1H-NMR-based metabolic profiling identifies non-invasive diagnostic and predictive urinary fingerprints in 5q spinal muscular atrophy

Figures

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  Fig. 1. Urinary 1H-NMR metabolic spectrum and possible confounders. A Representation of the complete urinary 1H-NMR metabolic spectrum of a healthy individual. Exemplary metabolites and close-ups of the aromatic region as well as the creatine and creatinine peaks are shown for illustrative purposes. TSP (trimethylsilylpropionic acid-d4 sodium salt) served as reference peak. The spectral regions highlighted in green were used for further analysis. B Plotting of different SMA subgroups vs. age-matched controls revealed age as a significant confounder emphasizing the importance of age-matched experimental setups. C No differences were observed concerning gender. D Quantification of the creatine-to-creatinine quotient showed significant differences between SMA subgroups and controls. To avoid variability due to creatinine metabolism, which was recently shown to be impaired in SMA [25], the spectral regions for creatine and creatinine were excluded prior to postprocessing in the following experiments. SMA: spinal muscular atrophy. CTRL: control

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  Fig. 2. Discrimination between SMA patients and healthy controls. PCA/CA classification and MCCV showed clear discrimination between the SMA group and an age-matched healthy control group. PCA/CA was performed on 1,000 variables from 0.5 to 10 ppm (exclusion: see Materials and Methods) with Expl. Variance of 99.9%. The Confusion Matrix is the result of 100 Monte-Carlo-Runs (MC) with 24-fold CrossValidation (CV). Space of discrimination is one representation of the modelling samples in 2-dimensions. □ represent the model-set and ○ represent the test-set. SMA: spinal muscular atrophy. CTRL: control

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  Fig. 3. Discrimination between SMA and DMD patients. PCA/CA classification and MCCV showed clear discrimination between the SMA group and the DMD group. PCA/CA was performed on 1,000 variables from 0.5 to 10 ppm (exclusion: see Materials and Methods) with Expl. Variance of 99.9%. The Confusion Matrix is the result of 100 Monte-Carlo-Runs (MC) with 15-fold CrossValidation (CV). Space of discrimination is one representation of the modelling samples in 2-dimensions. □ represent the model-set and ○ represent the test-set. SMA: spinal muscular atrophy. DMD: Duchenne muscular dystrophy

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  Fig. 4. Disease prediction. A PCA/CA classification and MCCV of the SMA 1, 2 and 3 group and an age-matched healthy control group showed clear discrimination between the SMA 1 and SMA 3 group with some overlap between the SMA 1 and 2 group as well as the SMA 3 group and controls. PCA/CA was performed on 1,000 variables from 0.5 to 10 ppm (exclusion: see Materials and Methods) with Expl. Variance of 99.9%. The Confusion Matrix is the result of 100 Monte-Carlo-Runs (MC) with sevenfold CrossValidation (CV). Space of discrimination is one representation of the modelling samples in 2-dimensions. B Expansion of 6% of overall 1H NMR spectra. The Kruskal–Wallis test of 1H NMR spectra of the different SMA types and controls revealed significant protein backgrounds in the aliphatic region as one of the major discriminants. Colored lines represent medians and colored areas correspond to variation (12.5–87.5% quantile) of 1H-NMR spectra. Spectral regions highlighted in black illustrate significant differences between the 4 groups (p-value < 0.01). SMA: spinal muscular atrophy. CTRL: control

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  Fig. 5. Early vs. late symptomatic SMA. A PCA/CA classification and MCCV of early symptomatic (SMA 1/2) vs. late symptomatic (SMA 3) group in relation to an age-matched control group of healthy individuals between 2 months and 19 years showed clear discrimination between early and late symptomatic SMA. B Similar results were returned after excluding children under 2 years rendering age bias as confounder unlikely. PCA/CA was performed on 1,000 variables from 0.5 to 10 ppm (exclusion: see Materials and Methods) with Expl. Variance of 99.5%. The Confusion Matrix is the result of 100 Monte-Carlo-Runs (MC) with sixfold CrossValidation (CV). Space of discrimination is one representation of the modelling samples in 2-dimensions. □ represent the model-set and ○ represent the test-set. SMA: spinal muscular atrophy. CTRL: control

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  Fig. 6. Prediction of disease severity in pre-symptomatic SMA. A Spectroscopic fingerprints of five pre-symptomatic SMA samples (determined as 1-1, 2-1, 3-1, 4-1, and 5-1) in comparison to SMA 1, SMA 2, SMA 3 and controls between 2 months and 19 years were consistent with the control group. B Comparison of pre-symptomatic SMA vs. early symptomatic SMA (SMA 1/2), late symptomatic SMA (SMA 3) and controls between 2 and 19 years returned similar results. C Age-matched comparison with SMA 1 and healthy controls between 2 and 18 months reveals a borderline fingerprint of patient SMA001 (1-1) showing some similarities to the SMA 1 profile. D Interestingly, follow-up urine samples from patient SMA001 after 6 months (1-2) and after 8 months (1-3), as well as a follow-up urine sample of patient SMA003 (3-1) after 4 months (3-2) confirm high similarities with the SMA 1 group. No changes were observed on a follow-up urinary sample of patient SMA002 (2-1) after 2.5 months (2-2). SMA: spinal muscular atrophy. CTRL: control