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Possible de novo CTG repeat expansion in the DMPK gene of a patient with cardiomyopathy
Journal of Clinical Neuroscience 17 (2010) 408–409
Contents lists available at ScienceDirect
Journal of Clinical Neuroscience journal homepage: www.elsevier.com/locate/jocn
Short Communication
Possible de novo CTG repeat expansion in the DMPK gene of a patient with cardiomyopathy Daisuke Furutama a,*,1, Nobuyuki Negoro a,1, Fumio Terasaki b, Kuniko Tsuji-Matsuyama a, Reiko Sakai c, Tamaki Maeda c, Toshifumi Tanaka c, Masaaki Hoshiga a, Tadashi Ishihara a, Nakaaki Ohsawa c, Toshiaki Hanafusa a a
First Department of Internal Medicine, Osaka Medical College, 2-7 Daigaku-cho, Takatsuki 569-8686, Japan Third Department of Internal Medicine, Osaka Medical College, Takatsuki, Japan c Aino Institute for Aging Research, Ibaraki, Japan b
a r t i c l e
i n f o
Article history: Received 4 August 2008 Accepted 8 June 2009
Keywords: Neurology Basic Neuroscience DMPK CTG repeat SP-PCR
a b s t r a c t CTG triplet repeats of ‘‘normal” length in the myotonic dystrophy protein kinase (DMPK) gene have been previously believed to be stable and new pathological expansion was not believed to occur. Here we report possible de novo CTG repeat expansion in the DMPK gene in a patient with cardiomyopathy, who was not diagnosed as having myotonic dystrophy type 1 (DM1) by conventional genetic tests. Ó 2009 Elsevier Ltd. All rights reserved.
A 73-year-old woman with a permanent pacemaker for complete atrioventricular block exhibited congestive heart failure. Physical examination and blood tests, including serum creatinine kinase were normal. Echocardiography revealed localized septal wall thinning at the basal side (Fig. 1A) with normal left ventricular contraction. Thallium 201 (201Tl) cardiac scintigraphy revealed multiple spotty defects in the septal, anterolateral, and inferoapical portions of the left ventricle. Most striking was the presence of multiple ventricular aneurysms detected by left ventriculography showing focal dyskinesis of the ventricular wall (Fig. 1B, C). An endomyocardial biopsy of the right ventricular septum demonstrated nodular fibro-fatty tissue in the subendocardial lesion (Fig. 1D). After gallium 67 (67Ga) cardiac scintigraphy and other examinations, she was diagnosed with idiopathic cardiomyopathy. Although this patient showed no abnormal neurological or electrophysiological findings, including myotonia, she had cardiac conduction block, cataracts, and three fetal losses of unknown cause, all of which are often seen in patients with myotonic dystrophy type 1 (DM1).1 Focal fatty degeneration of the cardiac ventricles can also be seen in patients with DM1.2 DM1 is genetically caused by the unstable expansion of the cytosine–thymine–guanine (CTG) * Corresponding author. Tel.: +81 726 83 1221; fax: +81 726 83 1801. E-mail address: [email protected] (D. Furutama). 1 These authors contributed equally to this work. 0967-5868/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved. doi:10.1016/j.jocn.2009.06.010
trinucleotide repeat in the 30 - untranslated region of the myotonic dystrophy protein kinase (DMPK) gene.1 Southern blotting (Fig. 1E) and conventional polymerase chain reaction (PCR) (Fig. 1F) analyses showed no CTG repeat expansion in her genomic DNA from peripheral blood mononuclear cells (PBMNC). However, using small pool PCR techniques (SP-PCR),3 we found the CTG repeat expansion in a fraction of her PBMNC (Fig. 1G). The CTG repeat expansion was not detected in an age-matched control’s genomic DNA (Fig. 1H). Surprisingly, the expanded alleles were more frequently detected in the cardiac muscle than in the PBMNC (Fig. 1I). We performed SP-PCR repeatedly with another genomic DNA sample from the patient, with another batch of PCR reagents and with ‘‘zero-DNA” negative controls.3 We also excluded the possibility of DNA contamination from other DM1 patient samples (data not shown). We then confirmed CTG repeat expansion in the PCR products by direct sequencing. The mutant allele in a small fraction of cells had more than 50 CTG repeats, whereas both normal alleles had 12 repeats. Unfortunately, there was not enough heart tissue to detect RNA foci of CTG repeat expanded transcripts by an RNA fluorescence in situ hybridization (FISH) study.4 We could not detect the triplet repeat instability in other loci, including SCA1, 2, 3, 6, DRPLA and Kennedy’s disease (data not shown), suggesting that the instability in the DM1 locus was not due to a functional defect in the control system for genome-wide repeat stability, such as mismatched repair genes.
