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Simple Repeat-Primed PCR Analysis of the Myotonic Dystrophy Type 1 Gene in a Clinical Diagnostics Environment

Simple Repeat-Primed PCR Analysis of the Myotonic Dystrophy Type 1 Gene in a Clinical Diagnostics... Hindawi Publishing Corporation Journal of Neurodegenerative Diseases Volume 2013, Article ID 857564, 8 pages http://dx.doi.org/10.1155/2013/857564 Research Article Simple Repeat-Primed PCR Analysis of the Myotonic Dystrophy Type 1 Gene in a Clinical Diagnostics Environment 1 1 1 1,2 Philippa A. Dryland, Elaine Doherty, Jennifer M. Love, and Donald R. Love Diagnostic Genetics, LabPlus, Auckland City Hospital, P.O. Box 110031, Auckland 1148, New Zealand School of Biological Sciences, University of Auckland, Private Bag 92019, Auckland 1142, New Zealand Correspondence should be addressed to Donald R. Love; donaldl@adhb.govt.nz Received 31 March 2013; Revised 18 September 2013; Accepted 19 September 2013 Academic Editor: Eng King Tan Copyright © 2013 Philippa A. Dryland et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Myotonic dystrophy type 1 is an autosomal dominant neuromuscular disorder that is caused by the expansion of a CTG trinucleotide repeat in the DMPK gene. eTh confirmation of a clinical diagnosis of DM-1 usually involves PCR amplification of the CTG repeat- containing region and subsequent sizing of the amplification products in order to deduce the number of CTG repeats. In the case of repeat hyperexpansions, Southern blotting is also used; however, the latter has largely been superseded by triplet repeat-primed PCR (TP-PCR), which does not yield a CTG repeat number but nevertheless provides a means of stratifying patients regarding their disease severity. We report here a combination of forward and reverse TP-PCR primers that allows for the simple and effective scoring of both the size of smaller alleles and the presence or absence of expanded repeat sequences. In addition, the CTG repeat- containing TP-PCR forward primer can target both the DM-1 and Huntington disease genes, thereby streamlining the work flow for confirmation of clinical diagnoses in a diagnostic laboratory. 1. Introduction severity of clinical presentation, and the age of onset. eTh se groupings are mild, classical, and congenital with repeat sizes Myotonic dystrophy type 1 (DM-1) is a multisystem disorder rangingfrom50to150repeats,100to1000repeats,and>2000 with a highly variable phenotypic expression. This autosomal repeats, respectively [3, 4]. Despite these repeat ranges, the dominant neuromuscular disorder has been reported to cause stratifying of patients into classical and congenital categories myotonia,progressivemuscleweakness,andatrophyinskele- can pose difficulties as some congenital patients carry CTG tal muscles, as well as cardiomyopathy, cataracts, and dys- repeats that overlap with the classic range. In addition, the function in the endocrine and central nervous systems [1, 2]. situation is further confounded by the recent identification of DM-1 is caused by a CTG trinucleotide repeat expansion a juvenile category with CTG repeats that lie at the high end in the 3 untranslated region of the myotonic dystrophy of the classic range but with a different clinical severity [ 5, 6]. protein kinase (DMPK)gene, whichislocated on chromo- The confirmation of a clinical diagnosis of DM-1 involves some 19q13.3. The number of repeats a patient carries is the molecular genetic testing of the DMPK gene. This associated with clinical severity. Repeats of 5–34 are found in genetic testing is commonly performed by PCR amplica fi tion unaffected individuals, and those that lie in the range of 35– of the CTG repeat-containing region of the DMPK gene, 49 are considered abnormal premutations such that carriers and subsequent determination of amplicon lengths using are at risk of having affected offspring with larger repeat capillary-based electrophoresis. This method is only able lengths. Patients with DM-1 are typically classified into three to identify patients that fall within the mild range, so any sample showing apparent homozygosity has usually involved overlapping groupings based on the number of repeats, the 2 Journal of Neurodegenerative Diseases P2 P1-FAM (CTG) (a) P3 P4CTG P1-FAM (CTG) (b) P4CTG P1-FAM (CTG) (c) P4CAG P2-FAM (CTG) (d) Figure 1: Location of primers for each of four amplifications of the CTG repeat region of the DMPK gene. a second round of testing by Southern blot (SB) analysis to Critically, recent evidence has shown that TP-PCR can detect the presence or absence of larger repeat lengths [3, 7]. lead to false negative results in 3%–5% of DM-1 cases. Warner et al. (1996) suggested the use of a triplet repeat- This outcome is due to sequence interruptions (comprising primed PCR (TP-PCR) to replace Southern blotting as a CCG,CTG,and GGC sequences) that liewithinthe 3 means of detecting hyperexpansions of trinucleotide repeat end of an expanded CTG repeat tract [2, 8, 9, 12], which sequences [8]. TP-PCR reduces the potential turnaround appear to prevent binding of the repeat (P4) primer. In time for a test from a week to one day, as well as the order to address this problem, Radvansky et al. (2011) has cost of testing [8, 9]. TP-PCR usually uses three primers described a bidirectionally labeled TP-PCR method in which (Figure 1): onethatisfluoresceinatedand flanksthe repeat amplification products are anchored at the 3 end of a CTG region (P1-FAM), a second that is complementary to the repeat expansion rather than the 5 end [11]. The effect of targeted repeat but carries a nonspecific tail sequence (P4), this redesign is that it overcomes the failure in detecting and a third that is identical to the nonspecific tail sequence expansion-positive patients carrying repeat interruptions. (P3). eTh P4CTG primer is added at 1/10th the concentration Despitethe abovedevelopments, we sought to simplify of P1-FAM and P3 primers, while the P3 primer is designed TP-PCR further by relying on only two rather than three to prevent progressive shortening of the amplicons during primers, refining the amplification conditions to be more PCR cycling [8]. TP-PCR produces a characteristic profile of aligned with those used routinely in a diagnostic laboratory, amplicons of increasing length, which differ by the length and having the flexibility to allow hyper-expansions to be of a repeat unit (3 bp), but of diminishing yield [10]. This detected in other triplet repeat disorders. profile