Figure 1. Novel G-insertion mutation identified in male with FXS symptoms.

1. Predicted effects of the G-insertion mutation on FMRP are schematized. The insertion site corresponds to the beginning of the RNA-binding RGG Box, where it alters the open reading frame and thus disrupts the domain entirely. The frameshift creates a novel peptide sequence [RKRTRRKRTWRRLQRKRRSLPNR] followed by a premature stop codon, causing the truncation of the FMRP C-terminus.
2. Photograph of male patient with FMR1^G-ins. allele. Typical physical and behavioral features of FXS were noted in the patient, who shows moderate to severe intellectual disability.
3. FMR1 mRNA levels were analyzed via RT–qPCR in EBV-transformed lymphocyte cells derived from the patient's blood samples. Patient cells showed a ˜60% decrease in FMR1 mRNA compared to control cells (*P = 0.010, 0.383 ± 0.110 SD, n = 3). Treatment with translational blocker puromycin restored FMR1 mRNA levels in patient cells (*P = 0.048, 0.383 ± 0.110 SD versus 1.25 ± 0.166 SD, n = 3), suggesting that the reduction of FMR1 mRNA in these cells is primarily due to nonsense-mediated decay. RT-qPCR reactions were run in triplicate in three independent experiments. Fold changes in FMR1 expression, normalized to HRPT expression, were calculated using the ΔΔC_T method, and analyzed statistically with a two-tailed t-test (GraphPad). Error bars represent mean values with SD.
4. Western blot analysis shows a significant decrease (> 90%) in FMRP protein levels in patient-derived cells (***P = 0.008, 0.13 ± 0.031 SD, n = 2), and the patient FMRP migrates slightly lower than the wild-type protein. Band intensities for the different cell lines were quantified, normalized for actin and analyzed statistically with a twot-tailed t-test (GraphPad). Error bars represent mean values with SD.