Five different Jordanian families with nephropathic cystinosis from different geographical locations in Jordan and one family from Sudan (Family 5) were investigated in this study for the CTNS mutation. Consanguinity was reported for all six of the families. All of the probands initially presented with Fanconi syndrome and rickets. Cystinosis was suggested by the presence of crystals in the cornea upon ophthalmological evaluation by age 2 years in most of the probands. The 57-kb deletion was not found in all of the families and that was evidenced by the presence of the 250-bp PCR product using the LDM2/exon 4 primers and the 266-bp PCR product by using the LDM1/D17S829 primers (data not shown). Amplification of the coding exons (3–12) for the CTNS gene followed by bi-directional DNA sequencing identified three polymorphisms, −294C>T, −180T>C and −118C>T in the CTNS promoter region, one silent (c.504G>A; p.Thr168Thr), one missense (c.779C>T; p.Thr26Ile) variants and two CTNS mutations: c.829dupA (Fig. 1A) and c.890G>A (Fig. 1B). The proband of family 2 in this study was compound heterozygous for both mutations, where c.829dupA was transmitted from the father and c.890G>A was contributed by the mother (Table 1). Both of the parents in family 6 were carriers of the c.829dupA mutation, and their affected children who were not available for testing were presumably homozygous for the same mutation. Both of the probands of Jordanian family 1 and Sudanese family 5 were homozygous for the c.890G>A mutation (Table 1). The C allele encoding Thr amino acid of the c.779C>T variant was in cis with the mutant alleles for both c.829dupA and c.890G>A mutations in the Jordanian families, whereas in the Sudanese proband, the T allele coding for Ile amino acid was in cis with mutant A allele for the c.890G>A mutation.

Fig. 1.

(A) DNA sequence chromatograms for heterozygous genotype (mother, family 3) of the c.829dupA mutation in exon 10 of the CTNS gene. (B) DNA sequence chromatograms for heterozygous genotype (mother, family 1) of the c.890G>A mutation in exon 11 of the CTNS gene. The arrows indicate the locations of the mutations.

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This is the first molecular study on Jordanian patients with cystinosis infantile nephropathic cystinosis. The 57-kb deletion was not found in any of the Jordanian probands using two different multiplex polymerase chain reactions. Our results agree with previous regional studies in which the 57-kb deletion was not found in Saudis, Egyptians, Emiratis, Turkish or Iranians.4,7–10 The 57-kb deletion was previously found in homozygous genotypes in 48 of 108 (44%) patients of European descendant living in America, and this deletion also accounts for 34% and 65% of the CTNS mutant alleles in patients of Spanish and German origin.18,22,29

In this study, we identified two frameshift mutations in the CTNS gene in five Jordanian families and one family from Sudan with a history of nephropathic cystinosis. The c.829dupA (p.Thr277Asnfs*19) mutation, which is located in exon 10, resulting in an amino acid substitution for residues 277–294 with the exception of the Phe residue at position 282 and a premature stop codon at residue 295 (Glu295X). The c.829dupA mutation was described in a compound heterozygous genotype with the 57-kb deletion in Belgium and was recently found in a homozygous genotype in three probands and one sibling from Egypt.7,30 The second mutation, c.890G>A mutation, which is located in exon 11, converts the Trp 297 TGG codon into a TAG stop codon (p.Trp297X). The c.890G>A was originally described in a heterozygous genotype in a male from South Italy with first-cousin parents; as well as for the c.779C>T variant that was found in 5 Italian patients.15 Both of the mutations in this study were found in the proband of family 2 as a compound heterozygous genotype that was not previously described. The finding of the compound heterozygous genotype is not usually expected in a consanguineous family. However, several examples have been reported in which two different mutations create compound heterozygotes in consanguineous families in Mediterranean populations. For example, Lebanese patients from an extended consanguineous family with Bardet-Biedl syndrome were found to be heterozygous for two missense mutations, p.Ser311Ala and p.Val11Gly, in the BBS10 gene.31 Similarly, in an inbred pedigree in an Israeli Arab family with congenital nephrotic syndrome, two patients carried both p.Gln380X and p.Cys721fs mutations in the NPHS1 gene as a compound heterozygous genotype.32 The case we report is less extreme in the sense that both mutations (p.Thr277Asnfs*19 and p.Glu295X) are seen in homozygous (four Jordanian families) and in heterozygous (one Jordanian family) living in different and distant locations; but founder effects likely account for their joint occurrence. This possibility could be further ascertained by additional genotyping and analyses of haplotype status in the probands and families.

