Severity of phenotype in cystinosis varies with mutations in the CTNS gene: predicted effect on the model of cystinosin

doi:10.1093/hmg/8.13.2507

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Severity of phenotype in cystinosis varies with mutations in the CTNS gene: predicted effect on the model of cystinosin

Human Molecular Genetics Pages 2507-2514 ©1999 Oxford University Press

Severity of phenotype in cystinosis varies with mutations in the CTNS gene: predicted effect on the model of cystinosin
Introduction
Results
   Patients
   Mutation screening
   Haplotype analysis
   Expression of mRNA
   Predicted structure of cystinosin and the effect of mutations
Discussion
Materials And Methods
   Clinical material and mutation detection
   Haplotype analysis
   RT-PCR
Acknowledgements
References

Severity of phenotype in cystinosis varies with mutations in the CTNS gene: predicted effect on the model of cystinosin

Marlene Attard⁺, Geneviève Jean¹^{, +}, Lionel Forestier¹, Stéphanie Cherqui¹, William van't Hoff, Michel Broyer², Corinne Antignac¹, Margaret Town^§

Nephrourology Unit, Institute of Child Health, University College London Medical School, 30 Guilford Street, London WC1N 1EH, UK, ¹INSERM U423, Tour Lavoisier, Hôpital Necker, Université René Descartes and ²Service de Néphrologie Pédiatrique, Hôpital Necker, 149 rue de Sèvres, 75015 Paris, France

Received August 2, 1999; Revised and Accepted September 22, 1999

Infantile nephropathic cystinosis is a rare, autosomal recessive disease caused by a defect in the transport of cystine across the lysosomal membrane and characterized by early onset of renal proximal tubular dysfunction. Late-onset cystinosis, a rarer form of the disorder, is characterized by onset of symptoms between 12 and 15 years of age. We previously characterized the cystinosis gene, CTNS, and identified pathogenic mutations in patients with infantile nephropathic cystinosis, including a common, ~65 kb deletion which encompasses exons 1-10. Structure predictions suggested that the gene product, cystinosin, is a novel integral lysosomal membrane protein. We now examine the predicted effect of mutations on this model of cystinosin. In this study, we screened patients with infantile nephropathic cystinosis, those with late-onset cystinosis and patients whose phenotype does not fit the classical definitions. We found 23 different mutations in CTNS; 14 are novel mutations. Out of 25 patients with infantile nephropathic cystinosis, 12 have two severely truncating mutations, which is consistent with a loss of functional protein, and 13 have missense or in-frame deletions, which would result in disruption of transmembrane domains and loss of protein function. Mutations found in two late-onset patients affect functionally unimportant regions of cystinosin, which accounts for their milder phenotype. For three patients, the age of onset of cystinosis was <7 years but the course of the disease was milder than the infantile nephropathic form. This suggests that the missense mutations found in these individuals allow production of functional protein and may also indicate regions of cystinosin which are not functionally important.

INTRODUCTION

Infantile nephropathic cystinosis (MIM 219800; Online Mendelian Inheritance in Man, http://www.ncbi.nlm.nih.gov/Omim ) is a rare, autosomal recessive disease caused by a defect in the transport of cystine across the lysosomal membrane resulting in intracellular cystine accumulation (1). Patients present in the first year of life with features of renal proximal tubular dysfunction (the renal Fanconi syndrome). Hence, this form of the disease is referred to as early-onset cystinosis. Without treatment, affected children suffer worsening growth retardation and develop end-stage renal failure by ~10 years (1). Late-onset (juvenile or adolescent type) cystinosis (MIM 219900) is a much rarer form of the disorder with an onset of symptoms usually between 12 and 15 years (1,2). These patients usually present with proteinuria and glomerular renal impairment but do not suffer from such profound tubular dysfunction nor growth retardation. Leukocyte cystine concentrations in untreated patients are generallyhigher in infantile nephropathic cystinosis than in late-onset cystinosis (2,3). However, characterization is not always clear cut and there are patients whose phenotype does not fit the classical description of either form. Complementation studies (4) and linkage analysis (5) have demonstrated that infantile and late-onset cystinosis are allelic, suggesting that phenotype would be determined predominantly by different mutations.

We previously identified the cystinosis gene, CTNS, which consists of 12 exons with the first methionine in exon 3. The gene is located on chromosome 17p and spans 23 kb of genomic DNA. CTNS encodes a protein of 367 amino acids which is predicted to be an integral lysosomal membrane protein with seven transmembrane domains. We demonstrated that the commonest mutation in cystinosis patients is an ~65 kb deletion, encompassing the 5[prime] end of the gene (6). On the basis of sequencing and PCR analysis, patients with this deletion have an identical 5[prime] breakpoint upstream of the CTNS gene and have a 3[prime] breakpoint in exon 10 of the gene (7,8). Individuals who are homozygous for this deletion are easily detected by PCR analysis and, to date, we have found that all such patients have classical infantile nephropathic cystinosis, with early onset of symptoms and severe disease. In a previous study, we screened cystinotic patients for the presence of mutations in seven of the 10 coding exons. We reported the full CTNS genotype (i.e. mutations on both alleles) for 38 individuals with nephropathic cystinosis (6). In addition to the ~65 kb deletion, we found 11 other truncating mutations. We have now screened the entire coding region of CTNS for mutations in 37 patients, including individuals with infantile nephropathic cystinosis, those with the rarer subtype of late-onset cystinosis and three patients whose phenotypes do not fit the classical definitions of cystinosis.

