Patients and methods

PATIENTS

From a survey of 107 British patients13 with generalised, multifocal, and segmental dystonia fulfilling the agreed criteria for primary torsion dystonia14 we identified 84 patients whose response to treatment with anticholinergic drugs was known. Of these, seven patients were studied, all of whom had obtained an excellent response to benzhexol at doses ranging from 15-120 mg daily such that dystonia was completely or virtually abolished. Two patients (4 and 5) had received a trial of levodopa treatment with no improvement in symptoms but were included in the study because it was not known if the dose given was sufficient to exclude a clinical response.

DNA SEQUENCING

DNA was extracted from peripheral blood lymphocytes using standard techniques. All six exons of the GCH1 gene were amplified separately using the polymerase chain reaction (PCR). PCR primers and conditions were as described previously.10

Purified products from the PCR reactions were sequenced using an automatic DNA sequencer (Applied Biosystems 373A). For exon 1, sequencing was performed with fluorescently labelled dye terminators using a T7 sequencing kit (Applied Biosystems). A Taq dye deoxy terminator cycle sequencing kit (Applied Biosystems) was used for sequencing of exons 2-6. Sequencing primers were as described previously.10 Both sense and antisense strands were sequenced for each sample. The Sequence Editor version 1.0.3 programme (Applied Biosystems) was used for detection of sequence variations from controls and the published sequence.

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Family trees of patients 2 and 7 showing GCH 1 genotypes. Index patients indicated by arrows. Wt= wild type allele.

RESTRICTION SITE ANALYSIS

The presence of mutations was verified by identification of gain or loss of restriction enzyme recognition sites created by point mutations. Restriction site analysis was then used to screen 210 ethnically matched control chromosomes for the presence of these mutations. Exons were amplified by PCR and the product digested with enzyme according to manufacturer’s instructions. Digestion products were separated by electrophoresis on a 3.2% agarose gel containing ethidium bromide.

Results

MOLECULAR GENETIC ANALYSIS

Three heterozygote mutations of the GCH1 gene were identified in two patients (table 1). All mutations resulted in changes in highly conserved regions of the gene.

In patient 2, a transversion in exon 6 resulted in a premature stop codon at position 224. A transition causing a proline to leucine amino acid change at codon 23 in exon 1 was also identified in this patient. These mutations create new Alu-I and Taq-I cleavage sites respectively. Restriction site analysis disclosed the codon 23 mutation to be present in one of 210 control chromosomes screened (in a male).The other mutation was not found in controls. The parental genotypes for patient 2 were determined by restriction site analysis, showing the Lys224Stop mutation to be present in the mother and the Pro23Leu variant in the father (figure). Both parents were asymptomatic and normal on examination (in the afternoon) by two neurologists.

A missense mutation in patient 7 resulted in an aspartic acid to asparagine amino acid change at residue 115 caused by a substitution in the last nucleotide of exon 1. The mutation would also be predicted to alter the splice site sequence at the exon 1-intron 1 boundary. This mutation abolishes a Dde-I restriction site and was not found in any controls. This patient was a sporadic case with no family history of dystonia. However, the patient’s father and brother also had the mutation but manifested no features of the disease (figure). An unaffected sister did not have the mutation.

No sequence variants were identified in the remaining five patients.

CLINICAL PHENOTYPE

Table 2 summarises the clinical characteristics of the patients studied. Dystonia started in the lower limb in all seven patients. The average age at onset was 8.7 years (range 5 to 20 years).

Dystonia progressed to become generalised in five patients and was segmental and multifocal in one patient each. The clinical features in both patients with GCH1 mutations were broadly similar to the group as a whole, with onset of dystonia in the lower limb in childhood, progressing to more generalised dystonia before treatment with benzhexol. The mean daily dose of benzhexol required to abolish symptoms was lower in patients harbouring GCH1 mutations (15 mg and 20 mg), when compared with patients without mutations (range 30-120 mg, mean 67 mg).

Patient 2 began to toe walk at the age of five. Within three years dystonia had spread to involve all limbs, the trunk, and the muscles of the neck. Dystonia was slightly less severe in the mornings. By the age of eight when treatment with benzhexol was started, she was still able to walk but required the use of a wheelchair for long distances. Within one week of starting treatment with benzhexol, dystonia was virtually abolished. At a dose of 20 mg daily there was no evidence of dystonia on clinical examination.

Patient 7 developed in-turning of the right foot at the age of seven. Over the subsequent two years she developed dystonia affecting both legs, the trunk, and the left hand and arm. There was no diurnal variation in symptoms. The response to benzhexol was apparent within four days and continued to develop over six weeks with almost complete abolition of dystonia, maintained at a dose of 15 mg daily. The patient has subsequently been switched from benzhexol to levodopa (Sinemet-plus three daily). On this regime she is asymptomatic with no dystonia on clinical examination.

Interestingly, both patients with GCH1 mutations were apparently sporadic cases, by contrast with four of the remaining five index patients, in whom the family history was compatible with autosomal dominant inheritance. The mother of patient 5 and the mother, brother, and daughter of patient 6 had primary torsion dystonia. In two patients (1 and 3), family members were asymptomatic but on examination were classified as affected on the basis of writer’s cramp, dystonic posturing of the outstretched hands, and pronounced postural tremor. No patients had Ashkenazi Jewish ancestry.

