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Becker Muscular Dystrophy
Electroretinogram in Duchenne/Becker Muscular Dystrophy Samuel I. Pascual Pascual, MD*, Jesus Molano, MD†, and Ignacio Pascual-Castroviejo, MD* The authors studied all cases with dystrophinopathy consecutively reviewed between May 1995 and December 1996 by means of electroretinography (ERG), which was recorded using skin eyelid electrodes and with standard flash stimulation. This methodology can detect the functional abnormalities associated with dystrophinopathies. The most valuable parameter is the ratio of B-wave amplitude to A-wave amplitude (B/A amplitude ratio), which was greater than 2 in all normal control patients (n 5 10) and nondystrophinopathic muscular dystrophy (MD) patients (n 5 2). It was less than 2 in 100% (n 5 16) of Duchenne muscular dystrophy (DMD) patients (mean ratio 0.73; range 0.4-1.26). It was less than 2 in 71% (n 5 7) of Becker muscular dystrophy (BMD) patients (mean ratio 1.12; range 0.88-1.37), and less than 2 in 50% (n 5 4) of definitive DMD carriers. Twenty-nine percent of BMD, 50% of DMD carriers, and the only case with asymptomatic dystrophinopathy had normal ratios (greater than 2). The differences between the mean ratios of control, DMD, and BMD groups were statistically significant, all of them with P < 0.001. ERG abnormalities of dystrophinopathies were associated with the more severe muscular phenotype but not with the presence or location of gene deletion. ERG is an easy and simple technique that is useful in cases of suspected dystrophinopathy with a nonconclusive molecular study. It is less useful in patients who are DMD carriers. © 1998 by Elsevier Science Inc. All rights reserved. Pascual Pascual SI, Molano J, Pascual-Castroviejo I. Electroretinogram in Duchenne/Becker muscular dystrophy. Pediatr Neurol 1998;18:315-320.
Introduction Dystrophin, a protein of 427 kd codified by the gene of Duchenne/Becker muscular dystrophy (DMD/BMD), has been located in its full transcription not only in striated
From the *Service of Neuropediatrics; †Molecular Genetics Unit; Service of Biochemistry; Hospital Infantil ‘‘La Paz’’; Madrid, Spain.
© 1998 by Elsevier Science Inc. All rights reserved. PII S0887-8994(97)00208-7 ● 0887-8994/98/$19.00
muscle but also in cardiac and smooth muscle, in cerebral tissue, and in the outer plexiform layer of the retina [1-3]. Partial transcriptions of the gene, of the carboxy terminus, have also been found in glial and Schwann cells and in kidney, liver, pancreas, and testis [1]. The role of dystrophin in the retina is not known. It has been located in the synaptic complex of the photoreceptorbipolar cell [2]. This complex, and Mu¨ller cells, are the structures implicated in the origin of the B-wave of the electroretinogram (ERG). Dystrophin can give stability to the cell membranes or affect the function of the ionic channels [2,4]. The normal ERG (Fig 1) is composed of two waves, the A-wave with negative polarity resulting from a hyperpolarity of photoreceptors and the larger B-wave, with positive polarity produced by function of bipolar and Mu¨ller cells [4,5]. In DMD/BMD the scotopic ERG is often altered. A B-wave with an amplitude less than normal or even absent can be recorded [4,6-9] with more impairment of rod function seen than cone function. The more altered item is the amplitude ratio between the B-wave and A-wave (B/A amplitude ratio), produced by stimuli of a bright white light after adaptation to darkness. Normally the B/A amplitude ratio is greater than 2. In DMD/BMD, often it is less than 1 (termed ERG negative). This abnormality is asymptomatic; patients do not complain of nictalopia or other visual disturbances, unlike patients who have other visual diseases with similar ERG abnormalities [4,5,9]. The ERG has been found abnormal in 50% [7], 62.5% [5], or 88% [6] of DMD/BMD patients using the usual methodology [10] of corneal electrodes and full-field Ganzfeld stimulation. Nevertheless, these technical conditions are not easily available and the lack of cooperation from children makes the exploration difficult without general anesthesia. In children the ERG can be recorded with reliable results using eyelid electrodes [11,12], although ampli-
Communications should be addressed to: Dr. Pascual Pascual: Servicio de Neuropediatrı´a; Hospital Infantil ‘‘La Paz’’; Po Castellana, 261; 28046-Madrid, Spain. Received July 2, 1997; accepted September 29, 1997.
Pascual Pascual et al: ERG in Dystrophinopathies 315
Figure 1. (A, B) ERGs of two normal patients. The amplitude of the B-wave is higher than the A-wave. ERGs in all figures were performed with a bright-white light flash. O.D. 5 right eye; O.I. 5 left eye.
