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Becker muscular dystrophy families – Detection of carrier status in symptomatic and asymptomatic female relatives
Neuromuscular Disorders 19 (2009) 108–112
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Neuromuscular Disorders journal homepage: www.elsevier.com/locate/nmd
Case report
Dystrophin gene analysis in Hungarian Duchenne/Becker muscular dystrophy families – Detection of carrier status in symptomatic and asymptomatic female relatives Henriett Pikó a, Viktor Vancsó a,b, Bálint Nagy b, Zoltán Bán b, Ágnes Herczegfalvi c, Veronika Karcagi a,* a b c
National Institute of Environmental Health, Department of Molecular Genetics and Diagnostics, Gyali út 2-6, H-1097 Budapest, Hungary Semmelweis Medical School, Clinic of Obstetrics and Gynaecology, Laboratory of Genetics, Budapest, Hungary Bethesda Children’s Hospital, Department of Neurology, Budapest, Hungary
a r t i c l e
i n f o
Article history: Received 28 July 2008 Received in revised form 20 October 2008 Accepted 29 October 2008
Keywords: Duchenne/Becker muscular dystrophy MLPA analysis Carrier detection Manifesting carriers
a b s t r a c t A comprehensive study of the Hungarian Duchenne/Becker muscular dystrophy (DMD/BMD) families is presented. Deletions in the hot spots regions were identified by multiplex PCR, whereas rare mutations were detected by Southern blot and multiplex ligation-dependent probe amplification (MLPA) techniques. DMD/BMD disease was confirmed and exact deletion borders were determined in 19 out of 135 affected males using multiplex PCR. Additional exons involved as well as rare exon deletions were identified by MLPA in 71 male patients, whereas duplications were observed in seven patients. In two DMD patients, the entire dystrophin gene and adjacent genes were deleted. Out of the 95 female relatives, 41 proved to be carriers, including three manifesting carrier females. Using MLPA method, a large portion of the Hungarian DMD/BMD patients and their female relatives were exactly genotyped. For the first time, the incidence and prevalence of asymptomatic and symptomatic female carriers in Hungary was estimated. Ó 2008 Elsevier B.V. All rights reserved.
1. Introduction Muscular dystrophies are a genetically heterogeneous group of degenerative muscle disorders, characterized by the progressive loss of muscle strength and integrity. There is marked clinical similarity among patients with genetically different diseases, suggesting that many of the gene products may participate in a common functional structure or biochemical cascade [1]. Duchenne/Becker muscular dystrophy (DMD/BMD) is a lethal/severe X-linked recessive disorder affecting 1 in 3500 (DMD) and 1 in 30,000 (BMD) male births [2]. It is caused by mutations in the dystrophin gene located on Xp21.2, which encodes a protein of the membrane cytoskeleton in skeletal muscle (dystrophin) [3–5]. Mutations responsible for the disease are deletions or duplications encompassing the 79 exons of the gene, which account for 60–65% and 5–10% of cases of DMD/BMD, respectively [5,6]; while the remaining cases, about 30–35%, are caused by single point mutations (nonsense or splice sites) or small rearrangements [2,7–10]. In BMD, the proportion of large deletions and duplications is higher, 85% [10]. Approximately one-third of the DMD/BMD patients originate through new mutations, while the rest are inherited through carrier mothers or arise from germ line mosaicism [11,12]. The main
* Corresponding author. Fax: +36 2150148. E-mail address: [email protected] (V. Karcagi). 0960-8966/$ - see front matter Ó 2008 Elsevier B.V. All rights reserved. doi:10.1016/j.nmd.2008.10.011
problem of the molecular genetic analysis of the DMD/BMD disease is the size and the wide mutation spectrum of the dystrophin gene. Although there are two hot spot regions, wherein the mutation frequencies are higher than in the other region, the relatively frequent point mutations can occur anywhere in the gene. Further complicating factor of the DMD/BMD mutation analysis is the presence of germ line and somatic mosaicism [12,13]. Females with muscular dystrophy symptoms are often considered as limb-girdle muscular dystrophy patients, although the frequency of DMD/BMD manifesting carriers might be higher as previously suggested [14,15]. There are few population data on the prevalence of carriers, especially on symptomatic carriers. In this study we compared the newly introduced MLPA technique to the formerly used quantitative Southern blot assay in female individuals who were assumed as possible carriers. On the other hand, MLPA was used as quantitative assessment of all 79 exons, in order to identify exact deletion borders and eventual duplication events in affected males. Here we report the genetic results of 135 affected males, three manifesting carrier women and 92 female relatives using different diagnostic approaches for DMD/BMD disease. 2. Materials and methods Patients affected with DMD/BMD were assessed in our laboratory between 2000 and 2008. There is no overlap with a previous
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study published in 1999 (31). In all of them the diagnosis was established through clinical and neurological examinations including family history, serum creatine kinase (CK) analysis, and where available, through histopathology of the muscle biopsy. Genomic DNA was extracted from peripheral blood leukocytes (Promega Wizard genomic DNA purification Kit, Promega Corporation, USA). DNA analyses included screening of deletions by multiplex PCR and detection of deletions/duplications borders using MLPA technique in the index case. Female relatives were tested for their carrier status using cDNA probes and MLPA analysis.
3. Results 3.1. Molecular diagnosis of index cases During the period of 2000–2008 March, 135 unrelated DMD and BMD Hungarian patients were sent to our laboratory for genetic confirmation of the disease. They were classified as non-ambulatory and ambulatory by the clinicians. Molecular analysis of the patients’ dystrophin gene was performed by multiplex PCR reactions, Southern blot analysis and MLPA technique. Results are summarised in Table 1.
