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Duchenne and Becker muscular dystrophies: an Estonian experience
Brain & Development 21 (1999) 244–247
Original article
Duchenne and Becker muscular dystrophies: an Estonian experience ¨ lvi Astra Talkop a, Tiina Klaassen b, Andres Piirsoo c, Valentin Sander d, Aita Napa e, U Ene Essenson e, Jaana Tammur b, Tiina Talvik f ,* a
Department of Pediatrics, Tartu University, Tartu, Estonia Institute of Molecular and Cell Biology, Tartu University, Tartu, Estonia c Laboratory of Cell Biology, Institute of General and Molecular Pathology, Tartu University, Tartu, Estonia d Pediatric Neurology Unit, Childrens’ Hospital of Tallinn, Tallinn, Estonia e Pediatric Neurology Unit, Children’s Hospital of the University Hospitals of Tartu, Tartu, Estonia f Department of Pediatrics, Children’s Hospital of the University Hospitals of Tartu University, 6 Lunini Street, Tartu 51014, Estonia b
Received 27 November 1997; received in revised form 2 January 1999; accepted 22 January 1999
Abstract The clinical and molecular features of 25 Duchenne (DMD), two intermediate (D/BMD) and three Becker (BMD) muscular dystrophy patients from 26 unrelated families were evaluated. Early psychomotor development was normal in patients with D/BMD and BMD. Learning to walk independently after 15 months of age was a risk sign of DMD in nine (36%) patients. Abnormality in crawling was seen in 13 (54%) patients with DMD. These boys demonstrated initial symptoms earlier than those who learned to crawl normally. Mental retardation was established in five (20%) patients with DMD. Deletions in the dystrophin gene were found in 11 families (48%). They were accumulated (9/11, 82%) in the distal region of the gene. 1999 Elsevier Science B.V. All rights reserved. Keywords: Duchenne muscular dystrophy; Becker muscular dystrophy; Dystrophin; Motor developmental delay; Risk sign
2. Patients
in the period between September 1st, 1994 and June 18th, 1998. Of these patients, 30 male patients with DMD/BMD from 26 unrelated families between the ages of 2 and 36.5 years (mean age 11.80 years) were included in the cohort. Diagnosis was based on clinical symptoms, the elevation of serum creatine kinase (CK) and on electromyography. In addition, in most cases one of the following conditions had to be fulfilled: (1) clear X-linked inheritance of a myopathy clinically compatible with DMD or BMD (2) the absence of dystrophin in patients with DMD and weak or patchy staining for dystrophin in patients with BMD in the muscle biopsy specimen (3) the presence of an intragenic deletion in the dystrophin gene. Patients were divided into three groups according to the criteria of the clinical severity of the disease as described by Dubowitz [10].
A retrospective and prospective study was performed on 44 patients with a definite or probable diagnosis of DMD/BMD
3. Methods
1. Introduction Duchenne (DMD) and Becker (BMD) muscular dystrophies are allelic X-linked progressive disorders caused by mutations in the dystrophin gene. Deletions in the dystrophin gene are found in 22–86% of patients with DMD/BMD [1–8]. Dystrophin testing (by either immunoblot or immunofluorescence) is capable of identifying at least 98% of patients with DMD/BMD [9]. The present study summarizes the Estonian experience of DMD/BMD with an emphasis on early clinical symptoms and dystrophin gene lesions.
* Corresponding author. Fax: +372-7-449-608.
