Abstract
Background: Hereditary motor and sensory neuropathy type 2C (HMSN2C, Charcot–Marie–Tooth 2C [CMT2C]) is an autosomal dominant motor and sensory neuropathy involving limb, diaphragm, vocal cord, and intercostal muscles.
Objective: To identify the chromosome localization for this disorder in one large American family of English and Scottish ethnicity.
Methods: Variable clinical severity led the authors to combine several approaches to accurately identify affected patients. Genome-wide two-point linkage analysis, high-definition mapping, and multipoint and recombinant haplotype analyses were performed. Mutation analysis of the triplet repeat region of ataxin-2 was also carried out.
Results: The initial genome-wide scan identified a region at 12q24, and fine mapping provided a maximal lod score of 4.73 (D12S1645 and D12S1583 at θ = 0.01 and 0, respectively). With multipoint analysis, a higher lod score of 5.17 was obtained and localized to the same region at 119.0 cM. Haplotype analysis narrowed the region to approximately 5.0 cM between D12S1646,D12S1330 and D12S105,D12S1339 (12q23.3-24.21). Ataxin-2, the gene responsible for spinocerebellar ataxia type 2 (SCA2), localizes to this region, but no triplet repeat expansion or point mutations within the repeat were found.
Conclusions: The gene for HMSN2C maps to 12q23-24. This region is associated with SCA2, scapuloperoneal spinal muscular atrophy, and congenital distal spinal muscular atrophy. Further studies are needed to demonstrate the specific gene alteration and its relationship with nearby genes.
Hereditary motor and sensory neuropathy type 2C (HMSN2C [MIM 606071], Charcot–Marie–Tooth disease type 2C [CMT2C]) is a rare autosomal dominant axonal form of peroneal muscular atrophy with progressive muscle weakness and atrophy of limb, diaphragm, vocal cord, and intercostal muscles with variable degrees of acral sensory loss.1 Among affected persons, the age at onset and clinical severity are variable. Individuals presenting in childhood tend to have severe disease and a shortened life span from respiratory failure and complications. Sensory loss is most apparent in severely affected patients. As with other forms of HMSN, sensory involvement may be mild and unassociated with positive neuropathic sensory symptoms, that is, pain, asleep numbness, and paresthesias. We previously provided evidence that HMSN2C is not genetically linked to HMSN2A (1P36.2) [MIM 118210], 2B (3q13-22) [MIM 600882], or 2D (7p14) [MIM 601472].2 A recent European kindred has also not shown linkage to varieties of HMSN2 or distal hereditary motor neuronopathy type VII (dHMN-VII; 2q14) [MIM 158580].3
Although classification of the inherited neuromuscular disorders ultimately now depends on specific molecular genetic derangements, careful characterization of the neurologic features, the nature of inheritance, the nerve conduction features, and the neuropathologic alterations is important for clinical classification and determination of severity and course. Additionally, clinical characterization provides for genotype–phenotype correlation important in understanding pathogenesis. Whereas diaphragmatic, vocal cord, and intercostal paralysis are distinguishing features of HMSN2C, these features can occur rarely in other HMSN diseases such as HMSN4A (CMT4A) [MIM 214400].4 Several autosomal dominant neuronopathies (motor neuron diseases) and distal myopathies may also have overlapping clinical features with HMSN2C, including peroneal muscle atrophy and pharyngeal or vocal cord paralysis. These disorders are without sensory nerve involvement and include dHMN-VII locus at 2q145 [MIM 158580] and one distal myopathy (vocal cord and pharyngeal dysfunction with distal myopathy [VCPDM]) locus at 5q316 [MIM 606070]. Despite the similarities in phenotype, various tissues (motor neurons, nerve fibers, muscle fibers, or their supporting cells) may be affected. An emerging literature also notes pharyngeal and vocal cord involvement in some spinocerebellar syndromes.7-11⇓⇓⇓⇓ Because spinocerebellar features are prominent, they are unlikely to be mistaken for HMSN disorders. Triplet repeat expansions in various genes account for the phenotype of spinocerebellar ataxia.12
We investigated the chromosomal localization for HMSN2C in a large kindred. The small size of other families has limited such investigation. Because of the known variability in clinical severity in this kindred, we used several approaches including composite scoring of nerve conduction abnormality to identify individuals as affected or unaffected.