2.1. Multiplex PCR 3.2. Carrier detection Two sets of PCR primers were used which cover 17 exons and the muscle specific promoter in the deletion hot spots. PCR and agarose gel electrophoresis was performed by standard protocols [16,17]. 2.2. Southern blot and cDNA probes Genomic DNA was digested with HindIII and BglII restriction enzymes, separately. Electrophoresis and membrane transfer was performed according to standard techniques. [18]. After blotting, the membrane was hybridized with a 32P-dCTP labelled cDNA probe (XJ10, 30-2, 30-1, 47-4, 7b8, or 63-1), using the random priming procedure [8,18–21]. For data evaluation, the Packard Instant Imager was applied to quantify the relative intensities of the signals. Intensities were corrected and calculated according to standard protocols [22–24]. DNA samples of female relatives were tested together with their affected offspring’s DNA, if available and fragment densities of the different exons were also compared to normal controls. 2.3. Multiple ligation-dependent probe amplification (MLPA) The MLPA DMD/BMD test kit (SALSA P034/035) was obtained from MRC-Holland, Amsterdam, The Netherlands. Analysis was performed according to the manufacturer‘s recommendations [25–27]. PCR products were run on an ABI model 3130 capillary sequencer (Applied Biosystems) using Genescan 500 size standards (Applied Biosystems). Individual peaks corresponding to each exon were identified with the Genescan software, based on the difference in migration relative to the size standards [25–27]. Peak areas of the amplified probes derived in this way were analyzed using a Microsoft Excel macro (Coffalyser, MRC-Holland) [28]. Phenotypes were checked in the database Leiden muscular dystrophy Pages (www.dmd.nl) according to the extension of the deletions/duplications on the basis of the frame shift hypothesis.
Out of the 146 families affected by DMD/BMD, in 47 families additional DNA samples of the female relatives were also sent, therefore we were able to offer carrier analysis. Direct diagnosis of female carriers was possible by Southern blot using specific cDNA probes when a deletion or duplication was detected in the index case. In 22 families where the index case deceased, MLPA analysis had to be performed. Carrier status was confirmed in nine females. In two female relatives of the same patient duplication events were detected (ex18ex21). All Southern blot results were confirmed by MLPA. In three girls presenting symptoms of unspecific muscular dystrophy, manifesting carrier status was detected (Table 2). Out of the 95 female relatives, including the three symptomatic girls, we were able to assess carrier status in 41 (43%), using cDNA and MLPA analyses. 3.3. Outcome of prenatal diagnoses using cDNA analysis and MLPA If the carrier status had been confirmed, prenatal diagnosis was offered. In total, prenatal molecular analyses were performed in 14 cases and, in three out of eight male foetuses, deletions in the dystrophin gene were confirmed. In the other five, unaffected male foetuses, four cases were analysed with multiplex PCR as the pathogenic mutations in the family was covered by this technique. However, in the fifth family, a duplication event was detected in the pregnant woman by MLPA, therefore, the prenatal testing had to be performed with the same technique. Female fetuses have not been tested for ethical considerations. 4. Discussion The spectrum of clinical severity in the different muscular dystrophies is very broad. Our aim was to establish a mutation spectrum for gross arrangements for Duchenne/Becker muscular
Table 1 Results of the dystrophin gene analysis in male patients. Number of patients tested
Type of mutation
Distribution of deletions/duplications
Phenotype
Mutation analysis using three different technique (PCR, cDNA analysis and MLPA) in 135 male patients
Deletions confirmed in 90 patients (66.7%)
In major hot spot region (ex44ex53): 61 patients (67.8%) In minor hot spot region (ex02ex20): 16 patients (17.8%) In both hot spot regions (large deletion): six patients (6.7%) Deletion outside of the major and minor hot spot regions: five patients (5.5%) In two patients (2.2%) the whole dystrophin gene was deleted – contiguous gene deletion syndrome (ARX gene present, IL1RAPL1, NROB1, GK, dystrophin genes deleted, PRRG1 gene present) ex08ex09 (two cases); ex12; ex21; ex44; ex45ex50; ex45ex49
44 DMD, 17 BMD 6 DMD, 10 BMD 3 DMD, 3 BMD 3 DMD, 2 BMD
4147C>T; Gln1383Stop
DMD
Duplication events in seven patients (5.2%) (Three of the seven patients with only one exon duplication) In two patients (twin brothers): pathogenic point mutation was detected
2 DMD
Seven patients with the milder DMD phenotype
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Table 2 Clinical symptomes and genetic results of the manifesing carriers. Family history
Clinical phenotype and DNA analysis
First manifesting female: The patient’s mother had an affected boy with DMD and an affected brother. Sister of the mother had two affected boys with DMD
Gowers’ sign positive CK values varied between 10590 and 32220 U/l Wheelchair dependent at the age of 14 Result of DNA analysis: Ex45-ex50 deletion in one copy of the dystrophin gene. Mother carries the same deletion without any symptoms Onset with 5 years CK values: 1577–1944 U/l Muscle pains and unable to run Result of DNA analysis: Ex10ex44 deletion in one copy of the dystrophin gene. Mother and grandmother carries the same deletion but they are asymptomatic 11 years old CK values: 12661–23000 U/l Calf hypertrophy Myogenic lesion by EMG Gait difficulties Result of DNA analysis: Ex49ex52 deletion in one copy of the dystrophin gene. Mother does not carry the mutation
Second manifesting female: The patient’s grandmother had an affected brother with DMD phenotype
Third manifesting female: No family history of muscular dystrophy
Fig. 1. Graphical overview of deletions and duplications. Number of cases is shown in corresponding brackets.
