Predictive markers useful in the treatment of fragile X syndrome (FXS)
An X chromosome and syndrome technology, applied in the field of individualized treatment, can solve problems such as limited efficacy and adverse side effects
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Embodiment 1
[0685] Example 1: Setting up a study to identify whether there is a fraction of patient
[0686] The embodiment of the present invention is used as a mGluR5 antagonist (-)-(3aR, 4S, 7aR)
[0687] - A follow-up study of a clinical trial whether methyl 4-hydroxy-4-m-tolylethynyl-octahydro-indole-1-carboxylate could provide beneficial treatment to individuals with FXS. This study was established to identify whether there is a subset of patients who respond to treatment with (-)-(3aR,4S,7aR)-4-hydroxy-4-m-tolylethynyl-octahydro-indole-1-carboxylate methyl ester. In an attempt to identify the subset of patients who respond to treatment with (-)-(3aR,4S,7aR)-4-hydroxy-4-m-tolylethynyl-octahydro-indole-1-carboxylate, a study To explore the relationship between FMR1 methylation / mRNA expression and (-)-(3aR,4S,7aR)-4-hydroxy-4-m-tolylethynyl-octahydro-indole-1-carboxylate efficacy in this study relationship between.
[0688] Clinical samples: Of the 30 patients who completed the cl...
Embodiment 2
[0730] Example 2: Restriction enzyme digestion based on Taqman probes for determining FMR1 promoter methylation after real-time PCR detection
[0731] Clinical samples: Of the 26 genomic DNA purified from patients who consented to the pharmacogenetic / pharmacogenomic assessment in Example 1, 12 had sufficient quantities for analysis by probe-based methylation assays.
[0732] Probe-based methylation detection:
[0733] The assay is based on the MethylScreen technology from Orion Genomics (St. Louis, MO). However, it combines methyl-selective restriction enzyme DNA treatment with TaqMan hydrolysis probe-based real-time PCR. Briefly, to assess the methylation status of the FMR1 promoter region, genomic DNA purified from EDTA anticoagulant blood was digested independently and consistently with McrBC and Hhal from NEB (Ipswich, MA) according to the manufacturer's instructions. , resulting in the following four conditions: 1) No enzyme digestion; 2) McrBC digestion; 3) HhaI digest...
Embodiment 3
[0735] Example 3: FMRP assay using time-lapse fluorescence resonance energy transfer (TR-FRET) immunodetection
[0736] The following antibodies were used for TR-FRET immunoassay: F4055 (Sigma, RTGKDRNQKKEKPDSVDG (SEQ ID NO: 7)); 2160 (Millipore; ITVAFENNWQPDRQIPFHD; SEQ ID NO: 8) and H00002332-M03 (Arnold Abnova; ATKDTFHKIKLDVPEDLRQMCAKEAAHKDFKKAVGAFSVTYDPENYQLVI; (SEQ ID NO: 9)).
[0737] Temperature-dependent signaling kinetics of human FMRP protein detection by F4055-H00002332-M03 antibody combination
[0738] HEK293T cells were transiently transfected with eGFP plastids (mock) or human FMRP plastids (transfected with FMRP). Cells were lysed in M-PER (Pierce) lysis buffer, 150 nM NaCl and protease inhibitors. 1 μg of total protein (5 μl) and 1 μl of antibody detection buffer were loaded into each low volume 384 well. The results are shown in figure 2 middle.
[0739] Temperature-dependent signaling kinetics of human FMRP protein detection by MAB2160-F4055 antibody co...
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