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Essential tremor (ET) is a common inherited movement disorder whose causes remain unknown. The presence of spontaneous tremor in murine mutants may provide clues into the pathogenesis of ET. SCN8A encodes the neuronal voltage gated sodium channel Nav1.6 that is widely expressed in the central nervous system. Several mutations Scn8a in the mouse result in congenital postural tremor of the extremities and head.
We screened SCN8A as a candidate gene in a cohort of 95 Caucasian patients with ET and a positive family history, including 48 patients with early onset in the first two decades of life. Early and adult onset ET subgroups did not differ in disease severity, but early onset patients had longer disease duration. Observed sequence variants were also screened in an ethnically matched control group.
We did not detect SCN8A mutations affecting amino acid sequence or splice sites in our cohort of ET patients.
Although mutations Scn8a cause congenital tremor in mice, mutations in the sequence of the exons and splice sites of human SCN8A do not appear to be a common cause of autosomal dominant essential tremor in Caucasian patients.
Essential tremor (ET) is one of the most common neurological disorders of adults, affecting 0.4% – 3.9% of the population . ET is characterized by bilateral, predominantly symmetrical, postural and/or kinetic tremor involving mostly the upper extremities, including head and voice tremor . ET is clinically heterogeneous, and additional movement abnormalities such as dystonia may be associated with postural or kinetic tremor . Patients with advanced ET also develop predominantly midline ataxia. Most reported pathologic changes are localized in the cerebellum, supporting the emerging view that that ET is primarily a cerebellar disorder .
ET appears to be inherited in a significant proportion of cases. A positive family history is present in 30% to 70% of ET cases, suggesting a strong genetic component . Childhood onset is observed in ~5% of ET patients, and among these early onset patients, approximately 80% belong families with autosomal dominant inheritance . There is considerable variation in age of onset within families, suggesting that childhood onset ET is unlikely to be a separate condition . Genetic loci responsible for ET in individual families have been mapped to chromosome 3q13, 2p24.1, 6p23, and 5q31.1–q33.1, but disease causing genes have not been conclusively identified. The Ser9Gly variant in the dopamine receptor gene DRD3 may increase susceptibility to the disease , but this finding was not replicated in a large cohort of patients . A SNP in the HS1-BP3 gene, which is located within the ET locus on chromosome 2p, is also likely to be a benign polymorphism .
Since the genetics of ET is fraught with difficulties, animal models with similar clinical phenotypes may be useful in identifying genes with a role in pathogenesis. Several mouse mutants of the voltage gated sodium channel gene Scn8a exhibit postural and kinetic tremor of the extremities, in combination with ataxia and dystonia . For example, mice with the medjolting mutant that alters the voltage dependence of the channel display a wide unsteady gait and a rhythmic tremor in the head and neck.
Voltage-gated sodium channel α subunits are large transmembrane proteins composed of four homologous domains with voltage sensor and pore regions. Voltage gated sodium channels are responsible for the initiation and propagation of neuronal action potentials. The nine orthologous mammalian sodium channels differ in biophysical properties, subcellular localization and tissue specificity. Mutations in different neuronal sodium channel genes are responsible for inherited forms of epilepsy, migraine, and pain disorders .
SCN8A encodes the voltage-gated sodium channel Nav1.6, one of the most abundant channels in the central and peripheral nervous system. Nav1.6 is localized at axonal initial segments, dendrites, and nodes of Ranvier in myelinated axons. Expression of Nav1.6 is required for repetitive firing of neuronal populations implicated in tremorogenesis, including cerebellar Purkinje neurons, cortical pyramidal neurons, and subthalamic nucleus neurons . Heterozygotes for a null mutation of human SCN8A exhibit ataxia and cognitive defects . Because tremor is a common feature of mice with mutations in Scn8a, we explored the role of the human gene as a candidate gene for autosomal dominant (AD) form of ET.
Every patient was diagnosed by a movement disorder neurologist (PH) and signed an informed consent, approved by the Institutional Review Board at Vanderbilt University. The cohort of unrelated Caucasian patients of a northern European ancestry was diagnosed with definite ET based on the following criteria: the presence of bilateral postural and kinetic arm tremor without prominent asymmetry lasting at least for more than five years, the absence of additional neurologic abnormalities, no history of exposure to tremorogenic drugs before the onset of symptoms, and no history or examination suggestive of psychogenic tremor or sudden onset with stepwise deterioration . Tremor was quantified using the NIH ET consortium grading and the rating scale from the Washington Heights-Inswood Genetic Study of Essential Tremor (WHIGET). We did not include patients with a significant asymmetry of right and left arm tremor severity to minimize a risk of misdiagnosis, such as an atypical dystonic tremor or Parkinson's disease. Patients with slight tremor (scores 0 and 1) were not included. Furthermore, the degree of disability was also judged by self-reporting of questions adapted from the Tremor disability questionnaire.
