Incidence of fragile X syndrome by newborn screening for methylated FMR1 DNA

Abstract

Fragile X syndrome (FXS) results from a CGG-repeat expansion that triggers hypermethylation and silencing of the FMR1 gene. FXS is referred to as the most common form of inherited intellectual disability, yet its true incidence has never been measured directly by large population screening. Here, we developed an inexpensive and high-throughput assay to quantitatively assess FMR1 methylation in DNA isolated from the dried blood spots of 36,124 deidentified newborn males. This assay displays 100% specificity and 100% sensitivity for detecting FMR1 methylation, successfully distinguishing normal males from males with full-mutation FXS. Furthermore, the assay can detect excess FMR1 methylation in 82% of females with full mutations, although the methylation did not correlate with intellectual disability. With amelogenin PCR used for detecting the presence of a Y chromosome, this assay can also detect males with Klinefelter syndrome (KS) (47, XXY). We identified 64 males with FMR1 methylation and, after confirmatory testing, found seven to have full-mutation FXS and 57 to have KS. Because the precise incidence of KS is known, we used our observed KS incidence as a sentinel to assess ascertainment quality and showed that our KS incidence of 1 in 633 newborn males was not significantly different from the literature incidence of 1 in 576 (p = 0.79). The seven FXS males revealed an FXS incidence in males of 1 in 5161 (95% confidence interval of 1 in 10,653-1 in 2500), consistent with some earlier indirect estimates. Given the trials now underway for possible FXS treatments, this method could be used in newborn or infant screening as a way of ensuring early interventions for FXS.

Figure 1

FMR1 Promoter Sequence Targeted for DNA Methylation Analysis The sequence of the FMR1 promoter from position −192 to position +59 is shown. The top line is the genomic reference (Ref.). The second line represents the sequence after sodium bisulfite treatment and PCR amplification if every cytosine in each CpG is methylated (Meth.). The third line represents the sequence after sodium bisulfite treatment and PCR amplification if every cytosine in each CpG is unmethylated (Unmeth.). The amplification primers are underlined, and the TaqMan probes are indicated by the shaded boxes. The CpGs targeted by the TaqMan probes are underlined and in bold. The transcription start site is indicated by the arrow.

Figure 2

Proportion of Methylated FMR1 DNA Detected in 25 Different FXS Males and Three KS Males Methylated FMR1 DNA was quantified by Q-MSP and is expressed as the percentage of methylated DNA (amount of methylated FMR1 DNA / amount of methylated FMR1 DNA + amount of unmethylated FMR1 DNA). The height of the bar corresponds to the mean amount of methylated FMR1 DNA from three measurements, with the error bars representing 1 SD.

Figure 3

Amplification Plots for 88 Male Dried Blood Spot Samples Tested Simultaneously via Q-MSP for Methylated FMR1 DNA Right panel: amplification plot for Fam-labeled TaqMan probe specific for methylated FMR1 DNA. Left panel: amplification plot for Hex-labeled TaqMan probe specific for unmethylated FMR1 DNA. Each line represents a single sample.

Figure 4

Quantification of Methylated FMR1 DNA in 33 Females Who Carry the Full-Mutation Allele The proportion of methylated FMR1 DNA was quantified by Q-MSP and is expressed as a percentage of total FMR1 DNA. Thirteen normal females with normal FMR1 alleles are represented by the green bars on the right-hand side of the graph. The 33 females carrying the full mutation are represented as follows: affected full mutation, red bars; unaffected full mutation, dark blue bars; and full mutation with no phenotypic information, white bars. The height of the bar corresponds to the mean amount of methylated FMR1 DNA from three measurements, with the error bars representing 1 SD. The normal range of methylation (normal female mean ± 2 SD) is represented by the shaded box.

Figure 5

Amplification Plots of Pools of 96 Male Dried Blood Spot Samples with or without a Mosaic Male with FXS The red lines represent the pooled sample containing one mosaic FXS male dried blood spot mixed with 95 dried blood spots from normal males. The blue lines represent the sample containing 96 dried blood spots from normal males. (A) Amplification plot for TaqMan probe specific for methylated FMR1 DNA. (B) Amplification plot for TaqMan probe specific for unmethylated FMR1 DNA.

Figure 6

Southern Blot with the Use of DNA Isolated from the Dried Blood Spot Card from One of the FXS Males Identified in the Screen, Sample 1419F10 Lane 1. normal male control. Lane 2. sample 1419F10. Lane 3. normal female control. Lane 4. FXS genomic DNA control. Lane 5. molecular weight marker.