inherited (neuro-)muscular disorders

- Morbus Duchenne / Becker- Limb girdle muscular dystrophy

Dystrophinopathien Muskeldystrophie DuchenneMuskeldystrophie Becker

Einschlusskörper-Myopathien Einschlusskörpermyopathie 2Einschlusskörpermyopathie 3Einschlusskörpermyopathie IBMPFD1Einschlusskörpermyopathie IBMPFD2Einschlusskörpermyopathie IBMPFD3

Envelopathien/Emery-Dreyfuss-Muskeldystrophien Emerinopathie; Laminopathie

Gliedergürteldystrophien Gliedergürtel-Muskeldystrophie-Typ 1AGliedergürtel-Muskeldystrophie-Typ 1BGliedergürtel-Muskeldystrophie-Typ 1CGliedergürtel-Muskeldystrophie-Typ 1DGliedergürtel-Muskeldystrophie-Typ 1EGliedergürtel-Muskeldystrophie-Typ 1FGliedergürtel-Muskeldystrophie-Typ 1GGliedergürtel-Muskeldystrophie-Typ 1HGliedergürtel-Muskeldystrophie-Typ 2AGliedergürtel-Muskeldystrophie-Typ 2BGliedergürtel-Muskeldystrophie-Typ 2CGliedergürtel-Muskeldystrophie-Typ 2DGliedergürtel-Muskeldystrophie-Typ 2EGliedergürtel-Muskeldystrophie-Typ 2FGliedergürtel-Muskeldystrophie-Typ 2GGliedergürtel-Muskeldystrophie-Typ 2HGliedergürtel-Muskeldystrophie-Typ 2IGliedergürtel-Muskeldystrophie-Typ 2JGliedergürtel-Muskeldystrophie-Typ 2KGliedergürtel-Muskeldystrophie-Typ 2LGliedergürtel-Muskeldystrophie-Typ 2MGliedergürtel-Muskeldystrophie-Typ 2NGliedergürtel-Muskeldystrophie-Typ 2OGliedergürtel-Muskeldystrophie-Typ 2PGliedergürtel-Muskeldystrophie-Typ 2QGliedergürtel-Muskeldystrophie-Typ 2RGliedergürtel-Muskeldystrophie-Typ 2S

Kardiomyopathien (>50 Formen) Dilatative Kardiomyopathie (MYBPC3)Dilatative Kardiomyopathie (LMNA)Dilatative Kardiomyopathie (SGCD)

Kongenitale Myasthenien Kongenitale Myasthenie (MUSK)Kongenitale Myasthenie (RAPSN)>6 weitere Formen

Kongenitale Myopathien Bethlem-MyopathieFukuyama-MDCMuscle-Eye-Brain diseaseWalker-Warburg SyndromWalker-Walker Syndrom Kongenitale Muskeldystrophie Typ 1BKongenitale Muskeldystrophie Typ 1CKongenitale Muskeldystrophie Typ 1DMyotubuläre MyopathieNemaline Myopathie 1Nemaline Myopathie 2Nemaline Myopathie 3 Nemaline Myopathie 4 Nemaline Myopathie 5Nemaline Myopathie 6Nemaline Myopathie 7Nemaline Myopathie 8Ullrich-Myopathie

Myofibrilläre Myopathien Myofibrilläre Myopathie 1Myofibrilläre Myopathie 2Myofibrilläre Myopathie 3Myofibrilläre Myopathie 4Myofibrilläre Myopathie 5Myofibrilläre Myopathie 6

Myotonien/Myot. Dystrophien Kongenitale Muskeldystrophie Typ 1AHypokaliämische periodische Paralyse 1Hypokaliämische periodische Paralyse 2Myotone Dystrophie 1Myotone Dystrophie 2Myotonia congenita ThomsenParamyotonia congenitaProximale myotone Myopathie DM2/PROMMMyotonia congenita Becker

Struktur-Myopathien Central-Core-MyopathieMultiminicore-MyopathieMaligne Hyperthermie 1-6Rigid-Spine-Syndrom

Weitere Muskeldystrophien Fazioskapulohumerale Muskeldystrophie 1Fazioskapulohumerale Muskeldystrophie 2Okulopharyngeale Muskeldystrophie

adinherited muscle disorders

pseudo-hypertrophy

lordosis,scapulae

alatae

Gower maneuvre

DMD / BMD: clinical aspects

DMD

• 1:3500 newborn boys

• age of onset: 2-4 years

• life expectation 20 J.

• progr. dystrophy skeletal muscles

• pseudohypertrophy of calves

• Creatin kinase in blood

• heart muscle affected, ECG!