enables the rapid identification of large pathogenic CTG repeats that cannot be amplified using primers that flank the repeat region. The main disadvantage of TP-PCR is that 2. Materials and Methods it does not enable the repeat number to be determined [11]. This disadvantage has no serious clinical implications in the Twenty-one genomic DNAs that had been analysed by con- case of myotonic dystrophy as the age of presentation plays ventional PCR and Southern blotting were used to validate a pivotal role in helping a diagnostic laboratory interpret the the proposed TP-PCR methodology. es Th e DNAs comprised implications of CTG repeats in excess of 150. seven unaffected control individuals with repeat sizes ranging Journal of Neurodegenerative Diseases 3 Table 1: Primer sequences. Primer name Primer sequence P1-FAM FAM-CTTCCCAGGCCTGCAGTTTGCCCATC P2 AACGGGGCTCGAAGGGTCCTTGTAGC P4CTG GGCGGTGGCGGCTGTTGCTGCTGCTGCTGC P3 GGCGGTGGCGGCTGTTG P2-FAM AACGGGGCTCGAAGGGTCCTTGTAGC P4CAG GGCGGTGGCGGCTGTTGCCAGCAGCAGCAGCAG from 4 to 27 and 14 samples carrying expanded CTG repeats termed HD3, had originally been designed as the reverse ranging from 54 to over 1000. Fifth-three further DNAs were primer for amplification of the CAG repeat region of the analysed in a diagnostic setting by conventional PCR and the Huntington disease (HD)gene[6]and compriseda3 (CTG) validated TP-PCR methodology. es Th e DNAs comprised of repeat and a 5 tail of a 17-base sequence immediately 25 unaffected controls with repeat sizes ranging from 4 to 27 downstream of the HD gene repeat. Amplicfi ation using the and 28 samples carrying expanded CTG repeats ranging from TP-PCR forward primer combination was undertaken in 50 to full expansions. the presence and absence of primer P3. The sequence of The initial 21 DNA samples were tested using four this primer corresponded to the 17-base 5 tail of primer variations of PCR amplification, as shown in Figure 1.The P4CTG. Finally, the TP-PCR reverse primer combination was first comprised conventional PCR using P1-FAM forward and used that comprised primers P2-FAM and P4CAG; the latter P2 reverseprimers.Thesecondwas aTP-PCRmethodusing primer was similar in sequence to primer P4CTG except that the P1-FAM forward primer with the P4CTG reverse primer the (CTG) repeat was replaced by a (CAG) repeat. 4 5 at 1/10 the concentration and the P3 reverse primer specific The amplification products of all PCRs were initially to the tail region of primer P4CTG (TP-PCR forward primer analysed by gel electrophoresis (Figure 2). Conventional PCR combination, together with primer P3). The third was the using primers flanking the CTG repeat region amplified up same as thesecondbut only used P1-FAM forwardand the to 150 CTG repeats, which would be appropriate to conrfi m P4CTG reverse primers at the same concentration (TP-PCR a clinical diagnosis of mild DM-1 (50–150 repeats). The TP- forward primer combination). eTh n fi al PCR method used PCR primer combinations (forward and reverse), with and the P2-FAM reverse primer with a P4CAG forward primer without the additional P3 primer, amplified both CTG alleles (TP-PCR reverse primer combination). Primer sequences in all patients (except those carrying expanded alleles in are shown in Table 1.Thethird andfourthPCR methods excess of 150 CTG repeats), but against high background (TP-PCR forward and TP-PCR reverse primer combinations) staining.Critically, thepresenceofthe P3 primer resulted were validated (in triplicate) and used in conjunction with in qualitatively weaker amplicfi ation of CTG repeat alleles conventional PCR to analyse 53 further DNA samples. compared to the absence of this primer (Figure 3). Each 25𝜇 L PCR comprised FastStart Reaction Bueff r The TP-PCR forward primer combination, with and without MgCl (Roche), 2 mM MgCl (Roche), GC-rich without P3, was unable to accurately amplify the character- 2 2 solution (Roche), 10 mM dNTP mix, 20𝜇 Mforward and istic ladder profile for two patients with expanded repeat reverse primers, 1U FastStart Taq DNA Polymerase (Roche), sequences resulting in false negative results; an example of and 50 ng of genomic DNA. The PCR amplification con- this is shown in Figure 4(a)(4). Amplification using the TP- ditions comprised an initial denaturation of 94 Cfor vfi e PCR reverse primer combination, without primer P3, was minutes then 35 cycles of denaturation at 94 C for 45 seconds, able to detect the presence of the expanded repeat sequence ∘ ∘ 󸀠 annealing at 70 C for 30 seconds with extension at 72 Cfor (Figure 4(b)(4)). Profiles of 3 -interrupted sequences are 30 seconds and a na fi l extension at 72 Cfor 10 minutes. eTh expected to show a characteristic ladder profile, with peaks conventional PCR used a longer extension time of 2 minutes increasing in length by 3 bp increments but diminishing in order to reduce the presence of split peaks, which were not in intensity with length. A break in this profile would be observed in TP-PCR profiles. PCR products were subjected to expected if the repeat was interrupted, aeft r which the ladder capillary electrophoresis using an Applied Biosystems model should be reinstated with diminishing yield associated with 3130xl Genetic Analyzer, and the data were analysed using increasing CTG repeat length. GeneMapper software. The converse to the above was also found in which a TP-PCR forward primer combination gave a characteristic hyper-expansion profile ( Figure 4(a)(3)), but the TP-PCR 3. Results reverse primer combination suggested interruptions at the 5 An initial validation study was undertaken using 21 genomic endofthe CTGrepeat(Figure 4(b)(3)), hence supporting the need to use both the TP-PCR forward and reverse primer DNA samples. Amplification of the CTG repeat region of the DMPK gene involved four different reactions. eTh rfi st combinations to detect repeat hyper-expansions. used primersP1-FAMand P2,which flank therepeat. eTh A complete characterisation of each DNA sample includ- second,designatedtheTP-PCRforwardprimercombination, ing age, sex, repeat length, disease classification, and methods comprised P1-FAM and P4CTG. The latter primer, also of analysis in the validation study is shown in Table 2 4 Journal of Neurodegenerative Diseases Table 2: Characterisation of samples, including age, sex, repeat length, analysis methods used, and disease classification. Analysis methods used Sample Age Sex Repeat length Disease classification PCR SB TP-PCR 11y7m F 7,11 √√ √ Unaeff cted 2 2 y F 11, 13 √√ √ Unaeff cted 33y