Fragile X associated primary ovarian insufficiency (FXPOI): ovarian phenotype and FMR1 RNA toxic gain of function in human granulosa cells

Abstract

OBJECTIVE: To examine the clinical ovarian phenotype and, by in vitro investigation, determine if RNA toxic gain of function mechanism might explain the development of fragile X-associated primary ovarian insufficiency (FXPOI). FMR1 encodes a selective RNA-binding protein FMRP and is expressed in granulosa cells. The FMR1 premutation (more than 55 but less than 200 CGG repeats) also puts individuals at risk for fragile X-associated tremor/ataxia syndrome (FXTAS). Drosophila and mouse model systems of the premutation support an RNA toxic gain of function mechanism in the neurodegeneration.DESIGN: Controlled.MATERIALS AND METHODS: Ovarian volumes were determined by transvaginal ultrasound in control women (N=41) and in women with confirmed FXPOI (N=12). We transfected (Lipofacamin-2000, Invitrogen) control and experimental plasmid DNA into human granulosa cell line COV-434 (pCMV-Control-GFP does not have CGG repeat at 5'-end of GFP cDNA; pCMV-88CGG-GFP has 88 CGG repeats; and pdCMV-88CGG-GFP does not have CMV promoter although it has 88 CGG repeats). Two days later we assessed cell viability by trypan blue exclusion.RESULTS: Median (range) total ovarian volume (cc) in women with FXPOI was significantly lower than control: 2 (1 to 11) vs 13 (5 to 29), p<0.0001]. In vitro, we found decreased percent cell viability for cells expressing GFP mRNA with 88 CGG repeats driven by a CMV promoter as compared to cells expressing GFP mRNA without CGG repeats (mean ± SD from five independent experiments: 73.2 ± 6.1 vs 83.0 ± 3.2, p<0.01).CONCLUSIONS: The mRNA containing FMR1 CGG repeats (88) is toxic and causes cell damage/death to human granulosa cells, similar to the proposed pathogenic role of the FMR1 premutation in neural cells. FMR1 RNA toxic gain of function may be a mechanism explaining the development of FXPOI and the accompanying smaller ovarian volume. OBJECTIVE: To examine the clinical ovarian phenotype and, by in vitro investigation, determine if RNA toxic gain of function mechanism might explain the development of fragile X-associated primary ovarian insufficiency (FXPOI). FMR1 encodes a selective RNA-binding protein FMRP and is expressed in granulosa cells. The FMR1 premutation (more than 55 but less than 200 CGG repeats) also puts individuals at risk for fragile X-associated tremor/ataxia syndrome (FXTAS). Drosophila and mouse model systems of the premutation support an RNA toxic gain of function mechanism in the neurodegeneration. DESIGN: Controlled. MATERIALS AND METHODS: Ovarian volumes were determined by transvaginal ultrasound in control women (N=41) and in women with confirmed FXPOI (N=12). We transfected (Lipofacamin-2000, Invitrogen) control and experimental plasmid DNA into human granulosa cell line COV-434 (pCMV-Control-GFP does not have CGG repeat at 5'-end of GFP cDNA; pCMV-88CGG-GFP has 88 CGG repeats; and pdCMV-88CGG-GFP does not have CMV promoter although it has 88 CGG repeats). Two days later we assessed cell viability by trypan blue exclusion. RESULTS: Median (range) total ovarian volume (cc) in women with FXPOI was significantly lower than control: 2 (1 to 11) vs 13 (5 to 29), p<0.0001]. In vitro, we found decreased percent cell viability for cells expressing GFP mRNA with 88 CGG repeats driven by a CMV promoter as compared to cells expressing GFP mRNA without CGG repeats (mean ± SD from five independent experiments: 73.2 ± 6.1 vs 83.0 ± 3.2, p<0.01). CONCLUSIONS: The mRNA containing FMR1 CGG repeats (88) is toxic and causes cell damage/death to human granulosa cells, similar to the proposed pathogenic role of the FMR1 premutation in neural cells. FMR1 RNA toxic gain of function may be a mechanism explaining the development of FXPOI and the accompanying smaller ovarian volume.