Fragile X–related element 2 methylation analysis may provide a suitable option for inclusion of fragile X syndrome and/or sex chromosome aneuploidy into newborn screening: a technical validation study

Early Detection of Fragile X Syndrome: Applications of a Novel Approach for Improved Quantitative Methylation Analysis in Venous Blood and Newborn Blood Spots

“…Using the MALDI-TOF mass spectrometry (MS)-based Epi-TYPER system, we have identified novel epigenetic markers for FXS, fragile X-related epigenetic elements 1 (FREE1) and 2 (FREE2), that are inversely correlated with FMRP expression in males and females with expanded FMR1 alleles (13,14 ). Using the MALDI-TOF MS EpiTYPER system, we have shown that methylation analysis of the CpG sites located within FMR1 intron 1 of FREE2 (positioned 3Ј of the expansion) is superior to methylation-sensitive Southern blot (used in current FXS diagnostics) and FMRP immunostaining in blood as a predictor of cognitive impairment and is related to X-inactivation skewing in FM females (15,16,17 ). To make FREE2 methylation analysis accessible for use in most diagnostic laboratories, we have developed a new real-time PCR-based method named methylation-specific-quantitative melt analysis (MS-QMA), which targets methylation of the same intronic sites.…”

“…To make FREE2 methylation analysis accessible for use in most diagnostic laboratories, we have developed a new real-time PCR-based method named methylation-specific-quantitative melt analysis (MS-QMA), which targets methylation of the same intronic sites. We then performed a large-scale validation of the method in previously clinically described cohorts (15,(17)(18)(19) and compared its performance attributes to those of the reference methods.…”

“…The patient cohort comprised 258 male and 427 female samples from individuals from birth to 82 years of age collected as part of previous studies (15,(17)(18)(19). Of these, formal cognitive assessments were performed on samples from 23 PM females, 21 FM females, and 3 high-functioning unmethylated FM (UFM) males [with full scale IQ (FSIQ) between 71 and 81] determined using the Wechsler intelligence test appropriate for chronological age as described in our previous publications (15,18,20 ).…”

Identification of Males with Cryptic Fragile X Alleles by Methylation-Specific Quantitative Melt Analysis

“…This created 560 DNA pools that were treated with sodium bisulfite with EZ-96 DNA Methylation-Gold™ (Zymo Research), as previously described (28 ). The bisulfite-converted DNA pools were then assayed with real-time PCR followed by HRM with MeltDoctor™ reagents in 10-L reactions on the ViiA™ 7 Real-Time PCR System, according to manufacturer's instructions (Life Technologies), with primers targeting specific CpG sites within the FREE2 region (Fig.…”

“…To determine the lower limit of detection for each assay used in this study, artificial NS/FM mosaic samples were created from high-quality DNA extracted from lymphoblast cell lines used in previous studies (17,28 ). Fully methylated FM (530 CGG) male DNA was mixed with NS (30 CGG) male DNA at different ratios.…”