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Proteomics of silent cerebral infarction in SCA
nial Doppler (TCD) scanning and blood transfusion reduces this by 90%.10 The implementation of TCD-based stroke prevention has led to a significant fall in overt strokes in many countries.11,12 SCI is more common than overt stroke with MRI showing relevant lesions in 13% of two year olds, 25% of six year olds, and 30-40% of 14 year olds.13
Although by definition SCIs are not associated with overt symptoms, they cause significant morbidity, includ- ing a reduction in IQ, defects in executive function, epilep- sy, and increased risk of further SCIs and overt strokes.13 Early detection of SCI is useful to allow cognitive assess- ment, educational support and therapeutic intervention. The Silent Cerebral Infarct Multi-Center Clinical Trial (SIT Trial), a randomised controlled study, showed that regular blood transfusion reduced the incidence of recurrent infarction in children with SCIs compared to the observa- tional arm.14
The pathophysiology of SCI is unclear. It is often stated that SCIs are caused by small vessel disease, although there is no direct evidence for this. There are no post- mortem studies looking at the histological changes corre- sponding to the MRI appearances of SCI, although older studies identified small necrotic lesions in the subcortex of the brain, possibly representing SCIs.15 It is also possible that SCIs are areas of demyelination, or linked to venous sinus thrombosis.13 SCIs may also be caused by difficulties maintaining constant blood flow to the brain, leading to watershed infarction precipitated by acute anemia or hypoxia.16 The lack of pathophysiological understanding makes it difficult to develop new therapeutic strategies. Here, we used an unbiased proteomic approach to identi- fy plasma proteins associated with SCI and potentially linked to the underlying pathophysiology.
It is not easy to identify children with or at increased risk of SCI. Baseline data from the SIT Trial showed that steady state hemoglobin <7.6g/dl, steady state systolic blood pressure >104mmHg and male sex were all associ- ated with increased risk of SCI, although a model combin- ing all three significant factors had weak predictive pow- ers, with a C statistic of about 0.6.8 Abnormal transcranial Doppler velocities are not reliably associated with SCI, although there is a possible association with stenosis of the cervical internal carotid artery.17 Neuropsychometry can detect some children with SCIs, but is time consuming and not easily available. MRI is currently the only way of diagnosing SCI, although before 7 years of age this usually requires sedation or general anesthetic, which carries sig- nificant risks in children with SCD and is unsuitable for screening. MRI is also not widely available in many African countries where SCA is most prevalent. It would therefore be beneficial to develop a simple, screening test for SCI, potentially based on blood testing.
Proteomics can detect, identify and quantify very low concentrations of proteins. For example, a recent study suggested that urine proteomics might help in the diagno- sis of acute stroke by detecting brain peptides released by ischemia.18 The presence of SCI may suggest small blood vessel disease and a tendency to infarct brain tissue, which is likely to be a chronic process, releasing detectable brain proteins into plasma. Plasma protein profiles could also differ between those with and without SCI because of causative pathological factors, including coagulopathy, inflammation, hypoxia and vasculopathy; these differ- ences may be acquired or inherited, and reflect genetic
predisposition to silent cerebral infarction. We investigat- ed the possibility that there might be differences in the plasma proteome between children with and without SCIs, with a view to understanding more about the patho- physiology of SCIs and identifying potentially useful bio- markers. We further investigated the hypothesis that genetic factors account for the differences in concentra- tions of the various proteins, using data from a genome wide association study (GWAS) in a separate cohort of patients with SCA and SCIs.
Methods
Patients and setting
The National Research Ethics Committee approved the study (reference 13/LO/0709). Children were recruited from clinics at King’s College Hospital and the Evelina Children’s Hospital. Approximately 1000 different children are seen annually in these clinics with a further 500 seen in outreach clinics.19 The aim was to recruit a total of 50 patients, with equal numbers of those with SCIs and controls with normal brain MRIs. Children known to have SCIs from previous MRI scans were selectively recruited. Eligible patients without previous MRIs were recruited sequential- ly in clinic, on the basis that 20-30% of these children would have SCIs, and combining the two groups would result in approximate- ly 25 children in each group. SCI and control groups were not specifically matched for age and gender. Inclusion criteria were: sickle cell anemia (HbSS), age 8-18 years old, normal or condition- al TCD velocities (<200cm/s) (10), able to tolerate MRI scan with- out sedation. Exclusion criteria were: history of overt stroke, blood transfusion in last 4 months, serious co-existing disorder (renal failure, HIV, hepatitis, malignancy, autoimmune disease, chronic infection, cardiac disease, long-term medication), acute complications requiring hospital attendance in last 3 months. Patients on hydroxyurea (HU) were recruited to both arms. Routine clinical and laboratory data were collected.
Brain MRI and neurological assessment
Brain MRIs were performed to identify the presence of SCIs using standardised definitions20 (see Online Supplementary Materials). On the day of blood sampling, all children underwent standardised neurological examination by experienced clinicians to document the absence of neurological abnormalities suggestive of an overt stroke. All children had routine TCD scans performed at least annually, and these were used in this study.
Proteomic analysis
Blood samples were collected on all children at the time of con- sent. A data-driven proteomics approach was used to discover protein changes in the plasma associated with SCI. We used an established workflow combining isobaric Tandem Mass Tags (TMT) reagents with off-gel fractionation, followed by high sensi- tivity rapid throughput mass spectrometry (MS) and LC-MS/MS. (details in Online Supplementary Materials). Relative amounts of each peptide were compared between the SCI and control groups, and P values were calculated for those peptides showing greater than 1.3 fold differences.
Targeted plasma analysis for biomarkers of neurological disease
Plasma samples were also analysed for 92 protein biomarkers known to be implicated in neurological disease, using a multi- plexed proximity extension assay21 (see Online Supplementary Materials for full list of biomarkers).
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