Phenotype of Charcot-Marie-Tooth disease Type 2.

OBJECTIVE: To investigate the clinical and electrophysiologic phenotype of Charcot-Marie-Tooth disease (CMT) Type 2 in a large number of affected families. METHODS: We excluded CMT Type 1, hereditary neuropathy with liability to pressure palsies, and CMT due to Cx32 gene mutations by DNA analysis. We performed genetic analysis of the presently known CMT Type 2 genes. RESULTS: Sixty-one persons from 18 families were affected. Ninety percent of patients were able to walk with or without the help of aids. Proximal leg muscle weakness was present in 13%. Asymmetrical features were present in 15%. Normal or brisk knee reflexes were present in 36%. Extensor plantar responses without associated spasticity occurred in 10 patients from eight families. Only three causative mutations were identified in the MFN2, BSCL2, and RAB7 genes. No mutations were found in the NEFL, HSPB1, HSPB8, GARS, DNM2, and GDAP1 genes. CONCLUSIONS: At group level, the clinical phenotype of Charcot-Marie-Tooth disease (CMT) Type 2 is uniform, with symmetric, distal weakness, atrophy and sensory disturbances, more pronounced in the legs than in the arms, notwithstanding the genetic heterogeneity. Brisk reflexes, extensor plantar responses, and asymmetrical muscle involvement can be considered part of the CMT Type 2 phenotype. The causative gene mutation was found in only 17% of the families we studied.

Charcot-Marie-Tooth disease due to a de novo mutation of the RAB7 gene.

We report a 32-year-old patient with Charcot-Marie-Tooth (CMT2B) including foot ulcerations. Genetic analysis identified a de novo mutation in the small GTP-ase late endosomal RAB7 gene, consisting of a c.471G>C, p.Lys157Asn missense mutation. This observation strongly supports the hypothesis that RAB7 mutations are responsible for CMT2B.

[Homocysteine, lipoprotein (a): risk factors for coronary heart disease]. / Homocystéine, lipoprotéine (a): acteurs de risque de l'athérosclérose coronarienne.

Our data suggest that the hyperhomocysteinemia and/or increased plasma level of lipoprotein Lp(a) are risk factors for coronary heart disease. We investigated 178 patients who underwent complete cardiac examination comprising coronary angiography and biological analysis (CT, HDL-c, LDL-c, TG, and apoAI, apoB, homocysteine and Lp(a)). Patients presenting a significant stenosis of the coronary artery ( 50% of the vascular lumen) were considered as cases (113 patients). Those without stenosis or with non-significant stenosis (< 50% of the vascular lumen) were used as controls (65 subjects). Homocysteinemia was significantly higher in cases than in control subjects (8.26 mol/L (2.34 versus 17.85 (2.34, p < 0.001). A strong association between coronary heart disease and homocystein has been found (Eta(2) = 0.76). The OR were 0.16 when homocystein level was lower than 15 mol/L, and 27.78 when homocysteine level was upper than or equal to 15 mol/L. The RR was 5.16 (95% IC = 3.66-6.66, p < 0.001). Even though there was a significant correlation between tabagic impregnation and homocysteinemia (Spermann's rho = 0.37, p < 0.05), there was no interactive effect between these two factors and coronary disease (Wald khi2 = 0.086, p > 0.05). Therefore, no association was found between homocyteinemia and other coronary heart disease risk factors. The Lp(a) levels were significantly higher in cases than in controls subjects (188 (84 mg/L in control subjects versus 590 (199 in cases, p < 0.001). A stronger relationship was noted between coronary heart disease and Lp(a) (Eta (2) = 0.66). The OR were 0.09 when lipoprotein (a) levels were lower than 350 mg/L, and 5,88 when Lp(a) levels were higher than or equal to 350 mg/L. The estimate RR was 6.47 (95% IC = 4.39-8.55, p < 0.001). The level of Lp(a) was positively correlated with the severity of coronary heart disease (Spermann's rho = 0.95, p < 0.001). A weak correlation between Lp(a) and LDL-c was observed (Spermann's rho = 0.12, p = 0.048). But the multivariate analysis didn't show interactive effect between these two factors and coronary disease (khi2 de Wald = 0.264, p > 0.05). No association was noted between Lp(a) and the others risk factors. Moreover, a positive correlation between the levels of homocysteine and those of Lp(a) was found (Spermann's rho = 0.54, p < 0.001). In contrast their effect on coronary heart disease seems to be independant (Wald khi2 = 2.957, p > 0.05). Thus, these two parameters appear as independant risk factors for coronary heart disease.

