- Email: [email protected]
Late-onset CMT2 associated with a novel missense mutation in the cytoplasmic domain of the MPZ gene
Clinical Neurology and Neurosurgery 112 (2010) 798–800
Contents lists available at ScienceDirect
Clinical Neurology and Neurosurgery journal homepage: www.elsevier.com/locate/clineuro
Case report
Late-onset CMT2 associated with a novel missense mutation in the cytoplasmic domain of the MPZ gene Hirotaka Shimizu a , Nobuyuki Oka b , Toshitaka Kawarai a,∗ , Koichiro Taniguchi a , Naoki Saji a , Makoto Tadano a , Giorgio Bernardi c,d , Antonio Orlacchio c,d , Yasushi Kita a a
Department of Neurology, Hyogo Brain and Heart Centre, Saisho-Ko 520, Himeji City 670-0981, Hyogo Prefecture, Japan Department of Neurology, N.H.O. Minami-Kyoto National Hospital, Joyo, Kyoto, Japan Laboratorio di Neurogenetica, CERC-IRCCS Santa Lucia, Rome, Italy d Dipartimento di Neuroscienze, Università di Roma “Tor Vergata”, Rome, Italy b c
a r t i c l e
i n f o
Article history: Received 24 May 2009 Received in revised form 23 July 2010 Accepted 31 July 2010 Available online 25 August 2010 Keywords: MPZ gene Charcot-Marie-Tooth type 2 Missense mutation PKC alpha substrate motif
a b s t r a c t Phenotypic variations have been reported in Charcot-Marie-Tooth disease type 2 (CMT2) including ageat-onset, disease progression and severity. Sporadic cases with CMT2 have also been demonstrated by genetic test. We here report a patient with late-onset CMT2 without family history, who developed gait disturbance at the age of 68. Sequence analysis revealed a novel heterozygous Arg198Gly mutation in the cytoplasmic domain of the major peripheral myelin protein zero (MPZ). The mutation is located in the protein kinase C (PKC) alpha substrate motif (RSTK) of MPZ, presumably leading to the loss of PKC-mediated phosphorylation in adhesion. Routine genetic test for CMT is not recommended for every patient with late-onset peripheral neuropathy without known causes, however, the genetic test may be taken into consideration if the patient shows a clinical phenotype similar to that of CMT, and the possibility of a de novo mutation cannot be excluded. © 2010 Elsevier B.V. All rights reserved.
1. Introduction
2. Case report
Myelin protein zero (MPZ) is a structural protein of peripheral myelin, which has been predicted to contain a single putative transmembrane domain, glycosylated, immunoglobulin-like extracellular domain, and a basic intracellular domain. MPZ functions as a homophilic adhesion molecule, which promotes compaction of adjacent leaflets in peripheral nerve myelin [1–7]. MPZ mutations cause hereditary disorders such as Charcot-Marie-Tooth neuropathy type 1B (CMT1B), Dejerine-Sottas syndrome (DSS), congenital hypomyelinating neuropathy (CHN) and axonal CMT phenotype (CMT2). Autosomal dominant inheritance is more likely to occur familial cases; however, sporadic cases with de novo mutations were also detected by sequence analyses [1,2]. The clinical heterogeneity observed in hereditary neuropathy caused by MPZ mutation might be attributed to the nature and the position of mutations. We performed a clinico-genetic study of sporadic case with late-onset CMT2 disease carrying a novel missense mutation Arg198Gly in MPZ. Our report would contribute to further understanding of the pathogenesis in CMT2.
