CLINICAL UTILITY OF OPTIMIZED FRAGILE X PROTEIN ANALYSIS

Abstract

The fragile X mental retardation 1 (FMR1) gene-specific protein product, fragile X protein (FXP), is critical for brain development. An unstable trinucleotide repeat expansion (cytosine-guanine-guanine [CGG] repeats >200) and subsequent abnormal methylation of FMR1 located on the X chromosome results in fragile X syndrome (FXS) and markedly reduced FXP levels. There are few studies demonstrating relationships between FXP levels and phenotypic expression. Identifying these relationships may be critical to planning clinical care and understanding development over time. Here, we optimized a method to reliably and reproducibly detect FXP at extremely low values in whole blood samples to examine the relationship between FXP levels and relevant clinical features in FXS. Fifty-two individuals with full-mutation FXS (28 males [FXS(M)] and 24 females [FXS(F)]) aged 11 to 45 years completed a battery of psychosocial, neurocognitive, and electrophysiological measures. Participant whole blood samples, obtained via venipuncture, were spotted onto Whatman Bloodstain cards. Dried blood spots were hole-punched from the cards, and proteins were eluted. The eluate was analyzed in triplicate against a 9-point standard curve generated from a recombinant protein, via a Luminex-based immunoassay, to determine participants’ FXP concentration. Higher FXP levels related to higher IQ scores, less severe stereotyped behaviors, hyperactivity, speech abnormalities, and lower resting-state gamma power across sexes. Unexpectedly, higher FXP levels were associated with more severe social anxiety in FXS(M) and more severe depressive symptoms in FXS(F). In a subset of individuals who completed an investigational drug study (n = 12), only FXS(M) with complete absence of FMRP demonstrated increased Repeatable Battery for the Assessment of Neuropsychological Status (RBANS) verbal memory and reduced Aberrant Behavior Checklist (ABC) irritability. Our optimized FXP analysis, which allowed us to reproducibly identify FXP at extremely low values, provides new evidence of the link between FXP production and clinical phenotypic expression in FXS. Of note, we identify a subset of patients—males without any FXP—who demonstrated significant improvement on verbal memory and irritability following a 2-week course of an investigational drug, suggesting that optimized FXP analysis may be a highly useful tool in treatment planning and trial stratification.