0387-7604/99/$ - see front matter PII: S03 87-7604(99)000 16-9
Efforts were made to identify all affected patients by
1999 Elsevier Science B.V. All rights reserved.
245
U.A. Talkop et al. / Brain & Development 21 (1999) 244–247
means of telephone calls and a letter circulated to general practitioners, pediatricians, and neurologists all over Estonia once a year throughout the study period. From 1993 it was agreed that all children with suspected muscular disorders were referred to the child neurology unit of the Children’s Hospital, University Hospitals, Tartu. Further information was disseminated through lectures, seminars, and the presentation of papers at medical congresses throughout the study period. Taking into account the severity of the disease, it is highly probable that all patients reach a doctor anyway. ¨ .A.T. and V.S. Patients were examined by two authors (U or T.T). The data about early motor and speech development and initial symptoms were collected from patients or their
parents according to the study protocol. General intelligence was tested in eleven boys using the Wechsler Pre-School and Primary Scale of Intelligence (revised), or the Leiter International Performance Scale. Other patients were classified as mentally retarded if they had not been able to attend normal school owing to mental subnormality. Blood samples for DNA analysis were obtained from 26 patients from 23 unrelated families. Dystrophin gene deletion analysis was done using 25 exon multiplex PCR (exons 3, 4, 6, 8, 12, 13, 16, 17, 19, 25, 32, 34, 41, 42, 43, 44, 45, 47, 48, 50, 51, 52, 60, Pm and Pb (Baylor Medical College, Houston, USA; Research Genetics, USA), as described previously [11,12]. Altogether, these PCR assays allow rapid detection of over 98% of deletions in the dystrophin gene [13].
Table 1 Main characteristics of patients with DMD/BMD Case
Age (y)
Crawling
Walking (m)
Presenting IQ (test) symptom (age)
Wheelchairbound (age)
DNA (exons)
DYS 1, 2, 3
Diagnosis
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 Median SD
2* 6.5 7.5* 8 10 11 11 11 10.5(–) 12 12* 12* 6 17.5 9.5 9 9.5 5* 13.5 8.5 19 17.5 19 17.5* 8.5 15* 4* 22.5* 6.5 36.5 10.25 6.79
Absent 11 m ‘Spy’ Rolling Normal 10 m Normal Normal Normal Absent ‘Spy’ Normal Absent Little Little Normal 11 m Normal ‘Spy’ 11 m Normal
14 22 15 17 15 16 16 18 13 15 15 13 12 14 16 18 24 14 24 13 12 14 12 13 11 12 15 12 12
1y2m 6m 1y3m 1y5m 1y6m 1y6m 5y 2y 1y1m 1y3m 1y3m 1y1m 1y 1y2m 1y 1 y 10 m 1y 3y 2y 8m 1y 1y2m 11 m 2y 6y 2y6m 1y3m 1y3m 1y 2y 1.25 1.31
No No No No No No 10.5 y 10 y 10.5 y 9y 8.5 y 11.5 y No 10 y No 7.5 y No No 9y No 9y Yes 10 y 9.5 y No 15 y No 16 y No No 9.75 2.51
No del No del 48-52(59) 44-51 44 45-47 48-50 no del no del no del 48-52(59) no del no del 48-51 49-50 no del no del no del (7)8-16 no del NP NP 49-52(59) NP 3-19(24) no del no del NP no del (46)47-48
NP Neg Neg Neg Neg/gr + Neg NP NP Neg NP Neg Neg Neg NP Neg Neg Neg Neg NP Neg NP NP NP NP NP Neg NP NP Weak NP
DMD DMD DMD DMD DMD DMD DMD DMD DMD DMD DMD DMD DMD DMD DMD DMD DMD DMD DMD DMD DMD DMD DMD DMD DMD D/BMD D/BMD BMD BMD BMD
Normal Normal Normal Normal Normal Normal Normal Normal
15 3.78
Normal Normal (W) sl MR (W) Normal mo MR (L) Normal (L) Normal Normal Normal Normal MR Normal sl MR (L) Normal bo MR (L) bo MR (L) sl MR (L) bo MR (L) Normal Normal (L) Normal Normal Normal Normal Normal (L) Normal Normal Normal Normal (W) Normal
*More than one boy with DMD/BMD in the family (cases 1 and 18, cases 3 and 11 were cousins; cases 12 and 16 were brothers; case 28 was an uncle to case 27 and he also had a cousin with D/BMD; case 24 had had a grand-cousin with DMD), – = adopted; crawling = characterization of the way and age of learning to crawl; normal = learning to crawl before the age of 10 months; absent = absence of crawling; ‘spy’ = crawling like a scout (dragging oneself forward with hands without help from hands); little = crawling only for a short interval; rolling = moving on the floor rolling oneself over one side; walking = age of independent walking; age = age at last check-up; y = years; m = months; W = the Wechsler Pre-School and Personality Scale of Intelligence-Revised; L = the Leiter International Performance Scale; sl MR = slight mental retardation; mo MR = moderate mental retardation and autism; bo MR = borderline mental retardation; DNA (exon) = site of deletion; (exon number) = precise boundary of deletion was not confirmed because screening of all DMD gene exons was not feasible; no del = no deletion; NP = no probes; DYS 1, 2, 3 = dystrophin antibodies to rod-domain, C-terminal domain, and Nterminal domain respectively; pos = positive staining with dystrophin antibodies; neg = negative staining with dystrophin antibodies; neg/gr + = mostly negative staining, but some positive staining in few fiber groups with DYS 2 and DYS 3 antibodies; weak = weak staining with dystrophin antibodies in comparison to staining with spectrin antibody.
