Figure 2.

MS-QMA testing of: (A) U.S.A. cohort males with: DD negative by standard FXS testing (n = 189), FM and confirmed FXS diagnosis (n = 22), males with Klinefelter syndrome (n = 6), one CGG microduplication with normal karyotype. Australian cohort included male reference samples with normal CGG size including typically developing (IQ > 70) controls (n = 42) and newborn blood spots from the general population (n = 89), as well as 2 FM female and 8 FM male positive controls from and one Klinefelter syndrome patient samples. Note: “+ve” = positive; “−ve” = negative. Confirmatory testing was performed using AmplideX mPCR, and MALDI-TOF MS EpiTYPER system FMR1 methylation analysis. Male control reference range indicated in light blue (mean ± 2 standard deviations): 2 ± 0.01%. Below 2% methylation positive calls were made from raw data assessment of MS-QMA derivative curves. (B) U.S.A. cohort females included: DD negative by standard FXS testing (n = 90), FM and confirmed FXS diagnosis (n = 8), males with Klinefelter syndrome. Australian cohort included female reference samples with normal CGG size including typically developing controls (IQ > 70; n = 33) and newborn blood spots from the general population (n = 95). (C) Percentage and the number of MS-QMA positive, as well as the total number of cases tested for the U.S.A. cohort in each allelic group for males and females. Note: DD = developmental delay referral; RTT = Rett syndrome; AS = Angelman syndrome; true positives: FXS males and females confirmed to have FM alleles by both AmplideX CGG screening assay and mPCR. Female control reference range indicated in light blue (mean ± 2 standard deviations): 27 ± 9%, or between 18% and 37% representing the normal amount of FREE2 methylation attributed to X chromosome inactivation, consistent with the range reported in previous studies¹².