dystrophy in Hungary. In this work mutations in the dystrophin gene were identified in 99 affected males and in 41 carrier female relatives. In 66.7% of the clinically assumed DMD/BMD patients, deletions were detected. This finding corresponds well to the results of several publications on different populations [29,30]. The patients represented two phenotypes: 70% DMD (severe form) and 30% BMD (mild form) according to the comprehensive genetic results (www.dmd.nl). According to our results, there are two regions of the dystrophin gene, which are prone to deletions in the Hungarian population: 67.8% of the detected deletions were located in the major hot spot region including exons 44–53 and 17.8% of the detected deletions were in minor hot spots (including exons 2–20). This distribution pattern corresponds well to the data published in the Leiden database (63% in major hot spot and 18% in minor hot spot region), representing a large portion of the European patients [9]. This distribution is overall similar to a previously reported study on a different Hungarian cohort of patients that exclusively used multiplex PCR (31). In contrast, the patients reported here were genotyped with cDNA probes and MLPA studies and the mutation spectrum maybe more comprehensive. Deletion and duplication breakpoints were located mainly in intron 44 (43%) in agreement with that several authors have previously shown: intron 44 is the most frequent site for rearrangements in the dystrophin gene (Fig. 1) [8,31,32]. Further
breakpoints, although less pronounced, were located within introns 50 (11.3%) and 52 (9.3%). The most frequent deletion encompassed ex45ex50 in the Hungarian population; eight of the patients carried this mutation causing the DMD phenotype. Seven of the deletions are not published in the Leiden muscular dystrophy pages (www.dmd.nl): ex01ex30del, ex04ex09del (two cases), ex06ex12del, ex08ex30del, ex10ex12del, ex22ex41del, ex61ex64del. In this study we detected nine extending deletions (ex01ex30, ex01ex44, ex05ex44, ex08ex30, ex08ex47, ex18ex44, ex22ex41, two cases with ex13ex44) and two total gene deletion events. In five cases, the deletion in the dystrophin gene was identified only by MLPA analysis (ex22ex41, ex30, ex32, ex61, ex61ex64). In additional seven cases (5.2% of the patients), duplications were present in the dystrophin gene. Duplication frequency was highest near the 50 end of the gene in all previous studies, with a duplication of exon 2 being the single most common duplication identified [33,34]. In contrary, in the Hungarian duplication cases, rearrangements were spread through almost the entire gene (exons involved: ex08ex09 (two cases), ex12, ex21, ex44, ex45ex49, ex45ex50 and additionally, ex18ex21 resulting from carrier analysis in one family) (Fig. 1). The frequency corresponds well to the estimation of the duplication frequency of 5–10% in DMD patients [33–35], although a recent study found an unusual
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high rate of duplications (21% of mutation positive patients) [36]. All of the duplications have already been published in the Leiden muscular dystrophy Pages. The detected small rearrangements, especially the one- and two-exon duplications lead to DMD phenotype. In the case with ex45ex49dup, BMD phenotype was predicted but there was a discrepancy with the more severe clinical course of the disease. These findings indicate that for clinical diagnosis, duplications should be treated with special care, and without further analysis the reading frame rule should not be applied [36,37]. The reading frame is violated in about 9% of the cases published in the Leiden DMD mutation database [37]. Recent extensive study in BMD patients found a high rate of exceptions (30%) to the reading frame rule [36]. In our study, some of the deletion cases were also exceptional; two of our patients had such in-frame deletions which did not follow the reading frame hypothesis (ex05ex44, ex06ex12). These patients had relatively severe course of the disease with early onset, high CK values (8870–18,200), severe gait difficulties and also severe ventilation problems. Unfortunately, protein analysis was not available. These deletions involve part of the actin-binding domain and part of the central rod domain [38]. There were two interesting cases with severe forms of muscular dystrophy and additional symptoms in which the whole dystrophin gene was missing. One of the patients had also glycerol kinase deficiency and adrenal insufficiency and the other patient showed only somatic and mental retardation. These entire deletions were detected by multiplex PCR and were confirmed by MLPA and both cases represented a contiguous gene deletion syndrome, as neighbouring genes in addition to the dystrophin were also deleted. The lack of the dystrophin gene caused Duchenne muscular dystrophy, the GK gene deletion was responsible for the glycerol kinase deficiency, the NROB1 gene deletion caused adrenal hyperplasia (AHC) syndrome and the IL1RAP gene deletion should be associated with mental retardation in these two children. Although the same genes were involved in both children there were some phenotypic differences indicating that the breakpoints were not equal in the two children. The results fit very well into the few described cases with complex glycerol kinase deficiency in different ethnic groups involving similar phenotypes and genotypes [39–42]. In both families, the mothers carried the same deletion as detected by MLPA. In this work we analysed 95 female relatives by the quantitative MLPA technique and in 41 (43%) we confirmed the carrier status. As an additional important finding we identified three manifesting carrier females. In two of them there was a family history of DMD/ BMD disease, therefore, the clinical assumption of the symptomatic carrier status of the girls were straightforward. Interestingly, genetic analyses of the affected male patients were not performed beforehand and the DMD diagnosis was confirmed through the female patients. In these two families, we confirmed the mother’s carrier status, as well. The third young female patient might represent a de novo mutation event and was originally misdiagnosed having limb-girdle muscular dystrophy (Table 2). The number of manifesting carriers represents 3% of all females in the tested Hungarian DMD/BMD families, which is similar to other population studies [14,15]. These findings emphasize the importance of the occurrence of symptomatic DMD/BMD carriers which should always be considered in the diagnostic panel of muscular dystrophies. One-third of the DMD/BMD patients result from a new mutation, therefore a large proportion of the patients are isolated cases [8,9,12,16]. In our family studies, 47 mothers were analysed as asymptomatic deletion or duplication carriers and 19 of them were detected as non-carriers of the mutations, although gonadal mosaicism should also be considered [12]. The incidence of female carriers in the Hungarian DMD/BMD population is in good correlation
