Brain & Development 37 (2015) 880–886 www.elsevier.com/locate/braindev
Original article
Founder mutation causes classical Fukuyama congenital muscular dystrophy (FCMD) in Chinese patients Haipo Yang a, Kazuhiro Kobayashi b, Shuo Wang a, Hui Jiao a, Jiangxi Xiao c, Tatsushi Toda b, Xiru Wu a, Hui Xiong a,⇑ b
a Department of Pediatrics, Peking University First Hospital, Beijing 100034, China Division of Neurology/Molecular Brain Science, Kobe University Graduate School of Medicine, Kobe 650-0017, Japan c Department of Radiology, Peking University First Hospital, Beijing 100034, China
Received 12 November 2014; received in revised form 8 February 2015; accepted 26 February 2015
Abstract Purpose: Fukuyama congenital muscular dystrophy (FCMD) is a congenital muscular dystrophy rarely reported outside Japan. Here, we report three patients with Fukuyama congenital muscular dystrophy (FCMD) in China who shared a similar clinical phenotype and 3-kb insertion in the FKTN 30 untranslated region. Methods: Immunofluorescence staining was undertaken on muscle biopsies from three patients using alpha dystroglycan antibody (IIH6). Genomic DNA from patients and parents was extracted from peripheral blood leukocytes. Polymerase chain reaction and DNA sequencing were employed to analyze the exons and surrounding intron sequences of the fukutin (FKTN) gene to detect mutations. Haplotype analysis was also performed on each patient and their parents. Results: All patients had delayed mental and motor development, febrile convulsions, muscle weakness, and moderate to significant raised levels of serum creatine kinase (7000–11,160 U/L, 25–60 normal). Brain MRI scans showed micropolygyria and extensive dysplasia in the white matter and brainstem. Electromyography revealed myopathic changes. Muscle immunofluorescence studies demonstrated reduced IIH6 staining. Genetic testing showed compound heterozygous mutations of FKTN. Cases 1 and 2 had a c.139C>T (p.Arg47*) heterozygous mutation. Case 3 had a c.346C>T (p.Gln116*) heterozygous mutation. Conclusion: All patients had a heterozygous 3-kb insertion in the FKTN 30 untranslated region. Haplotype analyses suggested that these patients had the same haplotype as Japanese patients. Ó 2015 The Japanese Society of Child Neurology. Published by Elsevier B.V. All rights reserved.
Keywords: Fukuyama congenital muscular dystrophy; FKTN; 3-kb insertion; Haplotype analysis; Founder mutation
1. Introduction Fukuyama congenital muscular dystrophy (FCMD) is a congenital muscular dystrophy inherited in an autosomal recessive pattern, and is found mostly in individuals of Japanese descent. It was first described by Fukuyama ⇑ Corresponding author. Tel.: +86 (10) 83573238; fax: +86 (10) 66530532. E-mail address: [email protected] (H. Xiong).
in 1960. The incidence ranges from 6.2 to 11.9 per 100,000 in Japan. Although FCMD is the second most common form of muscular dystrophy after Duchenne muscular dystrophy in Japan [1], it is rarely reported outside of Japan. The characteristics of FCMD include congenital onset hypotonia and weakness with contractures of the hips, knees, and interphalangeal joints. Other features include motor and speech retardation, as well as intellectual disability despite the relative preservation of social skills. Intelligence-quotient scores in most FCMD
http://dx.doi.org/10.1016/j.braindev.2015.02.010 0387-7604/Ó 2015 The Japanese Society of Child Neurology. Published by Elsevier B.V. All rights reserved.
H. Yang et al. / Brain & Development 37 (2015) 880–886
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patients lie between 30 and 50. Seizures (febrile or afebrile) occur in almost half of the cases, in association with abnormal findings on electroencephalography [2]. Usually, the maximum level of motor function achieved is sitting, and most FCMD patients do not acquire the ability to walk. A static course of FCMD is observed until early childhood, when diffuse and extensive muscle wasting begins along with progressive joint contractures and pseudohypertrophy of the calves and forearms. Patients usually become bedridden before the age of 10 years due to generalized muscle atrophy and joint contractures. Most affect patients die by the age of 20 years. Ophthalmologic abnormalities (including visual impairment and retinal abnormalities) are observed. If present, retinal abnormalities are mild. Brain MRI findings include the following: an irregular or “cobblestone” brain surface; enlargement of the lateral ventricles; abnormal white matter signal; mild dysplasia in the cerebellum and brainstem; and cerebellar cysts. Cardiac involvement progresses slowly and, after 10 years of age, myocardial fibrosis may appear [3]. Muscle biopsy changes are consistent with muscular dystrophy, and immunofluorescence demonstrates the absence or reduction of IIH6. FCMD has been reported to involve peripheral nerves, but the relationship between the hypoglycosylation of a-DG and peripheral nerve involvement is not clear [4]. Therefore, patients typically present at birth with hypotonia (muscle weakness), abnormal brain structures detected by brain imaging and mild eye abnormalities. A Japanese founder mutation, a homozygous retrotransposal insertion in the 30 UTR of fukutin, accounts for 90% of all Japanese patients with FCMD. Here, we report the cases of three Chinese patients with a clinical presentation of classic FCMD and a heterozygous mutation of this founder mutation.
were designed from the fukutin (FKTN) genomic sequence to amplify each exon and the surrounding intron sequences (primer sequences are available upon request). After detecting only one mutation by direct DNA sequencing, we undertook a three primer-PCR method, as described by Watanabe et al. [5]. We also designed a primer pair to detect the genomic sequence downstream of the 3-kb insertion (forward position ins2795–2814, 50 -ATTAAGGGCGGTGCAAGATG-30 ; reverse position c.4469–4488, 50 -GAGAGAAGGAGGCAAACTGG-30 ). Cycling conditions were identical to those of the three primer-PCR method. PCR products were analyzed by electrophoresis on 2% agarose gels.
