Optimization, validation and initial clinical implications of a Luminex-based immunoassay for the quantification of Fragile X Protein from dried blood spots

Fragile X Syndrome (FXS) is caused by a trinucleotide expansion leading to silencing of the FMR1 gene and lack of expression of Fragile X Protein (FXP, formerly known as Fragile X Mental Retardation Protein, FMRP). Phenotypic presentation of FXS is highly variable, and the lack of reproducible, sensitive assays to detect FXP makes evaluation of peripheral FXP as a source of clinical variability challenging. We optimized a Luminex-based assay to detect FXP in dried blot spots for increased reproducibility and sensitivity by improving reagent concentrations and buffer conditions. The optimized assay was used to quantify FXP in 187 individuals. We show that the optimized assay is highly reproducible and detects a wide range of FXP levels. Mosaic individuals had, on average, higher FXP levels than fully methylated individuals, and trace amounts of FXP were consistently detectable in a subset of individuals with full mutation FXS. IQ scores were positively correlated with FXP levels in males and females with full mutation FXS demonstrating the clinical utility of this method. Our data suggest trace amounts of FXP detectable in dried blood spots of individuals with FXS could be clinically relevant and may be used to stratify individuals with FXS for optimized treatment.