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Human Molecular Genetics, 2004, Vol. 13, No. 13 1361-1371
DOI: 10.1093/hmg/ddh152
Human Molecular Genetics, Vol. 13, No. 13 © Oxford University Press 2004; all rights reserved

Molecular pathogenesis of cystinosis: effect of CTNS mutations on the transport activity and subcellular localization of cystinosin

Vasiliki Kalatzis1,*, Nathalie Nevo1,{dagger}, Stéphanie Cherqui1,{dagger}, Bruno Gasnier3 and Corinne Antignac1,2

1Inserm U574 and 2Department of Genetics, Hôpital Necker-Enfants Malades, 149 rue de Sèvres, 75015 Paris, France and 3CNRS UPR 1929, Institut de Biologie Physico-Chimique, 13 rue Pierre et Marie Curie, 75005 Paris, France

Received March 10, 2004; Accepted April 26, 2004


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Cystinosis is an inherited disorder characterized by defective lysosomal efflux of cystine. Three clinical forms (infantile, juvenile and ocular cystinosis) have been described according to the age of onset and severity of the symptoms. The causative gene, CTNS, encodes a seven transmembrane domain protein, cystinosin, which we recently identified as a H+-driven cystine transporter using an in vitro transport assay. In this study, we explored the relationship between transport activity and intracellular localization of cystinosin mutants and their associated clinical phenotype. Thirty-one pathogenic mutations (24 missense mutations, seven in-frame deletions or insertions) were analysed. Most of the mutations did not alter the lysosomal localization of cystinosin, although three partially mislocalized the protein independently of its C-terminal sorting motif, thus confirming the presence of an additional sorting mechanism. Sixteen of 19 mutations associated with infantile cystinosis abolished transport, whereas three of five mutations associated with juvenile or ocular forms strongly reduced transport, in agreement with the milder clinical phenotype. Five atypical, unclassified or misclassified mutations could be clarified using the transport data and additional genetic information. Overall, our data demonstrate that, excluding premature termination of cystinosin, impaired transport is the most frequent cause of pathogenicity, with infantile cystinosis generally resulting from a total loss of activity. Thus the transport assay could be used as a prognostic tool when novel mutations are identified.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Lysosomes are filled with hydrolases involved in the degradation of diverse macromolecules. Specific transporters in the lysosomal membrane then export the hydrolysed products to the cytosol. Abnormal lysosomal storage can occur in two ways: a defective hydrolase can result in the accumulation of an undegraded macromolecule or a defective transporter can result in the accumulation of a degraded substrate. The former scenario gives rise to over 40 different clinical disorders termed ‘lysosomal storage disorders’ (LSD), classified according to the macromolecule that accumulates (1). The latter causes a subset of LSD referred to as lysosomal transport disorders (reviewed in 2), such as sialic acid storage diseases (3) and cystinosis.

Cystinosis (MIM 219800), an autosomal recessive monogenic disease with a low incidence (1 in 200 000 live births), is characterized by an intra-lysosomal accumulation of cystine. There exists a spectrum of disease phenotypes, but affected individuals are generally grouped into three clinical forms, based on the age of onset and severity of the symptoms (4). The most severe form, infantile cystinosis, generally appears between 6 and 12 months with a proximal renal tubulopathy (the Fanconi syndrome characterized by fluid and electrolyte loss, poor growth and rickets) that, in the absence of treatment, leads to end stage renal disease (ESRD) by ~10 years. Within the first 2 years of age, corneal cystine crystals also give rise to a severe and painful photophobia. Continuous widespread cystine accumulation eventually leads to retinal, endocrinological, hepatic, gastrointestinal, muscular and neurological anomalies. Juvenile cystinosis (MIM 219900) is generally characterized by an adolescent onset of photophobia and glomerular renal impairment, but not necessarily renal Fanconi syndrome. Ocular non-nephropathic cystinosis (MIM 219750) is characterized solely by an adult onset of mild photophobia without renal anomalies. Atypical forms, where individuals present with various clinical features of severity between infantile and juvenile cystinosis also have been recently described (5,6).

