1. MPZ mutations associated with deafness and abnormal pupillar reaction in Czech CMT2 patients, but also in HMSN III patients. P. Seeman 1, K. Huehne 2, R. Mazanec 3, B. Rautenstrauss 2, V. Beneš jr. 1, P. Šušlíková 1, O. Keller 4. 1- Dept of Child Neurology, 3 - Dept of Neurology 2nd School of Medicine, Charles University Prague, 2- Institute of Human Genetics, University Erlangen, 4- Dept of Neurology Thomayer University Hospital Prague.

2. Different HMSN phenotypes associated with MPZ mutations. Charcot – Marie – Tooth type 1B (demyelinating) Dejerine – Sottas neuropathy (DSN) Congenital hypomyelination neuropathy (CHN) Charcot – Marie – Tooth type 2 (axonal) - „new“

3. Deafness in CMT Deafness was reported in some of the CMT2 families with MPZ mutations (Thr124Met) Deafness was not observed in HMSN type III patients or families associated with MPZ mutations

4. Myelin protein zero (MPZ).

5. 173bp 307bp 278bp 309bp exon 1 exon 2 exon 3exon 4 exon 5 Myelin protein zero gene. exon 6

6. Axonal - CMT 2 phenotype due to mutation in myelin gene (MPZ). Recent findings of MPZ mutations in CMT2 patients and families with distinct phenotype (Ser44Phe, Asp61Gly, Asp75Val, Ile99Thr, Tyr119Cys, Thr124Met, Gln141stop, …) axonal type of CMT - EMG and nerve biopsy late onset polyneuropathy abnormal pupillar reaction – slow or absent deafness - usually after the onset of neuropathy

7. Czech family with axonal CMT beginning with deafness

8. Family F.

9. 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, deaffness now abnormal pupillar reaction (Argyll-Robertson pupilles, Adie pupilles) before the onset of the neuropathy fully normal fysical abilities untill 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 severe distal muscle atrophies in mother and grandfather whorsening of electrophysiological findings correlated with higher age in the family – axonal loss and later demyelination

11. distal atrophies and weakness

12. no pes cavus, severe footdrop pronounced hand muscles atrophies

13. Electrophysiological (EMG, VEP, BAEP) findings I. Motor and sensory nerve conduction velocities Patient Motor nerve Sensory nerve DML CMAP MCV SCV SNAP (ms) (mV) (m/s) (m/s) (uV) 645 Tibial none Sural none Grandf. Median 9.4 0.1 29 Median none 622 Tibial none Sural none moth) Median 4.8 0.4 30 Median 14 7 623 Tibial 6.2 10.3 40 Sural 47 16 boy) Median 3.5 13.2 45 Median 47 26

14. Electrophysiological (EMG, VEP, BAEP) findings II. Brainstem auditory evoked potentials (monoaural,rarefaction click, 10 Hz ) Patient Side Latencies of waves /ms/ I III V I-III III-V I-V 645 L no response G R no response 622 L 2.36 4.44 6.29 2.08 1.85 3.93 M R 2.30 4.32 none 2.02 none none 623 L 1.68 3.92 5.58 2.24 1.66 3.90 B R 1.98 4.15 6.11 2.17 1.96 4.13 Upper limits of normal range of BAEPs in our laboratory / +- 2 SD /: Latencies : I 2.04 ms III 4.16 ms V 6.06 ms I – III 2.16 ms III – V 2.04 ms I – V 4.15 ms

15. Electrophysiological (EMG, VEP, BAEP) findings III. Visually evoked potentials (pattern reversed, monocular, 2 Hz, 16 ´´) Latencies of waves / ms / N 75 P 100 N 145 Patient Side 645 Left 79 104 136 Right 79 105 137 622 L 68 100 127 R 67 100 124 623 L 76 124 189 R 76 116 193 Limits of normal range of VEPs in our laboratory / +- 2 SD / Latencies : N 75 / 67 – 84 ms / P 100 / 94 – 116 ms / N 145 / 125 – 155 ms / Amplitude: N 75 / P 100 5.4 uV

16. Results from the mother (nr. 622) VEP – normal latencies BAEP – prolonged latencies - peripheral Audiometry – profound perception hearing loss brain MRI – normal, only diffuse nonspecific white matter changes in both hemispheres CSF – normal protein and normal cell count muscle biopsy – neurogenic lesion, fiber hypertrophy and atrophy nerve biopsy – done in 1993, axonal loss, axonal lesion

17. 290 A>T (Glu97Val) in MPZ gene

18. Nerve and muscle biopsy in nr. 622 (mother) Normal control nervemuscle

19. MRI in nr. 622 (mother)

21. Dejerine Sottas neuropathy and MPZ mutation Deafness was usually not reported in DSS patients – (but the reported patients were usually children or young persons, few data about adult DSS patiens and their hearing status)

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

23. Family K. Mother (44y.) and son (18y.) severely affected (HMSNIII 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 CSF - ? 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 - extremely prolonged opening reaction (opening)

24. Family K. mother son

25. Family K. motherson

26. Conclusions Is deafness a common feature associated with MPZ mutations, but later in life (in adult age) ? Do also other HMSN III or DSN patients develop hearing loss in adulthood ? In our CMT2 family F. (Glu97Val), deafness was by far the first symptom of the disease – before any signs of HMSN Testing of the functional effect of the newly discovered mutations with CMT2 phenotype in expression systems should clarify the mechanism leading to axonal demage resulting from some MPZ mutation Only the location in the extracellular domain alone, do not explain really different phenotypes resulting from mutation of the neibour aminoacids ( Lys96Glu – CMT1, Glu97Val – CMT2, Arg98Cys – DSN, Arg98His - Ile99Thr – CMT2) some other mechanism is more probable.