Evaluation of Fragile X Mental Retardation Protein Levels in NICU Infants

Background: Fragile X syndrome (FXS) is a most common form of inherited intellectual disability with a single gene effect. At least 30% of individuals with FXS are also diagnosed with autism spectrum disorder (ASD). FXS is caused by CGG repeat expansions in X-linked gene FMR1, resulting in reduction or absence of the gene product, fragile X mental retardation protein (FMRP). Typically, FMRP is expressed early during fetal development and a substantial amount of the protein is detectable at birth. Although reduction of FMRP is associated with FXS, scientific efforts to rescue Fmrp expression in Fmr1 KO mice reported to result in increased hyperactivity/anxiety-like behaviors when the protein is greatly overexpressed. Profiling the developmental expression of FMRP may help to establish physiologic and diagnostic guidelines. Neonatal intensive care unit (NICU) graduates are at risk for neurodevelopmental delay and are more frequently diagnosed with ASD later in life than the general population.

Objectives: The current study surveyed the levels of FMRP in NICU infants as an at-risk population for ASD.

Methods: Newborn dried blood spots (DBS) were collected at the Richmond University Medical Center, Staten Island NY. Research DBS card collection for consented participants was timed to the collection of the state-mandated newborn card. The levels of FMRP and the FMR1 gene CGG repeat number were assessed. This study examined the levels of FMRP expression in the peripheral blood of infants from a well nursery (WN) (N=324) and the NICU (N= 75) with normal FMR1 genotypes. We used the immuno-based qFMRP assay to evaluate the protein levels in extracts from 3mm DBS punches using anti-FMRP 6B8 mAb (BioLegend) - R477 polyclonal antibody duo and abbreviated recombinant FMRP standard for protein quantification. Detected FMRP was further normalized to the number of white blood cells (WBC).

Results: Analysis of FMRP data for the two cohorts shows elevated FMRP levels in NICU newborns (mean_NICU 4.23 pg/10³ WBC) compared to the well nursery infants (mean_WN 3.90 pg/10³ WBC). In the NICU cohort the FMRP data distribution failed a normality test due to the greater proportion of the infants presenting with either reduced or elevated levels of FMRP expression, resulting in an asymmetric and more heavily tailed data distribution (skewness 1.336 and kurtosis 2.774).

Conclusions: The FMRP levels among age matched NICU and a well nursery newborns showed small but significant differences (p < 0.05) in mean values. Further recruitment and analysis is necessary for more definitive conclusions. Patterning the data for the presence of diagnostic commonalities that would correlate with the increased frequency of the contrasting levels of FMRP in NICU newborns is expected to refine this study’s conclusions.