The gene underlying cystinosis, CTNS, is located on 17p13 (7) and contains 12 exons, the first two of which are non-coding (8). The most common mutation associated with cystinosis is a 57 kb deletion (8,9) that removes the 5' region of the gene upstream of, and including, exon 10. CTNS mutations have been identified in all forms of the disease and are primarily situated in the coding region (5,6,8,10–14), although three mutations also have been reported in the undelineated promoter region (15). It has been hypothesized that the underlying differences between the clinical forms is because individuals with infantile cystinosis harbour ‘severe’ mutations (i.e. loss of function) on both alleles, whereas individuals with milder forms of cystinosis harbour a specific, non-severe mutation (i.e. never observed in cases of infantile cystinosis) either on both alleles or in association with a severe allele (compound heterozygotes) (5,11,12).

CTNS encodes a 367 amino acid protein named cystinosin (8). Cystinosin is a lysosomal membrane protein (16) with a seven transmembrane domain (TM) topology. The C-terminal tail is predicted to be oriented towards the cytosol and the highly glycosylated N-terminal region towards the lysosomal lumen. Two signals have been identified as playing a role in the lysosomal sorting of cystinosin: a classic tyrosine-based GYDQL motif situated in the C-terminal tail and a novel conformational signal situated in the fifth inter-TM loop (16). Owing to the relative inaccessibility of the lysosomal lumen, we expressed cystinosin at the plasma membrane, by deletion of the GYDQL motif, and measured the cellular uptake of cystine at acidic external pH to assay its ‘lysosomal cystine efflux’ activity. This in vitro model, equivalent to giant, inside-out lysosomes, showed that cystinosin is the lysosomal cystine transporter and that its activity is driven by the H+ electrochemical gradient of the lysosomal membrane (17).

This expression assay now represents a unique tool for the functional analysis of CTNS mutations that do not truncate the protein. We previously showed that the missense mutation G308R, situated in the sixth TM and identified in several individuals with infantile cystinosis (5,10), is sufficient to abolish the transport activity of cystinosin without altering its subcellular localization (17). This observation accounted for the severe phenotype associated with G308R and indicated a critical role for the sixth TM. We report here a study of 31 missense CTNS mutations or in-frame deletions/insertions associated with the various forms of cystinosis, and their effect on the subcellular localization and function of cystinosin. This work provides an insight into the regions of the protein critical to its transport activity, as well as into the relationship between the molecular and clinical phenotypes of the disease.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Two assays were performed in parallel to investigate the molecular effects of the CTNS mutations. To analyse the effect on the lysosomal localization of the protein, cystinosin–EGFP fusion constructs containing the mutation of interest were expressed in HeLa and Martin-Darby Canine Kidney (MDCK) cells. The wild-type fusion protein localizes to late endosomes and lysosomes in these cells (Fig. 1A and B) (16). To assay the effect on cystine transport, mutations were introduced into an expression plasmid encoding cystinosin deleted in its C-terminal targeting motif (cystinosin–{Delta}GYDQL). COS-7 cells expressing wild-type cystinosin–{Delta}GYDQL are able to take up [35S]L-cystine from the extracellular medium (17).



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  		Figure 1. Effect of cystinosis-associated mutations on the subcellular localization of cystinosin. EGFP-fused cystinosin constructs bearing the mutation of interest, were transiently expressed in HeLa (A, C, E, G, I, K) and MDCK (B, D, F, H, J, L) cells. For HeLa cells, the cystinosin–EGFP fluorescence (left and right) is compared with the immunoreactivity of the endogenous epithelial membrane antigen (EMA) (middle and right). Scale bar=20 µm. For MDCK cells, the cystinosin–EGFP fluorescence (left and right) is compared with the immunoreactivity of the endogenous late endosomal/lysosomal marker LAMP-2 (middle and right). Scale bar=10 µm. (A, B) Wild-type cystinosin–EGFP exhibits an intracellular, punctate distribution corresponding to late endosomes and lysosomes. (C, D) Deletion of the C-terminal GYDQL lysosomal sorting motif from wild-type cystinosin–EGFP induces a partial mislocalization to the plasma membrane with a signal remaining in the late endosomes/lysosomes. (E, F) IVFD343–346del, associated with infantile cystinosis, induces a partial mislocalization of cystinosin–EGFP to the plasma membrane, in addition to the endosomal/lysosomal localization. (G, H) Deletion of the GYDQL motif from cystinosin–IVFD343–346del–EGFP completely redirects the protein to the plasma membrane. (I, J) N323K does not alter the late endosomal/lysosomal localization of cystinosin–EGFP. (K, L) Deletion of the GYDQL motif from cystinosin–N323K–EGFP partially mislocalizes the protein to the plasma membrane, as is the case for wild-type cystinosin–EGFP.