D. Furutama et al. / Journal of Clinical Neuroscience 17 (2010) 408–409
409
Fig. 1. (A) Long-axis echocardiography showing marked localized thinning of the septal wall at the basal side (arrowhead). (B, C) Left ventriculography, 30 degree right anterior oblique view, revealing multiple aneurysms (arrowheads) in the both the diastolic and systolic phases. (D) An endomyocardial biopsy of the right ventricular septum demonstrated nodular fibro-fatty tissue in the subendocardial lesion (haematoxylin and eosin, 100). (E) Southern blot analysis with cDNA25 (p5B1.4)5 probe (‘‘P”) after digestion with EcoRI or BglI. Because of the Alu polymorphism, EcoRI digested 9.6 kb band was observed only in the control sample (‘‘C”). (F) Conventional polymerase chain reaction (PCR) using myotonic dystrophy (DM)-A and DM-BR primers.3 (G, I) Small pool (SP-PCR) analysis3 with 10 ng (left 6 lanes) or 1 ng (right 6 lanes) of genomic template DNA using the synthesized 40 repeats of CTG oligonucleotide probe revealed expanded alleles (arrows) in (G) the patient’s peripheral blood mononuclear cells (PBMNC) and (I) cardiac muscle cells. Because cardiac muscle tissue was fixed with formaldehyde, paraffin-embedded and genomic DNA was partially broken down, it seemed that the PCR amplification was less effective than usual. (H) Genomic DNA of age-matched control’s PBMNC as template. To exclude contamination from the template DNA, water was used as a ‘‘template” for a negative ‘‘zero” control, in all SP-PCRs3 (data not shown).
Our results suggest that the ‘‘new” expansion arose in only a fraction of cells and the patient could have DM1-associated cardiomyopathy. The repeat number is variable between tissues in a single DM1 patient, whereas it has been believed that CTG repeats of normal length, up to 37 repeats, are stable between tissues and generations.1 One possible explanation for the pathogenesis is that isolated cardiomyopathy developed because a relatively large number of mutated cells existed in her heart. However, it is unclear whether this frequency, up to 10%, of the expanded allele in cardiac muscle is sufficient to cause the cardiomyopathy. Although possibly coincidental, it could raise the possibility of a relationship between the phenotype and mutation. Because cardiac muscle cells are post-mitotic and non-proliferative in adults, there might have been more cells with the expanded allele prior to the development of fatty degeneration. We propose that it could be clinically important to add DM1 to the list of differential diagnoses for ‘‘idiopathic” cardiomyopathy. This should stimulate further research into genetic background (including de novo DM1 mutation), in patients with cardiomyopathy, cataract or other isolated DM1 symptoms.
Acknowledgement We thank Dr T. Ashizawa (Department of Neurology, University of Florida) for his valuable comments. References 1. Harper PS. Myotonic dystrophy. 3rd ed. London: WB Saunders; 2001. 2. Vignaux O, Lazarus A, Varin J, et al. Right ventricular MR abnormalities in myotonic dystrophy and relationship with intracardiac electrophysiologic test findings: initial results. Radiology 2002;224:231–5. 3. Monckton DG, Wong LJC, Ashizawa T, et al. Somatic mosaicism, germline expansions, germline reversions and intergenerational reductions in myotonic dystrophy males: small pool PCR analyses. Hum Mol Genet 1995;4:1–8. 4. Mankodi A, Logigian E, Callahan L, et al. Myotonic dystrophy in transgenic mice expressing an expanded CUG repeat. Science 2000;289:1769–72. 5. Buxton J, Shelbourne P, Davies J, et al. Detection of an unstable fragment of DNA specific to individuals with myotonic dystrophy. Nature 1992;355:547–8.
