Mutations in TMEM76* cause mucopolysaccharidosis IIIC (Sanfilippo C syndrome)

Abstract

Mucopolysaccharidosis IIIC (MPS IIIC, or Sanfilippo C syndrome) is a lysosomal storage disorder caused by the inherited deficiency of the lysosomal membrane enzyme acetyl-coenzyme A: alpha -glucosaminide N-acetyltransferase (N-acetyltransferase), which leads to impaired degradation of heparan sulfate. We report the narrowing of the candidate region to a 2.6-cM interval between D8S1051 and D8S1831 and the identification of the transmembrane protein 76 gene (TMEM76), which encodes a 73-kDa protein with predicted multiple transmembrane domains and glycosylation sites, as the gene that causes MPS IIIC when it is mutated. Four nonsense mutations, 3 frameshift mutations due to deletions or a duplication, 6 splice-site mutations, and 14 missense mutations were identified among 30 probands with MPS IIIC. Functional expression of human TMEM76 and the mouse ortholog demonstrates that it is the gene that encodes the lysosomal N-acetyltransferase and suggests that this enzyme belongs to a new structural class of proteins that transport the activated acetyl residues across the cell membrane.

Figure 1.

Four families from the Czech Republic used in the linkage and mutation analyses. Fully blackened symbols indicate individuals with MPS IIIC; arrowheads indicate probands. Measurements in seven obligate heterozygotes from these pedigrees (mean ± SD 11.6 ± 1.5 nmol/h/mg) and 89 controls not known to be related to members of the pedigree (mean ± SD 24.4 ± 5.7 nmol/h/mg) were used to establish N-acetyltransferase activity ranges for heterozygotes (symbols with blackened inner circle) and normal homozygotes (open symbols). An individual was assigned to a class if his or her enzyme activity was within 2 SDs of the class, unless the value was within the overlap of the upper end of the obligate heterozygotes and the lower end of the controls. Individuals with values within the open interval 13.0–14.6 nmol/h/mg were classified as unknown (symbols with gray inner circle). A symbol with a question mark (?) indicates that no material was available for the enzyme assay. DNA was available for individuals with ID numbers, and N-acetyltransferase activity measurements in white blood cells are shown below the ID numbers.

Figure 2.

Multipoint linkage analysis of MPS IIIC on chromosome 8. A, Multipoint LOD scores in an 8.9-cM interval from two sets of families. Symbols above the marker names indicate the map position. Marker names are listed in the correct order but may be displaced from the symbols for visibility. The dashed line is based on families genotyped in Montreal, and the dotted line on families genotyped in Prague. Straight lines next to marker names indicate that the markers were typed in both data sets. Triangles pointing down indicate markers typed only in the Montreal data set, and triangles pointing up indicate markers typed only in the Prague data set. For the Montreal data, the SimWalk2 run with the highest likelihood is shown. TMEM76 lies between D8S1115 and D8S1460, and, according to the March 2006 freeze of the human genome sequence from the University of California–Santa Cruz Genome Browser, the order is D8S1115–(500 kb)–TMEM76–(800 kb)–centromere–(200 kb)–D8S1460. B, Multipoint LOD scores from the Montreal data from four runs of SimWalk2, version 2.91, showing the variation between runs.

Figure 3.

Predicted amino acid sequence of the TMEM76 protein. Amino acid sequence alignment of Homo sapiens TMEM76 with orthologs from Mus musculus (cloned sequence), Canis familiaris (GenBank accession number XP_539948.2), Bos taurus (XP_588978.2), Rattus norvegicus (XP_341451.2), and Pan troglodytes (XP_519741.1) by use of BLAST. All cDNA sequences are predicted except the sequence for M. musculus. The identical residues are boxed, the residues with missense mutations in patients with MPS IIIC are shown in red, and the amino acid changes are indicated above the sequence. The first 67 aa of the human sequence shown as black on yellow comprise the predicted signal peptide. The predicted transmembrane domains in the human sequence are shown as black on turquoise. The topology model^– strongly predicts that the N-terminus is inside the lysosome and the C-terminus is outside. Four predicted N-glycosylation sites are shown as black on pink, and the predicted motifs for the lysosomal targeting, as black on green.

Figure 4.

Volcano plot of genes located within the MPS IIIC candidate region, showing significantly reduced expression of the TMEM76 gene in white blood cells of two patients with MPS IIIC: AIV.8 and BIII.5. The natural logarithm of the probability that the gene is differentially expressed (Log odds) is plotted as a function of the logarithm of the gene-expression log₂ fold change (Log fold change) between the patient and control samples.

Figure 5.

Northern-blot analysis of TMEM76 mRNA in human tissues. A 12-lane blot containing 1 μg of poly A+ RNA per lane from various adult human tissues was hybridized with a [³²P]-labeled 220-bp cDNA fragment corresponding to exons 8–10 of the TMEM76 gene or β-actin, as described in the Material and Methods section.

Figure 6.

Functional expression of human and mouse TMEM76 protein. A, The full-size human and mouse TMEM76 coding sequences subcloned into pCMV-Script, pCMV-Tag4A, and pEGFP-N3 vectors were expressed in COS-7 cells and in cultured skin fibroblasts from a patient with MPS IIIC. The cells were harvested 48 h after transfection, and N-acetyltransferase activity was measured in the homogenates of TMEM76-transfected and mock-transfected fibroblast or COS-7 cells by use of the artificial fluorometric substrate 4-methylumbelliferyl-β-d-glucosaminide. Values represent means ± SD of four independent experiments. B, The intracellular localization of TMEM76 was studied by expressing the fusion protein of the mouse TMEM76 with EGFP. Before fixation, the cells were treated for 45 min with 50 nM lysosomal marker, LysoTracker Red DND-99 dye. Slides were analyzed on an LMS 510 Meta confocal microscope (Zeiss). Magnification ×1000. The image was randomly selected from 30 studied panels, all of which showed a similar localization of TMEM76-EGFP. The fluorescence of EGFP was not quenched as it would have been if the fluorophore had been exposed to the acidic lysosomal microenvironment, confirming that the C-terminus of TMEM76 faces the cytoplasmic side of the lysosomal membrane.