A locus for an axonal form of autosomal recessive Charcot-Marie-Tooth disease maps to chromosome 1q21.2-q21.3.

Charcot-Marie-Tooth disease (CMT) is a heterogeneous group of disorders that affect the peripheral nervous system. Three loci are known for the autosomal dominant forms of axonal CMT (CMT2), but none have yet been identified for autosomal recessive axonal CMT (ARCMT2). We have studied a large consanguineous Moroccan ARCMT2 family with nine affected sibs. The onset of CMT was in the 2d decade in all affected individuals who presented with a severe motor and sensory neuropathy, with proximal muscle involvement occurring in some patients. After exclusion of known loci for CMT2 and for demyelinating ARCMT2, a genomewide search was performed. Evidence for linkage was found with markers on chromosome 1q. The maximum pairwise LOD score was above the threshold value of 3.00, for markers D1S514, D1S2715, D1S2777, and D1S2721, and it reached 6.10 at the loci D1S2777, D1S2721, and D1S2624, according to multipoint LOD-score analysis. These markers defined a region of homozygosity that placed the gene in a 4.4-cM interval. Moreover, a recombination event detected in an unaffected 48-year-old individual excludes the D1S506 marker, thereby reducing the interval to 1.7 cM. In addition, the P0 gene, an attractive candidate because of both its location on chromosome 1q and its role in myelin structure, was excluded by physical mapping and direct sequencing.

The first de novo mutation of the connexin 32 gene associated with X linked Charcot-Marie-Tooth disease.

X linked Charcot-Marie-Tooth disease (CMTX) is a hereditary motor and sensory neuropathy caused by mutations in the connexin 32 gene (Cx32). Using the SSCP technique and direct sequencing of PCR amplified genomic DNA fragments of the Cx32 gene from a Moroccan patient and her relatives, we identified the first de novo mutation of the Cx32 gene, consisting of a deletion of a G residue at position 499 in the Cx32 open reading frame. This previously unreported mutation produces a frameshift at position 147 in the protein and introduces a premature stop codon (TAG) at nucleotide 643, which results in the production of a truncated Cx32 molecule. This mutation illustrates the risk of an erroneous diagnosis of autosomal recessive CMT, especially in populations where consanguineous unions are frequent, and its consequences for genetic counselling, which can be avoided by molecular analysis.

William L. McGuire Memorial Symposium. 1,25(OH)2D3 modulation of mammary tumor cell growth in vitro and in vivo.

The biological role of 1,25(OH)2D3 in controlling Ca++ homeostasis in the body has been identified and widely investigated for a long time. More recently its effect in regulating cell proliferation or differentiated activity was described in a variety of normal and malignant cells. The present study was carried out to investigate the different aspects and biological mechanisms of this activity and to determine if the use of 1,25(OH)2D3 in the treatment of breast cancer patients could be considered. It is found that 1,25(OH)2D3 reduces the proliferation of MCF-7 and BT-20 cells lines regardless of their sex steroid receptor status. This effect is related to the concentration, from 10(-12) M to 10(-8) M. Its amplitude is less in other cell lines, but it opposes the EGF-induced increase of proliferation. It is observed that the proliferation rate of MCF-7 and BT-20 cells is increased when these tumor cells are cocultured with fibroblasts derived from breast tumor biopsies and that 1,25(OH)2D3 reverses this process. Moreover, experiments on DMBA induced mammary tumors in Sprague Dawley rats found that 1,25(OH)2D3 given at non toxic doses reduces significantly the tumor proliferation. These data showed that 1,25(OH)2D3 at low doses is effective on the proliferation of BT-20 and MCF-7 cells and on the paracrine growth stimulatory effect observed in the presence of fibroblasts. They suggest that 1,25(OH)2D3 or related synthetic molecules which are less active on Ca++ metabolism could be useful in the treatment of breast cancer patients.

