Hereditary Motor and Sensory Neuropathy Type 2C Is Genetically Distinct From Types 2B and 2D FREE

HEREDITARY MOTOR and sensory neuropathy (HMSN; also called Charcot-Marie-Tooth disease, or CMT) comprises a group of phenotypically similar inherited disorders of the peripheral nervous system.¹ HMSN type 1 (HMSN 1) represents a genetically heterogeneous group of autosomal-dominant demyelinating hypertrophic neuropathies with characteristically low peripheral nerve conduction velocities. The HMSN 1 loci have been mapped to chromosome region 17p11.2-p12 (HMSN 1A) and 1q22-q23 (HMSN 1B).

Hereditary motor and sensory neuropathy type 2 (HMSN 2) is a genetically heterogeneous group of axonal (neuronal) neuropathies with normal or slightly low nerve conduction velocities, also usually inherited as autosomal dominant traits. Hentati et al² and Loprest et al³ have demonstrated that the autosomal dominant HMSN 2 families that they studied did not have abnormal alleles at the HMSN 1A or HMSN 1B loci. Othmane et al⁴ have reported 3 families with dominantly inherited HMSN 2 linked to chromosome region 1p35-p36 (HMSN 2A) and also 3 other pedigrees with HMSN 2 that did not link to these loci, demonstrating genetic heterogeneity within HMSN 2. Subsequently, Kwon et al⁵ have reported a family with dominantly inherited HMSN 2 linked to markers mapped to chromosome region 3q13-q22 (HMSN 2B). Then Ionasescu et al⁶ described a family with dominantly inherited HMSN 2 linked to 7p14 (HMSN 2D).

Hereditary motor and sensory neuropathy type 2C is a clinical variety of HMSN 2 characterized by motor and sensory involvement of the limbs and progressive weakness of the vocal cords, diaphragm, and intercostal muscles.⁷ We have reported 2 unrelated HMSN 2C families⁷ ; the larger of these kindred was genetically distinct from HMSN 2A.⁸ In the present study we tested whether the HMSN 2C phenotype in this family is linked to the HMSN 2B or HMSN 2D loci.

The clinical and electrophysiologic features of the kinship studied here have been described.⁷ Autosomal dominant inheritance was evident from at least 1 occurrence of male-to-male transmission based on the clinical examination. Affected persons had weakness and atrophy of peroneal and hand muscles, and decreased or absent deep tendon reflexes of the limbs. Their motor nerve conduction velocities in the median nerve were within the normal range. Sensory involvement of the feet and absent or decreased sensory nerve action potentials occurred in severely affected persons. Of 12 affected subjects, 9 had clinical evidence of diaphragmatic or vocal-cord paresis or paralysis. The severity of clinical symptoms in affected persons was variable; phenotypes did not differ by sex.

In the present study, the neuropathic status of 33 subjects in the larger kinship, including 12 affected individuals and 11 individuals at risk, were investigated (Figure 1). Clinically unaffected offspring who were at risk but younger than 26 years were excluded. After informed consent was given, 20 mL of blood was obtained by venipuncture from the family members. Continuing lymphoblast cell lines were established by Epstein-Barr virus transformation,⁹ and genomic DNA was isolated using a nucleic acid extraction kit (Isoquick; Microprobe, Bothell, Wash).

Genotyping was performed for DNA microsatellite markers D3S1558, D3S1769, GGAA8B03, D3S1267, D3S1551, D3S1290, GATA4A10, and D3S1744. These markers map to the chromosome region 3q13-q22 and are linked to the HMSN 2B phenotype.⁵ Genotyping also was performed for microsatellite markers mapped to 7p14 (D7S1808, D7S1869, D7S435, and D7S1806), linked to the HMSN 2D phenotype,⁶ and for markers situated proximally (D7S2201) and distally (D7S526 and D7S1830) to 7p14. The markers (Research Genetics, Huntsville, Ala) were amplified by polymerase chain reaction in a final volume of 15 µL containing 50 ng of genomic template DNA, 2 pmol of each oligonucleotide microsatellite primer, 200 µM concentrations of dATP, dGTP, and dTTP, 50 mM of dCTP (Boehringer Mannheim, Indianapolis, Ind), 0.5 µCi of α-³²P dCTP (Amersham Life Science, Arlington Heights, Ill), and 0.5 U of Taq DNA polymerase (Boehringer Mannheim) in a 1 × Taq buffer containing 10-mmol/L Trishydrochloride (pH 8.3 at 20°C), 50-mmol/L potassium chloride, and 1.5-mmol/L magnesium chloride. Amplification was performed for 35 cycles of denaturation at 94°C for 15 seconds, annealing at 55°C for 1 minute, and extension at 72°C for 15 seconds, with a final elongation cycle at 72°C for 10 minutes. After 10 µL of loading buffer (95% formamide, 10-mmol/L EDTA, 0.1% bromophenol blue, and 0.1% xylene cyanol) was added, the amplified products were denatured at 94°C for 10 minutes and then cooled rapidly to 4°C and placed on ice. Three microliters of each sample was electrophoresed on 6% denaturing polyacrylamide sequencing gels at 75 W for 2 to 3 hours. Gels were vacuum dried and autoradiographed overnight at −70°C. Base-pair sizes of the polymerase chain reaction products were calculated using a double-strand DNA cycle sequencing system (Gibco BRL, Grand Island, NY).

