Download Neurogenetics

Neurogenetics and
DNA laboratory
Pavel Seeman
CMT team Prague
Neurogenetics
„neurology of the whole family“
for clinicians : ask about and
investigate the relatives !
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)
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
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
PMP 22
chromosome 17 p 11.2
PMP 22
Normal
chromosome 17 p 11.2
PMP 22
chromosome 17
p 11.2
chromosome 17
p 11.2
PMP 22
PMP 22
CMT 1 A
PMP 22
chromosome 17
p 11.2
HNPP
chromosome 17
p 11.2
PMP 22
chromosome 17
p 11.2
chromosome 17
p 11.2
PMP 22
CMT 1A
DSS, CHN
HNPP
Protein 0
chromosome 1q22q23
chromosome 1q22q23
Protein 0
CMT1B
DSS,CHN,
CMT2
connexin 32
chromosome X
CMTX
chromosome X
connexin 32
Detection of the most common
mutation in CMT in DNA laboratory
Set 58°C
Set 55°C
now: 15 microsatellites
markers located within the
duplicated CMT1A region
Detection of point mutations in
further genes in patients with
excluded CMT1A/HNPP
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
In patients where we have
enough clinical data !
Czech CMT families 1997 - 2003
548 - families tested
1271 – individuals
mutation detected in
total 238 families
140
309
268 individual gene tests
69
CMT1A duplication
HNPP deletion
Cx32
1
20
8
MPZ
PMP22
still unknown
CMT1A: 258 individuals, HNPP: 131 individuals
CMT families with enough
clinical data – 408 families
still
unknown
42%
CMT1A
duplicat.
34%
in 140 families out of
548 not enough
clinical data were
provided
408 families with
sufficient clinical data
HNPP
PMP22 MPZ Cx32
5%
0%
2%
deletion
17%
Modes of inheritance in Czech
CMT patients cohort
In 392 families – unrelated patients with suficient family information
recessive
(AR)
1%
sporadic
45%
dominant:
AD + XD
54%
Gene testing – sequencing in
CMT patients
without the CMT1A duplication
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 )
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
Mutation Glu208Lys in Cx32 in 6 large Czech
families is due to a founder efect.
Marker
Haplotypes
Distance
Family K
Family B-S
Family Z-U
Family S-B-V Famiy S-D-H
Family S
DXS1053
5
2
5
2
1
51.000 kbp
DXS8083
5
1
2
2
2
23.000 kbp
DXS1111
3
1
1
1
1
2400 kbp
DXS453
3
1
1
1
1
1000 kbp
DXS8107
1
1
1
1
1
800 kbp
GJB1
Glu208Lys
Glu208Lys
Glu208Lys
Glu208Lys
Glu208Lys
Glu208Lys
0 bp
SNP;
rs1997625, T>C
SNP;
rs752081, A>G
DXS8060
C
C
C
C
C
C
1 kbp
G
G
G
G
G
G
5 kbp
4
1
1
1
1
2500 kbp
DXS1225
4
1
1
1
1
7000 kbp
DXS1197
1
1
1
1
1
8000 kbp
DXS8020
2
1
2
1
1
28.000 kbp
DXS1206
2
5
1
2
2
54.000 kbp
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
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 – hotspot
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
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
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 (>5 %), one is
Gly113Ser in a family with very mild CMT
Many polymorfisms incl. Ile92Val, Thr78Thr
Two different phenotypes caused
by P0/MPZ mutation
Czech family with axonal
CMT beginning with deafness
Family F.
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
no pes cavus, severe footdrop
pronounced hand muscles atrophies
Audiograms
mother
(622)
son
(623)
Electrophysiology
Axonal polyneuropathy – very low
amplitudes with preserved NCVs
Nerve and muscle biopsy in nr. 622
(mother)
nerve
muscle
Normal
control
290 A>T (Glu97Val) in MPZ
gene
Dejerine Sottas neuropathy and
MPZ mutation
Deafness as late symptom in DSN patients
Family K.
Mutation Arg98Cys
- neighbour
aminoacid to the
previous family
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
Family K.
mother
son
Family K.
mother
no foot deformities
son
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
Larger families with unknown mutation
– linkage studies –
new candidate genes discoveries
HMN II and 12q24 locus
CMT196
14
8
20
7
9
10
11
12
13
15
17
16
18
19
23
55
21
22
24
25
26
27
32
1
2
28
31
29
3
30
47
53
48
49
50
51
52
34
33
35
38
40
36
37
39
54
42
41
43
45
44
46
4
5
6
Linkage analysis to localize possible new „CMT genes“
AD CMT family, CMT1A, MPZ excluded
Linkage analysis to localize possible new „CMT genes“
AD-CMT family,
CMT1A, MPZ, PMP22 mutations excluded
electrophysiology intermediate