Genetic diagnosis and patient registry of Hungarian DMD

and SMA patients in collaboration with TREAT-NMD

and CARE-NMD

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

Veronika Karcagi1, Márta Garami1, Henriett Pikó1, László Tímár2,Ágnes

Herczegfalvi3

EAMDA, 42nd Annual General Meeting, Budapest, October 4-7. 2012

Overview

1. Duchenne/Becker muscular dystrophy

(DMD/BMD)

2. Spinal muscular atrophy (SMA)

3. TREAT-NMD Consortium and patient

registries

4. CARE-NMD Consortium and care standards

for DMD

Duchenne/BeckerMuscular 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

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 of the gene (ex02- ex10 and ex44-ex52)

• Intragenic duplications (5-8 %)

• Point mutations and splicing errors (30-35 %)

1/3 of cases are de novo mutations

High ratio of germinal/somatic mosaicism

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

Molecular analyses of the dystrophin gene and of the protein

Multiplex PCR reaction (2x9 exons simultanously amplified in two reactions; Beggs and Chamberlain), since 2001.

Southern blot using cDNA probes (XJ10,7b8,30.2, 30.1, 47.4, 60.1) since 2002.

MLPA: Multiplex Ligation-dependent Probe Amplification (79 exons and promoter analysed; MRC Holland), since 2006.

Immunohistochemistry of muscle biopsy (LMU Munich, H. Lochmüller, Molnar MJ, SE Clinic for Neurology)

Western blot technique (LMU Munich, H. Lochmüller, Molnar MJ, SE Clinic for Neurology)

Sequencing of the dystrophin gene (Univ.Würzburg, Leiden, Ferrara)

Multiplex PCR analysis

MLPA analysis

Dystrophin gene ex45-ex46 deletion

Dystrophin gene ex51-ex55 duplication

Sample 66/3, affected boy, deletion Dp427m-ex44 (Score:-0,715)

NMD-CHIP array analysis in the dystrophin gene

Sample 66/2, carrier mother, deletion Dp427m-ex44 (Score:-0,281)

NMD-CHIP array analysis in the dystrophin gene

318 DMD/BMD male patients

analysed

in 119 patients no mutations found

in 199 patients mutations confirmed

in 163 patients deletions found

in 23 patients duplications

found

in 13 patients point mutations

confirmed

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)

Results – male patients

150 female relatives analysed

105 mothers

15 males out of 40 prenatal

cases

10 affected5 unaffected

Results – female relatives, prenatal diagnosis

40 sisters/cousins5 daughters

48 non-carrier mothers found

57 carrier mothers found

5 manifesting carriers (2 de novo cases)

17 non-carrier sisters/cousins

found

12 cases confirmed by MLPA without having the sample of index

patient

11 carrier sisters/cousins

found

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

2.Prevalence: 1/6000, carrier frequency 1/35

Classification of spinal muscular atrophies

type onset movements life span

Werdnig- Hoff mann (SMA I . acute)

< 3 months

doesn't sit

< 2 year

Fried- Emery (SMA I I . intermedier)

> 6 months

sits unaided, cannot stand

> 4 years, but short

Kugelberg- Welander (SMA I I I . chronic)

> 3 years

able to walk

small decrease

Adult form >30 years able to walk normal (SMA IV.)

SMA region and pathogenic mutations

• SMA determinant gene: SMN (survival motor neuron)

• 5q13 locus: 500 kb element with inverted duplication

homologue copies of SMN, 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

Genetic diagnostic tests at NIEH

1. Direct mutation screening:

• PCR and RFLP; SMN1 gene exon 7 (and 8) deletion

• Homozygous state = genetic confirmation of SMA

• Not suitable for carrier testing

2. Haplotype analysis:

• Polymorph microsatellite markers in 5q13 flanking the SMN gene

• Always performed in prenatal diagnosis

3. Quantitative real-time PCR

Hungarian SMA patientsIn 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:Phenotyp

eNumber of patients

Point mutations

SMA I. 1

SMA II. 5 1 e5 S230L

SMA III. 12 3 e5 S230L

e6 Y272C

e2a S28S

Total 18

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 analyses from 118 families were

offered with the outcome of 144 healthy and

56 affected fetuses

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TREAT-NMD

A network of excellence to catalyse research infrastructure globally

Advancing diagnosis, care and treatment for people with neuromuscular diseases around the world

