Autosomal dominant Charcot-Marie-Tooth disease type 1A and hereditary neuropathy with liability to pressure palsies: detection of the recombination in Slovene patients and exclusion of the potentially recessive Thr118Met PMP22 point mutation

Charcot-Marie-Tooth disease type 1 (CMT1) and hereditary neuropathy with liability to pressure palsies (HNPP) are the most frequent autosomal dominantly inherited disorders of the peripheral nervous system. The recessive inheritance is observed only exceptionally. Unequal crossing-over of misaligned flanking CMT1A-REP elements on chromosome 17p11.2 is the most frequent cause of the CMT1A duplication and of the reciprocal deletion in HNPP patients. Recently a recombination was noted. In our study 71 Slovene CMT1 patients from 36 families, 12 HNPP patients from 6 families and their 31 healthy relatives were analysed for the presence of these recombination mutations. In 29 of 36 unrelated CMT1 (81%) and in all 6 unrelated HNPP patients the duplication or the deletion, on chromosome 17p11.2-12 was detected. In 26 out of 29 duplication patients (CMT1A) (90%) a 3.2 kb EcoRI/SacI duplication junction fragment was observed. The analogous 7.8 kb EcoRI/EcoRI deletion junction fragment was found in 4 out of 6 unrelated HNPP deletion patients (67%). Overall we found a recombination mutation inside the in 86% of unrelated Slovene CMT1A and HNPP patients. One hundred and thirty-six DNA samples of the CMT1 and HNPP patients and of the healthy controls were negative for the potentially recessive Thr118Met PMP22 amino acid substitution. Dominantly inherited CMT1A duplications and HNPP deletions on chromosome 17p11.2 are thus, as in most other European countries, the most common mutations in Slovene CMT1 and HNPP patients. No signs of polymorphism or of potentially recessive mutation were found at the specific Thr118Met PMP22 site.     

Gene dosage effects in hereditary peripheral neuropathy. Expression of peripheral myelin protein 22 in Charcot-Marie-Tooth disease type 1A and hereditary neuropathy with liability to pressure palsies nerve biopsies

A duplication of a 1.5-Megabase genomic region encompassing the gene for the peripheral myelin protein 22 (PMP22) is found on chromosome 17p11.2-12 in Charcot-Marie-Tooth disease type 1A (CMT1A), whereas the reciprocal deletion is associated with hereditary neuropathy with liability to pressure palsies (HNPP). Since most CMT1A patients harbor three copies of the PMP22 gene, and most HNPP patients carry only a single copy, a gene dosage effect has been proposed as a mechanism for both diseases. We have analyzed the steady-state expression of PMP22 protein in sural nerve biopsies from three CMT1A and four HNPP patients. Quantitative immunohistochemical determination showed that PMP22 protein expression relative to that of myelin protein zero and myelin basic protein was increased in all CMT1A patients and reduced in all HNPP patients, as compared with biopsy samples of patients with normal PMP22 gene expression. These data demonstrate that both neuropathies result from an imbalance of PMP22 protein expression.     

