 3.1 Genetic findings
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The CAG repeat lengths in SCA3, SCA6, SCA7, SCA17 and DRPLA genes were within the normal range in three groups of subjects (Table 1).
In HD gene we found several intermediate length alleles (27-35 CAG repeats) with similar
U
frequencies in all groups: 3% in MSA-C, 4% in MSA-P, and 2% in controls (Table 1).
N
In SCA1, we identified an intermediate allele in a MSA-C subject (36 CAG repeats, allele frequency
A
0.4%). The digestion of the PCR product with SfaNI enzyme showed the presence of CAT interruption
patient (allele frequency 0.4%).
M
in the SCA1-36 CAG allele. An intermediate SCA2 allele with 31 CAG repeats was found in a MSA-P
ED
In addition, we found a pathological expanded SCA2 allele with 36 CAG repeats (allelic
PT
frequency=0.4%) in a patient who was initially misdiagnosed as MSA-C (Table 1 and Figure 1). No SCA1 and SCA2 intermediate or expanded alleles were observed in controls.
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3.2 Allelic distribution of CAG repeat length The distribution of CAG repeat length was similar in MSA-C, MSA-P and controls in all the tested genes except for the SCA1 gene (Figure 1, Table 1).
A
In fact, MSA-C patients had a higher mean of CAG repeat length in SCA1 gene (28.5 ±1.6) compared to MSA-P (27.8 ±1.7) and to controls (27.6 ± 1.3) (p<0.0001). This finding was also confirmed after excluding the MSA-C subject carrying the SCA1 intermediate allele with 36 CAG (p<0.0001) (Table 2).
5
MSA-C patients carried a higher percentage of the SCA1 large normal allele with 30 repeats (χ2 test <0.0001) in comparison with both MSA-P patients and controls. Large normal SCA1 alleles (31-to-34 repeats) were also more represented in MSA-C and MSA-P subjects in comparison with controls, without reaching statistical significance (Table 2). To test the hypothesis that the large SCA1 alleles (30-34 CAG) in MSA-C patients could have an influence on the earlier age at onset observed in this
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group, we performed a linear regression analysis including the two variables, but no significant
3.3 Clinical features of MSA-C patient with SCA1 36 CAG
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correlation was found (ρ=-0.020, p=0.89).
The MSA-C patient with an intermediate SCA1 repeat length allele (36 CAG with CAT interruption)
U
presented gait and postural instability at the age of 64. Familiar history was unremarkable, and brain
N
MRI showed cerebellar atrophy. During the course of the disease the patient also presented autonomic
A
failure with erectile dysfunction and orthostatic decrease of systolic blood pressure of 30 mm Hg
M
within 3 minutes of standing, confirmed at the Tilt-test examination after 2 minutes of orthostatism. The patient did not complain of overt urinary dysfunctions. The clinical record reported the presence
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ED
of sleep disorders characterized by noisy sleep and limb movements.
3.4 Clinical features of MSA-P patient with SCA2 31 CAG
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The MSA-P patient carrying an intermediate SCA2 allele (31 CAG) was a 77 year old woman presenting with bradykinesia, rigidity and postural instability. These symptoms were poorly responsive to levodopa therapy. She also had constipation and urinary urgency. Episodic REM Sleep
A
Behavior Disorder (RBD) was referred at baseline visit and also during follow-up. Brain MRI showed moderate diffuse supratentorial cortical atrophy, cerebellar atrophy and bilateral atrophy of the putamen. Five years after the onset of symptoms, she had moderate dysarthria, dysphagia for both liquid and solid food, and urinary incontinence. She died after 8 years of disease duration.
6
3.5 Clinical features of patient with SCA2 (36 CAG) The patient carrying an expanded SCA2 allele (36 CAG) presented gait unsteadiness at the age of 68. Later, he developed mild dysarthria, motor incoordination, and mild parkinsonian signs, such as camptocormia, mild bradykinesia, and impaired postural response. He also complained urinary urgency, constipation, and restless leg syndrome. MRI showed the "hot cross bun" sign in the pons, as
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well as cerebellar and brainstem volume reduction. A 99mTc SPET showed hypometabolism at the cerebellar cortex. When the genetic screening revealed the correct diagnosis of SCA2 disease, the
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family history was further inquired and we found that the patient's mother, deceased for cancer at age 72, was suffering from an undefined gait disorder.
