The Expanding World of Myotonic Dystrophies: How Can They Be Detected?

Abstract

Myotonic dystrophy (DM) comprises at least two genetically distinct forms, both of which are caused by expansions of microsatellite repeats. The expansion of a CTG repeat in the DMPK gene leads to the first genetic form (DM type 1), and the expansion of a CCTG repeat in the ZNF9 gene causes the second genetic form of the disease (DM type 2). In both cases, the repeat units may expand to several thousand repeats, and the number of repeats in the expanded alleles shows a high degree of meiotic and somatic instability. The unprecedented size of expansions and their dynamic nature still represents a diagnostic challenge, which has been facilitated using different methods and modifications since the identification of the underlying mutations of these disorders. Here, we present an overview of the basic methods described for the purpose of identification of the DM type 1 and DM type 2 expansions and discuss particular modifications and improvements implemented to extend the detection ranges of these methods. Our review focuses on the advantages and disadvantages of the methods based on Southern blot analysis, polymerase chain reaction amplification, and in situ hybridization techniques and also on the possibilities of preimplantation and prenatal genetic testing.

Introduction

Myotonic dystrophy (DM) is the most common autosomal dominant neuromuscular disorder in adults, and it comprises at least two genetically distinct forms. The underlying mutation of the first genetic form of the disease, myotonic dystrophy type 1 (DM1; OMIM#160900), was identified in 1992 as an unstable expansion of the (CTG)_n repeat tract in the 3′-untranslated region of the DMPK (dystrophia myotonica protein kinase; OMIM*605377) gene in the chromosomal region 19q13.3 (Brook et al., 1992; Fu et al., 1992; Mahadevan et al., 1992). The number of CTG repeats is highly polymorphic, in both healthy individuals and DM1 patients. According to the DNA testing guidelines of the International Myotonic Dystrophy Consortium, healthy individuals have alleles with between 5 and 35 CTG repeats, whereas in patients with clinical signs of DM1, the number of repeats varies from 50 to several thousands. Alleles with between 35 and 49 CTG repeats are without known clinical symptoms; however, they are considered as premutations, with the potential of expanding into the mutation range during gametogenesis (The International Myotonic Dystrophy Consortium, 2000). Different patterns of highly unstable CCG, CTC, and GGC sequence interruptions in the 3′ end of the expanded CTG repeat tract have been reported in one allele of a Caucasian individual with unknown clinical status (Leeflang and Arnheim, 1995) and in a cohort of patients of Czech (Musova et al., 2009), Dutch, and French origins (Braida et al., 2010). These interruptions were identified in almost 5% of expanded alleles among the Czech (Musova et al., 2009) and in ∼3%-4% among the French patients (Braida et al., 2010).

The mutation responsible for the second genetic form of the disease (myotonic dystrophy type 2; DM2; OMIM#602668) was linked in 1998 to chromosomal region 3q21.3 (Ranum et al., 1998) and identified as an unstable expansion of the (CCTG)_n repeat tract in the first intron of the ZNF9 gene (Zinc Finger Protein 9; OMIM*116955) (Liquori et al., 2001). The (CCTG)_n repeat is a part of a complex repetitive motif (TG)_n(TCTG)_n(CCTG)_n, and each of the elements is highly polymorphic, even in healthy individuals. Healthy range alleles with a complex repeat tract from 94 to 217 bp in length have been identified, and the largest identified allele contained 32 CCTG repeats (Bachinski et al., 2009). Expanded alleles containing CCTG repeats between 75 and more than ∼11,000 were reported, with a mean of ∼5000 (Liquori et al., 2001). The CCTG repeat tract is generally interrupted by GCTG, TCTG, or ACTG motifs in normal alleles, whereas it is uninterrupted in premutation and expanded alleles (Liquori et al., 2001; Bachinski et al., 2003, 2009).