Subjects and methods.
The clinical and electrophysiologic features of this American kindred of English and Scottish descent have been published.1 Most affected patients were not able to give a specific age at onset of their disease, it having begun insidiously. Several reported the onset of their symptoms as an altered voice or “a kind of shortness of breath” or “asthma.” Weakness and atrophy of the hand muscles were the second most frequently reported symptom, and leg muscle weakness was demonstrated (often without symptoms). The propositus (IV-58) and several others were affected early in infancy or childhood, but many were mildly affected, including her mother (III-67) and grandmother (II-17) (figure 1). Clinical examination and electrophysiologic testing demonstrated unequivocal sensory fiber involvement in most affected persons.
Figure 1. The degree of clinical severity was variable among persons in the studied kindred with hereditary motor and sensory neuropathy type 2C. Shown is the proband IV-58, whose neuropathy impairment score (NIS) was 164 points; her mother, III-67, had an NIS of 50 points.
As reported earlier, the nerve conduction abnormalities are similar to those described for HMSN2 (motor and sensory nerve fibers are affected, nerve conduction velocity values are normal, low normal, or slightly abnormal when motor and sensory amplitudes are sufficiently preserved to adequately assess conduction velocity). Needle electromyographic examination was characteristic of a neurogenic process with large motor unit potentials with reduced recruitment in a length-dependent pattern. Nerve biopsy or postmortem tissue is not available for study.
Because of the variability in age at onset, we revisited this family on several occasions over a span of about 10 years and performed detailed studies especially on cases with questionable involvement. To improve identification of persons as affected or unaffected, we utilized 1) a standard sum score of neuropathic impairment (neuropathy impairment score [NIS]),13 2) speech and laryngeal evaluation and phrenic nerve stimulation in some patients, 3) lineage with affected progenitors and descendants, 4) age, and 5) composite score of attributes of nerve conduction.14 We chose a composite score of nerve conduction attributes to increase the certainty of assignment of persons within the kindred as affected or unaffected. In generalized polyneuropathies, composite series of nerve conductions are more sensitive, representative, and concordant with overall neuropathic impairment than are single attributes.15 All patients gave informed consent as reviewed by our institutional review board.
Clinical evaluations, transformed lymphoblast cell lines, and DNA were available on 73 individuals. Patients designated as affected had 1) varying degrees of voice, intercostal, and limb muscle weakness typical of the disorder and an NIS of ≥2 points; and 2) composite nerve conductions at or above the 97.5th percentile of normal values or unequivocally abnormal phrenic nerve stimulation responses or weakness of vocal cords (based on direct visualization and examination of speech). To use composite scores, the abnormalities in amplitudes, velocities, and latencies of nerve conductions must be in the same upper or lower tail of the normal distribution. For a number of reasons outlined previously,15 we expressed all abnormalities in the upper tail of the distribution. If needle electromyography was done, fibrillation and neurogenic motor unit potentials were supportive of the diagnosis. Also designated as affected were persons (without overt manifestations) in a lineage having both an affected parent and child. The NIS of affected individuals varied from 8 (IV-50) to 164 (IV-58) points, which are both unequivocally abnormal.13
Persons designated as putatively unaffected 1) were older than 23 years, were without neuropathic symptoms, and had an NIS of <2 points; and 2) had summated nerve conduction scores at <50th percentile of normal.