tudes of the waves are not comparable with those recorded with corneal electrodes. This report tries to confirm that the abnormalities of the B-wave in DMD/BMD can be detected using eyelid electrodes and with simple flash stimulation and then describes the characteristic ERG responses in comparison with those of normal control patients, patients with nondystrophinopathic muscular dystrophies, and female carriers of DMD dystrophy. Material and Methods Four different groups of patients were studied (Table 1). All the patients studied, or their parents, were informed of the objective and characteristics of the tests and gave their written consent. Group 1. Twenty-four patients with dystrophinopathies comprised group 1. Included were all the patients with dystrophinopathy revised consecutively in the neuropediatric service between May 1995 and December 1996. They consisted of 16 patients with DMD (group 1a), aged 2-16 years; seven patients with BMD (group 1b), and one patient with elevated levels of creatine kinase (CK) and asymptomatic dystrophinopathy (AD) (group 1c). The diagnosis of muscular dystrophy was made in every patient by clinical examination, electromyography, and muscular enzyme testing (CK, aldolase). A muscle biopsy was performed in 18 patients (13 DMD and 5 BMD), and a molecular study in 22 (Table 1). Every DMD and BMD patient had a clinical examination, EMG, and CK levels compatible with their diagnosis. The three DMD patients without biopsy demonstrated gene deletions. The only two BMD patients without biopsy and without gene deletion were 5- and 13-year-old boys who manifested mild muscular weakness. Both were still able to walk, and both had a myopathic EMG and raised CK (levels greater than 20 times the maximum normal value) (Table 1). Group 2. Group 2 included two patients, 7- and 10-year-old girls with limb-girdle muscular dystrophy ‘‘Duchenne-like’’ (MD), one of them with adhaline deficiency. Group 3. Four DMD definitive carriers were in group 3; three were mothers of some patients in group 1. One had a son with DMD and a daughter with a raised CK level more than 20 times the maximum normal value; the daughter was the fourth patient in this group; another woman had two sons with DMD; and the third mother had a son with DMD and she exhibited a mild generalized weakness and raised CK level. Group 4. The control group included ten healthy patients without visual or muscular disorders, aged 9 months to 15 years, all of whom had normal visual evoked potentials. They were chosen from among patients studied by brainstem-evoked potentials or were healthy relatives of other patients without visual abnormalities or muscular disorders.
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ERG Procedure. All patients were studied with ERG recorded by skin electrodes placed in both lower eyelids. The stimulus was a standard flat flash placed 5 to 10 cm away from the eyes to stimulate the entire visual field. The light energy was of 0.4 J, controlled by Retinophot (Braintronic) and Sapphire Premiere, (Medelec-Vickers Medical, New York, NY) equipment, averaging at least 50 stimuli. Each study was repeated at least twice (two tests in both eyes). The patients’ pupils were pharmacologically dilated. Sedation with chloral hydrate was used when necessary. The types of stimuli were (1) scotopic conditions after 5 minutes of dark adaptation using a white bright light at 0.3 Hz, 50 stimuli, repeated twice and blue light flashes at 0.5 Hz, 50 stimuli, repeated twice (to stimulate mainly rods) and (2) photopic conditions, after 5 minutes of bright light adaptation, 30 Hz of white bright flashes (flicker ERG), repeated twice. Before our ERG study, every patient was studied with visual-evoked potentials (VEPs) with monocular and binocular patterns or goggles stimuli (if no cooperation possible) before pupil dilatation. From the VEP results, absolute latencies of N70, P100, and N150 and amplitudes of N70/P100 and P100/N150 were analyzed. From the ERG results, separate responses of the eyes in each test were analyzed; annotations were made of absolute latencies of A-wave and B-wave, A-wave amplitude (from onset to top of wave), B-wave amplitude (from top of A to bottom of B), and the B/A amplitude ratio calculated. Molecular Genetic Analysis. Genomic DNA was extracted from peripheral blood lymphocytes by the standard phenol/chloroform method. Identification of deletions at the dystrophin gene was carried out by Multiplex polymerase chain reaction amplification of two set of nine exons as described by Chamberlain et al. [13] and Beggs et al. [14], with some modifications, as follows: dimethylsulfoxid was omitted from the reaction mixture and the amplification program for the two sets of nine exons was the same at one cycle at 95°C for 5 minutes, 29 cycles of denaturation at 94°C for 30 seconds, annealing at 57°C for 45 seconds, and DNA extension at 65°C for 4 minutes. DNA fragments were electrophoresed on 1.5% agarose gels and stained with ethidium bromide. The statistical significance was calculated with mean value differences between samples (Student t test), and considered when P , 0.05.
Results None of the patients demonstrated visual disturbances, except for some mild hypermetropic, myopic, or astigmatic abnormalities. No one suffered partial or complete night blindness or other conditions that could explain the B-wave ERG abnormalities. VEPs were normal in every patient, without significant differences between eyes. Flicker ERGs were also
Table 1.
Patient ADA AGJ AGV BCJ DMJ EVD GLA GMI MFM MGJ MRA PGR
CJL GRF OPF USR CLR GGA MDE DSD GNS HBI LHI MRA PCD PZJ FMM GPC SMD BCM RPM MRM BCA
Patients studied
