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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