Neurogenetics and DNA laboratory
description
Transcript of Neurogenetics and DNA laboratory
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 CMTGrants of Ministery of Health of Czech RepublicAll 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
PMP 22
chromosome 17 p 11.2
chromosome 17 p 11.2
Normal
PMP 22
PMP 22 PMP 22
CMT 1 A
chromosome 17 p 11.2 chromosome 17 p 11.2
PMP 22
HNPP
PMP 22
PMP 22
chromosome 17 p 11.2
chromosome 17 p 11.2
CMT 1A DSS, CHN HNPP
chromosome 17 p 11.2
chromosome 17 p 11.2
Protein 0
Protein 0 chromosome 1q22-q23
chromosome 1q22-q23
CMT1B DSS,CHN, CMT2
chromosome X CMTX
connexin 32
connexin 32
chromosome X
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
309
69
140
208
1
CMT1A duplication
HNPP deletion
Cx32
MPZ
PMP22
still unknown
548 - families tested1271 – individuals
mutation detected in total 238 families
268 individual gene tests
CMT1A: 258 individuals, HNPP: 131 individuals
CMT families with enough clinical data – 408 families
still
unknown
42%
HNPP
deletion
17%
CMT1A
duplicat.
34%
Cx32
5%MPZ
2%
PMP22
0%
in 140 families out of 548 not enough clinical data were provided
408 families with sufficient clinical data
Modes of inheritance in Czech CMT patients cohort
dominant:
AD + XD
54%
sporadic
45%
recessive
(AR)
1%
In 392 families – unrelated patients with suficient family information
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.
Haplotypes
Marker
Family K Family B-S Family Z-U Family S-B-V Famiy S-D-H Family S
Distance
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
C C C C C C 1 kbp
SNP; rs752081, A>G
G G G G G G 5 kbp
DXS8060 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) geneinvestigated: 80 families without CMT1A duplicationmutation found in 8 families (10 %)4x axonal form a 4x demyelinating severe formArg98Cys mutation 2x – in two families de-novo – hot-spot
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
mother(622)
son(623)
Audiograms
Electrophysiology
Axonal polyneuropathy – very low amplitudes with preserved NCVs
Nerve and muscle biopsy in nr. 622 (mother)
Normal control
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 sonno foot deformities
Other, recently discovered genes in CMT(to be screened in the future in „unknown“ patients)
PRX - AR demyelGDAP1 - AR - demyel., axonal and intermediar.MTMR2 - AR – focally folded myelinNEFL - AD - axonal and demyel LITAF/SIMPLE - CMT1C - AD, demyel. LMNA - AR - axonalRAB 7 - AD – axonal with ulcersNDRG1 - HMSN Lom - BulgariaGAN - giant axonal neuropathy NTRK 1 - HSAN IVSPTLC1 - HSN I
Larger families with unknown mutation – linkage studies –
new candidate genes discoveries
CMT196
14 7 20
8 9 10 11 15 16 19 55 21
12 13 17 18 22
23
24 25 1
26 27 2 29 3
3028
32
31 53 33
47 48 49
50 51
52 34 35
38
36 37
39
54 42 43 44
40 41 45 46 4
5 6
HMN II and 12q24 locus
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 excludedelectrophysiology intermediate