 
Effect of mutations on localization
The mutations examined in this study are listed according to the clinical phenotype to which they are associated (Table 1). Twenty-seven of the 31 mutations showed a late endosomal/lysosomal localization (Table 2; Fig. 1I and J and data not shown). Three mutations, Q222R, IVFD343–346del and DVVF346–349del, led to a partial expression of cystinosin at the plasma membrane (Fig. 1E and F and data not shown). Deletion of the GYDQL motif completely relocalized these mutant proteins to the plasma membrane (Fig. 1G and H and data not shown), in contrast to the wild-type protein and other mutants, which are partially relocalized by this deletion (Fig. 1C, D, K and L and data not shown). Finally, no fluorescence was observed upon transfection of cystinosin–EGFP harbouring the mutation M1I, suggesting that the fusion protein is not translated.


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  		Table 1. List of cystinosis mutations studied according to the associated phenotype
 

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  		Table 2. Effect of each mutation on the subcellular localization and cystine transport activity of cystinosin
 
Effect of mutations on cystine transport
None of the mutants tested transported cystine significantly at neutral pH (data not shown), therefore, only data obtained at acid pH are presented.

Infantile cystinosis.
Of the 19 mutations associated with typical cases of infantile cystinosis, 14 are missense mutations and five are in-frame deletions or insertions. Seventeen of these mutations are situated within (I133F, S141F, L158P, G169D, W182R, Q222R, S270del, D305Y, G308R, L338P, G339R, IVFD343–346del, DVVF346–349del and DVEF349–350ins) or immediately adjacent to (D205N, D205del and S298N) the TMs. Of the remaining two mutations, N288K was situated in the middle of the fifth inter-TM loop and M1I affected the methionine start codon.

Consistent with the subcellular localization data, no cystine transport was observed with M1I (Table 2 and Fig. 2), further suggesting the absence of an alternative initiation codon. Q222R, IVFD343–346del and DVVF346–349del, which partially altered the subcellular localization of cystinosin, abolished cystine transport. This was also true for S141F, L158P, G169D, S270del, D205N, D205del, N288K, D305Y, G308R, L338P, G339R and DVEF349–350ins, which did not affect the lysosomal localization of cystinosin. As cystinosin is still present at the lysosomal membrane in these cases, the lack of transport was attributed to an inability to transport cystine, rather than to a decreased cell surface expression of cystinosin–{Delta}GYDQL in our functional assay, as confirmed for the G308R mutation (17). In contrast, the three remaining mutations, I133F, S298N and W182R, retained transport activity. I133F (not a causative mutation according to recent data, see Discussion) and S298N did not significantly alter cystine transport (92±16 and 77±21% of wild-type cystinosin–{Delta}GYDQL activity, respectively), whereas W182R reduced the uptake to 34±5.9% of cystinosin–{Delta}GYDQL activity (Table 2 and Fig. 2).



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  		Figure 2. Effect of cystinosis-associated mutations on the transport activity of cystinosin. Cystine transport is expressed as a percentage of wild-type cystinosin–{Delta}GYDQL activity. Error bars correspond to the SEM (the number of determinations is given in Table 2). Diamonds, triangles and the circle represent the mutations associated with infantile, juvenile and ocular cystinosis, respectively. Squares represent the four mutations associated with atypical forms of the disease. Three mutations that could not be classified unambiguously to a clinical phenotype are omitted from the graph. Note that the mutations S298N and W182R allow a significant level of cystine transport that contrasts with the severe phenotype. This is also the case for I133F, indicated in italics because recent data suggest that this mutation is in fact not causative (see Discussion). Conversely, the mutations N323K and K280R abolish cystine transport, despite their association to a milder juvenile phenotype. The same holds true for S139F, which is associated with an atypical form that is less severe than the infantile, but more severe than the juvenile phenotype.

