UTILIZATION OF NUCLEOTIDE PROBES FOR THE MEASUREMENT OF SPECIFIC MESSENGER RNA: APPLICATION FOR MOLECULAR DIAGNOSIS OF AUTOSOMAL RECESSIVE SPINAL MUSCULAR ATROPHY

ABSTRACT

Spinal muscular atrophy (SMA) is a lethal autosomal recessive disease. SMA is characterized by degeneration of motor neurons in the spinal cord, causing progressive weakness of the limbs and trunk, followed by muscle atrophy. The gene most highly associated with SMA is the survival motor neuron (SMN) gene. This paper describes the results concerning the development of a quantitative method for the molecular diagnosis of SMA by measuring the amount of cytosolic mRNA from human muscle cells. The procedures using radioactive material and the Enzyme-Linked Immunosorbent Assay (ELISA) non-radioactive method were developed using ³²P-dCTP labeled and biotinylated nucleotide probes, respectively; the results obtained demonstrate that the measurement of specific mRNA could be used as a quantitative method for the molecular diagnosis of SMA. There was a perfect concordance of the results obtained between the procedure using radioactive material, the ELISA method and the single-strand conformation polymorphism (SSCP) analysis regarding negative and positive SMA samples. All values obtained for the control group were significantly greater than the ones obtained for the SMA positive samples (33–76% in radioactive method and 38–54% in ELISA method). The methods developed in this study may be applicable to the diagnosis (detection of homozygous and heterozygous deletions in exons 7 and 8 of the SMN gene) and the control of mRNA concentrations in future gene therapy of patients with SMA.

Keywords:

• Nucleotide probes
• Molecular diagnosis
• Autosomal recessive spinal muscular atrophy
• Radioactive label
• Biotin label
• Enzyme-Linked Immunosorbent Assay

ACKNOWLEDGMENTS

This work was supported by grants from the Association Française contre les Myopathies and the Agence Nationale de la Valorisation et l’Aide à la Recherche.