Abstract
BackgroundThe 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.MethodsHere, 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.ResultsAnalysis 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.ConclusionsThe 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.
Highlights
The fragile X syndrome (FXS) results from mutation of the FMR1 gene that prevents expression of its gene product, fragile X mental retardation protein (FMRP)
We recently reported the development of a simple, accurate, and inexpensive capture immunoassay that determines the level of FMRP in dried blood spots (DBS) as well as in lymphocytes and other tissues [12]
NY State newborn DBS samples We applied the qFMRP immunoassay to 1,000 male and 1,000 female newborn DBS that had been stored for only five weeks
Summary
The fragile X syndrome (FXS) results from mutation of the FMR1 gene that prevents expression of its gene product, FMRP. Fragile X syndrome, the most common inherited cause of intellectual disability, results from mutation of the FMR1 gene on the X chromosome that disrupts expression of the fragile X mental retardation protein (FMRP) [1,2]. There are very rare cases in which the fragile X syndrome is due to point mutation or deletion of the FMR1 gene [3,4,5,6,7], the most common fragile X mutation eliminates FMRP expression through expansion of a CGG repeat in the 5’-untranslated region of FMR1 to FMR1 alleles are sorted into four size categories based on their stability upon transmission: normal (up to 44 CGG repeats): intermediate (45-54 repeats); premutation (55-200 repeats); and full mutation (>200 repeats). FMRP expression is reduced as triplet repeat length increases from the normal range and is eliminated by a process analogous to X inactivation when the repeat exceeds approximately 200 copies [9,10]
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