2. Patients and methods
Three patients and their parents were genotyped with the polymorphic microsatellite marker cen-D9S2105(FCMD)-D9S2170-D9S2171-D9S2107-tel, as described previously [6].
2.1. Patients Written informed consent was obtained from the parents of the three subjects. Diagnostic testing involved a muscle biopsy, brain MRI and blood samples from both the patient and their parents. The research protocol from this study was reviewed and approved by the Ethics Committee of Peking University First Hospital (Beijing, China). The clinical and neuroradiological findings of the three patients are summarized in Table 1. 2.2. Polymerase chain reaction (PCR) and sequencing analysis Genomic DNA from the peripheral blood samples of patients and their parents was extracted using phenol– chloroform and precipitated with isopropanol. Primers
2.3. Immunofluorescent analyses Open biopsy of the gastrocnemius muscle was undertaken. Tissue was pre-cooled with isopentane and then frozen in liquid nitrogen. Frozen sections (8 lm) of specimens of muscle tissue were fixed with 4% paraformaldehyde at room temperature for 10 min. Non-specific binding was reduced by 30-min incubation with 10% goat serum (Jackson ImmunoResearch, West Grove, PA, USA) in phosphate-buffered saline (PBS). Two monoclonal antibodies, IIH6, containing the sugar chain of a-dystroglycan (a-DG; 1:200, Chemicon, Temecula, CA, USA), and merosin (1:4000, mAb1922; Chemicon International, Temecula, CA, USA), were used. They were diluted and incubated at 4 °C overnight. They were then washed three times with PBS. Rabbit anti-mouse fluorescent antibody (1:200 dilutions) was used to incubate the frozen sections at room temperature for 1 h. The result was observed using a Fluoview Viewer ver1.6b (Olympus, Tokyo, Japan). 2.4. Haplotype analysis
3. Results 3.1. Findings regarding clinical phenotype 3.1.1. Case 1 The details of this patient have been reported in our previous paper [7]. He is now an 11-year-old male. At 10 years of age, he had a bout of severe pneumonia, and has been bedbound since then. 3.1.2. Case 2 Case 2 was a 2.5-year-old female born to a G2P1 mother by normal spontaneous vaginal delivery, with full-term, birth weight of 3.2 kg. The mother’s first
Normal 7760 No eye involvement
Neonatal Sit
Neonatal Sit
F/2 y
F/1.5 y
2
3
+
+
+
–
–
+++ ++ ++
–
–
Normal 7000 Left-eye strabismus +
+
–
–
++
+
+
+
–
Normal Eye movement slow 11,160
+
CC
+++ – ++ ++ ++ + + + + Neonatal Sit M/11 y 1
+++
FKTN CK (U/L) Echocardiogram a-DG Eye Motor Contractures MR Febrile MRI ability seizures Lis VC WM CD CCyst BS Age at onset Sex/ age Case number
Table 1 Clinical and genetic characteristics of the three patients.
y, year; M, male; F, female; MR, mental retardation; Lis, lissencephaly; VC, ventriculomegaly; WM, white-matter abnormality; CD, cerebellar dysplasia; CCyst, cerebellar cysts; BS, brainstem involvement; CC, corpus callosum involvement; a-DG, a-dystroglycan; NA, not available.
H. Yang et al. / Brain & Development 37 (2015) 880–886 Absent c.139C>T, p. R47X c.4375_4376insAB185332.1 Absent c.139C>T, p. R47X c.4375_4376insAB185332.1 NA c.346C>T, P.Q116X c.4375_4376insAB185332.1
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pregnancy had resulted in a first trimester miscarriage. Fetal ultrasound revealed a left-sided hydrocephalus. During infancy, the patient had considerable difficulties with feeding, decreased cry and weakness. At 1 year of age, she could control her head movements and was able to sit unsupported and distinguish between acquaintances at 14 months. Starting at the age of 11 months of age, she began to have febrile convulsions approximately 5–6 times per day. Video electroencephalography showed diffuse low-amplitude h–b waves with continuous b-rhythms in the bilateral occipital and temporal areas. She showed some signs of heat sensitivity with seizures developing after a prolonged bath. Physical examination revealed left-eye strabismus, muscle weakness and the absence of tendon reflexes. The Babinski sign was negative. The serum level of CK was 7000–8000 U/L. Electromyography revealed a myopathic pattern. Brain MRI demonstrated the malformation of micropolygyria, lissencephaly, extensive dysplasia of white matter, dysplasia of the brainstem and cerebellum, cerebellar cysts, and enlarged ventricles (Fig. 1). 3.1.3. Case 3 Case 3 was a 17-month-old female. She was born to a G5P2 mother by cesarean delivery at 42 weeks, with a birth weight of 3.1 kg. She had one unaffected sibling. Similar to the other cases, this patient had significant feeding difficulties and decreased cry during infancy. She could laugh at 3 months, but could not kick a bed quilt at 5 months. She could control her head movements at 8 months and sit unsupported at 15 months. Recently, she had a febrile convulsion. Her parents noted that she often has colds.
Fig. 1. Cranial MRI scans of the three patients. The observable changes are cerebellar dysplasia, white-matter changes, micropolygyria, lissencephaly and extensive dysplasia in the white matter, dysplasia in the brainstem, cerebellar cysts and extensive ventricles.