Diagnosis DMD DMD DMD DMD DMD DMD DMD DMD DMD DMD DMD DMD DMD DMD DMD DMD BMD BMD BMD BMD BMD-Down BMD BMD AD MD Adhaliopathic MD Normal Normal Normal Normal Normal Normal Normal Normal Normal Normal DMD-Carrier DMD-Carrier DMD-Carrier DMD-Carrier
Age (years)
Weakness
CK
EMG
6 16 4 3 5 7 4 12 16 15 5 11 12 9 2 12 13 11 13 12 5 5 21 9 7 10 8 8 0 11 1 0 10 30 8 1 30 38 39 6
Severe Severe Moderate Mild Moderate Severe Moderate Severe Severe Severe Moderate Severe Severe Severe Mild Severe Mild Mild Mild Mild Mild Mild Moderate No Moderate Moderate No No No No No No No No No No No No Mild No
. 310 . 310 . 310 . 310 . 310 . 310 . 310 . 310 . 310 . 310 . 310 . 310 . 310 . 310 . 310 . 310 . 310 . 310 . 310 . 310 . 310 . 310 . 310 . 310 . 310 . 310
Myopatic Myopatic Myopatic Myopatic Myopatic Myopatic Myopatic Myopatic Myopatic Myopatic Myopatic Myopatic Myopatic Myopatic Myopatic Myopatic Myopatic Myopatic Myopatic Myopatic Myopatic Myopatic Myopatic Normal Myopatic Myopatic
Normal 38 320
Muscular Biopsy Yes Yes No Yes Yes Yes Yes Yes Yes Yes Yes Yes No No Yes Yes No Yes Yes Yes No Yes Yes No Yes Yes No No No No No No No No No No No No No No
Delection
Ex 8-19 Ex 3-44 No Ex 48-51 No Ex 47-end Ex 51 No No Ex 45-48 Ex 17 Ex 17 Ex 45 No No No No No No No No Ex 48
Probable No Probable
ERG B/A Amplitude Ratio 0.73 1.04 0.68 0.22 0.65 0.98 0.79 0.85 1.01 0.65 0.45 0.50 0.44 0.40 1.02 1.26 1.12 1.13 1.37 3.81 5.56 0.88 1.12 3.08 184.00 5.50 5.38 13.30 4.75 11.90 3.49 3.96 8.07 2.66 2.66 5.42 1.15 1.51 22.30 4.89
Abbreviations: AD 5 Asymptomatic dystrophinopathy B/A 5 B-wave/A-wave BMD 5 Becker muscular dystrophy CK 5 Creatine kinase results in reference to the normal maximum value DMD 5 Duchenne muscular dystrophy Down 5 Down syndrome (21 trisomy) EMG 5 Electromyogram ERG 5 Electroretinogram MD 5 Muscular dystrophy nondystrophinopathic
normal. The scotopic white light ERG was, like in other reported studies, more useful than the blue light study in distinguishing among the groups. Results presented here are those of the scotopic white light studies (Tables 1-3). Latencies of the A- and B-waves were normal. Amplitudes of both waves were highly variable, without group differences. Amplitudes are smaller and less reliable when registered with eyelid electrodes than with corneal electrodes. However, the B/A amplitude ratio to white flash stimuli
was very different among the groups (Table 3 and Figs 1-2). None of the patients in group 4 (normal control) or group 2 (MD) had a B/A amplitude ratio of less than 2 in the average of the four tests performed (two tests for each eye). By contrast, every patient in group 1a (DMD) had a mean B/A amplitude ratio of less than 2. Only one of the 64 tests performed on the 16 patients had a B/A amplitude ratio of more than 2 (2,11). The mean ratio for each test and each eye of the 16 patients ranged from 0.68 to 0.82 (Table 3). These results were significantly different from
Pascual Pascual et al: ERG in Dystrophinopathies 317
Table 2.
ERG Results: latencies and amplitudes
Group
Latency A (ms) Mean S.D.
Test
Control (n 5 10)
LE, T1 LE, T2 RE, T1 RE, T2 LE, T1 LE, T2 RE, T1 RE, T2 LE, T1 LE, T2 RE, T1 RE, T2
Dystrophinopathies (n 5 24)
Nondystrophinopathic MD (n 5 2)
19.7 20 20.1 20.2 19.7 20.6 19.8 20.2 19.5 19.1 19.5 19.1
2.3 3.2 2.6 3.1 5.3 6.5 5.1 5.6 4.3 4.3 4.3 4.3
Latency B (ms) Mean S.D. 43.8 44.4 43.6 43.8 41.9 41.5 41.1 41.1 47.8 47.3 48.2 45.9
3 4 4.7 4.2 10.4 9.7 9.2 9.2 5.7 5.7 6.3 6.3
Amplitude A (uV) Mean S.D.
Amplitude B (uV) Mean S.D.
8.9 9.5 9 9.1 23.8 23 24.7 22.8 0.1-37 5-25 0.3-5.4 3-17
30.6 31.5 27.6 28.3 24.6 24.7 24.3 24.3 54-73 55-64 50-74 57-65
8.8 8.6 12.9 13 19.2 18.2 18.7 18.9
15.5 16.5 13.8 15 16.7 16.7 17 17.3
Abbreviations: ERG 5 Electroretinogram LE 5 Left eye MD 5 Muscular dystrophy RE 5 Right eye T1, T2 5 Test 1, test 2
Table 3.
B/A Amplitude ratio in ERG
Group
B/A Amplitude Ratio Ratio <1 Max. (%)
Ratio <1.5 (%)
Ratio >2 (%)
Test
Mean
S.D.
Min.
Control (n 5 10)
LE, T1 LE, T2 RE, T1 RE, T2 Average
7 5.3 7.8 4.6 6.2
7 5.6 9.5 2.6 3.6
1.5 1.7 2.7 1.5 2.7
12 21.5 34 11 13.3
0 0 0 0 0
0 0 0 0 0
80 90 100 90 100
DMD (n 5 16)
LE, T1 LE, T2 RE, T1 RE, T2 Average
0.7 0.7 0.75 0.82 0.73
0.3 0.3 0.5 0.4 0.3
0.2 0.2 0.1 0.3 0.2
1.3 1.7 2.1 1.6 1.3
81 88 81 75 75
100 94 94 100 100
0 0 6 0 0
BMD (n 5 7)*
LE, T1 LE, T2 RE, T1 RE, T2 Average
1.05 1.23 0.95 1.26 1.12
0.4 0.3 0.2 0.2 0.3
0.8 0.9 0.7 1 0.9
1.8 1.6 1.3 1.4 1.4
57 28 43 0 0
57 57 71 57 71
29 29 29 29 29
Nondystrophinopathic MD (n 5 2)
LE, T1 LE, T2 RE, T1 RE, T2
2 2.5 13.6 3.8
542 9.5 169 17.7
0 0 0 0
0 0 0 0
100 100 100 100
272 6 91 10.7
Statistical Significance vs DMD vs BMD* (P) (P) ,0.05 ,0.1 NS ,0.1 NS ,0.01 ,0.001
,0.05 ,0.1 NS ,0.1 NS ,0.05 ,0.001 ,0.05 ,0.01 NS ,0.01 ,0.001
* In the BMD group there are two subgroups, one (n 5 5) with a low B/A amplitude ratio. The table data of mean, S.D., minimum and maximum values, and statistical significance refer to these five patients. The data about proportion (%) of cases with ratios of less than 1, less than 1.5, and more than 2 refer to the whole group of seven patients. Abbreviations: Average 5 Average of the four tests of each patient B/A 5 B-wave/A-wave BMD 5 Becker muscular dystrophy DMD 5 Duchenne muscular dystrophy LE 5 Left eye MD 5 Muscular dystrophy Max. 5 Maximum Min. 5 Minimum RE 5 Right eye T1, T2 5 Test 1, test 2
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Figure 2. (A-F) ERGs of four children with Duchenne muscular dystrophy. The latencies and absolute amplitudes of A- and B-waves are normal, but the B/A amplitude ratio is less than 1 (ERG negative) (A-C, F). ERG from a patient with Becker muscular dystrophy. The B/A amplitude ratio is 1.12 (D). Normal ERG from a boy with asymptomatic dystrophinopathy (E). O.D. 5 right eye; O.I. 5 left eye.
