Evaluation of SMN protein, transcript, and copy number in the biomarkers for spinal muscular atrophy (BforSMA) clinical study

Abstract

Background: The universal presence of a gene (SMN2) nearly identical to the mutated SMN1 gene responsible for Spinal Muscular Atrophy (SMA) has proved an enticing incentive to therapeutics development. Early disappointments from putative SMN-enhancing agent clinical trials have increased interest in improving the assessment of SMN expression in blood as an early "biomarker" of treatment effect.

Methods: A cross-sectional, single visit, multi-center design assessed SMN transcript and protein in 108 SMA and 22 age and gender-matched healthy control subjects, while motor function was assessed by the Modified Hammersmith Functional Motor Scale (MHFMS). Enrollment selectively targeted a broad range of SMA subjects that would permit maximum power to distinguish the relative influence of SMN2 copy number, SMA type, present motor function, and age.

Results: SMN2 copy number and levels of full-length SMN2 transcripts correlated with SMA type, and like SMN protein levels, were lower in SMA subjects compared to controls. No measure of SMN expression correlated strongly with MHFMS. A key finding is that SMN2 copy number, levels of transcript and protein showed no correlation with each other.

Conclusion: This is a prospective study that uses the most advanced techniques of SMN transcript and protein measurement in a large selectively-recruited cohort of individuals with SMA. There is a relationship between measures of SMN expression in blood and SMA type, but not a strong correlation to motor function as measured by the MHFMS. Low SMN transcript and protein levels in the SMA subjects relative to controls suggest that these measures of SMN in accessible tissues may be amenable to an "early look" for target engagement in clinical trials of putative SMN-enhancing agents. Full length SMN transcript abundance may provide insight into the molecular mechanism of phenotypic variation as a function of SMN2 copy number.

Trial registry: Clinicaltrials.gov NCT00756821.

Figure 1. Modified Hammersmith Functional Motor Scale versus age by SMA cohort.

Scores for the MHFMS were well-distributed by age across the enrollment cohorts. It should be noted that not all control individuals achieved a score of 40 on the scale, while all Type I SMA subjects were assigned scores of zero in the assessment.

Figure 2. SMN2 copy numbers in SMA and Control subjects.

SMN2 copy number is lower in Controls than it is in SMA subjects, in whom copy number is related to type.

Figure 3. SMN2 copy number relationship to age, and by SMA Type or Control.

Subjects with each SMA type are broadly distributed across the age range, with the exception of type I SMA for whom there is some bias to younger age. As a consequence, SMN2 copy numbers are also broadly distributed. Values have been plotted with a small y-axis offset to avoid overlap of values.

Figure 4. SMN transcript concentrations in SMA and Control subjects.

A,C: SMN2-FL, SMN-FL and Total SMN transcripts generally increase with SMA Type. SMN-FL (A) is a sum of SMN1-FL (present only in healthy controls) and SMN2-FL. B: SMN-Δ7 expression levels are lower in Type I patients compared to other SMA Types but they are similar to that of Controls. D: Ratios of SMN2-FL to SMN- Δ7 differ between SMA Types and Controls, however differences between SMA Types are absent with the exception of Type II versus Type III patients. E: GAPDH transcript levels are elevated in SMA Type I and Controls relative to Type II and III patients.

Figure 5. SMN protein levels in SMA and Control Subjects.

While SMN protein levels are lower in SMA relative to Control subjects, protein levels by SMA type are not statistically different from each other.