(E) Methods
Louis Rating Scale
A neurologist specializing in movement disorders (blinded to the genotype data) rated the severity of tremor in ET patients during sustained arm extension and five activities (pouring water, drinking water, using a spoon, finger-to-nose maneuver, and drawing a spiral) with each arm (6 items with each arm and 12 items total). The tremor was rated using a 4-point scale: 0 (no visible tremor), 1 (low amplitude or intermittent tremor), 2 (tremor of moderate amplitude, and clearly oscillatory, and usually present), 3 (large amplitude tremor). A total tremor score (range = 0-36 [severe tremor]) was assigned to each ET patient based on the 0-3 ratings for 12 items.
Reference
Louis ED, Ford B, Lee H, Andrews H. Does a screening questionnaire for essential tremor agree with the physician's examination? Neurology 1998;50: 1351-1357.
Genetic Analysis
Primers and probes were designed using the Primer Express 1.5 software. The TaqMan MGB probes contain minor groove binder, nonfluorescent quencher (NFQ) and a reporter dye. The amplification reactions were carried out in an ABI Prism 7700 Sequence Detection System (Applied Biosystems) with two initial steps (50°C for 2 min, followed by 95°C for 10 min) and 40 cycles of a two step PCR (95°C for 15 sec, 60°C for 1 min). After PCR, the allelic specific fluorescence was measured on the ABI Prism 7700 Sequence Detection System (Applied Biosystems) using the Sequence Detection Systems 1.7 software for allelic discrimination. Sequence analysis was used to confirm the genotypes for representative samples. Dideoxy chainterminator sequencing was carried out according to the manufacturers’instructions (BigDye, Applied Biosystems, Warrington, UK) andthe products were electrophoresed on an ABI 3100 automated DNAsequencer (Applied Biosystems).
Statistical Analysis
The genotype and allele frequency between ET and controls was compared using the chi-square test. Both ET and control groups were tested for Hardy-Weinberg Equilibrium. Multivariate analysis with diseased group (ET or controls) as the dependent variable and age, gender and genotype as the independent variables was carried out. A multivariate ordinal logistic regression was also done amongst ET to determine whether family history of ET was a predictor for genotype (AA/AG/GG) with adjustment for sex and age of onset of ET. Correlation of the ET severity score with the genotype and allele was calculated. In the family studies, we provided descriptive statistics on whether there was possible cosegregation of the G allele with ET phenotype. As the families genotyped were small in size, the genotype and allele data from the various families were pooled together for analysis. The frequency of the genotypes and alleles were then compared between affected and unaffected members by chi-square test. Statistical significance was defined at p<0.05.
Power Calculation
Based on the 20% difference of the GG genotype between ET and controls reported in the initial French study (6), our sample size for the case control study has >95% power to detect such a difference at alpha=0.05.
Recruitment of Study subjects
The diagnosis of classic ET was made based on the recommendations of the Consensus Statement of the Movement Disorders Society in 1998. All ET patients evaluated at our center had a normal thyroid function test. Separately, the authors also examined family members of ET patients who gave a positive family history and their DNA samples and clinical data were also collected. Controls were healthy individuals physically examined by the authors at the Health Screening Unit of the same hospital. None of the controls had any evidence to suggest presence of ET.