De novo mutation in DMD gene in a patient with

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Nov 26, 2013 - Abstract We report an unusual case of a patient with two combined X-linked diseases, severe hemophilia A (HA) and Duchenne muscular ...

Int J Hematol DOI 10.1007/s12185-013-1488-4


De novo mutation in DMD gene in a patient with combined hemophilia A and Duchenne muscular dystrophy Lana Strmecki • Petra Hudler • Majda Benedik-Dolnicˇar • Radovan Komel

Received: 30 May 2013 / Revised: 26 November 2013 / Accepted: 26 November 2013 Ó The Japanese Society of Hematology 2013

Abstract We report an unusual case of a patient with two combined X-linked diseases, severe hemophilia A (HA) and Duchenne muscular dystrophy (DMD), of which only HA was hereditary. There was no family history of muscular dystrophy. Genetic analysis revealed that HA was caused by the hereditary coagulation factor VIII (F8) intron 22 inversion (distal/type I inversion), whereas DMD was caused by a de novo deletion in the dystrophin gene. This is the first report of a patient with two severe X-linked diseases, of which only HA was hereditary. Despite the fact that the probability of acquiring two X-linked abnormalities, one hereditary and one de novo, is extremely low, the emergence of such cases indicates that genetic testing for distinct X-linked diseases could be of importance in patients with hereditary hemophilia. Keywords DMD  Duchenne muscular dystrophy  Factor VIII  Hemophilia  X-linked diseases

L. Strmecki Department of Biochemistry, University of Oxford, Oxford, UK L. Strmecki  P. Hudler (&)  R. Komel (&) Faculty of Medicine, Medical Centre for Molecular Biology, Institute of Biochemistry, University of Ljubljana, Vrazov trg 2, 1000 Ljubljana, Slovenia e-mail: [email protected] R. Komel e-mail: [email protected] M. Benedik-Dolnicˇar Department of Paediatrics, National Haemophilia Centre, University Medical Centre, Ljubljana, Slovenia

Abbreviations APCC Activated prothrombin complex concentrates CK Creatine kinase DMD Duchenne muscular dystrophy DMD gene Dystrophin gene HA Hemophilia A PCC Prothrombin complex concentrates

Introduction Hemophilia A (HA) is a recessive X-linked disorder, characterized by the absence or an insufficient amount of functional factor VIII (FVIII, coded by F8 gene) [1]. Patients affected with severe form of the disease suffer from spontaneous bleeding in joints, muscles, and soft tissue. Approximately half of the severe cases of HA are caused by the F8 intron 22 inversions, distal/type I [2, 3]. Duchenne muscular dystrophy (DMD) is a recessive X-linked form of muscular dystrophy, which results in muscle degeneration and eventual death. The disease is caused by mutations in the dystrophin (DMD) gene, which codes structural protein within muscle tissue that provides structural stability to the dystroglycan complex (DGC) of the cell membrane [4]. The most common type of diseasecausing mutation is the deletion of one or more exons [5]. The absence of the protein in muscle cells results in muscle cell necrosis, followed by progressive muscular degeneration and wasting [6]. Affected boys are usually confined to a wheelchair before the age of 12 and die in their late teens or early twenties [4]. A combination of two hereditary chromosome X-linked disorders is considered a rare event. HA occurs in approximately 1 in 5,000 males (2.0 per 10,000). According to


L. Strmecki et al.

WHO, DMD is also considered a rare disease, with an estimated incidence of 1 in 3,300 newborn males worldwide (3.0 per 10,000). We report an extremely rare event, a case of combined severe HA, caused by hereditary F8 intron 22 inversion (distal/type I), and DMD, caused by the de novo mutation.

Methods The patient The proband, a 26-year-old male of Iranian descent with severe HA had arrived to Slovenia at age 4 and had at that time already developed FVIII inhibitors [highest level was 115 Bethesda units (BU)/ml which cross-reacted with porcine FVIII (pFVIII) with a titer of 64 BU/ml]. In rare minor acute bleedings, he has been successfully treated with inactive prothrombin complex concentrates (PCC), however, when he had intracranial hemorrhage (after trauma), he received activated PCC (APCC) and recombinant FVIIa (rFVIIa) concentrates without complications. He did not receive immune tolerance induction. At age 5, the proband manifested weakness of the neck muscles and had a raised CK level of 7 lcat/L (normal levels for men: up to 2.85 lcat/L). At age 7 his CK level was 62 lcat/L. He was wheelchair bound from age 10. The CK values were considerably lower in the last years and he had only 4 bleedings in the last 5 years. The patient died at the age 26 due to complications associated with DMD. The proband had an elder sibling with HA, but without DMD and FVIII inhibitors. There was no history of consanguinity and no history of muscular dystrophy in the family. Genetic analyses DNA was isolated from peripheral blood and DMD gene was investigated with multiplex polymerase chain Fig. 1 Analysis of DMD gene deletions showing a deletion from exons 45 to 52 in patient HA142 (P) and amplified gene from his mother HA139, used as negative control (C)