RESULTS

Patients

We have included in this study 12 patients in whom we had previously detected only one mutation; 11 of these are heterozygous for the ~65 kb CTNS deletion and one patient (P18) is hemizygous for a nonsense mutation (6). We have also included 18 patients in whom we had not detected any mutations (6) and seven new cystinosis patients. From this cohort of 37 patients, 32 presented with features of the renal Fanconi syndrome in the first year of life and are classified as having infantile nephropathic cystinosis. Two patients (L36 and L47) were classified as having late-onset cystinosis; both had been asymptomatic and had grown well but on routine examination were found to have proteinuria and chronic renal impairment at the ages of 12.5 and 14.5 years, respectively. Three unrelated patients have phenotypes which are not typical of either early- or late-onset cystinosis. Patient L18, who presented at 3 years of age with rickets, and patient P41, who presented at 6 years of age with vomiting, were found to have a renal Fanconi syndrome but did not receive cysteamine. In contrast to patients with classical infantile nephropathic cystinosis, who without specific treatment develop end-stage renal failure at ~10 years (1), patient L18 had plasma creatinine concentrations within the normal range for age at 18 years and patient P41 had a creatinine clearance of 70 ml/min/1.73 m² (normal >80 ml/min/1.73 m²) at age 22 years. Patient L16 developed symptoms at 19 months of age, did not have a full Fanconi syndrome and was able to tolerate only very small doses of cysteamine. He developed end-stage renal failure at the age of 16 years.

Mutation screening

The seven new cystinosis patients were screened by PCR in order to detect the presence of the ~65 kb deletion. None of these individuals was homozygous, but three were found to be heterozygous for the deletion. DNA samples from 37 patients were analysed by single strand conformation polymorphism (SSCP) and direct sequencing in order to detect mutations in the CTNS gene. Where possible, DNA from parents and other family members was analysed, and in every case the mutation was found to segregate with the disease. For missense mutations, a panel of at least 50, ethnically matched, unaffected individuals was screened. No mutations were detected on control chromosomes. Of the 14 patients shown to be heterozygous for the ~65 kb deletion, we detected a second mutation in all except four individuals. Of the remaining 23 patients, mutations were detected on both alleles for 19 individuals, a single mutation was found in one individual and in three patients no mutations were detected using this screening strategy. Tables 1 and 2 show a total of 24 different mutations found in 34 unrelated cystinosis patients. In addition to the ~65 kb deletion and the premature stop previously found in patient P18, we found eight different truncating and seven missense mutations, four deletions and four altered splice sites. Six of the mutations recur in more than one family in this study, and eight mutations have been reported previously (9). Table 3 summarizes the 16 recurrent mutations that have been observed in cystinotic patients to date. Although cystinosis is a recessive disorder, we have recorded numbers of patients rather than individual chromosomes or alleles since, in these families, homozygosity frequently is due to consanguinity, and this is not always admitted. Therefore, Table 3 indicates which mutations are recurrent; however, the numbers are likely to be an underestimate of the actual frequency of the mutations in cystinosis patients. Throughout this study, we have used the recommended nomenclature system for human gene mutations (10), with the exception of nucleotide numbering, which is the same as previously described (6). The dinucleotide insertion (CG) at position 1036, detected in two patients (L1 and L28), and the deletion (ACG) at position 953, detected in one patient (P8), occur in short tandem repeats and we have denoted these mutations at the most 3[prime] nucleotide interval: 1036-1038insCG and 953-955delACG. Therefore, these mutations are likely to be identical to the previously reported 1033insCG and 950delACG, respectively (9).

Table 2. Mutation analysis in patients with late-onset or non-classical cystinosis

Patient ID Nucleotide change Amino acid change Type of mutation Consequence of mutation

Late-onset L47 537-557del21^a ITILELP67-73del In-frame deletion 7 amino acids deleted from N-terminal domain

Not found ? ? ?

L36 801-10C->G PCS154-155ins In-frame insertion 3 amino acids added to cytosolic domain

801-10C->G PCS154-155ins In-frame insertion 3 amino acids added to cytosolic domain

Non-classical L18 463G->A V42I Missense Amino acid change in N-terminal domain

463G->A V42I Missense Amino acid change in N-terminal domain

P41 755C->T S139F Missense Amino acid change in TM1

985-986insA^a Frameshift at 216, E227X Out-of-frame insertion Truncated 227 residue protein

L16 1080delC^a Frameshift at 247, M252X Out-of-frame deletion Truncated 252 residue protein

1375G->A D346N Missense Amino acid change in TM7

^aPreviously reported by Shotelersuk et al. (9).

Table 3. Recurrent mutations

Mutation No. of patients

Town et al. (6) This study Shotelersuk et al. (9) Total

Del exons 1-10 37 3 48 88

357-360delGACT 4 0 5 9

399-400delTG 2 0 0 2

537-557del21 0 1 3 4

622G->T 2 0 0 2

721C->T 1 0 1 2

753G->A 2 0 14 16

900+1delG 1 0 1 2

953-955delACG 0 1 1 2

985-986insA 0 1 1 2

1080delC 0 1 1 2

1035-1036insC 0 6 2 8

1036-1037insCG 0 2 1 3

1261G->A 0 4 2 6

1310-12G->A 0 2 0 2

1354G->A 0 5 1 6

Fourteen of the mutations reported here are novel. Ten of these were found in patients with infantile nephropathic cystinosis, and four occur in patients with late-onset cystinosis or with a non-classical disease phenotype.

