Cystinosis is a rare autosomal-recessive lysosomal storage disease caused by mutations in the CTNS gene that encodes the cystine transporter, cystinosin, which leads to lysosomal cystine accumulation. The most common form of cystinosis, the nephropathic or infantile type, is characterized by renal failure at the age of 10 years and other systemic complications and early corneal cystine crystal deposition. The Cornea is a common site for crystals accumulation. This is manifested by photophobia, the major ocular symptom in patients with cystinosis . Deposits affect conjunctiva and retinal pigment epithelium (RPE) too. They can also be deposited in the iris, ciliary body, choroid and lens capsule.
This work was conducted as a straight continuation of studies carried out in other Tunisian cystinosis patients and their families .
Eight different Tunisian families with cystinosis were investigated in this study for both the molecular profile (CTNS mutations) and the ophthalmic examination. Consanguinity between first and second degree was reported in eight families. All of the probands initially presented with Fanconi syndrome. Cystinosis was suggested by the presence of crystals in the cornea in most of studied patients. Of note, the corneal crystal deposition starts a similar significant early age for all the studied patients, at the periphery of the cornea, with progression to the center and the posterior stroma , leading to corneal erosions, scarring, and neovascularization. They can also be deposited in the iris, ciliary body, choroid and lens capsule. However, in these Tunisian patients, the deposits affect only conjunctiva and retinal pigment epithelium (P1, P4, P6, P6 and P7) according to several studies .
In the current study, molecular analysis showed that none of the Tunisian patients had the 57-kb deletion. We could described four previously identified mutations: 20-kb deletion, p.G308R missense mutation, p.G258fs frameshift mutation; c.829 duplication; and three novel mutations including two missense mutations: p.G88K and p.S139Y and one splice site mutation IVS 8+7delC. All of the patients carried a homozygous CTNS mutation probably due to the high parental consanguinity rate.
This deletion was described for the first time in the homozygous state in a Tunisian patient who showed early onset cystinosis with severe features . The functional test of the 20-kb deletion could not be further characterized in this study. The studied patient (P1) presented the same clinical profile of her previously analyzed brother . Additionally, the clinical profile of this patient was in agreement with several studies showing that the large deletion was always associated with the severe phenotype, resulting in cystinosin expression defect .
The P3 patient was found homozygous for the described frameshift mutation which is located in exon 10, resulting in a small deletion of 23 nucleotides (c.771_793del), predicted to introduce premature termination of glycopeptides at 288 residue. This reported micro deletion is located in the sixth putative transmembrane domaine TM6. This mutation occurs at residues which are conserved between cystinosin and the transmembrane protein of yeast of C. elegance . Furthermore, it has been noted that for the affected conserved residues or residues located in TMs of many other membrane protein, the transport activity is less tolerant , thus is similar with the G285, which is considered a conserved residue. Besides, this G285 mutation induces a loss cystin transport activity in the studied patient (P3) who presented crytals accumulation in the anterior stromal corneal. Besides, the cysteamine eye drops treatment during few months could at any age because direct placement of cysteamine solution on the cornea can dissolve cystine crystals in less than a year . This mutation was identified for the first time in a patient presenting a classical infantile neuphropathic cystinosis  similar to the patient studied (P3).
Both P6 and P7 patients with infantile nephropathic cystinosis were homozygous for the c.829dupA mutation. The c.829dupA is the most common mutation detected in Egyptian patients with infantile nephropathic cystinosis  but it has been reported only once in a heterozygous European patient .
The pathogenicity of this mutation caused by a single-base duplication and predicted to introduce a premature stop codon downstream that could also trigger nonsense-mediated decay. This is consistent with the severe phenotype observed in the unrelated patients (P6 and P7). The ophthalmologic examination showed diffuse crystals and retinal atrophy in the two patients. Furthermore, the lack of cystine transport activity of the CTNS- Glu227* mutant may be associated with the frameshift mutation which is located at the 5th inter-transmembrane loop containing the PQ motif, required for H+ and cystine co-transport .
