Splice site, frameshift, and chimeric GFAP mutations in Alexander disease

Abstract

Alexander disease (AxD) is a usually fatal astrogliopathy primarily caused by mutations in the gene encoding glial fibrillary acidic protein (GFAP), an intermediate filament protein expressed in astrocytes. We describe three patients with unique characteristics, and whose mutations have implications for AxD diagnosis and studies of intermediate filaments. Patient 1 is the first reported case with a noncoding mutation. The patient has a splice site change producing an in-frame deletion of exon 4 in about 10% of the transcripts. Patient 2 has an insertion and deletion at the extreme end of the coding region, resulting in a short frameshift. In addition, the mutation was found in buccal DNA but not in blood DNA, making this patient the first reported chimera. Patient 3 has a single-base deletion near the C-terminal end of the protein, producing a short frameshift. These findings recommend inclusion of intronic splice site regions in genetic testing for AxD, indicate that alteration of only a small fraction of GFAP can produce disease, and provide caution against tagging intermediate filaments at their C-terminal end for cell biological investigations.

Figure 1

Relative amount of mutant transcript. The ratio of wild type to mutant transcripts was determined by comparing the band intensities obtained by PCR of patient cDNA (far right lane) with those amplified from wild type and mutant plasmids mixed at different ratios (lanes 2–7). The results indicate that the level of the truncated transcript is about 12-fold lower than the wild type transcript.

Figure 2

Polymerization properties of wild type and mutant proteins. Expression plasmids encoding wild type (WT) GFAP and/or mutant GFAPs predicted for Patient 1 (Δex4) or Patient 2 (I/D/F) were transiently transfected into SW13vim⁻ cells and the cells stained two days later for GFAP as described in Materials and Methods. A, WT; B, Δex4; C, 1:1 WT:Δex4; D, 8:1 WT:Δex4; E, I/D/F.

Figure 3

Effect of the ratio of wild type GFAP (WT) to Patient 1 mutant GFAP (Δex4) on filament assembly in transfected SW13vim⁻ cells. SW13vim⁻ cells were transfected with different ratios of WT to Δex4 expression plasmids, while the total amount of plasmid was held constant. Cells were then stained for GFAP, and the patterns classified as a normal filamentous network (cf Fig. 3A), thick filaments (Fig. 3D & E), diffuse staining (background in 3C) or aggregates (3B & C). The percentages of each pattern for a minimum of 150 cells are shown for the different WT to Δex4 ratios. *The difference compared with WT is significant (p<0.01) by Chi Square test.

Figure 4

Mutation present in Patient 2. The mutation involves deletion of 8 bp (green) and insertion of 3 bp (red), resulting in a frameshift that changes the last two amino acids of wild type GFAP from VM to DR and adds 11 additional residues before encountering a stop codon.

Figure 5

MRI for Patient 3. The MRI at 6 months (A–E) shows an abnormally high signal in the frontal white matter, the head of the caudate nucleus, the putamen, globus pallidus (B) and dentate nucleus (A). The frontal lobes are atrophic (A). Contrast enhancement of the dentate nucleus (C), frontal white matter (D, E), head of the caudate nucleus (D), the putamen (D), and the hypothalamus (E) is shown. Follow-up MRI at the age of 12 months (F) shows extensive cystic degeneration of the frontal white matter.

Figure 6

Mutation present in Patient 3. The predicted coding change resulting from the c.1249delG mutation is shown. The G that is deleted is shown in the black box in the wild type sequence.