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U.A. Talkop et al. / Brain & Development 21 (1999) 244–247
Muscle biopsies were obtained using the open method from the vastus lateralis muscle or the rectus femoris muscle in 16 patients from 14 unrelated families. For immunohistochemical investigation, frozen sections were used. In all specimen, dystrophin expression was immunolocalized with monoclonal antibodies DYS 1, DYS 2, DYS 3 (Novocastra, UK). As a control we used an anti-spectrin monoclonal antibody (Novocastra, UK). The computer program Statistica was used to make calculations of basic statistics and Spearman rank order correlations.
4. Results We investigated 30 male patients from 26 unrelated families, whose main characteristics are given in Table 1. There was a positive family history of DMD/BMD in five (20%) families. Data about achieving different milestones in motor development was mostly described as normal, although the anamnesis in most cases lacked the precise ages of these accomplishments. To this rule there were two exceptions in the DMD group, namely concerning independent walking and crawling. Crawling was described as normal in 11 (45.8%) patients. Abnormality in crawling was described in 13 (54.2%) boys (Table 1): like a ‘spy’ (dragging oneself forward with hands without help from legs), as rolling, as occurring only for a short interval, absence of crawling, and delayed age (at 10 and 11 months of age) of crawling. The severity of diagnosis was positively correlated with crawling abnormalities (r = 0.41, P , 0.05). At the age of 15 months, 16 (64%) of our DMD patients were able to walk independently. The mean age of learning to walk independently was 15.8 months. Speech delay was defined by failure to use two or more words together in sentences before the age of 3 years. Only one patient (case 5) learned to speak in sentences later than normal (at the age of 3 year 10 m) (data not shown). Mental retardation was established in five (20%) boys with DMD (Table 1). A correlation between mental retardation and a delayed age of learning to speak (r = 0.41, P , 0.05) was established. Clumsiness, frequent fallings and difficulties in rising were the most often-described initial symptoms in DMD patients. Myalgias, inability to keep up with their peers, and frequent falls were the presenting symptoms in BMD patients. The age at presenting symptom was inversely correlated with crawling abnormalities (r = −0.43; P , 0.05). A deletion was proven in 12 patients from 11 unrelated families (47.8%). Most of the discovered deletions (9/11, 81.8%; the exact location of each is given in Table 1) were located in the major ‘hot spot’ region between exons 44 and 52. No correlation was found between the severity of the disease and the location or extent of deletion.