• mental retardation not obligatory

• smooth muscles involved as well

BMD

• 1:35000 newborn boys

• milder symptoms, esp. childhood

• rarely late learning to walk

• >20 y difficult to run, climb steps

• in mid-life unable to walk

• Creatin kinase in blood

• often learning disabilities

• own children

Dystrophin protein

alternative Dystrophin mRNA species:

1. alternative promotors

2. alternative splicing

3. alternative polyadenylation start

DMD / BMD: „self-cure“ by alternative mRNAs ?

promotors Dystrophin gene

B, brain M, muscle P, PurkinjeR, retinaB,K, brain and kidneyS, Schwann cellsG, general

Dystrophin and interacting proteins

DMD / BMD: genetics

• X chromosomal recessive

• Xp21; 2.3 Mb; 79 exons

• 14 kb transcript; different tissue specif. isoforms

• 1/3 point mutations

• 2/3 deletions/ rearrangements (5% insertions)

• 1/3 new mutations

• Deletionen sehr variabel

• DMD: meist Leserahmen -

• BMD: meist Leserahmen +

DMD / BMD: deletions

Exon 44

hotspot for deletions

intron 44

Dystrophin mutations

Dystrophin gene variations http://www.dmd.nl/DMD_deldup.html; refreshed 1.3.2017

variation n = position

5‘ UTR coding intronic 3‘ UTR

substitutions 20248 120 4000 15926 202

deletions 2521 3 635 1668 215

duplications 2119 6 213 1541 359

insertions 465 2 25 434 4

insertion + deletion 100 59 41

2 variations in 1 allele 200

complex + unknown 175 2

25828 131 4932 19612 780

DMD / BMD: point mutations

• BMD patients mostly missense mutations in N

or C terminus or point mutations at splice

sites without affecting reading frame

• DMD patients mostly nonsense mutations, spread

all over the gene

DMD / BMD diagnosis

DNA sequence analysis

• MLPA, in situ hybridisation deletions

• WAVE (SSCP) gel electrophoresis point mutations

Protein analysis

• Western blotting

• immunohistochemistry

RNA analysis

• RT-PCR /agarose gel electrophoresis del. + dupl. / rearrangements

• Protein Truncation Test (PTT) nonsense mutations

protein (427kD): 0.002 % of muscle mass

therapy DMD: symptomatic, no causal gene replacement therapy so far yet

dystrophinopathy

gene therapy approachesgene augmentation

via insertional or episomal vectors

genome editingvia programmable nucleases

exon skippingvia antisense oligonucleotides

target-site specific modification unexplored delivery method, off-target mutagenesis

expression of cDNA (µdystrophin or µUTRN) random integration, low transduction efficiency

systematic delivery feasible transient and off-target effects

chromosomal DNA

genomic DNA

pre-mRNA

mature mRNA

protein

„mis-“translation of stop codons

PTC124 read-through systematic delivery feasible

• nonsense mutations translation stop

- therapy: PTC124 read-through

stop codon „mis“-translated

- Dystrophin production in cell culture

- improved muscle function in mouse model

- clinical phase I / II studies

• exon skipping with AON (antisense

oligonucleotide)

- clinical studies in progress

PTC124 (Ataluren) + AON

dystrophinopathystem cell therapy

muscular dystrophy Golden Retriever

escaper affected

escaper affected

chromos. 24 Jagged 1

candidateregion

Jagged 1overexpression

dystrophic zebrafish

rescued zebrafish

modifier gene effect

Dystrophin427 kD

Dp260

Dp140

Dp116

Dp71

ab3

ab2ab1

ab1

ab2

Actin binding rod domain

ß-Dystroglycan/Syntrophin binding

proteinhomo-logies

compensatorymechanisms+therapeuticapproaches

up-regulation of compensatory proteins

membrane stabilisation

up-regulation of compensatory proteins

α-dystroglycan glycosilation

Integrin Dystrophin

UtrophinActin

Actin

suppression of premature stop codon via PTC 124

normalstop codon

„normal“protein

substituted amino acid

ribosome

mRNA

spliceosome

wildtype protein

exon skipped

DMD deletion truncated

TGA

exon a exon b exon cintron a intron bU1

U2

U2

U1

sf sf

exon a exon b exon c +intron a intron b

splicing

exon a exon b exon cintron a intron bU1

U2

sf sf

exon a exon c +exon b

intron asf

sso

Sso – mediated splice switching

mutation

X chromosome inactivation

XpXm zygote

XpXm XpXm

Xp Xm XpXm

early divisions

random X inactivation

Xp Xm XpXm

mosaic

inactivation patterntransmitted to all daughter cells

M. Duchenne/Becker ♀♀

1 : 50 000 000 asymmetric X inactivation 45X 2 mutations

XX mutation carrier: cardiomyopathy?

