Two novel missense mutations in the myelin protein zero gene causes Charcot-Marie-Tooth type 2 and Déjérine-Sottas syndrome

Patients

Norwegian patients with CMT disease were analyzed for mutations in the MPZ gene. Affected were interviewed and had a neurological examination. The Neuropathy Impairment Score (NIS) and Charcot-Marie-Tooth disease neuropathy score (CMTNS) were used [14, 15]. Nerve conduction velocities were recorded with surface electrodes, while needles were used for electromyography (EMG).

Genetics

DNA was extracted from leucocytes using QIAgen FlexiGene kit (Düsseldorf, Germany). The sequences were derived from the National Center for Biotechnology Information (NP_000521 and NM_000530, MPZ). The coding region of the MPZ gene as well as exon and intron junctions were amplified using Eppendorf hotmaster taq polymerase. The sequencing was carried out using forward, reverse and internal primers, and BigDye Terminator kit version 1.1 Applied Biosystems (Life Technologies, Carslbad, CA, USA). Sequencing was performed using Applied Biosystems ABI3130x1 Genetic analyzer and aligned with the Sequencher programme (Gene Codes Corporation, Ann Arbor, MI). Numbering nomenclature used for the mutations includes the leader peptide of 29 amino acids.

Statistical analysis

SPSS for Windows version 14.0 was used for statistical analysis.

Ethics

The study was approved by the Regional Committees for Medical Research Ethics.

Phenotype

Missense mutations in the MPZ gene account for about 2% of patients with CMT type 1 [18]. The phenotype varies considerably from early to late age at onset. Clinically missense mutations in the MPZ gene can cause congenital hypomyelinating neuropathy (CH), DSS, CMT type 1, CMT type 2 and intermediate CMT [6, 16–22]. The phenotypes CH and DSS are characterized by early onset and severe symptomatology, while the other phenotypes have a later age at onset and milder symptomatology. Our patients had phenotypes compatible with DSS and CMT type 2.

Phenotype related to MPZ protein structure

Our patient with DSS had a Gly123Asp amino acid substitution. Another patient with DSS had a Gly123Cys, while a patient with CMT type 1 had a Gly123Ser amino acid substitution [6, 18]. Details about the patient with CMT type 1 are unfortunately not available. Ser has a structure that is much more similar to Gly than both Asp and Cys. Thus, the Gly123Ser amino acid change may cause less conformational changes of P₀ than the Gly123Asp and Gly123Cys amino acid change. Similarly, missense mutations in the codon that include residue number 124 can cause CMT type 1 or CMT type 2 [19, 21, 22]. This is probably due to the characteristics of the specific amino acid substitution and the conformational changes of the MPZ protein. Our patients with CMT type 2 had Asp35Asn amino acid substitution. Asp and Asn has very similar structure and probably only causes minor conformational changes of P₀, even though Asp is negatively charged and Asn is polar. Asp35Tyr causes intermediate CMT, CMT type 1 and CMT type 2 [16, 17]. Tyr, a polar amino acid, has a hydroxyphenyl group that replace Asp charged side chain. The conformational change is likely to be small.

HeLa cells tranfected with two late onset and two early onset MPZ mutations showed that the two late onset mutations had normal transportation of the P₀ to the cell membrane and moderately reduced MPZ-mediated intercellular adhesion, while the two early onset mutations had different patterns [23]. One early onset type was correctly glycosylated and trafficked to the plasma membrane, but intercellular adhesion was strongly affected, while the other early onset type caused by deletion in the gene showed that the transcripted P₀ was retained within the cytoplasm with secondary reduced adhesion. The Ser63del causes a demyelinating neuropathy in transgenic mice, similar to the Ser63del causes a CMT type 1 in humans [24–26]. Interestingly, the P₀ Ser63del is retained in the endoplasmic reticulum, and it fails to be incorporated into myelin [24, 25]. These are very interesting mechanisms which are not directly related to the conformational change of the P₀. The retained P₀ might be the mechanism that causes congenital hypomyelinating neuropathy (CH). The literature provides several examples on allelic heterogeneity, i.e. mutations in one gene can cause different phenotypes. An example is mutations in the CACNA1A gene, which causes familial hemiplegic migraine (FHM-1), episodic ataxia (EA-2) or spinocerebellar ataxia (SCA-6) [27, 28]. FHM-1 is characterized by sensory and motor aura followed by headache. The FHM-1 families show both intra- and interfamilial variability. Affected from some FHM-1 families experience severe hemiplegic attacks, cerebral oedema and life threatening coma due to minor head trauma. The more common Charcot-Marie-Tooth genes identified so far show both locus and allelic heterogeneity. The allelic heterogeneity in the MPZ gene is characterized by different types of neuropathy, in contrast to different disorders caused by mutations in the CACNA1A gene. At present it is anticipated that four P₀ proteins form a tetramer and link to a tetramer of the opposed membrane. To our knowledge expression of the MPZ alleles is unknown. If one allele is expressed per cell, it will either be a wild type or changed P₀. However, if both alleles are expressed equally, tetramer assembly would statistically be in the order 1:16, 4:16, 6:16, 4:16, 1:16 for the different combinations of wild type and changed P₀. Thus, only 1:256 (1:16²) will be normal tetramers that can link together. A sural nerve biopsy of a patient with CH caused by a mutation in P₀ presented a characteristic picture of non-myelinated and poorly myelinated axons with basal lamina onion bulbs and lack of myelin breakdown products [29]. The histology supports that both alleles, i.e. the wild type and mutated allele, are expressed in each Schwann cell. If only one allele was expressed per cell, one would expect to observe both normal and abnormal Schwann cell histology, i.e. normal and abnormal myelination along the axon. The assembly of different tetramers may also explain the intrafamilial variation of symptoms and age at onset [16, 24]. Even a concordant monozygotic twin pair was described with different progression, classified as Roussy-Lévy variant of CMT1 and CMT1, respectively [30]. We cannot exclude the importance of environmental factor with certainty. However we do not think the environment has a crucial role given proper nutrition and exclusion of other neuropathy inducing factors.