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Physical examination showed a myopathic facies, muscle weakness and the absence of tendon reflexes. The serum level of CK was 7765 U/L. Electromyography revealed a myopathic pattern. Brain MRI showed abnormal white-matter signals in the bilateral frontal region, abnormal signals in the bilateral putamen, cerebellar hypoplasia and cerebellar cysts (Fig. 1). The patient had normal vision and no abnormal eye findings. 3.2. Immunofluorescence staining Hematoxylin and eosin staining of biopsy tissue was performed and demonstrated findings that were consistent with a dystrophic disease, including muscle degeneration, increased fiber size diameter and increased endomysial connective tissue and adipose cells. Immunofluorescence showed the absence of a-DG glycosylation (Fig. 2). 3.3. Mutation analyses Direct sequencing of the FKTN exons and the flanking introns was undertaken. Cases 1 and 2 were found to have a c.139C>T (p.Arg47*) heterozygous nonsense mutation, and Case 3 had a c.346C>T (p. Gln116X) heterozygous nonsense mutation. Three-primer PCR revealed that all three patients had the FKTN heterozygous 3-kb insertion (Fig. 3). 3.4. Haplotype analyses Haplotype analyses revealed that Cases 1 and 2 had the same haplotype, and Case 1 was reported by Xiong et al. in 2009 [7]. For Case 2, the paternallyderived mutation had the 138-192-147-183 haplotype
Fig. 3. The founder 3-kb insertion analysis in the 30 untranslated region. PCR analyses of the pedigree of the patients’ family, bearing the founder 3-kb retrotransposal insertion allele. A 382-bp PCR product (lower); 382-bp bands arise from the founder insertion. 1 Negative control; 2 positive control (Case 1); 3 Case 2; 4 Case 2’s father; 5 Case 2’s mother; 6 Case 3; 7 Case 3’s father; 8 Case 3’s mother.
and the maternally-derived mutation was 130-201-157183. Case 3 had a different haplotype, where the paternally-derived mutation had the 142-196-147-193 haplotype and the maternally-derived mutation had the 138-193-153-195 haplotype (Fig. 4). 4. Discussion FKTN is located on chromosome 9 and has 10 exons. This gene is expressed in various tissues in normal
Fig. 2. Immunofluorescence staining. (a) Hematoxylin and eosin (H&E) staining of skeletal muscle of normal control serial sections. (b) Immunofluorescent staining of skeletal muscle tissue of the normal control with an anti-a-dystroglycan sugar antibody. (c) Immunofluoresent staining of skeletal muscle tissue of the normal control with anti-merosin antibody. (d) H&E staining of skeletal muscle of Case 2 shows dystrophic changes: variability in the size of muscle fibers, fibrosis and fat replacement. (e) Immunofluorescent staining of the skeletal muscle tissue of Case 2 with an anti-a-dystroglycan sugar antibody, which shows the absence of a-DG glycosylation. (f) Immunofluoresent staining of the skeletal muscle tissue of Case 2 with anti-merosin antibody, which was normal.
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Fig. 4. Haplotype analyses showing that P1 and P2 had the same haplotype. The 3-kb insertion mutation had the 138-192-147-183 haplotype and the c.139C>T, p.R47X mutation had the 130-201-157-183 haplotype. P3 had a different haplotype, where the paternally-derived c.346C>T, p. The Q116X mutation had the 142-196-147-193 haplotype and the maternally-derived 3-kb insertion mutation had the 138-193-153-195 haplotype.
individuals. In Japan, 90% of genetic changes for the FKTN 30 untranslated region are 3-kb repetitive sequences of homozygous insertions [8]. FCMD is the first known human disease that involves the ancestral insertion of a retrotransposon into a causative gene [9]. The 3-kb insert stops the expression of 38 C-terminal amino acids of fukutin, instead of containing 129 amino acids derived from the SVA sequence [10]. FKTN mutations can also lead to milder limb-girdle muscular dystrophy [11] and more severe Walker–Warburg syndrome (WWS) phenotypes [12,13]. All of the three patients in this report had neonatal hypotonia, muscle weakness, and mental retardation. Brain MRI showed different degrees of structural abnormalities, including micropolygyria, cerebellar dysplasia, extensive dysplasia of white matter and cerebellar cysts. Cases 1 and 2 had the same mutation, but they had some different clinical manifestations. Compared with Case 2, motor delay was more profound in Case 1 with the ability to sit unsupported only gained at age 5 years, compared with 14 months in Case 2. The slow eye movement in Case 1 may have been due to mental retardation at the onset of the disease and then the involvement of
ocular muscles. All three cases suffered febrile convulsions, but they occurred earlier and more frequently in Case 2. In FCMD, seizures associated with fevers have been reported previously [14]. It is interesting to note that Japanese patients who carry these two compound heterozygous mutations have a more severe phenotype and never acquire the ability to sit unsupported. A prior case study in a 7-year-old Korean child describes the same mutations found in Case 3; the Korean child was able to roll over but exhibited myopia and epilepsy [15]. A different FKTN mutation in the same base pair location (c.345_346GC>CT, p.Gln116X) has been reported previously in a patient with WWS. This patient presented with congenital hypotonia, ocular abnormalities and hydrocephalus in association with gyrus abnormalities and an absence of the corpus callosum, and died at 4.5 months of age [16]. Severe ocular abnormalities were not observed in our patients in agreement with prior descriptions of FCMD [17]. The differences in clinical severity in our patients may be due to both environmental factors and genetic modifiers. FKTN encodes the protein fukutin. The specific function of fukutin has not been elucidated, but has been
H. Yang et al. / Brain & Development 37 (2015) 880–886
postulated to play an important role in a-DG glycosylation. FKTN mutations can lead to abnormal neuronal migration and is thought to play a role in synaptic plasticity. Many children with FCMD have mental retardation and epilepsy [18]. Fukutin localizes to the Golgi apparatus, and its localization changes with certain FKTN mutations [19]. Localization can be rescued with curcumin treatment in FKTN missense mutations [20]. With respect to the 3-kb insertion, antisense oligonucleotide-targeted combination of the splicing site can correct the abnormal expression of fukutin protein, and could be a new direction for FCMD treatment [10]. We undertook haplotype analysis of blood samples from family members. For Cases 1 and 2, both of the chromosomes of the probands had the same haplotype as those of Japanese patients. The retrotransposon was concordant with the founder haplotype represented as 138-192-147-183 (the most common kind of haplotype in Japanese patients), which suggested that both mutant alleles were derived from the same founder mutation as Japanese patients [1]. Case 3 had a different haplotype, the nonsense mutation c.346C>T (p.Q116X), 142-196147-193, which has also been reported [1,21]. There are nine types of haplotype for the 3-kb insertion mutation in Japanese patients. Although the haplotype 138-193153-195 in Case 3 has not been reported previously, we propose that it carries the 138-component and speculate that the site of the haplotype may change across generations as a result of the exchange of genetic material between homologous chromosomes during meiosis. China is close to Japan in terms of geography and race, but the 3-kb insertion has seldom been reported in China. In 2000, Jong et al. described three patients who had the typical clinical, myopathological and neuroradiological characteristics of FCMD, but full mutational analyses of FKTN revealed that neither a 3-kb insertion nor a point mutation was found [22]. In 2005, Watanabe et al. assessed the 3-kb insertion in 766 Chinese individuals but did not detect insertion alleles [5]. He ruled out the possibility that a single ancestor bearing an insertion chromosome migrated to Japan from Korea or mainland China and concluded that FCMD carriers are rare outside of Japan. In our study, all three patients had a FKTN nonsense mutation and the 3-kb insertion and the same haplotype as those of Japanese patients, indicating that both mutant alleles were derived from the same founder as Japanese patients. This suggests that segments of the Chinese and Japanese populations may have a recent common ancestor. It is well known that FKTN mutations have been reported in other countries other than Japan, but the classical manifestation of FCMD and a similar haplotype have not been reported elsewhere. Our study widens the distribution of the 3-kb insertion founder mutation; further investigations of the origin of 3-kb insertion founder mutation would be helpful.