 
Juvenile cystinosis.
Of the four mutations associated with juvenile cystinosis, three are missense mutations situated immediately adjacent to the fifth TM (K280R) or within the inter-TM loops oriented towards the lysosomal lumen (P200L and N323K), and one is an insertion of three amino acids in the first inter-TM loop (PCS154–155ins). P200L and PCS154–155ins led to a low level of cystine transport (15±4.5 and 9.2±2.1% of cystinosin–{Delta}GYDQL activity, respectively) (Table 2 and Fig. 2). However, K280R and N323K abolished cystine transport, in contrast to the associated juvenile phenotype. To examine whether these mutations induced a low level of activity undetected in our assay, the cystinosin constructs were expressed for 72 h (as compared with 48 h in the standard protocol) and cystine uptake time was extended to 20 min (compared with 10 min). These conditions significantly improved the signal-to-noise ratio of the wild-type cystinosin–{Delta}GYDQL leading to a mean transport activity of 1200±73% above background (n=6), compared with 640±50% in the standard protocol (17). However, we did not detect cystine transport by these juvenile cystinosis-associated mutants. We also examined whether the lack of transport activity resulted from a lack of cell surface expression of the cystinosin–{Delta}GYDQL constructs used in the transport assay. For this, constructs bearing K280R and N323K in addition to the GYDQL deletion and EGFP tag were expressed in HeLa and MDCK cells (Fig. 1I–L and data not shown). However, these recombinant proteins, which also did not lead to cystine uptake (data not shown), partially localized to the plasma membrane, as does wild-type cystinosin–{Delta}GYDQL–EGFP (16). Thus, despite the juvenile phenotype associated with these mutations, K280R and N323K apparently abolish the transport activity of cystinosin in our assay.

Ocular cystinosis.
The missense mutation G197R, which is associated with several cases of ocular cystinosis and situated in the second inter-TM loop, led to a low level of cystine transport: 20±7.8% of wild-type cystinosin–{Delta}GYDQL activity (Table 2 and Fig. 2).

Atypical cystinosis.
Four missense mutations associated with a combination of symptoms that could not be classified as either infantile or juvenile cystinosis, were also studied (Table 2 and Fig. 2). G110V (6) and V42I (5), localized in the N-terminal region, did not significantly alter cystine transport activity (120±27 and 97±4.1% of cystinosin–{Delta}GYDQL activity, respectively), whereas D346N (5), situated in the seventh TM, moderately altered this activity (61±11%). In contrast, S139F (5) abolished cystine transport and, as observed for the juvenile K280R and N323K mutations, increased times of protein expression and cystine uptake did not give significantly different results. Furthermore, cystinosin–S139F–{Delta}GYDQL–EGFP partially localized to the plasma membrane (and did not induce cystine uptake), demonstrating that the absence of activity was not due to the lack of surface expression. We thus conclude that S139F impairs the transport capacity of cystinosin, whereas G110V, V42I and D346N do not, or only partially, alter this capacity.

Unclassified mutations.
Three mutations that could not be classified into any group with certainty were also studied (Table 2). The missense mutations N177T and I260T are situated in the second TM and fourth inter-TM loop, respectively, and the seven amino-acid deletion ITILELP67–73del in the N-terminal region. N177T abolished cystine transport, I260T moderately (74±13%) altered this activity and ITILELP67–73del reduced cystine transport to 19±6.1% of cystinosin–{Delta}GYDQL activity.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
MATERIALS AND METHODS
 REFERENCES
 
The development of a functional assay for cystinosin (17) provides a tool for analysing the pathogenesis of cystinosis at the molecular level. For this, we studied 31 mutations associated with the different clinical forms of cystinosis for their effect on transport activity and subcellular localization.

Overall, for the three defined forms of cystinosis (infantile, juvenile and ocular), the transport data tend to correlate with the clinical data: of the 18 infantile mutations (excluding M1I which prevents translation), 15 abolish cystine transport. Conversely, three of the five mutations associated with juvenile or ocular forms display ~9–20% of wild-type activity. Therefore, the hypothesis that infantile cystinosis is caused by two loss-of-function alleles, whereas patients with milder forms are homozygous or compound heterozygotes for a specific, partially active allele (5,11,12), holds true in most cases. However, there are some exceptions. Six mutations retain a full or substantial (>30% of wild-type) level of activity, despite their association to a severe phenotype (infantile and atypical). Conversely, for two other mutations associated with juvenile cystinosis, no transport activity could be detected. The lack of transport in the S139F mutant is also surprising because the carrier, reported as atypical due to a late onset of renal Fanconi syndrome at 3 years, only presents with moderate chronic renal insufficiency at 22 years of age (5). These discrepancies will be discussed later.