A genome-wide linkage screening was performed on 41 individuals (see figure 2) (17 affected, 17 unaffected, 7 unaffected and unrelated spouses). A total of 382 microsatellite markers were used, spaced at approximately 10-cM intervals, but not including chromosome X, given the clear male-to-male transmission of disease. The markers were obtained from the ABI Prism linkage-mapping set (LMS-10; Applied Biosystems, Weiterstadt, Germany) and were run on 4.0% denaturing polyacrylamide gels on the ABI 377 DNA sequencer. Two-point linkage analyses were calculated by the Linkage program, assuming a rare susceptibility allele (frequency 0.001) and an autosomal dominant mode of inheritance. To allow for potential ambiguities of the relationship between the underlying genotype and affection status, penetrance was assumed to be 0.1 for noncarriers and 0.9 for carriers. Marker allele frequencies were estimated from our data, and equal recombination fractions for males and females were assumed. Two-point limit-of-detection (lod) scores were calculated using the MLINK program of Linkage. Multipoint analysis was calculated by the Vitesse Program and haplotypes16 inferred by SIMWALK2.17 To confirm and refine the location of this region at chromosome 12q, we identified additional polymorphic microsatellite markers between D12S78 and D12S79, spanning a 12.8-cM distance with an average 2-cM density (table). Amplification and sequencing across the triplet repeat in exon-1 of ataxin-2 were via standard techniques. All primer sequences were identified from data available from the University of California at Santa Cruz working draft of the human genome (http://genome.ucsc.edu, April 2002 freeze).
Figure 2. Shown is the studied kindred with hereditary motor and sensory neuropathy type 2C. Clinical evaluations, transformed lymphoblast cell lines, and DNA were available on 73 individuals. Genetic analysis was performed on 44 indicated individuals.
Table 1 Parametric two-point lod scores for 12 markers of chromosome 12q
Results.
By revisiting the kindred on several occasions over time and using the composite score approach, five additional affected persons were identified as compared with ones identified in earlier publications.1,2⇓ Of the 18 clinically felt to be affected, all but 2 had varying degrees of vocal cord, diaphragm, or intercostal weakness. These weaknesses were often subtle, and in several persons a kind of hoarseness in voice was the only feature. On initial genome-wide linkage analysis, a possible region of linkage was found at 12q with two-point lod scores: D12S346 (lod score = 1.99, θ = 0.1), D12S78 (lod score = 1.48, θ = 0.1), D12S79 (lod score = 1.97, θ = 0.1), D12S86 (lod score = 1.31, θ = 0.2), and D12S324 (lod score = 1.53, θ = 0.1). Two-point lod scores of >1 were also seen at D11S901 (lod score = 1.13, θ = 0.01), and D11S968 (lod score = 1.719, θ = 0.1), but their flanking markers were not suggestive of linkage. High-definition markers between D12S78 and D12S79, spanning 12.8 cM, demonstrated a maximum two-point lod score of 4.73 at markers D12S1645 and D12S1583, with recombination fractions of θ = 0.01 and 0, respectively. Two-point lod scores of >3 were seen for markers D12S84, D12S1339, D12S1645, D12S1583, D12S1646, and D12S369 (see the table). Multipoint analysis established a slightly higher lod score of 5.17 at the region 119.0 cM utilizing markers D12S78, D12S1583, D12S1646, D12S79, and D12S86 (figure 3). Attempts to increase the number of markers in the multipoint analysis to include D12S1330 and D12S105 were unsuccessful, given the size and nature of the pedigree. The results, however, co-localize with the two-point linkage data and provide for an increased lod score.
Figure 3. Multipoint lod scores utilizing five markers at 12q24 (D12S78, D12S1583, D12S1646, D12S29, D12S86). The peak multipoint lod score 5.177 occurred between D12S78 and D12S1583, at position 119.0 cM.