Inherited neuropathies: Charcot-Marie-Tooth disease and related disorders

Collectively, the inherited disorders of peripheral nerves represent a common group of neurological diseases and are frequently encountered in the clinical setting. Recent advances in molecular genetics have not only provided improved diagnosis and counselling, but may ultimately lead to specific, rational therapies for the various forms of inherited neuropathy. Charcot-Marie-Tooth neuropathy type 1 (CMT1) is a genetically heterogeneous group of chronic demyelinating polyneuropathies with loci mapping to chromosome 17p (CMT1A), chromosome 1q (CMT1B), the X chromosome (CMTX) and to another unknown autosome (CMT1C). CMT1A is most often associated with a tandem 1.5-megabase (Mb) duplication in chromosome 17p11.2-12, or may occasionally result from a point mutation in the peripheral myelin protein 22 (PMP22) gene. CMT1B is associated with point mutations in the myelin protein zero (P0) gene. The molecular defect in CMT1C is unknown. CMTX is associated with defects in the connexin 32 gene. Charcot-Marie-Tooth neuropathy type 2 (CMT2) is an axonal neuropathy, also of undetermined cause. One locus for CMT2 has been assigned for chromosome 1p (CMT2A). Dejerine-Sottas disease is a severe, infantile-onset, demyelinating polyneuropathy which may be associated with point mutations in the P0 or PMP22 genes. Hereditary neuropathy with liability to pressure palsies (HNPP) is an autosomal dominant disorder that results in a recurrent, episodic demyelinating neuropathy. HNPP is associated with a 1.5-Mb deletion in chromosome 17p11.2-12 and may result from reduced expression of the PMP22 gene. CMT1A and HNPP are apparent reciprocal duplication/deletion syndromes originating from unequal cross-over during germ-cell meiosis.     

Clinical and pathological correlations in Charcot-Marie-Tooth neuropathy type 1A with the 17p11.2p12 duplication: a cross-sectional morphometric and immunohistochemical study in twenty cases

In a cross-sectional, clinical, and morphometric analysis we assessed the correlation between the clinical and pathological evolution of disease in 20 unrelated patients of various ages affected by Charcot-Marie-Tooth neuropathy type 1A (CMT1A) with the 17p11.2p12 (peripheral myelin protein 22, PMP22) duplication. The severity of neurologic deficits and slowing of motor conduction velocity at the median nerve did not vary significantly with the patients' age. The amount of demyelination was significantly higher below 15 years than in older age groups; in contrast, myelinated fiber and onion bulb densities were similar at all ages. The results indicate that in duplicated CMT1A, the pathological process develops early in life and progresses little during the course of the disease. Younger patients had lower g-ratio values, suggesting that the trigger of demyelination in early years could be a hypermyelination, resulting from PMP22 overexpression. Yet none of the 20 patients examined had immunohistochemical evidence of altered PMP22 expression. The early onset and development of the disorder make it difficult to detect PMP22 overdosage in nerve biopsies.     

A 1.5-Mb deletion in 17p11.2-p12 is frequently observed in Italian families with hereditary neuropathy with liability to pressure palsies

Hereditary neuropathy with liability to pressure palsies (HNPP) is an autosomal dominant disorder characterized by recurrent mononeuropathies. A 1.5-Mb deletion in chromosome 17p11.2-p12 has been associated with HNPP. Duplication of the same 1.5-Mb region is known to be associated with Charcot-Marie-Tooth disease type 1 (CMT1A), a more severe peripheral neuropathy characterized by symmetrically slowed nerve conduction velocity (NCV). The CMT1A duplication and HNPP deletion appear to be the reciprocal products of a recombination event involving a repeat element (CMT1A-REP) that flanks the 1.5-Mb region involved in the duplication/deletion. Patients from nine unrelated Italian families who were diagnosed with HNPP on the basis of clinical, electrophysiological, and histological evaluations were analyzed by molecular methods for DNA deletion on chromosome 17p. In all nine families, Southern analysis using a CMT1A-REP probe detected a reduced hybridization signal of a 6.0-kb EcoRI fragment mapping within the distal CMT1A-REP, indicating deletion of one copy of CMT1A-REP in these HNPP patients. Families were also typed with a polymorphic (CA)n repeat and with RFLPs corresponding to loci D17S122, D17S125, and D17S61, which all map within the deleted region. Lack of allelic transmission from affected parent to affected offspring was observed in four informative families, providing an independent indication for deletion. Furthermore, pulsed-field gel electrophoresis analysis of SacII-digested genomic DNA detected junction fragments specific to the 1.5-Mb HNPP deletion in seven of nine Italian families included in this study. These findings suggest that a 1.5-Mb deletion on 17p11.2-p12 is the most common mutation associated with HNPP.     