U
4. Discussion
N
MSA clinical diagnosis may be challenging due to the lack of definite biomarkers and the wide
A
clinical overlap with other neurodegenerative disorders [4]. In particular, several diseases caused by
M
trinucleotide expansions in coding regions of various genes, such as spinocerebellar ataxias subtypes and Huntington disease, may share common phenotypic characteristics with MSA patients. For this
ED
reason, genetic testing for SCAs, including SCA1, 2, 3, 6, 17 and DRPLA could be important in the
PT
differential diagnosis of MSA patients without a family history. In this study we analyzed the CAG repeat length in the dominant polyglutamine disease genes in a large cohort of Italian MSA patients
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with the aim of determining the prevalence of CAG expansions and the possible role of the repeat length on MSA phenotype. With the exception of SCA1 alleles, we found that the CAG repeat size distributions for most of the
A
autosomal dominant polyglutamine diseases were similar among MSA patients and controls. Our findings were consistent with those previously reported in other cohort of patients with ataxia [10] or Parkinson disease [11] and in healthy controls. Interestingly, we found that SCA1 large normal alleles (30-35 CAG) were more frequent in the MSA-C group compared to both MSA-P and controls. This finding has not been previously reported in MSA-C. 7
Differences in the CAG repeat length of SCAs normal alleles have been reported in Caucasians and Japanese ethnic groups. In fact, Caucasian showed higher SCA1 and SCA2 CAG repeats length in large normal alleles (SCA1>30 repeats, SCA2>22 repeats) as compared to Japanese. This could contribute to the generation of fully expanded alleles and reflect the variable prevalence of the two SCA diseases in different populations [12].
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It is known that the larger size of normal SCA1 alleles can lower the age at onset of symptoms in SCA1 and SCA6 affected patients [13]. In our study, the group of MSA-C patients had a significantly
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younger age at onset a compared to MSA-P group. A similar observation was also described in a recent prospective study in American MSA patients, showing different ages at examination but similar natural histories among MSA subgroups [14]. In other studies no age differences were observed in
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large cohorts of European [15] and American [2] patients.
N
In our work no significant correlation was found between the younger age at onset of MSA-C patients
A
and longer SCA1 alleles. However, an enriched distribution of SCA1 large normal alleles (30-35
M
CAG) was found in MSA-C patients, suggesting that large normal SCA1 alleles might have a role as a risk factor or potential phenotype modifier in MSA.
ED
Further investigation in larger population-based MSA-C cohorts is needed to confirm this hypothesis.
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In our study we also identified a SCA1 intermediate length allele in a patient with a clinical diagnosis of possible MSA-C. This subject carried an interrupted SCA1 allele with 36 CAT/CAG repeats. A few
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ataxic patients with this type of intermediate allele have been previously identified [9]. In the series described by Zühlke et al., subjects with 35-38 triplet (interrupted by CAT) showed a clinical phenotype consisting of cerebellar ataxia, but lacking of the additional characteristic features of SCA1
A
[9]. Up to now, literature supports a pathogenic role only for the SCA1 alleles containing a pure CAG sequence of at least 39 repeats. Our patient presented some clinical and neuroimaging features in overlap with both SCA1 and MSA-C diseases, however the presence of CAT interruptions in a SCA1 intermediate allele did not support the diagnosis of SCA1.
8
A clinical overlap was also encountered in the case of the MSA-P subjects carrying intermediate SCA2 allele. Parkinsonism with autonomic disturbances has been previously recognized as part of the phenotypic spectrum in SCA2 not only in patients carrying fully expanded alleles (≥ 34 CAG) [16], but also in a subject showing a SCA2 intermediate allele with 32 CAG [17]. Intermediate SCA2 alleles with ≥31 CAG were associated with an increase risk of amyotrophic lateral sclerosis (ALS) [18],
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however their specific pathogenic role in other neurodegenerative disorders is not yet fully determined [19].
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In our sample we found a pathologic trinucleotide expansions in SCA2. The patient carrying this allele (CAG 22/36) was initially misdiagnosed as possible MSA-C. At recruitment for this study, the patient seemed to meet the diagnostic criteria for possible MSA-C, supporting the observation of a potential
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clinical overlap between the two neurodegenerative disorders, particularly in the early phase of the
N
diseases [3, 5]. Only a posteriori anamnestic evaluation identified the presence of analogous
M
history in the differential diagnosis of MSA.
