Abstract
Mutations in TUBB4A have been identified to cause a wide phenotypic spectrum ranging from hereditary generalized dystonia with whispering dysphonia (DYT4) to the leukodystrophy hypomyelination syndrome with atrophy of the basal ganglia and cerebellum (H-ABC). To test for the contribution of TUBB4A mutations in different ethnicities (Spanish, Italian, Korean, Japanese), we screened 492 isolated dystonia cases for mutations in this gene and for the first time determined TUBB4A copy number variations in 336 dystonia patients. A potentially pathogenic rare 3bp-in-frame deletion was found in a patient with cervical dystonia but no copy number variations were detected in this study, suggesting that TUBB4A mutations exceedingly rarely contribute to the etiology of isolated dystonia.
Keywords: TUBB4A, Dystonia, Leukodystrophy, H-ABC
1. Introduction
Mutations in TUBB4A (chr19:6,494,319–6,502,584) have been identified to cause a wide spectrum of neurological diseases ranging from hereditary generalized dystonia with whispering dysphonia (DYT4) [1,2] to the more severe leukodystrophy hypomyelination syndrome with atrophy of the basal ganglia and cerebellum (H-ABC) [3]. To date, only one large Australian family has been described with dystonia unequivocally linked to the missense variant NM006087.3:c.4C > G in the TUBB4A gene [1,2]. However, at present, screening of dystonia patients has mainly been restricted to Caucasian samples and neither revealed the NM006087.3:c.4C > G DYT4-associated mutation, nor any novel TUBB4A variant [1,2,4,5]. To further elucidate whether TUBB4A mutations are limited to a single ethnicity (here Caucasian), as may be the case for rare variants [6], we additionally genotyped samples of patients of Asian descent. Furthermore, we here also examined for the first time the contribution of gene dosage alterations in TUBB4A in a large dystonia sample.
2. Methods
For TUBB4A Sanger sequencing of all 4 exons and exon/intron boundaries (primer sequences used as published [2]), we included 37 Korean, 191 Japanese, 101 Italian and 163 Spanish patients with isolated dystonia, all of whom were diagnosed with focal, segmental or generalized dystonia (Table 1) and were negative for mutations in exon 5 of the TOR1A gene. For gene dosage analysis, we included 336 dystonia patients with an age at onset of ≤30 years and/or a positive family history, and patients diagnosed with spasmodic dysphonia. The multiethnic samples could not be included due to limited DNA amounts.
Table 1.
Clinical and demographic data of patients included in the study.
| Population | All patients | Focal dystonia | Segmental dystonia | Generalized dystonia | |
|---|---|---|---|---|---|
| Korean | No. of patients | 37 | 25 | 4 | 8 |
| Female | 31 | 21 | 4 | 6 | |
| AAO | 40.0 (±22.6) | 49.5 (±16.5) | 23.5 (±28.0) | 18.5 (±18.9) | |
| Japanese | No. of patients | 191 | 127 | 37 | 27 |
| Female | 89 | 54 | 21 | 14 | |
| AAO | 40.1 (±15.6) | 39.7 (±14.3) | 46.2 (±14.4) | 35.7 (±19.4) | |
| Italian | No. of patients | 101 | 95 | 6 | 0 |
| Female | 57 | 53 | 4 | 0 | |
| AAO | 46.8 (±15.8) | 46.3 (±15.7) | 53.5 (±17.4) | 0 | |
| Spanish | No. of patients | 163 | 162 | 0 | 1 |
| Female | 122 | 121 | 0 | 1 | |
| AAO | 51.3 (±14.8) | 51.3 (±14.9) | 0 | 53.0 |
AAO – age at onset.
To detect copy number variations, TaqMan copy number assays (Hs02186957_cn, Hs00446221_cn, and Hs02319378_cn (Applied Biosystems)) for exons 2, 3 and 4 of the TUBB4A gene were used. Due to the short length of exon 1, a forward (5′–CCAGCCCCCTCCATCATC-3′), a reverse primer (5′-GGGCACGCGTCACG-3′) and a FAM- and NFQ-Quencher-labeled probe (5′–CCGGTCACCCTCCCGCTCC-3′) against the non-coding region of exon 1 were designed. TaqMan Copy Number Reference Assay (Applied Biosystems) targeting the telomerase reverse transcriptase (TERT) was used as reference.
3. Standard protocol approvals, registrations, and patient consents
The study was approved by the local ethics committee at the University of Lübeck (04–155). All participants gave written informed consent for participation in the research study.
4. Results
TUBB4A screening in isolated dystonia patients from different populations (Korean, Japanese, Italian, Spanish) (Table 1) revealed one rare, in-frame-deletion at position NM006087.3:c.1015_1017del (rs756023196) (NP_006078.2:p.S339del) in a male Italian patient with cervical dystonia and an age at onset of 21 years (Supplementary Fig. S1). Unfortunately, this patient was lost to follow-up and it was not possible to assess disease progression after his last examination aged 35 years. This variant was found in one allele among 66,714 alleles of European (Non-Finnish) samples (MAF 1.499e-05) in the Exome Aggregation Consortium Browser (ExAC at http://exac.broadinstitute.org/) and has a CADD PHRED score of 14.95 (CADD score calculation at http://cadd.gs.washington.edu).
