Abstract
Abnormalities in dopamine neurotransmission are thought to underlie the generation of dystonic movements. The authors performed a case-control allelic association study in patients with the focal dystonia blepharospasm, using polymorphisms in the dopamine receptor and transporter genes. Allele 2 of a DRD5 dinucleotide repeat was significantly associated with blepharospasm. This may indicate a pathogenic role for this receptor.
Blepharospasm is a primary focal dystonia affecting the orbicularis oculi muscles, usually beginning in middle age. Initial symptoms include eye irritation and frequent blinking, progressing to involuntary spasms of eyelid closure. In severe cases, this can lead to functional blindness. The cause of focal dystonia remains unclear, but there are several lines of evidence suggesting that abnormal CNS dopamine neurotransmission underlies the pathogenesis of dystonic movements.
It has long been recognized that neuroleptic medications (dopamine receptor antagonists) cause tardive dystonia,1 and experimental work with primates has shown that treatment with the toxin 1-methyl-4-phenyl-1,2,3,6 tetrahydropyridine (MPTP), which selectively destroys dopamine producing neurons, causes transient dystonia.2
Two generalized forms of dystonia also provide evidence for involvement of the dopaminergic system. Dopa-responsive dystonia shows a marked and sustained response to levodopa and is due to mutations in the genes for either the guanosine triphosphate cyclohydrolase 1 or tyrosine hydroxylase enzymes, both of which are needed for dopamine synthesis.3 The DYT1 gene causing early-onset primary generalized dystonia is preferentially expressed in the dopaminergic cells of the substantia nigra pars compacta (SNPC).4 In addition, some patients with dystonia myoclonus have been found to carry a missense change in the dopamine receptor DRD2 gene on chromosome 11.5
A recent rat model has given some insight into the pathogenesis of blepharospasm.6 A combination of both cell destruction in the SNPC and a lesion weakening the orbicularis oculi muscle can lead to a condition resembling blepharospasm in humans. Destruction of dopaminergic cells was achieved by injections of 6 hydroxydopamine and weakening of orbicularis oculi by sectioning the zygomatic branch of the facial nerve. Both of these lesions individually led to trigeminal reflex blink hyperexcitability, but only when both were performed on the same animal did spontaneous spasms of eyelid closure occur. This model suggested that a dopaminergic lesion leads to susceptibility to developing blepharospasm, which only becomes apparent following a secondary event.
We have recently performed an allelic association study with polymorphisms in dopamine receptor and transporter genes in a cohort of patients with primary cervical dystonia and control subjects and found a link with a microsatellite in the D5 dopamine receptor gene.7 The current article extends this work to patients with blepharospasm to examine whether there is an association in other forms of primary focal dystonia.
Methods.
The study was undertaken following ethical committee approval from the Royal Free Hospital. Eighty-eight patients with blepharospasm were recruited. All had been diagnosed with primary blepharospasm for at least 1 year by a neurologist with a specialist interest in movement disorders. Patients with secondary dystonia (according to the criteria of Marsden and Harrison8) were excluded, as were those with dystonia affecting sites other than the eyes. Patients with affected relatives also were excluded to avoid the possible confounding effect of additional disease loci. All patients were British Caucasians. 100 control subjects were recruited from patients attending the Royal Free Hospital for nonneurologic conditions. They were matched for age, sex, and ethnicity.
Ten published polymorphisms within the dopamine transporter gene and dopamine receptor genes D1–5 were studied.7 Genomic DNA was extracted from blood samples by standard methods and the polymorphisms were amplified by PCR in a total volume of 25 μL using 100 ng of DNA and 20 pmol of each primer. Statistical analysis was performed using χ2. Hardy–Weinberg equilibrium was assessed in the control population.
Results.
The male-to-female ratio in both the blepharospasm and control groups was equal at 1:4. The mean ages of the control group (60.6 years, SD 17.3) and the blepharospasm group (63.74 years, SD 9.49) were not significantly different. The mean age at onset of blepharospasm in the blepharospasm group was 55.83 years.
There was no significant association between control and patient allele frequencies for polymorphisms in the D1, D2, D3, D4, and dopamine transporter (DT) genes (tables 1 and 2⇓). For the D1.1 single-nucleotide polymorphism (SNP), analysis suggested an association with genotype (see table 1), but this was not supported by analysis of either allele frequency or of the D1.8 SNP.
Results for allelic association for blepharospasm and single-nucleotide polymorphisms in D1–3 receptor genes
Allelic association between blepharospasm and microsatellites in the dopamine receptor and DAT genes
Significant association was found, however, for allele 2 of the dinucleotide repeat in the D5 receptor gene (p = 0.009)(see table 2). This was overrepresented in the blepharospasm group. Hardy–Weinberg equilibrium was demonstrated in the control population for all polymorphisms studied.
Discussion.
We found a possible association between blepharospasm and a microsatellite repeat in the D5 dopamine receptor gene on chromosome 4. Allele 2 was significantly more common in the blepharospasm group compared with the control group. This association was also seen in our study of patients with cervical dystonia, and again allele 2 was overrepresented in the patient group.7 The significance of this current finding in blepharospasm is that the same association has now been identified in two independent cohorts of patients with focal dystonia.
It is unlikely that the dinucleotide repeat itself has a functional effect on the D5 receptor, but these data may indicate that allele 2 is in linkage disequilibrium with another polymorphism either within or outside of the D5 gene that does impart such an effect. D5 receptor messenger RNA has been identified within the substantia nigra pars compacta,9 so a functional variant could influence basal ganglia dopaminergic pathways. We suggest that there is a haplotype associated with allele 2 of the D5 receptor microsatellite that confers susceptibility to developing focal dystonia. This functional dopaminergic susceptibility may lead to dystonia after an appropriate secondary stimulus, similar to the rat model of blepharospasm.
Further studies are required in independent cohorts of patients, preferably from different ethnic groups, to confirm these findings, and identification of other polymorphisms in and around the D5 receptor gene that may have a functional effect is needed.
Acknowledgments
Supported by the Dystonia Society (UK).
Acknowledgment
The authors thank all of the patients who participated in this study.
- Received June 18, 2001.
- Accepted September 26, 2001.
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