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May 01, 1996; 46 (5) Articles

Linkage and mutation analysis of Charcot‐Marie‐Tooth neuropathy type 2 families with chromosomes 1p35‐p36 and Xq13

V. Timmerman, P. De Jonghe, P. Spoelders, S. Simokovic, A. Löfgren, E. Nelis, J. Vance, J.-J. Martin, C. Van Broeckhoven
First published May 1, 1996, DOI: https://doi.org/10.1212/WNL.46.5.1311
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Abstract

A locus for autosomal dominant Charcot-Marie-Tooth disease type 2 (CMT2A) was assigned by linkage analysis to chromosome 1p35–p36. We examined 11 unrelated CMT2 families for linkage to CMT2A using short tandem repeat (STR) polymorphisms. Only one family showed suggestive evidence for linkage to 1p35–p36. Further, because of an overlap in electrophysiologic data between CMT2 and CMTX female patients, we screened 6 of 11 CMT2 families compatible with dominant X-linkage for mutations in the connexin 32 (Cx32) gene at Xq13. There was a Cx32 mutation in one family, whereas another family showed suggestive evidence for Xq13 linkage upon analysis with STR polymorphisms. Our results suggest that the CMT2A locus is a minor locus for CMT2, additional linkage studies are needed to localize other CMT2 loci, and Cx32 mutations may be the underlying genetic defect in some CMTS families.

Charcot-Marie-Tooth disease (CMT) represents a group of clinical and genetical heterogeneous disorders affecting the peripheral nervous system. 1,2 Most CMT patients belong to families in which the disease segregates according to an autosomal dominant inheritance pattern. 3,4 There are reports of families with an X-linked or recessive mode of inheritance as well as sporadic patients. 4 CMT is clinically characterized by progressive weakness and atrophy of distal muscles of both lower and upper extremities. The severity of the disease varies among patients from almost no symptoms to severe foot-drop and high arched feet. Neurophysiological, as well as histopathologic criteria, differentiates CMT disease into three forms of peroneal muscular atrophy syndrome. CMT type 1 (CMT1) or hereditary motor and sensory neuropathy type 1 (HMSN I) is the hypertrophic form of CMT, because there are extensive segmental de- and remyelination and onion bulb formations on peripheral nerve biopsies. CMT type 2 (CMT2) or HMSN II is the neuronal form of CMT because of axonal degeneration without segmental demyelination of peripheral nerves. 3,4 In the spinal form of CMT or distal hereditary motor neuropathy (distal HMN), the axons of motor anterior horn neurons seem to be primarily affected, and the sensory neurons are spared. 5

According to Harding and Thomas, 6 CMT1 is associated with nerve conduction velocities (NCVs) in the motor median nerve less than 38 m/s. Reduced NCVs in CMT1 patients is a 100% penetrant phenotype and provides a reliable diagnosis of CMT1 in at-risk individuals even in early childhood when the symptoms are not yet apparent. 3 NCV limits in CMTS patients are less well defined. Harding and Thomas 6 considered motor NCVs higher than 38 m/s the hallmark of CMT2. Dyck et al. 4 used a more restrictive definition for CMT2 (i.e., normal or slightly reduced NCVs), but they did not specify the lower NCV limit. CMT2 defined on electrophysiological criteria is not a genetic entity because both autosomal dominant or recessive mode of inheritance are observed. Also, female patients or carriers in X-linked CMT families often meet the CMT2 criteria. 7 Furthermore, there are CMT patients with intermediate NCV values, making the electrophysiological classification of CMT indefinable.8,9 CMT2 is also characterized by low amplitudes of compound muscle action potentials, whereas sensory nerve action potentials (SNAPs) are often absent. Harding and Thomas 10 further showed that the essential distinguishing feature between CMT2 and distal HMN is the presence of normal SNAPs in distal HMN.

Genetic studies using positional cloning strategies have improved the clinical, histopathologic, and electrophysiological classification of CMT disease, especially of CMT1 disease. Autosomal dominant CMT1 is a genetically heterogeneous disorder with at least three loci: a frequent CMT type 1A locus (CMT1A) located at chromosome 17p11.2, a less frequent CMT type 1B locus (CMT1B) located at chromosome 1q22–923, and a third unassigned CMT type 1C locus (CMT1C) not linked to 17p11.2 nor 1q22–q23. 4 There was absolute cosegregation of CMT1A with a 1.5-Mb tandem duplication in chromosome 17p11.2 in 70.7% of CMT1 families, indicating that the duplication is the major disease causing mutation. 11–14 The peripheral myelin protein 22 gene (PMP-22) was located within the CMT1A duplication, 15–18 and the identification of different mutations in the PMP-22 gene in nonduplicated CMT1A patients proved that PMP-22 is the CMT1A gene. 19–22 The CMT1B gene was the major peripheral myelin protein zero gene (P0) because there were distinct mutations in Po in several CMT1B patients. 23–29 In dominant X-linked CMT1 families (CMT1X), investigators detected 22 mutations in the gap junction protein gene connexin 32 (Cx32) located at chromosome Xq13.30-33 Additionally, we recently observed single base changes in the Cx32 gene in eight unrelated patients with a diagnosis compatible with CMT1. 34

