Prenatal diagnosis of Charcot-Marie-Tooth disease type 1A by multicolor in situ hybridization.

Genetic heterogeneity within the most common genetic neuropathy, Charcot-Marie-Tooth disease (CMT) results in about 70% slow nerve conduction CMT1 and 30% normal nerve conduction CMT2. Autosomal dominant CMT1A on chromosome 17p11.2 represents about 70% of CMT1 cases and about 50% of all CMT cases. Three different size CMT1A duplications with variable flanking breakpoints were characterized by multicolor in situ hybridization and confirmed by pulsed field gel electrophoresis and quantitative polymerase chain reaction (PCR) amplification. These different size duplications result in the same CMT1A phenotype confirming that trisomy of a normal gene region results in CMT1A. The smallest duplication does not include the 409 locus used previously to screen for CMT1A duplications. Direct analysis of interphase nuclei from fetuses and at-risk patients by multicolor in situ hybridization to a commonly duplicated CMT1A probe is informative more often than polymorphic PCR analysis, faster than pulsed field gel electrophoresis (PFGE), and faster, more informative, and more reliable than restriction enzyme analysis. CMT1B restriction enzyme analysis of CMT pedigrees without CMT1A is expected to diagnose another 8% of at-risk CMT1 patients (total: 78%).

Asian hereditary neuropathy patients with peripheral myelin protein-22 gene aneuploidy.

Japanese hereditary neuropathy with liability to pressure palsy (HNPP) patients have a deletion of one peripheral myelin protein-22 (PMP22) gene region in distal chromosome band 17p11.2 as do Caucasian patients. Japanese and Asiatic Indian CMT1A patients have a PMP22 gene duplication that results in Charcot-Marie-Tooth disease type IA (CMT1A; HMSNIA) in patients of European and Middle Eastern ancestry. About 70% of Japanese CMT1 patients have a PMP22 duplication as do Caucasians, while Japanese CMT1B, CMT2 and Dejerine-Sottas patients to not have PMP22 gene region aneuploidy. Although HNPP and CMT1A genotypes are generated simultaneously by unequal recombination that results in PMP22 gene aneuploidy in each daughter cell, only 3 Japanese HNPP probands with PMP22 deletion from a large patient population were referred to a single center compared to 18 referred CMT1A probands with PMP22 duplication. This lower HNPP frequency more likely reflects lower HNPP reproductive fitness than patient ascertainment bias because disease severity and variation in severity is about the same in CMT1A and HNPP patients and because all patients of both types were referred regardless of disease severity. These results, along with an apparently high de novo CMT1A mutation rate, suggest that common ancestors of Japanese, Asian Indians, and Caucasians carried PMP22 geneflanking sequences that enhance unequal crossing over.

Point mutation analysis of archived cytogenetic slide DNA.

Archived Giemsa-stained cytogenetic slide repositories represent valuable DNA resources for medical, scientific, and forensic studies. Sequencing readily identified a Charcot-Marie-Tooth disease point mutation in a 209-bp PCR amplified product. With optimal PCR primers and amplification conditions, our protocol quickly and reliably isolated sufficient DNA for at least 12 independent PCR amplification reactions for forensic and medical applications from single slides up to 5 years old.

Myelin protein zero gene mutated in Charcot-Marie-tooth type 1B patients.

Autosomal dominant of Charcot-Marie-Tooth disease (CMT), whose gene is type 1B (CMT1B), has slow nerve conduction with demyelinated Schwann cells. In this study the abundant peripheral myelin protein zero (MPZ) gene, MPZ, was mapped 130 kb centromeric to the Fc receptor immunoglobulin gene cluster in band 1q22, and a major MPZ point mutation was found to cosegregate with CMT1B in one large CMT1B family. The MPZ point mutation in 18 of 18 related CMT1B pedigree 1 patients converts a positively charged lysine in codon 96 to a negatively charged glutamate. The same MPZ locus cosegregates with the CMT1B disease gene in a second CMT1B family [total multipoint logarithm of odds (lod) = 11.4 at theta = 0.00] with a splice junction mutation. Both mutations occur in MPZ protein regions otherwise conserved identically in human, rat, and cow since these species diverged 100 million years ago. MPZ protein, expressed exclusively in myelinated peripheral nerve Schwann cells, constitutes > 50% of myelin protein. These mutations are anticipated to disrupt homophilic MPZ binding and result in CMT1B peripheral nerve demyelination.

Multicolor fluorescence in situ hybridization and pulsed field electrophoresis dissect CMT1B gene region.

