Fragile X protein in newborn dried blood spots
Tatyana Adayev1, Giuseppe LaFauci2, Carl Dobkin3
1Department of Developmental Biochemistry, New York State Institute for Basic Research in Developmental Disabilities, 1050 Forest Hill Road, Staten Island, New York, 10314, USA. tatyana.adayev@opwdd.ny.gov.
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.
Insights
Fragile X syndrome (FXS) can be identified in newborns using a quantitative assay for Fragile X Messenger Protein (FMRP) in dried blood spots (DBS). This method also effectively screens aged DBS for FXS, even after long-term storage.
Area of Science:
- Biochemistry
- Genetics
- Neonatal Screening
Background:
- Fragile X syndrome (FXS) is caused by FMR1 gene mutations, leading to reduced Fragile X Messenger Protein (FMRP).
- Previous studies used a Luminex-based immunoassay (qFMRP) on dried blood spots (DBS) to characterize FMRP levels in various FMR1 genotypes.
Purpose of the Study:
- To establish the variability of FMRP expression in a larger population of newborns.
- To evaluate the impact of long-term storage on the qFMRP assay's effectiveness.
Main Methods:
- Quantified FMRP in 2,000 fresh newborn DBS using qFMRP.
- Assayed 74 aged newborn DBS (stored 7-84 months) to assess long-term storage effects.
- Analyzed DNA from samples with low FMRP levels to identify associated alleles.
Main Results:
- Fresh newborn DBS showed a wide FMRP distribution, with mean levels ~7-fold higher than in adults; no FXS cases were detected.
- DNA analysis of low FMRP samples identified individuals within the normal range, including a female with an FMR1 premutation.
- Aged DBS had significantly lower mean FMRP levels (<30% of fresh DBS), but qFMRP still successfully identified FXS individuals.
Conclusions:
- Newborn DBS possess sufficient FMRP levels for FXS identification in both males and females using the qFMRP assay.
- The qFMRP assay is a viable screening tool for aged DBS, effective for samples stored up to four years.
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