those of the control group (P , 0.001). No differences between patients with and without deletion were seen nor between patients with first exons deletion and patients with a deletion in the middle part of the gene (Table 1). In group 1b (BMD), five patients (71%) had a B/A amplitude ratio of less than 2 in all four tests. The mean B/A amplitude ratios of the four tests of these five patients were between 0.88 and 1.37 (Table 3), values that are between those of the DMD group and normal control group, and significantly different from both of them (P , 0.001). The other two BMD patients (29%) had normal B/A amplitude ratios, similar to the control group (Table 1). There were no differences in muscular weakness, onset or progression of symptoms, and serum CK or aldolase levels between these two patients and the five BMD patients with a low B/A amplitude ratio. The only patient with asymptomatic dystrophinopathy, carrier of a deletion of exon 48, had a normal B/A amplitude ratio (Table 1). In group 3 the DMD definitive carriers, half of them had a low B/A amplitude ratio (mean 1.15 and 1.5), with results between 0.92 and 1.73 in individual tests, whereas the other two had normal B/A amplitude ratios with mean values of 22.5 and 4.9, respectively (one of them manifested muscle weakness, and the other was the daughter of a DMD carrier who had a low B/A amplitude ratio).
Discussion Other diseases with abnormalities of the B-wave and negative ERGs have been reported: X-linked or autosomal-recessive congenital stationary night blindness, Aland Island eye disease, the enhanced S-cone syndrome, Goldmann-Favre syndrome, a special type of retinitis pigmentosa, carriers of some types of retinitis pigmentosa, cone dystrophy, optic neuropathies, diabetic retinopathy, other retinal vascular disorders, and toxic retinopathies (quinine, vincristine, trimetoprim-sulfamethoxazol, carbamazepine, digoxine, and corticoids) [5]. ERG abnormalities of dystrophinopathies, although similar to these disorders, are asymptomatic. Our study confirms the existence of asymptomatic retinal dysfunction in dystrophinopathies and that this dysfunction can be well registered with eyelid electrode scotopic ERG after stimulation with a standard flash, conditions that are simpler and easier than corneal electrodes and full-field Ganzfeld stimulation. Usually the abnormality does not affect the latencies or absolute amplitudes of the A- and B-waves (amplitudes are greatly variable with this method), but it does affect the B/A amplitude ratio. Although this is an open study, it is composed of all the patients with dystrophinopathies consecutively monitored in our neuropediatric service,
Pascual Pascual et al: ERG in Dystrophinopathies 319
without bias, and demonstrates that the ERG abnormality is constant in DMD, in which the mean B/A amplitude ratio (average of the four tests of both eyes) is always lower than 1.5, and even that in 75% of patients is lower than 1 (negative ERG). Normal patients and patients with nondystrophinopathy muscular dystrophies obtain B/A amplitude ratios greater than 2. In previous reports, normal values of B/A amplitude ratios are also greater than 2 [11,12]. Of the patients with BMD, 71% demonstrated ERG abnormalities, with B/A amplitude ratio of less than 1.5 (mean 1.12, range 0.88-1.37), which is significantly superior to those of DMD. The other 29%, and also the only patient with asymptomatic dystrophinopathy, had normal ratios. There were no clinical differences among BMD patients with normal and low ratios. Half of the DMD definitive carriers have intermediate ratio values between normal and the ratios of DMD patients, and the other half have normal ratios. These results indicate the existence of a parallelism of dystrophin deficit in muscle and retinal dysfunction. Other authors have found a relation between ERG abnormalities and gene deletion (more frequent ERG abnormalities in patients with deletion in the middle part of the gene) [5,6], although the number of patients in each group is low. Another study [7] does not confirm this relation but finds more frequent abnormalities among the patients with a deletion than among those without deletion. Fitzgerald et al. [4] did not find an association between severity of muscle weakness and ERG abnormalities. On the contrary, our study, and the one recently published by Girlanda et al. [15] do not find any relation between ERG abnormalities and gene deletion or their site, but they do demonstrate a clear association with muscular phenotype. We conclude that our study confirms that this method of ERG examination is very sensible for detection of ERG abnormalities associated with DMD/BMD and thus is preferable to the conventional corneal electrode and fullfield Ganzfeld stimulation because of its simplicity and safety in children.