With regards to subcellular localization, only three mutations, Q222R, IVFD343–346del and DVVF346–349del, which are associated with infantile cystinosis, partially relocalize cystinosin to the plasma membrane. We have previously shown that the sorting of cystinosin to lysosomes requires two sorting signals: a classical tyrosine-based motif (GYDQL) in the C-terminal tail and a conformational signal delineated to a region of the fifth inter-TM loop (16). Mutation of either signal results in partial redirection to the plasma membrane, whereas a complete relocalization is achieved by mutating both signals. Although deletions in the seventh TM could have prevented exposure of the GYDQL motif to the cytosolic compartment, this motif is recognized by the cytosolic sorting machinery in the IVFD343–346del and DVVF346–349del mutants. This is demonstrated by the fact that deletion of the GYDQL motif results in the complete relocalization of these mutants to the plasma membrane. Thus, IVFD343–346del and DVVF346–349del probably interfere with the second sorting signal. The same observations were made with Q222R, which is in the third TM. Taken together, these data suggest that the second sorting signal is contained within the overall conformation of cystinosin, rather than restricted to the linear sequence in the fifth inter-TM loop (16). An attractive, though speculative, mechanistic possibility for this second signal could be that cystinosin dynamically interacts with another lysosomal protein that possesses a sorting motif. Such an interaction would drive cystinosin partially to the lysosome in the absence of the GYDQL motif.

Predictive value of the transport assay
Before considering the discrepancies between biochemical and clinical data, it is important to assess the level of confidence we can place in our functional assay. First, transport data are in good agreement with topological and phylogenetic information on cystinosin: as observed for many other membrane proteins, transport activity is less tolerant of amino acid substitutions when they affect conserved residues or residues located in TMs. Among 23 substitutions analysed (excluding M1I), 14 abolish transport, and all but one (N323) of these essential residues are strictly conserved across eukaryotic species (Fig. 3). With respect to the position on the topological model, 12 of the 14 deleterious substitutions are located within or at the border of the TMs; the remaining two, N288K and N323K, are located in the last cytosolic and lumenal loops, respectively (Fig. 4).



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  		Figure 3. Amino acid sequence alignment of eukaryotic cystinosin homologues. Orthologous amino acid sequences from Homo sapiens (SWISSPROT accession no. O60931), Mus musculus (accession no. P57757), Drosophila melanogaster (accession no. Q9VCR7), Caenorhabditis elegans (accession no. Q09500), Saccharomyces cerevisiae (accession no. P17261) and Arabidopsis thaliana (accession no. P57758) were aligned using the CLUSTAL W software (21). Black shading indicates amino-acid identities and grey indicates similarities. The position of missense mutations that abolish cystine transport are indicated by filled circles, and those that allow partial or total cystine transport are indicated by open circles. Overlining indicates the positions of the TMs.

 


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  		Figure 4. Position of each class of transport-altering mutations on the topological model of cystinosin. Mutations are categorized into three classes according to whether they abolish (red symbols), severely inhibit (<30% of wild-type; blue symbols) or only moderately, or do not, alter (≥30% of wild-type; green symbols) cystine transport. Stars represent point mutations, dashes correspond to deletions of 1 amino acid, and triangles indicate insertions of three (position 154) or four (position 349) amino acids. Black circles represent the predicted N-glycosylation sites. Circles with a light blue centre represent the lysosomal sorting signals: the GYDQL motif in the C-terminal tail and the critical residuals of the conformational signal in the fifth inter-TM. Numbers correspond to the position of predicted N-glycosylation sites and of the extremities of TMs.

 
Second, from a more quantitative perspective, the value of 20% of wild-type transport activity obtained for the ocular G197R mutation is probably relevant, as it is consistent with previous biochemical data on cystinotic lysosomes. Indeed, cystine transport is reduced 2-fold in lysosomes from leukocytes of individuals heterozygous for an infantile mutation (22), implying that a residual activity of 50% should be clinically silent. Moreover, similar measurements made on lysosomes from patients with ocular cystinosis yielded a cystine transport corresponding to 19±10% of wild-type, whereas values for those with infantile cystinosis were in the 0–5% range (23). Therefore, our assay provides a good estimate of the effect of mutations on transport. In the future, its sensitivity should be increased to allow precise comparison of mutants with low residual activities (e.g. G197R versus P200L and PCS154–155ins).