The patient, a 69-year old man, was referred to our clinic for gait disturbance due to muscle weakness, which appeared at the age of 68. He walked with a steppage gait, but he was able to walk without help. He was born healthy to non-consanguineous parents and grew up without any developmental delay. He belonged to an athletic club in his high school days. The patient was able to climb a mountain up to the age of 65 without weakness and reported that his peroneal and calf muscles had become thin over the last few years. The examination showed no skeletal abnormalities apart from pes cavus, and no muscle hypertrophy was observed. However, prominent muscle wasting was present in the calf muscle and the intrinsic hand muscle. There was distal lower limb weakness; the manual muscle testing score was 3/5 for the tibialis anterior and gastrocnemius muscles. The patient could not walk on heels or tiptoes. Fasciculation was not observed in the affected muscles. Vibration sensation was impaired, but somatic sensations – including light touch and pinprick perception – were preserved also at the distal lower limbs. Tendon reflexes were absent at the lower limbs and decreased at the upper limbs. No pupillary abnormalities, autonomic nervous dysfunction, or cerebellar ataxia were reported. His parents passed away in their late seventies and they never showed gait disturbance. He had three brothers aged in their sixties and no children. Clinical investiga-
∗ Corresponding author. Tel.: +81 79 293 3131; fax: +81 79 295 8199. E-mail address: [email protected] (T. Kawarai). 0303-8467/$ – see front matter © 2010 Elsevier B.V. All rights reserved. doi:10.1016/j.clineuro.2010.07.020
H. Shimizu et al. / Clinical Neurology and Neurosurgery 112 (2010) 798–800
799
Table 1 Electrophysiological studies in the patient with an axonal form of CMT associated with a novel mutation. NCS
DL (ms)
MCV (m/s)
CMAP (mV)
SCV (m/s)
SNAP amplitude (V)
Segment, normal value Right median Left median
2.3–4.6 3.6 4.0
Elbow to wrist, 51–65 44.1 42.6
4.5–19.0 11.2 10.1
Wrist to the 2nd digit, 53.2–71.2 48.6 48.6
28.7–86.3 15.5 13.7
Segment, normal value Right ulnar Left ulnar
2.1–3.2 3.4 3.3
Elbow to wrist, 50–69 47.2 43.1
9.0–22.0 9.5 9.6
Wrist to the 4th digit, 59.7–74.9 43.8 46.1
11.4–89.4 7.7 9.7
Segment, normal value
4.2–6.5
5.0–21.5
Right tibial Left tibial
10.5 11.6
Popliteal fossa to the medial malleolus, 41–55 26.7 24.7
0.40 0.42
SEP
Erb (N9) (ms)
Cervical (N13) (ms)
Cortical (N19) (ms)
Right median Left median
14.9 14.4
19.7 19.3
24.5 24.7
NCS, nerve conduction study; CMAP, compound motor action potential; SEP, somatosensory evoked potentials; SCV, sensory conduction velocity; DL, distal latency; SNAP, sensory nerve action potential; MCV, motor conduction velocity. Sural SNAP was not evoked in NCS. Peroneal CMAP was not evoked in NCS. Cortical N40 was not evoked in SEP. Normal values are cited from previous publication [18].
tions showed that none of his brothers had foot deformity or gait disturbance. Results of nerve conduction studies and somatosensory evoked potentials are described in Table 1, which shows axonal dysfunction especially in the legs. Electromyography showed no active denervation, but rather a chronic denervation in the affected muscles. Selective loss of large myelinated fibers was shown by light microscopic analysis of biopsied specimens from his right sural nerve (Fig. 1). The density of large (diameter > 7 m) and small (≤7 m) myelinated nerve fibers was 510/m2 and 5810/m2 respectively (control 3340/m2 and 4470/m2 ). Regenerating clusters were frequent, quantified at 240/m2 , but onion-bulb formation was absent. Rare myelin ovoids were present, and no myelin debris was observed. Ultrastructural examination showed some bands of Bungner and numerous regenerating clusters of nerve fibers. A few fibers with unusual redundant myelin sheaths were present (Fig. 1B), but uncompacted myelin lamella were not observed. Excessively folded myelin sheaths were also not observed. A teased nerve fiber study showed no tomacula and segmental demyelination; remyelination was rare (figure not shown). Laboratory examinations and pathological analysis of sural nerve biopsy excluded paraneoplastic polyneuropathy, impaired glucose tolerance neuropathy, polyneuropathy associated with vitamin deficiency, chronic inflammatory demyelinating polyneu-
ropathy, vasculitic polyneuropathy, polyneuropathy associated with anti-MAG IgM antibodies, arsenic neuropathy, sarcoidal polyneuropathy and familial amyloid polyneuropathy. Considering the previous reports of sporadic cases with de novo mutations in CMT genes [1,2], genetic test was performed in PMP22 and MPZ. Chromosome 17p11.2-p12 duplication – commonly seen in CMT1A – was excluded by pulsed field gel electrophoresis. However, sequence analyses of the MPZ gene showed a A>G exchange at nucleotide 679 (numbering using the A of the translation initiation codon as +1, NCBI accession number P25189). This results in a heterozygous arginine to glycine substitution at codon 227 (Arg227Gly) or codon 198 (Arg198Gly) in conventional description in which the initial 29 amino acid leader peptide is not included [1]. The case report describes the identified Arg198Gly mutation. The nucleotide substitution was absent in 200 control chromosomes from unrelated Japanese individuals. 3. Discussion Neither the missense mutation Arg198Gly nor the nucleotide substitution was listed in the public databases (http://molgenwww.uia.ac.be/CMTMutations and http://www.ncbi.nlm.gov/snp). An amino acid substitution at the same position, Arg198Ser, was previously reported [1,6]. A patient carrying the missense muta-
Fig. 1. (A) Toluidine blue stained transverse semithin section showing a marked reduction in the number of large myelinated fibers and numerous regenerating clusters (black arrows). Onion-bulb formation was absent. (B) Electron micrograph showing that myelin loops are partly non-compact in a few fibers (white arrow). Uncompacted myelin lamella were not observed.
