Vol. 51 No. 1/2004 273–280 QUARTERLY Communication

The myelin protein zero gene (MPZ) coding for the most abundant protein of the peripheral myelin was shown to be mutated in Charcot-Marie-Tooth type 1B disease (CMT1B). Later on MPZ mutations have been shown in axonal type of CMT (CMT2). Recently three novel MPZ gene mutations were reported in congenital hypomyelinating neuropathy (CHN). In contrast to the previously reported studies, focused on CMT1B disease, we aimed to analyze the coding and promoter sequences of the MPZ gene in a group of patients with three CMT phenotypes i.e.: CMT1, CMT2 and CHN. Over 500 PCR products were screened by single strand conformation polymorphism analysis (SSCP) and heteroduplex analysis (HA). In one CMT2 family we founded the E56K mutation in the MPZ gene and in one CHN patient the T124K substitution was detected. In agreement with previously reported studies we conclude that MPZ gene screening should be performed for wide phenotype spectrum of CMT.

gene in two families, in patients suffering from demyelinating type of Charcot-Marie-Tooth disease linked to the 1q22-q23 locus (CMT1) (Hayasaka et al., 1993).By contrast to the CMT1A disease caused by submicroscopical duplication in the 17p11.2-p12region, CMT1 caused by mutations in the MPZ gene was designed as CMT1B (Raeymaekers et al, 1991;Bird et al., 1982).Three years after the first description of the MPZ mutations segregating with CMT1B disease, the Gln215stop mutation was reported in patient suffering from congenital hypomyelinating neuropathy (CHN) (Warner et al., 1996).Recently, two novel mutations in the MPZ gene were found in patients suffering from congenital hypomyelination neuropathy (Lupski et al., 2003).Although MPZ protein has previously been shown to be expressed in the myelinating Schwann cells, the Ser44Phe mutation was detected in a large Sardinian family consisting of patients suffering from the axonal type of Charcot-Marie-Tooth disease (CMT2) (Marrosu et al., 1997).Up to now, over ninety mutations have been reported in the MPZ gene.The majority of them segregate with CMT1 disease.Nineteen mutations have been shown to segregate with more severe form of CMT1, designed as Dejerine-Sottas-syndrome (DSS).The mutations were reported in congenital hypomyelination neuropathy (CHN) and in CMT2 disease (http://molgen-www.uia.ac.be/CMTMutations).

FAMILIES AND PATIENTS
The study included a total of 46 CMT patients (Table 1).Based on electrophysiological and morphological criteria, 15 patients were diagnosed with CMT1 (motor median nerve conduction velocity (MNCV) < 38 m/s), 23 had CMT2 (MNCV > 38 m/s), 4 had CHN (sural nerve biopsy revealing thin myelin sheet and MNCV < 6 m/s) and 4 patients had an unspecified peripheral neuropathy based on clinical records only.
From only 9 families the possible trait of inheritance could be deduced from the pedigree analysis.By contrast, CMT diagnosis in 24 families was established in the sporadic cases.The autosomal dominant trait of inheritance was observed in 8 pedigrees, whereas "possible" autosomal recessive (AR) trait was apparent in two families.Although, no paternity testing was performed, we tended to find an AR trait, if at least two individuals were CMT affected and their parents were healthy.In the family F16 the autosomal dominant CMT2 transmission was present within three generations.In family F1 two sons of the healthy parents were CMT affected, suggesting the AR trait of inheritance.The patients included in this study represent four CMT phenotypes.Firstly, CMT1 disease was diagnosed in 13 families.In 11 families CMT2 was observed.Congenital hypomyelination neuropathy was observed in 4 families, in 4 individuals.
After denaturation for 5 min, at 95°C, samples were rapidly loaded on ice.Heteroduplex analysis (HA) of the MPZ gene was performed with primers used in SSCP.
Samples (3 ml) were mixed with DNA-loading buffer (saccharose, 0.5 M EDTA, 10 M Tris, Bromophenol blue) and denaturated for 5 min at 95°C.For renaturation, samples were placed at 65°C for 45 min.Samples were loaded on non-denaturing 6% acrylamide gel (acrylamid/bis-acrylamide, 99:1, w/w) at 8 W for 20 h.After electrophoresis gels were silver stained.DNA fragments disclosing different, from wild, migration pattern were direct sequenced.The Prism Sequenase Terminator Double Stranded Sequencing Kit was used.Enzyme restriction analysis was performed to confirm nucleotide changes in family F6.