Establishing the etiological diagnosis in patients with clinical suspicion of muscular dystrophies is highly relevant, not only for the application of correct therapeutic approaches, but also for the life and family planning decisions and prognostic approaches for patients. Because the clinical signs of DM1 and DM2 are heterogeneous, their symptoms can be overlapping with other neuromuscular disorders and their age of onset is extremely variable, and differential diagnosis based on clinical examination can be uncertain (Botta et al., 2006). Therefore, genetic testing plays an important role in making an accurate diagnosis in myotonic dystrophies, because it allows direct detection of the (CTG)_n or (CCTG)_n expansion (The International Myotonic Dystrophy Consortium, 2000). It also allows preimplantation, prenatal diagnosis (PND), and preclinical testing in asymptomatic subjects, and it can identify individuals with alleles in the premutation range.

Although the identification of the (CTG)_n or (CCTG)_n expansion on the DNA level is highly important, mutation analysis of these expansions poses a challenge for molecular diagnostic methods. It is complicated by the immense length of the expanded alleles (Cheng et al., 1996; Liquori et al., 2001) and by the extremely stable secondary structures forming inside the repetitive GC-rich sequence (Pearson and Sinden, 1996), which interfere with conventional polymerase chain reaction (PCR) amplifications. Although several improvements have been introduced to overcome this challenge, there is still no single method that would reliably identify and size all ranges of expanded alleles in neither the DM1 nor DM2 locus (Carson, 2009).

Here, we present a brief overview of the methods and modifications that have been introduced for expansion detection since the identification of the DM1 and DM2 causative mutations. We will discuss the possibilities of using Southern blot hybridization, PCR-based analyses, in situ hybridization techniques, and linked genetic markers analysis, and the possibilities of preimplantation and prenatal molecular testing.

Southern Blot Analysis of Enzymatically Digested Genomic DNA

Although Southern blot analysis (SBA) of enzymatically digested genomic DNA is a time-consuming method, is unsuitable for automation, and requires a large amount of intact, high-molecular-weight genomic DNA (Falk et al., 2006; Carson, 2009), it represents a widely used method for the identification and sizing of extremely large expansions in the molecular genetic testing of DM1 and DM2 (Brook et al., 1992; Fu et al., 1992; Shelbourne et al., 1993; The International Myotonic Dystrophy Consortium, 2000; Liquori et al., 2001; Day et al., 2003). In general, expanded alleles can be detected as single bands, multiple bands, or smears (Day et al., 2003). When the expanded allele shows a smear, the size of the allele with the highest density should be reported (The International Myotonic Dystrophy Consortium, 2000).

Although conventional SBA provides results accurate enough for the detection of large DM1 expansions, it is unsuitable for the identification of premutated alleles and alleles with small expansions (Brunner et al., 1992; Petronis et al., 1996). Moreover, its failure has been reported in ∼20% of DM2-positive samples (Bachinski et al., 2003; Day et al., 2003). Increased quality of results and better resolution can be obtained by using field inversion electrophoresis (Bachinski et al., 2003), pulsed-field gel electrophoresis (Jakubiczka et al., 2004), and restriction enzymes that cut closer to the repeat region (Erginel-Unaltuna and Akbas, 2004). A more efficient transfer of high-molecular-weight DNA to nylon membranes can be achieved by incubating the gel in HCl before transferring, which breaks the high-molecular-weight DNA into smaller pieces (Carson, 2009). Although only as a supportive method, semiquantitative SBA has also been reported to measure the dosage of the detected healthy range fragment (Jakubiczka et al., 2004).

Nakamori et al. reported more sensitive detection (even from as little as 20-50 ng of input DNA) with the use of digoxigenin-labeled short CAG- or CCTG-repeat-specific locked nucleic acids probes, together with the elimination of the majority of flanking sequences using four-base restriction enzymes. However, detection of alleles with less than ∼750 CTG repeats was possible only after enrichment of the target region by rolling circle amplification before SBA (Nakamori et al., 2009).

PCR-Based Molecular Testing

Conventional PCR amplification with primers closely flanking the DM1/DM2 repeat region is suitable for the exclusion of DM1 or DM2 diagnosis only if both nonexpanded alleles are detected, because large expansions are refractory to amplification. A detection limit of ∼80-100 CTG repeats is common in DM1 (Brunner et al., 1992; Warner et al., 1996; Carson, 2009), whereas only alleles in the healthy range are amplifiable in DM2 using conventional PCR (Bachinski et al., 2009; Carson, 2009).