Discussion

Primary torsion dystonia is an autosomal dominant disorder with reduced (30-40%) penetrance and variable expression.13Genetic linkage analysis has identified one locus, DYT 1, on the distal long arm of chromosome 9 in one large non-Jewish and several Jewish families.15 16 However, a significant proportion of non-Jewish families with primary torsion dystonia are not linked to the DYT 1 locus, indicating the existence of other genetic loci for primary torsion dystonia.17

We identified three novel mutations in the GCH1 gene in two out of seven patients diagnosed with primary torsion dystonia, all of whom showed an excellent response to anticholinergic drug therapy. These findings indicate that these two patients have a variant of dopa responsive dystonia.

The mutations in patient 2 are of particular interest. This patient is a compound heterozygote with Lys224Stop inherited from her mother and Pro23Leu inherited from her father. The non-sense mutation found in exon 6 creating a stop codon would be predicted to result in premature truncation of the GCH1 protein with loss of the C terminal 10% of the protein. It is interesting to note that a missense mutation causing a conservative amino acid change (a change to a biochemically similar amino acid) has been identified in the same codon in another British family with an unusual presentation of dopa responsive dystonia resembling athetoid cerebral palsy.10 The Pro23Leu mutation in the paternal GCH1 allele was found in one of 210 control chromosomes examined. There are two possible explanations for this finding: either Pro23Leu could represent a rare variant in the population, or alternatively, the mutation in combination with a second mutation in the maternal allele may be sufficient to reduce GCH1 activity below a critical threshold whereas neither alone is sufficient to cause disease in either of the carrier parents. Pro23Leu results in a non-conservative amino acid change in a highly conserved region of the gene which suggests that it is a pathogenic rather than neutral mutation. All GCH1 mutations so far identified have been private mutations, so our finding of two apparently unrelated persons (patient 2 and one control) with the same mutation is unusual but may reflect the fact that many previous studies have not screened controls for the presence of mutations. Furthermore, the control subject in whom the mutation was found was male and he may therefore be a non-manifesting carrier of dopa responsive dystonia similar to the father of patient 2 who is the transmitting carrier of the mutation Very reduced penetrance is the rule rather than the exception in this condition and Ichinoseet al have shown that asymptomatic male gene carriers have higher enzyme activities than affected females with the same mutation.12 Homozygote missense mutations of GCH1 result in undetectable enzyme activity and patients typically present in infancy with hyperphenylalaninaemia, severely retarded development, abnormal muscle tone, and convulsions.18

The parents of patient 2 are both asymptomatic gene carriers with no manifestations of dopa responsive dystonia. This is the first report of compound heterozygote mutations of GCH1 in a patient with dystonia, and therefore raises the possibility that dystonia due to GCH1 deficiency may be inherited as an autosomal recessive trait in a minority of cases. The patient continues to maintain an excellent response to benzhexol with complete abolition of dystonia, and has not switched to levodopa at her own request.

Two male relatives of patient 7 (Asp115Asn) are non-manifesting carriers. Interestingly, this mutation may disrupt a splice donor site as well as changing the amino acid at residue 23. G->A mutations at the −1 position of the exon-intron boundary have been shown to disrupt RNA transcription in some other diseases.19-22 Splice site mutations within introns have been previously described in dopa responsive dystonia and RNA studies are underway in this patient.23 24 Recently levodopa was substituted for benzhexol in this patient with a good response.

Five of our patients had no identifiable mutation within the coding region of the GCH1 gene. This does not exclude the possibility that these patients have a variant of dopa responsive dystonia as up to half of the cases of clinically definite dopa responsive dystonia have no identified GCH1 mutation.25 Mutations outside the coding region of GCH1 or in a regulatory gene, or gene coding for another enzyme of the tetrahydrobiopterin synthesis pathway could explain such cases. The absence of diurnal variation of symptoms and the higher dose of benzhexol required to produce a response in patients without identified mutations support the supposition that these patients do not have dopa responsive dystonia but rather a variant of primary torsion dystonia. It has been suggested that patients with primary torsion dystonia respond more slowly to anticholinergic drugs than patients with dopa responsive dystonia as well as showing a generally poorer response and needing higher doses.6 The difficulty in distinguishing between primary torsion dystonia and dopa responsive dystonia on the basis of clinical features alone makes a therapeutic trial of levodopa mandatory.

Not all patients with clinically definite dopa responsive dystonia respond to anticholinergic drugs.5 Nygaard et al found that 17 patients with dopa responsive dystonia had previously tried an anticholinergic drug with varying degrees of success.6 Overall, most patients with dopa responsive dystonia derived some benefit from anticholinergic drugs, but in most, levodopa therapy was judged superior in terms of both efficacy and adverse effects. Anticholinergic therapy is therefore not a substitute for levodopa in dopa responsive dystonia.

In summary, we have identified three novel GCH1 mutations in two patients with anticholinergic responsive dystonia, suggesting that a variant of dopa responsive dystonia underlies dystonia in a minority of patients with apparent primary torsion dystonia. Our findings highlight the difficulty of distinguishing clinically between primary torsion dystonia and dopa responsive dystonia. A therapeutic trial of levodopa is therefore essential in all cases of dystonia with onset in childhood or early adult life even in the absence of any diurnal fluctuation of symptoms or family history. A favourable response to anticholinergic drugs in patients with what seems to be primary torsion dystonia should alert clinicians to the possibility that such patients have a levodopa responsive dystonia.