RESULTS
Over 500 PCR products corresponding with six exons of the MPZ gene have been analysed by SSCP and HA.In 17 PCR products SSCP (17) and HA (12) revealed an altered migration pattern.DNA sequencing was per-formed in 7 individuals.In one family RFLP analysis was performed (Table 2).
In patient F6MJ, the SSCP and HA of the exon 3 of the MPZ gene showed an altered mobility of the PCR fragment.Sequence analysis of exon 3 in this patient revealed a C to A base change, resulting in an amino acid substitution of Thr to Lys at the codon 124.(Fig. 1).
The SSCP analysis performed in parents of F6MJ did not reveal any mobility alteration suggesting that exon 3 of the MPZ gene in them was most probably not mutated.Due to the the MaeII restriction site loss, caused by heterozygous C(371)A transversion, an addi- tional 190 bp band was observed in patient F6MJ on RFLP analysis.RFLP analysis with MaeII endonuclease, performed in 10 control samples revealed only one 190 bp band (Fig. 2).The Thr124Lys mutation is a novel heterozygous substitution located in the exon 3 of the MPZ gene found in the 14-year-old boy diagnosed with congenital hypomyelination neuropathy (Kochanski et al., in press).
The SSCP analysis of the exon 2 of the MPZ gene in patient F16KB revealed an altered migration pattern.In the sequencing of the exon 2 of the MPZ gene an heterozygous G-A transition was detected leading to the Glu to Lys amino acid substitution at the codon 56 of the MPZ gene.The Glu56Lys mutation in the exon 2 of the MPZ gene was found in an 40 years-old female suffering from CMT2 disease (Kochanski et al., in press).
In 11 individuals (see Table 2) both SSCP and HA analysis of exon 6 of the MPZ gene revealed an altered migration pattern.The automated sequencing of the exon 6 of the MPZ gene disclosed an heterozygous C to T base change at position 684, which did not change the Ser at codon 228.In one healthy individual from family F23 (Fig. 3) the SSCP analysis of exon 6 of the MPZ gene disclosed an altered migration pattern, whereas HA did not reveal any migration changes.By sequencing of exon 6 of the MPZ gene the homozygous replacement of C to T at position 684 which did not change Ser at codon 228 was observed.In 11 individuals SSCP analysis of the promoter region of the MPZ gene disclosed three different migration pattern (Fig. 4).The HA of the MPZ promoter region did not reveal any migration changes.Sequencing analysis of MPZ promoter region was performed in two individuals (F32GA and F26DM) showing an altered SSCP migration pattern.In patient F32GA an homozygous single nucleotide G insertion was found at position -140 within   the CAAT sequence in the sense strand.The antisense strand sequencing did not revealed any abnormality in the analysed region.In patient F19WE the abnormal migration pattern was visible in SSCP analysis, whereas no migration changes were detected in HA.No sequence alteration was found in the exon 3 of the MPZ gene in the patient F19WE.

DISCUSSION
The screening of the MPZ gene revealed the presence of two novel mutations i.e.T124K and E56K.From the molecular genetics point of view, the T124K and E56K substitutions represent conservative amino acid changes located in the exons 2 and 3 of the MPZ gene corresponding with the extracellular part of the MPZ protein (Fig. 5).As far, the vast majority of the MPZ gene mutations have been reported in the extracellular part of the MPZ protein which is thought to be responsible for its adhesive properties (Filbin et al., 1990).
Interestingly the novel mutations were not found in the most common form of CMT1B disease associated with MPZ gene mutations but in the CMT2 disease and congenital hypomyelination neuropathy.
By contrast to the previously published MPZ gene screening's data, we decided to extend the spectrum of phenotypes for MPZ molecular genetic analysis.Besides the classical CMT1 phenotype associated with MPZ gene mutations, we analysed the MPZ gene in patients diagnosed with CMT2, and CHN.The reason for that was the wide spectrum of phenotypes associated with mutations in the