The CTG repeat number of amplifiable DMPK alleles can be determined from the amplicon length. In contrast, the CCTG repeat number in the ZNF9 gene cannot be directly determined, because this repeat can be amplified exclusively in combination with the adjacent polymorphic repeats of the complex tract. Sizing based on amplification from flanking primers thus leads to the combined length of all three repeats and the allele size is generally reported in base pairs instead of the exact number of the CCTG repeat (Carson, 2009). Amplicons can be detected by conventional polyacrylamide gel electrophoresis, the automated laser fluorescent DNA analysis system, and capillary electrophoresis. DM2 alleles differing by just 2 bp were reported to be not resolvable by conventional polyacrylamide gel electrophoresis of radioactively labeled PCR amplicons (Sallinen et al., 2004). If amplicons are detected using capillary electrophoresis, labeling both primers with different dyes (instead of labeling only one of the primers) can increase the reliability of the assay, because of the altered mobility shift of the complementary DNA strands observed after amplification of an allele containing CCG interruptions (Radvansky et al., in press).

Despite the fact that conventional PCR amplification can be used mainly for the detection of alleles in the healthy range, it still represents a useful initial test for both DM1 and DM2, because the heterozygosity of these loci in European populations was estimated to be ∼78%-83% for DM1 (Zerylnick et al., 1995; Tishkoff et al., 1998) and 85%-95% for DM2 (Liquori et al., 2001; Bachinski et al., 2003; Day et al., 2003).

Long-range PCR assays have been reported to improve PCR amplification possibilities. In addition to the combination of primers not closely flanking the repeat region, they also utilize several modifications, including special DNA polymerases (Cheng et al., 1996), PCR buffers and additives, such as long template additives (Cheng et al., 1996; Gennarelli et al., 1998; Bonifazi et al., 2004; Kakourou et al., 2010), GC-enhancer, Taq-stabilizer (Skrzypczak-Zielinska et al., 2009), dimethyl sulfoxide (Falk et al., 2006), glycerol (Kakourou et al., 2010), and partial substitution of 7-deaza-dGTP for dGTP (Cheng et al., 1996; Gennarelli et al., 1998; Bonifazi et al., 2004; Falk et al., 2006). In DM1 alleles, the optimal percentage of 7-deaza-dGTP varies from 20% to 90%, depending upon the expansion size and detection method. Although larger expansions were amplified more effectively using higher percentages of 7-deaza-dGTP, incorporation of a high level of 7-deaza-dGTP interfered with ethidium bromide staining (Cheng et al., 1996). Using deoxyinosine triphosphate did not lead to comparable results. Alternative dyes, such as SYBR Green I, propidium iodide, acridine orange, Hoechst No. 33258 on agarose gel, and silver nitrate on polyakrylamide gel, did not lead to better visualization results of large alleles (Cheng et al., 1996). The detection limit of 800-1700 CTG repeats has been reported after direct visualization of amplified alleles on gels (Cheng et al., 1996; Skrzypczak-Zielinska et al., 2009), or 2700-3700 CTG after transfer to nylon membranes and oligo-specific hybridization (Cheng et al., 1996; Gennarelli et al., 1998). In DM2, oligo-specific hybridization-based detection of PCR amplicons allowed visualization of expanded alleles up to 25 kb in length; however, their sizing was possible only in alleles smaller than 15 kb (∼3700 CCTG repeats) (Bonifazi et al., 2004). Using oligo-specific hybridization-based detection of PCR amplicons, a higher resolution in sizing of expanded alleles was reported in comparison to SBA of enzymatically digested genomic DNA in both DM1 (Gennarelli et al., 1998) and DM2 testing (Bonifazi et al., 2004).

Small-pool PCR amplification of restriction-digested and diluted genomic DNA has been used to resolve discrete bands in samples giving smeared or diffused bands by SBA. This approach increased the detection limit over 1300 CTG repeats with oligo-specific hybridization and autoradiography-based amplicon detection (Monckton et al., 1995; Wong et al., 1995).