A
neurological symptoms in his mother, emphasizing the importance of a correct and complete family
ED
We did not identify expanded alleles in other SCA genes. In a previous study on Korean MSA patients, high prevalence of expanded alleles (7.3%) was observed in SCA1, SCA2, SCA3, SCA6,
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SCA17 and DRPLA genes, with more than half of the mutations occurring in SCA17 gene [4]. Our findings in the SCA17 gene are in contrast with Kim et al., probably because of the different cutoff
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size used in the two studies. Kim et al., considered abnormal alleles with >40 repeats, whereas in our study we considered intermediate alleles with 43-48 repeats and expanded alleles ≥49 repeats. In the
A
cohort described by Kim and colleagues [4] 7 MSA patients carried 41 CAG repeats (2.7%), 3 patients had 42 repeats (1.2%), and 3 MSA subjects had respectively 43, 44, and 46 CAG. None had allele size above 49 repeats [4]. In our study, we identified 5 patients carrying 41 CAG (2%), 1 patient carrying 42 CAG (0.4%), and no SCA17 alleles with ≥43 CAG were found. If we consider that in Italy SCA1 and SCA2 are the most frequent SCA genotypes and that SCA17 mutation accounts for less than 1% of the cases [20], the lack of SCA17 intermediate alleles could be likely due to the limited sample of our 9
cohort. In addition a possible bias of our study is represented by the fact that we did not screened for other trinucleotide disorders mimicking MSA phenotype such as late onset Friedreich ataxia or Xlinked tremor and ataxia syndrome [1]. In conclusion, our study confirms the value of genetic testing for the dominant polyQ disorders in the differential diagnosis of MSA, and in particular of SCA1 and SCA2 genes in the Italian population.
IP T
The observation of an enriched distribution of SCA1 large normal alleles (30-35 CAG) in MSA-C needs to be further investigated in a large series of cases. The identification of intermediate or
SC R
expanded alleles in polyQ genes is crucial for a correct diagnosis and an appropriate genetic
U
counseling for patients and families.
A
N
Declarations of interest: none.
M
Aknowledgement
The DNA samples of MSA patients of the Parkinson Institute of Milan were obtained from the
ED
“Parkinson Institute Biobank” member of the Telethon Network of Genetic Biobank (http://biobanknetwork.telethon.it, project n. GTB12001) funded by TELETHON Italy, and supported
PT
by the ‘Fondazione Grigioni per il Morbo di Parkinson’. Genetic analyses of MSA and control
CC E
subjects, performed at the Foundation IRCCS Neurological Institute Carlo Besta, were partially supported by the Italian Ministry of Health (Grants GR-2013-02357821 to LN; and RF-2011-
A
02347420 to CM).
10
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14
Figure Legend Figure 1. Allelic distribution of CAG repeat within SCA1, 2, 3, 6, 7, 17, DRPLA and HD genes in MSA-C (blue bars) and MSA-P (red bars) patients. Vertical axes represent allelic frequencies (%) while horizontal axes represent number of CAG repeat units. Arrows indicates: (a) the intermediate SCA1 allele (CAGn=36), (b) the intermediate SCA2 allele (CAGn=31), and (c) the expanded SCA2 (CAGn=36)
in
MSA-C
patients.
SCA
indicates
spinocerebellar
ataxia;
A
CC E
PT
ED
M
A
N
U
SC R
dentatorubropallidoluysian atrophy; and HD, Huntington disease.
15
DRPLA,
IP T
allele
Table 1: Demographic and genetic characteristics of MSA patients and controls. MSA-C
MSA-P
Controls
Subjects N.
128
118
223
Total alleles N.
