The exact pathogenic mechanisms that cause myotonic dystrophy are still elusive. Previous expression studies for the DMPK gene have provided contradictory results, pos­sibly due to different experimental approaches [4-11]. In the present study, both attempted approaches of DMPK expression monitoring indicated an increase of the entire DMPK transcripts in DM patients compared to normal subjects in blood (about three-fold) and in muscle (about four-fold). Therefore, our data do not seem to support the possibility of an erroneous splicing effect of the (CTG)n expansion, as previously suggested [18]. Our findings, with the use of two RT-PCR approaches, were due to the increased amount of mutant DMPK transcripts, as other groups have previously observed [7,8,19]. This increased amount of mutant DMPK transcript may be due either to increased gene expression or a post-transcriptional mech­anism that results in the accumulation of DM mRNA molecules. The latter possibility is supported by evidence of nuclear retention of DM mRNA transcripts [19].

In contrast to the increased DMPK total mRNA level, we observed a decrease in the total RNA level of the DMAHP gene in DM patients, in accordance with previ­ous reports [20,21]. However, previous studies were involved with the expression of DMAHP in cultured myo­blasts and fibroblasts without monitoring the DMPK expression in the same samples. In this report, we found that there is a simultaneous reduction in DMAHP total RNA level and an increase of DMPK total RNA level in all DM patients studied, in comparison to normal controls. Our data seem to be in accordance with the observation that there is nuclear retention of DMPK transcripts but not of DMAHP [22].

In addition to the involvement of the DMAHP gene, the accumulating evidence of the unrelated DM phenotype of transgenic mice over-expressing or completely lacking DMPK [23,24], and a small number of known DM cases without an abnormal size allele [25-27] suggest that the (CTG)n repeat expansion might not be the sole defect causing DM. Since there were no reports in the literature of mutations in other genes of the DM locus (besides DMPK), we performed a CFLP analysis of the DMAHP gene but did not detect any nucleotide changes in 11 DM patients. If the DMAHP gene is indeed involved in DM pathogenesis, we think that our overall data, as well as those of others, support the hypothesis that this is mainly due to decrease of its expression, because of a local nucleosome stabilization effect which represses an enhancer element 5' flanking the gene [20]. However, it remains possible that the increase in DMPK total RNA level and the concomitant reduction in DMAHP total RNA level in muscle and blood cells of DM patients, may be due to post-transcriptional effects on both genes.

Although still unclear in its details, the molecular pathophysiology of DM seems to involve (in combination?) at least two novel pathogenic mechanisms (for review [28-30]). It appears that the (CTG)n expansion causes the complex multisystemic phenotypic characteristics of DM by reflecting the combined effect of the altered RNA processing and deficiency of the DMPK protein (muscle and endocrine abnormalities) and the haploin sufficient homeobox DMAHP gene (facial dysmorphology). In order to clarify the effect of the (CTG)n expansion in the expression of additional flanking genes, further studies of the expression of the DM9-N0 gene [31], at the 5' end of the DMPK gene, are necessary.

Figure 4. DMAHP total mRNA quantitation by RT-PCR. N: normal subject ; D: DM patient. a) In blood: PCR analysis was performed using primers for an internal control (globin gene) producing a 383 bp fragment, along with primers DMAHPF, DMAHPR for the DMAHP gene producing a 137 bp fragment (lower band). b) In skeletal muscle: PCR analysis was performed using primers for an internal control (tpi gene) producing a 227 bp fragment, along with primers DMAHPF, DMAHPR for the DMAHP gene producing a 137 bp fragment (lower band).