M 12,13 √√ √ Unaeff cted 45y F 5,14 √√ √ Unaeff cted 5 48 y F 11, 15 √√ √ Unaeff cted 6 85 y M 11, 26 √√ √ Unaeff cted 711m M 5,27 √√ √ Unaeff cted 8 72 y M 13, 57 √√ √ Mild 968y F 5,74 √√ √ Mild 10 34 y F 4, 77 √√ √ Mild 11 57 y M 11, 84 √√ √ Mild 12 47 y F 5, 120 √√ √ Mild-classical 13 45 y M 5, EXP (250) √√ √ Classical 14 57 y F 5, EXP (380) (I) √√ √ Classical 15 10 y M 12, EXP (500) √√ √ Classical 16 24 y M 14, EXP (550) √√ √ Classical 17 21 y F 12, EXP (800) √√ √ Classical 18 38 y F 22, EXP (1000–1500) √√ √ Classical 19 36 y M 5, EXP (1067–1600) √√ √ Classical 20 11 y F 5, EXP (400) √√ √ Classical 21 54 y F 24, EXP (260–490) (I) √√ √ Classical 22 2 M M 13, 13 √√ Unaeff cted 23 1 d M 5, 13 √√ Unaeff cted 24 24 y M 5, 11 √√ Unaeff cted 25 27 y M 5, 10 √√ Unaeff cted 26 6 M F 5, 5 √√ Unaeff cted 27 54 y F 14, 15 √√ Unaeff cted 28 52 y F 23, 27 √√ Unaeff cted 29 25 y F 5, 12 √√ Unaeff cted 30 23 y F 11, 13 √√ Unaeff cted 31 32 y M 5, 5 √√ Unaeff cted 32 23 y M 12, 14 √√ Unaeff cted 33 56 y M 8, 12 √√ Unaeff cted 34 16 y F 12, 20 √√ Unaeff cted 35 21 y F 11, 13 √√ Unaeff cted 36 32 y M 13, 14 √√ Unaeff cted 37 3 y F 5, 5 √√ Unaeff cted 38 1 y 8 m F 5, 13 √√ Unaeff cted 39 33 y F 5, 5 √√ √ Unaeff cted 40 16 y F 5, 13 √√ Unaeff cted 41 70 y F 5, 5 √√ Unaeff cted 42 23 y F 5, 13 √√ Unaeff cted 43 37 y M 5, 14 √√ Unaeff cted 44 57 y M 5, 13 √√ Unaeff cted 45 65 y F 5, 12 √√ Unaeff cted 46 30 y F 5, 14 √√ Unaeff cted 47 55 y M 15, 62 √√ Mild 48 61 y M 14, 89 √√ Mild 49 32 y M 26, 84 Mild √√ Journal of Neurodegenerative Diseases 5 Table 2: Continued. Analysis methods used Sample Age Sex Repeat length Disease classification PCR SB TP-PCR 50 53 y F 5, 81 √√ Mild 51 63 y F 15, 50 √√ Mild 52 54 y M 5, 97 √√ Mild 53 39 y F 13, 51 √√ Mild 54 73 y M 13, 56 √√ Mild 55 37 y F 5, 101 √√ Mild-classical 56 47 y M 5, 141 √√ √ Mild-classical 57 60 y F 13, 92–145 √√ √ Mild-classical 58 30 y M 5, 140 √√ √ Mild-classical 59 43 y M 5, EXP √√ Classical 60 6 y F 5, EXP √√ Classical 61 22 y M 5, EXP √√ Classical 62 27 y F 11, EXP √√ Classical 63 30 y M 5, EXP √√ Classical 64 55 y M 13, EXP (I) Classical √√ 65 3 y M 12, EXP √√ Classical 66 80 y F 10, EXP (I) √√ √ Classical 67 42 y F 5, EXP √√ Classical 68 50 y M 13, EXP √√ Classical 69 24 y F 21, EXP √√ Classical 70 42 y M 5, EXP √√ Classical 71 52 y F 12, EXP √√ Classical 72 72 y M 19, EXP (I) √√ Classical 73 29 y F 5, EXP √√ Classical 74 45 y F 5, EXP (I) √√ Classical Repeat lengths up to 150 repeats are shown, and expansions (EXP) are indicated where the specific size of the allele is no longer able to be sized accurate ly (in excess of 150 repeats). I indicates samples that have a 3 interruption using the TP-PCR forward primer combination. Ticks indicate which methods were used to analyse each sample. PCR: standard polymerase chain reaction; SB: Southern blot; TP-PCR: triplet repeat primed PCR. 1 23456789 10 11 12 13 14 15 16 17 18 19 20 21 22 (a) (b) (c) (d) Figure 2: Agarose gel electrophoresis of PCR products for each of four amplifications of the CTG repeat region of the DMPK gene. (a) Primers P1-FAM and P2; (b) primers P1-FAM and P4CTG (TP-PCR forward primer combination) with P3 primer; (c) TP-PCR forward primer combination; (d) P2-FAM and P4CAG (TP-PCR reverse primer combination). From left to right the gels show DNAs with repeat lengths of [7,11], [11,13], [12,13], [5,14], [11,15], [11,26], [5,27], [13,54], [5,74], [4,77], [11,84], [5,120], [5,250], [5,380], [12,500], [14,530], [12,700], [1000–1500], [1067–1600], [5,405], and [24,260–490]. 6 Journal of Neurodegenerative Diseases 0 100 200 300 400 0 100 200 300 400 0 100 200 300 400 8000 8000 8000 7000 7000 6000 6000 6000 5000 5000 4000 4000 4000 3000 3000 2000 2000 2000 1000 1000 0 0 0 0 100 200 300 400 0 100 200 300 400 0 100 200 300 400 8000 8000 7000 7000 7000 6000 6000 5000 5000 5000 4000 4000 3000 3000 3000 2000 2000 1000 1000 1000 0 0 0 100 200 300 400 0 100 200 300 400 0 100 200 300 400 8000 8000 8000 7000 7000 7000 6000 6000 6000 5000 5000 5000 4000 4000 4000 3000 3000 3000 2000 2000 2000 1000 1000 1000 0 0 0 (a) (b) (c) Figure 3: Electropherograms of PCR products for each of four amplifications of the CTG repeat region of the DMPK gene. (a) Primers P1-FAM and P2; ((b), (c)) TP-PCR forward primer combination with and without the P3 primer, respectively. From top to bottom the electropherograms are from DNAs with repeat lengths of [5,14], [11,84], and [5,1600]. (rows 1–21). A test study followed using 53 further DNA The P4CTG primer used in this study was designed samples that were amplified using conventional PCR and the against the human HD gene repeat locus, with a 5 sequence forward and reverse TP-PCR primer combinations (see rows that is complementary to that immediately downstream of 22–74 of Table 2). Conventional PCR amplification allowed the HD gene CAG repeat [10]. The intention of this specific the sizing of up to 150 CTG repeats, thereby confirming a tail region was to enhance the potential to amplify both the clinical diagnosis of mild DM. Taken together, the TP-PCR normal allele and the expanded alleles at the HD locus. This primer combinations were able to confirm the size of the primer contrasts with those used in other TP-PCR methods lower alleles identified in the conventional PCR amplification described in the literature. These methods use a nonspecific and detect the presence of (but not accurately size) expanded tail sequence to create a unique platform for the binding of alleles to distinguish between mild and classic DM. The primer P3 in order to enable sufficient amplification within a TP-PCR forward primer combination showed a clear peak CTG repeat hyper-expansion. Critically, as the trinucleotide at the end of the characteristic ladder profile for smaller repeat in both Huntington disease and myotonic dystrophy expansions within the mild range (Figure 5(a)). Four of the is a CTG sequence, a simple change of primer P1 to one that 53 DNA samples showed 3 interruptions in the TP-PCR is specicfi to the upstream of the HD gene repeat enables forward primer combination electropherograms; however, the use of the P4CTG primer for diagnostic testing of repeat the TP-PCR reverse primer combination electropherograms expansions in both DM-1 and HD. showed clear expanded profiles. eTh TP-PCR reverse primer Due to the presence of interruptions observed at both 󸀠 󸀠 combination was able to accurately size alleles within the the 3 and the 5 ends of the CTG repeat sequence in the unaeff cted range consistent with the conventional