Haplotype analysis

Wherever possible (22 cases), we typed DNA from patients and their families with the microsatellite loci, D17S1828 and D17S2167 (which flank the cystinosis gene) and D17S829 (an intragenic marker), in order to ascertain whether any of the CTNS mutations have occurred on a common haplotype. We have shown previously that a common haplotype is associated with the ~65 kb deletion (8). We observed 23 different haplotypes on 34 control chromosomes, and 25 different haplotypes on 38 cystinotic chromosomes that carry a mutation (other than the ~65 kb deletion) in CTNS. No significant differences were observed between the two groups and no common haplotypes were found for unrelated individuals with identical mutations.

Expression of mRNA

Samples (whole blood) suitable for RNA extraction were available from four of the patients included in this study and from eight patients previously shown to be homozygous for the ~65 kb CTNS deletion (6). RNA was reverse transcribed using a primer (CTRT1) derived from the 3[prime]-untranslated region (3[prime]-UTR) of CTNS. Using primers located within exon 11 (F12) and the 3[prime]-UTR of CTNS cDNA (R12), a fragment of CTNS, spanning exon 12 and extending 63 bases into the 3[prime]-UTR, was amplified from the reverse transcription product by PCR. In contrast to normal controls, no PCR products were obtained with RNA from patients who are homozygous for the ~65 kb CTNS deletion. Figure 1A shows the results observed with three of the homozygously deleted patients and with four patients who have other mutations in CTNS. Patients L24 (homozygous for a 3 bp deletion in exon 10), L7 and L27 (both heterozygous for the ~65 kb deletion and for a stop mutation in exon 10), and L36 (homozygous for a mutation predicted to create a new intron 7 3[prime] splice site) all produce an amplification product of the expected size, indicating that the gene is transcribed. For patient L36, the PCR was repeated using primers located within exons 7 (F79) and 9 (R79), and an amplification product of the expected size was detected (Fig. 1B), indicating that the splice site mutation, 801-10C->G, does not result in splicing out of exons 8 or 9. Sequence analysis across the junction of exons 7 and 8 (Fig. 1C) shows the addition of nine bases in the cDNA, CCCCTGCAG, which match the last nine bases of intron 7 (6).

Figure 1. Expression of CTNS mRNA. Patient RNA was reverse transcribed using a primer (CTRT1) derived from the 3[prime]-UTR of CTNS. The presence of CTNS mRNA in the patient sample was indicated by PCR amplification of fragments of the CTNS gene. (A) Amplification of CTNS exon 11 (3[prime] end), exon 12 and part of the 3[prime]-UTR. An amplification product (267 bp PCR fragment) is seen in: lanes 1 and 2, normal individuals; lane 7, patient L24 who is homozygous for an in-frame deletion in exon 10; lanes 8 and 9, patients L7 and L27 who are both heterozygous for the ~65 kb deletion and a stop mutation in exon 10; lane 10, patient L36 who is homozygous for a new exon 7-exon 8 splice site. Lanes 3-5, no amplification product was obtained with patients L3, L8 and L39, homozygous for the ~65 kb deletion; lane 6, standard 1 kb DNA marker. (B) Amplification of CTNS exons 7 (3[prime] end), 8 and 9 (5[prime] end). A PCR fragment (~273 bp) is seen for: lanes 1 and 2, controls; lane 6, patient L36, indicating that the new splice site does not result in splicing out of these exons. Lanes 3 and 4, patients who are homozygous for the ~65 kb deletion and do not give an amplification product; lane 5, standard 1 kb DNA marker. (C) Sequence analysis across the exon 7-exon 8 boundary shows that patient L36 has an in-frame (9 bp) insertion at this junction. The additional bases are indicated by a horizontal line above the sequence and match exactly the 3[prime]-terminal sequence of intron 7.

Predicted structure of cystinosin and the effect of mutations

A hypothetical structure for the protein, cystinosin, and its orientation across the lysosomal membrane is given in Figure 2. This representation is based on previous modelling with hydrophobicity algorithms, comparative analysis with the transmembrane proteins of yeast and Caenorhabditis elegans and observation of sequence motifs (6). Our final model shows a transmembrane protein with an uncleavable signal peptide, a potentially glycosylated lysosomal N-terminus and three other small lysosomal domains. There are four short cytosolic domains including the C-terminus which carries the lysosomal targeting motif (11). This orientation is consistent with the higher positive charge density (24%) on the cytosolic side compared with the lysosomal side (8%) of the membrane. The majority of cysteine residues are intramembranous with a single cysteine on a lysosomal domain and none on cytoplasmic domains so that no disulfide bonds can form on either side of the membrane.

Figure 2. The location of mutations on the predicted cystinosin protein structure. Amino acid residues are represented by open circles. The lysosomal membrane is represented by the two solid horizontal lines. The N-terminus and first 128 amino acids of cystinosin lie within the lysosomal lumen. The C-terminus and final 10 amino acids are cytosolic. An uncleavable signal peptide is shown by bold circles. The lysosomal targeting motif is located at amino acids 362-366, denoted by GYDQL. N-glycosylation sites, cysteine residues and positively charged residues are denoted by N, C and +, respectively. Amino acids that are mutated are indicated by their number in the protein sequence and are shown as closed (missense) or hatched (insertion) circles or by a horizontal bar (deletion). The missense mutations D305Y, G308R, L338P and G339R were found in patients with infantile nephropathic cystinosis. Three in-frame deletions are also associated with this severe form of cystinosis: D205del, S270del and IVFD343-346del. The mutations ITILELP67-73del and PCS154-155ins are associated with late-onset cystinosis, and missense mutations V42I, S139F and D346N are associated with non-classical phenotypes. Mutations ITILELP67-73del, D205del, G308R and G339R were reported previously by Shotelersuk et al. (9).

