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An intergenerational contraction of the CTG repeat in Japanese myotonic dystrophy
JOURNAL OF THE
NEUROLOGICAL SCIENCES ELSEVIER
Journal of the Neurological Sciences 139 (I 996) 48-5 1
An intergenerational contraction of the CTG repeat in Japanese myotonic dystrophy Ryusuke Matsumura a3* , Tadashi Namikawa b, Tetsuro Miki ‘, Tameko Kihira d, Hidehisa Yamagata ‘, Yukio Mano a, Tetsuya Takayanagi a a Department
Neurology, Nara Medical Unicersir?;, 840 Shijocho, Kashihara, Nam 634, Japan b Konishibashi Hospital, 240-I Tani, Sakurai, Nara, Japan ’ Department of Geriatric Medicine, Osaka Unil>ersity Medical School, 2-2 Yamadaoka, Suita, Osaka, Japan ’ Division of Neurological Diseases, Wakayama Medical College, 27.9 Bancho, Wokayama, Japan of
Received 9 August 1995; revised 7 December 1995; accepted 17 December 1995
Abstract We present the first report of a Japanese family with myotonic dystrophy (DM) that showed an intergenerational contraction of the CTG repeat. The size of the expanded CTG repeats was 3.2 kb for the father and 2.2 kb for the daughter, indicating that the expansion decreased during transmission from the father to the daughter. Despite the CTG repeat contraction, the daughter showed earlier age of onset than the father. However, she appeared to be less severely affected than the father. We discuss the correlation between the CTG repeat contraction and the clinical phenotype. The presence of the CTG repeat contraction in Japanese DM is important for genetic counseling of Japanese DM families. Kewordst
Myotonic dystrophy; CTG repeat: Contraction; Japanese; Anticipation;
1. Introduction Myotonic dystrophy (DM) is an autosomal dominant, multisystem disorder characterized primarily by myotonia, progressive muscle weakness and wasting, cardiac conduction defects and cataract (Harper, 1989). The clinical phe-
notype is highly variable, ranging from a mild, late onset to a severe congenital form (Harper, 1989). The mutation responsible for DM has been identified as an expansion of a CTG trinucleotide repeat in the 3’ untranslated region of a putative serine/threonine protein kinase gene on chromosome 19q13.3 (Brook et al., 1992; Buxton et al., 1992; Fu et al., 1992; Harley et al., 1992a; Mahadevan et al., 1992). In normal individuals, the number of CTG repeats ranges from 5 to 37 copies, while in affected individuals it ranges from 50 to thousands of copies (Brook et al., 1992; Brunner et al., 1992; Lavedan
z Corresponding author. Tel: + 8 I (7442) 2-30.51 Ext. 3417; Fax: + 8 I (7442) 4-6065. 0022-510X/96/$15.00 Published by Elsevier Science B.V. PII SOO22-5 10X(96)000 14-7
Paternal transmission; Genetic counseling
et al., 1993b). Expanded CTG repeats are unstable and usually increase in size in successive generations (Ashizawa et al., 1992; Harley et al., 1992b; Lavedan et al., 1993b; Mulley et al., 1993; Redman et al., 1993). This is thought to be the molecular basis for anticipation, a clinical phenomenon characterized by earlier age of onset and increasing severity of the disease in successive generations. However, in recent reports, a small number of offspring have shown a decrease in the CTG repeat size compared with their parents (Ashizawa et al., 1992, Ashizawa et al., 1994; Abeliovich et al., 1993; Brunner et al., 1993; Cobo et al., 1993; Hunter et al., 1993; Lavedan et al., 1993a; Mulley et al., 1993; O’Hoy et al., 1993; Redman et al., 1993; Shelboume et al., 1993). Especially, a systematic report of Ashizawa
et al. (1994) revealed
that 95 (6.4%)
of 1,489
DM parent-child pairs with different ethnic backgrounds showed such intergenerational contractions of the repeat. In that report, 80 Japanese parent-child pairs were examined, but no pair had the CTG repeat contraction. Here we present the first report of a Japanese DM family
with an intergenerational
contraction
of the CTG
R. Matsumura
et al. /Journal
of the
Neurological
Sciences
139 (1996)
repeat, and discuss the correlation between the contraction and clinical phenotype of this case.