Ben Othmane et al. 35 reported evidence for genetic linkage and genetic heterogeneity of CMT2, with the localization of one form (CMT2A) to chromosome 1p35p36.

In this study, we performed linkage analysis with lp35–p36 short tandem repeat (STR) markers in 11 unrelated families referred to our laboratory as CMT2 families based on clinical and electrophysiological examinations. We also screened the families without male-to-male transmission of the disease for mutations in the Cx32 gene and performed linkage studies with STR markers flanking the CMT1X gene.

Methods

Family data

We selected seven Belgian CMT2 pedigrees for linkage and mutation analysis (figure 1, A and B): CMT-W, CMT-28, CMT-48, CMT-56, CMT-61, PN-22, and PN-54. A section from the CMT2 family PN22 was studied 50 years ago by André-van Leeuwen. 36 The families were recruited in a research programme on CMT (CMT family numbers) or were referred to our laboratory for DNA diagnosis (PN family numbers). The patients belonging to these seven families were diagnosed with the typical CMT phenotype of progressive distal paresis and amyotrophy initially involving the lower limbs and later on spreading to the upper limbs. In all families, male and female patients were equally affected. Motor and sensory NCVs were measured by stimulation of the median, ulnar, and peroneal nerves and recorded with surface electrodes according to standard procedures. In each family, at least one patient had motor median or ulnar NCVs higher than 38 m/s. In our linkage studies, we also included four CMT2 families of different ethnic origin: CMT-E and CMT-83 are German families, CMT-32 is a Dutch family, and CMT71 is an Italian family. The extended pedigree of CMT-71 has been described as an intermediate form of CMT disease by Rossi et al. 9 using clinical, electrophysiological, and ultra-structural diagnostic methods. In family CMT-E, two male patients had motor median NCVs of 43 and 66 m/s. In family CMT-32, one patient had slowed motor median NCVs but normal sensory NCVs of the median and sural nerve. In family CMT-83, one patient had motor peroneal NCVs of 34 and 42 m/s. We never observed normal motor peroneal or sensory NCVs in our CMT1 patients (De Jonghe, unpublished data); therefore, we classified these latter two families as CMT2 families. Also, at least one patient in each of the 11 CMT2 families had signs of unequivocal sensory involvement on clinical examination, nerve conduction study, or nerve biopsy. Dominant inheritance of CMT disease was observed in all families, and autosomal dominant inheritance occurred only in CMT-E, CMT-32, CMT-48, CMT-71, and PN-54.

Figure

Figure 1. CMT2 families. (A) Individuals of which the DNA was analyzed are indicated with a number.

Figure

Figure 1. (continued). (B) The haplotypes of the chromosomes Xq13 and lp35–p36 loci are shown in families CMT56 and CMT71. The haplotypes segregating with the disease are boxed. Slashed = deceased, squares = males, circles = females, filled = affected individuals, blank = nonaffected individuals, half filled = disease status unknown.

DNA analysis

STR genotype analysis was performed at the chromosome 1p35–p36 loci DlS160 (MIT-MS48) and D1S170 (MIT-COS37), 37 D1S244 (AFM220yf4) and D1S228 (AFMl96xb4), 38 and at the chromosome Xql1.2-q21 loci DXYS1X (pDP34) and PGK1 (pHPGK-7C) 39,40 and DXS453 (Mfd66) 41 Genomic DNA, 0.15 μg, was amplified using oligonucleotide primers labeled with FAM or JOE fluorophores according to the protocol of Applied Biosystems Incorporation (ABI, Foster City, CA). PCR was performed in a 30-μL reaction volume containing 10 pmol of each primer and 0.1 U Goldstar Taq DNA polymerase (Eurogentec, Seraing, Belgium). The PCR amplifications were performed in the automated thermal cycler Techne PHC-3 (New Brunswick Scientific, Nijmegen, The Netherlands). An aliquot of 1 μL of each amplified product was mixed with 4 μL formamide and 0.5 μL fluorescent-labeled size standard GeneScan2500-ROX (ABI) and heated 4 minutes at 95°C. Denatured PCR products were loaded on 6% polyacrylamide sequencing gels and electrophoresed in the ABI automated DNA sequencer 373A. Finally, the data were collected and analyzed using the ABI GENESCAN 672 software, 42 which estimates the size of alleles and also computes a calibration curve that allows quantification of the signals based on peak heights and peak areas.