We included only subjects with a positive family history of tremor, defined by the presence of at least one affected living first degree relative who met diagnostic criteria for definite ET and was examined by the same neurologist (PH), and vertical transmission of the disease (parent to offspring) consistent with autosomal dominant (AD) inheritance. Because the tremor in Scn8a mutant mice is congenital with very early onset, we enriched the patient cohort with early onset ET defined as onset before the age of 18 years. Not all affected family members of these probands exhibited juvenile onset of disease. The age of tremor onset was self-reported by adults and obtained from parents, when available, for juvenile onset cases. Controls for determining SNP frequencies consisted of 178 neurologically normal, ethically matched individuals from the Coriell Institute (panels NDPT006 and NDPT009), as well as 431 previously described, ethnically matched patients with the following neurological disorders: ataxia (n=150); autism (n=91); epilepsy (n=95), and Charcot Marie Tooth disease (n=95). The 4 unique SNPs reported here were not observed in any of these ethnically matched individuals, demonstrating that they are not common polymorphisms in the Caucasian population.
Heteroduplex analysis was carried out by conformation sensitive gel electrophoresis (CSGE) as described previously . Twenty seven coding exons of SCN8A, including the alternatively spliced exons 5A and 5N, were amplified by PCR from genomic DNA using previously described primers. PCR products with altered mobility were sequenced by the University of Michigan Sequencing Core. Sequence analysis was carried out with Sequencher software (Gene Codes Corporation, Ann Arbor, Michigan). The effect of novel variants on splice enhancer sites was evaluated using ESEfinder (http://rulai.cshl.edu/cgi-bin/tools/ESE3/esefinder.cgi).
We included 95 ET patients (42 men, 53 women, average current age 53 ± 22 years) with mean WHIGET score 16.3 ± 3.7 (range = 6 to 25). The subgroup of early onset ET patients had average age of disease onset at 11± 7 years (23 men, 25 women, average current age 44 ± 13 years). The subgroup of adult onset ET had average age of onset = 49 ± 12 years (19 men, 28 women, average current age 62 ± 9 years). Disease severity did not differ between the early onset patients (mean WHIGET score 13.8 ± 4.7, range = 6 to 22) and the adult onset patients (mean WHIGET score 18.2 ± 6.8, range = 7 to 25). The two groups differed, as expected, in the duration of the disease (29 ± 9 years for early onset and 9.3 ± 4.4 years for late onset, p < 0.05). Within the subgroup of 45 probands with juvenile onset, 40 kindreds included first or second degree relatives with typical adult onset. Exclusively juvenile onset was observed in 5 small pedigrees containing only 2 or 3 affected individuals.
Five SNPs in SCN8A were observed in our cohort of 95 ET patients. None of these variants changed the amino acid sequence or consensus splice sites (Table 1). The three SNPs located in exons did not change any predicted exonic splice enhancer sites (ESEfinder, http://rulai.cshl.edu/cgi-bin/tools/ESE3/esefinder.cgi). The intron 5 SNP is located 19 bp upstream of the start of exon 6 and is not predicted to influence splicing. Each of these four SNPs were observed in a single ET patient and were not present in > 600 controls. The fifth SNP is located in intron 17 at a position 34 bp downstream of exon 17 and does not change any recognizable functional element. The intron 17 SNP was present in 6% of patients (6/95) and 2% of controls (6/178) (p= 0.12). Among the patients, the intron 17 SNP was present in 5/48 early onset cases and 1/47 late onset cases; this trend was not statistically significant (χ2 = 1.5; p<0.22).
Animal models with spontaneous tremor provide an opportunity for identification of causative ET genes. The postural and action extremities tremor in Scn8a mutant mice are associated with neurologic abnormalities such as dystonia and ataxia that are commonly present in ET patients. In spite of the phenotypic similarities, our results indicate that mutations in the SCN8A gene are not a common cause of autosomal dominant familial ET. We studied Caucasian individuals only and cannot exclude the possibility that this gene plays a more important role in patients with other ethnic backgrounds.
The relationship between the tremor phenotype in mutant mice and human ET is controversial . ET is clinically and genetically heterogeneous, and it is possible that tremor in mouse mutants could have a different pathology and pathophysiology. Although alcohol responsive tremor has been reported in mice lacking the GABAA receptor α1 subunit , genetic analysis of a large cohort of ET patients did not identify pathogenic variants in this gene . In spite of the negative outcome for SCN8A and the GABAA receptor, ligand and voltage gated ion channels remain important candidate genes for this disorder.
Inherited ET is characterized by vertical transmission. Autosomal dominant inheritance with reduced penetrance is the most widely accepted genetic model. However, many multigenerational families with a large number of affected individuals do not demonstrate linkage to a single genetic locus. Thus, autosomal dominant ET may have a complex mode of inheritance involving genetic interaction between multiple susceptibility genes . Age of onset also exhibits significant intrafamilial variability, consistent with the effects of genetic modifiers. Indeed, 93% of our kindreds with individuals suffering from a juvenile onset of the disease had also affected relatives with a later age of ET onset, arguing against the possibility that juvenile AD ET represents a genetically distinct subtype of ET, and further supporting the role of modifying genes . Interactions between mutations in different ion channel genes are known to modify epilepsy severity in mouse models , and may provide a model for polygenic ET.
Our data suggest that mutations of SCN8A are unlikely to be a major cause of autosomal dominant ET in Caucasian patients, but rare mutations may influence clinical expression in some patients.
Supported by NIH R01 NS34509 (MHM) and the Center for Genetics in Heath and Disease, University of Michigan (LMS). PH is supported by NIH K08 NS42743.