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Neurogenetics and DNA laboratory

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DNA laboratory in neurological departments at 2nd School of Medicine, ... Dept of Pediatric Otorhinolaryngology (Congenital Nonsyndromic Deafness Connexin 26) ... – PowerPoint PPT presentation

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Title: Neurogenetics and DNA laboratory


1
Neurogenetics and DNA laboratory
  • Pavel Seeman CMT team Prague

2
Neurogenetics neurology of the whole family
for clinicians ask about and investigate
the relatives !
3
DNA laboratory in neurological departments at 2nd
School of Medicine, Charles University and
University Hospital Motol Prague
Dept of Child Neurology(Hereditary
Neuropathies, Pelizaeus Merzbacher
disease,Nijmegen Breakage Syndrome)
Dept of Adult Neurology(Hereditary
Neuropathies)CMT
Dept of Pediatric Otorhinolaryngology(Congenital
Nonsyndromic Deafness Connexin 26)
4
DNA testing for CMT in Czech Rep
  • DNA testing for CMT in the Czech Republic
    available since 1997 still the only lab testing
    for CMT
  • Grants of Ministery of Health of Czech Republic
  • All DNA samples and patient data from CMT
    patients in one lab many advantages and great
    potential

5
Hereditary neuropathies Charcot-Marie-Tooth
diseases
  • Most common group of genetically caused
    neuromuscular diseases (1 2 500 5 000)
  • Genetically very heterogeneus group now
    mutations in 23 genes can cause CMT
  • Highly prevalent mutation is the CMT1A
    duplication / HNPP deletion on chromosome 17p
    incl. PMP22 gene
  • Challenge for the detection methods to small
    for cytogenetics, to big for molecular genetics
    and to close together for conventional metaphase
    FISH

6
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7
Detection of the most common mutation in CMT in
DNA laboratory
Set 58C
Set 55C
now 15 microsatellites markers located within
the duplicated CMT1A region
8
Detection of point mutations in further genes in
patients with excluded CMT1A/HNPP
In patients where we have enough clinical data !
  • Connexin 32 gen (GJB1) in CMTX
  • Myelin protein zero (MPZ) in severe
    demyelinating forms and in axonal form
  • PMP22 gene in demyelinating forms
  • LITAF gene in demyelinating dominant forms

9
Czech CMT families 1997 - 2003
548 - families tested1271 individuals mutation
detected in total 238 families
268 individual gene tests
CMT1A 258 individuals, HNPP 131 individuals
10
CMT families with enough clinical data 408
families
in 140 families out of 548 not enough clinical
data were provided 408 families with sufficient
clinical data
11
Modes of inheritance in Czech CMT patients cohort
In 392 families unrelated patients with
suficient family information
12
Gene testing sequencing in CMT patients
without the CMT1A duplication
13
Connexin 32 gen, GJB1
  • X- linked dominant
  • expressed in PNS as well as in CNS
  • mutations in Cx32 are the second most common
    cause of CMT (after CMT1A )

14
Connexin 32 gene, GJB1
  • Investigated 58 families without CMT1A
    duplication
  • Causal mutation found in 21 families (36,2 )
  • Among 46 familiar cases only 45,6
  • Families positive for Cx32 mutation were always
    large many members affected by CMT
  • One family, possibly a de-novo mutation
  • 6 families from 13 (46) carry the same
    mutation Glu208Lys haplotype analysis showed a
    founder efect all 6 large families have a
    common founder

15
Mutation Glu208Lys in Cx32 in 6 large Czech
families is due to a founder efect.
16
Myelin Protein Zero (MPZ) (P0) gene
  • expressed in PNS only in myelinated Schwann cells
    in central involvement
  • 4 clinical phenotypes due to a mutation in MPZ
    CMT1 classical (demyel.)