48-51
Subject
Subject
II-5
Ill-6
13kb[I
2. Case report The pedigree of the Japanese family is shown in Fig. 1.
49
4 12kb
9.8 kb ) * 8.6 kb
2. I. Subject II-S A Japanese man began to notice some stiffness and weakness of the hands at age 40 years. Around that time he developed gait disturbance. When examined at the age of 45, he had moderate weakness and wasting of the distal limb muscles and to a lesser extent the proximal limb muscles. His face showed a characteristic appearance, that is, wasting of the bitemporal, masseter and facial muscles, and frontal baldness. Marked weakness and wasting of the stemomastoids were observed. He displayed grip and percussion myotonia, and electromyography showed myotonic discharges in the intrinsic hand muscles. A highly arched palate and slurred speech characterized by a monotonous nasal voice were observed. He showed no intellectual deterioration but a personality pattern of inattention. Bilateral cataracts had been extracted at age 33. Serum creatine kinase was slightly elevated and serum immunogloblin G was decreased. Electrocardiography (ECG) demonstrated first-degree heart block, and head CT scanning revealed hyperostosis of the frontal bones. At age 47, he often fell unless he used a walking stick and he had some difficulty in rising from supine position. The same year he died of intracerebral hemorrhage. 2.2. Subject III-6 The daughter of II-5 was interviewed in detail at the age of 14 years. She had begun to note some stiffness of the hands at age 8. She had been an average student and completed high school. At the time of the study, she was 21 years of age and working satisfactorily as a waitress. She could lift heavy weights. However, she sometimes had some difficulty in masticating food. Her face showed some wasting of the bitemporal, masseters and facial muscles, but no frontal baldness. She had a highly arched palate but
I 7
II
Fig. 1. The pedigree of the Japanese family. Males are represented by squares, females by circles, and those affected clinically by solid symbols. Diagonal lines indicate deceased persons.
Fig. 2. Southern blot analysis of EcoRI-digested DNA from Subject II-5 and Subject III-6 with the p5B1.4 probe. The bracket shows a diffuse smear of bands whose average size is 13 kb in the DNA from Subject 11-5.
no dysarthria. Moderate weakness and wasting of the sternomastoids were observed. Muscle strength of the extremities was almost normal, but her grasping strength was slightly weak (right side, 17 kg; left side, 13 kg). She displayed grip and percussion myotonia. Serum creatine kinase was slightly elevated and serum immunogloblin G level was normal. ECG indicated first-degree heart block. Since a slit lamp examination had not been performed, it was unknown whether she had cataracts.
3. Southern blot analysis Blood samples were obtained from Subject II-5 at age 45 and Subject III-6 at age 14 with informed consent. Genomic DNA was extracted by standard procedures and digested with a restriction endonuclease, EcoRI. The digests were electrophoresed in a 0.7% agarose gel, then transferred onto a nylon membrane, and hybridized with the radiolabeled probe p5B 1.4 as described (Shelboume et al., 1992). The results are shown in Fig. 2. The father had one normal band (9.8 kb) and a diffuse smear of bands whose average size was 13 kb. The smear suggests mitotic instability of the CTG repeat in peripheral blood leukocytes (Lavedan et al., 1993b; Monckton et al., 1995; Wong et al., 1995). The daughter had one normal band (8.6 kb) and one expanded band of 12 kb. The normal 9.8 kb/8.6 kb bands result from an Alu element insertion/deletion polymorphism located 5 kb upstream from the CTG repeat (Shelboume et al., 1992; Mahadevan et al., 1993). It is known that the CTG repeat expansions in both Caucasian DM and Japanese DM occurred exclusively in the Ah insertion chromosomes (Harley et al., 1992a; Yamagata et al., 1992; Mahadevan et al., 1993). Therefore, the size of the expanded CTG repeats was calculated at 3.2 kb (about 1060 repeats) for the father and at 2.2 kb (about 730 repeats) for the daughter. Thus, the CTG repeat expansion decreased during transmission from the father to the daughter.
