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