Myoclonus–dystonia: clinical and genetic evaluation of a large cohort
- K Ritz1,2,
- M C F Gerrits3,
- E M J Foncke1,
- F van Ruissen2,
- C van der Linden4,
- M D I Vergouwen1,
- B R Bloem5,
- W Vandenberghe6,
- R Crols7,
- J D Speelman1,
- F Baas2,
- M A J Tijssen1
- 1Department of Neurology, Academic Medical Centre, University of Amsterdam, Amsterdam, The Netherlands
- 2Neurogenetic Laboratory, Academic Medical Centre, University of Amsterdam, Amsterdam, The Netherlands
- 3Department of Neurology, Bronovo Hospital, The Hague, The Netherlands
- 4Centre for Movement Disorders, St Lucas Hospital Ghent, Ghent, Belgium
- 5Department of Neurology, Radboud University Nijmegen Medical Centre, Donders Centre for Neuroscience, Nijmegen, The Netherlands
- 6Department of Neurology, University Hospital Leuven, Leuven, Belgium
- 7Department of Neurology, Middelheim Hospital, Antwerp, Belgium
- Dr M A J Tijssen, Department of Neurology H2-261, Academic Medical Centre, University of Amsterdam, PO Box 22660, 1100 DD Amsterdam, The Netherlands;
- Received 3 September 2008
- Revised 10 November 2008
- Accepted 12 November 2008
- Published Online First 9 December 2008
Background: Myoclonus–dystonia (M-D) is an autosomal dominant inherited movement disorder. Various mutations within the epsilon-sarcoglycan (SGCE) gene have been associated with M-D, but mutations are detected in only about 30% of patients. The lack of stringent clinical inclusion criteria and limitations of mutation screens by direct sequencing might explain this observation.
Methods: Eighty-six M-D index patients from the Dutch national referral centre for M-D underwent neurological examination and were classified according to previously published criteria into definite, probable and possible M-D. Sequence analysis of the SGCE gene and screening for copy number variations were performed. In addition, screening was carried out for the 3 bp deletion in exon 5 of the DYT1 gene.
Results: Based on clinical examination, 24 definite, 23 probable and 39 possible M-D patients were detected. Thirteen of the 86 M-D index patients carried a SGCE mutation: seven nonsense mutations, two splice site mutations, three missense mutations (two within one patient) and one multiexonic deletion. In the definite M-D group, 50% carried an SGCE mutation and one single patient in the probable group (4%). One possible M-D patient showed a 4 bp deletion in the DYT1 gene (c.934_937delAGAG).
Conclusions: Mutation carriers were mainly identified in the definite M-D group. However, in half of definite M-D cases, no mutation could be identified. Copy-number variations did not play a major role in the large cohort.
Myoclonus–dystonia (M-D) is a genetically heterogeneous movement disorder with an autosomal dominant inheritance. The disorder is clinically characterised by myoclonic jerks and dystonic movements, which often respond to alcohol.1 Psychiatric abnormalities can be part of the phenotype. A major gene locus maps to the epsilon-sarcoglycan gene (SGCE, DYT11)2 on human chromosome 7q21–22, which encodes a transmembrane protein that is widely expressed in the brain.3 4 The function of the protein remains to be elucidated. Various types of SGCE mutations have been reported in several familial and sporadic M-D cases, such as missense, nonsense and splice site mutations or deletions.2 5 6 A reduced penetrance was observed due to maternal imprinting.7 8 Little is known about genotype–phenotype associations, and still for many patients no mutation could be identified. Recent findings of large heterozygous deletions of the complete gene and single or multiple exons indicate the need for SGCE gene dosage analysis in M-D patients, as exon deletions or duplications might have been overlooked by standard procedures.9 10 It has been suggested that comprehensive clinical criteria for M-D patients will help to precede genotype–phenotype associations.10
In this study, we screened a large cohort of M-D patients for SGCE mutations, including exonic rearrangements, and grouped them according to recently published criteria.10 In addition, mutation analysis for the DYT1 3 bp deletion was performed, since an association between both proteins has been observed.11
After obtaining informed consent, a detailed history was taken and neurological examination and blood withdrawal were performed on 86 M-D index patients. They have all been tested for SGCE mutations in our centre, a national referral centre for M-D, between 2004 and the end of 2007. All index patients were examined and classified into three groups (by MAJT, EMJF, JDS) and three of them were seen by other experienced movement disorder neurologists (BRB, RC, WV). The classification was based on published criteria:10 definite M-D (early-onset myoclonus and dystonia or isolated myoclonus predominantly in the upper body half and a positive family history for myoclonus and/or dystonia), probable M-D (early-onset myoclonus and dystonia or isolated myoclonus predominantly in the upper body half) and possible M-D (jerky dystonia of neck or isolated jerky movements of variable distribution or signs of dystonia and/or myoclonus in the lower body half or no response to alcohol). The age of onset below 26 years was considered as early onset. Twenty-one additional M-D index patients were excluded, as they have not been seen by named neurologists or because sufficient clinical information was lacking. Previously published data of 31 M-D patients were included in this study (large heterozygous deletions had not been screened for in the previous study).12 Two patients were diagnosed in early childhood. We considered age at onset as 5 years for calculations. The association between age of onset and gender was analysed using Mann–Whitney U tests.
