Quantification of FMRP in Human and Mouse Tissues By Capture Immunoassays

Background:  The Fragile X syndrome is a leading inherited cause of ASD. The Fragile X syndrome is due to mutations of the FMR1 gene that result in the absence of fragile X mental retardation protein (FMRP).

Objectives:  To develop a screening test for FMRP.

Methods:  We have developed a rapid, highly sensitive method for quantifying FMRP from dried blood spots and lymphocytes. This assay uses two new antibodies mAb 6B8 (Biolegend) and R477, a bacterially expressed abbreviated FMRP standard, and a Luminex platform to quantify FMRP.

Results:  The assay readily distinguishes between samples from males with fragile X full mutations and samples from normal males. It also differentiates mosaic from non-mosaic full-mutation male samples. We have employed the assay to screen 2000 newborn dried blood spots (DBS) and present their distribution. We also applied the assay in a retrospective study of 76 newborn DBS that had been stored for an extended period and included full mutation males as well as normal individuals. We were able to correctly identify all 5 known male fragile X positive cases among samples stored up to 47 months.

Using mAb 5C2 (Biolegend) and R477, we have also developed a similar immunoassay for the quantification of Fmrp in mouse tissues. This assay was used to quantify Fmrp in brainstem, cerebellum, hippocampus, and cortex strains of mouse (C57 BL and FVB) in seven and ten week-old animals, showing developmental variation.

Conclusions:  This sensitive assay allows for the quantitation of FMRP for purposes of newborn screening. The assay will also allow studying the developmental variation of mouse Fmrp expression in different organs.