Intracellular transport, assembly, and degradation of wild-type and disease-linked mutant gap junction proteins.

More than 130 different mutations in the gap junction integral plasma membrane protein connexin32 (Cx32) have been linked to the human peripheral neuropathy X-linked Charcot-Marie-Tooth disease (CMTX). How these various mutants are processed by the cell and the mechanism(s) by which they cause CMTX are unknown. To address these issues, we have studied the intracellular transport, assembly, and degradation of three CMTX-linked Cx32 mutants stably expressed in PC12 cells. Each mutant had a distinct fate: E208K Cx32 appeared to be retained in the endoplasmic reticulum (ER), whereas both the E186K and R142W mutants were transported to perinuclear compartments from which they trafficked either to lysosomes (R142W Cx32) or back to the ER (E186K Cx32). Despite these differences, each mutant was soluble in nonionic detergent but unable to assemble into homomeric connexons. Degradation of both mutant and wild-type connexins was rapid (t(1/2) < 3 h) and took place at least in part in the ER by a process sensitive to proteasome inhibitors. The mutants studied are therefore unlikely to cause disease by accumulating in degradation-resistant aggregates but instead are efficiently cleared from the cell by quality control processes that prevent abnormal connexin molecules from traversing the secretory pathway.

The role of the gap junction protein connexin32 in the pathogenesis of X-linked Charcot-Marie-Tooth disease.

Mutations in the gene encoding the gap junction protein connexin32 (Cx32; beta 1) cause the X-linked form of Charcot-Marie-Tooth disease (CMTX), a common form of inherited demyelinating neuropathy. Cx32 is localized to the paranodes and incisures of myelinating Schwann cells, and probably participates in the formation of gap junctions at these locations, thereby allowing the diffusion of ions and small molecules directly across the myelin sheath. In transfected cells different CMTX mutations have different effects on the ability of the mutant protein to form functional gap junctions; some mutant proteins cannot be detected within the cell, other mutant proteins accumulate within the cell but do not reach the cell membrane, while other mutants reach the cell membrane and some of these form functional gap junctions. In transgenic mice two mutants, R142W and 175 frameshift, have similar effects on protein trafficking as in transfected cells: the R142W mutant protein remains in the perinuclear region and does not reach the paranodes or incisures, and the 175 frameshift protein cannot be detected. Thus, different CMTX mutations have different effects on Cx32 protein, and these differences may help to explain the phenotypic differences seen in CMTX kindreds.

Mammalian DNA mismatch repair.

DNA mismatch repair (MMR) is one of multiple replication, repair, and recombination processes that are required to maintain genomic stability in prokaryotes and eukaryotes. In the wake of the discoveries that hereditary nonpolyposis colorectal cancer (HNPCC) and other human cancers are associated with mutations in MMR genes, intensive efforts are under way to elucidate the biochemical functions of mammalian MutS and MutL homologs, and the consequences of defects in these genes. Genetic studies in cultured mammalian cells and mice are proving to be instrumental in defining the relationship between the functions of MMR in mutation and tumor avoidance. Furthermore, these approaches have raised awareness that MMR homologs contribute to DNA damage surveillance, transcription-coupled repair, and recombinogenic and meiotic processes.

Altered trafficking of mutant connexin32.

We examined the cellular localization of nine different connexin32 (Cx32) mutants associated with X-linked Charcot-Marie-Tooth disease (CMTX) in communication-incompetent mammalian cells. Cx32 mRNA was made, but little or no protein was detected in one class of mutants. In another class of mutants, Cx32 protein was detectable in the cytoplasm and at the cell surface, where it appeared as plaques and punctate staining. Cx32 immunoreactivity in a third class of mutants was restricted to the cytoplasm, where it often colocalized with the Golgi apparatus. Our studies suggest that CMTX mutations have a predominant effect on the trafficking of Cx32 protein, resulting in a potentially toxic cytoplasmic accumulation of Cx32 in these cells. These results and evidence of cytoplasmic accumulation of other mutated myelin proteins suggest that diseases affecting myelinating cells may share a common pathophysiology.

Connexin32 and X-linked Charcot-Marie-Tooth disease.

Mutations in the gap junction gene connexin32 (Cx32) cause the X-linked form of Charcot-Marie-Tooth disease, an inherited demyelinating neuropathy. More than 130 different mutations have been described, affecting all portions of the Cx32 protein. In transfected cells, the mutant Cx32 proteins encoded by some Cx32 mutations fall to reach the cell surface; other mutant proteins reach the cell surface, but only one of these forms functional gap junctions. In peripheral nerve, Cx32 is localized to incisures and paranodes, regions of noncompact myelin within the myelin sheath. This localization suggests that Cx32 forms "reflexive" gap junctions that allow ions and small molecules to diffuse directly across the myelin sheath, which is a thousandfold shorter distance than the circumferential pathway through the Schwann cell cytoplasm. Cx32 mutations may interrupt this shorter pathway or have other toxic effects, thereby injuring myelinating Schwann cells and their axons.

Mutagenicity of acridines in a reversion assay based on tetracycline resistance in plasmid pBR322 in Escherichia coli.

