kjlm The Korean Journal of Laboratory Medicine Korean J Lab Med 2383-5702 2383-5710 The Korean Society for Laboratory Medicine 10.3343/kjlm.2008.28.6.483 kjlm-28-483 Original Article Molecular and Clinical Characteristics of Myotonic Dystrophy Type 1 in Koreans KimSo YeonM.D. 1 KimJi YeonM.D. 2 KimGyoung PyoungM.T. 1 SungJung-JunM.D. 3 LimKyu SangM.T. 1 LeeKwang-WooM.D. 3 ChaeJong HeeM.D. 4 HongYoon-HoM.D. 5 SeongMoon-WooM.D. 6 ParkSung SupM.D. 1 2 Department of Laboratory Medicine, Seoul National University College of Medicine and Seoul National University Hospital, Seoul, Korea Seoul National University Hospital Clinical Research Institute, Seoul National University College of Medicine and Seoul National University Hospital, Seoul, Korea Department of Neurology, Seoul National University College of Medicine and Seoul National University Hospital, Seoul, Korea Department of Pediatrics, Seoul National University College of Medicine and Seoul National University Hospital, Seoul, Korea Department of Neurology, Boramae Hospital, Seoul National University College of Medicine, Seoul, Korea Department of Laboratory Medicine, National Cancer Center, Goyang, Korea Corresponding author: Sung Sup Park, M.D. Department of Laboratory Medicine, Seoul National University Hospital Clinical Research Institute, 28 Yeongeon-dong, Jongno-gu, Seoul 110-744, Korea Tel: +82-2-2072-3206, Fax: +82-2-747-0359 E-mail: sparkle@snu.ac.kr

∗This study was supported from the research fund of Seoul National University College of Medicine and Seoul National University Hospital (2002).

∗First three authors equally contributed to this work.

 022008 14022008 286 483492 01092008 30092008 01102008 Copyright © 2008 by the Korean Society for Laboratory Medicine 2008 This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/3.0) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited. Background

Myotonic dystrophy type 1 (DM1) is an autosomal-dominant muscular dystrophy caused by expansion of cytosine-thymine-guanine (CTG) trinucleotide repeats in the myotonic dystrophy protein kinase (DMPK) gene. The clinical features of DM1 are multisystemic and highly variable, and the unstable nature of CTG expansion causes wide genotypic and phenotypic presentations. The aim of this study was to characterize the molecular and clinical spectra of DM1 in Koreans.

 Methods

The CTG repeats of 283 Korean individuals were tested by PCR fragment analysis and Southern blot. The following characteristics were assessed retrospectively: spectrum of CTG expansions, clinical findings, genotype-phenotype correlation, anticipation, and genetic instability.

 Results

One-hundred twenty-four patients were confirmed as DM1 by molecular tests, and the CTG expansions ranged from 50 to 2,770 repeats (median 480 repeats). The most frequent clinical features were myotonia, muscular weakness, and family history. Patients with muscular weakness or dysfunction of the central nervous system harbored larger CTG expansions than those without each symptom (P<0.05). The age of onset was inversely correlated with the size of the CTG expansion (γ=-0.422, P<0.001). The instability of CTG expansion representing as the maximum difference between sibships was observed from 50 to 700 repeats in nine families. Clinical anticipation and the increase in CTG repeat were significantly higher in maternally transmitted alleles (P=0.002).

 Conclusions

Molecular genetic tests are not only essential for diagnosis, but also helpful for suggesting the spectrum and relationship between genotype and phenotype in Korean DM1 patients.

