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Rescue of human RET gene expression by sodium butyrate: a novel powerful tool for molecular studies in Hirschsprung
P Griseri1, G Patrone, F Puppo
1Laboratorio di Genetica Molecolare, Istituto G Gaslini, Genova, Italy.
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.
Insights
Scientists developed a new method to detect RET gene anomalies in Hirschsprung disease (HSCR) by activating RET expression in cells. This approach identified previously undetectable gene defects in HSCR patients.
Area of Science:
- Genetics
- Molecular Biology
- Developmental Biology
Background:
- The RET gene is crucial for neurocristopathies like Hirschsprung disease (HSCR).
- RET mRNA is typically undetectable in adult cells, limiting genetic anomaly detection.
- Conventional DNA screening misses many HSCR-related gene anomalies.
Purpose of the Study:
- To develop a protocol for activating RET expression in RET-negative cell lines.
- To enable direct investigation of RET mRNA for splicing defects and expression issues.
- To expand upon conventional gene mutation analysis for HSCR.
Main Methods:
- Utilized sodium butyrate (NaB), a histone deacetylase inhibitor, to induce RET expression.
- Tested NaB's effect on RET expression in lymphoblasts from HSCR patients and controls.
- Employed one-round reverse transcription-polymerase chain reaction (RT-PCR) on total RNA.
Main Results:
- Successfully activated RET expression in RET-negative lymphoblasts using NaB.
- Detected impaired RET expression and a splicing defect in two HSCR patients.
- Identified gene abnormalities missed by previous conventional DNA screening.
Conclusions:
- The developed protocol enables RET expression analysis in previously undetectable cases.
- This method can identify RET gene anomalies contributing to HSCR.
- Further application may clarify RET's role in HSCR pathogenesis and predisposition.
