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1 National Institute of Environmental Health, Dept. of Molecular Genetics and Diagnostics,

Genetic diagnosis and patient registry of Hungarian DMD and SMA patients in collaboration with TREAT-NMD and CARE-NMD. Veronika Karcagi 1 , Márta Garami 1 , Henriett Pikó 1 , László Tímár 2 ,Ágnes Herczegfalvi 3.

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1 National Institute of Environmental Health, Dept. of Molecular Genetics and Diagnostics,

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  1. Genetic diagnosis and patient registry of Hungarian DMD and SMA patients in collaboration with TREAT-NMD and CARE-NMD Veronika Karcagi1, Márta Garami1, Henriett Pikó1, László Tímár2,Ágnes Herczegfalvi3 1National Institute of Environmental Health, Dept. of Molecular Genetics and Diagnostics, 2National Institute of Children’s Health, Genetic Counselling Unit 3Semmeilweis Medical Univ.,1st Paediatric Clinic, Dept. of Neurology EAMDA, 42nd Annual General Meeting, Budapest, October 4-7. 2012

  2. Overview • Duchenne/Becker muscular dystrophy (DMD/BMD) • Spinal muscular atrophy (SMA) • TREAT-NMD Consortium and patient registries • CARE-NMD Consortium and care standards for DMD

  3. Duchenne/Becker Muscular Dystrophy • DMD/BMD caused by mutation of the dystrophin gene; XR • DMD incidence of 1 in 3500 live born males • BMD is a milder form with a later onset and slower clinical progression; incidence 1:30000

  4. Genetic background • The dystrophin gene (Xp21) has 79 coding exons, substantial amount of alternative splicing and at least seven tissue-specific promoters • Mutations • Deletions (60 %), in hot-spot regions of ofthegene (ex02- ex10 and ex44-ex52) • Intragenicduplications (5-8 %) • Pointmutations and splicingerrors (30-35 %) • 1/3 of casesare de novomutations • High ratio of germinal/somaticmosaicism • Phenotype and genotype correlations • in frame mutation: Becker phenotype (reduced dystrophin protein level or truncated dystrophin) • out of frame mutation:Duchenne phenotype (absence of dystrophin protein) • Point mutations mostly with stop codons – DMD phenotype

  5. Molecular analyses of the dystrophin gene and of the protein • Multiplex PCR reaction (2x9 exonssimultanouslyamplified in two reactions; Beggs and Chamberlain), since 2001. • Southern blotusingcDNAprobes (XJ10,7b8,30.2, 30.1, 47.4, 60.1) since 2002. • MLPA: Multiplex Ligation-dependentProbeAmplification (79 exons and promoter analysed; MRC Holland), since 2006. • Immunohistochemistryof muscle biopsy (LMU Munich, H. Lochmüller, Molnar MJ, SE ClinicforNeurology) • Western blottechnique (LMU Munich, H. Lochmüller, Molnar MJ, SE ClinicforNeurology) • Sequencing of thedystrophingene (Univ.Würzburg, Leiden, Ferrara)

  6. Multiplex PCR analysis

  7. MLPA analysis Dystrophin gene ex45-ex46 deletion Dystrophin gene ex51-ex55 duplication

  8. NMD-CHIP array analysis in the dystrophin gene Sample 66/3, affected boy, deletion Dp427m-ex44 (Score:-0,715)

  9. NMD-CHIP array analysis in the dystrophin gene Sample 66/2, carrier mother, deletion Dp427m-ex44 (Score:-0,281)

  10. Results – male patients 318DMD/BMD male patients analysed in 119 patients no mutations found in 199 patients mutations confirmed in23 patients duplications found in 13 patients point mutations confirmed in 163 patients deletions found in 149 patients deletions in hot-spot regions (ex44-ex52 and ex02-ex10) in 12 patients rare deletions (detected only by MLPA) 2 patients with contigousgene deletion syndrome (dystrophin + ARX, IL1RAP1l, NR0B1, GK, RPGR genes)

  11. Results – female relatives, prenatal diagnosis 15 males out of 40 prenatal cases 150female relativesanalysed 5 unaffected 10 affected 105 mothers 5 daughters 40 sisters/cousins 12 cases confirmed by MLPA without having the sample of index patient 48 non-carrier mothers found 57 carrier mothers found 5 manifesting carriers (2 de novo cases) 17 non-carrier sisters/cousins found 11 carrier sisters/cousins found

  12. Diagnostic criteria and prevalence of SMA 1. Typical clinical features • progressive symmetrical limb and trunk paralysis associated with muscular athrophy • Signs of denervation on EMG-ENG • Histopathology confirms denervation process in muscle biopses • Prevalence: 1/6000, carrier frequency 1/35

  13. Classification of spinal muscular atrophies

  14. SMA region and pathogenic mutations SMA determinant gene: SMN (survival motor neuron) 5q13 locus: 500 kb element with inverted duplication homologue copies ofSMN, NAIP and other genes SMN1 exon7 deletion/gene conversion in 96% of patients 4% compound heterozygous (deletion/gene conversion + point mutation) SMN2 responsible for the different phenotypes more SMN2 copies decrease disease severity