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5. Conclusions Here, we reported on the cases of three patients with the typical clinical manifestations of FCMD. Gene testing confirmed that all three patients had a 3-kb founder insertion mutation. Haplotype analysis suggested that Cases 1 and 2 had the same haplotype as Japanese patients. Case 3 had a different haplotype, but this may have arisen from the same ancestor founder mutation because the haplotype site can change between generations. This suggests that segments of the Chinese and Japanese populations may have a recent common ancestor. Author Contributions Conceived and designed the experiments: HPY KK HX. Performed the experiments: HPY SW HJ JXX. Analyzed the data: KK TT XRW. Contributed reagents/materials/analysis tools: HPY. Wrote the paper: HPY.
Acknowledgments We are in deep mourning for Prof. Fukuyama. We will never forget him and will let him live in our heart and continue his work. The authors are grateful to all the patients and their families who have participated in this study and thank Dr. Anne Rutkowski, MD, for her comments and criticisms on the manuscript. This work was supported by grants from the National Natural Science Foundation of China (81271400), and the National Basic Research Program of China (2012CB944602). References [1] Fukuyama Y, Ohsawa M. A genetic study of the Fukuyama type congenital muscular dystrophy. Brain Dev 1984;6:373–90. [2] Fukuyama Y, Osawa M, Suzuki H. Congenital progressive muscular dystrophy of the Fukuyama types – clinical, genetic and pathological considerations. Brain Dev 1981;3:1–29. [3] Saito, K. Fukuyama Congenital Muscular Dystrophy. GeneReviews Last Update: May 10; 2012. [4] Jang DH, Sung IY, Ko TS. Peripheral nerve involvement in Fukuyama congenital muscular dystrophy: a case report. J Child Neurol 2013;28:132–7. [5] Watanabe M, Kobayashi K, Jin F, Park KS, Yamada T, Tokunaga K, et al. Founder SVA retrotransposal insertion in Fukuyama-type congenital muscular dystrophy and its origin in Japanese and Northeast Asian populations. Am J Med Genet A 2005;138:344–8. [6] Kobayashi K, Nakahori Y, Mizuno K, Miyake M, Kumagai T, Honma A, et al. Founder-haplotype analysis in Fukuyama-type congenital muscular dystrophy (FCMD). Hum Genet 1998;103: 323–7. [7] Xiong H, Wang S, Kobayashi K, Jiang Y, Wang J, Chang X, et al. Fukutin gene retrotransposal insertion in a non-Japanese
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H. Yang et al. / Brain & Development 37 (2015) 880–886 Fukuyama congenital muscular dystrophy (FCMD) patient. Am J Med Genet A 2009;149A:2403–8. Godfrey C, Clement E, Mein R, Brockington M, Smith J, Talim B, et al. Refining genotype phenotype correlations in muscular dystrophies with defective glycosylation of dystroglycan. Brain 2007;130:2725–35. Kobayashi K, Nakahori Y, Miyake M, Matsumura K, KondoIida E, Nomura Y, et al. An ancient retrotransposal insertion causes Fukuyama-type congenital muscular dystrophy. Nature 1998;394:388–92. Taniguchi-Ikeda M, Kobayashi K, Kanagawa M, Yu CC, Mori K, Oda T, et al. Pathogenic exon-trapping by SVA retrotransposon and rescue in Fukuyama muscular dystrophy. Nature 2011;478:127–31. Puckett RL, Moore SA, Winder TL, Willer T, Romansky SG, Covault KK, et al. Further evidence of Fukutin mutations as a cause of childhood onset limb-girdle muscular dystrophy without mental retardation. Neuromuscul Disord 2009;19:352–6. Chang W, Winder TL, LeDuc CA, Simpson LL, Millar WS, Dungan J, et al. Founder Fukutin mutation causes Walker– Warburg syndrome in four Ashkenazi Jewish families. Prenat Diagn 2009;29:560–9. Cotarelo RP, Valero MC, Prados B, Pen˜a A, Rodrı´guez L, Fano O, et al. Two new patients bearing mutations in the fukutin gene confirm the relevance of this gene in Walker–Warburg syndrome. Clin Genet 2008;73:139–45. Yoshioka M, Higuchi Y. Long-term prognosis of epilepsies and related seizure disorders in Fukuyama-type congenital muscular dystrophy. J Child Neurol 2005;20:385–91. Lim BC, Ki CS, Kim JW, Cho A, Kim MJ, Hwang H, et al. Fukutin mutations in congenital muscular dystrophies with