5. Discussion Learning to walk later than 15 months of age, which is the age when 90% of children can walk well according to the Denver developmental scale [14], is a well-known risk symptom of DMD [10,15,16]. In our study, the late-walkers (36%) constituted a similar proportion to other studies (20– 30%) [15,16]. The earliest detectable risk sign, however, in over half (57%) of boys with DMD was abnormality in crawling. This is a new risk sign of DMD that has not, to our knowledge yet been mentioned earlier. In addition, patients with crawling abnormalities tended to present initial symptoms earlier. We suggest that for those baby boys, serum CK measurements or special screening in specialized centers should be performed. A delay in the development of language skills is considered to be a feature of DMD [5,10,17]. It was one of the presenting symptoms in 4% (1/25) of our DMD patients. Our data is in concordance with the study of Marshall et al., who discovered a delay in speech development in 3% of patients with DMD [16]. Hodgson et al. were able to determine a speech delay in 62% of patients [3]. This discrepancy can be attributed to different definitions of normal speech development (2 years for Hodgson et al., vs. 3 years this report) [3]. We were also able to confirm a correlation between speech delay and mental retardation. Our results can be related to the fact that the only boy with speech delay had the severest degree of mental retardation. Approximately one third of patients with DMD have mental impairment [5,10]. In our cohort we were able to establish mental impairment in five (20%) patients with DMD. Our three BMD patients had normal IQ, which is in concordance with the thesis, that mental retardation is rare in BMD [10]. Mental retardation is more common in boys with distal deletions [2,3,18]. Yet one must remember that patients with and without mental impairment may share the same deletion. We were able to detect deletions in 48% of cases. This proportion was similar to that described in analogous studies (42-52%) [1,2,4,6]. The accumulation of deletions (82%) in the major hot spot region between exons 44 and 52 was also observed. In most studies there is a tendency for 80–91% of deletions to be accumulated in the distal region of the dystrophin gene [1,6,7]. Recognizing the risk signs of DMD and applying DNA diagnostics and dystrophin analysis for precise diagnosis gives us the possibility to offer families an early and accurate prenatal diagnosis.
Acknowledgements We would like to thank all patients, their families, and their local doctors who participated in the study. We are also grateful to Professor A. Metspalu for his cooperation, criticism and useful remarks and to M.Sc.M. Thetloff for her
U.A. Talkop et al. / Brain & Development 21 (1999) 244–247
statistical advice. The research described in this publication was carried out in part with assistance from the Estonian Science Foundation (grants No. 269, 592, 1079, 2407, 3121), TARLA 0475 and DARLA 0502. References [1] Koenig M, Hoffman EP, Bertelson CJ, Monaco AP, Feener C, Kunkel LM. Complete cloning of the Duchenne muscular dystrophy (DMD) cDNA and preliminary genomic organization of the DMD gene in normal and affected individuals. Cell 1987;50:509–517. [2] Lindlo¨f M, Kiuru A, Ka¨a¨ria¨inen H, Kalimo H, Lang H, Pihko H, et al. Gene deletions in X-linked muscular dystrophy. Am J Hum Genet 1989;44:496–503. [3] Hodgson SV, Abbs S, Manzur A, Heckmatt JZH, Dubowitz V, Bobrow M. Correlation of clinical and deletion data in Duchenne and Becker muscular dystrophy, with special reference to mental ability. Neuromusc Disord 1992;4:262–276. [4] Ballo R, Viljoen D, Beighton P. Duchenne and Becker muscular dystrophy prevalence in South Africa and molecular findings in 128 persons affected. S Afr Med J 1994;84:494–497. [5] Bresolini N, Castelli E, Comi GP, Felisari G, Bardoni A, Perani D, et al. Cognitive impairment in Duchenne muscular dystrophy. Neuromusc Disord 1994;4:359–369. [6] Odinokova ON, Puzyrev VP, Radzhabaliev SF, Rakhmonov RA. Deletion analysis of the dystrophin gene in patients with Duchenne’s muscular dystrophy in Tajikistan (in Russian). Genetica 1996;32:1392–1395. [7] Singh V, Sinha S, Mishra S, Chaturvedi LS, Pradhan S, Mittal RD, et
[8]
[9]
[10] [11]
[12]
[13]
[14] [15] [16] [17] [18]