The “triplet” or “tetraplet” repeat-primed PCR assay (TP-PCR), originally developed by Warner et al. for DM1 and Huntington's disease, represents a robust and reliable PCR system that can rapidly identify the presence of expanded alleles for any disorder caused by repeat expansions. Although it is not able to determine the exact size of the repeats over a certain threshold, it can distinguish healthy homozygous and affected heterozygous samples with no length restrictions. TP-PCR uses a fluorescently labeled locus-specific flanking primer, a second primer that has a 3′ part complementary to the repeat sequence, and a 5′ tail common with the third primer that has no homology to known human sequences. Specificity is ensured by the locus-specific primer, whereas the repeat-specific primer amplifies from multiple priming sites within the repeat region. Such a primer combination produces a set of amplicons forming a characteristic ladder where the signal intensity diminishes gradually with increasing amplicon size. Amplicons in the ladder follow each other in a 3 bp periodicity in “triplet” (DM1) (Warner et al., 1996; Falk et al., 2006) or in a 4 bp periodicity in “tetraplet” (DM2) repeat-primed PCR (Bachinski et al., 2003; Day et al., 2003). Sizing of alleles in the healthy range is based on the number of amplicons, and thus it is not affected by alterations in the electrophoretic mobility. Unexpected sequence interruptions in DM1 expanded alleles may lead to altered TP-PCR results, because the repeat-specific primer cannot bind to the interrupted region (Musova et al., 2009; Braida et al., 2010). This may lead to a discontinuous or prematurely terminated signal and, therefore, to false-negative results and misdiagnosis. If such an interruption is located close to the 3′ end of a large CTG repeat, the discontinuous signal affects only the reverse directional TP-PCR, whereas the forward directional TP-PCR remains unaffected. If the interrupted allele is short, both the forward and reverse directional TP-PCR results will be affected (Radvansky et al., in press). On the other hand, the TP-PCR assay is the only generally used DM1 testing method that can identify such unexpected sequence interruptions, especially if it is performed simultaneously in both directions (Musova et al., 2009).

Amplicons are generally detected using a fluorescently labeled locus-specific flanking primer (Warner et al., 1996; Falk et al., 2006) or a primer that recognizes the tail part of the repeat-specific primer by capillary electrophoresis on a genetic analyzer (Bachinski et al., 2003; Sallinen et al., 2004). Day et al. (2003) used an internal probe and SBA to ensure specificity, because they experienced a high number of false-positive samples, with simple ultraviolet visualization or detection by automatic genetic analyzer.

For DM1, a sizing limit of up to 50-80 CTG repeats has been reported (Warner et al., 1996; Falk et al., 2006; Radvansky et al., in press), whereas for DM2 there were no specific data about the sizing limit, possibly because of the complexity of the CCTG motif.

Quantitative fluorescent PCR has recently been described for the molecular diagnosis of DM1 and it is performed with three primer sets. They flank the DMPK repeat region and two reference genes, in one multiplex reaction. One primer from each pair must be fluorescently labeled with the same fluorescent tag. Reference markers should contain neither large duplications nor deletions. Following capillary electrophoresis, amplicons resulting from the CTG repeat flanking primers are used for the identification and sizing of alleles amplifiable with conventional PCR (up to ∼100 CTG repeats). If only one allele is detected, the allele dosage ratios of DM1 amplicon to reference genes 1 and 2 can be used to determine whether the detected allele is in homozygous or heterozygous state. The allele dosage ratios of reference gene 1 to reference gene 2 can serve as the quality control. The exact sizing of the expanded nonamplifiable alleles is not possible by this method and it requires an additional analytical step (Skrzypczak-Zielinska et al., 2009).

In Situ Hybridization Techniques

Expanded DMPK or ZNF9 transcripts form several discrete nuclear foci in different types of cells of DM1 and DM2 patients (Taneja et al., 1995; Mankodi et al., 2001). Fluorescent in situ hybridization (FISH) has been utilized to determine the presence or absence of nuclear foci containing expanded CUG or CCUG RNA molecules in cells from interphase nuclei of trophoblast cells of DM1-predicted fetuses (Bonifazi et al., 2006) and also in muscle biopsy tissues of DM2 patients (Liquori et al., 2001). However, alleles with <100 CTG repeats do not form detectable foci (Bonifazi et al., 2006). Crosshybridization of the antisense CUG probe to CCUG foci in DM2 cells was reported by Liquori et al. (2001), but they did not find similar crosshybridization of the antisense CCUG probe to CUG foci in DM1 cells. Sense probes can be used for testing whether the probe hybridized to RNA or DNA (Liquori et al., 2001).