Chr. 4 der(4) X-Chr. der(X)

translocation t(X;4)(p21;q31)

autosomal dominant LGMD

form chromos. protein

LGMD1A 5q31 Myotilin

LGMD1B, ADEDMD... 1q21 Lamin A/C

LGMD1C 3p25 Caveolin 3

LGMD1E 7q DNAJB6

LGMD1F 7q32 Transportin 3

LGMD1G 4q21 HNRNPDL heterogenous nuclear ribonucleoprotein D-like protein

LGMD1H 3p23 ? {refreshed 1.3.2017}

Form Chromosom ProteinLGMD2A 15q Calpain 3LGMD2B 2p DysferlinLGMD2C-2F 13q, 17q, 4q, 5q SarkoglykansLGMD2G 17q TCAPLGMD2H 9q TRIM32LGMD2I 19q Fukutin-rel. proteinLGMD2J 2q TitinLGMD2K 9q POMT-1LGMD2L 11p ANO5LGMD2M 9q FKTNLGMD2N 14q POMT2LGMD2O 1p POMGNT1LGMD2P 3p DAG1LGMD2Q 8q PlectinLGMD2R 2q DESLGMD2S 4q TRAPPC11LGMD2T 3p GMPPBLGMD2U 7p21 ISPDLGMD2W 2q14 LIMS2LGMD2X 6q21 BVESLGMD2Y 1q25 TOR1AIP1LGMD2Z 3q13 POGLUT1

{refreshed 1.3.2017}

103 genes

Conclusions

gene diagnostics complex - mostly definitive result

gene analysis helps to understand causal pathogenesis? therapeutic options ?

multiple sclerosis (MS)a common disease

- pathogenesis

- clinics

- optimised therapy / prevention ?

- „inheritance“

„autoimmunity“environment

genetics„hygiene“MS

complex diseases

geneticvariations

locus 1, 2, 3…

bio-molecular interactions

ind

epen

den

t m

ain

eff

ects

; (n

on

-) a

dd

itiv

e

epigenetics, epistasis

gene expression ↑↓

non -additive

no / minimal effects

non -additiv

intrinsic factorsage, sex, generalhealth conditions…

# in

div

idu

als

symptoms

environment factorsfood, hygiene, infections toxins, medication…

MS

MS - pathogenesis

scarred myelinmyelin

sheath

nerve fiber

cell soma

healthynerve

MS -demyelination

• autoimmunity (?)

• neuro-degeneration (?)

• apoptosis (?)

ad de-/remyelination

shadow plaques, remyelinated

demyelinated

partial remyelination

symptoms

• sensory

• cognitive

• dysfunctions

diagnosis

• MRI

• Ig

• evoked potentials:

visual, auditory

T1, T2 MRI in MS

ad clinics

MS symptoms

- double vision / dizziness

- ataxia (cerebellar signs)

- motor disturbances

- sensible disturbances

age

patients

male MS

female MS

MS courses

time

relaps/ rem.

boutprogression

time

sec. chron. progressive

time

prim. progr.

remyelin

ation

MS - risksU

V in

tensity

0°

ttgctgtgtgaggcagaacctgcgggggcaggggcgggctggttccctggccagccattggcagagtccgcaggctagggctgtcaatcatgctggccggcgtggccccgcctccgccggcgcggccccgcctccgccggcgcacgtctgggacgcaaggcgccgtgggggctgccgggacgggtccaagatgga

cggccgctcaggttctgcttttacctgcggcccagagccccattcattgccccggtgctgagcggcgccgcgagtcggcccgaggcctccggggactgccgtgccgggcgggagaccgccatggcgaccctggaaaagctgatgaaggccttcgagtccctcaagtccttccagcagcagcagcagcagcagcag

cagcagcagcagcagcagcagcagcagcagcagcagcagcagcagcagcagcagcagcagcagcagcagcagcagcagcagcagcagcagcagcagcagcagcagcagcagcagcagcagcagcagcagcagcagcagcagcagcagcagcagcagcagcagcagcagcagcagcagcagcagcagcagcagcag

cagcagcagcagcagcagcagcagcagcagcagcagcagcaacagccgccaccgccgccgccgccgccgccgcctcctcagcttcctcagccgccgccgcaggcacagccgctgctgcctcagccgcagccgcccccgccgccgcccccgccgccacccggcccggctgtggctgaggagccgctgcaccgacca

aagaaagaactttcagctaccaagaaagaccgtgtgaatcattgtctgacaatatgtgaaaacatagtggcacagtctgtcagaaattctccagaatttcagaaacttctgggcatcgctatggaactttttctgctgtgcagtgatgacgcagagtcagatgtcaggatggtggctgacgaatgcctcaacaaa

gttatcaaagctttgatggattctaatcttccaaggttacagctcgagctctataaggaaattaaaaagaatggtgcccctcggagtttgcgtgctgccctgtggaggtttgctgagctggctcacctggttcggcctcagaaatgcaggccttacctggtgaaccttctgccgtgcctgactcgaacaagcaag

agacccgaagaatcagtccaggagaccttggctgcagctgttcccaaaattatggcttcttttggcaattttgcaaatgacaatgaaattaaggttttgttaaaggccttcatagcgaacctgaagtcaagctcccccaccattcggcggacagcggctggatcagcagtgagcatctgccagcactcaagaagg

acacaatatttctatagttggctactaaatgtgctcttaggcttactcgttcctgtcgaggatgaacactccactctgctgattcttggcgtgctgctcaccctgaggtatttggtgcccttgctgcagcagcaggtcaaggacacaagcctgaaaggcagcttcggagtgacaaggaaagaaatggaagtctct