50
R. Matsumura et al./ Journal
of the Neurological Sciences 139 (1996148-51
4. Discussion The increasing size of the CTG repeats in successive generations is thought to be the molecular basis for anticipation. Therefore, we expect that the CTG repeat contraction accompanies a later age of onset and less severe manifestations in the offspring, compared with the parent. However, in this family, the daughter has an earlier age of onset than the father. One possible explanation for this unexpected anticipation is the bias involved in ascertaining the age of onset. Some patients with DM become so well adjusted to the difficulty in relaxing their muscles that they may not complain of it or even realize that the symptom is abnormal (Harper, 1989). Myotonia is thus a symptom and sign that has to be actively sought. Since the father was not known to be a DM heterozygote at the time his symptoms developed but the daughter was known to be a possible mutation carrier, earlier diagnosis in the daughter might have been made. Another possible explanation for the unexpected anticipation is somatic mosaicism of the CTG repeat size. In this study, we determined the CTG repeat size in DNA isolated from peripheral blood leukocytes. However, the primarily affected tissue in DM is skeletal muscle. It is known that the CTG repeat size differs between skeletal muscle and leukocytes (Anvret et al., 1993; Ashizawa et al., 1993; Lavedan et al., 1993b; Thornton et al., 1994). Therefore, there is a possibility that the CTG repeat size in skeletal muscle of the daughter is larger than that in skeletal muscle of the father and this resulted in anticipation, while the repeat size in leukocytes of the daughter is smaller. Since muscle biopsies of the father and the daughter had not been carried out, we can not make the comparison of the CTG repeat sizes in muscle. The third possible explanation for this anticipation is pseudocontractions. By comparing individual patient samples collected at two different times, Wong et al. (1995) revealed that the CTG repeat size increased with age in some DM patients. Based on this age-dependent CTG expansion, Monckton et al. (1995) recently postulated the concept of pseudocontractions; that is, a true germline CTG expansion during transmission from the father to the daughter can be masked by a higher somatic expansion in the older father compared with the more limited somatic expansion in the younger daughter, resulting in the apparent intergenerational contraction. In the future, it will be interesting to see whether the CTG repeat size is larger in a blood sample from the daughter at age 45 than in that from the father at age 4.5 (3.2 kb). Surprisingly, Ashizawa et al. (1994) reported that approximately one half of 56 parent-child pairs with the CTG repeat contractions showed anticipation as in our case. Therefore, it remains possible that other genetic or environmental factors may cause anticipation regardless of the CTG repeat size.
At present the daughter appears to be less severely affected than the father despite her earlier age of onset. This may truly reflect the CTG repeat contraction. For accurate evaluation of the effects of the CTG repeat contraction on the clinical phenotype, it is necessary to see whether the daughter is less severely affected than the father when she reaches the age of 40 years. It is important to follow the clinical course as well as the CTG repeat size of the daughter. In this family, Subject II-2 and Subject III-2 were also examined. The CTG repeat size of Subject 11-2, whose age of onset was 30 years, and that of Subject III-2 with congenital DM were 1.2 kb and 5.2 kb, respectively. It should be noted that the contraction of the CTG repeat occurred in paternal transmission while the large expansion of the CTG repeat, which resulted in congenital DM, occurred in maternal transmission within the same family. The paternal and maternal transmissions appear to have distinct influences on the intergenerational changes of the CTG repeat size in DM. There may be a maternal factor that allows the CTG repeats to expand further and a paternal factor that restricts the expansion. So far the intergenerational contractions of the CTG repeats have been reported in DM populations from different ethnic backgrounds in Europe, North and South America, and Australia, but not in Japan (Ashizawa et al., 1994). In this family we demonstrate for the first time the contraction in Japanese DM, providing evidence that unstable CTG repeats in Japanese DM have the same characteristics as in Caucasian DM. The presence of the CTG repeat contraction in Japanese DM needs to be taken account of and is important for genetic counseling of Japanese DM families.
Acknowledgements This work was supported in part by a Grant-in-Aid for Intractable Disease, Ministry of Health and Welfare of Japan. We are grateful to Professor Richard F. Mayer, University of Maryland School of Medicine, Baltimore, MD, for critical reading of the manuscript.
