Elevated levels of FMR1 mRNA in carrier males: a new mechanism of involvement in the fragile-X syndrome

Abstract

Fragile-X syndrome is a trinucleotide-repeat-expansion disorder in which the clinical phenotype is believed to result from transcriptional silencing of the fragile-X mental retardation 1 (FMR1) gene as the number of CGG repeats exceeds approximately 200. For premutation alleles ( approximately 55-200 repeats), no abnormalities in FMR1-gene expression have been described, despite growing evidence of clinical involvement in premutation carriers. To address this (apparent) paradox, we have determined, for 16 carrier males (55-192 repeats), the relative levels of leukocyte FMR1 mRNA, by use of automated fluorescence-detection reverse transcriptase-PCR, and the percent of lymphocytes that are immunoreactive for FMR1 protein (FMRP). For some alleles with>100 repeats, there was a reduction in the number of FMRP-positive cells. Unexpectedly, FMR1 mRNA levels were elevated at least fivefold within this same range. No significant increase in FMR1 mRNA stability was observed in a lymphoblastoid cell line (160 repeats) derived from one of the carrier males, suggesting that the increased message levels are due to an increased rate of transcription. Current results support a mechanism of involvement in premutation carriers, in which reduced translational efficiency is at least partially compensated through increased transcriptional activity. Thus, diminished translational efficiency may be important throughout much of the premutation range, with a mechanistic switch occurring in the full-mutation range as the FMR1 gene is silenced.

Figure 1

Outline of analysis of FMR1 mRNA levels, by use of automated, fluorescence-detection RT-PCR, with peripheral blood leukocytes obtained from a male premutation carrier with 192 CGG repeats. A, Logarithm of the relative reporter (FAM) fluorescence intensities ΔR_n, for FMR1 (blackened circles) and GUS (unblackened circles) probes, plotted as a function of the cycle number (C) in the PCR reaction (total cellular RNA 5 ng/μl). For a set of curves, C_t is the cycle number at a given threshold value of probe fluorescence (see the Material and Methods section). ΔC_t is the difference in cycle number for a given fluorescence threshold. B, Plots of C_t and ΔC_t, as a function of total cellular RNA. C_t plots: blackened triangles denote the FMR1 probe, control (RP) cell line; unblackened triangles, GUS probe, RP cell line; unblackened circles, GUS probe, carrier male; blackened circles, FMR1 probe, carrier male. ΔC_t plots: gray-shaded circles, C_t(FMR1)-C_t(GUS), carrier male; gray-shaded triangles, C_t(FMR1)-C_t(GUS), RP cell line. ΔΔC_t (blackened squares) represents the corrected value for the difference in cycle number for FMR1 mRNA versus GUS mRNA and is used to determine relative mRNA levels (see the Material and Methods section).

Figure 2

Plot of FMR1 mRNA levels (relative to GUS mRNA [rel]), as a function of the CGG repeat number (n). nl, n<55, lp = 55 ⩽ n<100, and hp = 100 ⩽ n ⩽ 200. Horizontal lines represent the group (unweighted) means from table 1.

Figure 3

Plots of the increase in C_t values for FMR1 and GUS mRNAs, as a function of time after treatment of cell lines with 10 μg actinomycin D/ml. FMR1 (blackened symbols) and GUS (unblackened symbols) probes indicate results for both a normal control cell line, AG (squares), and a premutation cell line, MM (160 CGG repeats) (circles). Increasing C_t reflects decreasing mRNA level, with an increase of one unit representing a twofold decrease in probe-specific message.

Figure 4

Plot of %FMRP(+) lymphocytes, as a function of CGG-repeat number. The standard error associated with each point is ∼4%. The horizontal dotted line represents 1 SD below the mean value of the %FMRP(+) lymphocytes for males with nonexpanded alleles (Willemsen et al. 1997); dashed lines, 99% confidence limits for a second-order regression of the current data.