Measurement of occupied and non-occupied epidermal growth factor receptor sites in 216 human breast cancer biopsies.

The epidermal growth factor (EGF) is one of several growth factors involved in normal breast epithelial development and tumor proliferation. EGF and EGF-like peptide TGF alpha bind and activate the same membrane receptor protein. This receptor (EGF-R) has been recently studied in breast tumor biopsies and its detectability reported as a prognostic indicator. However, normal and tumor tissue themselves produce EGF and related peptides in variable amount. This suggests that the standard measurement of EGF-R by binding assay should reflect only the number of non-occupied receptor sites. Based on this observation, the presence of occupied sites (EGF-R2) has been assessed in 216 human mammary tumor biopsies simultaneously with the direct measurement of non occupied EGF receptor sites (EGF-R1) and the results compared to estrogen and progesterone receptor status (ER, PGR). EGF-R1 and EGF-R2 were evaluated by 2 separate (125I) EGF binding assays performed on 2 aliquots of tumor crude membrane fraction, the first one directly, the other after dissociation of the endogenously bound ligand. The validity of the method has been assessed on membrane fractions prepared from human placenta. It is shown that the dissociation does not modify the binding dissociation constant. ER and PGR were measured by the dextran coated charcoal method. Results greater than 10 fmol/mg of membrane or cytosol protein were considered as positive. It is found that EGF-R1 and EGF-R2 are detectable in 54 and 90% of the cases, indicating that EGF-R is masked by endogenous ligand in 36% of the tumors.(ABSTRACT TRUNCATED AT 250 WORDS)

1,25-Dihydroxyvitamin D3 receptors and human colon adenocarcinoma.

Epidemiological evidence suggests that dietary calcium and 1,25-dihydroxyvitamin D3 (1,25-(OH)2D3) might have a protective effect against colorectal cancers. Since the presence of receptors is required for steroid action, specific 1,25-(OH)2D3 receptors (RD3) were investigated in biopsies taken at different levels of the colon. The study involved 90 biopsies from patients operated on for colorectal adenocarcinoma. They were paired biopsies from adenocarcinoma tissue and adjacent normal mucosa. In addition, 26 normal intestinal mucosa biopsies from patients without cancer were examined. RD3 receptors were assayed in tissue extract by the dextran-coated charcoal technique and also characterized by sucrose density gradient sedimentation. Scatchard analysis showed a single class of specific high affinity-low capacity sites binding for 1,25-(OH)2D3. The incidence of RD3 was 86 per cent in normal mucosa (n = 77) and lower in carcinoma (n = 34), for which the incidence decreased significantly (P less than 0.001) from right colon (58 per cent) to left colon (37 per cent) and rectum (19 per cent). These data suggest that the normal colon is a potential target organ for 1,25-(OH)2D3 which might modulate calcium transport in the colon. Loss of receptivity to 1,25-(OH)2D3 is associated with malignant transformation.

Status of sex steroid hormone receptors in large bowel cancer.