References Abeliovich, D., Lerer, I., Pashut-Lavon, I., Shmueli, E., Raas-Rothschild, A. and Frydman, M. (1993) Negative expansion of the myotonic dystrophy unstable sequence. Am. J. Hum. Genet., 52: 1175-l 181. Anvret, M., Ahlberg, G., Grandell, U., Hedberg, B., Johnson, K. and Edstrom, L. (1993) Larger expansions of the CTG repeat in muscle compared to lymphocytes from patients with myotonic dystrophy. Hum. Mol. Genet., 2: 1397-1400. Ashizawa, T., Anvret, M., Baiget, M., Barcelo, J.M., Brunner, H., Cobo, A.M., Dallapiccola, B., Fenwick, R.G. Jr., Grandell, U., Harley, H., Junien, C., Koch, M.C., Korneluk, R.G., Lavedan, C., Miki, T., Mulley, J.C., Lopez de Munain, A., Novelli, G., Roses, A.D., Seltzer, W.K., Shaw, D.J., Smeets, H., Sutherland, G.R., Yamagata,H. and
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et al. /Journal
of the
Harper, P.S. (19941 Characteristics of intergenerational contractions of the CTG repeat in myotonic dystrophy. Am. J. Hum. Genet., 54: 414-423. Ashizawa, T., Dubel, J.R., Dunne, P.W., Dunne, C.J., Fu, Y-H., Pizzuti, A., Caskey, C.T., Boerwinkle, E., Perryman, M.B., Epstein, H.F. and Hejtmancik, J.F. (1992) Anticipation in myotonic dystrophy. II. complex relationships between clinical findings and structure of the GCT repeat. Neurology, 42: 1877- 1883. Ashizawa, T., Dubel, J.R. and Harati, Y. (1993) Somatic instability of CTG repeat in myotonic dystrophy. Neurology, 43: 2674-2678. Brook, J.D., McCurrach, M.E., Harley, H.G., Buckler, A.J., Church, D., Aburatani, H., Hunter, K., Stanton, V.P., Thirion, J-P., Hudson, T., Sohn, R., Zemelman, B., Snell, R.G., Rundle, S.A., Crow, S., Davies, J., Shelbourne, P., Buxton, J., Jones, C., Juvonen, V.. Johnson, K., Harper, P.S., Shaw, D.J. and Housman, D.E. (1992) 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, 68: 799-808. Brunner, H.G., Jansen, G., Nillesen, W., Nelen, M.R., de Die, C.E.M., Howeler, C.J., van Oost, B.A., Wieringa, B., Ropers, H-H. and Smeets, H.J.M. (1993) Brief report: reverse mutation in myotonic dystrophy. N. Engl. J. Med., 328: 476-480. Brunner, H.G., Nillesen, W., van Oost, B.A., Jansen, G., Wieringa, B., Ropers, H-H. and Smeets, H.J.M. (1992) Presymptomatic diagnosis of myotonic dystrophy. J. Med. Genet., 29: 780-784. Buxton, J., Shelbourne, P., Davies, J., Jones, C., Tongeren, T.V., Aslanidis, C., de Jong, P., Jansen, G., Anvret, M., Riley, B., Williamson, R. and Johnson, K. (1992) Detection of an unstable fragment of DNA specific to individuals with myotonic dystrophy. Nature, 355: 547548. Cobo, A.M., Baiget, M., Lopez de Munain, A., Poza, J.J., Emparanza, J.I. and Johnson, K. (1993) Sex-related difference in intergenerational expansion of myotonic dystrophy gene. Lancet, 341: 1159-l 160. Fu, Y-H., Pizzuti, A., Fenwick, R.G., King, J., Rajnarayan, S., Dunne, P.W., Dubel, J., Nasser, G.A., Ashizawa, T., de Jong, P., Wieringa, B., Korneluk, R., Perryman, M.B., Epstein, H.F. and Caskey, CT. (19921 An unstable triplet repeat in a gene related to myotonic muscular dystrophy. Science, 255: 1256-1258. Harley, H.G., Brook, J.D., Rundle, S.A., Crow, S., Reardon, W., Buckler, A.J., Harper, P.S., Housman, D.E. and Shaw, D.J. (1992a) Expansion of an unstable DNA region and phenotypic variation in myotonic dystrophy. Nature, 355: 545-546. Harley, H.G., Rundle, S.A., Reardon, W., Myring, J., Crow, S., Brook, J.D., Harper, P.S. and Shaw, D.J. (1992b) Unstable DNA sequence in myotonic dystrophy. Lancet, 339: 1125- 1128. Harper, P.S. (1989) Myotonic dystrophy. 2d ed, WB Saunders, London. Hunter, A.G.W., Jacob, P., O’Hoy, K., MacDonald, I., Mettler, G., Tsilfidis, C. and Komeluk, R.G. (1993) Decrease in the size of the myotonic dystrophy CTG repeat during transmission from parent to child: implication for genetic counseling and genetic anticipation, Am. J. Med. Genet., 45: 401-407. Lavedan, C., Hofmann-Radvanyi, H., Rabes, J.P., Roume, J. and Junien, C. (1993a) Different sex-dependent constraints in CTG length varia-