The inheritance of the same c.890A mutant allele in cis with the C allele of the c.779C>T variant in Jordanian and in cis with the T allele in Sudanese patients suggests an independent origin of the same mutation in the Arabic population. The association of the c.779C allele with both of the mutant alleles for c.890A and c.829dupA is an additional DNA marker that can be used for prenatal molecular diagnostics of cystinosis in Jordanian families.

Both the c.829dupA in exon 10 and c.890G>A in exon 11 resulted in a truncated cystinosin protein that, if stable, would lack the 6th and the 7th transmembrane domains and the five-amino-acid ‘GYDQL’ lysosomal sorting signal at the c-terminal tail of the protein. Additionally, the c.829dupA mutation resulted in eight amino acid substitutions within the first nine amino acids of the third cytoplasmic loop, which is composed of 19 amino acids (280–298) between the fifth and sixth potential transmembrane domains.24,25 The deletion of the first nine amino acids of this loop (amino acids 280–288) coupled with the deletion of the defined carboxyl ‘GYDQL’ motif resulted in the complete relocalisation of cystinosin-GFP to the plasma membrane without a signal remaining in the lysosomes.24,25 Therefore, both of the mutations that were identified in this study potentially resulted in the partial redirection of cystinosin to the plasma membrane, and the c.829dupA mutation may also result in the complete relocalisation of cystinosin to the plasma membrane.

The proband of family 4 who was heterozygous for the c.829dupA mutation inherited haplotype −294T/T, −180C/C and −118T/T for the three polymorphisms in the CTNS promoter region spanning −85 to −406 nucleotides relative to the CTNS +1 transcription site. The C-to-T substitution is 294bp upstream of the transcription site of the CTNS gene, which is part of the Sp1 consensus binding (−299 to -293) sequence in the CTNS promoter.24 An in vitro study using a luciferase vector reporter gene construct harbouring the −294T allele that was transfected transiently in human kidney tubular cells (HK2) showed a 30% reduction in luciferase transcriptional activity compared to −294C construct.28 Similarly, a functional study showed an 81% reduction in CAT reporter gene constructs harbouring the −295C mutant allele of the CTNS promoter.24 However, four patients carrying the −295G>C mutation were also carrying two allelic mutations in the coding region for the CTNS gene, which may contradict a role of promoter mutation in the disease.15 In proband 4, the second mutated allele may be within the CTNS intronic regions, which were not sequenced in this study, and this mutation may affect the splicing processes.16

There is evidence for the allelic heterogeneity of nephritic cystinosis disease in Arabic patients, as twenty-three known and new mutations in the CTNS mutation have been characterised in Arabic individuals thus far.4–8 These mutations spread across the CTNS gene, mainly in the coding exon number 3 and exons 6–12, and include several types, including missense, nonsense, frameshift and splicing mutations. Twelve of the 23 mutations were novel.4–8 Four mutations, including the two mutations that were described in this study (Thr277Asnfs*19 and p. Trp297), i.e., p. G308R and p.G339R, were characterised in more than one Arabic nationality and have also been characterised in other parts of the world.4,7,9,15,18,29,30

However, the c.681G>A; p.E227E mutation that substituted for the last base of exon 9 of the CTNS gene and created a new cryptic donor site was previously found exclusively in several patients from Middle Eastern countries but not in Jordan.4,7,9,10 Despite the notable prevalence of the c.681A mutant allele in homozygous and heterozygous genotypes in cystinosis patients in the Middle East, further haplotype analysis for chromosome 17 is required to determine whether this is a founder mutation in the region. The geographical distribution of the c.681G>A allele in several Middle Eastern populations was analysed by Soliman et al., who proposed that the origin of this mutation is in Eastern Iran.7 Similarly, putative founder mutations, such as c.753G>A and c.898-900_24del27, have been characterised in French Canada and in the western province of Brittany, France, respectively.33,34

In summary, we have identified two loss-of-function mutations in the CTNS gene in several Jordanian families and one family from Sudan. These two mutations extend the regional mutation database for the orphan disease cystinosis. Both of the mutations were originally described in heterozygous genotypes in European patients and have been found in Egyptians, Jordanians and Sudanese individuals. These results will facilitate the molecular genetic diagnosis of cystinosis in Jordan.

The authors declare no conflict of interest.