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defective glycosylation of dystroglycan in Korea. Neuromuscul Disord 2010;20:524–30. de Bernabe´ DB, van Bokhoven H, van Beusekom E, Van den Akker W, Kant S, Dobyns WB, et al. A homozygous nonsense mutation in the fukutin gene causes a Walker–Warburg syndrome phenotype. J Med Genet 2003;40:845–8. Hino N, Kobayashi M, Shibata N, Yamamoto T, Saito K, Osawa M, et al. Clinicopathological study on eyes from cases of Fukuyama type congenital muscular dystrophy. Brain Dev 2001;23:97–107. Hiroi A, Yamamoto T, Shibata N, Osawa M, Kobayashi M. Roles of Fukutin, the gene responsible for Fukuyama-type congenital muscular dystrophy, in neurons: possible involvement in synaptic function and neuronal migration. Acta Histochem Cytochem 2011;44:91–101. Xiong H, Kobayashi K, Tachikawa M, Manya H, Takeda S, Chiyonobu T, et al. Molecular interaction between fukutin and POMGnT1 in the glycosylation pathway of a-dystroglycan. Biochem Biophys Res Commun 2006;350:935–41. Tachikawa M, Kanagawa M, Yu CC, Kobayashi K, Toda T. Mislocalization of Fukutin protein by disease-causing missense mutations can be rescued with treatments directed at folding amelioration. J Biol Chem 2012;287:8398–406. Toda T, Kobayashi K. Fukuyama-type congenital muscular dystrophy: the first human disease to be caused by an ancient retrotransposal integration. J Mol Med 1999;77:816–23. Jong YJ, Kobayashi K, Toda T, Kondo E, Huang SC, Shen YZ, et al. Genetic heterogeneity in three Chinese children with Fukuyama congenital muscular dystrophy. Neuromuscul Disord 2000;10:108–12.
Original article
Founder mutation causes classical Fukuyama congenital muscular dystrophy (FCMD) in Chinese patients Haipo Yang a, Kazuhiro Kobayashi b, Shuo Wang a, Hui Jiao a, Jiangxi Xiao c, Tatsushi Toda b, Xiru Wu a, Hui Xiong a,⇑ b
a Department of Pediatrics, Peking University First Hospital, Beijing 100034, China Division of Neurology/Molecular Brain Science, Kobe University Graduate School of Medicine, Kobe 650-0017, Japan c Department of Radiology, Peking University First Hospital, Beijing 100034, China
Received 12 November 2014; received in revised form 8 February 2015; accepted 26 February 2015
Abstract Purpose: Fukuyama congenital muscular dystrophy (FCMD) is a congenital muscular dystrophy rarely reported outside Japan. Here, we report three patients with Fukuyama congenital muscular dystrophy (FCMD) in China who shared a similar clinical phenotype and 3-kb insertion in the FKTN 30 untranslated region. Methods: Immunofluorescence staining was undertaken on muscle biopsies from three patients using alpha dystroglycan antibody (IIH6). Genomic DNA from patients and parents was extracted from peripheral blood leukocytes. Polymerase chain reaction and DNA sequencing were employed to analyze the exons and surrounding intron sequences of the fukutin (FKTN) gene to detect mutations. Haplotype analysis was also performed on each patient and their parents. Results: All patients had delayed mental and motor development, febrile convulsions, muscle weakness, and moderate to significant raised levels of serum creatine kinase (7000–11,160 U/L, 25–60 normal). Brain MRI scans showed micropolygyria and extensive dysplasia in the white matter and brainstem. Electromyography revealed myopathic changes. Muscle immunofluorescence studies demonstrated reduced IIH6 staining. Genetic testing showed compound heterozygous mutations of FKTN. Cases 1 and 2 had a c.139C>T (p.Arg47*) heterozygous mutation. Case 3 had a c.346C>T (p.Gln116*) heterozygous mutation. Conclusion: All patients had a heterozygous 3-kb insertion in the FKTN 30 untranslated region. Haplotype analyses suggested that these patients had the same haplotype as Japanese patients. Ó 2015 The Japanese Society of Child Neurology. Published by Elsevier B.V. All rights reserved.
Keywords: Fukuyama congenital muscular dystrophy; FKTN; 3-kb insertion; Haplotype analysis; Founder mutation
1. Introduction Fukuyama congenital muscular dystrophy (FCMD) is a congenital muscular dystrophy inherited in an autosomal recessive pattern, and is found mostly in individuals of Japanese descent. It was first described by Fukuyama ⇑ Corresponding author. Tel.: +86 (10) 83573238; fax: +86 (10) 66530532. E-mail address: [email protected] (H. Xiong).
in 1960. The incidence ranges from 6.2 to 11.9 per 100,000 in Japan. Although FCMD is the second most common form of muscular dystrophy after Duchenne muscular dystrophy in Japan [1], it is rarely reported outside of Japan. The characteristics of FCMD include congenital onset hypotonia and weakness with contractures of the hips, knees, and interphalangeal joints. Other features include motor and speech retardation, as well as intellectual disability despite the relative preservation of social skills. Intelligence-quotient scores in most FCMD
http://dx.doi.org/10.1016/j.braindev.2015.02.010 0387-7604/Ó 2015 The Japanese Society of Child Neurology. Published by Elsevier B.V. All rights reserved.
