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S. Tewari et al.
Data analysis
Statistical significance was assessed using appropriate tests to compare means with adjustments for multiple comparisons when appropriate. Statistical analyses were performed in the proteomics laboratory and medical statistics department of Public Health Sciences at King’s College London.
Genetic association of protein biomarkers with SCIs
The genes coding for plasma proteins which differed signifi- cantly between children with and without SCIs were identified using standard databases. Genetic association analysis was then undertaken in a separate adult cohort at KCH, evaluating the vari- ants within each gene and their association with SCI 22 (details in Online Supplementary Materials).
Results
A total of 55 patients consented to take part in the study, although no blood samples were taken from one, and three further patients did not have an MRI scan per- formed. Plasma from 54 patients underwent proteomic analysis, although only data from the 51 patients (22 female) with MRI results were analysed. In total, 19 (37%) had SCIs and 32 (63%) normal brain MRIs, deviating slightly from the aim of equal numbers in each group because of recruitment of children without previous MRIs, the majority of whom had normal brain imaging (Figure 1). A total of 15 children were identified as having SCIs from scans performed as part of this study, and four children were recruited as a result of previous MRI scans showing SCIs; in these four children, there was a median delay of 20 months (range 9 – 40 months) between MRI scan and blood sampling for proteomic analysis. One child
had conditional velocities and 50 normal velocities on TCD scanning.
Basic clinical and laboratory data
Relatively more males (13/29, 44%) than females (6/22, 27%) were in the SCI group, although this was not signif- icantly different (Chi square, P=0.199). There was no sig- nificant difference in HU use between the SCI and control groups: controls 11/32 (36%) on HU, SCI 7/19 (37%) on HU (Chi square, P=0.86). G6PD assays were available for 45 patients, and there was no association with SCI: G6PD deficiency was present in 3/29 (10%) controls and 2/16 (12%) with SCIs (Chi square, P=0.83). Basic laboratory and clinical characteristics are summarized in Table 1. Mean HbF levels were significantly lower (6.1% versus 8.6%, P=0.049), and mean systolic blood pressures signif- icantly higher (115 versus 108.6, P=0.027) in those with SCIs compared to controls.
Plasma proteomic analysis
A total of 4662 different peptides were identified, although only 1312 were present in all 51 patient samples, corresponding to 346 different proteins. Forty-four pep- tides from 13 different proteins were present at more than 1.3 times the concentration in the SCI patients compared to controls (Table 2), whereas 41 peptides from 4 proteins were more than 1.3 higher in the control population (Table 3). Proteins differentially expressed in the SCI group included those implicated in coagulation, lipid and inflammatory pathways.
Genetic association of identified biomarkers with SCI
We explored the possibility that differences in concen- trations of proteins (Tables 2 and 3) between SCI and con-
Figure 1: Diagram showing the flow of patients through the study.
haematologica | 2018; 103(7)