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with other studies [29,30]. For these comprehensive carrier analyses the introduction of MLPA assay in the Hungarian DMD/BMD diagnostic service was indispensable. Moreover, the MLPA technique proved to be especially useful in situations where the index patient deceased and no DNA sample was available for genetic confirmation of the disease. There were three efficient prenatal tests which were offered following successful carrier detection by MLPA analysis. All three ended with affected status of the foetuses showing the necessity of reliable quantitative assay in the diagnosis of Duchenne/Becker muscular dystrophy. Additionally, in two female relatives of a similarly deceased patient duplications in exons 18–21 were detected and in one of them prenatal analysis had to be performed. (Fortunately, the chorion sample did not carry the duplication). Our laboratory is the only one in Hungary, which introduced the molecular analysis for the more sophisticated mutation screening with cDNA and MLPA analyses for the Duchenne/Becker muscular dystrophy patients and their female relatives. Applying these new techniques, a more reliable molecular genetic diagnosis of the specific muscular dystrophy became available. This development provides not only the opportunity for proper genetic counselling and prenatal diagnosis for the affected families, but can also help to choose the correct therapeutic approach, where available. Acknowledgements This work was supported by the Hungarian OTKA (T038049) and EU FP6 NoE TREAT-NMD (036825) grants. The authors wish to thank for the Hungarian paediatricians and neurologists for the clinical diagnosis and referral of the patients and all the patients and their families with DMD/BMD disease for providing their DNA samples and personal data. The authors are grateful to Edit Gnotek, Éva Gönczi, Margit Czimbalmos and Mária Gogolák for their valuable technical help. The data on the point mutation of the twin brothers is especially acknowledged for Prof. Clemens Müller-Reible at Würzburg University, Institute of Human Genetics, Germany. Immunohistochemistry and Western blot analysis of dystrophin protein was kindly provided by Dr. Maggie Walter at the Department of Neurology, Friedrich-Baur-Institute, Ludwig Maximillian University, Munich, Germany. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.nmd.2008.10.011. References [1] Ehmsen J, Poon E, Davies K. The dystrophin-associated protein complex. J Cell Sci 2002;115(Pt 14):2801–3. [2] Roberts RG. Dystrophin, its gene, and the dystrophinopathies. Adv Genet 1995;33:177–231. [3] Hoffman EP, Brown RH, Kunkel LM. Dystrophin: the protein product of the Duchenne muscular dystrophy locus. Cell 1987;51:917–28. [4] Koenig M, Monaco AP, Kunkel LM. The complete sequence of dystrophin predicts a rod-shaped cytoskeletal protein. Cell 1988;53:219–28. [5] Den-Dunnen JT, Grootscholten PM, Bakker E. Topography of the Duchenne muscular dystrophy 8d0d9 gene: FIGE and cDNA analysis of 194 cases reveals 115 deletions and 13 duplications. Am J Hum Genet 1999;45:835–47. [6] Baumbach LL, Chamberlain JS, Ward PA, et al. Molecular and clinical correlations of deletions leading to Duchenne and Becker muscular dystrophies. Neurology 1989;39:465–74. [7] Koenig M, Beggs AH, Moyer M, et al. The molecular basis for Duchenne versus Becker muscular dystrophy: correlation of severity with type of deletion. Am J Hum Genet 1989;45:498–506. [8] Forrest SM, Cross GS, Speer A, et al. Preferential deletion of exons in Duchenne and Becker muscular dystrophies. Nature 1987;329:638–42. [9] Den Dunnen JT, Grootscholten PM, Bakker E, et al. Topography of the Duchenne muscular dystrophy (DMD) gene: FIGE and cDNA analysis of 194 cases reveals 115 deletions and 13 duplications. Am J Hum Genet 1989;45:835–47.
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[10] Beggs AH, Hoffman EP, Snyder JR, et al. Exploring the molecular basis for variability among patients with BMD: dystrophin gene and protein studies. Am J Hum Genet 1989;49:54–67. [11] Moser H. Duchenne muscular dystrophy: pathogenetic aspects and genetic prevention. Hum Genet 1984;66:17–40. [12] Bakker E, Veenema H, Den Dunnen JT, et al. Germinal mosaicism increases the recurrence risk for ‘‘new” Duchenne muscular dystrophy mutations. J Med Genet 1989;26:553–9. [13] Koenig M, Hoffman EP, Bertelson CJ, et al. Complete cloning of the Duchenne muscular dystrophy (DMD) cDNA and preliminary genomic organization of the DMD gene in normal and affected individuals. Cell 1987;50:509–17. [14] van Essen AJ, Abbs S, Baiget M, et al. Parental origin and germline mosaicism of deletions and duplications of the dystrophin gene: a European study. Hum Genet 1992;88:249–57. [15] Hoffman EP, Pegoraro E, Scacheri P, et al. Genetic counseling of isolated carriers of Duchenne muscular dystrophy. Am J Med Genet 1996;63:573–80. [16] Chamberlain JS, Gibbs RA, Rainer JE, et al. Multiplex PCR for diagnosis of Duchenne muscular dystrophy. In: Innis MA, Gelfand DH, Sninsky JJ, White TJ, editors. PCR protocols: a guide to methods and applications. New York: Academic Press; 1990. p. 273–87. [17] Beggs AH, Koenig M, Boyce FM, et al. Detection of 98% of DMD/BMD gene deletions by polymerase chain reaction. Hum Genet 1990;86:45–8. [18] Russel DW, Sambrook J. Molecular cloning (A laboratory manual). 3rd ed. New York: Cold Spring Harbor Laboratory press; 2003. [19] Pegoraro E, Schimke RN, Arahata K, et al. Detection of new paternal dystrophin gene mutations in isolated cases of dystrophinopathy in females. Am J Hum Genet 1994;54:989–1003. [20] Darras BT, Blattner P, Harper JF, et al. Intragenic deletions in 21 Duchenne muscular dystrophy (DMD) families studied with the dystrophin cDNA: Location of breakpoints on Hind III and Bgl II exon-containig fragments maps, meiotic and mitotic origin of the mutation. Am J Hum Genet 1988;43:620–9. [21] Schouten JP, Mcelgum CJ, Waaijer R, et al. Relative quantification of 40 nucleic acid sequences by multiplex ligation-dependent probe amplification. Nucleic Acids Research 2002;30:e57. [22] Bushby KMD, Anderson LVB, editors. Muscular dystrophy. Methods and protocols. Humana Press; 2001. p. 53–84. [23] Lalic T, Vossen RH, Coffa J. Deletion and duplication screening in the DMD gene using MLPA. Eur J Hum Genet 2005;13:1231–41. [24] Gatta V, Scarciolla O, Caspari AR. Identification of deletion and duplications of the DMD gene in affected males and carrier females by multiple ligation probe amplification (MLPA). Hum Gent 2005;117:92–8. [25] Sellner LN, Taylor GR. MLPA and MAPH: new techniques for detection of gene deletions. Human Mutat 2004;23:413–9. [26] Chamberlain JS, Gibbs RA, Rainer JE, Caskey CT. Deletion screening of the Duchenne muscular dystrophy locus via multiplex DNA amplification. Nucleic Acids Res 1988;16:11141–56. [27] Cooper ST, Lo HP, North KN. Single section western blot. Improving the molecular diagnosis of the muscular dystrophies. Neurology 2003;61:93–7.