Sallinen et al. also tested the utility of chromogenic in situ hybridization on muscle sections and DNA fiber-FISH for the detection of DM2 expansion mutation. The sense probe detects the expanded allele on the DNA level as a single spot per myonucleus, whereas the antisense probe detects multiple RNA foci in myonuclei (Sallinen et al., 2004).

Analysis of Linked Genetic Markers

Several intragenic and extragenic polymorphic markers have been described in close proximity to DM1 and DM2 loci, and certain haplotypes have been identified, in which expanded DM1 and DM2 alleles can be generally found (Imbert et al., 1993; Neville et al., 1994; Bachinski et al., 2003; Liquori et al., 2003). The most studied among these markers is the Alu insertion/deletion polymorphism located in intron 8 of the DMPK gene (Mahadevan et al., 1993), from which the larger allele was found to be in complete allelic association with the pathological alleles causing DM1 in patients of European and Asian ancestry (Imbert et al., 1993; Mahadevan et al., 1993; Neville et al., 1994; Yamagata et al., 1996). This polymorphism can be typed by the three-primer-based allele-specific amplification originally described by Mahadevan et al. (1993). As the markedly smaller size of the deletion-specific PCR product can lead to mistyping of heterozygous samples through preferential amplification of this deletion allele, the protocol was further modified with a newly designed insertion-specific primer (Zerylnick et al., 1995) and high-resolution melting-based amplicon detection (Radvansky et al., 2010). Linkage disequilibrium between the DM2 mutation and an SNP located in the first intron of the ZNF9 gene has been also described in the Italian population. Segregation analysis of these polymorphisms can support the results of direct molecular tests for DM1 and DM2 expansions (Vallo et al., 2005; Botta et al., 2006).

Preimplantation Genetic Testing

The development of diagnostic tests for preimplantation genetic diagnosis is generally challenging, because they have to be fast enough to allow rapid transfer of biopsied in vitro fertilized embryos, extremely sensitive to detect the target sequence even from a single-cell, and sufficiently reliable and accurate to ensure that only unaffected embryos are transferred. Amplification failure, allelic dropout (ADO), and sample contamination represent problems that may result in misdiagnosis or diagnosis failure (Thornhill et al., 2005).

The first preimplantation genetic diagnosis tests for DM1 were based on the detection of the normal-sized allele of the affected parent, which had to be distinguishable from the two alleles of the healthy parent (Sermon et al., 1997). Using nested PCR and agarose gel evaluation, a low amplification efficiency and high ADO rate were reported (Sermon et al., 1997). The high rate of ADO was reduced by using fluorescent PCR and fragment analysis on an automated sequencer (Sermon et al., 1998, 2001). For partially informative or completely uninformative couples, additional analysis of linked or unlinked highly polymorphic short-tandem repeat markers can reduce the risk of misdiagnosis and also allow detection of ADO and chromosomal mosaicism (Dean et al., 2001; Piyamongkol et al., 2001). Linked markers can prove the diagnosis by back-up linkage analysis and they can detect DNA contamination in the sample (Dean et al., 2001), whereas nonlinked markers can only serve for contamination detection (Piyamongkol et al., 2001). Using more than one linked short-tandem repeat marker on either side of the CTG region can prevent misdiagnosis resulting from crossover between the CTG repeat and one of the studied markers (Kakourou et al., 2007). Further improvement was achieved by the successful application of TP-PCR at the single-cell level (Sermon et al., 2001; Kakourou et al., 2008), although problems with detection of homozygous alleles containing five CTG repeats have recently been reported (Kakourou et al., 2010). A modified assay combining the conventional amplification, TP-PCR amplification, and the amplification of a linked polymorphic marker in one multiplex PCR was implemented to increase the reliability of this assay. This modification allows simultaneous identification of the nonexpanded alleles, confirmation of the presence or absence of an expanded allele, and the detection of the phase allele of the linked marker to support the diagnosis and also the detection of contamination (Kakourou et al., 2010).