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al. Proportion and pattern of dystrophin gene deletions in North Indian Duchenne and Becker muscular dystrophy patients. Hum Genet 1997;99:206–208. Haider MZ, Bastaki L, Habib Y, Moosa A. Screening 25 dystrophin gene exons for deletions in Arab children with Duchenne muscular dystrophy. Hum Hered 1998;48:61–66. Hoffman EP, Kunkel LM, Angelini C, Clarke A, Johnson M, Harris JB. Improved diagnosis of Becker muscular dystrophy by dystrophin testing. Neurology 1989;39:1011–1017. Dubowitz V. Muscle disorders in childhood. London: Saunders, 1995. Chamberlain JS, Gibbs RA, Rainer JE, Caskey CT. Multiplex PCR for diagnosis of Duchenne muscular dystrophy. In: Innis M, Gelfand D, Sninski J, White T, editors. PCR: protocols and applications- a laboratory manual. New York: Academic Press, 1990: 272–281. Beggs AH. Multiplex PCR for identifying dystrophin gene deletions. In: Diacopoli NC, Haines JL, Korf BR, et al., editors. Current protocols in human genetics. New York: Wiley 1996:9.3.1.–9.3.19. Beggs AH, Koenig M, Boyce FM, Kunkel LM. Detection of 98% of DMD/BMD gene deletions by polymerase chain reaction. Hum Genet 1990;86:45–48. Frankenburg WK, Dodds JB. The Denver developmental screening test. J Pediatr 1971;79:181–191. O’Brien T, Sibert JR, Harper PS. Implications of diagnostic delay in Duchenne muscular dystrophy. Br Med J 1983;287:1106–1107. Marshall PD, Galasko CSB. No improvement in delay in diagnosis of Duchenne muscular dystrophy. Lancet 1995;345:590–591. Dubowitz V. Intellectual impairment in muscular dystrophy. Arch Dis Child 1965;40:296–301. Bushby KMD. Genetic and clinical correlations of Xp21 muscular dystrophy. J Inher Metab Dis 1992;15:551–564.
Original article
Duchenne and Becker muscular dystrophies: an Estonian experience ¨ lvi Astra Talkop a, Tiina Klaassen b, Andres Piirsoo c, Valentin Sander d, Aita Napa e, U Ene Essenson e, Jaana Tammur b, Tiina Talvik f ,* a
Department of Pediatrics, Tartu University, Tartu, Estonia Institute of Molecular and Cell Biology, Tartu University, Tartu, Estonia c Laboratory of Cell Biology, Institute of General and Molecular Pathology, Tartu University, Tartu, Estonia d Pediatric Neurology Unit, Childrens’ Hospital of Tallinn, Tallinn, Estonia e Pediatric Neurology Unit, Children’s Hospital of the University Hospitals of Tartu, Tartu, Estonia f Department of Pediatrics, Children’s Hospital of the University Hospitals of Tartu University, 6 Lunini Street, Tartu 51014, Estonia b
Received 27 November 1997; received in revised form 2 January 1999; accepted 22 January 1999
Abstract The clinical and molecular features of 25 Duchenne (DMD), two intermediate (D/BMD) and three Becker (BMD) muscular dystrophy patients from 26 unrelated families were evaluated. Early psychomotor development was normal in patients with D/BMD and BMD. Learning to walk independently after 15 months of age was a risk sign of DMD in nine (36%) patients. Abnormality in crawling was seen in 13 (54%) patients with DMD. These boys demonstrated initial symptoms earlier than those who learned to crawl normally. Mental retardation was established in five (20%) patients with DMD. Deletions in the dystrophin gene were found in 11 families (48%). They were accumulated (9/11, 82%) in the distal region of the gene. 1999 Elsevier Science B.V. All rights reserved. Keywords: Duchenne muscular dystrophy; Becker muscular dystrophy; Dystrophin; Motor developmental delay; Risk sign
2. Patients
in the period between September 1st, 1994 and June 18th, 1998. Of these patients, 30 male patients with DMD/BMD from 26 unrelated families between the ages of 2 and 36.5 years (mean age 11.80 years) were included in the cohort. Diagnosis was based on clinical symptoms, the elevation of serum creatine kinase (CK) and on electromyography. In addition, in most cases one of the following conditions had to be fulfilled: (1) clear X-linked inheritance of a myopathy clinically compatible with DMD or BMD (2) the absence of dystrophin in patients with DMD and weak or patchy staining for dystrophin in patients with BMD in the muscle biopsy specimen (3) the presence of an intragenic deletion in the dystrophin gene. Patients were divided into three groups according to the criteria of the clinical severity of the disease as described by Dubowitz [10].