We have used multicolor fluorescence in situ hybridization of banded chromosomes to orient Fc gamma RII and clone 1054 on a single early metaphase chromosome band (1q22) representing about 2% of the physical map of chromosome 1 in the Charcot-Marie-Tooth (CMT1B) gene region. These two cloned fragments are on the same partially digested 900-kb MluI fragment detected by pulsed field gel electrophoresis. When applied to data from an earlier study, multicolor in situ hybridization results further refined the CMT1B genetic location from an 18 cM interval to a 6 cM interval and the physical map from 15% of chromosome 1 to 3% of chromosome 1. Occasionally the three Fc gamma RII immunoglobulin receptor genes within the 200-kb region are resolved in individual metaphase chromatids.

Dual blastomere analysis improves reliability of preimplantation trembler mouse diagnosis.

Dual blastomere biopsy and independent blastomere analysis dramatically improved preimplantation diagnostic reliability as confirmed by testing the remaining biopsied eight-cell mouse embryo. The autosomal dominant trembler mouse point mutation was selected as a model for human preimplantation diagnosis because: (1) single cell assay failure is predicted to be the highest when testing autosomal dominant mutations; (2) point mutations represent the most common of all mutation categories and the most demanding mutation to assay reliably; and (3) the trembler mouse point mutation in peripheral myelin protein 22 (Pmp22) is a model of human Charcot-Marie-Tooth type 1A disease. Mathematical models predict our experimental results assuming amplification of 80% of each target allele as well as trembler sperm DNA contamination in 1 of 44 normal biopsied single blastomeres. Single blastomere analysis correctly predicted the genotype in only 84% of embryos that would have been implanted as normal. In contrast, when independent tests of both biopsied blastomeres agreed, test results were confirmed in 20 of 21 (95.2%) of the remaining six-cell biopsied embryos designated as normal. Thus, biopsied six-cell embryo confirmation demonstrated that dual biopsied blastomere analysis improved test reliability remarkably.

Multicolor in situ hybridization and linkage analysis order Charcot-Marie-Tooth type I (CMTIA) gene-region markers.

This study demonstrates a clear and current role for multicolor in situ hybridization in expediting positional cloning studies of unknown disease genes. Nine polymorphic DNA cosmids have been mapped to eight ordered locations spanning the Charcot-Marie-Tooth type 1 (CMT1A) disease gene region in distal band 17p11.2, by multicolor in situ hybridization. When used with linkage analysis, these methods have generated a fine physical map and have firmly assigned the CMT1A gene to distal band 17p11.2. Linkage analysis with four CMT1A pedigrees mapped the CMT1A gene with respect to two flanking markers (8B10-5 cM[LOD 5.2]-CMT1A-3.5 cM[LOD 5.3]-10E4). Additional loci were physically mapped and ordered by in situ hybridization and analysis of phase-known recombinants in CMT1A pedigrees. The order determined by multicolor in situ hybridization was 17cen-LEW301-8B10-5H5/6A9-VAW409- 5G7-6G1-4A11-VAW412-10E4-pter. Two ordered probes, 4A11 and 6G1, reside on the same 440-kb partial SfiI restriction fragment. These data demonstrate the ability of in situ hybridization to resolve loci within 0.5 Mb on early-metaphase chromosomes. Multicolor in situ hybridization also excluded the possibility of pericentric inversions in two unrelated patients with CMT1 and neurofibromatosis type 1. When used with pulsed-field gel electrophoresis, multicolor in situ hybridization can establish physical location, order, and distance in closely spaced chromosome loci.

Tomaculous neuropathy in chromosome 1 Charcot-Marie-Tooth syndrome.

We performed morphological and immunohistochemical studies on sural nerve biopsies from two members of a Charcot-Marie-Tooth type 1B family, in which a mutation of the P0 gene on chromosome 1 had been found. Biopsies showed a tomaculous neuropathy with loss of myelinated fibers and frequent small onion bulbs. Immunofluorescence with antibodies to P0 showed this protein to be present in tomaculous and non-tomaculous areas of the myelin sheath. The severity of the myelin abnormalities suggests that in this family Charcot-Marie-Tooth disease may result from a generalized disturbance of Schwann cells as a result of an abnormal P0 protein.

Chromosome 1 Charcot-Marie-Tooth disease (CMT1B) locus in the Fc gamma receptor gene region.

The Charcot-Marie-Tooth disease (hereditary motor and sensory neuropathy) loci have been reported to be on at least three chromosomes: 1 (CMT1B, HMSN1B), 17 (CMT1A), and X (CMTX). In this study multipoint linkage analysis of two Duffy-linked families given a combined LOD score of 8.65 to establish that the Duffy-linked CMT1B gene exists in the 18 centimorgan region between the antithrombin III gene and the Duffy/sodium-potassium ATPase loci. The simultaneous segregation of polymorphisms near the CMT1A locus on chromosome 17 excludes linkage to this chromosome region in both families. Polymorphic sites that flank the CMT1B gene have been subchromosomally localized to the proximal chromosome-1 long arm (1q21.2----1q25) by spot blot analysis of sorted chromosomes, polymorphic deletion analysis, in situ hybridization, and multipoint linkage analysis.