Prenatal Genetic Testing

PND for DM1 is possible by analysis of fetal cells obtained by invasive (chorionic villus sampling and amniocentesis) (Norman et al., 1989; Lavedan et al., 1991; Myring et al., 1992) or noninvasive techniques (trophoblast cells from the lower part of the uterine cavity, fetal DNA in maternal plasma) (Massari et al., 1996; Amicucci et al., 2000) and requires a reliable and fast assay to reduce the time required for DNA testing (Zuhlke et al., 2000). Analysis of DNA from both parents may be required to exclude maternal contamination in the fetal DNA sample and to verify the results (The International Myotonic Dystrophy Consortium, 2000). Before the gene responsible for DM1 has been identified, restriction fragment length polymorphisms that are genetically linked to DM1 were used for prenatal testing in families with DM1 (Meredith et al., 1986; Norman et al., 1989). This approach was strongly limited with the informativeness of the markers in a particular family, inadequate family structure, or the possibility of genetic recombination (Norman et al., 1989). After the identification of the underlying mutation, most of the previously discussed methods were implemented for purposes of PND, including SBA (Myring et al., 1992), PCR amplification with hybridization-based amplicon detection (Zuhlke et al., 2000), and RNA-FISH (Bonifazi et al., 2006). Confirmatory testing is possible using linked genetic markers (Brunner et al., 1992). The possibility of performing prenatal DM1 testing for detection of paternally inherited expanded alleles was also reported using free fetal DNA circulating in maternal plasma with nested-PCR, long-PCR, and SBA (Amicucci et al., 2000).

Allele Size Heterogeneity and Its Consequences on Molecular Diagnostic Testing

Somatic heterogeneity of the repeat number of expanded alleles is a characteristic and frequent feature of DM1 and DM2, which plays an important role in both molecular diagnostic testing and assessing the prognosis of these disorders. In the following section, relevant findings related to this somatic allele size heterogeneity of the expanded alleles will be discussed. However, it should be mentioned that in many cases the available data are discordant and enunciating precise conclusions at present is problematic.

The repeat numbers of the expanded DM1 and DM2 alleles were found to differ in different cells in the same tissues in the same individual. This expansion size heterogeneity, for example, in leukocytes, was found to increase with average allele size in patients over 20 years of age (Monckton et al., 1995; Wong et al., 1995) and also with increasing age of the DM1 patients (Wong et al., 1995; Martorell et al., 1998). Additionally, the average expansion size was also found to increase with increasing age in both DM1 and DM2 (Wong et al., 1995; Martorell et al., 1998; Liquori et al., 2001; Day et al., 2003).

Striking differences of expanded allele sizes were also reported between different tissues of the same individual, and larger DM1 expansions were generally identified in muscle tissues (Anvret et al., 1993; Lavedan et al., 1993; Jansen et al., 1994; Thornton et al., 1994; Monckton et al., 1995; Zatz et al., 1995) and sperm cells (Monckton et al., 1995) in comparison with leukocytes. The largest expansions were measured in kidney and cardiac muscle (Lavedan et al., 1993; Jansen et al., 1994; Thornton et al., 1994). Anvret et al. (1993) reported certain, but not exclusive, correlation between expansion sizes in the leukocytes and muscle tissues. Zatz et al. (1995), however, found increasing differences between expansion length in muscles and leukocytes with increasing patient age.