[28] Coffa J, van de Wiel MA, Diosdado B, Carvalho B, et al. MLPAnalyzer: data analysis tool for reliable automated normalization of MLPA fragment data. Cell Oncol 2008;30:323–35. [29] Asano J, Tomatsu S, Sukegawa K, et al. Gene deletions in Japanese patients with Duchenne and Becker muscular dystrophies: deletion study and carrier detection. Clin Genet 1991;39:419–24. [30] Baumbach LL, Chamberlain JS, Ward PA, et al. Molecular and clinical correlations of deletions leading to Duchenne and Becker muscular dystrophies. Neurology 1989;39:465–74. [31] Herczegfalvi A, Tóth G, Gyürüs P, et al. Deletion patterns of dystrophin gene in Hungarian patients with Duchenne/Becker muscular dystrophies. Neuromuscul Disord 1999;9:552–4. [32] Roberts RG, Barby TF, Manners E, et al. Direct detection of dystrophin gene rearrangements by analysis of dystrophin mRNA in peripheral blood lymphocytes. Am J Hum Genet 1991;49:298–310. [33] White S, Kalf M, Liu Q, et al. Comprehensive detection of genomic duplications and deletions in the DMD gene, by use of multiplex amplifiable probe hybridization. Am J Hum Genet 2002;71:365–74. [34] White SJ, Aartsma-Rus A, Flanigan KM, et al. Duplications in the DMD Gene. Hum Mutat 2006;27:938–45. [35] Hu X, Ray PN, Murphy E, et al. Duplicational mutation at the Duchenne muscular dystrophy locus: its frequency, distribution, origin and phenotype/ genotype correlation. Am J Hum Genet 1990;46:682–95. [36] Kesari A, Pirra LN, Bremadesma L, et al. Integrated DNA, cDNA, and protein studies in Becker muscular dystrophy show high exception to the reading frame rule. Hum Mutat 2008;29:728–37. [37] Aartsma-Rus A, Van Deutekom JC, Fokkema IF, et al. Entries in the Leiden Duchenne muscular dystrophy mutation database: an overview of mutation types and paradoxical cases that confirm the reading-frame rule. Muscle Nerve 2006;34:135–44. [38] Beggs AH, Hoffman EP, Snyder JR, et al. Exploring the molecular basis for variability among patients with Becker muscular dystrophy: dystrophin gene and protein studies. Am J Hum Genet 1991;49:54–67. [39] Romero NB, Récan D, Rigal O, et al. A point mutation in the glycerol kinase gene associated with a deletion in the dystrophin gene in a familial X-linked muscular dystrophy: non-contiguous gene syndrome involving Becker muscular dystrophy and glycerol kinase loci. Neuromuscul Disord 1997;7:499–504. [40] Jin H, Gardner RJ, Viswesvaraiah R, et al. Two novel members of the interleukin-1 receptor gene family, one deleted in Xp22. 1-Xp21.3 mental retardation. Eur J Hum Genet 2000;8:87–94. [41] Zhang YH, Huang BL, Niakan KK, et al. IL1RAPL1 is associated with mental retardation in patients with complex glycerol kinase deficiency who have deletions extending telomeric of DAX1. Hum Mutat 2004;24: 273. [42] Wheway JM, Yau SC, Nihalani V, et al. A complex deletion–inversion–deletion event results in a chimeric IL1RAPL1-dystrophin transcript and a contiguous gene deletion syndrome. J Med Genet 2003;40:127–31.
Contents lists available at ScienceDirect
Neuromuscular Disorders journal homepage: www.elsevier.com/locate/nmd
Case report
Dystrophin gene analysis in Hungarian Duchenne/Becker muscular dystrophy families – Detection of carrier status in symptomatic and asymptomatic female relatives Henriett Pikó a, Viktor Vancsó a,b, Bálint Nagy b, Zoltán Bán b, Ágnes Herczegfalvi c, Veronika Karcagi a,* a b c
National Institute of Environmental Health, Department of Molecular Genetics and Diagnostics, Gyali út 2-6, H-1097 Budapest, Hungary Semmelweis Medical School, Clinic of Obstetrics and Gynaecology, Laboratory of Genetics, Budapest, Hungary Bethesda Children’s Hospital, Department of Neurology, Budapest, Hungary
a r t i c l e
i n f o
Article history: Received 28 July 2008 Received in revised form 20 October 2008 Accepted 29 October 2008
Keywords: Duchenne/Becker muscular dystrophy MLPA analysis Carrier detection Manifesting carriers
a b s t r a c t A comprehensive study of the Hungarian Duchenne/Becker muscular dystrophy (DMD/BMD) families is presented. Deletions in the hot spots regions were identified by multiplex PCR, whereas rare mutations were detected by Southern blot and multiplex ligation-dependent probe amplification (MLPA) techniques. DMD/BMD disease was confirmed and exact deletion borders were determined in 19 out of 135 affected males using multiplex PCR. Additional exons involved as well as rare exon deletions were identified by MLPA in 71 male patients, whereas duplications were observed in seven patients. In two DMD patients, the entire dystrophin gene and adjacent genes were deleted. Out of the 95 female relatives, 41 proved to be carriers, including three manifesting carrier females. Using MLPA method, a large portion of the Hungarian DMD/BMD patients and their female relatives were exactly genotyped. For the first time, the incidence and prevalence of asymptomatic and symptomatic female carriers in Hungary was estimated. Ó 2008 Elsevier B.V. All rights reserved.