The known physical maps in the region identified by two-point linkage analysis provided probable localization of the gene for HMSN2C to an approximately 12-cM region between D12S79 and D12S78 at 12q23-24. Haplotype analysis (figure 4) further narrows the candidate region. Meiotic recombinants in Patients III-59 and III-67, who are clearly affected, have breakpoints between D12S1646,D12S1330 and D12S105,D12S1339, respectively, suggesting a region narrowed to approximately 5.0 cM (see figure 3). The data are consistent with the area identified by multipoint analysis. Several additional family members were added to the haplotype analysis in addition to those run in the initial linkage analysis. These family members had relative certainty of their affected and unaffected status (IV-51, III-71, III-73) (see figure 2). Each carried the predicted haplotypes based on their putative affected and unaffected status. Also of note is one individual classified as unaffected who carried the affected haplotype combination. We have been careful not to identify this person as affected or other persons as unaffected when they do not have the haplotype.
Figure 4. Haplotype analysis of one nuclear portion of the family where meiotic recombination suggests association of an approximately 5-cM region between D12S1646,D12S1330 and D12S105,D12S1583 (12q23-24) for hereditary motor and sensory neuropathy type 2C. The genotype of II-18 is inferred from the children. IV-53 is clinically unaffected.
We initially examined the three patients (II-17, III-67, IV-58), and one unaffected family member (III-73) for triplet repeats of ataxin-2 (CAG) and found the repeat length to be 21 and therefore not indicative of expansion in successive generations. These three individuals were chosen because of possible anticipation seen in each successive generation, II-17 (grandmother, presenting at 80 years old), III-67 (mother, presenting at 37 years old), and IV-58 (daughter, presenting at age 1 month).1 The repeat length of 21 (CAG) is consistent with that number found in 94% of normal Caucasian populations.18 The lack of abnormality was confirmed by manual sequencing of the expanded region in these patients. No point mutation or expansion was identified. The remaining family members were analyzed, and none had CAG expansions. We also analyzed the studied cohort with marker D12S1672, which is a polymorphic marker residing in intron 1 of ataxin-2. Unfortunately, this marker was not informative, as the entire family possessed the 281 allele.
Discussion.
Knowing whether individuals are clinically affected or will become clinically affected may be difficult but is essential in linkage analysis. The difficulties are especially true for axonal neuropathies such as HMSN2C with variable expression. By contrast, patients with inherited demyelinating HMSN, for example, type 1 (CMT1), are readily detected even at a young age by their low nerve conduction velocities. To enhance accuracy of assignment of persons in our kindred as affected or unaffected, we utilized quantitative clinical assessment (NIS) and a composite score of nerve conduction abnormality. Based on careful characterization of our kindred and a genome-wide linkage analysis, we have localized the disorder to 12q23-24. The haplotype analysis narrows the region to approximately 5.0 cM. All patients identified as affected had a conserved haplotype.
A number of known genes are found in the 5-cM region of interest. One candidate gene is ataxin-2, or SCA2, for spinocerebellar ataxia type 2 [MIM 183090].19,20⇓ Patients with SCA2 have expansions in a triplet (CAG) repeat in the ataxin-2 gene, and families show anticipation characteristic of triplet repeat diseases. We considered ataxin-2 as a candidate gene because of possible anticipation in our family, although variable expression is known to occur in varieties of HMSN,21,22⇓ which may mimic anticipation. The spinocerebellar degenerations also have neuropathy and peroneal atrophy, and some are known to have vocal cord involvement.7-10⇓⇓⇓ None of our patients had pyramidal, extrapyramidal tract, ocular, dementia, or other features of spinocerebellar degeneration. We did not find triplet expansion or point mutation within the triplet repeat of ataxin-2. Further work will be required to exclude alternative mutations in the SCA2 gene. In SCA6 [MIM 183086], micromutations of the calcium channel gene CACNL1A4 have resulted in familial hemiplegic migraine [MIM 141500] and episodic ataxia [MIM 108500], in contrast to the triplet expansions of the same gene, which produce the spinocerebellar phenotype.23,24⇓
Whereas the region linked to HMSN2C in this study is distinct from those identified in other HMSN syndromes,21,22⇓ several other lower motor predominant neuromuscular disorders have been linked to this region on chromosome 12 (figure 5). These include scapuloperoneal spinal muscular atrophy (SPSMA; 12q24.1-q24.31) [MIM 181405]25 and congenital distal spinal muscular atrophy (congenital distal SMA; 12q23-24) [MIM 600175]26; also noted is distal hereditary motor neuropathy type II (distal HMNII; 12q24) [MIM 158590],27 but this gene localizes approximately 10 cM telomeric to the one for HMSN2C and likely represents a distinct locus. Similarly, scapuloperoneal muscular dystrophy (SPMD) [MIM 181430] is about 20 cM centromeric to our identified region, but its localization is less well established with the greatest lod score being 2.95 (θ = 0.0) at D12S82.28 Patients with SPMD were felt to have a myopathy, based on clinical and histopathologic description.
