Spinal Muscular Atrophy after Nusinersen Therapy: Improved Physiology in Pediatric Patients with No Significant Change in Urine, Serum, and Liquor 1H-NMR Metabolomes in Comparison to an Age-Matched, Healthy Cohort

Abstract

Spinal muscular atrophy (SMA) is a genetically heterogeneous group of rare neuromuscular diseases and was until recently the most common genetic cause of death in children. The effects of 2-month nusinersen therapy on urine, serum, and liquor 1H-NMR metabolomes in SMA males and females were not explored yet, especially not in comparison to the urine 1H-NMR metabolomes of matching male and female cohorts. In this prospective, single-centered study, urine, serum, and liquor samples were collected from 25 male and female pediatric patients with SMA before and after 2 months of nusinersen therapy and urine samples from a matching healthy cohort (n = 125). Nusinersen intrathecal application was the first therapy for the treatment of SMA by the Food and Drug Administration (FDA) and the European Medicines Agency (EMA). Metabolomes were analyzed using targeted metabolomics utilizing 600 MHz 1H-NMR, parametric and nonparametric multivariate statistical analyses, machine learning, and modeling. Medical assessment before and after nusinersen therapy showed significant improvements of movement, posture, and strength according to various medical tests. No significant differences were found in metabolomes before and after nusinersen therapy in urine, serum, and liquor samples using an ensemble of statistical and machine learning approaches. In comparison to a healthy cohort, 1H-NMR metabolomes of SMA patients contained a reduced number and concentration of urine metabolites and differed significantly between males and females as well. Significantly larger data scatter was observed for SMA patients in comparison to matched healthy controls. Machine learning confirmed urinary creatinine as the most significant, distinguishing SMA patients from the healthy cohort. The positive effects of nusinersen therapy clearly preceded or took place devoid of significant rearrangements in the 1H-NMR metabolomic makeup of serum, urine, and liquor. Urine creatinine was successful at distinguishing SMA patients from the matched healthy cohort, which is a simple systemic novelty linking creatinine and SMA to the physiology of inactivity and diabetes, and it facilitates the monitoring of SMA disease in pediatric patients through non-invasive urine collection.

Keywords: 1H-NMR metabolomics; females; healthy control cohort; liquor; males; nusinersen; serum; spinal musular atrophy; urine.

Figure 1

A Partial least square discriminant analysis (PLSDA) of 1H-NMR metabolomes of SMA patients based on (A) urine, (B) serum, and (C) liquor before (red) and after (green) the 4th application of nusinersen. Ellipses designate 95% confidence intervals for each group. The differences are not significant (ensemble statistical approach (npMANOVA, MetaboAnalyst, JADBio)).

Figure 2

MetaboAnalyst heatmaps of urine 1H-NMR metabolomes representing existing differences between SMA cohort (before and after) and healthy cohort, for males (A) and females (B). The differences on metabolic level between the before-treatment (green) and after 4th application (red) groups were not significant due to the large variability in SMA samples. The most differentiating metabolites were selected by the PLSDA variable importance in projection (VIP) score, where decreasing metabolites were presented with negative values (blue color) and increasing metabolites were presented with positive values (red color). Individual data are presented in the Supplementary Material (Figures S7 and S8).

Figure 3

The significant differences in urine 1H-NMR metabolomic profiles between females and males in matched healthy cohorts. (A) PLSDA analysis and (B) heatmap analysis representing existing differences in the first 30 most important metabolites. (F—females (red), M—males (green)). The most differentiating metabolites were selected by the PLSDA VIP score, where decreasing metabolites had negative values (blue color) and increasing metabolites had positive values (red color).

Figure 4

Results of partial least square discriminant analysis (PLSDA) (A) and (B) Receiver Operating Characteristics curve of modeling of the data. A PLSDA-based ordination of 1H-NMR urine metabolomes: healthy (blue), before (red), and after (green) 4th application of nusinersen. Ellipses designate 95% confidence intervals for each group (A). (B) Receiver Operating Characteristic (ROC) curve for SMA patients obtained with model. The Just Add Data Bio (JADBIO) model is available as part of the electronic Supplementary Material for the classification of novel 1H-NMR metabolomic data.

Figure 5

Box-plots representing log10 transformed creatinine concentrations in all three groups (before treatment, after 4th application, healthy) in females and males separately. Original concentrations are presented in Figure S9.