Correlation of clinical and molecular features in spinal bulbar muscular atrophy

Patients diagnosed with SBMA were recruited through the British Neurological Surveillance Unit, an established collaborative research system run by the Association of British Neurologists. Consent was obtained directly from patients. Patients included in the register are followed in numerous centers across the United Kingdom, and DNA samples were obtained from local genetic laboratories with full informed consent where available. All participants in this study were interviewed by one research nurse specialist by telephone, with the exception of 2 patients who compiled the questionnaire by writing because of speech difficulties. There were no missing data for any of the patients who agreed to undergo interview. The questionnaire was composed of 54 questions designed to require a “yes/no” answer. Five questions, addressing the initial symptoms and major complaints, required a descriptive answer. For deceased patients, only age at onset and age at death data were included in the study.

The study was approved by the National Research Ethics Service of the UK National Health Service and local ethical committees. All patients received a patient information leaflet and signed a written informed consent form.

Genomic DNA was extracted from patient peripheral blood leukocytes using a FlexiGene DNA kit (Qiagen, Venlo, the Netherlands). AR alleles were amplified by PCR using GoTaq DNA polymerase (Promega, Madison, WI) and a FAM-label introduced by the forward primer. The primers used were previously described.³ PCR products were checked on a 4% (w/v) agarose gel and then electrophoresed on an ABI 3730xl DNA analyzer with a LIZ-500 size standard (Applied Biosystems, Foster City, CA). Fragment analysis was performed with GeneMapper software (version 4.0; Applied Biosystems).

The CAG repeat regions of patients with SBMA were amplified by PCR using REDtaq Polymerase (Sigma, St. Louis, MO) and the primers specified by La Spada et al.² (5′-GCCTGTTGAACTCTTCTGAGC-3′ and 5′-GCTGTGAAGGTTGCTGTTCCTC-3′). Reaction mixtures were made in accordance with the manufacturer's instructions and a touchdown PCR protocol was used to reduce nonspecific amplification. PCR cycling conditions were 30-second denaturing at 95°C, 30 seconds of annealing at 72°C (reduced by 0.5°C every cycle for 24 cycles), 40 seconds for extension at 72°C, then followed by a further 10 cycles of 30-second annealing at 95°C, 30-second annealing at 60°C, and 40 seconds of extension at 72°C. The PCR product was purified from the amplification mixture using microCLEAN PCR clean-up kit (Microzone, Haywards Heath, UK), and the repeat region of interest was sequenced using ABI Prism BigDye Terminator Cycle Sequencing Ready Reaction kit version 1.1 (Applied Biosystems, Foster City, CA). Fifteen-microliter sequencing reactions comprised 5 to 20 ng of DNA template, 1 µL BigDye mix, 5 µL Better Buffer (Microzone), 5 pmol of either the forward or reverse sequencing primer (same as PCR primers), 1 µL betaine, and were made up with an appropriate volume of nuclease free water. Cycling parameters for sequencing were set to 300 seconds at 95°C, 30-second denaturing at 96°C, 15-second annealing at 55°C, 240-second extension at 60°C, and repeated for 35 cycles. Sequences were precipitated in ethanol, ethylenediaminetetraacetic acid, and sodium acetate solution and further washed in 70% ethanol. Sequences were determined on a 3730xl DNA Analyzer capillary sequencer (Applied Biosciences) and analyzed with Geneious sequencing software (Biomatters, Auckland, NZ).

Fluorescent PCR fragment sizing was analyzed using Peak Scanner v1.0. The areas of the major peak and of all peaks corresponding to larger fragment sizes were calculated. Only peaks with an area >2% of major peaks were considered. We obtained a mosaicism index (MI) by dividing the area corresponding to all minor peaks by the major peak area. Cloning of the CAG repeat was performed as previously described.¹⁷

Linear regression analysis was performed using GraphPad Prism v5.03 (GraphPad Software, La Jolla, CA).

Since 2005, 61 patients with SBMA have consented to take part in the UK National Register for SBMA. Of a total of 51 living patients, 46 participated in the current study, of whom 44 were of European descent and 2 were of Indian descent. Five patients declined to undergo interview because of lack of interest. The available genetic and clinical data from 10 deceased patients were also included where relevant. The average age at the time of study was 57.6 years, and the average time from disease onset was 16.6 years. A positive family history was reported in 71.7% of participants, and 10.8% received a genetic diagnosis before symptom onset. The majority of patients (87%) were followed regularly by a neurologist, with yearly visits in most cases (table).

The average age of disease onset was 43.4 years (range 14–75 years). Lower limb weakness was the most common presenting symptom (86.7%), followed by upper limb weakness (22.2%), tremors (13.3%), and bulbar weakness (6.7%) (table). Onset of weakness was asymmetrical in 54.5% of cases, and in 70.8% of asymmetrical onset cases, weakness occurred on the dominant side first. Many patients (28.3%) underwent a muscle biopsy and 30% of patients received an alternative diagnosis in the first instance. The most frequent alternative diagnosis was spinal muscular atrophy (42.9%), followed by muscular dystrophy, Parkinson disease, myasthenia gravis, and amyotrophic lateral sclerosis (ALS) (table).

