High-throughput assessment of FMR1 and SNRPN methylation-based newborn screening using IsoPure and QIAcube HT systems

Aim
This study compared methylation-specific quantitative melt analysis of FMR1 and SNRPN methylation (mDNA) using automated bisulfite conversion by the magnetic-bead-based IsoPure and column-based QIAcube HT systems.
Methods
Two bisulfite conversion methods were assessed on 3.2 mm punches from the same archival blood spots stored at room temperature for >10 years of individuals with FMR1 premutation (n = 20), fragile X syndrome (FXS, n = 20), or chromosome 15 imprinting disorders (n = 50) and freshly made blood spots from 184 newborns from the general population. Performance criteria were: (i) diagnostic sensitivity and specificity for the conditions screened; (ii) reaction failure rate; (iii) variability in mDNA between groups.
Results
Both methods showed 100% sensitivity and specificity for differentiating FXS and individual chromosome 15 imprinting disorders. IsoPure showed reaction failure rates of 0.365% for SNRPN and 0.74% for FMR1 compared to 19.34% and 2.56%, for QIAcube HT, respectively, with most failed reactions originating from archival blood spots. IsoPure showed lower variability in mDNA values in the neurotypical and condition-specific ranges.
Conclusion
The IsoPure system showed superior performance especially on archival samples, with broader applications for screening and diagnostic testing requiring high-throughput mDNA analyses on materials of limited quantity and quality.