Discovery and verification of amyotrophic lateral sclerosis biomarkers by proteomics

Abstract

Recent studies using mass spectrometry have discovered candidate biomarkers for amyotrophic lateral sclerosis (ALS). However, those studies utilized small numbers of ALS and control subjects. Additional studies using larger subject cohorts are required to verify these candidate biomarkers. Cerebrospinal fluid (CSF) samples from 100 patients with ALS, 100 disease control, and 41 healthy control subjects were examined by mass spectrometry. Sixty-one mass spectral peaks exhibited altered levels between ALS and controls. Mass peaks for cystatin C and transthyretin were reduced in ALS, whereas mass peaks for posttranslational modified transthyretin and C-reactive protein (CRP) were increased. CRP levels were 5.84 +/- 1.01 ng/ml for controls and 11.24 +/- 1.52 ng/ml for ALS subjects, as determined by enzyme-linked immunoassay. This study verified prior mass spectrometry results for cystatin C and transthyretin in ALS. CRP levels were increased in the CSF of ALS patients, and cystatin C level correlated with survival in patients with limb-onset disease. Our biomarker panel predicted ALS with an overall accuracy of 82%.

Figure 1

(A) SELDI-TOF-MS relative intensity values of the 23.03 kDa peak for the 100 ALS and 41 HC CSF samples. (B) C-reactive protein absolute concentration values (ng/mL) measured by ELISA using 41 ALS and 20 age-matched HC samples. A line in each subject group denotes the mean value.

Figure 2

SELDI-TOF-MS analysis for CRP, cystatin C and CysGly-transthyretin levels in ALS, AD, HC, MS and DC subject groups. (A) Box plot of CRP levels (Kruskal-Wallis test across all groups, p = 0.002). The Mann-Whitney U test for pair-wise comparisons identified significant CRP alterations between ALS and HC (**, p = 0.002), ALS and AD (*, p = 0.012), and DC and HC (*, p = 0.014). (B) Box plot of cystatin C (Kruskal-Wallis test across all groups, p = 0.001). Cystatin C was significantly reduced in the ALS group when compared with the HC group (*, p = 0.002) or to the DC group (*, p = 0.01). Cystatin C levels in the AD group were significantly decreased when compared to the HC (p = 0.004) or DC (p = 0.01) groups. (C) Box plot of CysGly-transthyretin (Kruskal-Wallis test across all groups, p = 0.001). CysGly-transthyretin was significantly increased in ALS when compared to HC (*, p = 0.002) or MS (*, p = 0.049) groups. CysGly-transthyretin level in AD was increased over HC (p = 0.001). For all panels, the box represents the 25^th to 75^th quartile, the horizontal bar the median and the whiskers the range.

Figure 3

Cystatin C (13,377 Da) mass peak intensity correlated to ALS disease duration. Pearson correlation coefficient is r = -0.209 (p = 0.06).

Figure 4

Correlation of the cystatin C mass peak with survival, defined as the time from lumbar tap to patient death. Pearson correlation coefficient is r = 0.486 (p = 0.001).