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tion as explanation for congenital myotonic dystrophy. Lancet, 341: 237. Lavedan, C., Hofmann-Radvanyi, H., Shelboume, P., Rabes, J-P., Duros, C., Savoy, D., Dehaupas, I., Lute, S., Johnson, K. and Junien, C. (1993b) Myotonic dystrophy: size- and sex-dependent dynamics of CTG meiotic instability, and somatic mosaicism. Am. J. Hum. Genet., 52: 875-883. Mahadevan, M.S., Foitzik, M.A., Surh, L.C. and Korneluk, R.G. (19931 Characterization and polymerase chain reaction (PCR) detection of an Alu deletion polymorphism in total linkage disequilibrium with myotonic dystrophy. Genomics, 15: 446-448. Mahadevan, M.S., Tsiltidis, C., Sabourin, L., Shutler, G., Amemiya, C., Jansen, G., Neville, C., Narang, M., Barcel6, J., O’Hoy, K., Leblond, S., Earle-Macdonald, J., de Jong, P., Wieringa, B. and Korneluk, R.G. (19921 Myotonic dystrophy mutation: an unstable CTG repeat in the 3’ untranslated region of the gene. Science, 255: 1253-1255. Monckton, D.G., Wong, L-J.C., Ashizawa, T. and Caskey, C.T. (19951 Somatic mosaicism, germline expansions, germline reversions and intergenerational reductions in myotonic dystrophy males: small pool PCR analyses. Hum. Mol. Genet., 4: 1-8. Mulley, J.C., Staples, A., Donnelly, A., Gedeon, A.K., Hecht, B.K., Nicholson, G.A., Haan, E.A. and Sutherland, G.R. (1993) Explanation for exclusive maternal origin for congenital form of myotonic dystrophy. Lancet, 341: 236-237. O’Hoy, K.L., Tsilfidis, C., Mahadevan, M.S., Neville, C.E., Barcelb, J., Hunter, A.G.W. and Komeluk, R.G. (1993) Reduction in size of the myotonic dystrophy trinucleotide repeat mutation during transmission. Science, 259: 809-8 12. Redman, J.B., Fenwick, R.G., Fu, Y-H., Pizzuti, A. and Caskey, CT. (1993) Relationship between parental trinucleotide GCT repeat length and severity of myotonic dystrophy in offspring. J. Am. Med. Assoc., 269: 1960-1965. Shelboume, P., Davies, J., Buxton, J., Anvret, M., Blennow, E., Bonduelle, M., Schmedding, E., Glass, I., Lindenbaum, R., Lane, R., Williamson, R. and Johnson, K. (1993) Direct diagnosis of myotonic dystrophy with a disease-specific DNA marker. N. Engl. J. Med., 328: 47 l-475. Shelboume, P., Winqvist, R., Kunert, E., Davies, J., Leisti, J., Thiele, H., Bachmann, H., Buxton, J., Williamson, B. and Johnson, K. (19921 Unstable DNA may be responsible for the incomplete penetrance of the myotonic dystrophy phenotype. Hum. Mol. Genet., 1: 467-473. Thornton, C.A., Johnson, K. and Moxley, R.T.111. (1994) Myotonic dystrophy patients have larger CTG expansions in skeletal muscle than in leukocytes. Ann. Neurol., 35: 104-107. Wong, L-J.C., Ashizawa, T., Monckton, D.G., Caskey, C.T. and Richards, C.S. (19951 Somatic heterogeneity of the CTG repeat in myotonic dystrophy is age and size dependent. Am. J. Hum. Genet., 56: 114-122. Yamagata, H., Miki, T., Ogihara, T., Nakagawa, M., Higuchi, I., Osame, M., Shelboume, P., Davies, J. and Johnson, K. (19921 Expansion of unstable DNA region in Japanese myotonic dystrophy patients. Lancet, 339: 692.
