Biomarker for Spinal Muscular Atrophy: Expression of SMN in Peripheral Blood of SMA Patients and Healthy Controls

<div><p>Spinal muscular atrophy is caused by a functional deletion of SMN1 on Chromosome 5, which leads to a progressive loss of motor function in affected patients. SMA patients have at least one copy of a similar gene, SMN2, which produces functional SMN protein, although in reduced quantities. The severity of SMA is variable, partially due to differences in SMN2 copy numbers. Here, we report the results of a biomarker study characterizing SMA patients of varying disease severity. SMN copy number, mRNA and Protein levels in whole blood of patients were measured and compared against a cohort of healthy controls. The results show differential regulation of expression of SMN2 in peripheral blood between patients and healthy subjects.</p></div

Demographics of Enrolled SMA Patients.

<p>*<i>SMN2</i> copy number as reported by the patient and/or measured during the study. Copy number could not be determined for every patient.</p><p>Demographics of Enrolled SMA Patients.</p

Expression of SMN2 mRNA in SMA patients and healthy controls.

<p>SMN2 mRNA was isolated from blood and analyzed using qRT-PCR. Expression level is calculated using 2ˆ-deltaCp of the reference gene. There is a strong overlap of SMN2 mRNA in the different patient groups. Note that in the healthy controls SMN2 levels are lower than levels in patients with the same SMN2 copy number.</p

Correlation of protein and mRNA in SMA patients depend on Type.

<p>Significant correlation between expression levels of SMN protein and SMN2 mRNA in Type 1 and Type 2 patients but not in Type 3. Statistical analysis was done using Matlab 7.12. Protein levels are in ng/ml (x-axis) and mRNA (y-axis) Expression levels are calculated using 2ˆ-deltaCp of the reference gene.</p

SMN protein stability in whole blood: short term, long term, and freeze / thaw events.

<p>Whole blood of healthy subjects was used in the study. (A) SMN protein was measured in previously frozen, undiluted whole blood samples incubated at 4°C or at room temperature. (B) SMN protein was measured in undiluted whole blood samples of two subjects stored at -80°C or at -20°C. (C) SMN protein levels were measured in samples of two subjects that went through freeze-thaw cycles. *FDA acceptance criteria (below 85%).</p

SMN only detected in cerebral spinal fluid samples containing hemoglobin.

<p>The CSF samples obtained from healthy volunteers were concentrated prior to analysis, and the sensitivity of the SMN-ECL immunoassay was 0.3 pg/mL. Hemoglobin was measured using a hemoglobin immunoassay (Bethyl Laboratories E88-135). Approximately 3 pg of SMN correspond to 10,000 ng of hemoglobin in 1 mL of whole blood from a healthy adult. LLQ: lower limit of quantification.</p

Correlation of SMN protein levels between tissues.

<p>SMN protein levels were correlated in WT and C/C-allele SMA mice between (A) whole blood and brain and (B) whole blood and spinal cord.</p

SMN protein was found in various cell types in whole blood.

<p>Platelets, red blood cells, PBMCs and reticulocytes were separated by a fractionation procedure.</p

SMN protein levels in WT and C/C-allele SMA mice of different ages.

<p>SMN protein levels were measured by SMN-ECL at various days post-birth in (A) brain, (B) whole blood, (C) spinal cord, and (D) gastrocnemius muscle.</p

SMN protein levels in tissues of C/C-allele and WT mice measured by SMN-ECL and SMN-ELISA.

<p>Protein levels were measured in the spinal cord of C/C-allele and WT mice using (A) SMN-ECL and (B) SMN-ELISA. Both assays showed a statistically significant difference in SMN levels between WT and C/C-allele mice (p < 0.0001). (C) SMN protein levels in the whole blood of C/C-allele, WT and heterozygous mice measured by SMN-ECL.</p