reactions (PCR) of 18 DMD exons (3, 4, 6, 8, 12, 13, 17, 19, 43–45, 47–52, and 60) to detect deletion and/or duplication hot-spots. The reactions were performed in 3 sets (each reaction including 6 exons) according to conditions specified by Chamberlain et al. and Beggs et al. with modifications [7, 8]. DNA samples (200 ng) were amplified in 15 ll of PCR solution including: 9 pmol of each primer, 2.0 mM of each dNTP, 3.0 mM MgCl2, 1 9 PCR buffer (65 mM Tris–HCl pH 8.0, 16 mM (NH4)2SO4, 10 mM 2-mercapto-ethanol, 0.1 mg/ml bovine serum albumin) and 1.0 U of RTB DNA polymerase (AMED, Italy). After initial denaturation of 3 min at 95 °C, the fragments were amplified by 26 cycles of denaturation at 94 °C for 30 s and annealing/extension step at 65 °C for 4 min, followed by the final extension step of 6 min at 65 °C. PCR products were separated by agarose gel electrophoresis and stained with ethidium bromide. F8 gene was examined by southern blot analysis of BclIdigested DNA carried out using 0.9 kb EcoRI/SacI fragment from plasmid p482.6 as a probe, which was radiolabeled with 32 (P)a-dCTP using random hexameres (Promega, USA). The membranes (Hybond N?, GE Healthcare, USA) were autoradiographed at -70 °C for 1–3 days.

Results Electrophoretic analysis of the DMD gene showed a large deletion from exons 45 to 52 in proband HA142 (Fig. 1). No deletions were detected in proband’s mother (HA139) or brother (HA141) (data not shown). F8 gene analysis showed that the normal BclI genomic digestion pattern of 21.5, 16, and 14 kb DNA fragments was altered to 20, 17.5, and 14 kb both in patient HA142 and his affected brother HA141. The loss of the 21.5-kb fragment indicated the F8 intron 22 inversion, distal/type I. The patient’s mother was shown to be heterozygous for the inversion, whereas the father HA140 showed a normal band pattern (Fig. 2).

Combined HA and DMD

Fig. 2 Autoradiograph of a Southern blot showing the DNA pattern of F8 gene after digestion with BclI (N—control DNA of a healthy female HA139—mother (carrier of F8 intron 22 inversion, distal/type I), HA140—father (healthy male DNA), HA141—brother of case patient (F8 intron 22 inversion, distal/type I), HA142—patient (F8 intron 22 inversion, distal/type I))

Discussion We elucidated that HA was hereditary, since both siblings and mother carrier had the F8 intron 22 inversion, distal/ type I. Inhibitory antibodies to FVIII are the most serious treatment complication affecting the HA patients [9]. Our patient occasionally needed the FVIII and rFVIIa replacement therapy. Although he was a high responder patient, he never underwent standard immune tolerance induction to eradicate the inhibitors. However, he received rFVIIa, PCC or APCC therapies without complications. It is believed that the type of mutation, age, intensity of first exposure to FVIII, and the nature of the product used in therapy could be risk factors for inhibitor formation [10]. The siblings were both treated with the same FVIII concentrates and carry the same mutation, however, only the proband expressed inhibitors; therefore, these factors could not influence inhibitor development in the proband. The individual risk of developing an inhibitor is further influenced by inherited characteristics of the immune system such as the HLA class-II haplotype [11]. The proband and his brother shared the same maternal haplotype HLADRB1*1101/4/6/8/10/12/13/DQB1*0301/DQA1*0501, but differed in their paternal haplotype. Only the patient with inhibitors had the haplotype HLA-DRB1*1501/ DQB1*0502/DQA1*0102, which is almost identical with HLA-DRB1*1501/DQB1*0602/DQA1*0102 shown to be

associated with the presence of inhibitors in patients with F8 intron 22 inversion [11]. DMD in case patient was caused by a large deletion encompassing exons 45–52 of the DMD gene. His brother showed no symptoms of muscular degeneration and did not, as expected, carried the defect. As the deletion was not present in the patient’s mother, who also had normal CK levels, we propose that either the mother had gonadal mosaicism or the DMD mutation evolved de novo in the affected patient. It has been estimated that approximately one-third of the cases result from the de novo mutations [12]. The emergence of relatively frequent change in DMD gene in the patient could be linked to his genetic background, hereditary F8 intron 22 inversion (distal/type I) and specific HLA haplotype. Literature mining revealed only one such report, where both HA and DMD occurred in three relatives in the same family [13]. However, Konagaya et al. showed that the maternal grandmother and mother of the proband were carriers of both diseases, whereas in our case the DMD mutation had probably arisen de novo. Recently, there has been a new case report describing a patient with combined hemophilia B and DMD, however, the family genetic background was not known [14]. F8 and DMD genes are located on different arms of chromosome X and at this point we are not able to speculate what genetic or epigenetic mechanisms could influence the impairment of DMD gene. Nevertheless, in the light of our and previous evidence, the identification of these unusual cases adds valuable new information to medical knowledge and with increasing availability of genetic testing hemophilia patients could be simultaneously tested for DMD. Acknowledgments This work was supported by the grant J1-8762 provided by the Slovenian Research Agency to RK and LS. The authors would like to thank Dr. Gian Antonio Danieli and Dr. Maurizio Rosa for their contributions to molecular analysis of DMD gene. We also thank Dr. Jane Gitschier, Howard Hughes Medical Institute, University of California, San Francisco, for kindly supplying the plasmid p482.6 probe. Ethical standard The study was approved by the National Medical Ethics Committee of the Republic of Slovenia. The patient and the family members were informed about the purpose, procedures, meaning, limitations and possible consequences of these genetic analyses by the treating physician and signed consent was obtained. Conflict of interest

The authors declare no conflict of interest.

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