NEUROLOGICAL SCIENCES ELSEVIER
Journal of the Neurological Sciences 139 (I 996) 48-5 1
An intergenerational contraction of the CTG repeat in Japanese myotonic dystrophy Ryusuke Matsumura a3* , Tadashi Namikawa b, Tetsuro Miki ‘, Tameko Kihira d, Hidehisa Yamagata ‘, Yukio Mano a, Tetsuya Takayanagi a a Department
Neurology, Nara Medical Unicersir?;, 840 Shijocho, Kashihara, Nam 634, Japan b Konishibashi Hospital, 240-I Tani, Sakurai, Nara, Japan ’ Department of Geriatric Medicine, Osaka Unil>ersity Medical School, 2-2 Yamadaoka, Suita, Osaka, Japan ’ Division of Neurological Diseases, Wakayama Medical College, 27.9 Bancho, Wokayama, Japan of
Received 9 August 1995; revised 7 December 1995; accepted 17 December 1995
Abstract We present the first report of a Japanese family with myotonic dystrophy (DM) that showed an intergenerational contraction of the CTG repeat. The size of the expanded CTG repeats was 3.2 kb for the father and 2.2 kb for the daughter, indicating that the expansion decreased during transmission from the father to the daughter. Despite the CTG repeat contraction, the daughter showed earlier age of onset than the father. However, she appeared to be less severely affected than the father. We discuss the correlation between the CTG repeat contraction and the clinical phenotype. The presence of the CTG repeat contraction in Japanese DM is important for genetic counseling of Japanese DM families. Kewordst
Myotonic dystrophy; CTG repeat: Contraction; Japanese; Anticipation;
1. Introduction Myotonic dystrophy (DM) is an autosomal dominant, multisystem disorder characterized primarily by myotonia, progressive muscle weakness and wasting, cardiac conduction defects and cataract (Harper, 1989). The clinical phe-
notype is highly variable, ranging from a mild, late onset to a severe congenital form (Harper, 1989). The mutation responsible for DM has been identified as an expansion of a CTG trinucleotide repeat in the 3’ untranslated region of a putative serine/threonine protein kinase gene on chromosome 19q13.3 (Brook et al., 1992; Buxton et al., 1992; Fu et al., 1992; Harley et al., 1992a; Mahadevan et al., 1992). In normal individuals, the number of CTG repeats ranges from 5 to 37 copies, while in affected individuals it ranges from 50 to thousands of copies (Brook et al., 1992; Brunner et al., 1992; Lavedan
z Corresponding author. Tel: + 8 I (7442) 2-30.51 Ext. 3417; Fax: + 8 I (7442) 4-6065. 0022-510X/96/$15.00 Published by Elsevier Science B.V. PII SOO22-5 10X(96)000 14-7
Paternal transmission; Genetic counseling
et al., 1993b). Expanded CTG repeats are unstable and usually increase in size in successive generations (Ashizawa et al., 1992; Harley et al., 1992b; Lavedan et al., 1993b; Mulley et al., 1993; Redman et al., 1993). This is thought to be the molecular basis for anticipation, a clinical phenomenon characterized by earlier age of onset and increasing severity of the disease in successive generations. However, in recent reports, a small number of offspring have shown a decrease in the CTG repeat size compared with their parents (Ashizawa et al., 1992, Ashizawa et al., 1994; Abeliovich et al., 1993; Brunner et al., 1993; Cobo et al., 1993; Hunter et al., 1993; Lavedan et al., 1993a; Mulley et al., 1993; O’Hoy et al., 1993; Redman et al., 1993; Shelboume et al., 1993). Especially, a systematic report of Ashizawa
et al. (1994) revealed
that 95 (6.4%)
of 1,489
DM parent-child pairs with different ethnic backgrounds showed such intergenerational contractions of the repeat. In that report, 80 Japanese parent-child pairs were examined, but no pair had the CTG repeat contraction. Here we present the first report of a Japanese DM family
with an intergenerational
contraction
of the CTG
R. Matsumura
et al. /Journal
of the
Neurological
Sciences
139 (1996)
repeat, and discuss the correlation between the contraction and clinical phenotype of this case.