. Characteristics of CMT patients and their relatives, AD -autosomal dominant trait of inheritance, AR -autosomal recessive trait of inheritance
The patients in whom duplication of the PMP22 gene was found are marked by asterix.
MPZ gene.In 12 individuals the heterozygous S228S polymorphism was detected.Although SSCP analysis disclosed the altered migration pattern of exon 6 of the MPZ gene for heterozygous (11 samples) and homozygous (one sample) S228S polymorphism, no changes were observed in the HA for homozygous S228S polymorphism.Two types of mismatches have been detected by sequencing analysis.Firstly, in patients F16KB and F6MJ the G-T/A-C mismatch may be deduced from sequencing analysis.Interestingly, the same G-T/A-C mismatch found by HA in heterozygous individuals in the F17 family was not observed in the F16KB patient.Since the same G-T/A-C mismatch was located in exons 2 (F16KB) and 3 (F6) of the MPZ gene, it is possible that two different MPZ related sequences, "surrounding" the same mismatch, affected the HA sensitivity.
In patient F6MJ sequencing analysis revealed the C-T/A-G mismatch in the distal part of the PCR product corresponding with exon 3 of the MPZ gene.By contrast, the same type mismatch located in the central part of the exon 3 of the MPZ gene, 78 nucleotides away from that found in patient F6MJ was not detected in HA (Nelis et al., 1996).
Although, the mismatch position at the end of the PCR product was shown previously to result in a decreased HA sensitivity, our results indicate, that the more distally located changes are easier to detect than those located in the central portion of the PCR product (White et al., 1992).Therefore, it can be Eight different polymorphisms were reported in the coding sequence of the MPZ gene up to now.In this group, only the S228S polymorphism was reported in families originating from Spain, Belgium, United States and Russia (Nelis et al., 1994;Roa et al., 1996;Bort et al.,1997;Mersiyanova et al., 2000).The estimated frequency of the S228S polymorphism was 4.1% (Nelis et al., 1994).It seems that S228S is the most common MPZ sequence variant occurring in various populations.It is a subject of interest if S228S mutation may represent an effect of common ancestor or, if does reflect the presence of the potential MPZ gene "hot-spot" at codon 228.
Interestingly, we found one healthy individual with homozygous S228S polymorphism.Given, the 4.1% estimated frequency of S228S polymorphism, the frequency of homozygous S228S polymorphism may be calculated as 0.00166.In addition to the analysis of the coding sequence of the MPZ gene we also de-cided to screen its promoter sequence.To date, no mutations or polymorphisms have been detected in the promoter sequence of the MPZ gene.Interestingly, we observed three different patterns of the PCR fragments corresponding with MPZ gene promoter in the SSCP analysis.
The presence of the homozygous insertion in patient F32GA at the position -140, detected in the sequencing of the sense strand was not confirmed by the antisense strand sequencing.For this reason we tend to classify this homozygous guanosine insertion rather as the polymerase-induced-error than a novel variant of the MPZ gene promoter sequence.I am grateful to Prof. Dr. Irena Hausmanowa-Petrusewicz who enabled me to perfom molecular genetics analysis in the Neuromuscular Unit, Medical Research Centre, Polish Academy of Sciences.I would like to thank Dagmara Kabzinska MSc for support.Colours represent individual missense and silent mutation found in this investigation.Both missense mutations are located in evolutionary conserved region of the P 0 protein.

Figure 1 .
Figure 1.Schematic representation of the site of mutations and polymorphism in P 0 protein found in this study.Arrows indicate positions of mutations and polymorphism in P 0 amino-acid sequence.(EC, extracellular domain; TM, transmembrane domain; IC, intracellular domain).

Figure 2 .
Figure 2. Restriction fragment length polymorphism analysis (RFLP) was performed in family F6.From the left: 100 bp DNA leader; A, proband; B, proband's mother; C-F, negative controls.The size of alleles is indicated with arrows.

Figure 3 .
Figure 3. Pedigree of the CMT1 family (F23).Proband was marked with an arrow.III5 individual is homozygous for S(228)S, II3 and II4 are heterozygous for S(228)S polymorphism.

Figure 5 .
Figure 5. Amino-acid sequences comparison of myelin protein zero (P 0 ) of six species.

Table 2 . Overview of the mutations and polymorphisms detected by single stranded conformation polymorphism analysis (SSCP) and/or heteroduplex analysis in the MPZ gene said
that mismatch position within analysed sequence determines the HA sensitivity.