1. Introduction Muscular dystrophies are a genetically heterogeneous group of degenerative muscle disorders, characterized by the progressive loss of muscle strength and integrity. There is marked clinical similarity among patients with genetically different diseases, suggesting that many of the gene products may participate in a common functional structure or biochemical cascade [1]. Duchenne/Becker muscular dystrophy (DMD/BMD) is a lethal/severe X-linked recessive disorder affecting 1 in 3500 (DMD) and 1 in 30,000 (BMD) male births [2]. It is caused by mutations in the dystrophin gene located on Xp21.2, which encodes a protein of the membrane cytoskeleton in skeletal muscle (dystrophin) [3–5]. Mutations responsible for the disease are deletions or duplications encompassing the 79 exons of the gene, which account for 60–65% and 5–10% of cases of DMD/BMD, respectively [5,6]; while the remaining cases, about 30–35%, are caused by single point mutations (nonsense or splice sites) or small rearrangements [2,7–10]. In BMD, the proportion of large deletions and duplications is higher, 85% [10]. Approximately one-third of the DMD/BMD patients originate through new mutations, while the rest are inherited through carrier mothers or arise from germ line mosaicism [11,12]. The main
* Corresponding author. Fax: +36 2150148. E-mail address: [email protected] (V. Karcagi). 0960-8966/$ - see front matter Ó 2008 Elsevier B.V. All rights reserved. doi:10.1016/j.nmd.2008.10.011
problem of the molecular genetic analysis of the DMD/BMD disease is the size and the wide mutation spectrum of the dystrophin gene. Although there are two hot spot regions, wherein the mutation frequencies are higher than in the other region, the relatively frequent point mutations can occur anywhere in the gene. Further complicating factor of the DMD/BMD mutation analysis is the presence of germ line and somatic mosaicism [12,13]. Females with muscular dystrophy symptoms are often considered as limb-girdle muscular dystrophy patients, although the frequency of DMD/BMD manifesting carriers might be higher as previously suggested [14,15]. There are few population data on the prevalence of carriers, especially on symptomatic carriers. In this study we compared the newly introduced MLPA technique to the formerly used quantitative Southern blot assay in female individuals who were assumed as possible carriers. On the other hand, MLPA was used as quantitative assessment of all 79 exons, in order to identify exact deletion borders and eventual duplication events in affected males. Here we report the genetic results of 135 affected males, three manifesting carrier women and 92 female relatives using different diagnostic approaches for DMD/BMD disease. 2. Materials and methods Patients affected with DMD/BMD were assessed in our laboratory between 2000 and 2008. There is no overlap with a previous
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study published in 1999 (31). In all of them the diagnosis was established through clinical and neurological examinations including family history, serum creatine kinase (CK) analysis, and where available, through histopathology of the muscle biopsy. Genomic DNA was extracted from peripheral blood leukocytes (Promega Wizard genomic DNA purification Kit, Promega Corporation, USA). DNA analyses included screening of deletions by multiplex PCR and detection of deletions/duplications borders using MLPA technique in the index case. Female relatives were tested for their carrier status using cDNA probes and MLPA analysis.
3. Results 3.1. Molecular diagnosis of index cases During the period of 2000–2008 March, 135 unrelated DMD and BMD Hungarian patients were sent to our laboratory for genetic confirmation of the disease. They were classified as non-ambulatory and ambulatory by the clinicians. Molecular analysis of the patients’ dystrophin gene was performed by multiplex PCR reactions, Southern blot analysis and MLPA technique. Results are summarised in Table 1.
2.1. Multiplex PCR 3.2. Carrier detection Two sets of PCR primers were used which cover 17 exons and the muscle specific promoter in the deletion hot spots. PCR and agarose gel electrophoresis was performed by standard protocols [16,17]. 2.2. Southern blot and cDNA probes Genomic DNA was digested with HindIII and BglII restriction enzymes, separately. Electrophoresis and membrane transfer was performed according to standard techniques. [18]. After blotting, the membrane was hybridized with a 32P-dCTP labelled cDNA probe (XJ10, 30-2, 30-1, 47-4, 7b8, or 63-1), using the random priming procedure [8,18–21]. For data evaluation, the Packard Instant Imager was applied to quantify the relative intensities of the signals. Intensities were corrected and calculated according to standard protocols [22–24]. DNA samples of female relatives were tested together with their affected offspring’s DNA, if available and fragment densities of the different exons were also compared to normal controls. 2.3. Multiple ligation-dependent probe amplification (MLPA) The MLPA DMD/BMD test kit (SALSA P034/035) was obtained from MRC-Holland, Amsterdam, The Netherlands. Analysis was performed according to the manufacturer‘s recommendations [25–27]. PCR products were run on an ABI model 3130 capillary sequencer (Applied Biosystems) using Genescan 500 size standards (Applied Biosystems). Individual peaks corresponding to each exon were identified with the Genescan software, based on the difference in migration relative to the size standards [25–27]. Peak areas of the amplified probes derived in this way were analyzed using a Microsoft Excel macro (Coffalyser, MRC-Holland) [28]. Phenotypes were checked in the database Leiden muscular dystrophy Pages (www.dmd.nl) according to the extension of the deletions/duplications on the basis of the frame shift hypothesis.