To gain information on the clinical course of the disease, we investigated the onset of decline in activities of daily living (ADL). The earliest deficit noticed by patients was hand tremor at a median age of 35 years, followed by muscle weakness at a median age of 43. The sequential use of walking aids (handrail, cane, frame, and wheelchair), bulbar-related ADL milestones (dysarthria and dysphagia), and erectile dysfunction were reported between the ages of 50 and 60 years (figure 1A). Only one patient had undergone gastrostomy. Death occurred in 10 patients at a median age of 79. To investigate whether the SBMA course overlaps in cohorts with different genetic backgrounds, we plotted the median age of ADL milestone data from our study together with data from a large cohort (n = 223) described in Japan³ (figure 1B), and the data showed remarkable similarity.

Characteristic motor symptoms such as muscle cramps and muscle twitching were respectively described in 76.1% (n = 35) and 84.7% (n = 39) of patients, at a median age of 39.4 and 41.8 years. Onset of sensory symptoms, described as numbness or tingling, was noticed by 58.7% (n = 27) of patients, at a median age of 50 years. Breast enlargement was described by 73.9% of patients at a median age of 39.1 years (figure e-1 on the Neurology® Web site at Neurology.org).

Participants were evaluated at the time of study using the revised ALS Functional Rating Scale (ALSFRS-R), devised to monitor progression of motor neuron disease, which considers bulbar, limb, and respiratory weakness.¹⁸ For each item, scores range from 0 to 4, with 4 being unaffected. The average score was 38.2 (range 18–48), with 48 being the maximum possible score. The most severely affected activities related to mobility, walking and climbing stairs, and these items had an average score of 2.6 and 1.8, respectively. Bulbar and upper limb linked activities followed; the least affected items were those related to the respiratory function of patients (figure 2A). We have plotted our results in parallel with results from a cross-sectional study and show a similarity between the cohorts¹⁹ (figure e-2).

We also assessed the major subjective disease-related disabilities by asking participants to state their 3 major disabling issues. Impairment of mobility was the most frequent complaint (31%), followed by muscle weakness (22%), swallowing problems (14%), speech problems (7%), hand tremors (7%), breathing problems (5%), fatigue (3%), cramps (3%), and erectile dysfunction (3%) (figure 2B).

The comorbidity in our cohort consisted of 18% with diabetes and 28% had had infectious pneumonia.

All participants underwent genetic testing in different regional UK clinical genetics departments. Given the variability that can occur in performing fragment analysis,²⁰ we used data on CAG expansion sizing only when performed in our institute (n = 37) or in laboratories where the fragment analysis PCR was normalized by using a reference sample (n = 2) in order to obtain homogeneous data. Mean repeat size from 39 individuals was 46.03, ranging between 40 and 53.

We plotted the CAG repeat size in relation to age at disease onset and age at onset for the major ADL milestones and clinical features and performed regression analysis. CAG repeat size significantly inversely correlated with age at disease onset, use of a handrail, use of a cane, use of a wheelchair, and muscle weakness (p = 0.0035) (figure 3). Other clinical features, including hand tremor, muscle twitching, muscle cramps, sensory symptoms, dysarthria, dysphagia, breast enlargement, and erectile dysfunction showed trends for an inverse correlation, but did not reach statistical significance (figure e-3).

To assess whether CAG expansion size influences disease progression, we correlated CAG repeat number with time intervals occurring between ADL milestones and found no significant association (figure e-4).

Somatic instability of the repeat expansion is a feature common to the majority of microsatellite expansion disorders and has been shown to occur in numerous tissues in SBMA.²¹ Therefore, we investigated whether the degree of somatic mosaicism measured in blood correlated with disease clinical features.

When assessing the repeat size, using both fragment analysis and sequencing, we observed a major peak and 2 to 3 smaller peaks corresponding to both larger and smaller repeat expansions, as previously described (figure e-5B).²¹ All patients presented peaks on both sides of the major peak, while all tested samples with normal repeat range showed only peaks corresponding to smaller sizes (figure e-5A), which have previously been suggested to be PCR artifacts.²¹ To confirm the origin of the observed peaks, we PCR amplified the CAG expanded repeats and cloned and then sequenced the products, which revealed a single peak suggesting that sequencing produces no artifacts (figure 4A). We then reamplified the cloned product by PCR and observed the appearance only of peaks corresponding to smaller size repeats, thus confirming (1) that PCR amplification indeed contributes to generating smaller repeats by slippage, and (2) that the larger size peaks represent genuine somatic instability (figure 4A). Therefore, we considered only the peaks (1) corresponding to amplicons larger than the major peak, and (2) that had an area of >2% of the major peak. The number of peaks determined by somatic mosaicism in blood is fewer than that observed in other tissues, making the count of these not very informative. To grade the mosaicism in this setting, we calculated an MI by summing the area of all extra peaks and dividing by the area of the major peak (figure 4B). CAG repeat number and MI showed a positive correlation (p = 0.0014) (figure 4C), confirming results obtained in skin and muscle tissues.²¹ We then assessed whether the MI correlated to clinical features of the disease. The MI significantly correlated inversely with age at onset of disease, but correlation failed to reach significance for other clinical features and ADL milestones (figures 4D and e-6).