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H. Shimizu et al. / Clinical Neurology and Neurosurgery 112 (2010) 798–800
tion Arg198Ser showed a mild CMT1B phenotype, but no detailed features – including pathological and electrophysiological findings – were available [1,6]. The missense mutation Arg198Gly is located in the highly conserved protein kinase C (PKC) alpha substrate motif (198RSTK201) of MPZ. In this position, amino acids are highly conserved amongst MPZ homologous proteins, including Rattus norvegicus, Mus musculus, Gallus gallus and Bos taurus. MPZ protein expression harboring the missense mutation Arg198Ser in cultured cells showed a loss of MPZ-mediated adhesion [3]. Therefore, the missense mutation Arg198Gly was identified as pathogenic. MPZ dysfunction leads to lack of myelination or hypomyelination, which might affect Schwann cell-axon interactions and result in secondary axonal atrophy [8]. On the basis of the results obtained from pathological and electrophysiological studies, the patient was diagnosed with axonal CMT2. Genetic tests were not performed in other family members; therefore, it remains unknown whether this is a de novo or a low-penetrance mutation. A wide range of phenotypic variability was reported in patients carrying the MPZ mutation, including age-at-onset, disease progression and severity [1,2]. Such variability may be related to the dosage of MPZ protein or the position and nature of MPZ mutation. The severity of the clinical phenotype might depend on the dosage of MPZ protein. Moreover, it has been suggested that MPZ protein dosage might be regulated by the biological effect of the mutation (e.g. dominant negative effect) or by biological pathways such as the nonsense-mediated mRNA decay pathway leading to half-normal expression in MPZ protein [9–12]. In case of missense mutations or deletions, various mutational effects have been proposed. Two mutations, Lys236del and Asp35Asn, were identified in familial cases with late-onset CMT [13,14]. Both mutations are associated with intra-familial phenotypic variations or variable penetrance raging from an asymptomatic state to foot deformities. Lys236del mutation is more closely located to the carboxyl terminus of MPZ protein. Moreover, it has been suggested that an altered phosphorylation status – resulting in dysregulation of myelin compaction – might be involved in the pathogenesis [13]. At the same time, MPZ protein conformational changes have been suggested to be involved in pathological mechanisms in the case of Asp35Asn [14]. Taking into account recent evidence-based reviews and cost benefit analyses in late-onset polyneuropathy without known causes [15–17], a ‘shot gun’ approach for CMT mutations is not recommended. The genetic test should be considered for patients at risk of CMT disease. However, if the clinical phenotype is similar to that of CMT and a de novo mutation is reported, the genetic test might also be considered for the diagnosis of CMT disease. Acknowledgments This work was supported by grants from the Japanese Ministry of Health, Labor and Welfare (N.O.), the Comitato Telethon Fondazione Onlus, the Amministrazione Autonoma dei Monopoli di Stato (AAMS), and the Italian city of Gubbio (Grant No. GGP06209 to A.O.), as well as the Italian Ministry of Health (Grant Nos. EBRI1.O,
