The current work provides an explanation for the severe phenotype of MPS IIIC patients carrying missense mutations in the HGSNAT
gene. Altogether, we studied the activity and biogenesis of HGSNAT mutants having 21 amino acid substitutions previously identified in MPS IIIC families and affecting 8 of the 11 transmembrane segments of the enzyme as well as its luminal and cytosolic domains (). The missense mutations studied in this paper represent all of the currently identified missense HGSNAT mutations. The polymorphisms (P237Q, V481L, K523Q and A615T, shown in green in ) identified in MPS IIIC only in cis
either with a splice site mutation or with a missense mutation 
were previously reported by us to result in catalytically active protein 
. They were included in the current study because it was unclear whether they still could affect kinetic parameters or targeting of HGSNAT and therefore represent a clinically valuable phenotype. All variants were expressed as TAP-tagged proteins since no Western blotting or cellular immunostaining could be conducted using the patient fibroblasts due to a lack of specific antibodies against human HGSNAT. Our results show that the HGSNAT variants P237Q, V481L, K523Q and A615T are all correctly processed targeted to the lysosome and display full enzymatic activity. We conclude therefore that these four mutations represent rare polymorphisms in the HGSNAT
gene and do not have clinical significance thus confirming our previous hypothesis 
Distribution of missense mutations in HGSNAT protein.
Seventeen mutations (C76F, L137P, G262R, N273K, P283L, R344C, R344H, W403C, G424S, E471K, M482K, A489E, S518F, S539C, S541L, D562V and P571L, shown in red in ) result in production of misfolded HGSNAT protein that is abnormally glycosylated and not targeted to the lysosome. Seven of these mutations (G262R, P283L, W403C, M482K, A489E, S518F and P571L) are predicted to reside within the highly hydrophobic transmembrane domains of the protein. Four of them (G262R, W403C, M482K, A489E) introduce hydrophilic or charged residues inside the transmembrane domains which usually have a dramatic effect on the folding of the protein [reviewed in 18]
. Three other changes (P283L, S518F and P571L) result in replacement of hydrophilic residues for hydrophobic ones that also could destabilize the transmembrane helix.
Six mutations (R344C, R344H, E471K, S539C, S541L and D562V) are found adjacent to the predicted transmembrane domains either on the cytoplasmic (D562V) or on the lumenal (R344C, R344H, E471K, S539C, and S541L) side and 4 mutations (C76F, L137P, N273K, and G424S) reside inside the hydrophilic lumenal domains of the enzyme. In most cases these mutations are predicted to have a drastic effect on protein folding since they involve replacements with amino acids significantly different in hydrophobicity (C76F, D562V, S541L), charge (R344C/H, N273K, E471K) or size (C76F, L137P, G424S). Thus, enzyme folding defects due to missense mutations, together with nonsense-mediated mRNA decay seem to be the major molecular mechanisms underlying MPS IIIC.
For at least 5 of the above changes (N273K, R344C, R344H, S518F and S541L) the active conformation can be stabilized by the competitive inhibitor of HGSNAT glucosamine resulting in part of the enzyme pool being properly processed and targeted to the lysosomes. L137P and P283L mutants may also be stabilized by the glucosamine treatment, however this could not be verified experimentally because in the available patient cell lines they were present together with the responsive mutations S518F and R344C, respectively. Only one cell line carrying E471K and D562V mutations did not show a significant increase in N-acetyltransferase activity in response to glucosamine. Further structural studies are needed to fully understand the difference in the effect of glucosamine on these mutants.
Although the spectrum of mutations in MPS IIIC patients shows substantial heterogeneity, some of the missense mutations have a high frequency within the patient population. Importantly, the two mutations, R344C and S518F, responsive to glucosamine-mediated refolding account for 22.0% and 29.3%, respectively, of the alleles among the probands of Dutch origin 
. The S518F mutation has also been identified in a patient from Germany, while the R344C change was found in families from France, UK, Germany and Singapore. The responsive mutation R344H was found in 4 families from Eastern and Northern Europe (2 from Poland, one from Czech Republic and one from Finland) and the responsive mutation S541L was reported in 4 families from France, Ireland, Poland and Portugal. In general, the vast majority of patients is affected with at least one missense mutation interfering with the proper folding of the enzyme that could be partially rescued by the treatment of the cells with the competitive inhibitor of HGSNAT, glucosamine. We believe this makes MPS IIIC a good candidate for enzyme enhancement therapy [reviewed in 20]
, where active site-specific inhibitors are used as pharmacological chaperones to modify the conformation of the mutant lysosomal enzymes usually retained and degraded in the ER in order to increase the level of the residual activity to a point sufficient to reverse the clinical phenotype. Together with inhibitors of heparan sulfate synthesis, pharmacological chaperones could potentially reduce storage of this polymer in the central nervous system to levels sufficient to stop neuronal death and reverse inflammation.