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

Clinical data of SMA and Control Subjects.

*<p>ANOVA for continuous variables; Fisher exact test for categorical variables.</p><p>There were no significant differences in age or gender across the recruitment cohorts. A key goal of this study was to minimize the confounding correlation between present age and functional status. This goal was largely achieved, both overall and within SMA groups Type II and III, and to a partial extent, SMA Type I through a competitive recruitment plan managed through the data coordinating center at the New England Research Institutes (NERI). The Modified Hammersmith Motor Function Scale differentiated between SMA subjects and controls and between Type I, II and III subjects, as did respiratory support, reflecting current level of function. FVC and the nutritional assessment score significantly distinguished between SMA type; however, BMI proved to be far less discriminatory.</p

Top 20 univariate markers across all outcome measures.

<p>Each numeric entry indicates the number of statistical tests in which the described analyte was found to be a statistically significant biomarker when evaluated for regression against the given outcome measures or categorical characteristics: SMA Types, Disease Onset, Current Level of Function and Respiratory Support. Each outcome measure or characteristic inherently has a certain number of possible sub-categories available for pairwise statistical testing (in parentheses for each outcome or characteristic): 12 for SMA Types, 5 for Disease Onset, 15 for current level of function and 4 for Respiratory Support (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0035462#pone.0035462.s001" target="_blank">Table S1</a> for details). The Total denotes the sum of all outcomes for which the protein is a marker and indicates the overall strength of the relationship between the analyte and SMA values; the maximum possible number for Total is 36, as all the analytes above are also statistical significant regressors with the MHMS outcome. 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 listed in red are positive values and those in green negative values.*These are distinct isoforms detected by the LC/MS method.</p

Illustration of the top 5 markers as candidate biomarkers by Type.

<p>The natural log intensity of the protein abundance of CILP2, TNXB, COMP, ADAMTSL4 and CLEC3B are shown (Panels A-E) by Type. Error bars are expressed as standard errors.</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

Illustration of the top 5 markers as candidate biomarkers by MFMHS.

<p>The natural log intensity of the protein abundance of CILP2, TNXB, COMP, ADAMTSL4 and CLEC3B are shown by MHFMS (Panels A-E) across Types. Panels A-E again show a trend for type and MHFMS. Panels F-J shows the box plot distribution by type.</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

SMA and Controls SMN Transcripts, Protein and Copy Number.

<p>Mean and median values of SMN transcripts, protein and copy number for SMA Type I, II, III and Controls. Transcript values are presented as number of molecules per ng of total RNA (mol/ng).</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

Associations between SMN2 copy number, SMN2 full-length transcript, SMN protein and MHFMS.

<p>The “SMN2 copy number” and “SMN-FL transcript” columns include the partial correlation analysis for SMA+Controls, SMA (all SMA patients), Type I, Type II, Type III, Controls. The “MHFMS” column includes the analysis for Type II, Type III and Type II+Type III with exclusion from the analysis of subjects having minimum or maximum scores.</p>*<p>Correlation was only found for the SMA group (r = 0.33, p = 0.001) and Type II group (r = 0.41, p = 0.008).</p