PS05.11, PS05.21 and REG.17O to A.O.). We thank the patient and family members involved in this study. We also thank Ms Michela Renna (MA) for her language advice. Drs. Shimizu, Oka and Kawarai contributed equally to this work. Competing interests: None declared. Funding: Declared in the acknowledgments. Ethics Committee approval: The study was performed according to a protocol reviewed and approved by the Ethics Committee of the Hyogo Brain and Heart Centre. References [1] Shy ME, Jani A, Krajewski K, Grandis M, Lewis RA, Li J, et al. Phenotypic clustering in MPZ mutations. Brain 2004;127:371–84. [2] Reilly MM. Sorting out the inherited neuropathies. Pract Neurol 2007;7:93–105. [3] Gaboreanu AM, Hrstka R, Xu W, Shy M, Kamholz J, Lilien J, et al. Myelin protein zero/P0 phosphorylation and function require an adaptor protein linking it to RACK1 and PKC alpha. J Cell Biol 2007;177:707–16. [4] Menichella DM, Arroyo EJ, Awatramani R, Xu T, Baron P, Vallat JM, et al. Protein zero is necessary for E-cadherin-mediated adherens junction formation in Schwann cells. Mol Cell Neurosci 2001;18:606–18. [5] Xu W, Manichella D, Jiang H, Vallat JM, Lilien J, Baron P, et al. Absence of P0 leads to the dysregulation of myelin gene expression and myelin morphogenesis. J Neurosci Res 2000;60:714–24. [6] Xu W, Shy M, Kamholz J, Elferink L, Xu G, Lilien J, et al. Mutations in the cytoplasmic domain of P0 reveal a role for PKC-mediated phosphorylation in adhesion and myelination. J Cell Biol 2001;155:439–46. [7] Kamholz J, Awatramani R, Menichella D, Jiang H, Xu W, Shy M. Regulation of myelin-specific gene expression. Relevance to CMT1. Ann N Y Acad Sci 1999;883:91–108. [8] Hanemann CO, Gabreels-Festen AA. Secondary axon atrophy and neurological dysfunction in demyelinating neuropathies. Curr Opin Neurol 2002;15:611–5. [9] Wong MH, Filbin MT. Dominant-negative effect on adhesion by myelin Po protein truncated in its cytoplasmic domain. J Cell Biol 1996;134:1531–41. [10] Nagy E, Maquat LE. A rule for termination-codon position within introncontaining genes: when nonsense affects RNA abundance. Trends Biochem Sci 1998;23:198–9. [11] Inoue K, Khajavi M, Ohyama T, Hirabayashi S, Wilson J, Reggin JD, et al. Molecular mechanism for distinct neurological phenotypes conveyed by allelic truncating mutations. Nat Genet 2004;36:361–9. [12] Khajavi M, Inoue K, Wiszniewski W, Ohyama T, Snipes GJ, Lupski JR. Curcumin treatment abrogates endoplasmic reticulum retention and aggregationinduced apoptosis associated with neuropathy-causing myelin protein zero-truncating mutants. Am J Hum Genet 2005;77:841–50. [13] Sowden JE, Logigian EL, Malik K, Herrmann DN. Genotype–phenotype correlation in a family with late onset CMT and an MPZ lys236del mutation. J Neurol Neurosurg Psychiatry 2005;76:442–4. [14] Braathen GJ, Sand JC, Russell MB. Two novel missense mutations in the myelin protein zero gene causes Charcot-Marie-Tooth type 2 and Dejerine-Sottas syndrome. BMC Res Notes 2010;3:99. [15] England JD, Gronseth GS, Franklin G, Carter GT, Kinsella LJ, Cohen JA, et al. Evaluation of distal symmetric polyneuropathy: the role of laboratory and genetic testing (an evidence-based review). Muscle Nerve 2009;39:116–25. [16] Hughes RA, Umapathi T, Gray IA, Gregson NA, Noori M, Pannala AS, et al. A controlled investigation of the cause of chronic idiopathic axonal polyneuropathy. Brain 2004;127:1723–30. [17] Vrancken AF, Kalmijn S, Buskens E, Franssen H, Vermeulen M, Wokke JH, et al. Feasibility and cost efficiency of a diagnostic guideline for chronic polyneuropathy: a prospective implementation study. J Neurol Neurosurg Psychiatry 2006;77:397–401. [18] Kurokawa K, Tanaka E, Yamashita H, Kohriyama T, Mimori Y, Nakamura S. Ratios of nerve conduction parameters in proximal to distal limbs remain constant through the second to the eighth decades. Electromyogr Clin Neurophysiol 1998;38:169–76.