To determine the potential role of sex steroid hormones in the development of colorectal tumors in humans, specific androgen (AR), estrogen (ER), and progesterone (PGR) receptors were investigated in normal mucosa (NM) and in tumor (T) paired biopsy specimens from 94 patients. Androgen receptors were detected in 98% and 96% of NM and T samples, ER in 91% and 83% of NM and T biopsy samples, whereas PGR were detected only in 14% and 10% of NM and T specimens, respectively. These incidences are independent of the sex and age of the patients. They are not related to tumor localization, histologic grade, or stages of Dukes' classification. Scatchard analysis of labeled ligand binding indicated the existence of one single class of high affinity binding sites; the calculated dissociation constant (Kd) was 1.7 +/- 0.6 10(-9) molar concentration (M) for 5 alpha-dihydrotestosterone (DHT) and 0.6 +/- 0.3 10(-9) M for estradiol (E2). These values were identical in NM and T tissue for both AR and ER. The binding capacity for DHT was 148 +/- 67 and 93 +/- 43 fmol/mg of cytosol protein in NM and T tissues, respectively (P less than 0.05). The ER content was lower and similar in NM and T biopsy specimens: 19 +/- 9 and 18 +/- 10 fmol/mg protein, respectively. The PGR content was 10 +/- 4 in NM versus 17.5 +/- 6 fmol/mg protein in T specimens. It is observed that the elevated AR in normal mucosa is not related to any known function for androgens in the digestive tract. The receptor pattern observed in tumors does not support the hypothesis previously raised in the case of chemically induced colonic tumors in rodents.

Sex steroid and 1,25-dihydroxyvitamin D3 receptors in human colorectal adenocarcinoma and normal mucosa.

In order to determine the potential role of sex steroid and 1,25-dihydroxyvitamin D3 in the spreading of colorectal cancer, previously hypothesized from epidemiological and experimental data, specific androgen (AR, n = 94), estrogen (ER, n = 60), progesterone (PGR, n = 50), and 1,25-dihydroxyvitamin D3 receptors (VDR, (n = 111) were investigated in human colorectal adenocarcinoma (AC) and compared with the normal adjacent mucosa (NM). Scatchard analysis and competition studies of binding data did not reveal any difference between the biochemical behavior (affinity, specificity, and sedimentation coefficient) of the normal and tumoral tissue receptors. For ARs and ERs, high incidences were found (92 of 94 and 90 of 94 in NM versus 46 of 60 and 40 of 60 in AC, respectively) in both classes of tissues, while they were low for progesterone (7 of 50 and 5 of 50 in NM versus AC). While for sex steroid receptors the incidences did not vary with sex and age of the patients or the location and histopathological grade of the tumor, the VDR incidence was lower in AC (35 of 111) than in NM (99 of 111) and decreased significantly from the right colon to the rectum in adenocarcinoma. Binding capacities were similar in NM and AC for ERs and VDRs, whereas AR levels in NM were significantly higher than in AC. The expression of VDRs in some colorectal tumors suggests a possible clinical significance. No known function for sex steroid receptors is related to their presence in human colorectal tissues and their pattern in carcinoma does not support any hypothesis previously raised in the case of chemically induced colonic tumors in rodents.

Evidence of 1,25-dihydroxyvitamin D3-receptors in human digestive mucosa and carcinoma tissue biopsies taken at different levels of the digestive tract, in 152 patients.

Epidemiological and experimental data suggest that dietary calcium and 1,25-dihydroxyvitamin D3 (1,25-(OH)2D3) are protective against colorectal cancers, while their activity on colon mucosa still remains unknown. Since the presence of receptors is required for steroid action, specific 1,25-(OH)2D3 receptors were investigated in biopsies taken at different levels of the digestive tract from the oesophagus to the rectum and in pancreas. The total study involved biopsies from 152 patients. In 82% of the cases they were paired biopsies in adenocarcinoma tissue and in adjacent normal mucosa (NM). There were 120 operated on for colorectal adenocarcinoma (HCRA). 1,25-(OH)2D3 receptor was assayed in tissue extract by the dextran-coated charcoal (DCC) technique and also characterised by sucrose density gradient centrifugation. Scatchard analyses showed a single class of specific high affinity-low capacity sites binding for 1,25-(OH)2D3 with a Kd = 1.48 +/- 0.8 x 10(-10) M (n = 119). The sedimentation coefficient of the steroid receptor complex was approximately 3.2 S. The incidence of 1,25-(OH)2D3 receptors was significantly higher in NM (82.5%) than in HCRA (34.5%). In HCRA this incidence decreased from right colon (64.7%) to left colon (27.7%) and rectum (15%). All positive HCRA in left colon and rectum (16/76) were histologically well differentiated. The receptor content in NM and HCRA was in the same range: (median) 10-314 (58) and 13-175 (64) fmol/mg protein. These data suggest that 1,25-(OH)2D3 may modulate calcium transport in colon, as in the intestine. Also, loss of receptivity to 1,25-(OH)2D3 is observed as associated with malignant transformation of the human colorectal mucosa.