Out of the 146 families affected by DMD/BMD, in 47 families additional DNA samples of the female relatives were also sent, therefore we were able to offer carrier analysis. Direct diagnosis of female carriers was possible by Southern blot using specific cDNA probes when a deletion or duplication was detected in the index case. In 22 families where the index case deceased, MLPA analysis had to be performed. Carrier status was confirmed in nine females. In two female relatives of the same patient duplication events were detected (ex18ex21). All Southern blot results were confirmed by MLPA. In three girls presenting symptoms of unspecific muscular dystrophy, manifesting carrier status was detected (Table 2). Out of the 95 female relatives, including the three symptomatic girls, we were able to assess carrier status in 41 (43%), using cDNA and MLPA analyses. 3.3. Outcome of prenatal diagnoses using cDNA analysis and MLPA If the carrier status had been confirmed, prenatal diagnosis was offered. In total, prenatal molecular analyses were performed in 14 cases and, in three out of eight male foetuses, deletions in the dystrophin gene were confirmed. In the other five, unaffected male foetuses, four cases were analysed with multiplex PCR as the pathogenic mutations in the family was covered by this technique. However, in the fifth family, a duplication event was detected in the pregnant woman by MLPA, therefore, the prenatal testing had to be performed with the same technique. Female fetuses have not been tested for ethical considerations. 4. Discussion The spectrum of clinical severity in the different muscular dystrophies is very broad. Our aim was to establish a mutation spectrum for gross arrangements for Duchenne/Becker muscular
Table 1 Results of the dystrophin gene analysis in male patients. Number of patients tested
Type of mutation
Distribution of deletions/duplications
Phenotype
Mutation analysis using three different technique (PCR, cDNA analysis and MLPA) in 135 male patients
Deletions confirmed in 90 patients (66.7%)
In major hot spot region (ex44ex53): 61 patients (67.8%) In minor hot spot region (ex02ex20): 16 patients (17.8%) In both hot spot regions (large deletion): six patients (6.7%) Deletion outside of the major and minor hot spot regions: five patients (5.5%) In two patients (2.2%) the whole dystrophin gene was deleted – contiguous gene deletion syndrome (ARX gene present, IL1RAPL1, NROB1, GK, dystrophin genes deleted, PRRG1 gene present) ex08ex09 (two cases); ex12; ex21; ex44; ex45ex50; ex45ex49
44 DMD, 17 BMD 6 DMD, 10 BMD 3 DMD, 3 BMD 3 DMD, 2 BMD
4147C>T; Gln1383Stop
DMD
Duplication events in seven patients (5.2%) (Three of the seven patients with only one exon duplication) In two patients (twin brothers): pathogenic point mutation was detected
2 DMD
Seven patients with the milder DMD phenotype
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Table 2 Clinical symptomes and genetic results of the manifesing carriers. Family history
Clinical phenotype and DNA analysis
First manifesting female: The patient’s mother had an affected boy with DMD and an affected brother. Sister of the mother had two affected boys with DMD
Gowers’ sign positive CK values varied between 10590 and 32220 U/l Wheelchair dependent at the age of 14 Result of DNA analysis: Ex45-ex50 deletion in one copy of the dystrophin gene. Mother carries the same deletion without any symptoms Onset with 5 years CK values: 1577–1944 U/l Muscle pains and unable to run Result of DNA analysis: Ex10ex44 deletion in one copy of the dystrophin gene. Mother and grandmother carries the same deletion but they are asymptomatic 11 years old CK values: 12661–23000 U/l Calf hypertrophy Myogenic lesion by EMG Gait difficulties Result of DNA analysis: Ex49ex52 deletion in one copy of the dystrophin gene. Mother does not carry the mutation
Second manifesting female: The patient’s grandmother had an affected brother with DMD phenotype
Third manifesting female: No family history of muscular dystrophy
Fig. 1. Graphical overview of deletions and duplications. Number of cases is shown in corresponding brackets.
dystrophy in Hungary. In this work mutations in the dystrophin gene were identified in 99 affected males and in 41 carrier female relatives. In 66.7% of the clinically assumed DMD/BMD patients, deletions were detected. This finding corresponds well to the results of several publications on different populations [29,30]. The patients represented two phenotypes: 70% DMD (severe form) and 30% BMD (mild form) according to the comprehensive genetic results (www.dmd.nl). According to our results, there are two regions of the dystrophin gene, which are prone to deletions in the Hungarian population: 67.8% of the detected deletions were located in the major hot spot region including exons 44–53 and 17.8% of the detected deletions were in minor hot spots (including exons 2–20). This distribution pattern corresponds well to the data published in the Leiden database (63% in major hot spot and 18% in minor hot spot region), representing a large portion of the European patients [9]. This distribution is overall similar to a previously reported study on a different Hungarian cohort of patients that exclusively used multiplex PCR (31). In contrast, the patients reported here were genotyped with cDNA probes and MLPA studies and the mutation spectrum maybe more comprehensive. Deletion and duplication breakpoints were located mainly in intron 44 (43%) in agreement with that several authors have previously shown: intron 44 is the most frequent site for rearrangements in the dystrophin gene (Fig. 1) [8,31,32]. Further
breakpoints, although less pronounced, were located within introns 50 (11.3%) and 52 (9.3%). The most frequent deletion encompassed ex45ex50 in the Hungarian population; eight of the patients carried this mutation causing the DMD phenotype. Seven of the deletions are not published in the Leiden muscular dystrophy pages (www.dmd.nl): ex01ex30del, ex04ex09del (two cases), ex06ex12del, ex08ex30del, ex10ex12del, ex22ex41del, ex61ex64del. In this study we detected nine extending deletions (ex01ex30, ex01ex44, ex05ex44, ex08ex30, ex08ex47, ex18ex44, ex22ex41, two cases with ex13ex44) and two total gene deletion events. In five cases, the deletion in the dystrophin gene was identified only by MLPA analysis (ex22ex41, ex30, ex32, ex61, ex61ex64). In additional seven cases (5.2% of the patients), duplications were present in the dystrophin gene. Duplication frequency was highest near the 50 end of the gene in all previous studies, with a duplication of exon 2 being the single most common duplication identified [33,34]. In contrary, in the Hungarian duplication cases, rearrangements were spread through almost the entire gene (exons involved: ex08ex09 (two cases), ex12, ex21, ex44, ex45ex49, ex45ex50 and additionally, ex18ex21 resulting from carrier analysis in one family) (Fig. 1). The frequency corresponds well to the estimation of the duplication frequency of 5–10% in DMD patients [33–35], although a recent study found an unusual