ccttctgcagagcagcttgtccaggtttatgaactgacgttacatcatacacagcaccaagaccacaatgttgtgaccggagccctggagctgttgcagcagctcttcagaacgcctccacccgagcttctgcaaaccctgaccgcagtcgggggcattgggcagctcaccgctgctaaggaggagtctggtggc

cgaagccgtagtgggagtattgtggaacttatagctggagggggttcctcatgcagccctgtcctttcaagaaaacaaaaaggcaaagtgctcttaggagaagaagaagccttggaggatgactctgaatcgagatcggatgtcagcagctctgccttaacagcctcagtgaaggatgagatcagtggagagctg

gctgcttcttcaggggtttccactccagggtcagcaggtcatgacatcatcacagaacagccacggtcacagcacacactgcaggcggactcagtggatctggccagctgtgacttgacaagctctgccactgatggggatgaggaggatatcttgagccacagctccagccaggtcagcgccgtcccatctgac

cctgccatggacctgaatgatgggacccaggcctcgtcgcccatcagcgacagctcccagaccaccaccgaagggcctgattcagctgttaccccttcagacagttctgaaattgtgttagacggtaccgacaaccagtatttgggcctgcagattggacagccccaggatgaagatgaggaagccacaggtatt

cttcctgatgaagcctcggaggccttcaggaactcttccatggcccttcaacaggcacatttattgaaaaacatgagtcactgcaggcagccttctgacagcagtgttgataaatttgtgttgagagatgaagctactgaaccgggtgatcaagaaaacaagccttgccgcatcaaaggtgacattggacagtcc

actgatgatgactctgcacctcttgtccattgtgtccgccttttatctgcttcgtttttgctaacagggggaaaaaatgtgctggttccggacagggatgtgagggtcagcgtgaaggccctggccctcagctgtgtgggagcagctgtggccctccacccggaatctttcttcagcaaactctataaagttcct

cttgacaccacggaataccctgaggaacagtatgtctcagacatcttgaactacatcgatcatggagacccacaggttcgaggagccactgccattctctgtgggaccctcatctgctccatcctcagcaggtcccgcttccacgtgggagattggatgggcaccattagaaccctcacaggaaatacattttct

ttggcggattgcattcctttgctgcggaaaacactgaaggatgagtcttctgttacttgcaagttagcttgtacagctgtgaggaactgtgtcatgagtctctgcagcagcagctacagtgagttaggactgcagctgatcatcgatgtgctgactctgaggaacagttcctattggctggtgaggacagagctt

ctggaaacccttgcagagattgacttcaggctggtgagctttttggaggcaaaagcagaaaacttacacagaggggctcatcattatacagggcttttaaaactgcaagaacgagtgctcaataatgttgtcatccatttgcttggagatgaagaccccagggtgcgacatgttgccgcagcatcactaattagg

cttgtcccaaagctgttttataaatgtgaccaaggacaagctgatccagtagtggccgtggcaagagatcaaagcagtgtttacctgaaacttctcatgcatgagacgcagcctccatctcatttctccgtcagcacaataaccagaatatatagaggctataacctactaccaagcataacagacgtcactatg

gaaaataacctttcaagagttattgcagcagtttctcatgaactaatcacatcaaccaccagagcactcacatttggatgctgtgaagctttgtgtcttctttccactgccttcccagtttgcatttggagtttaggttggcactgtggagtgcctccactgagtgcctcagatgagtctaggaagagctgtacc

gttgggatggccacaatgattctgaccctgctctcgtcagcttggttcccattggatctctcagcccatcaagatgctttgattttggccggaaacttgcttgcagccagtgctcccaaatctctgagaagttcatgggcctctgaagaagaagccaacccagcagccaccaagcaagaggaggtctggccagcc

ctgggggaccgggccctggtgcccatggtggagcagctcttctctcacctgctgaaggtgattaacatttgtgcccacgtcctggatgacgtggctcctggacccgcaataaaggcagccttgccttctctaacaaaccccccttctctaagtcccatccgacgaaaggggaaggagaaagaaccaggagaacaa

gcatctgtaccgttgagtcccaagaaaggcagtgaggccagtgcagcttctagacaatctgatacctcaggtcctgttacaacaagtaaatcctcatcactggggagtttctatcatcttccttcatacctcaaactgcatgatgtcctgaaagctacacacgctaactacaaggtcacgctggatcttcagaac

agcacggaaaagtttggagggtttctccgctcagccttggatgttctttctcagatactagagctggccacactgcaggacattgggaagtgtgttgaagagatcctaggatacctgaaatcctgctttagtcgagaaccaatgatggcaactgtttgtgttcaacaattgttgaagactctctttggcacaaac

ttggcctcccagtttgatggcttatcttccaaccccagcaagtcacaaggccgagcacagcgccttggctcctccagtgtgaggccaggcttgtaccactactgcttcatggccccgtacacccacttcacccaggccctcgctgacgccagcctgaggaacatggtgcaggcggagcaggagaacgacacctcg