[Comparative study of the content of 1,25-dihydroxyvitamin D3 receptors in digestive mucosa and adenocarcinoma]. / Etude comparative du contenu en récepteurs de la 1,25-dihydroxyvitamine D3 dans des muqueuses et des adénocarcinomes digestifs.

We have studied and compared the 1,25-dihydroxyvitamin D3 receptor (RD3) content of 154 human digestive carcinoma with the normal mucosa one, removed at distance from the same surgical specimen. The distribution of biopsies is as follows: 5 oesophagus, 10 stomach, 6 small bowel, 35 right colon, 47 left colon, 40 rectum and 11 pancreas. RD3 were measured by the Dextran Coated Charcoal method and characterized by sucrose gradient ultracentrifugation. One single class of high affinity binding sites (kD = 1.5 +/- 0.7 x 10(-10) M) was defined, with a sedimentation coefficient of approximately 3.4 S. The RD3 was present in the 6 samples of small bowel and in 82% of whole normal mucosa, whatever their localization along the digestive tract and pancreas, while in the tumoral tissue, the RD3 was positive in only 32% of the cases. In these tumor specimens the incidence decreases from 64% in the right colon to 27% in the left colon and only 15% in the rectum (P less than 0.001). RD3 rates vary slightly with the localization and are of the same level in normal tissues and in tumors: 10-314 (median = 59) vs 13-175 (median = 64) (P greater than 0.005) respectively. No significative variations relating to age or sex of patients were found. However, all RD3 positive tumors from left colon and rectum were well differentiated histologically. These results show that the normal colonic mucosa is a potential target for 1,25-dihydroxyvitamin D3, which can play a role in the metabolism of calcium and other ions. They also suggest that vitamin D3 could be a modulator of colorectal cell growth and differentiation and its receptor is frequently lost during malignant transformation.

Specific receptors for 1,25-dihydroxyvitamin D3 (1,25-DR) and human colorectal carcinogenesis.

Because it is a common prerequisite for steroid responsiveness in target tissue, we investigated the presence of specific 1,25-DR in spontaneous human colorectal adenocarcinomas (ADC) and adjacent normal-appearing mucosa (NAM) from 23 operative specimens (12 male and 11 female patients). 1,25-DR was determined in cytosol by a DCC assay technique. 1,25-DR was present in 21 of 23 NAM and in only 4 of 23 HCRA. All positive ADC were well differentiated. Receptor content expressed in femtomoles/mg of protein (mean +/- SEM) was respectively 63.9 +/- 7.6 for right colon NAM and 51.3 +/- 12.9 for left colon or rectum NAM. When we compared all NAM specimens, receptor content was 56.7 +/- 8.0 femtomoles/mg of protein. No difference in 1,25-DR NAM level was observed between right colon and left colon or rectum. In adenocarcinoma the mean content was 66.5 +/- 14 fmoles/mg of protein. Scatchard analysis showed a single class of specific high-affinity saturable 1,25-DR with a dissociation constant (Kd) of 0.97 +/- 0.57 and 1.03 +/- 0.39 chi 10(-10) M in NAM and ADC respectively. These preliminary data represent the first demonstration of 1,25-DR throughout the entire human colon and indicate that the receptivity for this hormone is often lost during malignant transformation of the human colorectal mucosa. In addition, 1,25-DR could be a marker of differentiation in ADC. These preliminary results provide evidence supporting the addition of Vitamin D to the roster of developmental cancer chemopreventative agents.