PCR and DM-1 locus, neither the forward nor the reverse primer SB repeat scores where available (Figure 5(b)). combination can be considered reliable for clinical diagnostic testingalone,sotheyshouldbeusedintandem.Althoughnot shown here, it is possible to combine the TP-PCR forward 4. Discussion and reverse primer combinations in the same reaction using different labels in order to streamline diagnostic testing TP-PCR methods described in the literature use a P3 primer further. to prevent progressive shortening of the PCR products during subsequent cycles. eTh data presented above does not show On validation of the TP-PCR forward and reverse primer combination, these primers were successfully used in concert signs of progressive shortening of PCR products. There is adequate amplification across the repeat sequence to enable with conventional PCR in a diagnostic setting, thereby providing a simple and eeff ctive method to accurately size accurate scoring of alleles within the normal to mild disease alleles below 150 repeats and clearly indicate the presence of range and accurate scoring of the presence and absence of expansions within the classical and congenital ranges of expanded alleles. disease without primer P3. Also, the TP-PCRs without P3 In conclusion, the combination of the forward and reverse showed stronger amplicfi ation across the repeat sequence. TP-PCR primer combinations provide a simple and eeff ctive Journal of Neurodegenerative Diseases 7 40 80 120 160 200 240 280 320 360 400 440 480 40 80 120 160 200 240 280 320 360 400 440 480 8000 8000 6000 6000 4000 4000 (1) (1) 40 120 160 200 240 280 320 360 400 440 480 40 80 120 160 200 240 280 320 360 400 440 480 6000 6000 (2) (2) 40 80 120 160 200 240 280 320 360 400 440 480 40 80 120 160 200 240 280 320 360 400 440 480 8000 8000 Interruptions at 5 6000 6000 end of CTG repeat 4000 4000 (3) (3) 40 80 120 160 200 240 280 320 360 400 440 480 40 80 120 160 200 240 280 320 360 400 440 480 8000 8000 Interruptions at 3 6000 end of CTG repeat (4) (4) 40 80 120 160 200 240 280 320 360 400 440 480 40 80 120 160 200 240 280 320 360 400 440 480 4000 4000 2000 2000 0 0 (5) (5) (a) (b) Figure 4: Electropherograms of PCR products using the TP-PCR forward and reverse primer combinations. (a) and (b) show electropherograms derived from using the TP-PCR forward and reverse primer combinations, respectively. From top to bottom the electropherograms are from DNAs with repeat lengths of [5,14], [11,84], [5,380], [5,250], and [5,1600]. 0 100 200 300 400 500 600 700 800 900 60 70 80 90 100 110 120 130 140 150 500 10000 300 6000 5 repeats 10 repeats (a) (b) Figure 5: Electropherograms of PCR products using the TP-PCR forward and reverse primer combinations illustrating sizing capabilities. (a) and (b) show electropherograms derived from using the TP-PCR forward and reverse primer combinations, respectively. (a) shows an enlarged electropherogram image of sample 58 with repeat lengths 5 and 140; illustrating the ability to visualise the expanded allele peak, within the clinically mild range, at the right-hand end of the characteristic ladder profile. (b) shows two clear allele peaks illustrating the abilitytosizealleles within theunaeff cted rangewiththe TP-PCR reverseprimercombination. means of identifying the presence or absence of expanded References CTG repeat alleles in the DMPK gene. eTh same P4CTG [1] J. D. Brook, M. E. McCurrach, H. G. Harley et al., “Molecular primer has been shown to work both with myotonic dystro- basis of myotonic dystrophy: expansion of a trinucleotide phy and Huntington disease repeat sequences, streamlining (CTG) repeat at the 3 end of a transcript encoding a protein the work flow for these diseases. kinase family member,” Cell, vol. 68, no. 4, pp. 799–808, 1992. 8 Journal of Neurodegenerative Diseases [2] H.Petri,J.Vissing,N.Witting,H.Bundgaard,and L. Køber, “Cardiac manifestations of myotonic dystrophy type 1,” Inter- national Journal of Cardiology,vol.160,no. 2, pp.82–88, 2012. [3] M. Addis, M. Serrenti, C. Meloni, M. Cau, and M. A. Melis, “Triplet-primed PCR is more sensitive than Southern blotting- long PCR for the diagnosis of myotonic dystrophy type1,” Genetic Testing and Molecular Biomarkers,vol.16, no.12, pp. 1428–1431, 2012. [4] eTh International Myotonic Dystrophy Consortium (IDMC), “New nomenclature and DNA testing guidelines for myotonic dystrophy type 1 (DM1),” Neurology,vol.54, no.6,pp. 1218–1221, [5] B.Echenne,A.Rideau,A.Roubertie,G.Seb ´ ire, F. Rivier, and B. Lemieux, “Myotonic dystrophy type I in childhood. Long-term evolution in patients surviving the neonatal period,” European JournalofPaediatricNeurology,vol.12, no.3,pp. 210–223, 2008. [6] E. J. Kamsteeg, W. Kress, C. Catalli et al., “EMQN best practice guidelines and recommendations of myotonic dystrophy types 1and 2,” European Journal of Human Genetics,vol.20, no.12, pp. 1203–1208, 2012. [7] P. Ciotti, E. Di Maria, E. Bellone, F. Ajmar, and P. Mandich, “Triplet repeat primed PCR (TP PCR) in molecular diagnostic testing for Friedreich ataxia,” Journal of Molecular Diagnostics, vol. 6, no. 4, pp. 285–289, 2004. [8] J. P. Warner, L. H. Barron, D. Goudie et al., “A general method for the detection of large CAG repeat expansions by uo fl rescent PCR,” Journal of Medical Genetics, vol. 33, no. 12, pp. 1022–1026, [9] M. Falk, M. Vojt´ıˇskova, ´ Z. Luka´ˇs, I. Kroupova, ´ and U. Froster, “Simple procedure for automatic detection of unstable alleles in the myotonic dystrophy and Huntington’s disease loci,” Genetic Testing,vol.10, no.2,pp. 85–97, 2006. [10] J. M. Love,R.Marquis-Nicholson,R.C.Love, andD.R.Love, “Portable battery-operated rapid PCR amplification of the CAG repeat region of the Huntington disease locus,” Research Journal of Biology,vol.2,no. 6, pp.191–196,2012. [11] J. Radvansky, A. Ficek, G. Minarik, R. Palffy, and L. Kadasi, “Effect of unexpected sequence interruptions to conventional PCR and repeat primed PCR in myotonic dystrophy type 1 testing,” Diagnostic Molecular Pathology,vol.20,no.1,pp.48–51, [12] C. Braida,R.K.A.Stefanatos, B. Adam et al., “Variant CCG and GGC repeats within the CTG expansion dramatically modify mutational dynamics and likely contribute toward unusual symptoms in some myotonic dystrophy type 1 patients,” Human Molecular Genetics,vol.19, no.8,pp. 1399–1412, 2010. 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Simple Repeat-Primed PCR Analysis of the Myotonic Dystrophy Type 1 Gene in a Clinical Diagnostics Environment