ggatggtttgatgtcctccagaaagtgtctacccagttgaagacaaacctcacgagtgtcacaaagaaccgtgcagataagaatgctattcataatcacattcgtttgtttgaacctcttgttataaaagctttaaaacagtacacgactacaacatgtgtgcagttacagaagcaggttttagatttgctggcg

cagctggttcagttacgggttaattactgtcttctggattcagatcaggtgtttattggctttgtattgaaacagtttgaatacattgaagtgggccagttcagggaatcagaggcaatcattccaaacatctttttcttcttggtattactatcttatgaacgctatcattcaaaacagatcattggaattcct

aaaatcattcagctctgtgatggcatcatggccagtggaaggaaggctgtgacacatgccataccggctctgcagcccatagtccacgacctctttgtattaagaggaacaaataaagctgatgcaggaaaagagcttgaaacccaaaaagaggtggtggtgtcaatgttactgagactcatccagtaccatcag

gtgttggagatgttcattcttgtcctgcagcagtgccacaaggagaatgaagacaagtggaagcgactgtctcgacagatagctgacatcatcctcccaatgttagccaaacagcagatgcacattgactctcatgaagcccttggagtgttaaatacattatttgagattttggccccttcctccctccgtccg

gtagacatgcttttacggagtatgttcgtcactccaaacacaatggcgtccgtgagcactgttcaactgtggatatcgggaattctggccattttgagggttctgatttcccagtcaactgaagatattgttctttctcgtattcaggagctctccttctctccgtatttaatctcctgtacagtaattaatagg

ttaagagatggggacagtacttcaacgctagaagaacacagtgaagggaaacaaataaagaatttgccagaagaaacattttcaaggtttctattacaactggttggtattcttttagaagacattgttacaaaacagctgaaggtggaaatgagtgagcagcaacatactttctattgccaggaactaggcaca

ctgctaatgtgtctgatccacatcttcaagtctggaatgttccggagaatcacagcagctgccactaggctgttccgcagtgatggctgtggcggcagtttctacaccctggacagcttgaacttgcgggctcgttccatgatcaccacccacccggccctggtgctgctctggtgtcagatactgctgcttgtc

aaccacaccgactaccgctggtgggcagaagtgcagcagaccccgaaaagacacagtctgtccagcacaaagttacttagtccccagatgtctggagaagaggaggattctgacttggcagccaaacttggaatgtgcaatagagaaatagtacgaagaggggctctcattctcttctgtgattatgtctgtcag

aacctccatgactccgagcacttaacgtggctcattgtaaatcacattcaagatctgatcagcctttcccacgagcctccagtacaggacttcatcagtgccgttcatcggaactctgctgccagcggcctgttcatccaggcaattcagtctcgttgtgaaaacctttcaactccaaccatgctgaagaaaact

cttcagtgcttggaggggatccatctcagccagtcgggagctgtgctcacgctgtatgtggacaggcttctgtgcacccctttccgtgtgctggctcgcatggtcgacatccttgcttgtcgccgggtagaaatgcttctggctgcaaatttacagagcagcatggcccagttgccaatggaagaactcaacaga

atccaggaataccttcagagcagcgggctcgctcagagacaccaaaggctctattccctgctggacaggtttcgtctctccaccatgcaagactcacttagtccctctcctccagtctcttcccacccgctggacggggatgggcacgtgtcactggaaacagtgagtccggacaaagactggtacgttcatctt

gtcaaatcccagtgttggaccaggtcagattctgcactgctggaaggtgcagagctggtgaatcggattcctgctgaagatatgaatgccttcatgatgaactcggagttcaacctaagcctgctagctccatgcttaagcctagggatgagtgaaatttctggtggccagaagagtgccctttttgaagcagcc

cgtgaggtgactctggcccgtgtgagcggcaccgtgcagcagctccctgctgtccatcatgtcttccagcccgagctgcctgcagagccggcggcctactggagcaagttgaatgatctgtttggggatgctgcactgtatcagtccctgcccactctggcccgggccctggcacagtacctggtggtggtctcc

aaactgcccagtcatttgcaccttcctcctgagaaagagaaggacattgtgaaattcgtggtggcaacccttgaggccctgtcctggcatttgatccatgagcagatcccgctgagtctggatctccaggcagggctggactgctgctgcctggccctgcagctgcctggcctctggagcgtggtctcctccaca

gagtttgtgacccacgcctgctccctcatctactgtgtgcacttcatcctggaggccgttgcagtgcagcctggagagcagcttcttagtccagaaagaaggacaaataccccaaaagccatcagcgaggaggaggaggaagtagatccaaacacacagaatcctaagtatatcactgcagcctgtgagatggtg

gcagaaatggtggagtctctgcagtcggtgttggccttgggtcataaaaggaatagcggcgtgccggcgtttctcacgccattgctcaggaacatcatcatcagcctggcccgcctgccccttgtcaacagctacacacgtgtgcccccactggtgtggaagcttggatggtcacccaaaccgggaggggatttt