We detected mutations on two alleles in 25 of the patients with infantile nephropathic cystinosis, shown in Table 1. For 12 of these patients, both mutations are predicted to result in the absence of mRNA or in a severely truncated protein. These mutations include the ~65 kb deletion, frameshifts resulting in premature stop codons and splice site mutations. This includes patient P5 who is homozygous for a single base deletion which occurs near the end of the coding sequence. This mutation results in a frameshift such that substitution of amino acids from residue 331 onwards occurs. The change in the amino acid sequence in this region would disrupt the final transmembrane domain and destroy the lysosomal targeting motif. We also found six individuals with infantile nephropathic cystinosis who are heterozygous for a truncating mutation on one allele and a missense or in-frame deletion on the other allele. A further six patients with infantile nephropathic cystinosis are homozygous (as a result of consanguinity) and one is heterozygous (P23) for missense mutations and in-frame deletions. The missense mutations, at nucleotides 1252, 1261, 1352 and 1354, are predicted to cause non-conservative amino acid substitutions at residues 305, 308, 338 and 339, respectively. All are located in putative transmembrane domains, TM6 and TM7, and all occur at residues which are conserved between cystinosin and the transmembrane proteins of yeast and C.elegans (6). The 12 bp deletion in exon 12 (patient P23) would remove four amino acids (residues 343-346) within TM7. The 3 bp deletion in exon 9 (patient P46) and the 3 bp deletion in exon 10 (patient L24) are predicted to result in the loss of Asp205 adjacent to TM3 and the loss of Ser270 within TM5, respectively.

The genotypes for two patients with late-onset cystinosis are shown in Table 2. One patient (L47) has an in-frame deletion of 21 nucleotides which results in the loss of amino acids 67-73. The second individual (L36) is homozygous for a C->G transition in intron 7 which we have shown to create a new splice site, resulting in the addition of nine bases to the cDNA between exons 7 and 8. This mutation would be predicted to add three amino acids, proline-cysteine-serine, to the first cytosolic domain (Fig. 2) of cystinosin, immediately adjacent to TM2. Of three patients with a non-classical cystinotic phenotype (Table 2), two individuals, P41 and L16, are both heterozygous for a truncating mutation and a missense mutation at amino acids 139 and 346, and the third, L18, is homozygous for a missense mutation at amino acid 42.

DISCUSSION

In a previous study (6), we identified two major deletions of the cystinosis region and 11 other different mutations which result in frameshifts with downstream stop codons or in the abolition of a splice site. Shotelersuk et al. (9) identified 18 new mutations including four missense mutations and two in-frame deletions. In this study, we have identified 23 different mutations. One of these mutations, the major ~65 kb deletion, was identified in our previous study. Eight of these mutations were identified previously by Shotelersuk et al. (9). Fourteen of the mutations presented in this study are new.

The frequency of individual mutations observed in cystinosis patients is in agreement with the observations of Shotelersuk et al. (9). We have previously confirmed that the ~65 kb deletion seen in 76% of European patients is the result of a founder effect (8). The 753G->A transition, highlighted by the USA group (9) and observed in a total of 16 families, may also be due to a single, ancient mutation, but this will require confirmation by haplotype analysis. In addition, 14 other mutations have been observed in more than one family. The most frequent are the insertion of C between nucleotides 1035 and 1036, seen in eight patients, and the deletion of GACT observed in nine patients. These two mutations and eight other mutations all occur in regions of repeated nucleotides or in a CpG pair, and on a background of different haplotypes, suggesting that they are recurrent mutations due to slipped strand mispairing or methylation-mediated deamination, rather than a founder effect. The failure to detect mutations on 11 chromosomes is likely to be due to either insensitivity of the SSCP analysis or the presence of mutations in regulatory sequences.

Our predicted model of cystinosin suggests a specific orientation across the lysosomal membrane. The major lysosomal membrane proteins, such as the Lamps and Limps (11), have a similar orientation to cystinosin but these proteins tend to be more heavily glycosylated and have only one, two or four transmembrane domains. Lamp proteins also carry the lysosomal targeting motif at the cytosolic C-terminus. Cystinosin is similar in appearance to the models for other organelle membrane proteins. In particular, the G-protein-coupled, or adrenergic, receptors of endosomes, which have seven transmembrane domains, an extracellular N-terminus with one or more potential N-glycosylation sites and a cytoplasmic C-terminus (12). Also the integral membrane protein CLN3, which is mutated in Batten disease, has six transmembrane domains with short cytosolic and intraorganelle domains (13). It has been suggested that the seven membrane-spanning [alpha]-helices of the adrenergic receptors may form a `pocket' to accommodate binding of specific ligands (12). Although there is no significant homology between cystinosin and these receptors, the structural similarity may reflect the function of cystinosin as a membrane transporter or as part of a complex that binds cystine. In this study, we consider the effect and position of mutations in the CTNS gene on protein structure and compare these with the phenotype of the patients.

