Triplet-Primed PCR Is More Sensitive than Southern Blotting-Long PCR for the Diagnosis of Myotonic Dystrophy Type1

Abstract

Genetic testing of myotonic dystrophy type 1 (DM1) is very important because it enables the diagnosis and indicates the severity of the disease. Mutation analysis is based on the detection of the number of CTG triplets in the 3′ untranslated region of the myotonic dystrophy protein kinase (DMPK) gene. Sometimes it could be complicated by the presence of different patterns of repeat interruptions in the 5′ and 3′ ends of the expanded alleles recently described in about 3% to 5% of patients. To make molecular diagnosis easier and faster, the use of triplet-primed PCR (TP-PCR) for the detection of expansions in DM1 and other dynamic mutation diseases was proposed. Here we present the results of a retrospective study performed by TP-PCR on 100 subjects previously analyzed by Southern blotting-long PCR.

Introduction

Myotonic dystrophy type 1 (DM1; OMIM #160900) is an autosomal dominant multisystemic disorder showing a prevalence that ranges from 1:100000 to 3-15:100000. DM1 is caused by an expansion of a CTG triplet in the 3′ untranslated region of the myotonic dystrophy protein kinase (DMPK) gene. The gene is located on chromosome 19q13.3 and is expressed in skeletal muscle (Brook et al., 1992; Fu et al., 1992; Mahadevan et al., 1992). Normal individuals have 5 to 37 CTG repeats, while alleles containing more than 38 CTG are considered abnormal. DMPK alleles between 37 and 49 CTG are premutated alleles, and patients with these alleles are asymptomatic but at risk of having children with larger pathologically expanded repeats (IDMC, 2000). Alleles greater than 50 CTG are usually associated with symptomatic disease. Larger repeat size correlates with increased severity of symptoms and decreased age of onset (IDMC, 2000; Salehi et al., 2007). Recently, different patterns of repeat interruptions in the 5′ and 3′ ends of the expanded alleles have been described in about 3% to 5% of patients of Dutch, Czech, and French origins (Musova et al., 2009; Braida et al., 2010). Genetic testing of DM1 is very important because it can both confirm the diagnosis and give indications about the severity of the disease. Mutation analysis is based on the detection of the number of CTG triplets in the 3′ untranslated alleles of the DMPK gene. So far, no single method could be used to detect the entire range of alleles; therefore, diagnostic techniques include long PCR to detect normal alleles followed by blotting and hybridization with the (CTG)5 probe to detect expanded alleles (Gennarelli et al., 1998). These techniques are time-consuming and require careful evaluation of the results. To make molecular diagnosis easier and faster, the use of triplet-primed PCR (TP-PCR) for the detection of expansions in DM1 and other dynamic mutation diseases was proposed by Warner et al. (1996). TP-PCR uses triplet priming to produce a set of amplicons, characteristic of the expanded allele. The aims of this study were to verify the validity of this method to detect CTG expansion in patients showing DM signs and in relatives of DM1 patients and to evaluate the presence of interruptions in the CTG repeats in the Sardinian population.

Materials and Methods

Study subjects

We retrospectively analyzed 100 DNA samples belonging to Sardinian subjects referred to our laboratory as clinically suspected DM patients and DM1 relatives. Signed informed consent was obtained from all the subjects.

Genotyping

All DNA samples were previously analyzed with long PCR. The samples showing only one allele underwent to blotting followed by hybridization with (CTG)5 γATP labeled probe (Gennarelli et al., 1998). Successively, all 100 samples were analyzed using three different PCRs: conventional PCR using primers DM1for-FAM/DM1rev-VIC and two TP-PCRs performed in both directions according to Radvansky et al. (2011a, 2011b) using primers DM1-CTG-for/DM1-rev/P3, and DM1-for/DM1-CAG-rev/P3 (Fig. 1C) (Table 1). PCR products were separated on an ABI PRISM 3130 XL, and the data were analyzed using GeneMapper 3.7 software (Life Technologies). The same forward and reverse TP-PCR products in one patient (AB) were subjected to blotting followed by hybridization with (CTG)5γATP labeled probe (Southern blotting). The TP-PCRs were performed using P4-CC and P4-CTC as forward primers and Somy 4R as the reverse primer (Table 1).

FIG. 1.

(A) Analysis of myotonic dystrophy protein kinase (DMPK) repeat in patient AA by conventional PCR (A1) showing the normal allele; CTG triplet-primed PCR (TP-PCR) showing an interruption in the expanded allele (A2); CAG TP-PCR showing a CTG expanded allele (A3). (B) Sequencing of TP-PCR product showing the CCG interruptions (Underlined). (C) Location of primers for detection of CTG repeat in 3′ UTR DMPK.

Table 1.

