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. 1999 Jul 20;96(15):8562-6.
doi: 10.1073/pnas.96.15.8562.

Mucolipidosis IV consists of one complementation group

E Goldin  1 , A Cooney, C R Kaneski, R O Brady, R Schiffmann
Affiliations

Mucolipidosis IV consists of one complementation group

E Goldin et al. Proc Natl Acad Sci U S A. .

Abstract

Mucolipidosis IV (MLIV) is an autosomal recessive disorder of unknown etiology characterized by severe visual impairment and psychomotor retardation. Recently, there has been considerable interest in positional cloning of the MLIV gene. It is unknown whether MLIV is a genetically homogenous disorder. In this paper, we present experiments that determined whether the MLIV phenotype in fibroblasts could be corrected by fusing normal cells to MLIV cells and fusing fibroblasts from pairs of patients. All of our MLIV patients fulfilled several diagnostic criteria that we developed. In addition, we found high sensitivity to chloroquine in cultured fibroblasts from MLIV patients. We found that normal cells corrected the MLIV phenotype. Fusion products of normal and MLIV fibroblasts, but not MLIV fibroblasts themselves, were relatively protected against chloroquine selection. In addition, 74% of the normal-to-patient fusion products had reduced levels or total loss of MLIV characteristic autofluorescence. However, there was no complementation of the phenotype in fibroblast cultures from any of our MLIV patients, including those of non-Jewish ancestry. In fusion products of MLIV cultures from 24 patients, 90-100% of the cells remained autofluorescent. These results indicate that all of our known MLIV patients, regardless of ancestry or severity of the developmental defect, have a single mutated gene.

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Figures

Figure 1
Figure 1
Survival of normal and MLIV fibroblasts fusion products under chloroquine treatment. Normal and MLIV fibroblasts were fused as described in Materials and Methods. After 24 hr, cells were seeded in equal densities in two 2-chamber slides. The following day, the cells were treated with chloroquine for 24 hr and then washed and fixed for microscopy. One hundred surviving cells containing two or more nuclei were counted in each treatment, and the fraction of normal–normal (green), MLIV–MLIV (red), and normal–MLIV (orange) was recorded. The results of a representative experiment are presented. Chloroquine treatment almost eliminated MLIV–MLIV fusion products from the culture. Also, a small fraction of normal–MLIV fusion products disappeared in the higher chloroquine concentrations as indicated by the increased proportion of normal–normal cells.
Figure 2
Figure 2
Elimination of autofluorescence in fusion products of normal and MLIV fibroblasts. Normal (stained green) and MLIV (stained red) fibroblasts were fused as described in Materials and Methods. Four days after seeding on slides, cells were fixed and photographed. (A) A phase micrograph showing a normal cell (nucleus marked N), a normal–MLIV fusion product (nuclei marked N, M), and an MLIV cell (nucleus marked M). The large arrow points to the phase-dense material. (B) Fluorescence in the UV channel showing autofluorescence in MLIV cell and not in the fusion product. The autofluorescence colocalizes with the phase-dense material. (C) Fluorescence in the green wavelength. In the MLIV cell, only autofluorescent material appears green in that channel. (D) Fluorescence of the same cells in the red channel. The green nucleus is highly visible in the fused cell, whereas the red nucleus is less apparent because of postlabeling diffusion of the red dye.
Figure 3
Figure 3
Complementation of autofluorescence in MLIV fibroblast cultures. Cells from patients were fused with either one of the two index MLIV cultures, DMN96.73 (labeled 1) and DMN96.37 (labeled 2), as described in Materials and Methods. The results of counting 100 fusion products in each experiment are presented. The right bar presents the average results of fusing normal control and six different MLIV cell lines.
Figure 4
Figure 4
No complementation of phenotype by fusion of two MLIV fibroblasts lines. Two MLIV cultures (stained green and red) were fused as described in Materials and Methods. Four days after seeding on slides, cells were fixed and photographed under the microscope. (A) A phase micrograph showing a red cell (nucleus marked R) and a green and red fusion product (nuclei marked G and R); large arrows point to phase-dense material. (B) Autofluorescence shown in the UV channel. In the fusion product, fluorescence intensity seems even stronger than in the single nuclear cell. (C) The same cells at green wavelength. (D) The same cells at the red channel.
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