This study included 28 SMA patients with 185 visits and 28 gender- and age-matched healthy controls. Two SMA patients, aged 15 and 16, are adolescents, and the remaining 26 are adults aged 18 to 50. Of the total 28 patients, 13 cannot walk, with five being unable to sit independently and eight being unable to sit alone. Fifteen patients are capable of walking independently. Eight patients are classified as type 2, 17 as type 3, and three as type 4. Detailed characteristics of patients and controls are shown in Table 1.

Compared to healthy controls, SMA patients had significantly lower Crn values ​​(67.4 ± 14 vs. 23.7 ± 14.8 umol/L, p<0.0001) (Table 1). Based on the copy number of SMN2 gene, SMA patients were categorized into two groups: those with less than four copies and those with four or more copies of the SMN2 gene. Comparison of Crn values between ​​the two groups revealed that patients with SMN2 gene copies <4 had significantly lower Crn levels (Median: 12.5 umol/L, Q1-Q3: 8.75-23 umol/L, n = 18) compared to those with SMN2 gene copies ≥ 4 (Median: 36 umol/L, Q1-Q3: 27-47.5 umol/L, n = 10) (p = 0.0003) (Fig. 1A). Moreover, the Crn values ​​of both groups of SMA patients were significantly lower than those of gender- and age-matched healthy controls. Then, patients are categorized into two groups based on their motor function status: ambulant ones who can walk independently and non-ambulant ones who cannot walk independently (including individuals who can sit alone and those who cannot). Comparing the Crn values ​​between the two groups revealed that the Crn values ​​of non-ambulant patients (Median: 10 umol/L, Q1-Q3: 7.5–14 umol/L, n = 13) were significantly lower than those of ambulant ones (Median: 32 umol/L, Q1-Q3: 24–43 umol/L, n = 15) (p<0.0001) (Fig. 1B). Additionally, both groups of SMA patients exhibited significantly lower Crn values than healthy controls matched for gender and age.

Comparison of Crn values ​​between SMA patients and healthy controls. (A) The Crn values ​​of patients with SMN2 copies <4 were significantly lower than those with SMN2 copies ≥ 4. (B) The Crn values ​​of non-ambulant patients were significantly lower than those of ambulant ones. **: p<0.01;***: p<0.001; ***: p<0.0001; HC: Healthy controls

We employed partial correlation analysis to examine the relationship between Crn and disease severity indicators, including HFMSE, MRC, 6MWT, and FVC. We found that Crn has a positive correlation with HFMSE (p<0.0001, r = 0.884) and MRC scores (p<0.0001, r = 0.827), even after adjusting for age, sex, and BMI (Fig. 2A and B). This suggests that as Crn levels increase, there is an improvement in motor function scores and muscle strength among patients. Although the correlation between Crn and 6MWT (p = 0.069) was not statistically significant, we observed a trend of increasing Crn levels with higher 6MWT values. Furthermore, our analysis demonstrated a positive relationship between Crn concentration and FVC (p = 0.002, r = 0.730) after controlling for age, sex, and BMI (Fig. 2C). These results confirm the close connection between Crn levels and respiratory function. Finally, we analyzed the association between Crn and denervation by examining its relationship with ulner CMAP., We found a positive correlation between Crn and ulnar nerve CMAP (p<0.0001, r = 0.807) after adjusting for age, sex, and BMI (Fig. 2D). This indicates that increased Crn corresponds to an increase in the patient’s ulnar nerve CMAP amplitude.

Partial correlation of Crn levels to disease severity among adolescents and adults SMA corrected for age, sex, and BMI. Crn has a positive correlation with HFMSE (A), MRC (B), FVC (C), and CMAP (D) after correction for age, sex, and BMI. HFMSE, Hammersmith Functional Motor Scale Expanded; MRC, Medical Research Council Scale; FVC, forced vital capacity; CMAP, Compound Muscle Action Potential

Next, we used longitudinally measured Crn values​​ over a follow-up period of up to 2 years to explore the potential association between changing trends in Crn values and disease progression. Initially, we constructed a linear mixed-effects model by comparing the Crn values among patients with SMN2 copy number <4 and those with SMN2 copy number ≥ 4. After adjusting for sex, age, and BMI, our analysis revealed that patients with SMN2 copy number ≥ 4 exhibited significantly higher Crn levels (34.1 ± 3.75 umol/L) compared to patients with SMN2 copy number < 4 (17.2 ± 2.52 umol/L; p = 0.00145) (Fig. 3A and B). We further conducted a similar comparison between ambulant and non-ambulant patients. Following adjustment for sex, age, and BMI, we found that ambulant patients had more than twice the Crn level (32 ± 2.33 umol/L; p<0.0001) as non-ambulant patients (12.9 ± 2.38 umol/L) (Fig. 3C and D).

Comparison of creatinine using linear mixed model. Creatinine in patients with SMN2 copy numbers <4 (A) is significantly lower than that in patients with SMN2 copy numbers ≥ 4 (B). Creatinine in non-ambulant patients (C) is substantially lower than in ambulant patients (D). Each line represents data from different patients at various follow-up points

To elucidate the impact of nusinersen treatment on serum Crn levels and motor function in patients, we conducted a comprehensive analysis of Crn values at 6 months, 14 months, and 22 months post-treatment (Supplementary Figure S1), as well as changes in the HFMSE scores (Supplementary Figure S2). Our findings revealed that after 6 months of nusinersen treatment, stable or elevated Crn levels were observed in 50% (11/22) of patients; after 14 months, this percentage increased to 68.8% (11/16); and after 22 months, it further rose to 83.3% (5/6) (Supplementary Figure S1). Similarly, the HFMSE scores remained stable or increased in 93.3% (14/15), 100% (6/6), and 85.7% (6/7) of patients at 6 months, 14 months, and 22 months, respectively (Supplementary Figure S2). These results suggest that nusinersen can preserve or enhance motor function in adolescent and adult patients with SMA. Moreover, they reinforce the notion that serum Crn can serve as a reliable biomarker for disease severity in SMA patients.

We investigated the predictive capacity of Crn, conventional biomarkers (FVC, CMAP, and SMN2 copy), and BMI on the current motor function assessed by HFMSE, MRC, and 6MWT. When considered individually, Crn exhibited the highest explanatory variance as a predictor, reaching up to 83.5%, significantly superior to other biomarkers (FVC, CMAP, and SMN2 copy) (Fig. 4). Crn and SMN2 copies demonstrated predictive potential for all motor functions, including HFMSE, MRC, and 6MWT. However, FVC and CMAP were inadequate in predicting 6 MW in ambulant SMA patients. By combining Crn with FVC, SMN2 copy, and BMI, the explained variance of HFMSE could be increased to around 93% (Fig. 4A). In terms of predicting 6MWT, the combination of Crn and BMI enhanced the explained variance to around 64%, the combination of Crn and SMN2 copy to around 74%, and the combination of SMN2 copy and BMI to around 80% (Fig. 4C). To investigate the comparative efficacy of Crn, HFMSE, MRC, and 6MWT in evaluating motor function, we employed linear models to assess the correlation between Crn levels and these motor function measures. Our findings indicate that Crn exhibits similar or superior explained variance compared to the scales (Supplementary Figure S3).

Prediction of motor function scores using Crn, FVC, CMAP, SMN2 copy, and BMI. The explained variance of motor function scores, as assessed by HFMSE (A), MRC (B), and 6MWT (C), using each predictor separately, was highest using Crn. The x-axis represents the explained variance, with values closer to 1 indicating a higher degree of variance explained. Numbers behind the bars indicate the quantity of available data points for each prediction