Quantification of FMRP in Human and Mouse Tissues By Capture Immunoassay
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 new FMRP antibodies, a human specific mAb 6B8 (Biolegend) and rabbit polyclonal R477, a bacterially expressed abbreviated FMRP standard, and a Luminex platform to quantify FMRP.
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. Variable amount of FMRP are detected in typical individuals. In DBS samples, normalization of FMRP levels to the number of leukocytes reduced this variability and could allow to distinguish premutation carriers from typical individuals.
Using human and mouse detecting 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 the brainstem, cerebellum, hippocampus, and cortex two strains of mice (C57BL and FVB) in seven and ten week-old animals, showing developmental variations exist.
Conclusions: A rapid qualitative assay has been developed for the diagnosis of Fragile-X Syndrome. This sensitive assay allows for the screening of newborn infants using routinely collected dried blood spots. The assay will also allow further studies on variations of mouse Fmrp expression in different models and organs.