H. Yang et al. / Brain & Development 37 (2015) 880–886
881
patients lie between 30 and 50. Seizures (febrile or afebrile) occur in almost half of the cases, in association with abnormal findings on electroencephalography [2]. Usually, the maximum level of motor function achieved is sitting, and most FCMD patients do not acquire the ability to walk. A static course of FCMD is observed until early childhood, when diffuse and extensive muscle wasting begins along with progressive joint contractures and pseudohypertrophy of the calves and forearms. Patients usually become bedridden before the age of 10 years due to generalized muscle atrophy and joint contractures. Most affect patients die by the age of 20 years. Ophthalmologic abnormalities (including visual impairment and retinal abnormalities) are observed. If present, retinal abnormalities are mild. Brain MRI findings include the following: an irregular or “cobblestone” brain surface; enlargement of the lateral ventricles; abnormal white matter signal; mild dysplasia in the cerebellum and brainstem; and cerebellar cysts. Cardiac involvement progresses slowly and, after 10 years of age, myocardial fibrosis may appear [3]. Muscle biopsy changes are consistent with muscular dystrophy, and immunofluorescence demonstrates the absence or reduction of IIH6. FCMD has been reported to involve peripheral nerves, but the relationship between the hypoglycosylation of a-DG and peripheral nerve involvement is not clear [4]. Therefore, patients typically present at birth with hypotonia (muscle weakness), abnormal brain structures detected by brain imaging and mild eye abnormalities. A Japanese founder mutation, a homozygous retrotransposal insertion in the 30 UTR of fukutin, accounts for 90% of all Japanese patients with FCMD. Here, we report the cases of three Chinese patients with a clinical presentation of classic FCMD and a heterozygous mutation of this founder mutation.
were designed from the fukutin (FKTN) genomic sequence to amplify each exon and the surrounding intron sequences (primer sequences are available upon request). After detecting only one mutation by direct DNA sequencing, we undertook a three primer-PCR method, as described by Watanabe et al. [5]. We also designed a primer pair to detect the genomic sequence downstream of the 3-kb insertion (forward position ins2795–2814, 50 -ATTAAGGGCGGTGCAAGATG-30 ; reverse position c.4469–4488, 50 -GAGAGAAGGAGGCAAACTGG-30 ). Cycling conditions were identical to those of the three primer-PCR method. PCR products were analyzed by electrophoresis on 2% agarose gels.
2. Patients and methods
Three patients and their parents were genotyped with the polymorphic microsatellite marker cen-D9S2105(FCMD)-D9S2170-D9S2171-D9S2107-tel, as described previously [6].
2.1. Patients Written informed consent was obtained from the parents of the three subjects. Diagnostic testing involved a muscle biopsy, brain MRI and blood samples from both the patient and their parents. The research protocol from this study was reviewed and approved by the Ethics Committee of Peking University First Hospital (Beijing, China). The clinical and neuroradiological findings of the three patients are summarized in Table 1. 2.2. Polymerase chain reaction (PCR) and sequencing analysis Genomic DNA from the peripheral blood samples of patients and their parents was extracted using phenol– chloroform and precipitated with isopropanol. Primers
2.3. Immunofluorescent analyses Open biopsy of the gastrocnemius muscle was undertaken. Tissue was pre-cooled with isopentane and then frozen in liquid nitrogen. Frozen sections (8 lm) of specimens of muscle tissue were fixed with 4% paraformaldehyde at room temperature for 10 min. Non-specific binding was reduced by 30-min incubation with 10% goat serum (Jackson ImmunoResearch, West Grove, PA, USA) in phosphate-buffered saline (PBS). Two monoclonal antibodies, IIH6, containing the sugar chain of a-dystroglycan (a-DG; 1:200, Chemicon, Temecula, CA, USA), and merosin (1:4000, mAb1922; Chemicon International, Temecula, CA, USA), were used. They were diluted and incubated at 4 °C overnight. They were then washed three times with PBS. Rabbit anti-mouse fluorescent antibody (1:200 dilutions) was used to incubate the frozen sections at room temperature for 1 h. The result was observed using a Fluoview Viewer ver1.6b (Olympus, Tokyo, Japan). 2.4. Haplotype analysis
3. Results 3.1. Findings regarding clinical phenotype 3.1.1. Case 1 The details of this patient have been reported in our previous paper [7]. He is now an 11-year-old male. At 10 years of age, he had a bout of severe pneumonia, and has been bedbound since then. 3.1.2. Case 2 Case 2 was a 2.5-year-old female born to a G2P1 mother by normal spontaneous vaginal delivery, with full-term, birth weight of 3.2 kg. The mother’s first
Normal 7760 No eye involvement
Neonatal Sit
Neonatal Sit
F/2 y
F/1.5 y
2
3
+
+
+
–
–
+++ ++ ++
–
–
Normal 7000 Left-eye strabismus +
+
–
–
++
+
+
+
–
Normal Eye movement slow 11,160
+
CC
+++ – ++ ++ ++ + + + + Neonatal Sit M/11 y 1
+++
FKTN CK (U/L) Echocardiogram a-DG Eye Motor Contractures MR Febrile MRI ability seizures Lis VC WM CD CCyst BS Age at onset Sex/ age Case number
Table 1 Clinical and genetic characteristics of the three patients.
y, year; M, male; F, female; MR, mental retardation; Lis, lissencephaly; VC, ventriculomegaly; WM, white-matter abnormality; CD, cerebellar dysplasia; CCyst, cerebellar cysts; BS, brainstem involvement; CC, corpus callosum involvement; a-DG, a-dystroglycan; NA, not available.