Primer Sequences

Primer name	Primer sequence (5′→3′)
DM1for-FAM	GGG GCT CGAAGG GTC CTT GT
DM1rev-VIC	GTG CGT GGA GGA TGG AAC
DM1-CTG-for	AGC GGA TAA CAATTT CACACAGGA TGC TGC TGC TGC TGC TGC TG
P3	AGC GGA TAA CAATTT CACACAGGA
DM1-CAG-rev	AGC GGA TAA CAATTT CACACAGGA CAG CAG CAG CAG CAG CAG
P4-CTC	TAC GCA TCC CAGTTT GAG ACGTGC TGC TGC TGC TGC TC
P4-CC	TAC GCA TCC CAGTTT GAG ACG TGC TGC TGC TGC TG
Somy 4R	CGG GTT TGG CAAAAG CAAATT TCC CGA
DM1for	GGG GCT CGAAGG GTC CTT GT
DM1rev	GTG CGT GGA GGA TGG AAC

Sequencing

The TP-PCR products obtained by the use of P4-CC as forward primer and Somy 4R as reverse primer and TP-PCR product obtained with DM1for and DM1-CAG rev primers were sequenced using primer Somy4R and DM1-CAG-rev, respectively (Table 1) (Life Technologies) (Musova et al., 2009).

Results

Using the long PCR method followed by hybridization with (CTG)5 γATP-labeled probe, 54 samples out of 100 showed two fragments corresponding to normal alleles; the remaining 46 showed one fragment corresponding to the normal allele and one expanded fragment corresponding to the mutated allele. Utilizing the approach based on one conventional PCR and two bidirectional TP-PCRs in the same samples, we found that the results were concordant for 99 samples. A pattern of interruption was present in one DM1 sample (AA) (Fig. 1A). TP-PCR using primers DM1-CTG for and Somy4R, followed by sequencing with primer Somy4R, revealed a CCG interruption in the stretch of CTG in an AA patient (Fig. 1B).

One DNA sample (AB) belonging to an asymptomatic patient gave discordant results when reverse TP-PCR was performed. As shown in Figure 2A, with long PCR a single fragment in the normal range was detected (lane 1). Again, using conventional PCR, only one 160-bp fragment corresponding to a 4CTG allele was detected. The expanded fragment was not revealed by the forward TP-PCR, but only by reverse TP-PCR (Fig. 2B) even after Southern blotting as shown in Figure 2C. Sequencing analysis of TP-PCR products obtained with primers DM1-for and DM1-CAG-rev revealed a stretch of >70 CTG in patient AB. No TP-PCR products were obtained when P4-CTC and P4-CC were used as forward primers and Somy 4R as the reverse primer (Table 1).

FIG. 2.

Analysis of DMPK repeat in patient AB. (A) Southern blotting of long PCR product: lane 1, patient AB; lane 2, normal control; lane 3, mutated control. (B) Conventional PCR (1) showing the normal allele; CTG TP-PCR with no expanded allele (2); CAG TP-PCR showing a CTG expanded allele (3). (C) Southern blotting of CTG and CAG TP-PCRs: lanes 1, 4 patient AB; lanes 2, 5 normal control; lanes 3, 6 mutated control.

Discussion

Diagnostic testing of molecular pathologies due to dynamic mutations with possible large expansions, such as DM1, has been always troublesome. The reference method for DM1 has been long-range PCR followed by blotting and hybridization with radiolabeled probe to identify expansions up to 2500-3000 repeats (Gennarelli et al., 1998). This method is time-consuming and requires radioactive material, and sometimes amplification of large expansions could fail. The use of conventional PCR DM1for/DM1rev and TP-PCR has allowed these problems to be overcome, to rapidly detect large CTG expansions, and to exactly size the normal and premutated alleles. Radvansky et al. (2011b) suggested the need to perform TP-PCR in both directions to avoid false-negative results due to the presence of interruptions located in the 3′ end of CTG stretch. The DM1 locus was considered a pure CTG repeats on the basis of the sequencing of small alleles and small expanded alleles with the exception of one interrupted DMPK allele in a sperm donor described by Leeflang and Arnheim (1995). Only recently, it has been shown that interruptions in the CTG-expanded allele are quite frequent having been found in 3%-4% of French DM1 (Braida et al., 2010) and in 5% of Czech DM1 (Musova et al., 2009) and to have important consequences for molecular diagnosis of DM1 because they can give false-negative results. In our population, 1 out of 46 DM1 patients (∼2%) showed a CCG interruption.

In this study TP-PCR in both directions confirmed the diagnosis in 46 DM1 subjects and in 53 out of 54 samples previously found to be normal. One subject did not show any CTG expansion by using both long PCR followed by blotting and hybridization and by TP-PCR using DM1-CTGfor (Kakourou et al., 2010). Only when TP-PCR using DM1-CAG-rev was performed, an expanded fragment was obtained. TP-PCR carried out with primers P4-CTC and P4-CC (Musova et al., 2009) failed to detect the most common CCG and CTC interruptions. By these data AB, previously diagnosed as normal, was shown to contain an expanded CTG allele. The false-negative result obtained by long PCR could be explained by interruptions other than CCG and CTC, a repeat-flanking deletion, and an insertion of non-CTG sequences as recently described by Axford et al. (2011) in UR-ADM9 patient's tissues. In conclusion, the bidirectional TP-PCR assay in combination with conventional PCR seems to be sufficient to correctly establish the presence or absence of DM1-expanded alleles and more effective than long PCR followed by blotting and hybridization.

Footnotes

Acknowledgments

This work was supported by a grant of Regione Autonoma della Sardegna LR11 1990.

Author Disclosure Statement

No competing financial interests exist.

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