Heterozygous peripheral myelin protein 22-deficient mice are affected by a progressive demyelinating tomaculous neuropathy

Hereditary neuropathy with liability to pressure palsy (HNPP) is associated with a heterozygous 1.5 megabase deletion on chromosome 17 that includes the peripheral myelin protein (PMP) gene PMP22. We show that heterozygous PMP22 knock-out mice, which carry only one functional pmp22 allele and thus genetically mimic HNPP closely, display similar morphological and electrophysiological features as observed in HNPP nerves. As reported previously, focal hypermyelinating structures called tomacula, the pathological hallmarks of HNPP, develop progressively in young PMP22(+/0) mice. By following the fate of tomacula during aging, we demonstrate now that these mutant animals are also interesting models for examining HNPP disease mechanisms. Subtle electrophysiological abnormalities are detected in PMP22(+/0) mice >1 year old, and a significant number of abnormally swollen and degenerating tomacula are present. Thinly myelinated axons and supernumerary Schwann cells forming onion bulbs as fingerprints of repeated cycles of demyelination and remyelination are also encountered frequently. Quantitative analyses using electron microscopy on cross sections and light microscopy on single teased nerve fibers suggest that tomacula are intrinsically unstable structures that are prone to degeneration; however, the severity of morphological and electrophysiological abnormalities in PMP22(+/0) mice is variable. These combined findings are reminiscent of the disease progression in HNPP and offer a possible explanation about why some HNPP patients develop a chronic motor and sensory neuropathy later in life that resembles demyelinating forms of Charcot-Marie-Tooth disease by both morphological and clinical criteria.     

The human COX10 gene is disrupted during homologous recombination between the 24 kb proximal and distal CMT1A-REPs

The CMT1A-REPs are two large directly repeating DNA sequences located on chromosome 17p11.2-p12 flanking the region duplicated in patients with Charcot-Marie-Tooth disease type 1A (CMT1A) and deleted in patients with hereditary neuropathy with liability to pressure palsies (HNPP). We have sequenced two cosmids, c74F4 and c15H12, which contain the entire proximal and distal CMT1A-REPs and determined that these repeats are approximately 99% identical across a 24,011 bp region. In addition, both contain an exon of the human heme A:farnesyltransferase gene (COX10). Hybridization studies revealed that COX10 spans the distal CMT1A-REP, while the proximal CMT1A-REP contains an isolated COX10 'pseudo-exon'. There is also a COX10 hybridization signal on chromosome 10 which appears to represent a processed pseudogene. We propose that the distal CMT1A-REP represents the progenitor copy of COX10 exon VI which was duplicated with surrounding intronic sequences during mammalian genome evolution and that the HNPP deletion results in a COX10 null allele.     

Molecular basis of Charcot-Marie-Tooth neuropathy

Charcot-Marie-Tooth neuropathy (CMT) is the most common inherited peripheral neuropathy. CMT is classified into type types on the basis of pathological and electrophysiological findings: type 1(CMT1), characterized by decreased nerve conduction velocities and by "onion bulb" formation: type 2(CMT2), in which nerve conduction velocities are normal and "onion bulb" formations are rarely seen. CMT1 loci map to chromosome 17 (CMT1A), chromosome 1(CMT1B), another unknown autosome (CMT1C) and the X chromosome (CMTX). Recent work has identified the gene products corresponding to CMT1A, CMT1B and CMTX as peripheral myelin protein-22(PMP22), Po and connexin 32, respectively. Dejerine-Sottas disease has been identified as being caused by the mutation of PMP-22 or Po gene.     