 Myotonic dystrophy type 1 DMPK gene CTG Trinucleotide repeat expansion Polymerase chain reaction Southern blot Anticipation Instability Age of onset Korean REFERENCES Kurihara T New classification and treatment for myotonic disorders Intern Med 200544 102732 Brook JD McCurrach ME Harley HG Buckler AJ Church D Aburatani H Molecular basis of myotonic dystrophy: expansion of a trinucleotide (CTG) repeat at the 3′ end of a transcript encoding a protein kinase family member Cell 199268 799808 Machuca-Tzili L Brook D Hilton-Jones D Clinical and molecular aspects of the myotonic dystrophies: a review Muscle Nerve 200532 118 Day JW Ranum LP RNA pathogenesis of the myotonic dystrophies Neuromuscul Disord 200515 516 Harper PS Myotonic dystrophy 3rd ed. London WB Saunders 20014786 32449 The International Myotonic Dystrophy Consortium (IDMC) New nomenclature and DNA testing guidelines for myotonic dystrophy type 1 (DM1) Neurology 200054 121821 Deka R Majumder PP Shriver MD Stivers DN Zhong Y Yu LM Distribution and evolution of CTG repeats at the myotonin protein kinase gene in human populations Genome Res 19966 14254 Martorell L Monckton DG Sanchez A Lopez De Munain A Baiget M Frequency and stability of the myotonic dystrophy type 1 premutation Neurology 200156 32835 Shim SH Cho YH Choi SK Chung SR The CTG repeat polymorphisms of myotonic dystrophy (DM) gene in Korean population J Genet Med 19971 236 Choi BO SunWoo IN Kim SM Lee JS Lee EK Park KD Clinical significance of CTG repeat expansion in Korean myotonic dystrophy patients J Korean Neurol Assoc 199917 54853 (최병옥, 선우일남, 김승민, 이진성, 이은경, 박기덕 등. 한국인 근긴장성 이양증에서CTG 반복확장의임상적의의. 대한신경과학회지 1999;17: 548-53.) Shojasaffar B Moradin N Kahrizi K Cobo AM Najmabadi H CTG expansion & haplotype analysis in DM1 gene in healthy Iranian population Can J Neurol Sci 200835 2169 Pan H Lin HM Ku WY Li TC Li SY Lin CC Haplotype analysis of the myotonic dystrophy type 1 (DM1) locus in Taiwan: implications for low prevalence and founder mutations of Taiwanese myotonic dystrophy type 1 Eur J Hum Genet 20019 63841 Tishkoff SA Goldman A Calafell F Speed WC Deinard AS Bonne-Tamir B A global haplotype analysis of the myotonic dystrophy locus: implications for the evolution of modern humans and for the origin of myotonic dystrophy mutations Am J Hum Genet 199862 1389402 Culjkovic B Stojkovic O Vukosavic S Savic D Rakocevic V Apostolski S CTG repeat polymorphism in DMPK gene in healthy Yugoslav population Acta Neurol Scand 2002105 558 Marchini C Lonigro R Verriello L Pellizzari L Bergonzi P Damante G Correlations between individual clinical manifestations and CTG repeat amplification in myotonic dystrophy Clin Genet 200057 7482 Gharehbaghi-Schnell EB Finsterer J Korschineck I Mamoli B Binder BR Genotype-phenotype correlation in myotonic dystrophy Clin Genet 199853 206 Savic D Rakocvic-Stojanovic V Keckarevic D Culjkovic B Stojkovic O Mladenovic J 250 CTG repeats in DMPK is a threshold for correlation of expansion size and age at onset of juvenile-adult DM1 Hum Mutat 200219 1319 Brisson D Tremblay M Prevost C Laberge C Puymirat J Mathieu J Sibship stability of genotype and phenotype in myotonic dystrophy Clin Genet 200262 2205 Ashizawa T Anvret M Baiget M Barcelo JM Brunner H Cobo AM Characteristics of intergenerational contractions of the CTG repeat in myotonic dystrophy Am J Hum Genet 199454 41423 Amiel J Raclin V Jouannic JM Morichon N Hoffman-Radvanyi H Dommergues M Trinucleotide repeat contraction: a pitfall in prenatal diagnosis of myotonic dystrophy J Med Genet 200138 8502 Harley HG Rundle SA MacMillan JC Myring J Brook JD Crow S Size of the unstable CTG repeat sequence in relation to phenotype and parental transmission in myotonic dystrophy Am J Hum Genet 199352 116474 Figures and Tables

The schematic map of the DMPK gene with relative location of CTG repeats, exons near the repeats, primers, and probe used in this study. The CTG repeats are detected by PCR using primers 102-F and 101-R when the fragment is less than 500 bp. To detect large expansions of more than 500 bp, genomic DNA was digested with the restriction endonucleases EcoRI or PstI. The EcoRI digestion results in ~9 kb band without Alu element, or ~10 kb band with Alu insertion. The PstI digestion results in ~1.2 kb and ~0.8 kb bands. Probe pM10M-6 is ~1.4 kb fragment containing the CTG repeats. Each fragment size is from the allele with Alu insertion and (CTG)20 repeats.

Representative results from (A) PCR and fragment analysis and (B) Southern blot. (A) Chromatogram obtained (a) from a normal individual with heterozygous 12 and 17 CTG re- peats. (b) from a normal individual with homozygous five CTG repeats. (c) from a DM1 patient with one normal 13 CTG repeat in the upper panel and the expanded 129 repeats in the lower panel. The chromatogram in lower panel was obtained after longer time electrophoresis. (B) (a) an autoradiogram obtained from four individuals by Southern blot after EcoRI digestion, and (b) after PstI digestion. In autoradiogram (a), a normal allele of ~9 kb was observed in lanes 1, 2, and 4. Expanded mutant allele larger than ~10 kb was observed in lanes 1 and 2 (marked by asterisk). The expansion of CTG repeats was also suspected in lane 3 (marked by arrow), but not clearly discriminated from a normal band of ~10 kb length. In autoradiogram (b), two normal bands of ~0.8 kb and ~1.2 kb were observed in each lane, and an additional expanded band was observed in lanes 1, 2, and 3. The expanded alleles were estimated as ~690 repeats in lane 1 and ~980 repeats in lane 2. An expanded allele of 75 repeats observed in lane 3 was also determined by PCR and fragment analysis (data not shown). Therefore, these three patients were diagnosed as DM1. Lane 4 was an unaffected individual, heterozygous for 6 and 12 CTG repeats (data from PCR and fragment analysis, not shown).