Contents lists available at ScienceDirect
Clinical Neurology and Neurosurgery journal homepage: www.elsevier.com/locate/clineuro
Case report
Late-onset CMT2 associated with a novel missense mutation in the cytoplasmic domain of the MPZ gene Hirotaka Shimizu a , Nobuyuki Oka b , Toshitaka Kawarai a,∗ , Koichiro Taniguchi a , Naoki Saji a , Makoto Tadano a , Giorgio Bernardi c,d , Antonio Orlacchio c,d , Yasushi Kita a a
Department of Neurology, Hyogo Brain and Heart Centre, Saisho-Ko 520, Himeji City 670-0981, Hyogo Prefecture, Japan Department of Neurology, N.H.O. Minami-Kyoto National Hospital, Joyo, Kyoto, Japan Laboratorio di Neurogenetica, CERC-IRCCS Santa Lucia, Rome, Italy d Dipartimento di Neuroscienze, Università di Roma “Tor Vergata”, Rome, Italy b c
a r t i c l e
i n f o
Article history: Received 24 May 2009 Received in revised form 23 July 2010 Accepted 31 July 2010 Available online 25 August 2010 Keywords: MPZ gene Charcot-Marie-Tooth type 2 Missense mutation PKC alpha substrate motif
a b s t r a c t Phenotypic variations have been reported in Charcot-Marie-Tooth disease type 2 (CMT2) including ageat-onset, disease progression and severity. Sporadic cases with CMT2 have also been demonstrated by genetic test. We here report a patient with late-onset CMT2 without family history, who developed gait disturbance at the age of 68. Sequence analysis revealed a novel heterozygous Arg198Gly mutation in the cytoplasmic domain of the major peripheral myelin protein zero (MPZ). The mutation is located in the protein kinase C (PKC) alpha substrate motif (RSTK) of MPZ, presumably leading to the loss of PKC-mediated phosphorylation in adhesion. Routine genetic test for CMT is not recommended for every patient with late-onset peripheral neuropathy without known causes, however, the genetic test may be taken into consideration if the patient shows a clinical phenotype similar to that of CMT, and the possibility of a de novo mutation cannot be excluded. © 2010 Elsevier B.V. All rights reserved.
1. Introduction
2. Case report
Myelin protein zero (MPZ) is a structural protein of peripheral myelin, which has been predicted to contain a single putative transmembrane domain, glycosylated, immunoglobulin-like extracellular domain, and a basic intracellular domain. MPZ functions as a homophilic adhesion molecule, which promotes compaction of adjacent leaflets in peripheral nerve myelin [1–7]. MPZ mutations cause hereditary disorders such as Charcot-Marie-Tooth neuropathy type 1B (CMT1B), Dejerine-Sottas syndrome (DSS), congenital hypomyelinating neuropathy (CHN) and axonal CMT phenotype (CMT2). Autosomal dominant inheritance is more likely to occur familial cases; however, sporadic cases with de novo mutations were also detected by sequence analyses [1,2]. The clinical heterogeneity observed in hereditary neuropathy caused by MPZ mutation might be attributed to the nature and the position of mutations. We performed a clinico-genetic study of sporadic case with late-onset CMT2 disease carrying a novel missense mutation Arg198Gly in MPZ. Our report would contribute to further understanding of the pathogenesis in CMT2.