The predictive potential of our assay becomes evident when transport data for the three unclassified mutations ITILELP67–73del, N117T and I260T, the infantile mutation I133F and the atypical mutation G110V, are compared with clinical and genetic data. ITILELP67–73del has been described in homozygotes and compound heterozygotes with infantile cystinosis (10,14). However, we detected this mutation in the heterozygous state in two individuals with juvenile cystinosis: originally, in a case where the second mutated allele has not yet been identified (5) and, more recently, in another case, segregating with the 57 kb deletion (unpublished data). Our data showing that ITILELP67–73del leads to ~19% residual transport activity, as well as the fact that no other mutation associated with infantile cystinosis is located in the N-terminal region of cystinosin, further reinforce the conclusion that this mutation is associated with juvenile cystinosis. The confusion surrounding the clinical status of ITILELP67–73del illustrates the difficulties associated with classifying cystinosis patients into various clinical forms owing to heterogeneous criteria employed by clinicians world-wide.

N177T was also identified in an individual with juvenile cystinosis, and the second mutated allele has not yet been identified (6). The fact that N177T abolishes cystine transport suggests that it represents a severe mutation. This would be consistent with its position in the second TM, which houses two other infantile cystinosis-associated mutations that abolish cystine transport. Therefore, the second mutation segregating in this individual, which may lie in the regulatory regions of CTNS and hence so far escaped detection, is likely to be the mild mutation.

The last unclassified mutation, I260T, was identified in the homozygous state in conjunction with S141F in a patient with infantile cystinosis (unpublished data). S141F abolishes cystine transport, whereas I260T only moderately affects cystine transport (~74%). Subsequent to these results, I260T was reported as a polymorphism (refSNP ID: rs161400) in the public database of single nucleotide polymorphisms (http://www.ncbi.nlm.nih.gov/SNP). Therefore, only S141F contributes to the clinical phenotype of the patient.

I133F, identified in a French-Canadian population (18), has no effect on cystine transport, in apparent contradiction with its reported association to an infantile phenotype. However, recent data have revealed that I133F is not the causative mutation in these families (J. McGowan-Jordan, personal communication). This is consistent with the fact that we never detected this mutation in the French patients we have studied to date (108 patients), whereas we did detect the other missense mutation, L158P, identified in this same French-Canadian study (18).

Finally, the atypical mutation G110V was detected in the homozygous state in a patient who presented with early and severe features of the Fanconi syndrome and developed ESRD at 13 years. However, even in the absence of treatment, the only extra-renal disorders that she developed were mild ocular anomalies with photophobia occurring at 23 years. We show here that the G110V substitution does not affect cystine transport. However, this mutation involves the last nucleotide of exon 6 and hence doubles as a splice site mutation. Indeed, we have shown previously that it affects the splicing of exon 6 and leads to a frameshift (at amino acid 111) and a truncated protein (S124X) (6). Thus, our data demonstrate that it is not the amino acid substitution but the splicing defect that gives rise to the associated phenotype. These data support our hypothesis that inter-tissue variability in the expression of the splicing defect is responsible for the atypical character of cystinosis in this patient.

Discrepancies between molecular and clinical phenotypes
In contrast to the aforementioned mutations, seven of the mutations that we tested show significant discrepancies between transport and clinical data. There are two types of discrepancies: some mutations associated with a milder clinical phenotype abolish cystine transport, and others associated with a severe clinical phenotype have little effect on transport.

The first category consists of the atypical mutation S139F (5) and the juvenile mutations K280R and N323K (11). As mentioned earlier, we have delineated the cystine transport threshold for the appearance of clinical anomalies at 20% of wild-type. We cannot exclude the possibility that the lack of activity in these three mutants is only apparent and results from an insufficient sensitivity of our assay (e.g. transport activities ≤5% may be undetectable in our standard protocol). However, we doubled the sensitivity of our assay by increasing the duration of protein expression and cystine uptake, but did not detect residual activity in the mutants. In the case of the residues S139 and K280, the lack of transport in the mutants is consistent with the strict conservation of these amino acids during evolution (although K280R is a conservative change, an arginine is never observed in other eukaryotic sequences) (Fig. 3). Thus, an alternative possibility should be considered to explain this category of discrepancies. We speculate that, in vivo, cystinosin associates with another TM protein of the lysosome, and that this association stabilizes the conformation of the mutant S139F, K280R and N323K polypeptides. Such mutations would thus be less deleterious in the patient than in our in vitro assay, which is based on an ectopic expression of cystinosin at the plasma membrane. As mentioned earlier, a heteromeric association with another lysosomal protein would also explain the effect of other mutations on the intracellular localization of cystinosin.