Single stranded conformational polymorphism (SSCP) and sequence analysis

For SSCP analysis, the coding region of the Cx32 gene, exon 2, was amplified by touchdown PCR using three primer sets corresponding to nucleotides 54–77 ((2x32-1) and 336–359 (CX32-2) (i.e., part 1, 273–296 [Cx32-31 and 685–704 [Cx32-5]; part 2, 635–658 [Cx32-S1] and 919–938 [Cx32-A1]; part 3). 30 For the automated DNA sequence analysis of part 1 and part 2, PCR amplification was performed with primers (2x32-1 and Cx32-5 and sequencing was performed with primers Cx32-2 and Cx32-3. For sequencing part 3, the primers Cx32-3 and Cx32-A1 were used for PCR amplification and Cx32-S1 was used as sequencing primer. The SSCP and automated DNA sequencing methods are described elsewhere. 34

Linkage analysis

Two-point linkage studies were performed using the MLINK program of the FASTLINK computer package version 2.1. 43–46 CMT2 was assessed in the linkage study as an autosomal dominant trait in the 11 families for the chromosome lp35-p36 STR markers. The CMTX linkage was analyzed with the X-dominant condition in five families. In both studies, a CMT gene frequency of 1/10,000 and equal male and female recombination values were assumed. Twenty-nine patients had an age at onset ranging from the first to fifth decade, with a mean of 19.21 ± 11.4 years. Seven age-dependent penetrance classes were obtained from the family data and history according to the age at onset curve, 47 and a disease penetrance of 99.2% is reached at 50 years. In the linkage analysis, the asymptomatic at-risk individuals were assigned a probability of being a gene carrier at their age of clinical and electrophysiological examination. The allele frequencies of the lp35-p36 and Xq11.2-q21 STR markers were obtained from the Genome Data Base (http://gdbwww.gdb.org/). Further, we indicated the exclusion limits in cM, which are calculated from the recombination distances at which the LOD score (z) reaches the value −2, using the Haldanes mapping function. 48 The exclusion limit is the region on each side of the marker, from which the disease gene can be excluded.

Results

We first screened the CMT2 patients for the presence of the CMT1A duplication with the restriction fragment length polymorphic markers pVAW409R3a (D17S122) and pEW401HE (D17S61) on Msp I Southern blots according to the methods described by Raeymaekers et al.13 No density differences or triple alleles were observed with pVAW409R3a and pEW401HE, indicating that CMT in these CMT2 families is not caused by the CMT1A duplication mutation on 17p11.2. Additionally, the absence of the 500-kb CMT1A duplication junction fragment was confirmed with pVAW409R3a (D17S122) on Fsp I pulsed-field gel electrophoresis blots of CMT2 patients according to Timmerman et a1. 17

Next, we examined genetic linkage of CMT2 in the 11 pedigrees to 1p35–p36 STR markers. Negative or nonsignificant LOD scores were obtained in a two-point analysis with the 1p35–p36 loci D1S244, D1S160, D1S228, and D1S170, including both affected and unaffected individuals and in an analysis that included only affected individuals. In the affected-only linkage analysis, suggestive LOD scores were obtained with D1S244 (1.07 at 𝛉 = 0) and D1S170 (0.79 at 𝛉 = 0) in the Italian family CMT-71. In this family, slightly positive LOD scores were also obtained under the age-corrected model with the same markers. The slightly positive linkage results suggest that CMT2 disease in this family might be linked to chromosome 1p35–p36. Segregation analysis of the loci D1S244, D1S160, D1S228, and D1S170 in family CMT-71 is shown in figure 1B. The haplotype 9-3-6 is transmitted by patient II.11 to his affected son III.25 and affected grandson IV.28. Only allele 6 of the affected haplotype is present in patient IV.19, belonging to a different branch of family CMT-71 (figure 1B). Allele 6 corresponds to marker D1S288, the closest linked marker to CMT2A. 35 However, allele 6 is also present in the asymptomatic individual IV.20, aged 29 years at blood sampling, explaining the negative LOD scores obtained with marker D1S228 under the age-penetrance model. Because all but one patient in family CMT-71 had onset ages before age 20 years, 9 we cannot exclude the possibility that the positive linkage results reflect coincidental cosegregation rather than true linkage.