Contents lists available at ScienceDirect
Journal of Clinical Neuroscience journal homepage: www.elsevier.com/locate/jocn
Short Communication
Possible de novo CTG repeat expansion in the DMPK gene of a patient with cardiomyopathy Daisuke Furutama a,*,1, Nobuyuki Negoro a,1, Fumio Terasaki b, Kuniko Tsuji-Matsuyama a, Reiko Sakai c, Tamaki Maeda c, Toshifumi Tanaka c, Masaaki Hoshiga a, Tadashi Ishihara a, Nakaaki Ohsawa c, Toshiaki Hanafusa a a
First Department of Internal Medicine, Osaka Medical College, 2-7 Daigaku-cho, Takatsuki 569-8686, Japan Third Department of Internal Medicine, Osaka Medical College, Takatsuki, Japan c Aino Institute for Aging Research, Ibaraki, Japan b
a r t i c l e
i n f o
Article history: Received 4 August 2008 Accepted 8 June 2009
Keywords: Neurology Basic Neuroscience DMPK CTG repeat SP-PCR
a b s t r a c t CTG triplet repeats of ‘‘normal” length in the myotonic dystrophy protein kinase (DMPK) gene have been previously believed to be stable and new pathological expansion was not believed to occur. Here we report possible de novo CTG repeat expansion in the DMPK gene in a patient with cardiomyopathy, who was not diagnosed as having myotonic dystrophy type 1 (DM1) by conventional genetic tests. Ó 2009 Elsevier Ltd. All rights reserved.
A 73-year-old woman with a permanent pacemaker for complete atrioventricular block exhibited congestive heart failure. Physical examination and blood tests, including serum creatinine kinase were normal. Echocardiography revealed localized septal wall thinning at the basal side (Fig. 1A) with normal left ventricular contraction. Thallium 201 (201Tl) cardiac scintigraphy revealed multiple spotty defects in the septal, anterolateral, and inferoapical portions of the left ventricle. Most striking was the presence of multiple ventricular aneurysms detected by left ventriculography showing focal dyskinesis of the ventricular wall (Fig. 1B, C). An endomyocardial biopsy of the right ventricular septum demonstrated nodular fibro-fatty tissue in the subendocardial lesion (Fig. 1D). After gallium 67 (67Ga) cardiac scintigraphy and other examinations, she was diagnosed with idiopathic cardiomyopathy. Although this patient showed no abnormal neurological or electrophysiological findings, including myotonia, she had cardiac conduction block, cataracts, and three fetal losses of unknown cause, all of which are often seen in patients with myotonic dystrophy type 1 (DM1).1 Focal fatty degeneration of the cardiac ventricles can also be seen in patients with DM1.2 DM1 is genetically caused by the unstable expansion of the cytosine–thymine–guanine (CTG) * Corresponding author. Tel.: +81 726 83 1221; fax: +81 726 83 1801. E-mail address: [email protected] (D. Furutama). 1 These authors contributed equally to this work. 0967-5868/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved. doi:10.1016/j.jocn.2009.06.010
trinucleotide repeat in the 30 - untranslated region of the myotonic dystrophy protein kinase (DMPK) gene.1 Southern blotting (Fig. 1E) and conventional polymerase chain reaction (PCR) (Fig. 1F) analyses showed no CTG repeat expansion in her genomic DNA from peripheral blood mononuclear cells (PBMNC). However, using small pool PCR techniques (SP-PCR),3 we found the CTG repeat expansion in a fraction of her PBMNC (Fig. 1G). The CTG repeat expansion was not detected in an age-matched control’s genomic DNA (Fig. 1H). Surprisingly, the expanded alleles were more frequently detected in the cardiac muscle than in the PBMNC (Fig. 1I). We performed SP-PCR repeatedly with another genomic DNA sample from the patient, with another batch of PCR reagents and with ‘‘zero-DNA” negative controls.3 We also excluded the possibility of DNA contamination from other DM1 patient samples (data not shown). We then confirmed CTG repeat expansion in the PCR products by direct sequencing. The mutant allele in a small fraction of cells had more than 50 CTG repeats, whereas both normal alleles had 12 repeats. Unfortunately, there was not enough heart tissue to detect RNA foci of CTG repeat expanded transcripts by an RNA fluorescence in situ hybridization (FISH) study.4 We could not detect the triplet repeat instability in other loci, including SCA1, 2, 3, 6, DRPLA and Kennedy’s disease (data not shown), suggesting that the instability in the DM1 locus was not due to a functional defect in the control system for genome-wide repeat stability, such as mismatched repair genes.
