Rescue of human RET gene expression by sodium butyrate: a novel powerful tool for molecular studies in Hirschsprung disease

Abstract

Background:The RET gene encodes a tyrosine kinase receptor involved in different human neurocristopathies, such as specific neuroendocrine tumours and Hirschsprung disease (HSCR). Gene expression is developmentally regulated and the RET transcript is undetectable in most adult cells, including lymphocytes. The impossibility of performing functional studies on RET mRNA has to date limited the detection and characterisation of an indefinite proportion of gene anomalies that cannot be identified by conventional DNA genomic screening in HSCR cases. Aims: Development of a protocol suitable to activate RET expression in RET negative cell lines and therefore to investigate directly RET mRNA, extending the conventional gene mutation analysis to detection of splicing anomalies and impaired expression of the RET gene. Methods: The effect of sodium butyrate (NaB), a histone deacetylase inhibitor, on rescuing RET expression was tested by one round of reverse transcription-polymerase chain reaction from total RNA of treated lymphoblasts from both HSCR patients and control individuals. Results: Analysis of RET expression was possible by NaB treatment of RET negative cells, such as lymphoblasts. This treatment allowed us to detect impaired RET expression as well as a splicing defect in two HSCR patients previously believed to be devoid of any gene abnormality. Conclusions:The full application of the proposed protocol in most of the unexplained HSCR cases will allow us to establish the precise role of RET not only in causing but also in predisposing to HSCR pathogenesis.T he RET proto-oncogene (REarranged during Transfection) encodes a tyrosine kinase receptor predominantly expressed during neural crest development and involved in different human neurocristopathies.1 Germline RET mutations have been found in association with both inherited cancer syndromes, including multiple endocrine neoplasia type 2A (MEN2A), type 2B (MEN2B), and familial medullary thyroid carcinoma, and Hirschsprung disease (HSCR), a developmental disorder characterised by the absence of enteric neurones in variable lengths of the distal gastrointestinal tract.2 Loss of function, low penetrant RET mutations have been identified in only 10-40% of total HSCR cases while a small proportion of patients (5-10%) show alterations in other genes, generally related to the developmental programme of neural crest cells.3 4 By using sibpair analysis, a recent study has recognised three HSCR susceptibility loci at 3p21, 10q11, and 19q12 which may clarify the molecular basis of a great proportion of unexplained HSCR cases. 5 The locus in 10q11 probably corresponds to RET, confirming the central role played by this gene in HSCR pathogenesis. Taking into account the persistent failure to detect RET coding sequence mutations in a proportion of familial cases found to be linked to the gene, it has recently been suggested that genetic defects in introns and in the promoter region can contribute in some cases to the HSCR phenotype. [5][6][7] Moreover, several common polymorphi...

Effect of Clostridium butyricum against Microglia‐Mediated Neuroinflammation in Alzheimer's Disease via Regulating Gut Microbiota and Metabolites Butyrate

“…Our previous studies have shown that butyrate could exert an anti‐inflammation effect by decreasing the levels of TNF‐α, IL‐1β, and IL‐6 in the brain of cerebral ischemia/reperfusion injury mice . Butyrate could interfere with Aβ 1‐40 oligomerization and exert multiple effects against neuropsychiatric disorders as an inhibitor of histone deacetylase . Our previous studies also demonstrated that butyrate could exert neuroprotective effects on depression, Parkinson's disease, and traumatic brain injury in the model mice .…”

A common haplotype at the 5′ end of theRETproto‐oncogene, overrepresented in Hirschsprung patients, is associated with reduced gene expression

“…Lymphoblasts were used for testing RNA expression defects (mRNA splicing and total RET expression). To this end, NaB (B5887; Sigma-Aldrich, Milano, Italy) was dissolved in water (1 M stock solution), and delivered to the cells at a final concentration of 5 mM [Griseri et al, 2003]. Treatment was performed overnight.…”

“…Three neuroblastoma cell lines, characterized by different levels of RET expression, were used. In particular, SK-N-BE and SK-N-BE(2)C, representing distinct differentiation stages of the same cell line, display high levels of RET expression, which is more marked in the latter than in the former (T. Bachetti, unpublished data) (RET+ cells), while SK-N-MC are characterized by absence of RET transcription (RET-cells) [Griseri et al, 2003]. Luciferase activities in lysates of transfected cells were assayed and compared by analysis of variance (ANOVA).…”

Aberrant expression of LncRNA‐MIR31HG regulates cell migration and proliferation by affecting miR‐31 and miR‐31* in Hirschsprung's disease

“…According to genetic, epigenetic, and toxicology studies, both genetic factors and environmental factors contribute to HSCR. Many genes have been identified as being involved in the pathogenesis of HSCR, including RET, GDNF, and SOX10, which are only some components involved in the pathogenesis of HSCR. Our previous studies have also demonstrated that some small molecules, like microRNAs (miRNAs), are associated with the occurrence of HSCR …”