The patient P5, with classical nephropathic cystinosis, was homozygosity for a novel splicing mutation in CTNS, c.681+7delc. Several studies showed that CTNS splicing mutations have been described in all three clinical variants of cystinosis: classic nephropathic cystinosis , the intermediate variant, and the ocular form. The c.681+7delc mutation in patient P5 within the ocular form was one of the eighth splicing mutations reported among nephropathic cystinosis patients. Splicing mutations generally occur early in the CTNS coding region and generate premature stop codons implicating the obliteration of the putative C-terminal tyrosine based lysosomal targeting signal.
Patient P4 was homozygous for the novel missense mutation (p.Q88K) and showed the infantile cystinosis phenotype. Crystallographic analysis of the generated cystinosin 3D structure model demonstrates that most of the infantile cystinosis missense mutations are located in the transmembrane domains. In the literature, these mutations do not alter the lysosomal localization of cystinosin but abolish the cysteine transport . In fact, EGFP-fused cystinosin constructs bearing 19 missense mutations associated with infantile cystinosis, were transiently expressed in HeLa cell to determine the subcellular localization and cystine transport activity of cystinosin . The results suggest that the intralysosomal cystine accumulation is related to an inability to transport cystine rather than an expression defect. These mutations affect the normal folding of the cystinosin transmembrane domains in the phospholipid bilayer membrane resulting in infantile cystinosis phenotype.
On the other hand, missense mutations located in cystinosin regions oriented towards cytosol or lysosomal lumen region are usually associated with the juvenile or ocular phenotype. HeLa cell expression of these mutations show a low level of cystine transport compared to those associated to infantile patient cystinosis . In the present study, the novel mutation Gln88Lys which is buried in the lysosomal lumen, close to the lipid membrane region, represents an exception since it is associated with infantile cystinosis. The charge introduced by the mutated residue could disturb the interaction between the cystinosin N-terminal region and phospholipid bilayer polar head region, leading to impaired or limited transport activity which contrasts with the severe phenotype. About the second novel missense mutation Ser139Tyr situated in the first transmembrane helix, crystallographic analysis demonstrated that this mutation affects the hydrophobic interactions with the lipid bilayer polar region. Interestingly, another mutation has been previously reported in the same amino acid residue Ser139Phe and was associated with an atypical cystinosis phenotype. The carrier of this mutation, classified as atypical due to a late onset of renal Fanconi syndrome revealed only at the age of 3 years , compared to P8 in this study (Table 1). The physico-chemical properties of each mutated residue could explain the difference in phenotype expression between these cases.
Both the P2 patient and her sister (who died at 1 year and six months old) showed severe phenotype with growth retardation, renal tubular fonconi syndrome, polyuria, corneal cystine crystals and elevated leukocyte cystine levels. Mutational screening revealed a missense mutation p.G308R affecting a highly conserved amino acid residue among vertebrates, since it is essential for preserving a particular neutral charge in the sixth transmembrane domain of cystinosin. Based on these chemical properties, the positive charge introduced by the mutated residue Arg308 could affect the interaction between the transmembrane domain and the nonpolar tail region of the phospholipid bilayer membrane. Interestingly, COS cells expressing of cystinosin bearing the p.Gly308Arg mutation greatly affect the ability of the protein to transport cystine at the plasma membrane . In the literature, this mutation was detected in homozygous and compound heterozygous states in German and Swiss patients respectively and also associated with the severe infantile nephropathic phenotype.
The screening of polymorphisms in CTNS gene revealed that there are at least 13 polymorphisms detected and were associated with the severe infantile nephropatic form of disease, (Table 1). Here, we describe one novel polymorphisms in intron 10 within patient P5. The polymorphism was in the homozygous status.
It is noteworthy that the unclassified sequence change, p.I260T (refSNP ID: rs161400) co-segregates with all the identified mutations in the studied patients (P2, P4, P5, and P8) who presented the infantile form of cystinosis; as described in the literature . Thus, to understand the origin of a mutation, it is very useful to identify whether a certain recurrent mutation is associated with the same haplotype.