Charcot-Marie-Tooth disease type 2 associated with mutation of the myelin protein zero gene

Hereditary motor and sensory neuropathy (HMSN), or Charcot-Marie-Tooth disease (CMT), is a group of clinically and genetically heterogeneous peripheral nerve disorders.¹ The most common form, HMSN type I (HMSN I), can be subdivided into two genetically separated entities named CMT1A and CMT1B. CMT1A is due to mutations in the PMP22 gene on chromosome 17p11.2-12, usually represented by duplication of 1.5 megabases in the region containing the gene.² In addition, several point mutations in the PMP22 gene have been found in CMT1A families.^3-8

Mutations of the myelin protein zero (MPZ) gene have been associated with CMT1B^9-18 and with two more severe forms of hypertrophic polyneuropathies, Déjerine-Sottas disease(DSD)^18-20 and congenital hypomyelination(CH). The neuronal form of CMT or HMSN type II (HMSN II) is pathologically distinct from the HMSN I demyelinating form, because it is characterized by axonal degeneration with little evidence of peripheral nerve segmental demyelination or hypertrophic changes.¹ HMSN II is usually inherited in a dominant autosomal manner with variable onset, generally later than HMSN I.¹ Electrophysiologic studies have provided a useful tool for distinguishing between HMSN I, in which nerve conduction velocity (NCV) is always below 38 m/sec, and HMSN II, in which NCV is characteristically normal or slightly reduced.^1,21 Normal sensory nerve action potentials (SNAPs) differ between distal hereditary motor neuropathy and HMSN II, because in the latter SNAPs are often absent.²² From a genetic point of view, HMSN II is heterogeneous; linkage to 1p35-p36 (CMT2A),²³ 3q (CMT2B),²⁴ and 7p (CMT2D)²⁵ chromosomes has been reported. CMT2C with diaphragmatic and vocal cord weakness has not yet been mapped.²⁶ Most HMSN II families, however, do not link to any of the reported loci.²⁷MPZ is a type I membrane glycoprotein of 28 kDa present in the sheath of peripheral nerve MPZ, belonging to the immunoglobulin superfamily and having extracellular, transmembrane, and cytoplasmic domains.^28-30 MPZ gene expression is restricted to the peripheral nerve Schwann cell and is thought to hold extracellular leaflets of myelin together by homophilic interaction of its extracellular domains.³¹ The gene, mapped on chromosome 1q,^22-23 is composed of six exons spanning about 7 kb of DNA.²⁸

We report a large Sardinian family whose diagnosis of HMSN II was established on the basis of neurophysiologic criteria^1,21,22 in which a novel missense point mutation of the MPZ exon 2 sequence²⁸ was found. This TCC132TTC mutation, resulting in a serine to phenylalanine substitution at codon 44 of the MPZ extracellular domain, was present in the heterozygous state in 16 affected subjects but not in 6 healthy relatives or in 160 control chromosomes. This is the first example of an MPZ point mutation involved in HMSN II, suggesting that the spectrum of phenotype induced by mutation in this gene is wider than initially appreciated, ranging from HMSN I to HMSN II forms.

Methods. Subjects. We studied a large Sardinian family (figure 1), called P-B, in which 16 members showed clinical and electrophysiologic signs of HMSN II disease. Autosomal dominant inheritance was evident because affected subjects were present in all generations examined, along with male-to-male transmission. In symptomatic subjects, onset age ranged from 38 to 62 years. Clinical features are reported in table 1. Common early symptoms were calf cramps and difficulty standing on heels. Two subjects (40, age 76; and 215, age 68) were unable to walk without help at the time of examination. Neurologic examination showed distal hyporeflexia in most subjects and a mild pes cavus in some subjects. A remarkable calf atrophy was present in Subjects 40, 226, 225, and 214. Mean values of NCVs at the median nerve in 16 affected subjects was 42.3 m/sec (range, 57.3 to 0), whereas in 6 healthy relatives it was 54.9 m/sec (range, 56.5 to 53.8). In two asymptomatic patients (160 and 169), electromyography examination showed signs of chronic denervation in the distal leg muscles, consistent with axonal involvement. No abnormal spontaneous activity was observed in affected subjects. Electrophysiologic data from nine affected subjects who consented to extensive testing are detailed in table 2. Consent for a nerve biopsy was refused.

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Figure 1. Pedigree of family P-B. Only examined subjects are reported (this family included more than 150 individuals). Females are represented as circles and males as squares. Darkened figures are affected individuals. Deceased individuals are shown as cross-hatched symbols. The symbol ⁁ indicates clinically asymptomatic individuals.

Analysis for duplication and linkage with chromosome 17p11.2-12. Southern blot analysis was performed using peripheral blood genomic DNA digested with Msp I and probed with pVAW409R3a (locus D17S122),³² which maps within the CMT1A duplication region. Determination of (GT)n polymorphisms on D17S122 locus was studied as described.³³

DNA sequence analysis of the MPZ gene. Single-strand sequence analysis was performed using Sequenase kit version 2.0 (U.S. Biochemical, Cleveland, OH) according to the manufacturer's instructions and with primers flanking the six MPZ coding regions.³⁴