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Hindawi Publishing Corporation
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Copyright © 2013 Philippa A. Dryland et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
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2090-858X
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10.1155/2013/857564
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Abstract

Hindawi Publishing Corporation Journal of Neurodegenerative Diseases Volume 2013, Article ID 857564, 8 pages http://dx.doi.org/10.1155/2013/857564 Research Article Simple Repeat-Primed PCR Analysis of the Myotonic Dystrophy Type 1 Gene in a Clinical Diagnostics Environment 1 1 1 1,2 Philippa A. Dryland, Elaine Doherty, Jennifer M. Love, and Donald R. Love Diagnostic Genetics, LabPlus, Auckland City Hospital, P.O. Box 110031, Auckland 1148, New Zealand School of Biological Sciences, University of Auckland, Private Bag 92019, Auckland 1142, New Zealand Correspondence should be addressed to Donald R. Love; donaldl@adhb.govt.nz Received 31 March 2013; Revised 18 September 2013; Accepted 19 September 2013 Academic Editor: Eng King Tan Copyright © 2013 Philippa A. Dryland et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Myotonic dystrophy type 1 is an autosomal dominant neuromuscular disorder that is caused by the expansion of a CTG trinucleotide repeat in the DMPK gene. eTh confirmation of a clinical diagnosis of DM-1 usually involves PCR amplification of the CTG repeat- containing region and subsequent sizing of the amplification products in order to deduce the number of CTG repeats. In the case of repeat hyperexpansions, Southern blotting is also used; however, the latter has largely been superseded by triplet repeat-primed PCR (TP-PCR), which does not yield a CTG repeat number but nevertheless provides a means of stratifying patients regarding their disease severity. We report here a combination of forward and reverse TP-PCR primers that allows for the simple and effective scoring of both the size of smaller alleles and the presence or absence of expanded repeat sequences. In addition, the CTG repeat- containing TP-PCR forward primer can target both the DM-1 and Huntington disease genes, thereby streamlining the work flow for confirmation of clinical diagnoses in a diagnostic laboratory. 1. Introduction severity of clinical presentation, and the age of onset. eTh se groupings are mild, classical, and congenital with repeat sizes Myotonic dystrophy type 1 (DM-1) is a multisystem disorder rangingfrom50to150repeats,100to1000repeats,and>2000 with a highly variable phenotypic expression. This autosomal repeats, respectively [3, 4]. Despite these repeat ranges, the dominant neuromuscular disorder has been reported to cause stratifying of patients into classical and congenital categories myotonia,progressivemuscleweakness,andatrophyinskele- can pose difficulties as some congenital patients carry CTG tal muscles, as well as cardiomyopathy, cataracts, and dys- repeats that overlap with the classic range. In addition, the function in the endocrine and central nervous systems [1, 2]. situation is further confounded by the recent identification of DM-1 is caused by a CTG trinucleotide repeat expansion a juvenile category with CTG repeats that lie at the high end in the 3 untranslated region of the myotonic dystrophy of the classic range but with a different clinical severity [ 5, 6]. protein kinase (DMPK)gene, whichislocated on chromo- The confirmation of a clinical diagnosis of DM-1 involves some 19q13.3. The number of repeats a patient carries is the molecular genetic testing of the DMPK gene. This associated with clinical severity. Repeats of 5–34 are found in genetic testing is commonly performed by PCR amplica fi tion unaffected individuals, and those that lie in the range of 35– of the CTG repeat-containing region of the DMPK gene, 49 are considered abnormal premutations such that carriers and subsequent determination of amplicon lengths using are at risk of having affected offspring with larger repeat capillary-based electrophoresis. This method is only able lengths. Patients with DM-1 are typically classified into three to identify patients that fall within the mild range, so any sample showing apparent homozygosity has usually involved overlapping groupings based on the number of repeats, the 2 Journal of Neurodegenerative Diseases P2 P1-FAM (CTG) (a) P3 P4CTG P1-FAM (CTG) (b) P4CTG P1-FAM (CTG) (c) P4CAG P2-FAM (CTG) (d) Figure 1: Location of primers for each of four amplifications of the CTG repeat region of the DMPK gene. a second round of testing by Southern blot (SB) analysis to Critically, recent evidence has shown that TP-PCR can detect the presence or absence of larger repeat lengths [3, 7]. lead to false negative results in 3%–5% of DM-1 cases. Warner et al. (1996) suggested the use of a triplet repeat- This outcome is due to sequence interruptions (comprising primed PCR (TP-PCR) to replace Southern blotting as a CCG,CTG,and GGC sequences) that liewithinthe 3 means of detecting hyperexpansions of trinucleotide repeat end of an expanded CTG repeat tract [2, 8, 9, 12], which sequences [8]. TP-PCR reduces the potential turnaround appear to prevent binding of the repeat (P4) primer. In time for a test from a week to one day, as well as the order to address this problem, Radvansky et al. (2011) has cost of testing [8, 9]. TP-PCR usually uses three primers described a bidirectionally labeled TP-PCR method in which (Figure 1): onethatisfluoresceinatedand flanksthe repeat amplification products are anchored at the 3 end of a CTG region (P1-FAM), a second that is complementary to the repeat expansion rather than the 5 end [11]. The effect of targeted repeat but carries a nonspecific tail sequence (P4), this redesign is that it overcomes the failure in detecting and a third that is identical to the nonspecific tail sequence expansion-positive patients carrying repeat interruptions. (P3). eTh P4CTG primer is added at 1/10th the concentration Despitethe abovedevelopments, we sought to simplify of P1-FAM and P3 primers, while the P3 primer is designed TP-PCR further by relying on only two rather than three to prevent progressive shortening of the amplicons during primers, refining the amplification conditions to be more PCR cycling [8]. TP-PCR produces a characteristic profile of aligned with those used routinely in a diagnostic laboratory, amplicons of increasing length, which differ by the length and having the flexibility to allow