Repeat expansion and methylation-sensitive triplet-primed polymerase chain reaction for fragile X mental retardation 1 gene screening in institutionalised intellectually disabled individuals.

INTRODUCTION: Fragile X syndrome (FXS) is the most prevalent X-linked intellectual disability (ID) and a leading genetic cause of autism, characterised by cognitive and behavioural impairments. The hyperexpansion of a CGG repeat in the fragile X mental retardation 1 (FMR1) gene leads to abnormal hypermethylation, resulting in the lack or absence of its protein. Tools for establishing the diagnosis of FXS have been extensively developed, including assays based on triplet-primed polymerase chain reaction (TP-PCR) for detection and quantification of the CGG trinucleotide repeat expansion, as well as determination of the methylation status of the alleles. This study aimed to utilise a simple, quick and affordable method for high sensitivity and specificity screening and diagnosis of FXS in institutionalised individuals with ID. METHODS: A total of 109 institutionalised individuals at the Center for Social Rehabilitation of Intellectual Disability Kartini, Temanggung, Central Java, Indonesia, were screened in a three-step process using FastFrax™ Identification, Sizing and Methylation Status Kits. RESULTS: Two samples that were classified as indeterminate with respect to the 41-repeat control at the identification step were subsequently determined to be non-expanded by both sizing and methylation status analyses. Two samples classified as expanded at the identification step were determined to carry full mutation expansions > 200 repeats that were fully methylated using sizing and methylation status analyses, respectively, yielding a disease prevalence of 1.83%. CONCLUSION: Repeat expansion and methylation-specific TP-PCR is practical, effective and inexpensive for the diagnosis of FXS, especially in high-risk populations of individuals with ID of undetermined aetiology.

Regulation of the NRF2 transcription factor by andrographolide and organic extracts from plant endophytes.

The transcription factor NF-E2 Related Factor-2 (NRF2) is an important drug target. Activation of NRF2 has chemopreventive effects in cancer and exerts beneficial effects in a number of diseases, including neurodegenerative diseases, inflammatory diseases, hepatosteatosis, obesity and insulin resistance. Hence, there have been great efforts to discover and characterize novel NRF2 activators. One reported NRF2 activator is the labdane diterpenoid andrographolide. In this study, we identified the mechanism through which andrographolide activates NRF2. We showed that andrographolide inhibits the function of KEAP1, a protein that together with CUL3 and RBX1 forms an E3 ubiquitin ligase that polyubiquitinates NRF2. Andrographolide partially inhibits the interaction of KEAP1 with CUL3 in a manner dependent on Cys151 in KEAP1. This suggests that andrographolide forms Michael acceptor dependent adducts with Cys151 in KEAP1 in vivo, leading to inhibition of NRF2 ubiquitination and consequently accumulation of the transcription factor. Interestingly, we also showed that at higher concentrations andrographolide increases NRF2 protein expression in a Cys151 independent, but likely KEAP1 dependent manner, possibly through modification of other Cys residues in KEAP1. In this study we also screened secondary metabolites produced by endophytes isolated from non-flowering plants for NRF2-inducing properties. One of the extracts, ORX 41, increased both NRF2 protein expression and transcriptional activity markedly. These results suggest that endophytes isolated from non-flowering or other plants may be a good source of novel NRF2 inducing compounds.

Heteroaromatic 4-arylquinols are novel inducers of nuclear factor-erythroid 2-related factor 2 (Nrf2).

The induction of phase 2 and antioxidant enzymes via the transcription factor Nuclear factor-erythroid 2-related factor 2 (Nrf2) is an important chemopreventive strategy in cancer and neurodegenerative diseases. Nrf2 is mainly regulated at the level of its protein stability by the cytosolic protein Keap1, which functions as a substrate recruiting subunit of a Cullin3 E3 ubiquitin ligase to target Nrf2 for ubiquitination and subsequent degradation. Phase 2 inducing agents usually covalently modify cysteine residues in Keap1, leading to inhibition of Nrf2 ubiquitination. Quinols, which due to their Michael acceptor moiety react readily with cysteine residues in selective cellular proteins, are good candidates for potential Nrf2 inducing chemopreventive agents. Indeed, we found that similar to the known phase II inducer sulforaphane, the heteroaromatic 4-arylquinols PMX290 and PMX464 increase both Nrf2 protein concentrations and transcriptional activity. Interestingly, PMX290 had a much stronger effect on the Nrf2 protein concentration, but a weaker effect on Nrf2 transcriptional activity compared to PMX464. Given the marked effect of PMX290 on the Nrf2 protein concentration, we examined its effect on the interaction of Keap1 with its binding partners. While sulforaphane was found to decrease binding of Cullin3 to Keap1, PMX290 markedly increased the interaction between these two proteins in intact cells. PMX464, which increased Nrf2 protein only weakly, also had a much smaller effect on the binding between Keap1 and Cullin3. In conclusion, PMX290 is a novel phase 2 inducing agent which increases the interaction between Keap1 and Cullin3 and may inhibit Nrf2 ubiquitination via a novel mechanism.