Fragile X protein in newborn dried blood spots

Abstract

Background: The fragile X syndrome (FXS) results from mutation of the FMR1 gene that prevents expression of its gene product, FMRP. We previously characterized 215 dried blood spots (DBS) representing different FMR1 genotypes and ages with a Luminex-based immunoassay (qFMRP). We found variable FMRP levels in the normal samples and identified affected males by the drastic reduction of FMRP.

Methods: Here, to establish the variability of expression of FMRP in a larger random population we quantified FMRP in 2,000 anonymous fresh newborn DBS. We also evaluated the effect of long term storage on qFMRP by retrospectively assaying 74 aged newborn DBS that had been stored for 7-84 months that included normal and full mutation individuals. These analyses were performed on 3 mm DBS disks. To identify the alleles associated with the lowest FMRP levels in the fresh DBS, we analyzed the DNA in the samples that were more than two standard deviations below the mean.

Results: Analysis of the fresh newborn DBS revealed a broad distribution of FMRP with a mean approximately 7-fold higher than that we previously reported for fresh DBS in normal adults and no samples whose FMRP level indicated FXS. DNA analysis of the lowest FMRP DBS showed that this was the low extreme of the normal range and included a female carrying a 165 CGG repeat premutation. In the retrospective study of aged newborn DBS, the FMRP mean of the normal samples was less than 30% of the mean of the fresh DBS. Despite the degraded signal from these aged DBS, qFMRP identified the FXS individuals.

Conclusions: The assay showed that newborn DBS contain high levels of FMRP that will allow identification of males and potentially females, affected by FXS. The assay is also an effective screening tool for aged DBS stored for up to four years.

Figure 1

Distribution of FMRP levels in 2,000 newborn DBS from the NY State collection. The mean FMRP value was 44.8 pM; standard deviation 12.4 pM; skewness 1.11; kurtosis 3.175.

Figure 2

PCR analysis of DBS sample 2 in Table 1 , a female with a large premutation allele and a highly methylated normal allele. A: Capillary electrophoresis profile of PCR analysis. Arrows indicate alleles of 30, 161 and 167 CGG repeats. The 161 and 167 repeat alleles (arrows at right) represent a premutation allele. (Somatic mosaicism is presumably responsible for the bifurcation). B: Methylation analysis reference (no Hpa II digestion) PCR profile of DBS DNA. C: Methylation analysis PCR profile of Hpa II-digested DBS DNA. Comparison of B and C illustrates how each allele is protected from HpaII digestion by methylation. The premutation was split into two alleles and the analysis suggests that the larger, 167 repeat had a lower level of methylation than the smaller, 161 repeat allele. The 2 PCR products at approximately 0 CGG repeats represent internal controls for the methylation analysis.

Figure 3

Decline in detectable FMRP with DBS storage time. Samples from normal individuals are plotted according to duration of storage in months. The formula for the best fit trend line: y =21.903e^-0.028×; R² = 0.5509.

Figure 4

Distribution of FMRP levels in 59 newborn DBS from normal controls in the New South Wales archive. The mean FMRP value was 12.5 ± 6.5. Storage time for this sample set was ≤47 months.

Figure 5

Boxplot of FMRP values in 63 newborn DBS from New South Wales archive. Box-plot data are expressed as 25th to 75th percentile, median, and whiskers to 10^th and 90^th percentiles with outliers shown as circles. *P = ≤0.001, Mann-Whitney U-test. Storage time for this sample set was ≤47 months.