Psychiatric diagnosis has been assessed for seven of the 13 SGCE mutation carriers according to the DSM-IV axis 1 classification system using the Structural Clinical Interview DSM-IV diagnoses (SCID-I)13 or the Mini International Neuropsychiatric Interview (MINI) version 126.96.36.199 Psychiatric evaluations were performed by trained neuropsychologists.
DNA was extracted from white blood cells by standard procedures. All patients were sequenced for mutations in 11 exons (exon 1–12 except for exon 10, a rare splicing variant) and flanking intronic regions of the SGCE gene. Patients were also screened for the 3 bp deletion (c.904_906delGAG, NM_000113.2) in exon 5 of the DYT1 gene by direct sequencing. The sequence reactions were performed with the ABI big dye v3.1 chemistry and sequenced with an ABI 3730 capillary system (Applied Biosystems, Foster City, California).
Multiplex ligation-dependent probe amplification (MLPA) was performed using the commercially available probe set P099B (MRC Holland, Amsterdam, The Netherlands) according to the manufacturer’s instructions. All samples were tested in duplicate. Ratios between 0.8 and 1.2 were considered normal.
The double mutation was further characterised by restriction-enzyme digestion using MscI (NEB, Westburg, Leusden, The Netherlands). The recognition sequence of MscI (TGGCCA) encompasses both mutated nucleotides and only cleaves the wild-type sequence.
The detected deletion was narrowed by long-range PCR (Expand Long Template PCR System, Roche Diagnostics, Almere, The Netherlands) and by quantitative real-time PCR (FRET technology, LightCycler 480 instrument, Roche Diagnostics) (conditions are available upon request). The deletion was confirmed by sequencing on cDNA level. RNA was extracted from whole blood using the PAXgene system (Qiagen, Venlo, The Netherlands).
Eighty-six M-D patients were included in the present study and classified according to their clinical features: 24 patients as definite, 23 as probable and 39 as possible M-D (see tables 1, 2).
In 13 of the 86 M-D index cases, heterozygous SGCE mutations were identified. Seven cases have been reported previously (patients DE2, DE6, DE7, DE8, DE11, DE12, PR1).12 In addition, a splice site mutation in intron 2 (known mutation),15 four novel point mutations or small deletions, and one multiexonic deletion were detected (see table 1). We describe for the first time a patient with the M-D phenotype who carries two heterozygous mutations separated by only four bases in the SGCE gene (DE3, fig 1A). None of the tested relatives (son, five siblings, mother and two siblings of the father) carried the mutation, suggesting that both mutations are located on the same allele. This observation was confirmed by restriction-enzyme analysis; digestion of genomic DNA of a healthy subject with MscI resulted in two bands, and DNA of the index patient resulted in three bands deriving from the cut wild-type allele and the uncut mutated allele (fig 1A). In one patient, a heterozygous deletion of four exons was detected (c.exon6–9del, DE10). None of the patients carried the known 3 bp deletion in DYT1 gene. However, we identified a 4 bp deletion in exon 5 of the DYT1 gene (c.934_937delAGAG, NM_000113.2) leading to a frameshift and resulting in a premature stop codon at position 325 (p.R312FfsX325, PO1, fig 1C).
Characterisation of the exon 6–9 deletion
The heterozygous deletion of exon 6 to 9 in the SGCE gene was detected by MLPA and confirmed by quantitative real-time PCR. The deletion comprises at least 7.83 kb (data not shown). The deletion of exon 6 to 9 does not alter the open reading frame. Analysis of SGCE mRNA by RT-PCR confirmed the predicted exon 5–11 fusion product (fig 1B).
Clinical characteristics of the M-D mutation carriers
Twelve of the 24 definite (50%), one of the 23 probable (4%) and none of the 39 possible M-D cases carried a mutation in the SGCE gene. In addition, one possible M-D patient showed a mutation in the DYT1 gene.