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References [1] Ahn AH, Kunkel LM. The structural and functional diversity of dystrophin. Nat Genet 1993;3:283-91. [2] Pillers DM, Bulman DE, Weleber RG. Dystrophin expression in the human retina is required for normal function as defined by electroretinography. Nat Genet 1993;4:82-6. [3] Miyatake M, Miike T, Zhao J, Yoshioka K, Uchino M, Usuku G. Dystrophin localization and presumed function. Muscle Nerve 1991;14: 113-9. [4] Fitzgerald KM, Cibis GW, Giambrone SA, Harris D. Retinal signal transmission in Duchenne muscular dystrophy: Evidence for dysfunction in the photoreceptor/depolarizing bipolar cell pathway. J Clin Invest 1994;93:2425-30. [5] Jensen H, Warburg M, Sjo¨ O, Schwartz M. Duchenne muscular dystrophy: Negative electroretinograms and normal dark adaptation. Reappraisal of assignment of X linked incomplete congenital stationary night blindness. J Med Genet 1995;32:348-51. [6] Sigesmund DA, Weleber RG, Pillers DM, et al. Characterization of the ocular phenotype of Duchenne and Becker muscular dystrophy. Ophthalmology 1994;101:856-65. [7] Becker ID, Riddell DC, Dooley JM, Tremblay F. Correlation between electroretinogram findings and molecular analysis in the Duchenne muscular dystrophy phenotype. Br J Ophthalmol 1994;78:71922. [8] Tremblay F, De Becker I, Dooley JM, Riddell DC. Duchenne muscular dystrophy: Negative scotopic bright-flash electroretinogram but not congenital stationary night blindness. Can J Ophthalmol 1994; 29:274-9. [9] Tremblay F, De Becker I, Riddell DC, Dooley JM: Duchenne muscular dystrophy: Negative scotopic bright-flash electroretinogram and normal dark adaptation. Can J Ophthalmol 1994;29:280-3. [10] International Standardization Committee. Standard for clinical electroretinography. Arch Ophthalmol 1989;107:816-9 [11] Kriss A, Jeffrey B, Taylor D. The electroretinogram in infants and young children. J Clin Neurophysiol 1992;9:373-93. [12] Rodriguez Saez E, Otero Costas J, Moreno Montan˜es J, Relova JL. Electroretinographic changes during childhood and adolescence. Eur J Ophthalmol 1993;3:6-12. [13] Chamberlain JS, Gibbs RA, Ranier JE, Caskey CT. Multiplex PCR for the diagnosis of Duchenne muscular dystrophy. In: Innis M, Gelfand D, Sninsky JJ, White T, ed. PCR protocols: A guide to methods and applications. San Diego: Academic Press 1989:272-81. [14] Beggs AH, Koening M, Boyce FM, Kunkel LM. Detection of 98% of DMD/BMD deletions by PCR. Hum Genet 1990;86:45-8. [15] Girlanda P, Quartarone A, Buceti R, et al. Extra-muscle involvement in dystrophynopathies: An electroretinography and evoked potential study. J Neurol Sci 1997;146:127-32.
Introduction Dystrophin, a protein of 427 kd codified by the gene of Duchenne/Becker muscular dystrophy (DMD/BMD), has been located in its full transcription not only in striated
From the *Service of Neuropediatrics; †Molecular Genetics Unit; Service of Biochemistry; Hospital Infantil ‘‘La Paz’’; Madrid, Spain.
© 1998 by Elsevier Science Inc. All rights reserved. PII S0887-8994(97)00208-7 ● 0887-8994/98/$19.00
muscle but also in cardiac and smooth muscle, in cerebral tissue, and in the outer plexiform layer of the retina [1-3]. Partial transcriptions of the gene, of the carboxy terminus, have also been found in glial and Schwann cells and in kidney, liver, pancreas, and testis [1]. The role of dystrophin in the retina is not known. It has been located in the synaptic complex of the photoreceptorbipolar cell [2]. This complex, and Mu¨ller cells, are the structures implicated in the origin of the B-wave of the electroretinogram (ERG). Dystrophin can give stability to the cell membranes or affect the function of the ionic channels [2,4]. The normal ERG (Fig 1) is composed of two waves, the A-wave with negative polarity resulting from a hyperpolarity of photoreceptors and the larger B-wave, with positive polarity produced by function of bipolar and Mu¨ller cells [4,5]. In DMD/BMD the scotopic ERG is often altered. A B-wave with an amplitude less than normal or even absent can be recorded [4,6-9] with more impairment of rod function seen than cone function. The more altered item is the amplitude ratio between the B-wave and A-wave (B/A amplitude ratio), produced by stimuli of a bright white light after adaptation to darkness. Normally the B/A amplitude ratio is greater than 2. In DMD/BMD, often it is less than 1 (termed ERG negative). This abnormality is asymptomatic; patients do not complain of nictalopia or other visual disturbances, unlike patients who have other visual diseases with similar ERG abnormalities [4,5,9]. The ERG has been found abnormal in 50% [7], 62.5% [5], or 88% [6] of DMD/BMD patients using the usual methodology [10] of corneal electrodes and full-field Ganzfeld stimulation. Nevertheless, these technical conditions are not easily available and the lack of cooperation from children makes the exploration difficult without general anesthesia. In children the ERG can be recorded with reliable results using eyelid electrodes [11,12], although ampli-
Communications should be addressed to: Dr. Pascual Pascual: Servicio de Neuropediatrı´a; Hospital Infantil ‘‘La Paz’’; Po Castellana, 261; 28046-Madrid, Spain. Received July 2, 1997; accepted September 29, 1997.