hyper-expansions to be of a repeat unit (3 bp), but of diminishing yield [10]. This detected in other triplet repeat disorders. profile enables the rapid identification of large pathogenic CTG repeats that cannot be amplified using primers that flank the repeat region. The main disadvantage of TP-PCR is that 2. Materials and Methods it does not enable the repeat number to be determined [11]. This disadvantage has no serious clinical implications in the Twenty-one genomic DNAs that had been analysed by con- case of myotonic dystrophy as the age of presentation plays ventional PCR and Southern blotting were used to validate a pivotal role in helping a diagnostic laboratory interpret the the proposed TP-PCR methodology. es Th e DNAs comprised implications of CTG repeats in excess of 150. seven unaffected control individuals with repeat sizes ranging Journal of Neurodegenerative Diseases 3 Table 1: Primer sequences. Primer name Primer sequence P1-FAM FAM-CTTCCCAGGCCTGCAGTTTGCCCATC P2 AACGGGGCTCGAAGGGTCCTTGTAGC P4CTG GGCGGTGGCGGCTGTTGCTGCTGCTGCTGC P3 GGCGGTGGCGGCTGTTG P2-FAM AACGGGGCTCGAAGGGTCCTTGTAGC P4CAG GGCGGTGGCGGCTGTTGCCAGCAGCAGCAGCAG from 4 to 27 and 14 samples carrying expanded CTG repeats termed HD3, had originally been designed as the reverse ranging from 54 to over 1000. Fifth-three further DNAs were primer for amplification of the CAG repeat region of the analysed in a diagnostic setting by conventional PCR and the Huntington disease (HD)gene[6]and compriseda3 (CTG) validated TP-PCR methodology. es Th e DNAs comprised of repeat and a 5 tail of a 17-base sequence immediately 25 unaffected controls with repeat sizes ranging from 4 to 27 downstream of the HD gene repeat. Amplicfi ation using the and 28 samples carrying expanded CTG repeats ranging from TP-PCR forward primer combination was undertaken in 50 to full expansions. the presence and absence of primer P3. The sequence of The initial 21 DNA samples were tested using four this primer corresponded to the 17-base 5 tail of primer variations of PCR amplification, as shown in Figure 1.The P4CTG. Finally, the TP-PCR reverse primer combination was first comprised conventional PCR using P1-FAM forward and used that comprised primers P2-FAM and P4CAG; the latter P2 reverseprimers.Thesecondwas aTP-PCRmethodusing primer was similar in sequence to primer P4CTG except that the P1-FAM forward primer with the P4CTG reverse primer the (CTG) repeat was replaced by a (CAG) repeat. 4 5 at 1/10 the concentration and the P3 reverse primer specific The amplification products of all PCRs were initially to the tail region of primer P4CTG (TP-PCR forward primer analysed by gel electrophoresis (Figure 2). Conventional PCR combination, together with primer P3). The third was the using primers flanking the CTG repeat region amplified up same as thesecondbut only used P1-FAM forwardand the to 150 CTG repeats, which would be appropriate to conrfi m P4CTG reverse primers at the same concentration (TP-PCR a clinical diagnosis of mild DM-1 (50–150 repeats). The TP- forward primer combination). eTh n fi al PCR method used PCR primer combinations (forward and reverse), with and the P2-FAM reverse primer with a P4CAG forward primer without the additional P3 primer, amplified both CTG alleles (TP-PCR reverse primer combination). Primer sequences in all patients (except those carrying expanded alleles in are shown in Table 1.Thethird andfourthPCR methods excess of 150 CTG repeats), but against high background (TP-PCR forward and TP-PCR reverse primer combinations) staining.Critically, thepresenceofthe P3 primer resulted were validated (in triplicate) and used in conjunction with in qualitatively weaker amplicfi ation of CTG repeat alleles conventional PCR to analyse 53 further DNA samples. compared to the absence of this primer (Figure 3). Each 25𝜇 L PCR comprised FastStart Reaction Bueff r The TP-PCR forward primer combination, with and without MgCl (Roche), 2 mM MgCl (Roche), GC-rich without P3, was unable to accurately amplify the character- 2 2 solution (Roche), 10 mM dNTP mix, 20𝜇 Mforward and istic ladder profile for two patients with expanded repeat reverse primers, 1U FastStart Taq DNA Polymerase (Roche), sequences resulting in false negative results; an example of and 50 ng of genomic DNA. The PCR amplification con- this is shown in Figure 4(a)(4). Amplification using the TP- ditions comprised an initial denaturation of 94 Cfor vfi e PCR reverse primer combination, without primer P3, was minutes then 35 cycles of denaturation at 94 C for 45 seconds, able to detect the presence of the expanded repeat sequence ∘ ∘ 󸀠 annealing at 70 C for 30 seconds with extension at 72 Cfor (Figure 4(b)(4)). Profiles of 3 -interrupted sequences are 30 seconds and a na fi l extension at 72 Cfor 10 minutes. eTh expected to show a characteristic ladder profile, with peaks conventional PCR used a longer extension time of 2 minutes increasing in length by 3 bp increments but diminishing in order to reduce the presence of split peaks, which were not in intensity with length. A break in this profile would be observed in TP-PCR profiles. PCR products were subjected to expected if the repeat was interrupted, aeft r which the ladder capillary electrophoresis using an Applied Biosystems model should be reinstated with diminishing yield associated with 3130xl Genetic Analyzer, and the data were analysed using increasing CTG repeat length. GeneMapper software. The converse to the above was also found in which a TP-PCR forward primer combination gave a characteristic hyper-expansion profile ( Figure 4(a)(3)), but the TP-PCR 3. Results reverse primer combination suggested interruptions at the 5 An initial validation study was undertaken using 21 genomic endofthe CTGrepeat(Figure 4(b)(3)), hence supporting the need to use both the TP-PCR forward and reverse primer DNA samples. Amplification of the CTG repeat region of the DMPK gene involved four different reactions. eTh rfi st combinations to detect repeat hyper-expansions. used primersP1-FAMand P2,which flank therepeat. eTh A complete characterisation of each DNA sample includ- second,designatedtheTP-PCRforwardprimercombination, ing age, sex, repeat length, disease classification, and methods comprised P1-FAM and P4CTG. The latter primer, also of analysis in the validation study is shown in Table 2 4 Journal of Neurodegenerative Diseases Table 2: Characterisation of samples, including age, sex, repeat length, analysis methods used, and disease classification. Analysis methods