The second category of discrepancies concerns the infantile mutations W182R and S298N (10), and the atypical ones V42I and D346N (5). Unlike G110V, these mutations do not disrupt consensus splice site sequences located at exon–intron junctions, and therefore are not thought to affect splicing. However, the possibility that they may alter or introduce exonic splicing elements [such as enhancers or silencers; (24)] cannot be excluded. Similarly, it cannot be ruled out that, as discussed earlier for I133F, one or more of these mutations are not, in fact, causative mutations, in particular W182R, S298N and D346N, which were detected in the compound heterozygous state. Conversely, it is conceivable that other functional aspects than intracellular localization or transport are affected by these mutations. For example, the mutated cystinosin might be degraded at a higher rate in vivo, but this effect would be negligible in vitro when the protein is transiently overexpressed in COS cells. Alternatively, interactions with other components of the lysosomal matrix or lumen important for the biological function of cystinosin might be disrupted by the mutations in vivo. In this perspective, it might be interesting to note that V42 is adjacent to an N-glycosylation site at position 41, although its substitution to isoleucine is not predicted to prevent recognition by oligosaccharyltransferase (25). These hypotheses serve as a reminder that a simplified in vitro model cannot recapitulate all the subtleties and complexities at work in an entire organism. A further investigation of these discordant mutants could thus provide deeper insights into the molecular or cellular mechanisms that modulate disease severity.

In conclusion, this study shows that when the CTNS mutation does not prevent the synthesis of a full polypeptide, impaired transport is the most frequent cause of pathogenicity. Furthermore, the level of transport inhibition often correlates with the severity of symptoms: transport is abolished by 15 of the 17 mutations associated with classical infantile cystinosis (excluding M1I and I133F). This transport assay thus has potential prognostic interest when novel mutations are identified. On the other hand, the rarer cases of discrepancies between transport and clinical data might unravel unsuspected aspects of the pathogenic cascade.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
REFERENCES
 
CTNS mutations studied
Mutations that do not induce gross alterations of the reading frame (missense mutations and in-frame deletions or insertions) were studied exclusively (Table 1). Of a total of 31 such mutations, 19 were associated with infantile cystinosis, four with juvenile cystinosis, one with ocular cystinosis and four with atypical forms of the disease. The remaining three mutations could not be unambiguously associated to a clinical phenotype. Firstly, the missense mutation N177T was found in two sisters with juvenile cystinosis, but the mutation on the second allele has not yet been detected (6). N177T could thus represent the severe allele of a compound heterozygote. Secondly, I260T was detected on the same allele as S141F, in the homozygous state, in an individual with infantile cystinosis (unpublished data), raising the possibility that it is not a causative mutation. Finally, the in-frame deletion ITILELP67–73del was initially observed in a patient with juvenile cystinosis (5), but the mutation on the second allele was not identified, and other groups reported the same mutation, in the homozygous or compound heterozygous state, in individuals with infantile cystinosis (10,14). Recently, this mutation was observed in a second patient with juvenile cystinosis, harbouring the 57 kb deletion on the second allele (unpublished data).

Generation of constructs containing CTNS mutations
The introduction of all CTNS mutations was performed using the QuikChange site-directed mutagenesis kit (Stratagene) and verified by sequencing the region around the introduced mutation (primer sequences available upon request). For the localization studies, CTNS mutations were introduced into the pEGFP-N1 expression plasmid (Clontech) encoding a wild-type cystinosin–EGFP fusion protein (16). For the cystine transport assay, CTNS mutations were introduced into the pcDNA3.1/Zeo+ expression plasmid (Invitrogen) encoding cystinosin deleted in the C-terminal lysosomal sorting motif GYDQL [cystinosin–{Delta}GYDQL; (17)]. Additional constructs (cystinosin–{Delta}GYDQL–EGFP bearing the mutations Q222R, IVFD343–346del, DVVF346–349del, K280R, N323K and S139F) were also generated by site-directed mutagenesis and assayed for their effect on subcellular localization and transport activity.