Pascual Pascual et al: ERG in Dystrophinopathies 315
Figure 1. (A, B) ERGs of two normal patients. The amplitude of the B-wave is higher than the A-wave. ERGs in all figures were performed with a bright-white light flash. O.D. 5 right eye; O.I. 5 left eye.
tudes of the waves are not comparable with those recorded with corneal electrodes. This report tries to confirm that the abnormalities of the B-wave in DMD/BMD can be detected using eyelid electrodes and with simple flash stimulation and then describes the characteristic ERG responses in comparison with those of normal control patients, patients with nondystrophinopathic muscular dystrophies, and female carriers of DMD dystrophy. Material and Methods Four different groups of patients were studied (Table 1). All the patients studied, or their parents, were informed of the objective and characteristics of the tests and gave their written consent. Group 1. Twenty-four patients with dystrophinopathies comprised group 1. Included were all the patients with dystrophinopathy revised consecutively in the neuropediatric service between May 1995 and December 1996. They consisted of 16 patients with DMD (group 1a), aged 2-16 years; seven patients with BMD (group 1b), and one patient with elevated levels of creatine kinase (CK) and asymptomatic dystrophinopathy (AD) (group 1c). The diagnosis of muscular dystrophy was made in every patient by clinical examination, electromyography, and muscular enzyme testing (CK, aldolase). A muscle biopsy was performed in 18 patients (13 DMD and 5 BMD), and a molecular study in 22 (Table 1). Every DMD and BMD patient had a clinical examination, EMG, and CK levels compatible with their diagnosis. The three DMD patients without biopsy demonstrated gene deletions. The only two BMD patients without biopsy and without gene deletion were 5- and 13-year-old boys who manifested mild muscular weakness. Both were still able to walk, and both had a myopathic EMG and raised CK (levels greater than 20 times the maximum normal value) (Table 1). Group 2. Group 2 included two patients, 7- and 10-year-old girls with limb-girdle muscular dystrophy ‘‘Duchenne-like’’ (MD), one of them with adhaline deficiency. Group 3. Four DMD definitive carriers were in group 3; three were mothers of some patients in group 1. One had a son with DMD and a daughter with a raised CK level more than 20 times the maximum normal value; the daughter was the fourth patient in this group; another woman had two sons with DMD; and the third mother had a son with DMD and she exhibited a mild generalized weakness and raised CK level. Group 4. The control group included ten healthy patients without visual or muscular disorders, aged 9 months to 15 years, all of whom had normal visual evoked potentials. They were chosen from among patients studied by brainstem-evoked potentials or were healthy relatives of other patients without visual abnormalities or muscular disorders.
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ERG Procedure. All patients were studied with ERG recorded by skin electrodes placed in both lower eyelids. The stimulus was a standard flat flash placed 5 to 10 cm away from the eyes to stimulate the entire visual field. The light energy was of 0.4 J, controlled by Retinophot (Braintronic) and Sapphire Premiere, (Medelec-Vickers Medical, New York, NY) equipment, averaging at least 50 stimuli. Each study was repeated at least twice (two tests in both eyes). The patients’ pupils were pharmacologically dilated. Sedation with chloral hydrate was used when necessary. The types of stimuli were (1) scotopic conditions after 5 minutes of dark adaptation using a white bright light at 0.3 Hz, 50 stimuli, repeated twice and blue light flashes at 0.5 Hz, 50 stimuli, repeated twice (to stimulate mainly rods) and (2) photopic conditions, after 5 minutes of bright light adaptation, 30 Hz of white bright flashes (flicker ERG), repeated twice. Before our ERG study, every patient was studied with visual-evoked potentials (VEPs) with monocular and binocular patterns or goggles stimuli (if no cooperation possible) before pupil dilatation. From the VEP results, absolute latencies of N70, P100, and N150 and amplitudes of N70/P100 and P100/N150 were analyzed. From the ERG results, separate responses of the eyes in each test were analyzed; annotations were made of absolute latencies of A-wave and B-wave, A-wave amplitude (from onset to top of wave), B-wave amplitude (from top of A to bottom of B), and the B/A amplitude ratio calculated. Molecular Genetic Analysis. Genomic DNA was extracted from peripheral blood lymphocytes by the standard phenol/chloroform method. Identification of deletions at the dystrophin gene was carried out by Multiplex polymerase chain reaction amplification of two set of nine exons as described by Chamberlain et al. [13] and Beggs et al. [14], with some modifications, as follows: dimethylsulfoxid was omitted from the reaction mixture and the amplification program for the two sets of nine exons was the same at one cycle at 95°C for 5 minutes, 29 cycles of denaturation at 94°C for 30 seconds, annealing at 57°C for 45 seconds, and DNA extension at 65°C for 4 minutes. DNA fragments were electrophoresed on 1.5% agarose gels and stained with ethidium bromide. The statistical significance was calculated with mean value differences between samples (Student t test), and considered when P , 0.05.
Results None of the patients demonstrated visual disturbances, except for some mild hypermetropic, myopic, or astigmatic abnormalities. No one suffered partial or complete night blindness or other conditions that could explain the B-wave ERG abnormalities. VEPs were normal in every patient, without significant differences between eyes. Flicker ERGs were also
Table 1.