Figure 5. Neuromuscular diseases localizing to chromosome region 12q21-24 (from linkage data) including hereditary motor and sensory neuropathy type 2C (HMSN2C). Shown are the corresponding polymorphic markers and genetic position from pter centimorgans. SPMD = scapuloperoneal muscular dystrophy28; SPSMA = scapuloperoneal spinal muscular atrophy25; HMN2 = hereditary motor neuropathy27; congenital distal SMA = spinal muscular atrophy26; SCA2 = spinocerebellar ataxia. Precise chromosomal localization of D12S101 and D12S1366 is not available. Ataxin-2, the gene responsible for SCA2, localizes within the 5.0-cM region between D12S105 and D12S1330, the region implicated in HMSN2C.
The chromosomal regions for SCA2, SPSMA, and congenital distal SMA directly overlap the 5.0-cM region identified here for HMSN2C. The latter two diseases are characterized as spinal amyotrophies. Clinically they are distinguished by several features, most notably the absence of sensory involvement. Patients with SPSMA also have laryngeal palsy but with prominent scapular involvement.29 Patients with congenital distal SMA are without vocal cord involvement and have congenital nonprogressive deficit.30 Whether the genes responsible for these spinal amyotrophies are allelic to HMSN2C remains a possibility. Separation of the disorder in our kindred from the disorders of reported families with progressive spinal amyotrophies is based largely on our finding of sensory involvement. Therefore, we remain open to the possibility that a common genetic abnormality explains these varied phenotypes. Also described is a large Mongolian kindred, with a conserved haplotype and variable phenotype characterized by both spinal amyotrophy (dSMAV) [MIM 600794] and hereditary motor sensory neuropathy phenotype (CMT2D) [MIM 601472].31
Whereas we have identified the approximate location of the gene for HMSN2C (CMT2C), further studies are needed to demonstrate the specific gene alteration and its relationship with the other neuromuscular genes in this region.
Electronic database information. Online Mendelian Inheritance in Man (OMIM), http://www.ncbi.nlm.nih.gov/Omim/ (for HMSN2C [MIM 606071], HMSN2A [MIM 118210], HMSN2B [MIM 600882], HMSN2D [MIM 601472], HMSN4A (CMT4A) [MIM 214400], dHMNVII [MIM 158580], VCPDM [MIM 606070], SCA2 [MIM 183090], SCA6 [MIM 183086], episodic ataxia type 2 [MIM 108500], familial hemiplegic migraine [MIM 141500], SPSMA [MIM 181405], SPMD [MIM 181430], congenital distal SMA [MIM 600175], distal HMN II [MIM 158590], dSMAV [MIM 600794]). The distances and positions between the markers are based on the University of California at Santa Cruz working draft of the human genome (http://genome.ucsc.edu; April 2002 freeze).
Acknowledgments
Supported in part by the National Institute of Neurological Disorders and Strokes (NS36797), the Muscular Dystrophy Association, and the Mayo Gene Discovery Shared Resource (Mayo Comprehensive Cancer Center Grant) from the National Cancer Institute (CA15083).
Acknowledgment
The authors thank the affected family for their participation and Mary Lou Hunziker and Jim Tidwell for help in preparation of the manuscript and figure 2.
- Received November 8, 2002.
- Accepted December 26, 2002.
References
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