  15. Genetic diagnostic tests at NIEH • Direct mutation screening: • PCR and RFLP; SMN1 gene exon 7 (and 8) deletion • Homozygous state = genetic confirmation of SMA • Not suitable for carrier testing • Haplotype analysis: • Polymorph microsatellite markers in 5q13 flanking the SMN gene • Always performed in prenatal diagnosis • Quantitative real-time PCR

  16. Hungarian SMA patients In total, 670 patients were referred Genetically confirmed cases (329 patients out of 312 families) SMA I: 150+ SMA II: 66+ SMA III: 77+ Uncertain: 103 Non-SMA (other NMD): 238 Compound heterozygous patients:

  17. Hungarian SMA carriers and prenatal diagnosis • Carrier analysis was requested in 114 cases (family members of index patients and spouses) and 39 relatives were confirmed as carriers • Due to the efficient genetic counselling, 200 prenatal analysesfrom 118 families were offered with the outcome of 144 healthy and 56 affected fetuses

  18. TREAT-NMD Advancing diagnosis, care and treatment for people with neuromuscular diseases around the world A network of excellence to catalyse research infrastructure globally

  19. What is TREAT-NMD? • A “network of excellence” funded by the European Union (but with global collaborations) • Aims to help promising new treatments for neuromuscular diseases make the transition from the lab to the patient • Not a research project but an infrastructure project • Creating the “tools” for trial-readiness in the neuromuscular field • Helping researchers and expert centres collaborate better • Improving patient care worldwide • 21 core European partner organizations • Since 2012 TREAT-NMD Alliance, world-wide membership

  20. Suite of tools and resources to accelerate therapy delivery for NMD

  21. TREAT-NMD patient registries Globaldatabases of patients with the genetic and clinical data necessary for clinical trial feasibility and recruitment • Many benefits to registered patients • Feedback on standards of care and new research developments • Feeling a sense of “belonging” to a broader community • Not being left behind as clinical trials develop • A link to the research community • Many benefits to industry • Easy access to patient community • Clear concept of target market • Feasibility and planning of clinical trials • Recruitment of patients into clinical trials

  22. Patient registries for DMD, SMA The TREAT-NMD setup Patient self-report Curation! TREAT-NMDGlobalRegistry National registry Clinician / geneticist report

  23. Patient registries: legal/ethical best practice • Feedback to patients • Possibility of data withdrawal • Informed consent form • Pseudonymised (encrypted) data • Frequent updates of data The TREAT-NMD registries adhere to these principles via the TREAT-NMD registry charter

  24. How can the Eastern-European countries get involved? As doctors or patients/parents : • Register in the network of patient care and clinical trial sites: www.treat-nmd.eu/trialsites • Or register through the Hungarian national registry: www.treat-nmd.hu • Encourage patients to join the patient registry • Look out for the standards of care

  25. TREAT-NMD: an infrastructure for the neuromuscular field To prepare for clinical trials = trial readiness

  26. Standards of care -Family guide to SMA care • Consensus Statement for Standard of Care in Spinal Muscular Atrophy; Ching H. Wang et al., J Child Neurol 2007 • Created in collaboration with patient advocacy groups • Available online • Translations into Hungarian: www.treat-nmd.hu/images/stories/anyagok/sma_family_guide.pdf • Printed brochure also available, disseminated via the TREAT-NMD patientregistry, clinics and patient organizations

  27. Standards of care -New family guide to DMD care • Created in collaboration with patient advocacy groups • Printed brochure disseminated via advocacy groups, clinics and TREAT-NMD patient registries • Available online • Translations into multiple languages

  28. CARE-NMD ProjectDissemination and Implementation of the Standards of Care for Duchenne Muscular Dystrophy in Europe (7 partners, including Eastern countries)

  29. Overview CARE-NMD • CARE-NMD: DG Sanco Public Health funded project, 2010.-2013. • Bringing together leading care centers in a reference network for care of patients with DMD • Evaluate current treatment practices across Europe • Implement newly agreed international consensus care recommendations • Registration in national patient registries and care and trial Site Registry, respectively, will be encouraged. • Evaluate the impact of care on patients’ quality of life

  30. NIEH activity on TREAT-NMDRegistries and Dissemination of information • Registered DMD patients 110 • Registered SMA patients: 83 • Homepage www.treat-nmd.hu • News: events, clinical trials, latest research • Registration toolkits • Informed consent, self report-form (SMA, DMD) • We invite Hungarianpatientsfrom all over the word • FAQ • Downloads: DMD and SMA Family Guides, flyers – DMD, SMA • FaceBook page • MeetingswithHungarian DMD and SMA patientorganizations

  31. Within the frame of an international assessment 2011.

  32. Total number of received questionnaires: 67 Total number of evaluated questionnaires: 57 Filling out the questionnaire: n=57 The person with DMD on his own0% The parents of the person with DMD/other98,2% Both, the person with DMD and parents/other together1,8% Missing n= 0

  33. Basic information of participants Job 0%

  34. Daily activity Living with parents or with other relatives 100%

  35. Health information of participants

  36. Lost the ability to walk n=25 • mean age 9.84 yrs. • Range1,33-18,17 yrs. Sitting ability n=55 • Without support 92,7% …. • With support7,3% ….. • No sitting 0%

  37. Assisted devices

  38. Health care

  39. More information www.treat-nmd.eu www.treat-nmd.hu garami.marta@oki.antsz.hu karcagi.veronika@oki.antsz.hu

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