H. Yang et al. / Brain & Development 37 (2015) 880–886 Absent c.139C>T, p. R47X c.4375_4376insAB185332.1 Absent c.139C>T, p. R47X c.4375_4376insAB185332.1 NA c.346C>T, P.Q116X c.4375_4376insAB185332.1
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pregnancy had resulted in a first trimester miscarriage. Fetal ultrasound revealed a left-sided hydrocephalus. During infancy, the patient had considerable difficulties with feeding, decreased cry and weakness. At 1 year of age, she could control her head movements and was able to sit unsupported and distinguish between acquaintances at 14 months. Starting at the age of 11 months of age, she began to have febrile convulsions approximately 5–6 times per day. Video electroencephalography showed diffuse low-amplitude h–b waves with continuous b-rhythms in the bilateral occipital and temporal areas. She showed some signs of heat sensitivity with seizures developing after a prolonged bath. Physical examination revealed left-eye strabismus, muscle weakness and the absence of tendon reflexes. The Babinski sign was negative. The serum level of CK was 7000–8000 U/L. Electromyography revealed a myopathic pattern. Brain MRI demonstrated the malformation of micropolygyria, lissencephaly, extensive dysplasia of white matter, dysplasia of the brainstem and cerebellum, cerebellar cysts, and enlarged ventricles (Fig. 1). 3.1.3. Case 3 Case 3 was a 17-month-old female. She was born to a G5P2 mother by cesarean delivery at 42 weeks, with a birth weight of 3.1 kg. She had one unaffected sibling. Similar to the other cases, this patient had significant feeding difficulties and decreased cry during infancy. She could laugh at 3 months, but could not kick a bed quilt at 5 months. She could control her head movements at 8 months and sit unsupported at 15 months. Recently, she had a febrile convulsion. Her parents noted that she often has colds.
Fig. 1. Cranial MRI scans of the three patients. The observable changes are cerebellar dysplasia, white-matter changes, micropolygyria, lissencephaly and extensive dysplasia in the white matter, dysplasia in the brainstem, cerebellar cysts and extensive ventricles.
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Physical examination showed a myopathic facies, muscle weakness and the absence of tendon reflexes. The serum level of CK was 7765 U/L. Electromyography revealed a myopathic pattern. Brain MRI showed abnormal white-matter signals in the bilateral frontal region, abnormal signals in the bilateral putamen, cerebellar hypoplasia and cerebellar cysts (Fig. 1). The patient had normal vision and no abnormal eye findings. 3.2. Immunofluorescence staining Hematoxylin and eosin staining of biopsy tissue was performed and demonstrated findings that were consistent with a dystrophic disease, including muscle degeneration, increased fiber size diameter and increased endomysial connective tissue and adipose cells. Immunofluorescence showed the absence of a-DG glycosylation (Fig. 2). 3.3. Mutation analyses Direct sequencing of the FKTN exons and the flanking introns was undertaken. Cases 1 and 2 were found to have a c.139C>T (p.Arg47*) heterozygous nonsense mutation, and Case 3 had a c.346C>T (p. Gln116X) heterozygous nonsense mutation. Three-primer PCR revealed that all three patients had the FKTN heterozygous 3-kb insertion (Fig. 3). 3.4. Haplotype analyses Haplotype analyses revealed that Cases 1 and 2 had the same haplotype, and Case 1 was reported by Xiong et al. in 2009 [7]. For Case 2, the paternallyderived mutation had the 138-192-147-183 haplotype
Fig. 3. The founder 3-kb insertion analysis in the 30 untranslated region. PCR analyses of the pedigree of the patients’ family, bearing the founder 3-kb retrotransposal insertion allele. A 382-bp PCR product (lower); 382-bp bands arise from the founder insertion. 1 Negative control; 2 positive control (Case 1); 3 Case 2; 4 Case 2’s father; 5 Case 2’s mother; 6 Case 3; 7 Case 3’s father; 8 Case 3’s mother.
and the maternally-derived mutation was 130-201-157183. Case 3 had a different haplotype, where the paternally-derived mutation had the 142-196-147-193 haplotype and the maternally-derived mutation had the 138-193-153-195 haplotype (Fig. 4). 4. Discussion FKTN is located on chromosome 9 and has 10 exons. This gene is expressed in various tissues in normal
Fig. 2. Immunofluorescence staining. (a) Hematoxylin and eosin (H&E) staining of skeletal muscle of normal control serial sections. (b) Immunofluorescent staining of skeletal muscle tissue of the normal control with an anti-a-dystroglycan sugar antibody. (c) Immunofluoresent staining of skeletal muscle tissue of the normal control with anti-merosin antibody. (d) H&E staining of skeletal muscle of Case 2 shows dystrophic changes: variability in the size of muscle fibers, fibrosis and fat replacement. (e) Immunofluorescent staining of the skeletal muscle tissue of Case 2 with an anti-a-dystroglycan sugar antibody, which shows the absence of a-DG glycosylation. (f) Immunofluoresent staining of the skeletal muscle tissue of Case 2 with anti-merosin antibody, which was normal.
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Fig. 4. Haplotype analyses showing that P1 and P2 had the same haplotype. The 3-kb insertion mutation had the 138-192-147-183 haplotype and the c.139C>T, p.R47X mutation had the 130-201-157-183 haplotype. P3 had a different haplotype, where the paternally-derived c.346C>T, p. The Q116X mutation had the 142-196-147-193 haplotype and the maternally-derived 3-kb insertion mutation had the 138-193-153-195 haplotype.