A novel PMP22 point mutation causing HNPP phenotype: studies on nerve xenografts

BACKGROUND: Hereditary neuropathy with liability to pressure palsies (HNPP) in most cases is caused by a deletion in chromosome 17p11.2-12 or, rarely, mutations resulting in a functional loss of one copy of the peripheral myelin protein 22 (PMP22) gene. Point mutations that lie deep within transmembrane (TM) domains causing major structural changes in PMP22 are associated with severe neuropathy. METHODS: A 25-year-old asymptomatic woman with a normal neurologic examination volunteered as a control subject. Electrophysiologic studies showed multiple entrapment neuropathies, prompting a search for a genetic defect. In addition, sural nerve fascicles from the subject were grafted into the cut ends of the sciatic nerve of nude mice and studied at 2, 6, and 8 weeks and compared with controls. RESULTS: Direct sequencing of the PMP22 gene revealed a G-->A transition at position 202 in axon 3 of the PMP22 gene. To determine if this was a causative mutation rather than a polymorphism, 102 DNA samples from controls were studied; none showed a similar base pair change. In the nerve xenografts, there was a marked delay at the onset of myelination and an impairment in the regenerative capacity of the nude mice axons engulfed by the mutant human Schwann cells. The axon tips were enlarged and demonstrated neurofilament density increase. Neurofilament density distribution histograms were bimodal in xenografts as well as in the subject's sural nerve. CONCLUSION: This study provides unequivocal evidence that a base pair change causing a Val30Met substitution at the junction of the first TM domain and the extracellular loop of PMP22 results in the HNPP phenotype.     

Epidemiology of hereditary neuropathy with liability to pressure palsies (HNPP) in south western Finland

An epidemiological study of hereditary neuropathy with liability to pressure palsies (HNPP) was carried out in south western Finland, with a population of 435,000. The diagnosis was established in 69 patients from 23 unrelated families through family and medical history, clinical neurological and neurophysiological examinations and with documentation of the deletion at gene locus 17p11.2 in at least one member of each family. This gave a prevalence of at least 16/100,000, which is remarkably high. However, due to the insidious nature of HNPP, most probably it is still an underestimation. This is the first population-based prevalence figure reported for HNPP. The prevalence is somewhat lower than that obtained for CMT in the same population, which agrees with the proposal that HNPP and CMT 1A are reciprocal products of the same unequal crossing-over. The clinical pictures of our patients were, in general, similar to those previously described in HNPP.     

Video-assisted theloscopic electroincision of a high teat stenosis

The hereditary neuropathy Charcot-Marie-Tooth (CMT) type 1A is, in the majority of cases, caused by duplication of the gene for the peripheral myelin protein PMP22, which leads to abnormally increased PMP22 expression. Recent in vitro and in vivo data indicate a novel function of PMP22 in Schwann-cell growth and differentiation other than its role in myelination, and suggest that overproduction of PMP22 leads to a new Schwann-cell phenotype in CMT1A. Taking these data into account, we developed a new hypothesis on the pathogenesis of CMT1A neuropathy: that the defective myelin stability and turnover observed in the disease is caused by altered PMP22 gene dosage and its resultant effect on abnormal Schwann-cell growth and differentiation.     

Relationship between the plaque removal efficacy of a manual toothbrush and brushing force

The Charcot-Marie-Tooth disease type 1A (CMT1A) phenotype is most often associated with a 1.5 megabase (mb), tandem duplication of chromosome 17 band p12 (17p12). The prevailing hypothesis is that the demyelinating neuropathy results from a dosage effect of the peripheral myelin protein gene PMP22 which is included within this duplication. We present a patient with clinical and electrophysiological features of CMT1A in whom an extra PMP22 gene resulted from a rare unbalanced translocation of 17p to the X chromosome. This finding further supports the hypothesis of gene dosage as the basis for CMT1A. Moreover, this case highlights the importance of fluorescence in situ hybridization (FISH) as an alternative molecular technique in the diagnosis of CMT1A.     