Sma I enzyme restriction site analysis for the TCC132TTC MPZ mutation. Amplification of 0.5 µL of genomic DNA was performed using 50 pmol of each primer specific for exon 2,³⁴ 1 U of Taq DNA polymerase (GIBCO-BRL, Beverly, MA), and 0.2 mM of each dNTP (Pharmacia, Biotech, Uppsala, Sweden). Polymerase chain reaction buffer, 10 mM Tris-HCl pH 8.3, 50 mM KCl, 1.5 mM MgCl₂, and 0.1% gelatine (Perkin-Elmer, Branchburg, NJ) at a final concentration of 50 µL was used. Amplification conditions were 94°C for 1 minute, 57 °C for 1.5 minutes, 72 °C for 2 minutes, 30 cycles, final extension 10 minutes at 72 °C. Amplification generated a 127- and 182-bp fragment. Ten microliters of the product were digested overnight at 25 °C with 6 U of Sma I enzyme (Biolabs, Inchinnan, Scotland, UK) according to the manufacturer's instructions. Digested product was fractionated on a 6% polyacrilamide gel and visualized with ethidium bromide.

Results. Analysis for duplication and linkage with chromosome 17p11.2-12. Genetic analysis excluded the duplication of D17S122 locus by Southern blot of genomic DNA digested with Msp I and probed with pVAW409R3a.³² Linkage with the PMP22 gene was excluded by determination of (GT)n polymorphisms on D17S122 locus³³ (data not shown).

DNA sequence analysis of MPZ gene. Single-strand sequence analysis of genomic DNA was performed with primers flanking the six MPZ coding regions.³⁴ Sequencing revealed a missense point mutation TCC132TTC in exon 2²⁸ present in the heterozygous state in one affected individual (CMT 42) but absent in a healthy family member (CMT 218; figure 2).

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Figure 2. DNA sequence analysis of the MPZ gene. Single-strand sequence analysis showed TCC132TTC point mutation in exon 2 in the heterozygous state in affected Subject 42 but not in a healthy relative(Individual 218).

Sma I restriction enzyme analysis for the TCC132TTC MPZ mutation. The TCC132TTC point mutation abolished the 5′-CCCGGG-3′ restriction site of Sma I enzyme in one of two chromosomes, thus determining the presence in affected subjects of the expected 127- and 182-bp fragments and of an additional 309-bp fragment due to undigested DNA (figure 3). The TCC132TTC point mutation missense mutation was in total linkage with the HMSN II in our family because it was inherited in all affected members. To be sure that the TCC132TTC mutation was not a polymorphism, we performed restriction analysis in 80 ethnic-matched healthy individuals, all showing the 127- and 182-bp expected pattern.

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Figure 3. Sma I restriction enzyme analysis for the TCC132TTC MPZ mutation. Lane 7 is genomic predigested DNA from healthy Subject 3 (see figure 1) and lane 15 is genomic predigested DNA from affected Subject 200(seefigure 1). Lanes 1 through 6 and 9 through 14 are postdigested DNA from members of family B-P. The Sma I site is present in DNA from healthy relatives, generating a 127-bp and a 182-bp fragment (lanes 1, 9, 11, 13, and 14). The Sma I site appears in the heterozygous state in affected subjects (lanes 2 to 6, 10, and 12), resulting in one uncut fragment of 309 bp and in the two 127- and 182-bp fragments.

Discussion. In the present study we reported a large Sardinian family affected by an autosomal dominant HMSN II. Although the nerve biopsy was not performed, the electrophysiologic signs were entirely compatible with the clinical diagnosis of HMSN II. We found a novel TCC132TTC MPZ gene point mutation in total linkage with the HMSN II, which is likely to be responsible for the axonal neuropathy. This is the first report of HMSN II due to MPZ mutation. It is of interest to note that although in a few previous studies the genes involved in HMSN II were excluded from HMSN pedigrees,^23-27 some evidence of linkage to chromosome 1p was recently reported in a family with HMSN II.²⁷ The MPZ gene mutation found in our HMSN II family presented in this study suggests that the idea that the MPZ gene is not involved in hereditary axonal neuropathies should be reconsidered. The presence of MPZ point mutation in the phenotypically HMSN II family we reported suggests that axonal and demyelinating forms of HMSN may constitute a spectrum of genotypically related conditions. In addition, mutation in another myelin protein, connexin 32, can cause axonal and demyelinating neuropathy.²⁷

The phenotypic variability of CMT1B, DSD, and CH has been related to the position and nature of MPZ mutation.¹⁸ Analogously, the nature of the mutation may also account for some forms of HMSN II on the basis of the influence exerted from the myelin sheath of glial cells on the axon with which they are associated.³⁵ In the case of the HMSN II family described in this study, it can be hypothesized that an "axonal" function defect may arise from failure in axoglial interactions determined by the particular mutation of the MPZ protein. Because the expression of the MPZ gene is restricted to Schwann cells, the present family suggests that some mutations of the MPZ gene may not be so disruptive of myelin compactness as to cause relevant slowing of NCV, which occurs, however, in most cases of CMT1B, DSD, and CH. On the basis of these results, we suggest the inclusion of the MPZ gene in the genetic analysis of HMSN families. Moreover, results from genetic studies performed in demyelinating¹⁸ and axonal²⁷ inherited neuropathies, along with the present study, suggest that it might be more appropriate to classify hereditary neurophathies on the basis of the genetic defect along with clinical and electrophysiologic parameters in isolation.

Generation of animal models with specific MPZ mutations will aid in understanding the biological mechanisms underlying axonal-glial interactions in demyelinating and axonal forms of HMSN.

Acknowledgment

We are grateful to Dr. Enrico Fanni for his genealogic research.