A retrospective and prospective study was performed on 44 patients with a definite or probable diagnosis of DMD/BMD
3. Methods
1. Introduction Duchenne (DMD) and Becker (BMD) muscular dystrophies are allelic X-linked progressive disorders caused by mutations in the dystrophin gene. Deletions in the dystrophin gene are found in 22–86% of patients with DMD/BMD [1–8]. Dystrophin testing (by either immunoblot or immunofluorescence) is capable of identifying at least 98% of patients with DMD/BMD [9]. The present study summarizes the Estonian experience of DMD/BMD with an emphasis on early clinical symptoms and dystrophin gene lesions.
* Corresponding author. Fax: +372-7-449-608.
0387-7604/99/$ - see front matter PII: S03 87-7604(99)000 16-9
Efforts were made to identify all affected patients by
1999 Elsevier Science B.V. All rights reserved.
245
U.A. Talkop et al. / Brain & Development 21 (1999) 244–247
means of telephone calls and a letter circulated to general practitioners, pediatricians, and neurologists all over Estonia once a year throughout the study period. From 1993 it was agreed that all children with suspected muscular disorders were referred to the child neurology unit of the Children’s Hospital, University Hospitals, Tartu. Further information was disseminated through lectures, seminars, and the presentation of papers at medical congresses throughout the study period. Taking into account the severity of the disease, it is highly probable that all patients reach a doctor anyway. ¨ .A.T. and V.S. Patients were examined by two authors (U or T.T). The data about early motor and speech development and initial symptoms were collected from patients or their
parents according to the study protocol. General intelligence was tested in eleven boys using the Wechsler Pre-School and Primary Scale of Intelligence (revised), or the Leiter International Performance Scale. Other patients were classified as mentally retarded if they had not been able to attend normal school owing to mental subnormality. Blood samples for DNA analysis were obtained from 26 patients from 23 unrelated families. Dystrophin gene deletion analysis was done using 25 exon multiplex PCR (exons 3, 4, 6, 8, 12, 13, 16, 17, 19, 25, 32, 34, 41, 42, 43, 44, 45, 47, 48, 50, 51, 52, 60, Pm and Pb (Baylor Medical College, Houston, USA; Research Genetics, USA), as described previously [11,12]. Altogether, these PCR assays allow rapid detection of over 98% of deletions in the dystrophin gene [13].