We also performed SSCP analysis to screen for mutations in the coding region of Cx32. Only in family CMT-28 an altered SSCP pattern in part 1 of Cx32 exon 2, between primers Cx32-1 and Cx32-2, is observed in all patients and in one asymptomatic individual CMT-28 V.5 (data not shown). Direct PCR sequencing revealed a transversion mutation in codon 49 (TCT → TAT) resulting in an amino acid substitution of Ser → Tyr (figure 2). The single base change creates an Ssp I restriction site at position 205 according to the Cx32 cDNA sequence of Kumar and Gilula. 49 The maximal two-point LOD score between the mutation and the disease obtained in CMT-28 is 2.68 at 𝛉 = 0. Because no altered SSCP patterns for Cx32 were observed in the patients' DNA of families CMT-W, CMT-56, CMT-61, CMT-83, and PN-22, we also performed linkage analysis with STR markers flanking the Cx32 gene to exclude the CMTX1 region on chromosome Xq13. Negative or nonsignificant results were obtained with DXYS1X, PGK1, and DXS453 in a linkage study including both affected and unaffected individuals. In the two-point linkage analysis including only affected individuals, a maximum LOD score of 1.18 at 𝛉 = 0 is reached with DXYS1X in family CMT-56. Segregation analysis of the loci DXS453, PGK1, and DXYS1X in family CMT-56 is shown in figure 1B. The genotype of the grandparents can be deduced from the data obtained in the second generation. All affected males and females have the common disease haplotype 3-1-1. Two females (III.2 and III.7) can be considered as asymptomatic carriers. The nonaffected individuals II.2 and II.11 have also a 3-1-1 haplotype, but this could be the normal haplotype transmitted from the homozygous grandmother. Because the STR loci DXS453 and PGK1 are flanking the CMT1X gene, 30,50 we cannot exclude X-linkage in family CMT-56. For further information about obtaining the two-point linkage results, see Note at the end of the article.

Figure

Figure 2. Cx32 mutation in CMT28. A fraction of the Cx32 DNA sequence (part 1) is shown in patient V.4 in CMT-28. For sequence analysis of part 1 using the automated DNA sequencer 373A (ABI), PCR amplification was performed with primers Cx32-1 and Cx32-5 and sequencing was performed with primer Cx32-2. The amino acid residue representing codon 49 is mutated due to a single base change and is indicated in italics.

Discussion

Loprest et al. 51 and Hentati et al. 52 did the first exclusion mapping of CMT2 neuropathy in the chromosomal regions containing the CMT1A and CMT1B loci. Ben Othmane et al. 35 performed linkage analysis in six CMT2 families of which three were previously described by Loprest et al. 51 They obtained evidence for linkage in only one CMT2 pedigree (two-point LOD score of 4.41 at 𝛉 = 0.05) with D1S228. Multipoint linkage analysis in three CMT2 pedigrees suggested a most likely location for the CMT2 gene between D1S244 and D1S228. CMT2 demonstrated genetic heterogeneity because three other families showed no linkage with the chromosome 1p35–p36 markers. In this study, we performed a genetic linkage analysis in 11 nonrelated CMT pedigrees of which patients were clinically and electrophysiologically diagnosed with the CMT2 phenotype. Five families had autosomal dominant inheritance, and in six other families, male-to-male transmission did not occur. In 10 families, the loci D1S244, D1S160, D1S228, and D1S170 showed negative LOD scores and exclusion results. In one Italian CMT2 family, we obtained nonsignificant positive linkage results: however, additional linkage studies in the more extended pedigree of Rossi et al. 9 are needed to obtain more conclusive results. Our linkage results indicate that the CMT2 locus in our families is located elsewhere in the human genome and that the chromosome 1p35–p36 CMT2A locus of Ben Othmane et al. 35 is a minor locus for CMT2.