used Sample Age Sex Repeat length Disease classification PCR SB TP-PCR 11y7m F 7,11 √√ √ Unaeff cted 2 2 y F 11, 13 √√ √ Unaeff cted 33y M 12,13 √√ √ Unaeff cted 45y F 5,14 √√ √ Unaeff cted 5 48 y F 11, 15 √√ √ Unaeff cted 6 85 y M 11, 26 √√ √ Unaeff cted 711m M 5,27 √√ √ Unaeff cted 8 72 y M 13, 57 √√ √ Mild 968y F 5,74 √√ √ Mild 10 34 y F 4, 77 √√ √ Mild 11 57 y M 11, 84 √√ √ Mild 12 47 y F 5, 120 √√ √ Mild-classical 13 45 y M 5, EXP (250) √√ √ Classical 14 57 y F 5, EXP (380) (I) √√ √ Classical 15 10 y M 12, EXP (500) √√ √ Classical 16 24 y M 14, EXP (550) √√ √ Classical 17 21 y F 12, EXP (800) √√ √ Classical 18 38 y F 22, EXP (1000–1500) √√ √ Classical 19 36 y M 5, EXP (1067–1600) √√ √ Classical 20 11 y F 5, EXP (400) √√ √ Classical 21 54 y F 24, EXP (260–490) (I) √√ √ Classical 22 2 M M 13, 13 √√ Unaeff cted 23 1 d M 5, 13 √√ Unaeff cted 24 24 y M 5, 11 √√ Unaeff cted 25 27 y M 5, 10 √√ Unaeff cted 26 6 M F 5, 5 √√ Unaeff cted 27 54 y F 14, 15 √√ Unaeff cted 28 52 y F 23, 27 √√ Unaeff cted 29 25 y F 5, 12 √√ Unaeff cted 30 23 y F 11, 13 √√ Unaeff cted 31 32 y M 5, 5 √√ Unaeff cted 32 23 y M 12, 14 √√ Unaeff cted 33 56 y M 8, 12 √√ Unaeff cted 34 16 y F 12, 20 √√ Unaeff cted 35 21 y F 11, 13 √√ Unaeff cted 36 32 y M 13, 14 √√ Unaeff cted 37 3 y F 5, 5 √√ Unaeff cted 38 1 y 8 m F 5, 13 √√ Unaeff cted 39 33 y F 5, 5 √√ √ Unaeff cted 40 16 y F 5, 13 √√ Unaeff cted 41 70 y F 5, 5 √√ Unaeff cted 42 23 y F 5, 13 √√ Unaeff cted 43 37 y M 5, 14 √√ Unaeff cted 44 57 y M 5, 13 √√ Unaeff cted 45 65 y F 5, 12 √√ Unaeff cted 46 30 y F 5, 14 √√ Unaeff cted 47 55 y M 15, 62 √√ Mild 48 61 y M 14, 89 √√ Mild 49 32 y M 26, 84 Mild √√ Journal of Neurodegenerative Diseases 5 Table 2: Continued. Analysis methods used Sample Age Sex Repeat length Disease classification PCR SB TP-PCR 50 53 y F 5, 81 √√ Mild 51 63 y F 15, 50 √√ Mild 52 54 y M 5, 97 √√ Mild 53 39 y F 13, 51 √√ Mild 54 73 y M 13, 56 √√ Mild 55 37 y F 5, 101 √√ Mild-classical 56 47 y M 5, 141 √√ √ Mild-classical 57 60 y F 13, 92–145 √√ √ Mild-classical 58 30 y M 5, 140 √√ √ Mild-classical 59 43 y M 5, EXP √√ Classical 60 6 y F 5, EXP √√ Classical 61 22 y M 5, EXP √√ Classical 62 27 y F 11, EXP √√ Classical 63 30 y M 5, EXP √√ Classical 64 55 y M 13, EXP (I) Classical √√ 65 3 y M 12, EXP √√ Classical 66 80 y F 10, EXP (I) √√ √ Classical 67 42 y F 5, EXP √√ Classical 68 50 y M 13, EXP √√ Classical 69 24 y F 21, EXP √√ Classical 70 42 y M 5, EXP √√ Classical 71 52 y F 12, EXP √√ Classical 72 72 y M 19, EXP (I) √√ Classical 73 29 y F 5, EXP √√ Classical 74 45 y F 5, EXP (I) √√ Classical Repeat lengths up to 150 repeats are shown, and expansions (EXP) are indicated where the specific size of the allele is no longer able to be sized accurate ly (in excess of 150 repeats). I indicates samples that have a 3 interruption using the TP-PCR forward primer combination. Ticks indicate which methods were used to analyse each sample. PCR: standard polymerase chain reaction; SB: Southern blot; TP-PCR: triplet repeat primed PCR. 1 23456789 10 11 12 13 14 15 16 17 18 19 20 21 22 (a) (b) (c) (d) Figure 2: Agarose gel electrophoresis of PCR products for each of four amplifications of the CTG repeat region of the DMPK gene. (a) Primers P1-FAM and P2; (b) primers P1-FAM and P4CTG (TP-PCR forward primer combination) with P3 primer; (c) TP-PCR forward primer combination; (d) P2-FAM and P4CAG (TP-PCR reverse primer combination). From left to right the gels show DNAs with repeat lengths of [7,11], [11,13], [12,13], [5,14], [11,15], [11,26], [5,27], [13,54], [5,74], [4,77], [11,84], [5,120], [5,250], [5,380], [12,500], [14,530], [12,700], [1000–1500], [1067–1600], [5,405], and [24,260–490]. 6 Journal of Neurodegenerative Diseases 0 100 200 300 400 0 100 200 300 400 0 100 200 300 400 8000 8000 8000 7000 7000 6000 6000 6000 5000 5000 4000 4000 4000 3000 3000 2000 2000 2000 1000 1000 0 0 0 0 100 200 300 400 0 100 200 300 400 0 100 200 300 400 8000 8000 7000 7000 7000 6000 6000 5000 5000 5000 4000 4000 3000 3000 3000 2000 2000 1000 1000 1000 0 0 0 100 200 300 400 0 100 200 300 400 0 100 200 300 400 8000 8000 8000 7000 7000 7000 6000 6000 6000 5000 5000 5000 4000 4000 4000 3000 3000 3000 2000 2000 2000 1000 1000 1000 0 0 0 (a) (b) (c) Figure 3: Electropherograms of PCR products for each of four amplifications of the CTG repeat region of the DMPK gene. (a) Primers P1-FAM and P2; ((b), (c)) TP-PCR forward primer combination with and without the P3 primer, respectively. From top to bottom the electropherograms are from DNAs with repeat lengths of [5,14], [11,84], and [5,1600]. (rows 1–21). A test study followed using 53 further DNA The P4CTG primer used in this study was designed samples that were amplified using conventional PCR and the against the human HD gene repeat locus, with a 5 sequence forward and reverse TP-PCR primer combinations (see rows that is complementary to that immediately downstream of 22–74 of Table 2). Conventional PCR amplification allowed the HD gene CAG repeat [10]. The intention of this specific the sizing of up to 150 CTG repeats, thereby confirming a tail region was to enhance the potential to amplify both the clinical diagnosis of mild DM. Taken together, the TP-PCR normal allele and the expanded alleles at the HD locus. This primer combinations were able to confirm the size of the primer contrasts with those used in other TP-PCR methods lower alleles identified in the conventional PCR amplification described in the literature. These methods use a nonspecific and detect the presence of (but not accurately size) expanded tail sequence to create a unique platform for the binding of alleles to distinguish between mild and classic DM. The primer P3 in order to enable sufficient amplification within a TP-PCR forward primer combination showed a clear peak CTG repeat hyper-expansion. Critically, as the trinucleotide at the end of the characteristic ladder profile for smaller repeat in both Huntington disease and myotonic dystrophy expansions within the mild range (Figure 5(a)). Four of the is a CTG sequence, a simple change of primer P1 to one that 53 DNA samples showed 3 interruptions in the TP-PCR is specicfi to the upstream of the HD gene repeat enables forward primer combination electropherograms; however, the use of the P4CTG primer for diagnostic testing of repeat the TP-PCR reverse primer combination electropherograms expansions in both DM-1 and HD. showed clear expanded profiles. eTh TP-PCR reverse primer Due to the presence of interruptions observed at both 󸀠 󸀠 combination was able