Table 1 Main characteristics of patients with DMD/BMD Case
Age (y)
Crawling
Walking (m)
Presenting IQ (test) symptom (age)
Wheelchairbound (age)
DNA (exons)
DYS 1, 2, 3
Diagnosis
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 Median SD
2* 6.5 7.5* 8 10 11 11 11 10.5(–) 12 12* 12* 6 17.5 9.5 9 9.5 5* 13.5 8.5 19 17.5 19 17.5* 8.5 15* 4* 22.5* 6.5 36.5 10.25 6.79
Absent 11 m ‘Spy’ Rolling Normal 10 m Normal Normal Normal Absent ‘Spy’ Normal Absent Little Little Normal 11 m Normal ‘Spy’ 11 m Normal
14 22 15 17 15 16 16 18 13 15 15 13 12 14 16 18 24 14 24 13 12 14 12 13 11 12 15 12 12
1y2m 6m 1y3m 1y5m 1y6m 1y6m 5y 2y 1y1m 1y3m 1y3m 1y1m 1y 1y2m 1y 1 y 10 m 1y 3y 2y 8m 1y 1y2m 11 m 2y 6y 2y6m 1y3m 1y3m 1y 2y 1.25 1.31
No No No No No No 10.5 y 10 y 10.5 y 9y 8.5 y 11.5 y No 10 y No 7.5 y No No 9y No 9y Yes 10 y 9.5 y No 15 y No 16 y No No 9.75 2.51
No del No del 48-52(59) 44-51 44 45-47 48-50 no del no del no del 48-52(59) no del no del 48-51 49-50 no del no del no del (7)8-16 no del NP NP 49-52(59) NP 3-19(24) no del no del NP no del (46)47-48
NP Neg Neg Neg Neg/gr + Neg NP NP Neg NP Neg Neg Neg NP Neg Neg Neg Neg NP Neg NP NP NP NP NP Neg NP NP Weak NP
DMD DMD DMD DMD DMD DMD DMD DMD DMD DMD DMD DMD DMD DMD DMD DMD DMD DMD DMD DMD DMD DMD DMD DMD DMD D/BMD D/BMD BMD BMD BMD
Normal Normal Normal Normal Normal Normal Normal Normal
15 3.78
Normal Normal (W) sl MR (W) Normal mo MR (L) Normal (L) Normal Normal Normal Normal MR Normal sl MR (L) Normal bo MR (L) bo MR (L) sl MR (L) bo MR (L) Normal Normal (L) Normal Normal Normal Normal Normal (L) Normal Normal Normal Normal (W) Normal
*More than one boy with DMD/BMD in the family (cases 1 and 18, cases 3 and 11 were cousins; cases 12 and 16 were brothers; case 28 was an uncle to case 27 and he also had a cousin with D/BMD; case 24 had had a grand-cousin with DMD), – = adopted; crawling = characterization of the way and age of learning to crawl; normal = learning to crawl before the age of 10 months; absent = absence of crawling; ‘spy’ = crawling like a scout (dragging oneself forward with hands without help from hands); little = crawling only for a short interval; rolling = moving on the floor rolling oneself over one side; walking = age of independent walking; age = age at last check-up; y = years; m = months; W = the Wechsler Pre-School and Personality Scale of Intelligence-Revised; L = the Leiter International Performance Scale; sl MR = slight mental retardation; mo MR = moderate mental retardation and autism; bo MR = borderline mental retardation; DNA (exon) = site of deletion; (exon number) = precise boundary of deletion was not confirmed because screening of all DMD gene exons was not feasible; no del = no deletion; NP = no probes; DYS 1, 2, 3 = dystrophin antibodies to rod-domain, C-terminal domain, and Nterminal domain respectively; pos = positive staining with dystrophin antibodies; neg = negative staining with dystrophin antibodies; neg/gr + = mostly negative staining, but some positive staining in few fiber groups with DYS 2 and DYS 3 antibodies; weak = weak staining with dystrophin antibodies in comparison to staining with spectrin antibody.
246
U.A. Talkop et al. / Brain & Development 21 (1999) 244–247
Muscle biopsies were obtained using the open method from the vastus lateralis muscle or the rectus femoris muscle in 16 patients from 14 unrelated families. For immunohistochemical investigation, frozen sections were used. In all specimen, dystrophin expression was immunolocalized with monoclonal antibodies DYS 1, DYS 2, DYS 3 (Novocastra, UK). As a control we used an anti-spectrin monoclonal antibody (Novocastra, UK). The computer program Statistica was used to make calculations of basic statistics and Spearman rank order correlations.