The patient, a 69-year old man, was referred to our clinic for gait disturbance due to muscle weakness, which appeared at the age of 68. He walked with a steppage gait, but he was able to walk without help. He was born healthy to non-consanguineous parents and grew up without any developmental delay. He belonged to an athletic club in his high school days. The patient was able to climb a mountain up to the age of 65 without weakness and reported that his peroneal and calf muscles had become thin over the last few years. The examination showed no skeletal abnormalities apart from pes cavus, and no muscle hypertrophy was observed. However, prominent muscle wasting was present in the calf muscle and the intrinsic hand muscle. There was distal lower limb weakness; the manual muscle testing score was 3/5 for the tibialis anterior and gastrocnemius muscles. The patient could not walk on heels or tiptoes. Fasciculation was not observed in the affected muscles. Vibration sensation was impaired, but somatic sensations – including light touch and pinprick perception – were preserved also at the distal lower limbs. Tendon reflexes were absent at the lower limbs and decreased at the upper limbs. No pupillary abnormalities, autonomic nervous dysfunction, or cerebellar ataxia were reported. His parents passed away in their late seventies and they never showed gait disturbance. He had three brothers aged in their sixties and no children. Clinical investiga-
∗ Corresponding author. Tel.: +81 79 293 3131; fax: +81 79 295 8199. E-mail address: [email protected] (T. Kawarai). 0303-8467/$ – see front matter © 2010 Elsevier B.V. All rights reserved. doi:10.1016/j.clineuro.2010.07.020
H. Shimizu et al. / Clinical Neurology and Neurosurgery 112 (2010) 798–800
799
Table 1 Electrophysiological studies in the patient with an axonal form of CMT associated with a novel mutation. NCS
DL (ms)
MCV (m/s)
CMAP (mV)
SCV (m/s)
SNAP amplitude (V)
Segment, normal value Right median Left median
2.3–4.6 3.6 4.0
Elbow to wrist, 51–65 44.1 42.6
4.5–19.0 11.2 10.1
Wrist to the 2nd digit, 53.2–71.2 48.6 48.6
28.7–86.3 15.5 13.7
Segment, normal value Right ulnar Left ulnar
2.1–3.2 3.4 3.3
Elbow to wrist, 50–69 47.2 43.1
9.0–22.0 9.5 9.6
Wrist to the 4th digit, 59.7–74.9 43.8 46.1
11.4–89.4 7.7 9.7
Segment, normal value
4.2–6.5
5.0–21.5
Right tibial Left tibial
10.5 11.6
Popliteal fossa to the medial malleolus, 41–55 26.7 24.7
0.40 0.42
SEP
Erb (N9) (ms)
Cervical (N13) (ms)
Cortical (N19) (ms)
Right median Left median
14.9 14.4
19.7 19.3
24.5 24.7
NCS, nerve conduction study; CMAP, compound motor action potential; SEP, somatosensory evoked potentials; SCV, sensory conduction velocity; DL, distal latency; SNAP, sensory nerve action potential; MCV, motor conduction velocity. Sural SNAP was not evoked in NCS. Peroneal CMAP was not evoked in NCS. Cortical N40 was not evoked in SEP. Normal values are cited from previous publication [18].
tions showed that none of his brothers had foot deformity or gait disturbance. Results of nerve conduction studies and somatosensory evoked potentials are described in Table 1, which shows axonal dysfunction especially in the legs. Electromyography showed no active denervation, but rather a chronic denervation in the affected muscles. Selective loss of large myelinated fibers was shown by light microscopic analysis of biopsied specimens from his right sural nerve (Fig. 1). The density of large (diameter > 7 m) and small (≤7 m) myelinated nerve fibers was 510/m2 and 5810/m2 respectively (control 3340/m2 and 4470/m2 ). Regenerating clusters were frequent, quantified at 240/m2 , but onion-bulb formation was absent. Rare myelin ovoids were present, and no myelin debris was observed. Ultrastructural examination showed some bands of Bungner and numerous regenerating clusters of nerve fibers. A few fibers with unusual redundant myelin sheaths were present (Fig. 1B), but uncompacted myelin lamella were not observed. Excessively folded myelin sheaths were also not observed. A teased nerve fiber study showed no tomacula and segmental demyelination; remyelination was rare (figure not shown). Laboratory examinations and pathological analysis of sural nerve biopsy excluded paraneoplastic polyneuropathy, impaired glucose tolerance neuropathy, polyneuropathy associated with vitamin deficiency, chronic inflammatory demyelinating polyneu-
ropathy, vasculitic polyneuropathy, polyneuropathy associated with anti-MAG IgM antibodies, arsenic neuropathy, sarcoidal polyneuropathy and familial amyloid polyneuropathy. Considering the previous reports of sporadic cases with de novo mutations in CMT genes [1,2], genetic test was performed in PMP22 and MPZ. Chromosome 17p11.2-p12 duplication – commonly seen in CMT1A – was excluded by pulsed field gel electrophoresis. However, sequence analyses of the MPZ gene showed a A>G exchange at nucleotide 679 (numbering using the A of the translation initiation codon as +1, NCBI accession number P25189). This results in a heterozygous arginine to glycine substitution at codon 227 (Arg227Gly) or codon 198 (Arg198Gly) in conventional description in which the initial 29 amino acid leader peptide is not included [1]. The case report describes the identified Arg198Gly mutation. The nucleotide substitution was absent in 200 control chromosomes from unrelated Japanese individuals. 3. Discussion Neither the missense mutation Arg198Gly nor the nucleotide substitution was listed in the public databases (http://molgenwww.uia.ac.be/CMTMutations and http://www.ncbi.nlm.gov/snp). An amino acid substitution at the same position, Arg198Ser, was previously reported [1,6]. A patient carrying the missense muta-
Fig. 1. (A) Toluidine blue stained transverse semithin section showing a marked reduction in the number of large myelinated fibers and numerous regenerating clusters (black arrows). Onion-bulb formation was absent. (B) Electron micrograph showing that myelin loops are partly non-compact in a few fibers (white arrow). Uncompacted myelin lamella were not observed.