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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

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Suite of tools and resources to accelerate therapy delivery for NMD

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Global databases 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

TREAT-NMD patient registries

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Patient registries for DMD, SMA

Patient self-report

Clinician / geneticist report

National registry

TREAT-NMDGlobal

Registry

Curation!

The TREAT-NMD setup

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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

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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

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TREAT-NMD: an infrastructure for the neuromuscular field

To prepare for clinical trials = trial readiness

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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 patient registry, clinics and patient organizations

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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

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CARE-NMD Project

Dissemination and Implementation of the Standards of Care for Duchenne Muscular Dystrophy in Europe (7 partners, including Eastern countries)

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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

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NIEH activity on TREAT-NMDRegistries and Dissemination of information

Registered DMD patients 110 Registered SMA patients: 83 Homepage

www.treat-nmd.huo News: events, clinical trials, latest researcho Registration toolkits

– Informed consent, self report-form (SMA, DMD)– We invite Hungarian patients from all over the word – FAQ

o Downloads: DMD and SMA Family Guides, flyers – DMD, SMA

FaceBook page Meetings with Hungarian DMD and SMA patient organizations

Some results of Duchenne Care Survey in Hungary

Within the frame of an international assessment

2011.

Total number of received questionnaires: 67Total number of evaluated questionnaires: 57Filling out the questionnaire: n=57

The person with DMD on his own 0%The parents of the person with DMD/other 98,2% Both, the person with DMD and parents/other together 1,8% Missing n= 0

Basic information of participants

Main age: 10.53 Age range: 1,75-25,92

Current occupation of participants

• Nursery 19.3% ………………..• School 42.1%.....................• Special school 15.8%..........• Vocational training 8.8%...• University 1.8%.................

1

2

3

4

Job 0%

Daily activity

Personal assistant at home/nursery/school/job/university

• No 73.7% …………………………………..• not every day 12.3%.......................• for less than 6 hours per day 5.3%• 24 hours per day 8.8%..................

Living with parents or with other relatives 100%

1234

Age when the family first expressed concerns about motor development to their doctor

• mean age 3.21 yrs• range 0.42-8.17 yrs

Time from first concerns to diagnosis

• mean age 1.91 yrs • range 1- yrs

Health information of participants

Current stage of Duchenne Presymptomatic 10.9%

Early-ambulatory 34.5%

Late-ambulatory 3.6%

Early non-ambulatory 16.4%

Ambulatory 38.1%l

Non-ambulatory I 14.5%

Non-ambulatory II 16.4%

Non-ambulatory III 3.6%

Non-ambulatory 50.9%

Mobility

Lost the ability to walk n=25• mean age 9.84 yrs.• Range 1,33-18,17 yrs.Sitting ability n=55• Without support 92,7% ….• With support 7,3% …..• No sitting 0%

1

2

Assisted devicesWheelchair use

• Wheelchair 51.1%demanded 2.2%

• Electric wheelchair 44.4% demanded 8.9%

• Sitting support in wheelchair 10.0% demanded 10%Transfer device 9.8% demanded 29.3%

Bathroom equipment 31.0% demanded 23.8%

adjustable bed 16.7% demanded 23.8%

Computer to compensate functional loss 27.9% demanded 4.7%

Health care

Pulmonary care

Cardiac follow up

Physiotherapy

Scoliosis prevention, surgery

Psychology support, socio-economic issues

Steroid treatment

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www.treat-nmd.eu

www.treat-nmd.hu

garami.marta@oki.antsz.hu

karcagi.veronika@oki.antsz.hu

More information