individuals. In Japan, 90% of genetic changes for the FKTN 30 untranslated region are 3-kb repetitive sequences of homozygous insertions [8]. FCMD is the first known human disease that involves the ancestral insertion of a retrotransposon into a causative gene [9]. The 3-kb insert stops the expression of 38 C-terminal amino acids of fukutin, instead of containing 129 amino acids derived from the SVA sequence [10]. FKTN mutations can also lead to milder limb-girdle muscular dystrophy [11] and more severe Walker–Warburg syndrome (WWS) phenotypes [12,13]. All of the three patients in this report had neonatal hypotonia, muscle weakness, and mental retardation. Brain MRI showed different degrees of structural abnormalities, including micropolygyria, cerebellar dysplasia, extensive dysplasia of white matter and cerebellar cysts. Cases 1 and 2 had the same mutation, but they had some different clinical manifestations. Compared with Case 2, motor delay was more profound in Case 1 with the ability to sit unsupported only gained at age 5 years, compared with 14 months in Case 2. The slow eye movement in Case 1 may have been due to mental retardation at the onset of the disease and then the involvement of
ocular muscles. All three cases suffered febrile convulsions, but they occurred earlier and more frequently in Case 2. In FCMD, seizures associated with fevers have been reported previously [14]. It is interesting to note that Japanese patients who carry these two compound heterozygous mutations have a more severe phenotype and never acquire the ability to sit unsupported. A prior case study in a 7-year-old Korean child describes the same mutations found in Case 3; the Korean child was able to roll over but exhibited myopia and epilepsy [15]. A different FKTN mutation in the same base pair location (c.345_346GC>CT, p.Gln116X) has been reported previously in a patient with WWS. This patient presented with congenital hypotonia, ocular abnormalities and hydrocephalus in association with gyrus abnormalities and an absence of the corpus callosum, and died at 4.5 months of age [16]. Severe ocular abnormalities were not observed in our patients in agreement with prior descriptions of FCMD [17]. The differences in clinical severity in our patients may be due to both environmental factors and genetic modifiers. FKTN encodes the protein fukutin. The specific function of fukutin has not been elucidated, but has been
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postulated to play an important role in a-DG glycosylation. FKTN mutations can lead to abnormal neuronal migration and is thought to play a role in synaptic plasticity. Many children with FCMD have mental retardation and epilepsy [18]. Fukutin localizes to the Golgi apparatus, and its localization changes with certain FKTN mutations [19]. Localization can be rescued with curcumin treatment in FKTN missense mutations [20]. With respect to the 3-kb insertion, antisense oligonucleotide-targeted combination of the splicing site can correct the abnormal expression of fukutin protein, and could be a new direction for FCMD treatment [10]. We undertook haplotype analysis of blood samples from family members. For Cases 1 and 2, both of the chromosomes of the probands had the same haplotype as those of Japanese patients. The retrotransposon was concordant with the founder haplotype represented as 138-192-147-183 (the most common kind of haplotype in Japanese patients), which suggested that both mutant alleles were derived from the same founder mutation as Japanese patients [1]. Case 3 had a different haplotype, the nonsense mutation c.346C>T (p.Q116X), 142-196147-193, which has also been reported [1,21]. There are nine types of haplotype for the 3-kb insertion mutation in Japanese patients. Although the haplotype 138-193153-195 in Case 3 has not been reported previously, we propose that it carries the 138-component and speculate that the site of the haplotype may change across generations as a result of the exchange of genetic material between homologous chromosomes during meiosis. China is close to Japan in terms of geography and race, but the 3-kb insertion has seldom been reported in China. In 2000, Jong et al. described three patients who had the typical clinical, myopathological and neuroradiological characteristics of FCMD, but full mutational analyses of FKTN revealed that neither a 3-kb insertion nor a point mutation was found [22]. In 2005, Watanabe et al. assessed the 3-kb insertion in 766 Chinese individuals but did not detect insertion alleles [5]. He ruled out the possibility that a single ancestor bearing an insertion chromosome migrated to Japan from Korea or mainland China and concluded that FCMD carriers are rare outside of Japan. In our study, all three patients had a FKTN nonsense mutation and the 3-kb insertion and the same haplotype as those of Japanese patients, indicating that both mutant alleles were derived from the same founder as Japanese patients. This suggests that segments of the Chinese and Japanese populations may have a recent common ancestor. It is well known that FKTN mutations have been reported in other countries other than Japan, but the classical manifestation of FCMD and a similar haplotype have not been reported elsewhere. Our study widens the distribution of the 3-kb insertion founder mutation; further investigations of the origin of 3-kb insertion founder mutation would be helpful.
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5. Conclusions Here, we reported on the cases of three patients with the typical clinical manifestations of FCMD. Gene testing confirmed that all three patients had a 3-kb founder insertion mutation. Haplotype analysis suggested that Cases 1 and 2 had the same haplotype as Japanese patients. Case 3 had a different haplotype, but this may have arisen from the same ancestor founder mutation because the haplotype site can change between generations. This suggests that segments of the Chinese and Japanese populations may have a recent common ancestor. Author Contributions Conceived and designed the experiments: HPY KK HX. Performed the experiments: HPY SW HJ JXX. Analyzed the data: KK TT XRW. Contributed reagents/materials/analysis tools: HPY. Wrote the paper: HPY.
Acknowledgments We are in deep mourning for Prof. Fukuyama. We will never forget him and will let him live in our heart and continue his work. The authors are grateful to all the patients and their families who have participated in this study and thank Dr. Anne Rutkowski, MD, for her comments and criticisms on the manuscript. This work was supported by grants from the National Natural Science Foundation of China (81271400), and the National Basic Research Program of China (2012CB944602). References [1] Fukuyama Y, Ohsawa M. A genetic study of the Fukuyama type congenital muscular dystrophy. Brain Dev 1984;6:373–90. [2] Fukuyama Y, Osawa M, Suzuki H. Congenital progressive muscular dystrophy of the Fukuyama types – clinical, genetic and pathological considerations. Brain Dev 1981;3:1–29. [3] Saito, K. Fukuyama Congenital Muscular Dystrophy. GeneReviews Last Update: May 10; 2012. [4] Jang DH, Sung IY, Ko TS. Peripheral nerve involvement in Fukuyama congenital muscular dystrophy: a case report. J Child Neurol 2013;28:132–7. [5] Watanabe M, Kobayashi K, Jin F, Park KS, Yamada T, Tokunaga K, et al. Founder SVA retrotransposal insertion in Fukuyama-type congenital muscular dystrophy and its origin in Japanese and Northeast Asian populations. Am J Med Genet A 2005;138:344–8. [6] Kobayashi K, Nakahori Y, Mizuno K, Miyake M, Kumagai T, Honma A, et al. Founder-haplotype analysis in Fukuyama-type congenital muscular dystrophy (FCMD). Hum Genet 1998;103: 323–7. [7] Xiong H, Wang S, Kobayashi K, Jiang Y, Wang J, Chang X, et al. Fukutin gene retrotransposal insertion in a non-Japanese
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