Evaluation of SMN Protein, Transcript, and Copy Number in the Biomarkers for Spinal Muscular Atrophy (BforSMA) Clinical Study

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

Candidate Proteins, Metabolites and Transcripts in the Biomarkers for Spinal Muscular Atrophy (BforSMA) Clinical Study

Spinal Muscular Atrophy (SMA) is a neurodegenerative motor neuron disorder resulting from a homozygous mutation of the survival of motor neuron 1 (SMN1) gene. The gene product, SMN protein, functions in RNA biosynthesis in all tissues. In humans, a nearly identical gene, SMN2, rescues an otherwise lethal phenotype by producing a small amount of full-length SMN protein. SMN2 copy number inversely correlates with disease severity. Identifying other novel biomarkers could inform clinical trial design and identify novel therapeutic targets.To identify novel candidate biomarkers associated with disease severity in SMA using unbiased proteomic, metabolomic and transcriptomic approaches.A cross-sectional single evaluation was performed in 108 children with genetically confirmed SMA, aged 2-12 years, manifesting a broad range of disease severity and selected to distinguish factors associated with SMA type and present functional ability independent of age. Blood and urine specimens from these and 22 age-matched healthy controls were interrogated using proteomic, metabolomic and transcriptomic discovery platforms. Analyte associations were evaluated against a primary measure of disease severity, the Modified Hammersmith Functional Motor Scale (MHFMS) and to a number of secondary clinical measures.A total of 200 candidate biomarkers correlate with MHFMS scores: 97 plasma proteins, 59 plasma metabolites (9 amino acids, 10 free fatty acids, 12 lipids and 28 GC/MS metabolites) and 44 urine metabolites. No transcripts correlated with MHFMS.In this cross-sectional study, "BforSMA" (Biomarkers for SMA), candidate protein and metabolite markers were identified. No transcript biomarker candidates were identified. Additional mining of this rich dataset may yield important insights into relevant SMA-related pathophysiology and biological network associations. Additional prospective studies are needed to confirm these findings, demonstrate sensitivity to change with disease progression, and assess potential impact on clinical trial design.Clinicaltrials.gov NCT00756821

The Gene Ontology resource: enriching a GOld mine

The Gene Ontology Consortium (GOC) provides the most comprehensive resource currently available for computable knowledge regarding the functions of genes and gene products. Here, we report the advances of the consortium over the past two years. The new GO-CAM annotation framework was notably improved, and we formalized the model with a computational schema to check and validate the rapidly increasing repository of 2838 GO-CAMs. In addition, we describe the impacts of several collaborations to refine GO and report a 10% increase in the number of GO annotations, a 25% increase in annotated gene products, and over 9,400 new scientific articles annotated. As the project matures, we continue our efforts to review older annotations in light of newer findings, and, to maintain consistency with other ontologies. As a result, 20 000 annotations derived from experimental data were reviewed, corresponding to 2.5% of experimental GO annotations. The website (http://geneontology.org) was redesigned for quick access to documentation, downloads and tools. To maintain an accurate resource and support traceability and reproducibility, we have made available a historical archive covering the past 15 years of GO data with a consistent format and file structure for both the ontology and annotations

Top 20 univariate MHFMS markers across platforms.

<p>Table is ordered by lowest to highest Q-values. Only plasma markers are represented, as these were the markers with the lowest Q-values. Unknown metabolites were removed from this summary table but the full plasma and urine metabolite and proteomic datasets are available in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0035462#pone.0035462.s003" target="_blank">Tables S3</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0035462#pone.0035462.s004" target="_blank">S4</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0035462#pone.0035462.s005" target="_blank">S5</a>. AAA – Amino acid analysis; FFA – Free fatty acid; Q-VALUE – significance corrected for the effect of multiple comparisons; STD – Standard deviation; UCL – Upper 95% confidence limit; LCL – lower 95% confidence limit (on the value of slope). Slope values are given as positive and negative (-) values. *These are distinct isoforms detected by the LC/MS method.</p

Modified Hammersmith Functional Motor Scale versus age by SMA cohort.

<p>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.</p

SMN protein levels in SMA and Control Subjects.

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

SMN transcript concentrations in SMA and Control subjects.

<p>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.</p

Correlations of secondary measures with MHFMS and age for SMA subjects.

<p>Of the secondary outcome measures, only forced vital capacity (FVC) significantly correlated with the primary outcome measure – the Modified Hammersmith Motor Function Scale. While BMI is thought to have a strong effect on function in individuals affected by SMA, the correlation did not reach significance here. Not surprisingly, FVC and walking were negatively correlated with age.</p