Cell culture and transfection
For the localization studies, HeLa and MDCK cells were grown in minimal essential medium (Life Technologies) supplemented with 10% foetal calf serum (FCS) and 2 mM L-glutamine. Approximately 2x105 cells were cultured in 35 mm wells containing glass coverslips and transfections (with wild-type or modified cystinosin–EGFP) were carried out by incubating the cells with 2 µg plasmid and 4 µl FuGENE 6 (Roche Molecular Biochemicals) for 48 h, according to the manufacturer's recommendations. For the cystine transport assay, COS-7 cells were grown under 5% CO2 in glucose-rich Dulbecco's modified Eagle medium containing Glutamax-I (Life Technologies) supplemented with 7.5% FCS. Approximately 1x106 cells were transfected with 5 µg plasmid (wild-type or modified cystinosin–{Delta}GYDQL or, as a negative control, with pcDNA3.1/Zeo+) by electroporation as previously described (17). Electroporated cells were diluted into 2 ml of culture medium, divided into four aliquots and cultured in a 24-well plate.

Immunofluorescence studies
Transfected HeLa cells were washed twice with phosphate-buffered saline (PBS) on ice, and incubated with a 1 : 50 dilution [in 0.1% bovine serum albumin (BSA)/PBS] of the primary antibody EMA, directed against epithelial membrane antigen (DAKO), for 30 min at 4°C. Coverslips were then fixed with 4% paraformaldehyde for 20 min, incubated with 50 mM NH4Cl for 10 min and blocked using 1% BSA/10% donkey serum/PBS for 1 h at room temperature. Transfected MDCK cells were fixed with 4% paraformaldehyde for 20 min, incubated with 50 mM NH4Cl for 10 min and permeabilized using 0.1% Triton X-100 for 10 min. Coverslips were blocked in 1% BSA/0.1% saponin/10% donkey serum/PBS, and incubated with a 1 : 500 dilution (in 1% BSA/0.1% saponin/PBS) of the primary antibody AC17, directed against canine LAMP-2 (kindly provided by A. LeBivic, Marseille, France), for 1 h at room temperature. Finally, all coverslips were incubated using a 1 : 400 dilution (in 1% BSA/PBS) of donkey anti-mouse-Cy3 secondary antibody (Immunotech) for 1 h at room temperature, and mounted using Fluoroprep (bioMérieux). Fluorescence was visualized using a ZEISS LSM 510 laser scan microscope (Carl Zeiss) equipped with a plan apochromat 63.2x oil immersion objective and with a visible argon laser (488 nm) and two helium neon lasers (543 and 633 nm). Images were collected sequentially and processed using Adobe Photoshop 5.5.

Cystine uptake assay
Unless otherwise indicated, cystine uptake was assayed 48 h after transfection as previously described (17). Briefly, culture wells were washed twice with 500 µl uptake buffer A (5 mM D-glucose, 140 mM NaCl, 1 mM MgSO4, 20 mM potassium phosphate pH 7.4) and then incubated with 200 µl buffer B (identical to buffer A but with potassium phosphate adjusted to pH 5.6) supplemented with 40 µM or 1 µCi [35S]L-cystine (50–200 mCi/mmol; Amersham) for 10 min, unless otherwise stated. The cells were rinsed twice with 500 µl of chilled buffer A, lysed with 200 µl of 0.1 N NaOH and the accumulated radioactivity was counted by liquid scintillation in Emulsifier-Safe cocktail (Packard) using a Tri-Carb 2100 TR liquid scintillation analyser (Packard). Transport measurements were performed in triplicate and expressed as percentage of wild-type (cystinosin–{Delta}GYDQL) transport after subtracting the background uptake observed in mock-transfected cells. For each CTNS mutation, mean values±standard error of the mean (SEM) were derived from at least three independent transfections.


   ACKNOWLEDGEMENTS
 
This work was supported by Association Française contre les Myopathies, Vaincre les Maladies Lysosomales, Association pour le Développement du Rein Artificiel, Association pour l'Information et la Recherche sur les Maladies Génétiques Rénales and Fondation pour la Recherche Médicale (PhD grant to S.C.).


   FOOTNOTES
 
* To whom correspondence should be addressed at: Institut de Génétique Moléculaire de Montpellier, CNRS UMR 5535, 1919 Route de Mende, 34293 Montpellier, France. Tel: +33 467613674; Fax: +33 467040231; Email: kalatzis{at}igm.cnrs-mop.fr

{dagger} These authors contributed equally to the work. Back


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
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