Patient ADA AGJ AGV BCJ DMJ EVD GLA GMI MFM MGJ MRA PGR
CJL GRF OPF USR CLR GGA MDE DSD GNS HBI LHI MRA PCD PZJ FMM GPC SMD BCM RPM MRM BCA
Patients studied
Diagnosis DMD DMD DMD DMD DMD DMD DMD DMD DMD DMD DMD DMD DMD DMD DMD DMD BMD BMD BMD BMD BMD-Down BMD BMD AD MD Adhaliopathic MD Normal Normal Normal Normal Normal Normal Normal Normal Normal Normal DMD-Carrier DMD-Carrier DMD-Carrier DMD-Carrier
Age (years)
Weakness
CK
EMG
6 16 4 3 5 7 4 12 16 15 5 11 12 9 2 12 13 11 13 12 5 5 21 9 7 10 8 8 0 11 1 0 10 30 8 1 30 38 39 6
Severe Severe Moderate Mild Moderate Severe Moderate Severe Severe Severe Moderate Severe Severe Severe Mild Severe Mild Mild Mild Mild Mild Mild Moderate No Moderate Moderate No No No No No No No No No No No No Mild No
. 310 . 310 . 310 . 310 . 310 . 310 . 310 . 310 . 310 . 310 . 310 . 310 . 310 . 310 . 310 . 310 . 310 . 310 . 310 . 310 . 310 . 310 . 310 . 310 . 310 . 310
Myopatic Myopatic Myopatic Myopatic Myopatic Myopatic Myopatic Myopatic Myopatic Myopatic Myopatic Myopatic Myopatic Myopatic Myopatic Myopatic Myopatic Myopatic Myopatic Myopatic Myopatic Myopatic Myopatic Normal Myopatic Myopatic
Normal 38 320
Muscular Biopsy Yes Yes No Yes Yes Yes Yes Yes Yes Yes Yes Yes No No Yes Yes No Yes Yes Yes No Yes Yes No Yes Yes No No No No No No No No No No No No No No
Delection
Ex 8-19 Ex 3-44 No Ex 48-51 No Ex 47-end Ex 51 No No Ex 45-48 Ex 17 Ex 17 Ex 45 No No No No No No No No Ex 48
Probable No Probable
ERG B/A Amplitude Ratio 0.73 1.04 0.68 0.22 0.65 0.98 0.79 0.85 1.01 0.65 0.45 0.50 0.44 0.40 1.02 1.26 1.12 1.13 1.37 3.81 5.56 0.88 1.12 3.08 184.00 5.50 5.38 13.30 4.75 11.90 3.49 3.96 8.07 2.66 2.66 5.42 1.15 1.51 22.30 4.89
Abbreviations: AD 5 Asymptomatic dystrophinopathy B/A 5 B-wave/A-wave BMD 5 Becker muscular dystrophy CK 5 Creatine kinase results in reference to the normal maximum value DMD 5 Duchenne muscular dystrophy Down 5 Down syndrome (21 trisomy) EMG 5 Electromyogram ERG 5 Electroretinogram MD 5 Muscular dystrophy nondystrophinopathic
normal. The scotopic white light ERG was, like in other reported studies, more useful than the blue light study in distinguishing among the groups. Results presented here are those of the scotopic white light studies (Tables 1-3). Latencies of the A- and B-waves were normal. Amplitudes of both waves were highly variable, without group differences. Amplitudes are smaller and less reliable when registered with eyelid electrodes than with corneal electrodes. However, the B/A amplitude ratio to white flash stimuli
was very different among the groups (Table 3 and Figs 1-2). None of the patients in group 4 (normal control) or group 2 (MD) had a B/A amplitude ratio of less than 2 in the average of the four tests performed (two tests for each eye). By contrast, every patient in group 1a (DMD) had a mean B/A amplitude ratio of less than 2. Only one of the 64 tests performed on the 16 patients had a B/A amplitude ratio of more than 2 (2,11). The mean ratio for each test and each eye of the 16 patients ranged from 0.68 to 0.82 (Table 3). These results were significantly different from
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Table 2.
ERG Results: latencies and amplitudes
Group
Latency A (ms) Mean S.D.
Test
Control (n 5 10)
LE, T1 LE, T2 RE, T1 RE, T2 LE, T1 LE, T2 RE, T1 RE, T2 LE, T1 LE, T2 RE, T1 RE, T2
Dystrophinopathies (n 5 24)
Nondystrophinopathic MD (n 5 2)
19.7 20 20.1 20.2 19.7 20.6 19.8 20.2 19.5 19.1 19.5 19.1
2.3 3.2 2.6 3.1 5.3 6.5 5.1 5.6 4.3 4.3 4.3 4.3
Latency B (ms) Mean S.D. 43.8 44.4 43.6 43.8 41.9 41.5 41.1 41.1 47.8 47.3 48.2 45.9
3 4 4.7 4.2 10.4 9.7 9.2 9.2 5.7 5.7 6.3 6.3
Amplitude A (uV) Mean S.D.
Amplitude B (uV) Mean S.D.
8.9 9.5 9 9.1 23.8 23 24.7 22.8 0.1-37 5-25 0.3-5.4 3-17
30.6 31.5 27.6 28.3 24.6 24.7 24.3 24.3 54-73 55-64 50-74 57-65
8.8 8.6 12.9 13 19.2 18.2 18.7 18.9
15.5 16.5 13.8 15 16.7 16.7 17 17.3
Abbreviations: ERG 5 Electroretinogram LE 5 Left eye MD 5 Muscular dystrophy RE 5 Right eye T1, T2 5 Test 1, test 2
Table 3.
B/A Amplitude ratio in ERG
Group
B/A Amplitude Ratio Ratio <1 Max. (%)
Ratio <1.5 (%)
Ratio >2 (%)
Test
Mean
S.D.
Min.