The mutagenicity of a series of acridine compounds was studied in an assay based on the reversion of mutations in the tetracycline-resistance gene (tet) of plasmid pBR322 in Escherichia coli. Mutations that restore the tetracycline-resistant phenotype were detected in tetracycline-sensitive strains carrying mutant plasmids. Mutations that revert by +2, +1, -1 and -2 frameshift mutations and by base-pair substitutions were used to analyze the mutagenicity of two simple acridines, two acridine mustards, and a nitroacridine. The simple acridines (9-aminoacridine and quinacrine) effectively induced -1 frameshifts and weakly induced +1 frameshifts. The acridine mustards (quinacrine mustard and ICR-191) were more potent inducers of -1 and +1 frameshifts than the simple acridines. Reactive acridines, including both the mustards and the nitroacridine Entozon, were effective inducers of -2 frameshifts but the simple acridines were not. The two classes of reactive acridines differed from one another, in that the mustards were better inducers of +1 frameshifts than Entozon, whereas Entozon was a particularly potent inducer of -2 frameshifts. None of the compounds induced +2 frameshifts, and the induction of base-pair substitutions was negligible. These results confirm and extend studies showing that adduct-forming acridines are stronger frameshift mutagens than simple intercalating acridines and that the acridines differ from one another not only in overall mutagenic potency but also in the prevalence of different classes of frameshift mutations.

Connexin32 is a myelin-related protein in the PNS and CNS.

We have examined the expression of a gap junction protein, connexin32 (Cx32), in Schwann cells and oligodendrocytes. In peripheral nerve, Cx32 is found in the paranodal myelin loops and Schmidt-Lanterman incisures of myelinating Schwann cells, and the levels of Cx32 protein and mRNA change in parallel with those of other myelin-related genes during development, Wallerian degeneration, and axonal regeneration. In the central nervous system, Cx32 is found in oligodendrocytes and their processes, but not in compact myelin, and the levels of Cx32 protein and mRNA increase during development in parallel with those of the other myelin genes. Thus, Cx32 is expressed as part of the myelinating phenotype of both Schwann cells and oligodendrocytes, indicating that this gap junction protein plays in important role in the biology of myelin-forming cells.

Comparative mapping of canine and human proximal Xq and genetic analysis of canine X-linked severe combined immunodeficiency.

Parallel genetic analysis of animal and human genetic diseases can facilitate the identification and characterization of the causative gene defects. For example, canine X-linked severe combined immunodeficiency (SCID) is characterized by clinical, pathological, and immunological manifestations similar to the most common form of human SCID. To derive a canine syntenic map including genes that in humans are located in proximal Xq, near human X-linked SCID, poly(TG) polymorphisms were identified at the canine phosphoglycerate kinase (PGK) and choroideremia (CHM) loci. These plus a polymorphic poly(CAG) sequence in exon 1 of the canine androgen receptor gene (AR) were used to genotype members of the colony informative for X-linked SCID. No recombinations among SCIDX1, AR, PGK, or CHM were observed. Fluorescence in situ hybridization localized PGK and CHM to proximal Xq in the dog, in the same chromosomal location occupied by the human genes. Somatic cell hybrid analysis and methylation differences at AR demonstrated that female dogs carrying X-linked SCID have the same lymphocyte-limited skewed X-chromosome inactivation patterns as human carriers. These genetic and phenotypic findings provide evidence that mutations in the same gene, now identified as the gamma chain of the IL-2 receptor, cause canine and human X-linked SCID. This approach is an efficient method for comparative gene mapping and disease identification.

The interleukin-2 receptor gamma chain maps to Xq13.1 and is mutated in X-linked severe combined immunodeficiency, SCIDX1.

The gene encoding the gamma chain of the lymphocyte interleukin-2 receptor has been cloned and shown to be required to associate with the beta chain in order for IL-2 internalization and cell activation to occur (1). We considered this gene, IL2RG, a candidate for the X-linked form of severe combined immunodeficiency at the SCIDX1 locus, in which affected males have impaired lymphocyte development. Using fluorescence in situ hybridization and PCR amplification of somatic cell hybrid DNAs, we mapped IL2RG to human Xq13.1, a location within the SCIDX1 critical region established by linkage analysis. The 4.2 kb IL2RG gene was sequenced, and its genomic organization was elucidated. Seven of 19 transformed B-lymphocyte cell lines with independent SCIDX1 mutations had absent or minimal IL2RG mRNA. Unique point mutations were documented to be specifically associated with the disease and the carrier state in four unrelated affected males and their family members: one in a boy with no detectable IL2RG mRNA, in which the mutation ablated a splice donor site; one causing premature chain termination; and two causing distinct amino acid changes. The demonstration of impaired IL2RG mRNA expression in males with X-linked SCID and of unique point mutations in SCIDX1 pedigrees constitutes powerful evidence that the SCIDX1 gene is IL2RG. Noguchi et al. (2) have independently published IL2RG mapping to Xq13 and discovery of mutations in three affected males. The specific pathogenesis of IL2RG mutations and approaches to gene therapy can now be addressed in the X-linked form of SCID.

[Student enrollment in Canadian medical schools, 1976-77]. / Etudiants inscrits aux facultés de médecine du Canada, 1976-77.

The 1976-77 statistical study of medical school enrollment by the Association of Canadian Medical Colleges shows that total enrollment in Canadian medical schools had increased 103.8% since 1960-61, although the rate of increase had decreased to almost zero by 1976-77. Women accounted for 30.3% of the total enrollment in 1976-77 (for all years of the course), which represents an increase of more than 550% in the 17-year period; for the 16 schools the proportion ranged between 23.9% and 43.8%. Enrollment of foreign students had decreased from 340 in 1966-67 to 90 (1.2%) in 1976-77; 71 of the 90 students were American. For the entire nation the mean number of medical students per 10 000 population was 3.1, but in British Columbia the figure was only 1.5. Of the Canadian and landed immigrant students 94.5% were attending medical school in their home province.