256
236
446
Sex
52 M /76 F
49 M/69 F
116 M/107 F
Age at onset (years)
56.4 ± 7.7
60.3 ± 7.6
64.4 ± 7.3
62.5 ± 7.9
28.5 ± 1.6 [2536]
27.8 ± 1.7 [15-34]
27.6 ± 1.3 [19-33]
255 (99.6)
236 (100)
446 (100)
1 (0.4)
0
0
0
22.1 ± 1.1 [2136]
22.1 ± 0.8 [18-31]
22.1 ± 1.1 [12-30]
252 (99.6)
233 (99.6)
439 (98.4)
1 (0.4)
0
1 (0.4)
0
0
20.2 ± 5.1 [936]
20.2 ± 4.9 [10-34]
256 (100)
236 (100)
0
0
0
0
11.5 ±1.6 [7-14]
11.2 ± 1.9 [7-14]
256 (100) 0 0
236 (100) 0 0
9.8 ± 0.9 [9-13]
9.8 ± 0.9 [7-13]
254 (100)
236 (100)
0
0
0
0
37.1 ± 1.9 [2942]
36.9 ± 1.9 [29-41]
256 (100) 0
236 (100) 0
ED
SCA3
0
CC E
PT
Normal [≤44] Intermediate [4559] Expanded [≥61] SCA6
Normal [≤18] Intermediate [19] Expanded [≥20]
A
SCA7
Normal [≤27] Intermediate [2833] Expanded [≥34]
SCA17 Normal [≤42 ] Intermediate [43-
U N
0
A
Normal [≤26] Intermediate [3134] Expanded [≥35]
<0.0001 MSA-C vs. MSAP <0.0001 MSA-C vs. Controls ns MSA-P vs. Controls <0.0001 MSA-C vs. MSAP <0.0001 MSA-C vs. Controls ns MSA-P vs. Controls
SC R
61.6 ± 8.2
Normal [≤35] Intermediate [3639§] Expanded [≥pure 39] SCA2
<0.0001
M
SCA1 (N. CAG repeats)
ns
IP T
Age at examination (years)
P Value*
16
0
ns
ns
ns
ns
ns
0
17.4 ± 3.5 [928]
17.9 ± 3.3 [11-30]
254 (100)
236 (100)
0
0
0
0
18.5 ± 3.1 [928]
18.5 ± 3.3 [9-32]
18.2 ± 3.2 [8-33]
254 (96.8)
227 (96.2)
438 (98.2)
8 (3.2)
9 (3.8)
8 (1.8)
0
0
Normal [≤35] Intermediate [3648] Expanded [≥49] HD Normal [≤26] Intermediate [2735] Expanded [≥36]
0
Values show mean ± SD, [range] or
(%).§=
ns
ns
interrupt 39 CAG. *P values were assessed using: t-test
SC R
DRPLA
0
IP T
48] Expanded [≥49]
(age at onset MSA-C vs MSA-P); ANOVA (age at examination in the three groups), WilcoxonKruskal-Wallis test (allelic distribution). In bold statistically significant p values.
Abbreviations: N, number; MSA-C, multiple system atrophy - cerebellar type; MSA-P, multiple
U
system atrophy - parkinsonian type; vs, versus; M, male; F, female; ns, not significant (p≥0.05);
A
CC E
PT
ED
M
A
N
SCA, spinocerebellar ataxia; DRPLA, dentatorubropallidoluysian atrophy; HD, Huntington disease.
17
Table 2: SCA1 repeat length distribution in alleles from MSA- C, MSA-P and control subjects. Number of CAG repeats Tot. 15
19
20
21
24
25
26
27
28
29
30
31
32
33
34
N. allel es
0
2
0
2
3
6
24
182
158
45
20
2
Allelic frequency
0
0.
0
0.
0.
1.
5.
40.
35.
10.
4.5
0.
(%)
0.
5
0.
5
7
4
4
8
4
1
8.6
5
χ2 (z score)
48
1.
48
1.
0.
0.
1.
4.0
0.1
0.3
1
3.
15
16
77
92
6
1
4
15
MSA-C
1
1
0
0.
0.
0
2
2
0.95
2.
1.
71
97
63
SC R
N° alleles
IP T
Controls
0
0
0
0
5
8
62
74
42
44
8
5
5
1
N° alleles
0
0
0
0
0
2.
3.
24.
29.
16.
17.
3.
2.
2.
0.4
Allelic frequency
0.
0.
0.
0.
1.
0
2
4
1
5
3
2
0
0
0.38
(%)
27
54
27
54
36
0.
0.
8.3
2.1
7.0
22.
3.
1.
5.
01
52
4
6
89
79
93
06
0
1
0
2
N° alleles
0.
0
0.
0
0.
Allelic frequency
4
0.
(%)
2.
50
χ2 (z score)
22
N
7
6
85
91
16
16
5
4
1
1
3.
2.
36.
35.
6.8
6.8
2.
1.
0.
0.4 0.49
ED
1
4
0.
9
0
5
0
6
3.8
0.8
1
7
4
2.
50
0.
1.
1.
0.0
1.1
3
6
0.
0.
0.
34
34
5
2
39
87
33
22
1
2
1
2
5
18
38
329
323
103
80
15
10
7
2
%
0.
0.
0.
0.
0.
1.
4.
35.
34.
11.
8.6
1.
1.
0.
0.2
1
2
1
2
5
9
1
2
5
0
6
1
8
CC E
43
Tot. N. alleles
PT
patients
A
MSA-P
3
M
χ2 (z score)
U
0
patients
446
254
236
936
In bold statistically significant p value obtained with χ2 test. The MSA-C patient carrying a SCA1
A
intermediate allele (CAGn=27/36) has been excluded from the statistical analysis. Abbreviations: N, number; MSA-C, multiple system atrophy - cerebellar type; MSA-P, multiple system atrophy parkinsonian type.
18