8. Optical genome mapping analysis of FMR1 expansions in fragile X syndrome

      Fragile X syndrome (FXS) is associated with cognitive impairment and intellectual disability. Nearly all cases of FXS are associated with an expansion of a CGG triplet repeat in the FMR1 gene. Phenotype severity is often correlated with the expansion size; thus, accurate sizing of the CCG repeat array is crucial. The repetitive nature of these regions presents difficulties: 1. PCR is unable to traverse through long repeats; 2. sequencing has limited read lengths; 3. Southern blot may be inaccurate, time consuming, and expensive. Optical genome mapping (OGM) has the potential to address some of these shortcomings. OGM utilizes images of ultra-long DNA molecules, labeled at specific sequence motifs linearized in nanochannel arrays and can be used for structural and copy number variation calling and absence of heterozygosity analysis. Bionano Genomics has developed a targeted analysis workflow for analysis of FMR1. To evaluate the capability of measuring repeat arrays consistent with normal (<45 repeats), premutation (55-200 repeats), and full mutation (>200 repeats), we analyzed the FMR1 repeats in 75 samples. We also analyzed the repeat region on 20 phenotypically normal control subjects. We observed alleles consistent with annotation across the entire range of repeat counts. Analytical sensitivity was measured at 97% with 100% PPV for expansions >200 repeats. The largest expansion we detected was ∼1000 repeats. In the control samples, we measured repeat counts below the full mutation cutoff in all cases. Repeatability studies were carried out to show analytical consistency and to establish minimum molecule quality requirements. EnFocus™ analysis report provides pass/fail for QC metrics for input data quality as well as analytic measurement quality using internal control regions of the genome on each autosome. OGM performance for FMR1 repeat lengths analyzed here show a much higher dynamic range compared to PCR and higher precision compared to Southern blot.
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