Nicholson and Nash 7 demonstrated that in CMTX families, the affected females, in contrast to affected males, can have normal or intermediate NCVs resembling those obtained in CMT2 patients. These results suggested that CMT2 families without male-to-male transmission may in fact be X-linked CMT families. Furthermore, because the frequency of X-linked CMT seems to be high and accounts for 10% of all cases 53 and mutations in Cx32 occur in several X-linked CMT families, 30–33 the possible involvement of Cx32 mutations has to be considered in CMT families with a diagnosis of CMT2. Therefore, we analyzed the six CMT2 families without male-to-male inheritance of the syndrome for mutations in the Cx32 gene by SSCP analysis. In family CMT-28, we identified a single base mutation in codon 49 of Cx32 exon 2, changing the Ser amino acid to a Tyr amino acid. Codon 49 is located in the extracellular region between the first and second transmembrane domain of the Cx32 gap junction protein. Based on the hydropathicity plot of the Cx32 protein, 54 a change of hydrophobicity can be predicted in the mutated protein. To our knowledge, others have not yet described this mutation that together found 30 different Cx32 gene mutations in 36 unrelated CMT1X patients. 30–34 Our observation indicates that CMT-28 is not a CMT2 family but a CMT1X family in which a Cx32 mutation segregates. We also performed linkage analysis with chromosome X STR markers in five CMT2 families that did not show altered SSCP patterns in the Cx32 gene. The linkage results and segregation analysis obtained with STR markers flanking the Cx32 gene suggested the possible involvement of an X-linked mutation in family CMT-56. However, further linkage studies with Xq13 STR markers have to be performed to obtain more conclusive results. Also, because SSCP analysis does not detect mutations in 100% of the cases, we will sequence the Cx32 gene and regulatory regions in family CMT-56.

In this study, we diagnosed CMT families as CMT2 families when at least one patient had a median or ulnar motor NVC higher than 38 m/s. Motor median or ulnar NCVs in the seven Belgian CMT2 families are given in figure 3. The NCVs varied between 25 and 56 m/s for the seven males and between 30 and 57 m/s for the 10 females (figure 3, including CMT-28). In contrast, in our population of autosomaldominant CMT1 patients, we always observed median motor NCV values less than 37 m/s. The CMT1 patients either had a 1.5-Mb tandem duplication or a PMP-22 mutation (CMT1A subtype) or a P0 mutation (CMT1B subtype). In nine CMT1X patients with a mutation in the Cx32 gene 34 (De Jonghe et al., unpublished results), the median motor NCV values varied between 26 and 42 m/s in the six males and between 28 and 48 m/s in the three females (figure 3, excluding CMT-28). In the CMT2 families, two patterns are observed. In some families, NCVs were always higher than 38 m/s, whereas in other families we found intermediate NCVs. These observations indicate that when selecting CMT2 families for a genome-wide search for CMT2 genetic loci, special attention should be given to these families that do not show male-to-male transmission because they may be CMT1X families as evidenced by the identification of a Cx32 mutation in family CMT-28.

Figure

Figure 3. Motor median and ulnar NCVs in CMT2 and CMT1X patients. The NCV values of the motor median nerve (in PN54 motor ulnar nerve; in CMT28 motor ulnar and median nerve) in affected individuals with CMT2 (○, □) are compared with those of CMT1X patients (•, ▪). The horizontal line at 38 m/s represents the NCV upperlimit for CMT1 patients, according to Harding and Thomas. 6 The vertical dotted line separates the CMT2 and CMT1X patients. The CMT1X patients from families PN56, PN100, PN235, PN212, and PN234 have been described elsewhere, 34 and the CMT1X patients in family CMT28 were identified in this study. Squares = males; circles = females.

Acknowledgments

We are grateful to the patients and their relatives for their kind cooperation in our research project. Patients were examined and referred to our laboratory by Drs. B. Horsthemke, H. Hauss, P. Vieregge, F.H. De Bruijne, J.S. Struik, M. Villanova, A. Rossi, G. Guazzi, and by one of us (P.D.J.). Technical assistance was provided by H. Backhovens.

Footnotes

  • ↵Note: Readers can obtain 4 pages of supplementary material from the National Auxiliary Publications Service, c/o Microfiche Publications, PO Box 3513, Grand Central Station, New York, NY 10163-3513. Request document no. 05308. Remit with your order (not under separate cover), in US funds only, $7.75 for photocopies or $4.00 for microfiche. Outside the United States and Canada, add postage of $4.50 for the first 20 pages and $1.00 for each 10 pages of material thereafter, or $1.75 for the first microfiche and $.50 for each fiche thereafter. There is a $15.00 invoicing charge on all orders filled before payment.

  • Supported in part by a grant of the National Fund for Scientific Research (NFSR), a concerted action of the Flemish Ministry of Education, Belgium and the Muscular Dystrophy Association, Tucson, AZ. V.T. and E.N. are research assistants of the NFSR, Belgium.

    Received August 8, 1995. Accepted in final form September 8, 1995.

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