Determination of gene dosage at the PMP22 and androgen receptor loci by quantitative PCR

Although many genetic diseases are caused by the presence of point mutations in respective genes, an increasing number of diseases are known to be caused by gene copy number changes. We report the development of a rapid and reliable PCR-based method for quantitation of gene copy number with sufficient sensitivity to detect single copy changes without the use of radioactive or fluorescent labeling. The sensitivity of this technique has been demonstrated by the detection of the DNA duplication or deletion occurring in two inherited peripheral neuropathies, Charcot-Marie-Tooth type 1A (CMT1A) and hereditary neuropathy with liability to pressure palsies (HNPP), that are caused by a reciprocal duplication or deletion event on chromosome 17p11.2-12. This method relies on the comparison of the amount of PCR product generated from a potentially duplicated or deleted target sequence with the amount of product generated from a disomic reference gene. The value of this ratio (target PCR product:reference PCR product) indicates whether the target sequence is duplicated, deleted, or unchanged. Using primers from within a duplicated or deleted region (PMP22 gene and EW401) and from within a reference region (NF1 gene), we tested 50 CMT1A, 30 HNPP, and 50 unaffected individuals for the presence of a DNA duplication or deletion. Target:reference ratios of 1.58, 1.02, and 0.56 were detected for the CMT1A, unaffected, and HNPP groups, respectively. Thus, differentiation of the three groups of individuals was on the basis of gene copy number. This technique was successfully used to detect the difference in the X chromosome copy number between males and females (target:reference ratios of 1.1 and 2.3, respectively). This approach to the detection of DNA duplications and deletions is sensitive, accurate, and has potential applications in the quantitation of changes in gene copy number associated with diseases characterized by such chromosomal alterations.     

Correlation between PMP-22 messenger RNA expression and phenotype in hereditary neuropathy with liability to pressure palsies

Hereditary neuropathy with liability to pressure palsies (HNPP) is associated with a deletion in chromosome 17p11.2, which includes the gene for the peripheral myelin protein 22 (PMP-22). A "gene dosage" effect is probably the mechanism underlying HNPP, but the amount of PMP-22 mRNA in sural nerves of HNPP patients is highly variable and the role of PMP-22 underexpression in impairing myelination has yet to be clarified. We have studied 6 genetically proven HNPP patients, to evaluate the relationship between PMP-22 mRNA levels, and clinical, neurophysiological, and neuropathological findings. Underexpression of PMP-22 mRNA correlates with disease severity and with mean axon diameter and g ratio, but not with myelin thickness, number of "tomacula," or nerve conduction parameters. Our findings further confirm that underexpression of PMP-22 is the main pathogenetic mechanism underlying the severity of clinical symptoms and signs in HNPP. Smaller axons in sural nerves of HNPP patients with lower PMP-22 levels suggests that underexpression of PMP-22 may also affect axon development.     

Charcot-Marie-Tooth disease with intermediate motor nerve conduction velocities: characterization of 14 Cx32 mutations in 35 families

Charcot-Marie-Tooth disease can be inherited either autosomal dominantly or recessively or linked to the X chromosome. X-linked dominant Charcot-Marie-Tooth disease (CMTX) is a sensorimotor peripheral neuropathy in which males have usually more severe clinical symptoms and decreased nerve conduction velocities than do females. CMTX is usually associated with mutations in exon 2 of the connexin 32 (Cx32) gene. DNA from 35 unrelated CMT patients, without the 17p11.2 duplication, but with median nerve conduction between 30 and 40 m/s, were tested for the presence of Cx32 mutations. The entire coding sequence of the Cx32 gene was explored using a rapid nonradioactive technique to detect single-strand conformation polymorphisms (SSCP) on large PCR fragments. Thirteen abnormal SSCP profiles were detected and characterized by sequencing. In addition, systematic sequencing of the entire Cx32 coding region in the remaining index cases revealed another mutation that was not detected by SSCP. A total of 14 mutations were found, five of which were not previously reported. These results demonstrate the high frequency (40%) of mutations in the coding region of the Cx32 gene in CMT patients with intermediate MNCV, without 17p11.2 duplications. Most of these mutations (93%) can be detected by SSCP.     