Cystinosis has been described as having three forms: infantile (early onset) nephropathic (MIM 219800), late onset (MIM 219900) and benign (MIM 219750). In this study, 12 out of 25 individuals with infantile nephropathic cystinosis have truncating mutations on both alleles which are likely to result in a complete loss of protein. This is consistent with our previous results (6) and the work of Shotelersuk et al. (9) who reported a spectrum of mutations in patients with infantile nephropathic cystinosis. Although the function of cystinosin has yet to be defined, these mutations in the CTNS gene are consistent with the very severe phenotype of the disease and with the observations that patients have completely defective lysosomal cystine transport whereas obligate heterozygotes exhibit 50% of normal transport activity.

In this study, and previously (6,9), all patients found to have two truncating mutations have infantile nephropathic cystinosis. However, among patients with this severe form of the disease, we found four different missense mutations, two of which (G308R and G339R) had been reported previously in patients with infantile nephropathic cystinosis (9). In this study, five individuals (P16, L2, L29, P2 and P28) are all homozygous for these missense mutations. Five patients are heterozygous for one of these missense mutations, with a truncating mutation on the other allele, either the 65 kb deletion (P34, P42 and P15), or a frameshift with a premature termination (L28) or a disrupted splice site (P19). Although these are missense mutations, they each result in substitution of an amino acid that is conserved between cystinosin and the transmembrane proteins of yeast and C.elegans (6). These substitutions occur in putative transmembrane domains, and are likely to cause severe structural disruption to the protein. Since this protein is involved in cystine transport across the lysosomal membrane, then disruption to its orientation or localization would be predicted severely to reduce or ablate the function of cystinosin. For each of the 10 cases mentioned above, the genotype is consistent with loss of protein function and, therefore, is consistent with the severe, infantile nephropathic phenotype of these patients. Similarly, the in-frame deletions described for patients P23, L24 and P46 all occur at residues that are conserved between the three species. The deletion of D205 (P46) would result in the loss of a highly conserved residue and an acidic charge at a point where the protein is predicted to enter the membrane. Interestingly, this deletion was described in a patient (case 13) from the American-based cohort (9) who is homozygous for the mutation and also has early-onset cystinosis, but with some less severe features. Patient P46 is heterozygous for this mutation and for the ~65 kb deletion, which would account for the more severe phenotype. Patient P23 is heterozygous for an amino acid substitution (G308R) and a deletion (IVFD343-346del). G308R, as discussed above, is predicted to disrupt a transmembrane domain, and the loss of I-V-F-D from within a transmembrane domain (TM7) would also be expected to completely disrupt the [alpha]-helix. Patient L24 is homozygous for a deletion (S270del); the loss of the hydroxyl group on this conserved serine residue might block interactions within the membrane. As postulated for the missense mutations that occur in transmembrane domains, disruption, disorientation or mis-localization of a transmembrane protein is consistent with a loss of function and with the severe, infantile nephropathic phenotype of these patients.

In contrast, the phenotype of patients L36 and L47 is typical of late-onset cystinosis. For patient L36, the C->G mutation at nucleotide 801-10 results in the formation of an alternative splice site upstream of the normal site. Sequencing across the exon 7-8 boundary in cDNA showed the homozygous insertion of nine bases which would result in the addition of three amino acids, P-C-S, at a point immediately adjacent to the second transmembrane domain. Although this occurs in a conserved region, the first cytosolic domain is very small (normally four amino acids) and consequently may not be functionally important. The addition of the imino group of proline at this point might be expected to cause disruption to the folding of the polypeptide chain but is unlikely to enter or interfere with the transmembrane domain. For patient L47, an in-frame deletion results in the removal of seven amino acids from a region of cystinosin towards the N-terminus. This mutation would result in the loss of one out of the seven potential N-glycosylation sites within the first lysosomal luminal domain, but the region is unlikely to be a functionally important part of the protein. Three infantile nephropathic patients were identified previously (9) as homozygous for this mutation; in two cases (no details provided for the third) the phenotype was milder than expected for the classical disease and some expression of CTNS was observed. Although these mutations may affect the structure or function of the cystinosin protein, for patients L36 and L47 there is likely to be some protein product and hence some residual cystine transport activity, which would account for their milder phenotypes.

When the four missense mutations and three in-frame deletions associated with infantile nephropathic cystinosis are placed on our model of cystinosin, these mutations are found to affect conserved amino acids within transmembrane domains and are predicted to cause severe disruption of the protein. Of the two mutations associated with late-onset cystinosis, one is found near the N-terminus and the other adds three residues to a cytosolic domain. Although functional studies will be required to confirm this, we believe that the location of these mutations and the associated phenotype lends support for our model of cystinosin.

In contrast to these two clearly defined forms of this disorder, it has been recognized that there are individuals for whom the progression of the disease does not conform to the classical definitions. Three of the patients in this study (L16, L18 and P41) presented with cystinosis at an age which is below that typical for late-onset patients but with a clinical course that was milder than most patients with the classical infantile nephropathic form. Patient L18 is homozygous for a missense mutation that occurs in the non-conserved region of cystinosin towards the N-terminus. This part of the protein is predicted to lie within the lumen of the lysosome and the mutation is adjacent to, but would not affect, a potential N-glycosylation site. As with the late-onset patient (L47), the location of the mutation for patient P18 is therefore consistent with a milder phenotype. Patients L16 and P41 are both heterozygous for a severely truncating mutation and a missense mutation affecting an amino acid in a transmembrane domain. Presumably the missense mutations permit the production of some functional protein, which would account for the milder phenotypes. It is interesting that the mutation for patient P41 occurs in the first transmembrane domain since none of the amino acid substitutions associated with infantile nephropathic cystinosis, to date, are found in this domain. This suggests that functionally it may be a less important part of cystinosin. For patient L16, the G->N substitution in TM7 may not be disruptive, since in the yeast transmembrane protein the corresponding residue is also an asparagine and is compatible with the formation of a transmembrane domain. These data suggest a continuum of severity of the disorder (rather than discrete subtypes) resulting from different mutations of the gene.