ggcacagcattccctgagatccccgtggagttcctccaggaaaaggaagtctttaaggagttcatctaccgcatcaacacactaggctggaccagtcgtactcagtttgaagaaacttgggccaccctccttggtgtcctggtgacgcagcccctcgtgatggagcaggaggagagcccaccagaagaagacaca

gagaggacccagatcaacgtcctggccgtgcaggccatcacctcactggtgctcagtgcaatgactgtgcctgtggccggcaacccagctgtaagctgcttggagcagcagccccggaacaagcctctgaaagctctcgacaccaggtttgggaggaagctgagcattatcagagggattgtggagcaagagatt

caagcaatggtttcaaagagagagaatattgccacccatcatttatatcaggcatgggatcctgtcccttctctgtctccggctactacaggtgccctcatcagccacgagaagctgctgctacagatcaaccccgagcgggagctggggagcatgagctacaaactcggccaggtgtccatacactccgtgtgg

ctggggaacagcatcacacccctgagggaggaggaatgggacgaggaagaggaggaggaggccgacgcccctgcaccttcgtcaccacccacgtctccagtcaactccaggaaacaccgggctggagttgacatccactcctgttcgcagtttttgcttgagttgtacagccgctggatcctgccgtccagctca

gccaggaggaccccggccatcctgatcagtgaggtggtcagatcccttctagtggtctcagacttgttcaccgagcgcaaccagtttgagctgatgtatgtgacgctgacagaactgcgaagggtgcacccttcagaagacgagatcctcgctcagtacctggtgcctgccacctgcaaggcagctgccgtcctt

gggatggacaaggccgtggcggagcctgtcagccgcctgctggagagcacgctcaggagcagccacctgcccagcagggttggagccctgcacggcgtcctctatgtgctggagtgcgacctgctggacgacactgccaagcagctcatcccggtcatcagcgactatctcctctccaacctgaaagggatcgcc

cactgcgtgaacattcacagccagcagcacgtactggtcatgtgtgccactgcgttttacctcattgagaactatcctctggacgtagggccggaattttcagcatcaataatacagatgtgtggggtgatgctgtctggaagtgaggagtccaccccctccatcatttaccactgtgccctcagaggcctggag

cgcctcctgctctctgagcagctctcccgcctggatgcagaatcgctggtcaagctgagtgtggacagagtgaacgtgcacagcccgcaccgggccatggcggctctgggcctgatgctcacctgcatgtacacaggaaaggagaaagtcagtccgggtagaacttcagaccctaatcctgcagcccccgacagc

gagtcagtgattgttgctatggagcgggtatctgttctttttgataggatcaggaaaggctttccttgtgaagccagagtggtggccaggatcctgccccagtttctagacgacttcttcccaccccaggacatcatgaacaaagtcatcggagagtttctgtccaaccagcagccatacccccagttcatggcc

accgtggtgtataaggtgtttcagactctgcacagcaccgggcagtcgtccatggtccgggactgggtcatgctgtccctctccaacttcacgcagagggccccggtcgccatggccacgtggagcctctcctgcttctttgtcagcgcgtccaccagcccgtgggtcgcggcgatcctcccacatgtcatcagc

aggatgggcaagctggagcaggtggacgtgaaccttttctgcctggtcgccacagacttctacagacaccagatagaggaggagctcgaccgcagggccttccagtctgtgcttgaggtggttgcagccccaggaagcccatatcaccggctgctgacttgtttacgaaatgtccacaaggtcaccacctgctga

gcgccatggtgggagagactgtgaggcggcagctggggccggagcctttggaagtctgtgcccttgtgccctgcctccaccgagccagcttggtccctatgggcttccgcacatgccgcgggcggccaggcaacgtgcgtgtctctgccatgtggcagaagtgctctttgtggcagtggccaggcagggagtgtc

tgcagtcctggtggggctgagcctgaggccttccagaaagcaggagcagctgtgctgcaccccatgtgggtgaccaggtcctttctcctgatagtcacctgctggttgttgccaggttgcagctgctcttgcatctgggccagaagtcctccctcctgcaggctggctgttggcccctctgctgtcctgcagtag

aaggtgccgtgagcaggctttgggaacactggcctgggtctccctggtggggtgtgcatgccacgccccgtgtctggatgcacagatgccatggcctgtgctgggccagtggctgggggtgctagacacccggcaccattctcccttctctcttttcttctcaggatttaaaatttaattatatcagtaaagaga

ttaattttaacgaactctttctatgcccgtgtaaagtatgtgaatcgcaaggcctgtgctgcatgcgacagcgtccggggtggtggacagggcccccggccacgctccctctcctgtagccactggcatagccctcctgagcacccgctgacatttccgttgtacatgttcctgtttatgcattcacaaggtgac

tgggatgtagagaggcgttagtgggcaggtggccacagcaggactgaggacaggcccccattatcctaggggtgcgctcaactgcagcccctcctcctcgggcacagacgactgtcgttctccacccaccagtcagggacagcagcctccctgtcactcagctgagaaggccagccctccctggctgtgagcagc