4. Results We investigated 30 male patients from 26 unrelated families, whose main characteristics are given in Table 1. There was a positive family history of DMD/BMD in five (20%) families. Data about achieving different milestones in motor development was mostly described as normal, although the anamnesis in most cases lacked the precise ages of these accomplishments. To this rule there were two exceptions in the DMD group, namely concerning independent walking and crawling. Crawling was described as normal in 11 (45.8%) patients. Abnormality in crawling was described in 13 (54.2%) boys (Table 1): like a ‘spy’ (dragging oneself forward with hands without help from legs), as rolling, as occurring only for a short interval, absence of crawling, and delayed age (at 10 and 11 months of age) of crawling. The severity of diagnosis was positively correlated with crawling abnormalities (r = 0.41, P , 0.05). At the age of 15 months, 16 (64%) of our DMD patients were able to walk independently. The mean age of learning to walk independently was 15.8 months. Speech delay was defined by failure to use two or more words together in sentences before the age of 3 years. Only one patient (case 5) learned to speak in sentences later than normal (at the age of 3 year 10 m) (data not shown). Mental retardation was established in five (20%) boys with DMD (Table 1). A correlation between mental retardation and a delayed age of learning to speak (r = 0.41, P , 0.05) was established. Clumsiness, frequent fallings and difficulties in rising were the most often-described initial symptoms in DMD patients. Myalgias, inability to keep up with their peers, and frequent falls were the presenting symptoms in BMD patients. The age at presenting symptom was inversely correlated with crawling abnormalities (r = −0.43; P , 0.05). A deletion was proven in 12 patients from 11 unrelated families (47.8%). Most of the discovered deletions (9/11, 81.8%; the exact location of each is given in Table 1) were located in the major ‘hot spot’ region between exons 44 and 52. No correlation was found between the severity of the disease and the location or extent of deletion.
5. Discussion Learning to walk later than 15 months of age, which is the age when 90% of children can walk well according to the Denver developmental scale [14], is a well-known risk symptom of DMD [10,15,16]. In our study, the late-walkers (36%) constituted a similar proportion to other studies (20– 30%) [15,16]. The earliest detectable risk sign, however, in over half (57%) of boys with DMD was abnormality in crawling. This is a new risk sign of DMD that has not, to our knowledge yet been mentioned earlier. In addition, patients with crawling abnormalities tended to present initial symptoms earlier. We suggest that for those baby boys, serum CK measurements or special screening in specialized centers should be performed. A delay in the development of language skills is considered to be a feature of DMD [5,10,17]. It was one of the presenting symptoms in 4% (1/25) of our DMD patients. Our data is in concordance with the study of Marshall et al., who discovered a delay in speech development in 3% of patients with DMD [16]. Hodgson et al. were able to determine a speech delay in 62% of patients [3]. This discrepancy can be attributed to different definitions of normal speech development (2 years for Hodgson et al., vs. 3 years this report) [3]. We were also able to confirm a correlation between speech delay and mental retardation. Our results can be related to the fact that the only boy with speech delay had the severest degree of mental retardation. Approximately one third of patients with DMD have mental impairment [5,10]. In our cohort we were able to establish mental impairment in five (20%) patients with DMD. Our three BMD patients had normal IQ, which is in concordance with the thesis, that mental retardation is rare in BMD [10]. Mental retardation is more common in boys with distal deletions [2,3,18]. Yet one must remember that patients with and without mental impairment may share the same deletion. We were able to detect deletions in 48% of cases. This proportion was similar to that described in analogous studies (42-52%) [1,2,4,6]. The accumulation of deletions (82%) in the major hot spot region between exons 44 and 52 was also observed. In most studies there is a tendency for 80–91% of deletions to be accumulated in the distal region of the dystrophin gene [1,6,7]. Recognizing the risk signs of DMD and applying DNA diagnostics and dystrophin analysis for precise diagnosis gives us the possibility to offer families an early and accurate prenatal diagnosis.
Acknowledgements We would like to thank all patients, their families, and their local doctors who participated in the study. We are also grateful to Professor A. Metspalu for his cooperation, criticism and useful remarks and to M.Sc.M. Thetloff for her
U.A. Talkop et al. / Brain & Development 21 (1999) 244–247
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