The identification of mutations in the CTNS gene in patients with infantile nephropathic and late-onset cystinosis confirms earlier work which suggested that these diverse forms were allelic. Our results from patients with clearly defined infantile or late-onset phenotypes lend support to our model of cystinosin. Patients with two truncating mutations or mutations affecting conserved amino acids associated with transmembrane regions of the protein have the severe, infantile nephropathic cystinotic phenotype. The late-onset phenotype, with generally milder symptoms and a better prognosis for the patient, is associated with mutations that are located in functionally less important regions of the protein. We have also identified mutations in patients whose clinical course cannot easily be classified into the usual phenotypes. These individuals have at least one mutation which, we speculate, will result in the production of some functional protein. We currently are investigating the function of the protein and quantifying the effect of mutations on cystinosin activity. We hope that this work will allow us to predict more accurately the phenotypic effect of a given mutation, which may have important prognostic implications for the patients.

MATERIALS AND METHODS

Clinical material and mutation detection

Blood samples were obtained after informed consent from families of patients with infantile nephropathic and late-onset cystinosis, diagnosed according to standard criteria (1). In order to identify those patients who have the ~65 kb deletion, genomic DNA was amplified by PCR using primers which flank the breakpoint region. The details of these primers, PCR conditions and the results obtained are as previously described (8). In order to detect individuals who are heterozygous for the deletion, samples which produced an amplification product with the breakpoint primers were also tested using primers which amplify exons 3 and 10 of CTNS. These primer sequences and PCR conditions were as previously published (6). For samples found to be heterozygous for the ~65 kb deletion, or where a deletion fragment was not detected, the entire coding region of the CTNS gene was then screened for mutations. SSCP analysis (14) was carried out on the complete CTNS coding sequence and splice sites. We previously had designed and optimized a set of SSCP analysis primers, so that the entire coding region can be tested rapidly (6), except for the primers to exon 12 which are as follows: 12F, 5[prime]-CTCAGGAGGTGCCAACCTAA-3[prime] and 12R, 5[prime]-GGTGAGGCCTTCCCCAGCAG-3[prime]. In order to characterize individual mutations, direct sequencing was performed using Dye terminator chemistry (PE Biosystems, UK) for each exon displaying an abnormal SSCP band pattern.

Haplotype analysis

Genomic DNA was amplified with fluorescently labelled primers to microsatellite loci D17S2167, D17S2168 and D17S829. Primer sequences, PCR, electrophoresis and data analysis were as previously described (15-17).

RT-PCR

Total RNA was extracted from lymphocytes by standard methods (18). Reverse transcription was carried out using the primer CTRT1 (5[prime]-TGGCCCCAGGAGCAGAGTGG-3[prime]) according to standard protocols (18). PCR primers were selected from within CTNS cDNA. A 267 bp fragment between exon 11 and the 3[prime]-UTR of CTNS is amplified by primers F12 (5[prime]-GGCAACGTGC- TCCTGGACTT-3[prime]) and R12 (5[prime]-GGTGAGGCCTTCCCCAG-3[prime]). A 273 bp fragment spanning exon 8 is amplified by primers F79 (5[prime]-CGCCATTAGCATCATAAACC-3[prime]) and R79 (5[prime]-CGCGTGCAGGCTGAAGAAGA-3[prime]).

Following denaturation at 94°C for 2 min, PCR was with 30 cycles of 10 s at 94°C, 30 s at the annealing temperature of 64°C (F12/R12) or 58°C (F79/R79) and 30 s at 72°C, and final extension at 72°C for 5 min.

ACKNOWLEDGEMENTS

We thank the patients, families and physicians who have contributed to this project. We gratefully acknowledge Professor B. Winchester for advice. This study was supported, in the UK, by the National Kidney Research Fund and the Kidney Research Aid Fund and, in France, by the Association Vaincre les Maladies Lysosomales, INSERM (program APEX), the Association pour l'Utilisation du Rein Artificiel and the Association Française contre les Myopathies.

REFERENCES

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2. Goldman, H., Scriver, C.R., Aaron, K., Delvin, E. and Canlus, Z. (1971) Adolescent cystinosis: comparisons with infantile and adult forms. Paediatrics, 47, 979-988.

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4. Pellett, O.L., Smith, M.L., Greene, A.A. and Schneider, J.A. (1988) Lack of complementation in somatic cell hybrids between fibroblasts from patients with different forms of cystinosis. Proc. Natl Acad. Sci. USA, 85, 3531-3534. MEDLINE Abstract

5. The Cystinosis Collaborative Research Group(1995) Linkage of the gene for cystinosis to markers on the short arm of chromosome 17. Nature Genet., 10, 246-248. MEDLINE Abstract

6. Town, M., Jean, G., Cherqui, S., Attard, M., Forestier, L., Whitmore, S.A., Callen, D.F., Gribouval, O., Broyer, M., Bates, G.P., van't Hoff, W. and Antignac, C. (1998) A novel gene encoding an integral membrane protein is mutated in nephropathic cystinosis. Nature Genet., 18, 319-324. MEDLINE Abstract