Progesterone stimulates the activity of the promoters of peripheral myelin protein-22 and protein zero genes in Schwann cells

To understand better the mechanisms by which progesterone (PROG) promotes myelination in the PNS, cultured rat Schwann cells were transiently transfected with reporter constructs in which luciferase expression was controlled by the promoter region of either the peripheral myelin protein-22 (PMP22) or the protein zero (P0) genes. PROG stimulated the P0 promoter and promoter 1, but not promoter 2, of PMP22. The effect of PROG was specific, as estradiol and testosterone only weakly activated promoters. Dose-response curves for stimulation of both promoter constructs by PROG were biphasic. RU486, a PROG antagonist, did not abolish the effect of PROG, but stimulated promoter activities by itself. In the human carcinoma cell line T47D expressing high levels of PROG receptor, PROG did not stimulate the P0 and PMP22 promoters, whereas the promoter region of the mouse mammary tumor virus was fully activated. Thus, the activation by PROG of promoter activity of two peripheral myelin protein genes is Schwann-cell specific.     

Hereditary thermosensitive neuropathy: an autosomal dominant disorder of the peripheral nervous system

We report the clinical and electrophysiologic characteristics of eight patients (four men and four women) with a hereditary neuropathy with probable thermosensitivity (HTN) of autosomal dominant inheritance. Patients presented reversible episodes of ascending muscle weakness, paresthesiae, and areflexia apparently triggered by an elevation of body temperature over 38.5 degrees C. Mean age at onset was 13 +/- 12 (SD; range 6 to 43). Four patients had suffered up to five attacks. EMG and pathologic findings were compatible with a reversible demyelinating neuropathy such as Guillain-Barré syndrome. We excluded loci causing other hereditary demyelinating neuropathies, such as Charcot-Marie-Tooth disease type I (CMT type I) and hereditary neuropathy with liability to pressure palsies (HNPP), by linkage analysis; thus, HTN is not allelic to either CMT type I or to HNPP.     

Intermediate nerve conduction velocities define X-linked Charcot-Marie-Tooth neuropathy families

Three genetic loci for the Charcot-Marie-Tooth (CMT) syndromes with slow motor nerve conduction velocities (hereditary motor and sensory neuropathy: HMSN type I) have been mapped to chromosomes 1 (CMT1B), 17 (CMT1A), and the X chromosome (CMTX). The clinical features of these three CMT subgroups are similar. To determine whether any clinical features distinguish CMTX families, the range of clinical findings and motor nerve conduction velocities were examined in two large CMTX families, the range of clinical findings and motor nerve conduction velocities were examined in two large CMTX families with CMTX proven by linkage to X-chromosome markers. CMTX males had more wasting and weakness than CMTX females or individuals with CMT1A. Patellar reflexes were more often retained in CMTX. Motor nerve conduction velocities were faster than in CMT1A. Intermediate-range median nerve conduction velocities were present in CMTX females (45 +/- 9 m/sec; range, 26 to 61 m/sec). These velocities were significantly faster than those for CMT1A females (22 +/- 8 m/sec, p < 0.0001). Median nerve conduction velocities in CMTX males (31 +/- 6 m/sec) were significantly slower than in CMTX females and faster than in CMT1A males (20 +/- 6 m/sec, p < 0.0001). The combination of slow conduction velocities in affected males (< 40 m/sec) and intermediate-range median motor conduction velocity results (> 40 m/sec) in affected or obligate carrier females is a useful distinguishing feature to separate CMTX from CMT1A, as intermediate conduction velocities are not present in autosomal-dominant dominant CMT1A families. This feature defines possible CMTX families for linkage studies. Families with no male-to-male inheritance of the syndrome, slow motor nerve conductions in affected males, and normal or intermediate-range conduction velocities in carrier females should be considered to be X-linked CMT families.