48-51
Subject
Subject
II-5
Ill-6
13kb[I
2. Case report The pedigree of the Japanese family is shown in Fig. 1.
49
4 12kb
9.8 kb ) * 8.6 kb
2. I. Subject II-S A Japanese man began to notice some stiffness and weakness of the hands at age 40 years. Around that time he developed gait disturbance. When examined at the age of 45, he had moderate weakness and wasting of the distal limb muscles and to a lesser extent the proximal limb muscles. His face showed a characteristic appearance, that is, wasting of the bitemporal, masseter and facial muscles, and frontal baldness. Marked weakness and wasting of the stemomastoids were observed. He displayed grip and percussion myotonia, and electromyography showed myotonic discharges in the intrinsic hand muscles. A highly arched palate and slurred speech characterized by a monotonous nasal voice were observed. He showed no intellectual deterioration but a personality pattern of inattention. Bilateral cataracts had been extracted at age 33. Serum creatine kinase was slightly elevated and serum immunogloblin G was decreased. Electrocardiography (ECG) demonstrated first-degree heart block, and head CT scanning revealed hyperostosis of the frontal bones. At age 47, he often fell unless he used a walking stick and he had some difficulty in rising from supine position. The same year he died of intracerebral hemorrhage. 2.2. Subject III-6 The daughter of II-5 was interviewed in detail at the age of 14 years. She had begun to note some stiffness of the hands at age 8. She had been an average student and completed high school. At the time of the study, she was 21 years of age and working satisfactorily as a waitress. She could lift heavy weights. However, she sometimes had some difficulty in masticating food. Her face showed some wasting of the bitemporal, masseters and facial muscles, but no frontal baldness. She had a highly arched palate but
I 7
II
Fig. 1. The pedigree of the Japanese family. Males are represented by squares, females by circles, and those affected clinically by solid symbols. Diagonal lines indicate deceased persons.
Fig. 2. Southern blot analysis of EcoRI-digested DNA from Subject II-5 and Subject III-6 with the p5B1.4 probe. The bracket shows a diffuse smear of bands whose average size is 13 kb in the DNA from Subject 11-5.
no dysarthria. Moderate weakness and wasting of the sternomastoids were observed. Muscle strength of the extremities was almost normal, but her grasping strength was slightly weak (right side, 17 kg; left side, 13 kg). She displayed grip and percussion myotonia. Serum creatine kinase was slightly elevated and serum immunogloblin G level was normal. ECG indicated first-degree heart block. Since a slit lamp examination had not been performed, it was unknown whether she had cataracts.
3. Southern blot analysis Blood samples were obtained from Subject II-5 at age 45 and Subject III-6 at age 14 with informed consent. Genomic DNA was extracted by standard procedures and digested with a restriction endonuclease, EcoRI. The digests were electrophoresed in a 0.7% agarose gel, then transferred onto a nylon membrane, and hybridized with the radiolabeled probe p5B 1.4 as described (Shelboume et al., 1992). The results are shown in Fig. 2. The father had one normal band (9.8 kb) and a diffuse smear of bands whose average size was 13 kb. The smear suggests mitotic instability of the CTG repeat in peripheral blood leukocytes (Lavedan et al., 1993b; Monckton et al., 1995; Wong et al., 1995). The daughter had one normal band (8.6 kb) and one expanded band of 12 kb. The normal 9.8 kb/8.6 kb bands result from an Alu element insertion/deletion polymorphism located 5 kb upstream from the CTG repeat (Shelboume et al., 1992; Mahadevan et al., 1993). It is known that the CTG repeat expansions in both Caucasian DM and Japanese DM occurred exclusively in the Ah insertion chromosomes (Harley et al., 1992a; Yamagata et al., 1992; Mahadevan et al., 1993). Therefore, the size of the expanded CTG repeats was calculated at 3.2 kb (about 1060 repeats) for the father and at 2.2 kb (about 730 repeats) for the daughter. Thus, the CTG repeat expansion decreased during transmission from the father to the daughter.