7. Anikster, Y., Lucero, C., Touchman, J.W., Huizing, M., McDowell, G., Shotelersuk, V., Green, E.D. and Gahl, W.A. (1999) Identification and detection of the common 65-kb deletion breakpoint in the nephropathic cystinosis gene (CTNS).Mol. Genet. Metab., 66, 111-116. MEDLINE Abstract

8. Forestier, L., Jean, G., Attard, M., Cherqui, S., Lewis, C., van't Hoff, W., Broyer, M., Town, M. and Antignac, C. (1999) Molecular characterization of CTNS deletions in nephropathic cystinosis, development of a PCR-based detection assay. Am. J. Hum. Genet., 65, 353-359. MEDLINE Abstract

9. Shotelersuk, V., Larson, D., Anikster, Y., McDowell, G., Lemons, R., Bernardini, I., Guo, J., Thoene, J. and Gahl, W. (1998) CTNS mutations in an American-based population of cystinosis patients. Am. J. Hum. Genet., 63, 1352-1362. MEDLINE Abstract

10. Antonarkis, S.E. and the Nomenclature Working Group (1998) Recommendations for a nomenclature system for human gene mutations. Hum. Mutat., 11, 1-3. MEDLINE Abstract

11. Hunziker, W. and Geuze, H.J. (1996) Intracellular trafficking of lysosomal membrane proteins. Bioessays, 18, 379-389. MEDLINE Abstract

12. Lefkowitz, R.J. and Caron, M.G. (1988) Adrenergic receptors. Models for the study of receptors coupled to guanine nucleotide regulatory proteins. J. Biol. Chem., 263, 4993-4996. MEDLINE Abstract

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15. Dib, C., Faure, S., Fizames, C., Samson, D., Drouot, N., Vignal, A., Millasseau, P., Marc, S., Hazan, J., Seboun, E., Lathrop, M., Gyapay, G., Morisette, J. and Weissenbach, J. (1996) A comprehensive map of the human genome based on 5,264 microsatellites. Nature, 380, 152-154. MEDLINE Abstract

16. McDowell, G.A., Isogai, T., Tanigami, A., Hazelwood, S., Ledbetter, D., Polymeropoulos, M.H., Lichter-Konecki, U., Konecki, D., Town, M.-M., van't Hoff, W., Weissenbach, J. and Gahl, W.A. (1996) Fine mapping of the cystinosis gene using an integrated genetic and physical map of a region within human chromosome band 17p13. Biochem. Mol. Med., 58, 135-141. MEDLINE Abstract

17. Jean, G., Fuchshuber, A., Town, M., Gribouval, O., Schneider, J.A., Broyer, M., van't Hoff, W., Niaudet, P. and Antignac, C. (1996) High resolution mapping of a gene for cystinosis using combined biochemical and linkage analysis. Am. J. Hum. Genet., 58, 535-543. MEDLINE Abstract

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+These authors contributed equally to this work
§To whom correspondence should be addressed. Tel: +44 171 905 2196; Fax: +44 171 916 0011; Email: m.town{at}ich.ucl.ac.uk

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Patient ID	Nucleotide change	Amino acid change	Type of mutation	Consequence of mutation
L6; L25; L30; L34	65 kb deletion	-	Del exons 1-10	No transcription
	Not found	?	?	?
P45	65 kb deletion	-	Del exons 1-10	No transcription
	762delC	Frameshift at 142, V146X	Premature stop	Truncated 146 residue protein
P8	65 kb deletion	-	Del exons 1-10	No transcription
	898-900+24del27	-	5[prime] splice site IVS8	Truncated 187 residue protein
P46	65 kb deletion	-	Del exons 1-10	No transcription
	953-955delACG^a	D205del	In-frame deletion	One amino acid deleted adjacent to TM3
L7; L27; P14	65 kb deletion	-	Del exons 1-10	No transcription
	1035-1036insC^a	Frameshift at 233, E295X	Out-of-frame insertion	Truncated 295 residue protein
L1	65 kb deletion	-	Del exons 1-10	No transcription
	1036-1037insCG^a	Frameshift at 233, I253X	Out-of-frame insertion	Truncated 253 residue protein
P34	65 kb deletion	-	Del exons 1-10	No transcription
	1252G->T	D305Y	Missense	Amino acid change in TM6
P42	65 kb deletion	-	Del exons 1-10	No transcription
	1261G->A^a	G308R	Missense	Amino acid change in TM6
P15	65 kb deletion	-	Del exons 1-10	No transcription
	1354G->A^a	G339R	Missense	Amino acid change in TM7
P18	721C->T	Q128X	Premature stop	Truncated 128 residue protein
	1035-1036insC^a	Frameshift at 233, E295X	Out-of-frame insertion	Truncated 295 residue protein
P26	1020+1G->A	-	5[prime] splice site IVS9	Truncated 127 residue protein
	1035-1036insC^a	Frameshift at 233, E295X	Out-of-frame insertion	Truncated 295 residue protein
L28	1036-1037insCG^a	Frameshift at 233, I253X	Out-of-frame insertion	Truncated 253 residue protein
	1354G->A^a	G339R	Missense	Amino acid change in TM7
P30	1035-1036insC^a	Frameshift at 233, E295X	Out-of-frame insertion	Truncated 295 residue protein
	1035-1036insC^a	Frameshift at 233, E295X	Out-of-frame insertion	Truncated 295 residue protein
L19	1109-1131del 23	Frameshift at 258, N288X	Out-of-frame deletion	Truncated 288 residue protein
	1109-1131del 23	Frameshift at 258, N288X	Out-of-frame deletion	Truncated 288 residue protein
L24	1146-1148delCTC	S270del	In-frame deletion	Amino acid deleted from TM5
	1146-1148delCTC	S270del	In-frame deletion	Amino acid deleted from TM5
P16	1261G->A^a	G308R	Missense	Amino acid change in TM6
	1261G->A^a	G308R	Missense	Amino acid change in TM6
P19	1261G->A^a	G308R	Missense	Amino acid change in TM6
	1310-12G->A	-	3[prime] splice site IVS11	Truncated 323 residue protein
P23	1261G->A^a	G308R	Missense	Amino acid change in TM6
	1366-1377del12	IVFD343-346del	In-frame deletion	Four amino acids deleted from TM7
P13	1310-12 G->A	-	3[prime] splice site IVS11	Truncated 323 residue protein
	1310-12 G->A	-	3[prime] splice site IVS11	Truncated 323 residue protein
P5	1331del G	Frameshift at 331, L366X	Out-of-frame deletion	Truncated 366 residue protein
	1331del G	Frameshift at 331, L366X	Out-of-frame deletion	Truncated 366 residue protein
L2	1352T->C	L338P	Missense	Amino acid change in TM7
	1352T->C	L338P	Missense	Amino acid change in TM7
L29; P2; P28	1354G->A^a	G339R	Missense	Amino acid change in TM7
	1354G->A^a	G339R	Missense	Amino acid change in TM7