800
H. Shimizu et al. / Clinical Neurology and Neurosurgery 112 (2010) 798–800
tion Arg198Ser showed a mild CMT1B phenotype, but no detailed features – including pathological and electrophysiological findings – were available [1,6]. The missense mutation Arg198Gly is located in the highly conserved protein kinase C (PKC) alpha substrate motif (198RSTK201) of MPZ. In this position, amino acids are highly conserved amongst MPZ homologous proteins, including Rattus norvegicus, Mus musculus, Gallus gallus and Bos taurus. MPZ protein expression harboring the missense mutation Arg198Ser in cultured cells showed a loss of MPZ-mediated adhesion [3]. Therefore, the missense mutation Arg198Gly was identified as pathogenic. MPZ dysfunction leads to lack of myelination or hypomyelination, which might affect Schwann cell-axon interactions and result in secondary axonal atrophy [8]. On the basis of the results obtained from pathological and electrophysiological studies, the patient was diagnosed with axonal CMT2. Genetic tests were not performed in other family members; therefore, it remains unknown whether this is a de novo or a low-penetrance mutation. A wide range of phenotypic variability was reported in patients carrying the MPZ mutation, including age-at-onset, disease progression and severity [1,2]. Such variability may be related to the dosage of MPZ protein or the position and nature of MPZ mutation. The severity of the clinical phenotype might depend on the dosage of MPZ protein. Moreover, it has been suggested that MPZ protein dosage might be regulated by the biological effect of the mutation (e.g. dominant negative effect) or by biological pathways such as the nonsense-mediated mRNA decay pathway leading to half-normal expression in MPZ protein [9–12]. In case of missense mutations or deletions, various mutational effects have been proposed. Two mutations, Lys236del and Asp35Asn, were identified in familial cases with late-onset CMT [13,14]. Both mutations are associated with intra-familial phenotypic variations or variable penetrance raging from an asymptomatic state to foot deformities. Lys236del mutation is more closely located to the carboxyl terminus of MPZ protein. Moreover, it has been suggested that an altered phosphorylation status – resulting in dysregulation of myelin compaction – might be involved in the pathogenesis [13]. At the same time, MPZ protein conformational changes have been suggested to be involved in pathological mechanisms in the case of Asp35Asn [14]. Taking into account recent evidence-based reviews and cost benefit analyses in late-onset polyneuropathy without known causes [15–17], a ‘shot gun’ approach for CMT mutations is not recommended. The genetic test should be considered for patients at risk of CMT disease. However, if the clinical phenotype is similar to that of CMT and a de novo mutation is reported, the genetic test might also be considered for the diagnosis of CMT disease. Acknowledgments This work was supported by grants from the Japanese Ministry of Health, Labor and Welfare (N.O.), the Comitato Telethon Fondazione Onlus, the Amministrazione Autonoma dei Monopoli di Stato (AAMS), and the Italian city of Gubbio (Grant No. GGP06209 to A.O.), as well as the Italian Ministry of Health (Grant Nos. EBRI1.O,
