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.

No abstract

Fragile X syndrome (FXS) is the most common inherited intellectual disability. FXS is caused by a trinucleotide repeat expansion in the 5′ untranslated region of the FMR1 gene, which leads to gene methylation, transcriptional silencing, and lack of expression of Fragile X Messenger Riboprotein (FMRP). Currently available FXS therapies are inefficient, and the disease severity is highly variable, making it difficult to predict disease trajectory and treatment response. We and others have recently shown that a subset of full-mutation, fully-methylated (FM–FM) males with FXS express low amounts of FMRP which could contribute to phenotypic variability. To better understand the underlying mechanisms, we developed a sensitive qRT-PCR assay to detect FMR1 mRNA in blood. This assay reproducibly detects trace amounts of FMR1 mRNA in a subset of FM–FM males, suggesting that current Southern Blot and PCR determination of FM–FM status is not always associated with complete transcriptional silencing. The functional relevance of trace-level FMR1 mRNA is confirmed by showing a positive correlation with cognitive function; however, phenotypic variability is not fully explained by FMR1 expression. These results corroborate the need for better molecular assays for FXS diagnosis and encourage studies to elucidate the factors contributing to the phenotypic variability of FXS.

Background: Fragile X syndrome (FXS) is the most common inherited form of intellectual disability affecting 1 in 4,000 males and 1 in 6-8,000 females. FXS is caused by a trinucleotide expansion in the 5’UTR of the Fragile X Mental Retardation (FMR1) gene which in full mutation carriers (>200 repeats) leads to hypermethylation and transcriptional silencing of the 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 molecular markers explaining or predicting this variability are lacking. Recent studies suggest that trace amounts of FXP can be detected even in fully methylated individuals and may have clinical relevance; however, the lack of available reproducible, sensitive assays to detect FXP in peripheral tissue makes evaluation of peripheral FXP as a source of clinical variability challenging. Methods: 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 (101 males, 86 females; 0-78.4 years) including 35 typically developing controls (24 males, 11 females), 103 individuals carrying full mutations (70 males, 33 females), and 49 individuals with premutations (7 males, 42 females). A subset of these individuals showed repeat number or methylation mosaicism. We investigated the clinical relevance of peripheral FXP levels by examining its relationship with general intellectual functioning in a subset of individuals with available IQ scores. Results: 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 peripheral FXP levels in male and female individuals with full mutation FXS. Conclusions: We demonstrate that our optimized Luminex-based assay to detect FXP is reproducible, highly sensitive, and related to the core intellectual disability phenotype. Further, our data suggest that 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. Future studies are needed with larger sample sizes, evaluating FXP across development and expanded analysis of the relevance of FXP levels for behavioral and electrophysiological phenotypes in FXS.

BackgroundFragile X syndrome (FXS) is the most common inherited form of intellectual disability affecting 1 in 4,000 males and 1 in 6-8,000 females. FXS is caused by a trinucleotide expansion in the 5’UTR of the Fragile X Mental Retardation (FMR1) gene which in full mutation carriers (>200 repeats) leads to hypermethylation and transcriptional silencing of the 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 molecular markers explaining or predicting this variability are lacking. Recent studies suggest that trace amounts of FXP can be detected even in fully methylated individuals and may have clinical relevance; however, the lack of available reproducible, sensitive assays to detect FXP in peripheral tissue makes evaluation of peripheral FXP as a source of clinical variability challenging. MethodsWe 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 (101 males, 86 females; 0-78.4 years) including 35 typically developing controls (24 males, 11 females), 103 individuals carrying full mutations (70 males, 33 females), and 49 individuals with premutations (7 males, 42 females). A subset of these individuals showed repeat number or methylation mosaicism. We investigated the clinical relevance of peripheral FXP levels by examining its relationship with general intellectual functioning in a subset of individuals with available IQ scores. ResultsWe 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 peripheral FXP levels in male and female individuals with full mutation FXS. ConclusionsWe demonstrate that our optimized Luminex-based assay to detect FXP is reproducible, highly sensitive, and related to the core intellectual disability phenotype. Further, our data suggest that 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. Future studies are needed with larger sample sizes, evaluating FXP across development and expanded analysis of the relevance of FXP levels for behavioral and electrophysiological phenotypes in FXS.