Neurogenetics and DNA laboratory

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Neurogenetics and DNA laboratory

• Pavel Seeman CMT team Prague

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Neurogenetics neurology of the whole family
for clinicians ask about and investigate
the relatives !
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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)
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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

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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

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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
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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

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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
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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
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Modes of inheritance in Czech CMT patients cohort
In 392 families unrelated patients with
suficient family information
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Gene testing sequencing in CMT patients
without the CMT1A duplication
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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 )

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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

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Mutation Glu208Lys in Cx32 in 6 large Czech
families is due to a founder efect.
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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
  

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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

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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

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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

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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

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Two different phenotypes caused by P0/MPZ mutation
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Czech family with axonal CMT beginning with
deafness
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Family F.
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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

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no pes cavus, severe footdrop
pronounced hand muscles atrophies
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Audiograms
mother(622)
son(623)
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Electrophysiology

• Axonal polyneuropathy very low amplitudes with
  preserved NCVs

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Nerve and muscle biopsy in nr. 622 (mother)
nerve
muscle
Normal control
Normal control
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290 AgtT (Glu97Val) in MPZ gene
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Dejerine Sottas neuropathy and MPZ mutation

• Deafness as late symptom in DSN patients

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Family K.

• Mutation Arg98Cys - neighbour aminoacid to the
  previous family

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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

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Family K.
son
mother
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Family K.
mother
son
no foot deformities
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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

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