D. Furutama et al. / Journal of Clinical Neuroscience 17 (2010) 408–409
409
Fig. 1. (A) Long-axis echocardiography showing marked localized thinning of the septal wall at the basal side (arrowhead). (B, C) Left ventriculography, 30 degree right anterior oblique view, revealing multiple aneurysms (arrowheads) in the both the diastolic and systolic phases. (D) An endomyocardial biopsy of the right ventricular septum demonstrated nodular fibro-fatty tissue in the subendocardial lesion (haematoxylin and eosin, 100). (E) Southern blot analysis with cDNA25 (p5B1.4)5 probe (‘‘P”) after digestion with EcoRI or BglI. Because of the Alu polymorphism, EcoRI digested 9.6 kb band was observed only in the control sample (‘‘C”). (F) Conventional polymerase chain reaction (PCR) using myotonic dystrophy (DM)-A and DM-BR primers.3 (G, I) Small pool (SP-PCR) analysis3 with 10 ng (left 6 lanes) or 1 ng (right 6 lanes) of genomic template DNA using the synthesized 40 repeats of CTG oligonucleotide probe revealed expanded alleles (arrows) in (G) the patient’s peripheral blood mononuclear cells (PBMNC) and (I) cardiac muscle cells. Because cardiac muscle tissue was fixed with formaldehyde, paraffin-embedded and genomic DNA was partially broken down, it seemed that the PCR amplification was less effective than usual. (H) Genomic DNA of age-matched control’s PBMNC as template. To exclude contamination from the template DNA, water was used as a ‘‘template” for a negative ‘‘zero” control, in all SP-PCRs3 (data not shown).
Our results suggest that the ‘‘new” expansion arose in only a fraction of cells and the patient could have DM1-associated cardiomyopathy. The repeat number is variable between tissues in a single DM1 patient, whereas it has been believed that CTG repeats of normal length, up to 37 repeats, are stable between tissues and generations.1 One possible explanation for the pathogenesis is that isolated cardiomyopathy developed because a relatively large number of mutated cells existed in her heart. However, it is unclear whether this frequency, up to 10%, of the expanded allele in cardiac muscle is sufficient to cause the cardiomyopathy. Although possibly coincidental, it could raise the possibility of a relationship between the phenotype and mutation. Because cardiac muscle cells are post-mitotic and non-proliferative in adults, there might have been more cells with the expanded allele prior to the development of fatty degeneration. We propose that it could be clinically important to add DM1 to the list of differential diagnoses for ‘‘idiopathic” cardiomyopathy. This should stimulate further research into genetic background (including de novo DM1 mutation), in patients with cardiomyopathy, cataract or other isolated DM1 symptoms.
Acknowledgement We thank Dr T. Ashizawa (Department of Neurology, University of Florida) for his valuable comments. References 1. Harper PS. Myotonic dystrophy. 3rd ed. London: WB Saunders; 2001. 2. Vignaux O, Lazarus A, Varin J, et al. Right ventricular MR abnormalities in myotonic dystrophy and relationship with intracardiac electrophysiologic test findings: initial results. Radiology 2002;224:231–5. 3. Monckton DG, Wong LJC, Ashizawa T, et al. Somatic mosaicism, germline expansions, germline reversions and intergenerational reductions in myotonic dystrophy males: small pool PCR analyses. Hum Mol Genet 1995;4:1–8. 4. Mankodi A, Logigian E, Callahan L, et al. Myotonic dystrophy in transgenic mice expressing an expanded CUG repeat. Science 2000;289:1769–72. 5. Buxton J, Shelbourne P, Davies J, et al. Detection of an unstable fragment of DNA specific to individuals with myotonic dystrophy. Nature 1992;355:547–8.