50
R. Matsumura et al./ Journal
of the Neurological Sciences 139 (1996148-51
4. Discussion The increasing size of the CTG repeats in successive generations is thought to be the molecular basis for anticipation. Therefore, we expect that the CTG repeat contraction accompanies a later age of onset and less severe manifestations in the offspring, compared with the parent. However, in this family, the daughter has an earlier age of onset than the father. One possible explanation for this unexpected anticipation is the bias involved in ascertaining the age of onset. Some patients with DM become so well adjusted to the difficulty in relaxing their muscles that they may not complain of it or even realize that the symptom is abnormal (Harper, 1989). Myotonia is thus a symptom and sign that has to be actively sought. Since the father was not known to be a DM heterozygote at the time his symptoms developed but the daughter was known to be a possible mutation carrier, earlier diagnosis in the daughter might have been made. Another possible explanation for the unexpected anticipation is somatic mosaicism of the CTG repeat size. In this study, we determined the CTG repeat size in DNA isolated from peripheral blood leukocytes. However, the primarily affected tissue in DM is skeletal muscle. It is known that the CTG repeat size differs between skeletal muscle and leukocytes (Anvret et al., 1993; Ashizawa et al., 1993; Lavedan et al., 1993b; Thornton et al., 1994). Therefore, there is a possibility that the CTG repeat size in skeletal muscle of the daughter is larger than that in skeletal muscle of the father and this resulted in anticipation, while the repeat size in leukocytes of the daughter is smaller. Since muscle biopsies of the father and the daughter had not been carried out, we can not make the comparison of the CTG repeat sizes in muscle. The third possible explanation for this anticipation is pseudocontractions. By comparing individual patient samples collected at two different times, Wong et al. (1995) revealed that the CTG repeat size increased with age in some DM patients. Based on this age-dependent CTG expansion, Monckton et al. (1995) recently postulated the concept of pseudocontractions; that is, a true germline CTG expansion during transmission from the father to the daughter can be masked by a higher somatic expansion in the older father compared with the more limited somatic expansion in the younger daughter, resulting in the apparent intergenerational contraction. In the future, it will be interesting to see whether the CTG repeat size is larger in a blood sample from the daughter at age 45 than in that from the father at age 4.5 (3.2 kb). Surprisingly, Ashizawa et al. (1994) reported that approximately one half of 56 parent-child pairs with the CTG repeat contractions showed anticipation as in our case. Therefore, it remains possible that other genetic or environmental factors may cause anticipation regardless of the CTG repeat size.
At present the daughter appears to be less severely affected than the father despite her earlier age of onset. This may truly reflect the CTG repeat contraction. For accurate evaluation of the effects of the CTG repeat contraction on the clinical phenotype, it is necessary to see whether the daughter is less severely affected than the father when she reaches the age of 40 years. It is important to follow the clinical course as well as the CTG repeat size of the daughter. In this family, Subject II-2 and Subject III-2 were also examined. The CTG repeat size of Subject 11-2, whose age of onset was 30 years, and that of Subject III-2 with congenital DM were 1.2 kb and 5.2 kb, respectively. It should be noted that the contraction of the CTG repeat occurred in paternal transmission while the large expansion of the CTG repeat, which resulted in congenital DM, occurred in maternal transmission within the same family. The paternal and maternal transmissions appear to have distinct influences on the intergenerational changes of the CTG repeat size in DM. There may be a maternal factor that allows the CTG repeats to expand further and a paternal factor that restricts the expansion. So far the intergenerational contractions of the CTG repeats have been reported in DM populations from different ethnic backgrounds in Europe, North and South America, and Australia, but not in Japan (Ashizawa et al., 1994). In this family we demonstrate for the first time the contraction in Japanese DM, providing evidence that unstable CTG repeats in Japanese DM have the same characteristics as in Caucasian DM. The presence of the CTG repeat contraction in Japanese DM needs to be taken account of and is important for genetic counseling of Japanese DM families.
Acknowledgements This work was supported in part by a Grant-in-Aid for Intractable Disease, Ministry of Health and Welfare of Japan. We are grateful to Professor Richard F. Mayer, University of Maryland School of Medicine, Baltimore, MD, for critical reading of the manuscript.
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