PS05.11, PS05.21 and REG.17O to A.O.). We thank the patient and family members involved in this study. We also thank Ms Michela Renna (MA) for her language advice. Drs. Shimizu, Oka and Kawarai contributed equally to this work. Competing interests: None declared. Funding: Declared in the acknowledgments. Ethics Committee approval: The study was performed according to a protocol reviewed and approved by the Ethics Committee of the Hyogo Brain and Heart Centre. References [1] Shy ME, Jani A, Krajewski K, Grandis M, Lewis RA, Li J, et al. Phenotypic clustering in MPZ mutations. Brain 2004;127:371–84. [2] Reilly MM. Sorting out the inherited neuropathies. Pract Neurol 2007;7:93–105. [3] Gaboreanu AM, Hrstka R, Xu W, Shy M, Kamholz J, Lilien J, et al. Myelin protein zero/P0 phosphorylation and function require an adaptor protein linking it to RACK1 and PKC alpha. J Cell Biol 2007;177:707–16. [4] Menichella DM, Arroyo EJ, Awatramani R, Xu T, Baron P, Vallat JM, et al. Protein zero is necessary for E-cadherin-mediated adherens junction formation in Schwann cells. Mol Cell Neurosci 2001;18:606–18. [5] Xu W, Manichella D, Jiang H, Vallat JM, Lilien J, Baron P, et al. Absence of P0 leads to the dysregulation of myelin gene expression and myelin morphogenesis. J Neurosci Res 2000;60:714–24. [6] Xu W, Shy M, Kamholz J, Elferink L, Xu G, Lilien J, et al. Mutations in the cytoplasmic domain of P0 reveal a role for PKC-mediated phosphorylation in adhesion and myelination. J Cell Biol 2001;155:439–46. [7] Kamholz J, Awatramani R, Menichella D, Jiang H, Xu W, Shy M. Regulation of myelin-specific gene expression. Relevance to CMT1. Ann N Y Acad Sci 1999;883:91–108. [8] Hanemann CO, Gabreels-Festen AA. Secondary axon atrophy and neurological dysfunction in demyelinating neuropathies. Curr Opin Neurol 2002;15:611–5. [9] Wong MH, Filbin MT. Dominant-negative effect on adhesion by myelin Po protein truncated in its cytoplasmic domain. J Cell Biol 1996;134:1531–41. [10] Nagy E, Maquat LE. A rule for termination-codon position within introncontaining genes: when nonsense affects RNA abundance. Trends Biochem Sci 1998;23:198–9. [11] Inoue K, Khajavi M, Ohyama T, Hirabayashi S, Wilson J, Reggin JD, et al. Molecular mechanism for distinct neurological phenotypes conveyed by allelic truncating mutations. Nat Genet 2004;36:361–9. [12] Khajavi M, Inoue K, Wiszniewski W, Ohyama T, Snipes GJ, Lupski JR. Curcumin treatment abrogates endoplasmic reticulum retention and aggregationinduced apoptosis associated with neuropathy-causing myelin protein zero-truncating mutants. Am J Hum Genet 2005;77:841–50. [13] Sowden JE, Logigian EL, Malik K, Herrmann DN. Genotype–phenotype correlation in a family with late onset CMT and an MPZ lys236del mutation. J Neurol Neurosurg Psychiatry 2005;76:442–4. [14] Braathen GJ, Sand JC, Russell MB. Two novel missense mutations in the myelin protein zero gene causes Charcot-Marie-Tooth type 2 and Dejerine-Sottas syndrome. BMC Res Notes 2010;3:99. [15] England JD, Gronseth GS, Franklin G, Carter GT, Kinsella LJ, Cohen JA, et al. Evaluation of distal symmetric polyneuropathy: the role of laboratory and genetic testing (an evidence-based review). Muscle Nerve 2009;39:116–25. [16] Hughes RA, Umapathi T, Gray IA, Gregson NA, Noori M, Pannala AS, et al. A controlled investigation of the cause of chronic idiopathic axonal polyneuropathy. Brain 2004;127:1723–30. [17] Vrancken AF, Kalmijn S, Buskens E, Franssen H, Vermeulen M, Wokke JH, et al. Feasibility and cost efficiency of a diagnostic guideline for chronic polyneuropathy: a prospective implementation study. J Neurol Neurosurg Psychiatry 2006;77:397–401. [18] Kurokawa K, Tanaka E, Yamashita H, Kohriyama T, Mimori Y, Nakamura S. Ratios of nerve conduction parameters in proximal to distal limbs remain constant through the second to the eighth decades. Electromyogr Clin Neurophysiol 1998;38:169–76.