Marked within and between tissue allele size heterogeneity may complicate the prognostic possibilities of the progression and severity of the disease based on sizing the expanded allele in individual patients. Age-dependent increase of the DM1 expansion size was not observed in muscle tissues (Anvret et al., 1993; Thornton et al., 1994), suggesting that expansion sizing in muscle tissues may be a better predictor of disease severity in comparison to leukocytes. This observation of Anvret et al., however, was questioned by Wong et al. (1995), whereas Zatz et al. (1995) reported no significant correlation between expansion sizes in muscles and the age of onset of DM1. On the other hand, the size of the expansion in leukocytes was found to be a good, although not absolute, indicator of clinical severity (Zatz et al., 1995; Gennarelli et al., 1996; Marchini et al., 2000; Salehi et al., 2007), at least in young patients (Martorell et al., 1998) or in patients with small expansions (Hamshere et al., 1999; Marchini et al., 2000; Savic et al., 2002). In DM2, no significant correlation between the age of onset and the expansion size has so far been observed. Marchini et al. (2000) emphasized that individual clinical manifestations of DM1 should be evaluated for correlation with measured expansion sizes instead of the overall severity of the disease estimated by grouping distinct clinical manifestations. It has also been suggested that in such correlation studies the lower boundary of somatic distribution in leukocytes, that is, the bottom of the smear, can better represent the progenitor allele size than the midpoint value of the smear (Marchini et al., 2000; Martorell et al., 2000).

Within tissue, repeat length heterogeneity may have a considerable impact on molecular diagnostic testing, because in the analyzed sample it can lead to the presence of several discrete bands (Liquori et al., 2001) or a smeared signal after SBA or PCR, instead of one discrete band (Brook et al., 1992; Fu et al., 1992; Mahadevan et al., 1992; Liquori et al., 2001; Day et al., 2003; Bonifazi et al., 2004). For example, when using SBA, extreme allele size heterogeneity can even lead to failure of the detection of expanded alleles in ∼20% of DM2 patients (Day et al., 2003). The smears are most likely the results of a simultaneous analysis of a heterogeneous collection of cells where the size of expanded alleles differs. The repeat length variation causing smeared bands can be characterized in detail by using small-pool PCR (Monckton et al., 1995; Wong et al., 1995). Differences between tissues appear to have no effect on differential diagnosis based on expansion detection at the DNA level, because the presence of the expanded allele, although differently sized, has been proven in all studied tissues of the affected individuals.

Several studies have demonstrated that somatic mosaicism in DM1 is age dependent, tissue specific, and expansion biased, and therefore, the measured allele size is highly dependent on the age of the patients at sampling and on the tissue from which the isolated DNA is analyzed. Moreover, it has to be considered that several reported correlations are not absolute and refer only to a large group of patients and not to individual ones. Therefore, precise prediction of the phenotype with respect to the severity and the age of onset of the symptoms based on repeat sizes in individual DM1 patients is not recommended (The International Myotonic Dystrophy Consortium, 2000), or at least it is challenging and requires special caution in genetic counseling (Prior, 2009).

Conclusions

As different diagnostic assays offer different advantages in DM1 and DM2 testing (Table 1), it can be concluded that for the selection of the appropriate assay, the exact purpose of the testing should be considered. Several factors may influence the decision, such as, whether the test is aimed only at proving the presence or absence of the expanded alleles, or if there is a demand for exact sizing of the expanded alleles. The time required for the analyses and also the amount and quality of the DNA samples should be also taken into consideration. In both DM1 and DM2, the same principles can be applied in choosing the appropriate diagnostic procedures. Conventional PCR amplification represents a useful first analytic step in all cases. If two healthy range alleles are detected, the DM diagnosis can be excluded. Although alleles with premutations or small mutations can be also identified, the detection limit can vary, depending on the reaction conditions. If only one allele is detected, further analysis is required. If exact allele sizing is not necessary, two PCR-based assays can be used. The quantitative fluorescent PCR-based approach is the single current method that utilizes conventional PCR and simultaneously identifies the presence or absence of the expanded alleles. However, its accuracy and reliability has not been further proven since its very recent description. The repeat-primed PCR assay was initially also designated as a single method sufficient for DM1 testing; however, its combination with conventional amplification, or its bidirectional use, or their simultaneous combinations are recommended. On the other hand, the vulnerability of the repeat-primed assay to the presence of sequence interruptions makes it the only suitable tool for the detection of such interruptions. If sizing of the alleles is required, SBA of enzymatically digested genomic DNA and long-range PCR amplifications are still recommended, and both of these are suitable for the detection of expanded alleles in different ranges. In general, smaller expansions are not detected by SBA, whereas extremely large alleles are not detectable by long-range PCR. Not only several modifications, but also combinations of these methods have been described to improve their detection ranges, sensitivity, and accuracy. Although modifications of long-range PCR protocols are in many cases labor intensive and time consuming, their sensitivity also allows detection where the amount of available DNA is insufficient for SBA of digested genomic DNA. Additional tests using the analysis of linked genetic markers or in situ hybridization may be used when the results of the more generally used methods are not conclusive, or if they need to be supported by independent methods.