A linkage analysis was carried out between chromosome 3q13-q22 markers and the HMSN 2C phenotype in the large kinship described above. A linkage analysis was also performed between 7p14 markers and the HMSN 2C phenotype. Pairwise limit of detection (LOD) scores (Z values) were calculated, under the assumption of single-gene autosomal-dominant inheritance, using the MLINK option of the computer program LINKAGE (version 5.1; http://linkage.rockefeller.edu/soft/list.html) as described by Lathrop et al.^{10 - 11} A gene frequency of 0.0001 was assumed for the mutant HMSN 2C allele; no phenocopies were assumed, and penetrance was assumed to be 0.99 for heterozygotes and 1.0 for homozygotes. Male and female recombination fractions were assumed to be equal. Marker allele frequencies for white populations were obtained from the Genome Data Base (accessed June 1999; http://gdbwww.gdb.org/).

Multipoint location scores for the HMSN 2B locus were determined for the chromosome region 3q13-q22 using the LINKMAP option of LINKAGE, using published genetic map information from the Cooperative Human Linkage Center ^{12 - 13} and Généthon¹⁴ as in a previous report.⁵ Multipoint location scores for the HMSN 2D locus were determined for the chromosome region 7p14 using the same computer program, employing published genetic map information from the Genome Data Base. Haldane's mapping function was used to convert recombination fractions (θ) to genetic distances (centimorgan [cM]). Multipoint LOD scores were computed as the logarithm of the ratio of the likelihood with the disease locus at a specific recombination fraction from the test locus (θ=0.00-0.49) and the likelihood with the disease locus placed in an unlinked state (θ=0.50).

To assess how informative the data of our kindred were for linkage studies, simulation calculations were carried out by the computer program SIMLINK.¹⁵ The maximum attainable LOD score at a recombination fraction of 0 with fully informative linkage markers including all persons at risk was 7.7 (average LOD score, 6.1).

The results of pairwise analyses between HMSN 2C phenotype and 8 markers from the region of the HMSN 2B locus are given in Table 1: all of the 2-point LOD scores were negative. A multipoint linkage analysis testing with the HMSN 2C locus against the fixed genetic map of chromosome region 3q13-q22 markers is presented in Figure 2. The multipoint LOD scores in the region of these markers were less than −2, excluding the HMSN 2C locus from this region.

Ionasescu et al⁶ have reported that the HMSN 2D family showed the highest 2-point LOD score (4.83) at D7S435, and the highest multipoint LOD score (6.89) at the point 0.5 cM proximal from D7S435 and 4.6 cM distal to D7S1808. The results of pairwise analysis with 7 markers near the HMSN 2D locus are given in Table 1. No evidence for linkage was seen in this region. The results of multipoint linkage analysis with the HMSN 2C locus tested against a fixed genetic map of these markers are presented in Figure 3; the multipoint LOD scores in the region of these markers were less than −2 between points 9.7 cM proximal to D7S1801 and 10.1 cM distal to D7S435, which excludes the HMSN 2C locus from this region.

Three different disease loci have been established for autosomal dominant HMSN 2. Three families in the United States and 2 Japanese families with HMSN 2 have shown linkage with chromosome region 1p35-p36 (HMSN 2A),^{4 ,16} and an Italian family also has shown evidence suggestive of linkage to this locus.¹⁷ On the other hand, 3 of 6 families in the United States with HMSN 2 and 10 of 11 European families with HMSN 2 have been shown to lack linkage to the HMSN 2A locus^{4 ,17} ; in the aggregate these findings demonstrated genetic heterogeneity in HMSN 2. Subsequently, the disease locus in a family with HMSN 2 in the United States has been mapped to chromosome region 3q13-q22 (HMSN 2B),⁵ and then a European family with HMSN 2 has shown suggestive evidence for linkage to the same locus.¹⁸ The third disease locus for HMSN 2 has been mapped to chromosome region 7p14 (HMSN 2D) in a single family in the United States.⁶ Clinically HMSN 2A shows a motor-dominant pattern with more pronounced weakness in the lower than the upper extremities.¹¹ Hereditary motor and sensory neuropathy type 2B is characterized by a marked sensory disturbance in the distal lower extremities resulting in ulceration in the feet,^{18 - 19} while HMSN 2D shows more severe motor and sensory disturbance in the upper than in the lower extremities.⁶

In comparison with other forms of HMSN 2, the HMSN 2C family we studied here had unique clinical features in addition to motor and sensory involvement of the limbs, including progressive paresis of the vocal cords, diaphragm, and intercostal muscles. Yoshioka et al⁸ have shown this family's abnormality to be genetically distinct from HMSN 2A. In the present study, we found no evidence to support linkage of the HMSN 2C phenotype to either the HMSN 2B locus on chromosome 3 or the HMSN 2D locus on chromosome 7.

Our results provide further evidence of genetic heterogeneity in HMSN 2. At least 4 genetic varieties of HMSN 2 seem to have been confirmed: HMSN 2A (1p35-p36), HMSN 2B (3q13-q22), HMSN 2D (7p14), and HMSN 2C (not linked to any of these loci). A linkage study employing DNA markers from multiple autosomes will be needed to map the HMSN 2C locus.

Accepted for publication April 12, 1999.

This study was supported in part by research grants obtained from the Muscular Dystrophy Association of America, Tucson, Ariz (grant 12), the National Institute of Neurological Disorders and Stroke (grant 14304), and the Kawamura fund, Gifu City, Japan. We thank Amie Bissonnett, BS, for her expert assistance in computer programming.

Reprints: Peter James Dyck, MD, Peripheral Neuropathy Research Center, Mayo Clinic, 200 First St SW, Rochester, MN 55905 (e-mail: dyck.peter@mayo.edu).