    - DSS or HMSN III- early onset
    severe demyel. - congenital
    hypomyelination (CHN)very early onset
    - CMT 2 (axonal) late onset

17
MPZ (P0) gene
  • investigated 80 families without CMT1A
    duplication
  • mutation found in 8 families (10 )
  • 4x axonal form a 4x demyelinating severe form
  • Arg98Cys mutation 2x in two families de-novo
    hot-spot

18
PMP22 gene
  • expression only in PNS no signs of central
    involvement
  • dominant mutations heterozygotes are affected
  • phenotype CMT1 classical -
    Dejerine Sottas or congenital hypomyelination
  • PMP22 mutations are rare worldwide

19
PMP 22 gene
  • all coding exons sequenced in 33
    families/unrelated patients without CMT1A
    duplication
  • mutation found in 1 patient only ( 3)
    sporadic case, congenital hypomyelination (CHN)
    de-novo mutation

    Ser72Leu
  • point mutations in PMP22 are rare

20
LITAF 1/ SIMPLE gene
  • Lipopolysacharide-Induced Tumor necrosis Alfa
    Factor - Small Integral Mebrane Protein of the
    Lysosome/late Endosome
  • 3 coding exons, 486 bp, 161 AA
  • Street et al. 2003 Neurology 60 22-26
    Mutation in LITAF/SIMPLE in CMT1C disease.
  • 46 CMT1 families tested
  • 2 causal mutations detected (gt5 ), one is
    Gly113Ser in a family with very mild CMT
  • Many polymorfisms incl. Ile92Val, Thr78Thr

21
Two different phenotypes caused by P0/MPZ mutation
22
Czech family with axonal CMT beginning with
deafness
23
Family F.
24
Family F.
  • hearing loss and deafness as the first symptom of
    CMT disease - at the late teens in the
    grandfather and in the mother - progressive
    hearing loss, deafness now
  • abnormal pupillar reaction before the onset of
    the neuropathy
  • fully normal physical abilities until the end of
    3rd decade
  • late onset of polyneuropathy of axonal type -
    slow progression at the grandfather but quite
    fast at the mother, 12 years old boy clinically
    still unaffected
  • whorsening of electrophysiological and clinical
    findings correlated with higher age in the family
    axonal loss and later demyelination

25
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26
no pes cavus, severe footdrop
pronounced hand muscles atrophies
27
Audiograms
mother(622)
son(623)
28
Electrophysiology
  • Axonal polyneuropathy very low amplitudes with
    preserved NCVs

29
Nerve and muscle biopsy in nr. 622 (mother)
nerve
muscle
Normal control
Normal control
30
290 AgtT (Glu97Val) in MPZ gene
31
Dejerine Sottas neuropathy and MPZ mutation
  • Deafness as late symptom in DSN patients

32
Family K.
  • Mutation Arg98Cys - neighbour aminoacid to the
    previous family

33
Family K.
  • Mother (44y.) and son (18y.) severely affected
    (HMSN III or DSN), no other affected members in
    the family
  • early onset (3 y.) with hypotonia, delayed motor
    milestones, scoliosis, both affected never
    achieved normal independent gait
  • distal weakness and atrophies, areflexia, rather
    nonprogressive course
  • extremely decreased MNCV ( 8-10 m/s), absent SNAP
  • hypertrofic demyelinating neuropathy (with onion-
    bulbs) in the sural nerve biopsy in the mother
  • in the mother from the age of 25 y. - hearing
    loss, presently sensorineural hearing loss
    bilateral ( up to 70 dB)both affected showed
    abnormal pupillar reaction

34
Family K.
son
mother
35
Family K.
mother
son
no foot deformities
36
Other, recently discovered genes in CMT(to be
screened in the future in unknown patients)
  • PRX - AR demyel
  • GDAP1 - AR - demyel., axonal and intermediar.
  • MTMR2 - AR focally folded myelin
  • NEFL - AD - axonal and demyel
  • LITAF/SIMPLE - CMT1C - AD, demyel.
  • LMNA - AR - axonal
  • RAB 7 - AD axonal with ulcers
  • NDRG1 - HMSN Lom - Bulgaria
  • GAN - giant axonal neuropathy
  • NTRK 1 - HSAN IV
  • SPTLC1 - HSN I

37
Larger families with unknown mutation linkage
studies new candidate genes discoveries
HMN II and 12q24 locus
38
Linkage analysis to localize possible new CMT
genes
AD CMT family, CMT1A, MPZ excluded
39
Linkage analysis to localize possible new CMT
genes
AD-CMT family,CMT1A, MPZ, PMP22 mutations
excludedelectrophysiology intermediate
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