Table 1.

Characteristic Features of the Techniques Used for Myotonic Dystrophy Type 1/Myotonic Dystrophy Type 2 Molecular Testing

Technique	Modification	Advantages	Disadvantages	Effective range	References
SBA	Conventional	Ability to detect and size the majority of DM1/DM2 expanded alleles	Laborious, time consuming, unable to identify small expansions (and ∼20% of expanded DM2 alleles); sensitive to allele size heterogeneity; requires large amount of intact genomic DNA	All expanded alleles (except very small expansions and those with high degree of somatic mosaicism)	Brook et al. (1992); Mahadevan et al. (1992); Brunner et al. (1992); Petronis et al. (1996); Day et al. (2003); Bachinski et al. (2003); Carson (2009)
	FIGE, PFGE (with REs cutting closer to the repeat region)	Increased quality; better resolution	Laborious; time consuming	Not specified	Bachinski et al. (2003); Jakubiczka et al. (2004)
	Gel incubation in HCl prior to transferring	More efficient transfer of high-molecular-weight DNA to nylon membranes	Laborious; time consuming	Not specified	Carson (2009)
	LNA probes (with four-base REs)	Increased sensitivity	RCA is needed to detect alleles with <750 CTG repeats	>750 CTG	Nakamori et al. (2009)
PCR	Conventional	Simple; rapid; possibility to extend the effective range with special additives	Detects only healthy alleles and small expansions	<100 CTG	Brunner et al. (1992); Bachinski et al. (2009); Carson (2009)
	TP-PCR	Simple; rapid; sizing of small alleles and identification of the presence/absence of expanded alleles of all size ranges in one reaction	Unable to size >50-80 CTG; sensitive to interruptions	Sizing <50-80 CTG; identifies the presence/absence without length limitation	Warner et al. (1996); Falk et al. (2006); Day et al. (2003); Bachinski et al. (2003)
	LR-PCR	Simple; rapid; higher resolution in sizing; possibility to extend the effective range with special additives	Visualization of larger alleles requires HBD	<800-1700 CTG (direct visualization); <2700-3700 CTG (HBD); <3700 CCTG (HBD)	Cheng et al. (1996); Skrzypczak-Zielinska et al. (2009) Cheng et al. (1996); Gennarelli et al. (1998) Bonifazi et al. (2004)
	SP-PCR	Resolves discrete bands in samples giving smeared bands in SBA	Several reactions/sample; requires HBD	<1300 CTG (HBD)	Monckton et al. (1995)
	QF-PCR	Sizing of small alleles and identification of the presence/absence of expanded alleles of all size ranges in one reaction	Unable to size >100 CTG; requires multiplex reaction	Sizing <100 CTG; identifies the presence/absence without length limitation	Skrzypczak-Zielinska et al. (2009)
ISH		Identifies the presence/absence of expansion on RNA level	Laborious; time consuming; unable to size expanded alleles	Detection of alleles with >100 CTG	Liquori et al. (2001); Bonifazi et al. (2006)

DM, myotonic dystrophy; FIGE, field inversion electrophoresis; HBD, hybridization-based detection; ISH, in situ hybridization; LNA, locked nucleic acids; LR-PCR, long-range PCR; PCR, polymerase chain reaction; PFGE, pulsed-field gel electrophoresis; QF-PCR, quantitative fluorescent PCR; RCA, rolling circle amplification; REs, restriction enzymes; SBA, Southern blot analysis of enzymatically digested genomic DNA; SP-PCR, small-pool PCR; TP-PCR, “triplet” or “tetraplet” repeat-primed PCR.

Footnotes

Disclosure Statement

No competing financial interests exist.

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