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high rate of duplications (21% of mutation positive patients) [36]. All of the duplications have already been published in the Leiden muscular dystrophy Pages. The detected small rearrangements, especially the one- and two-exon duplications lead to DMD phenotype. In the case with ex45ex49dup, BMD phenotype was predicted but there was a discrepancy with the more severe clinical course of the disease. These findings indicate that for clinical diagnosis, duplications should be treated with special care, and without further analysis the reading frame rule should not be applied [36,37]. The reading frame is violated in about 9% of the cases published in the Leiden DMD mutation database [37]. Recent extensive study in BMD patients found a high rate of exceptions (30%) to the reading frame rule [36]. In our study, some of the deletion cases were also exceptional; two of our patients had such in-frame deletions which did not follow the reading frame hypothesis (ex05ex44, ex06ex12). These patients had relatively severe course of the disease with early onset, high CK values (8870–18,200), severe gait difficulties and also severe ventilation problems. Unfortunately, protein analysis was not available. These deletions involve part of the actin-binding domain and part of the central rod domain [38]. There were two interesting cases with severe forms of muscular dystrophy and additional symptoms in which the whole dystrophin gene was missing. One of the patients had also glycerol kinase deficiency and adrenal insufficiency and the other patient showed only somatic and mental retardation. These entire deletions were detected by multiplex PCR and were confirmed by MLPA and both cases represented a contiguous gene deletion syndrome, as neighbouring genes in addition to the dystrophin were also deleted. The lack of the dystrophin gene caused Duchenne muscular dystrophy, the GK gene deletion was responsible for the glycerol kinase deficiency, the NROB1 gene deletion caused adrenal hyperplasia (AHC) syndrome and the IL1RAP gene deletion should be associated with mental retardation in these two children. Although the same genes were involved in both children there were some phenotypic differences indicating that the breakpoints were not equal in the two children. The results fit very well into the few described cases with complex glycerol kinase deficiency in different ethnic groups involving similar phenotypes and genotypes [39–42]. In both families, the mothers carried the same deletion as detected by MLPA. In this work we analysed 95 female relatives by the quantitative MLPA technique and in 41 (43%) we confirmed the carrier status. As an additional important finding we identified three manifesting carrier females. In two of them there was a family history of DMD/ BMD disease, therefore, the clinical assumption of the symptomatic carrier status of the girls were straightforward. Interestingly, genetic analyses of the affected male patients were not performed beforehand and the DMD diagnosis was confirmed through the female patients. In these two families, we confirmed the mother’s carrier status, as well. The third young female patient might represent a de novo mutation event and was originally misdiagnosed having limb-girdle muscular dystrophy (Table 2). The number of manifesting carriers represents 3% of all females in the tested Hungarian DMD/BMD families, which is similar to other population studies [14,15]. These findings emphasize the importance of the occurrence of symptomatic DMD/BMD carriers which should always be considered in the diagnostic panel of muscular dystrophies. One-third of the DMD/BMD patients result from a new mutation, therefore a large proportion of the patients are isolated cases [8,9,12,16]. In our family studies, 47 mothers were analysed as asymptomatic deletion or duplication carriers and 19 of them were detected as non-carriers of the mutations, although gonadal mosaicism should also be considered [12]. The incidence of female carriers in the Hungarian DMD/BMD population is in good correlation
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with other studies [29,30]. For these comprehensive carrier analyses the introduction of MLPA assay in the Hungarian DMD/BMD diagnostic service was indispensable. Moreover, the MLPA technique proved to be especially useful in situations where the index patient deceased and no DNA sample was available for genetic confirmation of the disease. There were three efficient prenatal tests which were offered following successful carrier detection by MLPA analysis. All three ended with affected status of the foetuses showing the necessity of reliable quantitative assay in the diagnosis of Duchenne/Becker muscular dystrophy. Additionally, in two female relatives of a similarly deceased patient duplications in exons 18–21 were detected and in one of them prenatal analysis had to be performed. (Fortunately, the chorion sample did not carry the duplication). Our laboratory is the only one in Hungary, which introduced the molecular analysis for the more sophisticated mutation screening with cDNA and MLPA analyses for the Duchenne/Becker muscular dystrophy patients and their female relatives. Applying these new techniques, a more reliable molecular genetic diagnosis of the specific muscular dystrophy became available. This development provides not only the opportunity for proper genetic counselling and prenatal diagnosis for the affected families, but can also help to choose the correct therapeutic approach, where available. Acknowledgements This work was supported by the Hungarian OTKA (T038049) and EU FP6 NoE TREAT-NMD (036825) grants. The authors wish to thank for the Hungarian paediatricians and neurologists for the clinical diagnosis and referral of the patients and all the patients and their families with DMD/BMD disease for providing their DNA samples and personal data. The authors are grateful to Edit Gnotek, Éva Gönczi, Margit Czimbalmos and Mária Gogolák for their valuable technical help. The data on the point mutation of the twin brothers is especially acknowledged for Prof. Clemens Müller-Reible at Würzburg University, Institute of Human Genetics, Germany. Immunohistochemistry and Western blot analysis of dystrophin protein was kindly provided by Dr. Maggie Walter at the Department of Neurology, Friedrich-Baur-Institute, Ludwig Maximillian University, Munich, Germany. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.nmd.2008.10.011. References [1] Ehmsen J, Poon E, Davies K. The dystrophin-associated protein complex. J Cell Sci 2002;115(Pt 14):2801–3. [2] Roberts RG. Dystrophin, its gene, and the dystrophinopathies. Adv Genet 1995;33:177–231. [3] Hoffman EP, Brown RH, Kunkel LM. Dystrophin: the protein product of the Duchenne muscular dystrophy locus. Cell 1987;51:917–28. [4] Koenig M, Monaco AP, Kunkel LM. The complete sequence of dystrophin predicts a rod-shaped cytoskeletal protein. Cell 1988;53:219–28. [5] Den-Dunnen JT, Grootscholten PM, Bakker E. Topography of the Duchenne muscular dystrophy 8d0d9 gene: FIGE and cDNA analysis of 194 cases reveals 115 deletions and 13 duplications. Am J Hum Genet 1999;45:835–47. [6] Baumbach LL, Chamberlain JS, Ward PA, et al. Molecular and clinical correlations of deletions leading to Duchenne and Becker muscular dystrophies. Neurology 1989;39:465–74. [7] Koenig M, Beggs AH, Moyer M, et al. The molecular basis for Duchenne versus Becker muscular dystrophy: correlation of severity with type of deletion. Am J Hum Genet 1989;45:498–506. [8] Forrest SM, Cross GS, Speer A, et al. Preferential deletion of exons in Duchenne and Becker muscular dystrophies. Nature 1987;329:638–42. [9] Den Dunnen JT, Grootscholten PM, Bakker E, et al. Topography of the Duchenne muscular dystrophy (DMD) gene: FIGE and cDNA analysis of 194 cases reveals 115 deletions and 13 duplications. Am J Hum Genet 1989;45:835–47.
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