TUBB4A exon copy numbers were unremarkable in all tested 336 dystonia patients.
5. Discussion
Considering the influence of ethnicity on mutation frequencies, we screened ~500 multiethnic patients with isolated dystonia and found one rare, potentially pathogenic in-frame deletion (NM006087.3:c.1015_1017delAGC) in an Italian patient with cervical dystonia and no mutations in our Asian patients. This finding is reminiscent of the situation in DYT-Tor1A (DYT1) dystonia, where it has already been shown that in-frame deletions (NM_000113.2:c.907_909delGAG) can be a major cause of dystonia [7,8].
Unfortunately, the patient in our study was lost to follow-up for additional confirmation. In-silico analyses using the CADD PHRED score of 15 did not unequivocally confirm this variant as being pathogenic. Nevertheless, to finally evaluate pathogenicity of the TUBB4A variant, further in-vitro analyses are needed.
Although previous studies have highlighted the contribution of gene dosage changes to the mutational spectrum of several genes causative of movement disorders [9], no copy number variations were detected in the TUBB4 gene in this study.
Our data as well as other studies suggest that mutations in TUBB4A exceedingly rarely contribute to the etiology of isolated dystonia [4,5].
Consequently, routine genetic testing of TUBB4A in individuals with isolated dystonia is not recommended but mutational analysis in patients with complex forms of dystonia may be warranted in order to further explore the role of this gene and of microtubule dysfunction in these disorders.
Supplementary Material
Acknowledgments
CK is the recipient of a career development award from the Hermann and Lilly Schilling Foundation and receives funding from the DFG (KL1134/13-1).
Appendix A. Supplementary data
Supplementary data related to this article can be found at http://dx.doi.org/10.1016/j.parkreldis.2017.06.001.
Footnotes
Financial disclosures
| Author | Financial disclosures |
|---|---|
| Franca Vulinovic | FV reports no disclosures. |
| Susen Schaake | SS reports no disclosures. |
| Aloysius Domingo | AD is a recipient of a scholarship from the German Academic Exchange Service (DAAD). He also received grants from the MDS-AOS and the Collaborative Center for X-linked Dystonia-Parkinsonism at Massachussetts General Hospital. |
| Kishore R Kumar | KRK is a recipient of a National Health and Medical Research Council of Australia (NHMRC) Early Career Fellowship and the Douglas Piper Fellowship from the Royal North Shore Hospital Scholarship Program. He was awarded a Ramsay Research and Teaching Fund Knowledge Discovery Project (Biomedical Research) and a Bushell Travelling Fellowship in Medicine or the Allied Sciences (The Royal Australasian College of Physicians Foundation). He receives honoraria from UCB Australia Pty Ltd and Novartis Pharmaceuticals. |
| Giovanni Defazio | GD reports no disclosures |
| Pablo Mir | PM reports no disclosures |
| Kristina Simonyan | KS serves on the Medical and Scientific Advisory Council of the Dystonia Medical Research Foundation and has funding from NIH. |
| Laurie J Ozelius | LJO serves on the scientific advisory board fort he National Spasmodic Dysphonia Association, The Benign Essential Blepharospasm Research Association, The Tourette Association of America and the Alternating Hemiplegia of Childhood Foundation. She receives funding from NIH and the Foundation for Dystonia Research and patent royalties from Athena Diagnostics Inc. |
| Norbert Brüggemann | NB is funded by the Collaborative Center for X-linked Dystonia-Parkinsonism. He received travel grants from Ipsen, Merz and St. Jude Medical. NB received research funds from Medtronic and St. Judes Medical. |
| Sun Ju Chung | SJC is funded by a grant of the Korea Healthcare Technology R & D Project, Ministry of Health & Welfare, Republic of Korea (HI14C2206). |
| Aleksandar Rakovic | AR reports no disclosures. |
| Katja Lohmann | KL receives funding from the German Research Foundation and the Dystonia Coalition. |
| Christine Klein | CK is an Associate Editor of Annals of Neurology and Movement Disorders and a member of the editorial board of Neurology and has served as editor of the “Continuum Issue Neurogenetics 2008” and as faculty at the annual meetings of the American Academy of Neurology since 2004. She serves as a medical advisor to Centogene. She is the recipient of a career development award from the Hermann and Lilly Schilling Foundation. She is funded by the Deutsche Forschungsgemeinschaft, the European Union, and the Possehl Foundation and received institutional support from the University of Lübeck for genetics research. |
Conflict of interest
On behalf of all authors, the corresponding author states that there is no conflict of interest.
Funding disclosures for this study
The project was funded by the Hermann and Lilly Schilling Foundation and the German Research Foundation (to CK; KL-1134/13-1; to AR: RA 2614/1-1) and R01DC011805 (to KS).
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