to accurately size alleles within the the 3 and the 5 ends of the CTG repeat sequence in the unaeff cted range consistent with the conventional PCR and DM-1 locus, neither the forward nor the reverse primer SB repeat scores where available (Figure 5(b)). combination can be considered reliable for clinical diagnostic testingalone,sotheyshouldbeusedintandem.Althoughnot shown here, it is possible to combine the TP-PCR forward 4. Discussion and reverse primer combinations in the same reaction using different labels in order to streamline diagnostic testing TP-PCR methods described in the literature use a P3 primer further. to prevent progressive shortening of the PCR products during subsequent cycles. eTh data presented above does not show On validation of the TP-PCR forward and reverse primer combination, these primers were successfully used in concert signs of progressive shortening of PCR products. There is adequate amplification across the repeat sequence to enable with conventional PCR in a diagnostic setting, thereby providing a simple and eeff ctive method to accurately size accurate scoring of alleles within the normal to mild disease alleles below 150 repeats and clearly indicate the presence of range and accurate scoring of the presence and absence of expansions within the classical and congenital ranges of expanded alleles. disease without primer P3. Also, the TP-PCRs without P3 In conclusion, the combination of the forward and reverse showed stronger amplicfi ation across the repeat sequence. TP-PCR primer combinations provide a simple and eeff ctive Journal of Neurodegenerative Diseases 7 40 80 120 160 200 240 280 320 360 400 440 480 40 80 120 160 200 240 280 320 360 400 440 480 8000 8000 6000 6000 4000 4000 (1) (1) 40 120 160 200 240 280 320 360 400 440 480 40 80 120 160 200 240 280 320 360 400 440 480 6000 6000 (2) (2) 40 80 120 160 200 240 280 320 360 400 440 480 40 80 120 160 200 240 280 320 360 400 440 480 8000 8000 Interruptions at 5 6000 6000 end of CTG repeat 4000 4000 (3) (3) 40 80 120 160 200 240 280 320 360 400 440 480 40 80 120 160 200 240 280 320 360 400 440 480 8000 8000 Interruptions at 3 6000 end of CTG repeat (4) (4) 40 80 120 160 200 240 280 320 360 400 440 480 40 80 120 160 200 240 280 320 360 400 440 480 4000 4000 2000 2000 0 0 (5) (5) (a) (b) Figure 4: Electropherograms of PCR products using the TP-PCR forward and reverse primer combinations. (a) and (b) show electropherograms derived from using the TP-PCR forward and reverse primer combinations, respectively. From top to bottom the electropherograms are from DNAs with repeat lengths of [5,14], [11,84], [5,380], [5,250], and [5,1600]. 0 100 200 300 400 500 600 700 800 900 60 70 80 90 100 110 120 130 140 150 500 10000 300 6000 5 repeats 10 repeats (a) (b) Figure 5: Electropherograms of PCR products using the TP-PCR forward and reverse primer combinations illustrating sizing capabilities. (a) and (b) show electropherograms derived from using the TP-PCR forward and reverse primer combinations, respectively. (a) shows an enlarged electropherogram image of sample 58 with repeat lengths 5 and 140; illustrating the ability to visualise the expanded allele peak, within the clinically mild range, at the right-hand end of the characteristic ladder profile. (b) shows two clear allele peaks illustrating the abilitytosizealleles within theunaeff cted rangewiththe TP-PCR reverseprimercombination. means of identifying the presence or absence of expanded References CTG repeat alleles in the DMPK gene. eTh same P4CTG [1] J. D. Brook, M. E. McCurrach, H. G. Harley et al., “Molecular primer has been shown to work both with myotonic dystro- basis of myotonic dystrophy: expansion of a trinucleotide phy and Huntington disease repeat sequences, streamlining (CTG) repeat at the 3 end of a transcript encoding a protein the work flow for these diseases. kinase family member,” Cell, vol. 68, no. 4, pp. 799–808, 1992. 8 Journal of Neurodegenerative Diseases [2] H.Petri,J.Vissing,N.Witting,H.Bundgaard,and L. Køber, “Cardiac manifestations of myotonic dystrophy type 1,” Inter- national Journal of Cardiology,vol.160,no. 2, pp.82–88, 2012. [3] M. Addis, M. Serrenti, C. Meloni, M. Cau, and M. A. Melis, “Triplet-primed PCR is more sensitive than Southern blotting- long PCR for the diagnosis of myotonic dystrophy type1,” Genetic Testing and Molecular Biomarkers,vol.16, no.12, pp. 1428–1431, 2012. [4] eTh International Myotonic Dystrophy Consortium (IDMC), “New nomenclature and DNA testing guidelines for myotonic dystrophy type 1 (DM1),” Neurology,vol.54, no.6,pp. 1218–1221, [5] B.Echenne,A.Rideau,A.Roubertie,G.Seb ´ ire, F. Rivier, and B. Lemieux, “Myotonic dystrophy type I in childhood. Long-term evolution in patients surviving the neonatal period,” European JournalofPaediatricNeurology,vol.12, no.3,pp. 210–223, 2008. [6] E. J. Kamsteeg, W. Kress, C. Catalli et al., “EMQN best practice guidelines and recommendations of myotonic dystrophy types 1and 2,” European Journal of Human Genetics,vol.20, no.12, pp. 1203–1208, 2012. [7] P. Ciotti, E. Di Maria, E. Bellone, F. Ajmar, and P. Mandich, “Triplet repeat primed PCR (TP PCR) in molecular diagnostic testing for Friedreich ataxia,” Journal of Molecular Diagnostics, vol. 6, no. 4, pp. 285–289, 2004. [8] J. P. Warner, L. H. Barron, D. Goudie et al., “A general method for the detection of large CAG repeat expansions by uo fl rescent PCR,” Journal of Medical Genetics, vol. 33, no. 12, pp. 1022–1026, [9] M. Falk, M. Vojt´ıˇskova, ´ Z. Luka´ˇs, I. Kroupova, ´ and U. Froster, “Simple procedure for automatic detection of unstable alleles in the myotonic dystrophy and Huntington’s disease loci,” Genetic Testing,vol.10, no.2,pp. 85–97, 2006. [10] J. M. Love,R.Marquis-Nicholson,R.C.Love, andD.R.Love, “Portable battery-operated rapid PCR amplification of the CAG repeat region of the Huntington disease locus,” Research Journal of Biology,vol.2,no. 6, pp.191–196,2012. [11] J. Radvansky, A. Ficek, G. Minarik, R. Palffy, and L. Kadasi, “Effect of unexpected sequence interruptions to conventional PCR and repeat primed PCR in myotonic dystrophy type 1 testing,” Diagnostic Molecular Pathology,vol.20,no.1,pp.48–51, [12] C. Braida,R.K.A.Stefanatos, B. Adam et al., “Variant CCG and GGC repeats within the CTG expansion dramatically modify mutational dynamics and likely contribute toward unusual symptoms in some myotonic dystrophy type 1 patients,” Human Molecular Genetics,vol.19, no.8,pp. 1399–1412, 2010. 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