ctccactgtgtccagagacatgggcctcccactcctgttccttgctagccctggggtggcgtctgcctaggagctggctggcaggtgttgggacctgctgctccatggatgcatgccctaagagtgtcactgagctgtgttttgtctgagcctctctcggtcaacagcaaagcttggtgtcttggcactgttagt

gacagagcccagcatcccttctgcccccgttccagctgacatcttgcacggtgaccccttttagtcaggagagtgcagatctgtgctcatcggagactgccccacggccctgtcagagccgccactcctatccccaggacaggtccctggaccagcctcctgtttgcaggcccagaggagccaagtcattaaaat

ggaagtggattctggatggccgggctgctgctgatgtaggagctggatttgggagctctgcttgccgactggctgtgagacgaggcaggggctctgcttcctcagccctagaggcgagccaggcaaggttggcgactgtcatgtggcttggtttggtcatgcccgtcgatgttttgggtattgaatgtggtaagt

ggaggaaatgttggaactctgtgcaggtgctgccttgagacccccaagcttccacctgtccctctcctatgtggcagctggggagcagctgagatgtggacttgtatgctgcccacatacgtgagggggagctgaaagggagcccctgctcaaagggagcccctcctctgagcagcctctgccaggcctgtatga

ggcttttcccaccagctcccaacagaggcctcccccagccaggaccacctcgtcctcgtggcggggcagcaggagcggtagaaaggggtccgatgtttgaggaggcccttaagggaagctactgaattataacacgtaagaaaatcaccattcttccgtattggttgggggctcctgtttctcatcctagctttt

tcctggaaaagcccgctagaaggtttgggaacgaggggaaagttctcagaactgttgctgctccccacccgcctcccgcctcccccgcaggttatgtcagcagctctgagacagcagtatcacaggccagatgttgttcctggctagatgtttacatttgtaagaaataacactgtgaatgtaaaacagagccat

tcccttggaatgcatatcgctgggctcaacatagagtttgtcttcctcttgtttacgacgtgatctaaaccagtccttagcaaggggctcagaacaccccgctctggcagtaggtgtcccccacccccaaagacctgcctgtgtgctccggagatgaatatgagctcattagtaaaaatgacttcacccacgcat

atacataaagtatccatgcatgtgcatatagacacatctataattttacacacacacctctcaagacggagatgcatggcctctaagagtgcccgtgtcggttcttcctggaagttgactttccttagacccgccaggtcaagttagccgcgtgacggacatccaggcgtgggacgtggtcagggcagggctcat

tcattgcccactaggatcccactggcgaagatggtctccatatcagctctctgcagaagggaggaagactttatcatgttcctaaaaatctgtggcaagcacccatcgtattatccaaattttgttgcaaatgtgattaatttggttgtcaagttttgggggtgggctgtggggagattgcttttgttttcctgc

tggtaatatcgggaaagattttaatgaaaccagggtagaattgtttggcaatgcactgaagcgtgtttctttcccaaaatgtgcctcccttccgctgcgggcccagctgagtctatgtaggtgatgtttccagctgccaagtgctctttgttactgtccaccctcatttctgccagcgcatgtgtcctttcaagg

ggaaaatgtgaagctgaaccccctccagacacccagaatgtagcatctgagaaggccctgtgccctaaaggacacccctcgcccccatcttcatggagggggtcatttcagagccctcggagccaatgaacagctcctcctcttggagctgagatgagccccacgtggagctcgggacggatagtagacagcaat

aactcggtgtgtggccgcctggcaggtggaacttcctcccgttgcggggtggagtgaggttagttctgtgtgtctggtgggtggagtcaggcttctcttgctacctgtgagcatccttcccagcagacatcctcatcgggctttgtccctcccccgcttcctccctctgcggggaggacccgggaccacagctgc

tggccagggtagacttggagctgtcctccagaggggtcacgtgtaggagtgagaagaaggaagatcttgagagctgctgagggaccttggagagctcaggatggctcagacgaggacactcgcttgccgggcctggccctcctgggaaggagggagctgctcagaatgccgcatgacaactgaaggcaacctgga

aggttcagggcccgctcttcccccatgtgcctgtcacgctctggtgcagtcaaaggaacgccttcccctcagttgtttctaagagcagagtctcccgctgcaatctgggtggtaactgccagccttggaggatcgtggccaacgtggacctgcctacggagggtgggctctgacccaagtggggcctccttgccc

aggtctcactgctttgcaccgtggtcagagggactgtcagctgagcttgagctcccctggagccagcagggctgtgatgggcgagtcccggagccccacccagacctgaatgcttctgagagcaaagggaaggactgacgagagatgtatatttaattttttaactgctgcaaacattgtacatccaaattaaag

ggaaaaaatggaaaccatcaat

MS – a multifactorial disease

demyelination(symptoms)

auto-immunity

immunegenes

neurogenes

genet. pre-disposition

Psoriasis

Depress.

Schizophr.