Mutation	No. of patients
	Town et al. (6)	This study	Shotelersuk et al. (9)	Total
Del exons 1-10	37	3	48	88
357-360delGACT	4	0	5	9
399-400delTG	2	0	0	2
537-557del21	0	1	3	4
622G->T	2	0	0	2
721C->T	1	0	1	2
753G->A	2	0	14	16
900+1delG	1	0	1	2
953-955delACG	0	1	1	2
985-986insA	0	1	1	2
1080delC	0	1	1	2
1035-1036insC	0	6	2	8
1036-1037insCG	0	2	1	3
1261G->A	0	4	2	6
1310-12G->A	0	2	0	2
1354G->A	0	5	1	6

				A. Servais, V. Moriniere, J.-P. Grunfeld, L.-H. Noel, J.-M. Goujon, B. Chadefaux-Vekemans, and C. Antignac Late-Onset Nephropathic Cystinosis: Clinical Presentation, Outcome, and Genotyping Clin. J. Am. Soc. Nephrol., January 1, 2008; 3(1): 27 - 35. [Abstract] [Full Text] [PDF]

				E. Levtchenko, A. de Graaf-Hess, M. Wilmer, L. van den Heuvel, L. Monnens, and H. Blom Altered status of glutathione and its metabolites in cystinotic cells Nephrol. Dial. Transplant., September 1, 2005; 20(9): 1828 - 1832. [Abstract] [Full Text] [PDF]

				V. Kalatzis, N. Nevo, S. Cherqui, B. Gasnier, and C. Antignac Molecular pathogenesis of cystinosis: effect of CTNS mutations on the transport activity and subcellular localization of cystinosin Hum. Mol. Genet., July 1, 2004; 13(13): 1361 - 1371. [Abstract] [Full Text] [PDF]

				S. Cherqui, C. Sevin, G. Hamard, V. Kalatzis, M. Sich, M. O. Pequignot, K. Gogat, M. Abitbol, M. Broyer, M.-C. Gubler, et al. Intralysosomal Cystine Accumulation in Mice Lacking Cystinosin, the Protein Defective in Cystinosis Mol. Cell. Biol., November 1, 2002; 22(21): 7622 - 7632. [Abstract] [Full Text] [PDF]

				V. Kalatzis and C. Antignac Cystinosis: from gene to disease Nephrol. Dial. Transplant., November 1, 2002; 17(11): 1883 - 1886. [Full Text] [PDF]

				J. M. Geelen, L. A. H. Monnens, and E. N. Levtchenko Follow-up and treatment of adults with cystinosis in the Netherlands Nephrol. Dial. Transplant., October 1, 2002; 17(10): 1766 - 1770. [Abstract] [Full Text] [PDF]

				R. Kleta and W. A. Gahl Cystinosis: Antibodies and Healthy Bodies J. Am. Soc. Nephrol., August 1, 2002; 13(8): 2189 - 2191. [Full Text] [PDF]

				W. A. Gahl, J. G. Thoene, and J. A. Schneider Cystinosis N. Engl. J. Med., July 11, 2002; 347(2): 111 - 121. [Full Text] [PDF]

				V. KALATZIS, S. CHERQUI, G. JEAN, B. CORDIER, P. COCHAT, M. BROYER, and C. ANTIGNAC Characterization of a Putative Founder Mutation that Accounts for the High Incidence of Cystinosis in Brittany J. Am. Soc. Nephrol., October 1, 2001; 12(10): 2170 - 2174. [Abstract] [Full Text] [PDF]

				Y. Anikster, F. Lacbawan, M. Brantly, B. L. Gochuico, N. A. Avila, W. Travis, and W. A. Gahl Pulmonary Dysfunction in Adults With Nephropathic Cystinosis Chest, February 1, 2001; 119(2): 394 - 401. [Abstract] [Full Text] [PDF]

				C. P. Ponting, R. Mott, P. Bork, and R. R. Copley Novel Protein Domains and Repeats in Drosophila melanogaster: Insights into Structure, Function, and Evolution Genome Res., December 1, 2001; 11(12): 1996 - 2008. [Abstract] [Full Text] [PDF]