INCREASE OF DMPK AND DECREASE OF DMAHP GENE EXPRESSION IN MUSCLE AND BLOOD OF MYOTONIC DYSTROPHY PATIENTS COMPARED TO NORMAL SUBJECTS
Chronopoulou P1, Yapijakis C1, Karadimas C1, Panas M1, Manta P1, Cariolou M2, Vassilopoulos D1
*Corresponding Author: Christos Yapijakis, D.M.D., M.S., Ph.D., Clinical and Molecular Neurogenetics Unit, Department of Neurology, University of Athens Medical School, Eginition Hospital, Athens 11528, Greece; Tel: +30-10-728-9125; Fax: +30-1-881-1243; E-mail: cyapijakis_ua_gr.yahoo.com
page: 29

RESULTS

DMPK gene expression. Total RNA from muscle and blood samples of seven DM patients and three control subjects was used for expression studies of the DMPK gene with two RT-PCR approaches. The first approach included the amplification of exons 9 and 10 as a cis internal control, in addition to amplifying the 3'UTR of the DMPK gene. A multiplex RT-PCR was performed using primers RT3, RT4 and RT5 for the region of exon 9-intron 9-exon 10, as well as primers DM101, DM102 for the CTG repeat region,and primers RT1, RT2 for the region 3' flanking the CTG repeat (Fig. 1). The last set of primers was chosen in order to monitor the effect of the (CTG)n expansion on the transcription at the CpG island. With the conditions used, all fragments were RT-PCR amplified in all samples (including the normal CTG repeat alleles of the patients and control subjects) with two exceptions: a) the long expanded trinucleotide repeats (>200 copies) of DM patients, and b) the DNA fragment corresponding to the intron 9-exon 10 region, with the use of primers RT3 and RT5 (Fig. 1). We explain these observations as indications of expression of both the DM and the normal allelic genes and correct splicing out of intron 9 in the mutant transcript. After normalization, quantitative multiplex RT-PCR revealed an increased amount of all studied regions of the DMPK mRNA in DM patients compared to normal controls (Fig. 2, Table 2). The observed relative amount of all fragments was comparable in all samples of DM patients, therefore we exclude any effect on splicing or variable transcription of the middle and the end of the gene.

The second RT-PCR approach to studying DMPK gene expression included use of trans internal controls, such as the housekeeping gene of tpi and the tissue specific gene of b-globin, in muscle and blood, respectively. Total RNA was RT-PCR amplified using DMPK primers RT1 and RT2, that flank the 3' end of the CTG expansion and lie within the CpG island (Fig. 1). We observed a three-fold increase of DMPK gene expression in blood, and a four-fold increase in muscle of DM patients compared to normal controls, after normalization (Fig. 3, Table 2).

In order to examine whether our findings were due to the increased amount of mutant DMPK transcripts, we searched for normal and mutant transcripts with different allele-specific Bpm I polymorphisms. We were able to demonstrate that this was the case in two of the DM patients who were heterozygotes for the Bpm I polymorphism; therefore, they had distinguishable amounts of normal and mutant DMPK mRNAs.

DMAHP gene expression. The same total RNA sam­ples of DM patients and normal subjects were studied for DMAHP gene expression using primers DMAHPF and DMAHPR from exon A of the gene (Fig. 1). As trans internal controls, we again used the tpi gene for muscle and the b-globin gene for blood. After normalization, a decrease of the DMAHP expression in blood and in muscle (about three-fold and two-fold, respectively) of DM patients compared to normal controls was observed (Fig. 4, Table 2).

CFLP analysis of the DMAHP gene in DM patients. In order to examine the possibility of existing point muta­tions or polymorphisms in the DMAHP gene, that would either be in linkage disequilibrium with the (CTG)n expanded alleles or otherwise affect the DM phenotype, we decided to perform CFLP analysis in the coding region of the gene. We analyzed all three exons and exon/intron junction sites of the DMAHP gene in DNA samples isolated from blood of the same DM patients and four other DM cases using primers PD1F-PD1R, PD2F-PD2R and PD3F-PD3R (Fig. 1). We detected no nucleotide changes, excluding the above-mentioned possibility.

 

 

 

Figure 2. Radioactive multiplex RT-PCR products, using total RNA from skeletal muscle and primer couples RT1-RT2, DM101-DM102, and RT3-RT4, electrophoresed on a 6% polyacrylamide gel. N: Normal subject; DM: DM patient.

 

 

 

 

Figure 3. DMPK total mRNA quantitation by RT-PCR. N: normal subject ; D, D1 and D2: DM patients. a) In blood: PCR analysis was performed using primers for an internal control (globin gene) producing a 383 bp fragment, along with primers RT1 and RT2 for the DMPK gene producing a 152 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 RT1 and RT2 for the DMPK gene producing a 152 bp fragment (lower band).

 

 

Table 2. DMPK and DMAHP expression in blood and muscle samples of DM patients compared to normal controls [a: in the cis RT-PCR method, the ratio of the observed amount of each DNA fragment of the patients was normalized against the corresponding normal control (the value of which was assigned as 1.0)] [b: in the trans RT-PCR method the amount of each observed DNA fragment was normalized against the RT-PCR product of the control b-globin genes, and tpi for blood and muscle, respectively (the value of which was assigned as 1.0)]. DM: DM patients, N: normal controls.

 

a: DMPK Expression (ratios observed by cis RT-PCR)

Gene Region

Primers

N

DM

Exon 9-exon 10

RT4; RT3

1

3.6 0.4

Intron 9-exon 10

RT5; RT3

0

0

Normal (CTG)n allele

DM101; DM102

1

3.5 0.5

3'UTR

RT1; RT2

1

3.8 0.4

b: DMPK and DMAHP Expression (ratios observed by trans RT-PCR)

Blood: Gene

N

DM

  DMPK (3'UTR)

1.1 0.2

3.1 0.3

  DMAHP (exon 1)

0.9 0.2

0.3 0.1

Muscle: Gene

N

DM

  DMPK (3'UTR)

1.2 0.2

4.3 0.4

  DMAHP (exon 1)

1.1 0.2

0.5 0.2




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