Diabetes

… …

Asthma

Cardiac

Cancer

Parkinson

MS↓ inheritable

↑ inheritable

MS

↑ concordant EZ

(30-40%)

many genes involved

→ complex

↓ influence individ. genes:

protect (HLA-DRB1*01)

predispose (DRB1*15)

genetic research in MS

1972 2000 20122006

1. MS-HLA

association

report

human genome

project

internatl. HapMap project

60 MS-assoc. loci

GWAS

exome sequencing:

CYP27B1 as rare

variant MS-assoc.

contribution to heritability in MS

0

2

4

6

8

10

12

%

0

0,02

0,04

0,06

0,08

0,1

0,12

0,14

0,16

0,18

0,2

%

effect by variation in 1 gene

Osteoarthritis -

Asthma -

Psoriasis(RUNX)

Kardiovascul. -

Parkinson -

Mult. Sclerosis -

Rheum. Arthrit. -

IBD MC: CARD15

HLA association for common diseases ?

HLA assoc. effect by variation in 1 gene

Osteoarthritis -

Asthma -

Psoriasis (RUNX)

Kardiovascul. () -

Parkinson () -

MS -

Rheum. Arthrit. -

IBD MC: CARD15

ad immunology: antigen presentation

MS - genetic susceptibility

genetic contributions- MS linkage + association HLA-DRB1*1501- locus- and allelic heterogenity- dosis effect- complex allelic interactions in trans- modifier effects

ad HLA-DRB1*15

• HLA influences MS manifestation

i.e. Jersild, Svejgaard, Fog (1972) LANCET 1: 1240

• parental transmission DRB1*15 in MSRamagopalan (2008) HUM GENET 122: 661

→ ♀ > ♂ transmission:

♀ offspring DRB1*15+ → 1.39 x RR

twins discordant for MS

twins

type of twins critical stage placenta placenta

dichorionic

diamniotic

dichorionic

diamniotic

monochorionic

diamniotic

monochorionic

monoamniotic

dizygotic

2 fertilised eggs

2 placentas

2 chorions, 2 amnions

monozygotic

1 fertilised egg →

fission before blastocyst

2 placentas

monozygotic

1 fertilised egg → inner

embryonic cells separate

before amniogenesis

1 placenta, 2 amnions

monozygotic

1 fertilised egg → inner

embryonic cells separate

before amniogenesis

1 placenta, 2 amnions

monozygotic

1 fertilised egg → embryonic

cells separate before

embryonic axis develops

1 placenta, 1 amnion

fertilisation

egg sperm

preblastocyst

blastocyst

post-implantation

blastocyst

► chromosomal mosaicism(s)

► post-zygotic gene mutation(s)

► mtDNA differences

► different epigenetic profiles

- skewed X inactivation

- genomic imprinting

► epigenetic regulation of gene expression

not really completely identical twins

patient

samples

genes

time

n>100

n>100000

MS - susceptibility genes

Where is the missing heritability in MS ?

• rare variants• common variants• epigenetics• gene/gene interactions• pathway involvement• gene/environment interactions• copy number variants

0

2

4

6

8

10

12

14

16

18

20

empirical MS recurrence risk for relatives

%

MS - susceptibility genesMSGC-WTCCC 2011 (p < 10-8; n=29) incl. earlier studies (n=23)

gene region odds ratio

HLA-DRB*1501 3.08

HLA-DRB*1303 2.43

HLA-DRB*0301 1.26

HLA-DRB*0801 1.18

HLA-A*0201 0.73

GALC (GPR65) 1.22

CD86, PTGER4 1.21

CD58 1.18

TNFSF14, MMEL1(TNFRSF14), TMEM39A 1.16

EVI5, CLEC16A 1.15

SP140, IL22RA2 1.14

chromosome 3, MYB, TAGAP, CBLB 1.13

VCAM1, PVT1, MALT1, CYP24A1, RGS1, IL2RA, CD6, TNFRSF1A, IRF8 1.12

chromosome 2, PLEK, MERTK, EOMES, IL12B, ZNF767, MYC, ZFP36L1, BATF, MPV17L2, DKKL1, IL7R, IL7, CYP27B1, KIF21B 1.11

THEMIS, HHEX, MAPK1, SCO2, OLIG3, ZMIZ1, STAT3, TYK2, CD40 1.10

BACH2, CLECL1, IL12A 1.09

MPHOSPH9 1.08

MS - pharmacogenomics

gene(s) method samples

[cases/controls]

replication

HALPN1, GPC5 response IFNß

100K (pooled DNA)

206 rrMS patients:99 responders vs. 107 non-resp.

joint analysis: original cohort

+ 81 rrMS patients

HALPN1,GPC5

association study

199 MS patients:55 responders vs. 79 non-resp.

no replicationreplication

GRIA3response IFNß

500K (pooled DNA)

106 rrMS patients:53 responders vs. 53 non-resp.

49 resp. vs.45 non-responders

summary MS genetics

- environmental factors: latitude, UVR, vitamin D; EBV; smoking; hygiene hypothesis

- genetic susceptibility (major) factor in pathogenesis

- number of susceptibility loci certainly ↑↑↑:combinations of a limited number of loci in susceptible allelic state herald susceptibility