All 13 SGCE mutation carriers reported an early age of onset (mean age 9 (SD 5) years, range 1–16 years) with no significant difference between male and female mutation carriers (p = 0.15). Twelve of them had a positive family history (92%) with paternal transmission in 75% (9/12). No parental blood was available for the other three patients DE2, DE8 and DE9. The single patient with a negative family history (probable M-D) showed a de novo mutation (PR1). Initial dystonic symptoms were restricted to cervical dystonia and remained focal in most cases. In three patients upper and lower limbs were involved. Initial myoclonic symptoms presented mainly in the upper part of the body and at a later stage in the trunk, neck and upper as well as lower limbs. Alcohol alleviated the symptoms in 10 of 11 M-D patients. In two patients this information was not available. Evidence for psychiatric comorbidity was observed in all 13 SGCE mutation carriers by history with depression as the main feature (eight cases). Psychiatric assessment was performed for seven cases confirming our observations. Clinical details of SGCE mutation negative cases are summarised in table 2.
In the present study, we screened 86 M-D index patients for mutations in the SGCE gene, including a screen for genomic rearrangements, and for the 3 bp deletion in the DYT1 gene. All patients were classified according to previously published criteria.10
We identified mutations in the SGCE gene in 50% of the definite (12/24), 4% of the probable (1/23) and none in the possible M-D group. All mutation carriers exhibit myoclonic and dystonic features. Myoclonus was responsive to alcohol in most of the cases as reported in several studies.2 15 17 18 Psychiatric abnormalities were found in all mutation carriers by history and confirmed with formal testing in seven. Paternal transmission was observed in 75% of mutation carriers, in the remaining carriers no parental blood was available, and there was one de novo mutation. Our findings are in line with a previously published study observing mutation carriers in 60% (6/10) of definite, 20% (3/15) of probable and no mutation in possible M-D cases.10 We report many different types of mutations, including nonsense, missense, splice site, frameshift to stop mutations, and one large in-frame deletion. The majority of mutations (seven cases with nonsense or frameshift to stop mutations) may result in a null allele due to nonsense mediated decay.19 There is evidence that missense mutations in the SGCE gene lead to protein degradation and thus also to loss of function.11 Recently, an association between gender and age at onset has been reported in mutation carriers with girls having an earlier age of onset.20 We could not confirm these findings in our small number of gene positive cases. Further studies are required to study the influence of gender and age at onset.
In only one patient we identified exonic rearrangements, an in-frame deletion of exon 6 to 9. This patient was diagnosed as definite M-D, with facial tics as an additional symptom. It remains elusive whether the deletion will lead to protein degradation or whether there is an association between the tics and the mutated protein. One previous study reported the phenotypic association of M-D and Tourette syndrome in a family, but they excluded SGCE mutations including exonic rearrangements.21 Eleven heterozygous exonic or whole gene deletions of the SGCE gene have been previously detected in M-D patients.6 9 10 22 23 Most of the reported single or multiple exon deletions lead to a frameshift and premature termination. Only one large in-frame deletion has been reported so far (c.exon2del), but it is unknown whether this patient has additional symptoms.10 In all cases with a deletion of the whole SGCE gene, the deletion included neighbouring genes. This is concordant with additional non-motor symptoms of these patients like joint problems or osteoporosis.
In order to exclude that SGCE mutation negative cases carry the DYT1 deletion (c.904_906delGAG), we screened all patients for this mutation. We did not identify the 3 bp deletion in our patients. However, a 4 bp deletion in exon 5 of the DYT1 gene (c.934_937delAGAG) was detected in a possible M-D patient displaying myoclonic and dystonic features, a negative family history, late onset and showing mild signs of Parkinson’s disease (PD). This mutation has been previously described in a control case.24 This putative healthy control showed no symptoms during a routine examination by a general practitioner at the age of 39 years and refused further examination. There is no evidence for involvement of DYT1 mutations in PD.24 25 Recently, an association between both proteins, DYT1 and SGCE, has been reported, suggesting a role for DYT1 in the recognition and processing of misfolded SGCE.11 It remains unclear whether this deletion plays a role in the development of the parkinsonian or M-D symptoms.
In summary, SGCE mutations are predominantly identified in the definite M-D group. This is in line with previous findings. However, patients with a typical M-D phenotype and a young-onset with a negative family history as well as patients with a typical M-D phenotype, late onset but a positive family history should also be considered for genetic testing, as mutation carriers with a late age of onset have been reported in a Dutch M-D pedigree.16 Possible M-D cases do not need to be tested for SGCE mutations. Screening for exon rearrangements did not increase the number of mutation carriers significantly. For 50% definite M-D cases, no mutation could be identified, suggesting the involvement of other genes, still not identified pathogenic mutations in the SGCE promoter or non-coding regions, or epigenetic defects. The 3 bp deletion in the DYT1 gene is not involved in the pathogenesis of M-D. It remains unclear whether the detected in-frame deletion of four exons plays a role in the development of tics in our definite M-D patient.
The authors would like to thank the patients for participating in this study.
Funding: This study has been supported by Nederlandse Organisatie voor Wetenschappelijk Onderzoek (NWO) VIDI (project 016.056.333 to KR and MAJT).
Competing interests: None.
Patient consent: Obtained.