Pedigrees of 18 Korean DM1 families with two or more patients. Identifier number of each family is indicated above, and the CTG repeats number in the DMPK gene is below each symbol of family member.

Circle, female; square, male; diamond, offspring with no information about sex; filled symbol, symptomatic; open symbol, asymptomatic; shaded symbol, affected status not known; P, fetus in pregnant status.

The distribution of 430 normal alleles with different CTG repeats in the DMPK gene. Histogram is derived from the analysis of 94 normal alleles from 47 normal control subjects, 239 normal alleles from 120 unrelated subjects suspicious as DM1 but harbored no expanded alleles of 50 or more CTG repeats, and 97 normal alleles from 97 unrelated DM1 patients diagnosed by molecular analysis.

The CTG repeat expansion and the age of onset in 83 DM1 patients. Two factors show an inverse correlation.

The size of CTG repeats expansion for (A) mother-offspring, and (B) father-offspring pairs. Anticipation was more evident in maternally-transmitted alleles. Contraction occurred only in paternally-transmitted alleles. Diagonal lines mean that the same allele size was expected between parent and offspring. Open circles located below the diagonal line indicate contraction of CTG repeats in offspring.

The distribution of CTG expansions in 125 Koreans

Molecular diagnosis Clinical phenotype∗ CTG repeat N
Premutation (mutable normal)   35-49 1
DM1 Mild 50-99 9
    100-149 13
  Classic 150-999 82
    1,000-1,999 19
  Congenital >2,000 1
    Total DM1 patients 124

Phenotypes were based on the classification by International Myotonic Dystrophy Consortium (IDMC): mild phenotype, 50 to ~150 repeats; classic phenotype, ~100 to ~1,000; congenital phenotype, >2,000[6]. Abbreviations: CTG, cytosine-thymine-guanine; DM1, myotonic dystrophy type 1.

Clinical characteristics in Korean DM1 patients confirmed by molecular tests

Clinical findings N∗ Posivite Negative P Comment
N (%) CTG expansion median (range) N CTG expansion median (range)
Myotonia 105 84 (80.0) 495 (50-2,770) 21 750 (73-1,500) >0.05  
Muscular weakness 105 81 (77.1) 560 (50-2,770) 24 280 (73-1,420) 0.019  
Family history 88 46 (52.3) 665 (118-2,770) 42 470 (110-1,420) >0.05  
Muscular atrophy 105 44 (41.9) 540 (110-1,700) 61 480 (50-2,770) >0.05  
Frontal baldness 105 35 (33.3) 550 (50-1,700) 70 480 (73-2,770) >0.05  
CNS dysfunction/developmental delay 35 17 (48.6) 1,190 (230-2,770) 18 410 (110-1,790) 0.005  
Cataract 56 24 (42.9) 638 (120-2,770) 32 655 (110-1,400) >0.05  
Endocrine abnormalities 44 17 (38.6) 510 (120-990) 27 450 (50-1,350) >0.05  
Cardiac abnormalities 76 26 (34.2) 590 (120-1,420) 50 530 (110-2,770) >0.05  
Electromyography 72 69 (95.8) 510 (110-2,770) 3 550 (230-1,240) >0.05  
Muscle biopsy 23 19 (82.6) 560 (140-2,770) 4 295 (120-530) >0.05  
Creatine kinase 50 17 (34.0) 620 (140-1,700) 33 550 (120-1,500) >0.05 Median 228.0 Range 68.0-1,044.0 (IU/L)

The number of patients whose clinical information for each symptom was available. For the clinical manifestations that require specific tests, only patients who undergone tests were analyzed.

Abbreviations: CNS, central nervous system; See Table 1.

The instability in CTG repeat expansion assessed by comparison between siblings in nine DM1 families

Family identifier Parent allele (repeats) Parent sex Sib1 Sib2 Sib3 Sib4 Maximal difference (repeat)
Repeats Onset Repeats Onset Repeats Onset Repeats Onset
1 690 M 430 A 480 A - - - - 50
2 Unidentified F 620 A 570 A - - - - 50
5 Unidentified U 170 A 637 A - - - - 467
7 Unidentified U 118 N 95 N 390 A - - 295
8 Unidentified M 180 U 80 U - - - - 100
10 73 M 1,030 J 1,140 J - - - - 110
16 57 M 170 A 94 A 670 A 490 A 576
17 Unidentified U 1,350 A 1,700 A - - - - 350
18 Unidentified U 230 A 930 A - - - - 700

Abbreviations: M, male; F, female; A, adult-onset; N, asymptomatic; J, juvenile-onset; U, unknown; See Table 1.