Control (n 5 10)
LE, T1 LE, T2 RE, T1 RE, T2 Average
7 5.3 7.8 4.6 6.2
7 5.6 9.5 2.6 3.6
1.5 1.7 2.7 1.5 2.7
12 21.5 34 11 13.3
0 0 0 0 0
0 0 0 0 0
80 90 100 90 100
DMD (n 5 16)
LE, T1 LE, T2 RE, T1 RE, T2 Average
0.7 0.7 0.75 0.82 0.73
0.3 0.3 0.5 0.4 0.3
0.2 0.2 0.1 0.3 0.2
1.3 1.7 2.1 1.6 1.3
81 88 81 75 75
100 94 94 100 100
0 0 6 0 0
BMD (n 5 7)*
LE, T1 LE, T2 RE, T1 RE, T2 Average
1.05 1.23 0.95 1.26 1.12
0.4 0.3 0.2 0.2 0.3
0.8 0.9 0.7 1 0.9
1.8 1.6 1.3 1.4 1.4
57 28 43 0 0
57 57 71 57 71
29 29 29 29 29
Nondystrophinopathic MD (n 5 2)
LE, T1 LE, T2 RE, T1 RE, T2
2 2.5 13.6 3.8
542 9.5 169 17.7
0 0 0 0
0 0 0 0
100 100 100 100
272 6 91 10.7
Statistical Significance vs DMD vs BMD* (P) (P) ,0.05 ,0.1 NS ,0.1 NS ,0.01 ,0.001
,0.05 ,0.1 NS ,0.1 NS ,0.05 ,0.001 ,0.05 ,0.01 NS ,0.01 ,0.001
* In the BMD group there are two subgroups, one (n 5 5) with a low B/A amplitude ratio. The table data of mean, S.D., minimum and maximum values, and statistical significance refer to these five patients. The data about proportion (%) of cases with ratios of less than 1, less than 1.5, and more than 2 refer to the whole group of seven patients. Abbreviations: Average 5 Average of the four tests of each patient B/A 5 B-wave/A-wave BMD 5 Becker muscular dystrophy DMD 5 Duchenne muscular dystrophy LE 5 Left eye MD 5 Muscular dystrophy Max. 5 Maximum Min. 5 Minimum RE 5 Right eye T1, T2 5 Test 1, test 2
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Figure 2. (A-F) ERGs of four children with Duchenne muscular dystrophy. The latencies and absolute amplitudes of A- and B-waves are normal, but the B/A amplitude ratio is less than 1 (ERG negative) (A-C, F). ERG from a patient with Becker muscular dystrophy. The B/A amplitude ratio is 1.12 (D). Normal ERG from a boy with asymptomatic dystrophinopathy (E). O.D. 5 right eye; O.I. 5 left eye.
those of the control group (P , 0.001). No differences between patients with and without deletion were seen nor between patients with first exons deletion and patients with a deletion in the middle part of the gene (Table 1). In group 1b (BMD), five patients (71%) had a B/A amplitude ratio of less than 2 in all four tests. The mean B/A amplitude ratios of the four tests of these five patients were between 0.88 and 1.37 (Table 3), values that are between those of the DMD group and normal control group, and significantly different from both of them (P , 0.001). The other two BMD patients (29%) had normal B/A amplitude ratios, similar to the control group (Table 1). There were no differences in muscular weakness, onset or progression of symptoms, and serum CK or aldolase levels between these two patients and the five BMD patients with a low B/A amplitude ratio. The only patient with asymptomatic dystrophinopathy, carrier of a deletion of exon 48, had a normal B/A amplitude ratio (Table 1). In group 3 the DMD definitive carriers, half of them had a low B/A amplitude ratio (mean 1.15 and 1.5), with results between 0.92 and 1.73 in individual tests, whereas the other two had normal B/A amplitude ratios with mean values of 22.5 and 4.9, respectively (one of them manifested muscle weakness, and the other was the daughter of a DMD carrier who had a low B/A amplitude ratio).
Discussion Other diseases with abnormalities of the B-wave and negative ERGs have been reported: X-linked or autosomal-recessive congenital stationary night blindness, Aland Island eye disease, the enhanced S-cone syndrome, Goldmann-Favre syndrome, a special type of retinitis pigmentosa, carriers of some types of retinitis pigmentosa, cone dystrophy, optic neuropathies, diabetic retinopathy, other retinal vascular disorders, and toxic retinopathies (quinine, vincristine, trimetoprim-sulfamethoxazol, carbamazepine, digoxine, and corticoids) [5]. ERG abnormalities of dystrophinopathies, although similar to these disorders, are asymptomatic. Our study confirms the existence of asymptomatic retinal dysfunction in dystrophinopathies and that this dysfunction can be well registered with eyelid electrode scotopic ERG after stimulation with a standard flash, conditions that are simpler and easier than corneal electrodes and full-field Ganzfeld stimulation. Usually the abnormality does not affect the latencies or absolute amplitudes of the A- and B-waves (amplitudes are greatly variable with this method), but it does affect the B/A amplitude ratio. Although this is an open study, it is composed of all the patients with dystrophinopathies consecutively monitored in our neuropediatric service,
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without bias, and demonstrates that the ERG abnormality is constant in DMD, in which the mean B/A amplitude ratio (average of the four tests of both eyes) is always lower than 1.5, and even that in 75% of patients is lower than 1 (negative ERG). Normal patients and patients with nondystrophinopathy muscular dystrophies obtain B/A amplitude ratios greater than 2. In previous reports, normal values of B/A amplitude ratios are also greater than 2 [11,12]. Of the patients with BMD, 71% demonstrated ERG abnormalities, with B/A amplitude ratio of less than 1.5 (mean 1.12, range 0.88-1.37), which is significantly superior to those of DMD. The other 29%, and also the only patient with asymptomatic dystrophinopathy, had normal ratios. There were no clinical differences among BMD patients with normal and low ratios. Half of the DMD definitive carriers have intermediate ratio values between normal and the ratios of DMD patients, and the other half have normal ratios. These results indicate the existence of a parallelism of dystrophin deficit in muscle and retinal dysfunction. Other authors have found a relation between ERG abnormalities and gene deletion (more frequent ERG abnormalities in patients with deletion in the middle part of the gene) [5,6], although the number of patients in each group is low. Another study [7] does not confirm this relation but finds more frequent abnormalities among the patients with a deletion than among those without deletion. Fitzgerald et al. [4] did not find an association between severity of muscle weakness and ERG abnormalities. On the contrary, our study, and the one recently published by Girlanda et al. [15] do not find any relation between ERG abnormalities and gene deletion or their site, but they do demonstrate a clear association with muscular phenotype. We conclude that our study confirms that this method of ERG examination is very sensible for detection of ERG abnormalities associated with DMD/BMD and thus is preferable to the conventional corneal electrode and fullfield Ganzfeld stimulation because of its simplicity and safety in children.
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