Novel truncating mutation in the CTNS gene in an Egyptian family with cases of infantile nephropathic cystinosis and congenital heart malformations

Al-Haggar, Mohammada; Taranta, Annab; El-Hawary, Amanya; Al-Said, Afafa; Shaban, Abdel-Aziza; Wahba, Yahyaa

Middle East Journal of Medical Genetics:
doi: 10.1097/01.MXE.0000414810.01450.3c
Original articles

Introduction: Cystinosis is an autosomal recessive disorder characterized by lysosomal cystine accumulation throughout the body. We report on an Egyptian family with multiple cases of infantile nephropathic cystinosis occurring in association with congenital heart malformations, aiming to add some knowledge to the clinical spectrum of the disorder.

Participants and methods: A family that had two cases of proximal renal disorder was enrolled. A clinical-laboratory work up was performed for each family member after obtaining written informed consent and included complete urinalysis including chromatography for amino acids, renal function tests, estimation of serum electrolytes, arterial blood gases, alkaline phosphatase and parathormone levels, skeletal radiographs, renal ultrasound, and slit lamp examination. Because of the accidental discovery of a systolic murmur in the young proband, echocardiography was performed for the two cases. The clinical diagnosis of cystinosis was confirmed and sequencing analysis of the CTNS gene was carried out.

Results: Urine showed excessive amino acid excretion, and was positive for reducing sugars. Both probands showed aminoaciduria, glucosuria, corneal cystine crystals on slit lamp examination, radiological evidences for rickets, and cardiac defects on echocardiographic evaluation (septal defects in the younger sib and mitral and aortic incompetence in the older sib). Mutational analysis indicated a novel nonsense mutation (c.734 G>A); homozygous in probands but heterozygous in the parents. This mutation substitutes tryptophan by a premature stop codon at the position 245 in cystinosin (W245X).

Discussion: To the best of our knowledge, the detection of congenital heart defects in two patients with severe infantile cystinosis who had a novel truncating CTNS mutation has never been reported in the literature. This may suggest the future necessity for the cardiac assessment of any similar case, especially those with novel truncating CTNS mutations.

Author Information

aDepartment of Pediatrics, Genetics Unit, Mansoura University Children’s Hospital, Mansoura, Egypt

bDepartment of Nephrology and Urology, Division of Nephrology, Bambino Gesù Children’s Hospital and Research Institute, Rome, Italy

Correspondence to Mohammad Al-Haggar, MD, Department of Pediatrics, Genetics Unit, Mansoura University Children’s Hospital (MUCH), PO 35516, 31 Bank Misr Street, Mansoura, Egypt Tel: +20 502 310 661/+20 502 211 948/+20 111 171 5350; fax: +20 502 234 092; e-mail:

Received February 19, 2012

Accepted March 31, 2012

Article Outline
Back to Top | Article Outline


Cystinosis is an autosomal recessive disorder characterized by an accumulation of the amino acid cystine in lysosomes throughout the body. It occurs with a frequency of approximately one in 100 000–200 000 and has been found worldwide in all ethnic groups (Gahl et al., 2002). Cystinosis accounts for 5% of chronic childhood renal failure (Middleton et al., 2003).

The CTNS gene responsible that encodes the lysosomal cystine carrier protein (cystinosin) was cloned in 1998 and was mapped to the short arm of chromosome 17 (p13) (Town et al., 1998). Depending on the age at presentation and the degree of disease severity, there are three clinical forms of cystinosis: (a) an infantile nephropathic form (OMIM 219800), which is the most frequent and most severe form, (b) a juvenile nephropathic form (OMIM 219900), which is a late onset and milder form, and (c) an adult form (OMIM 219750), the most benign form, which is non-nephropathic, but purely ocular. All three forms of the disease are caused by mutations of the CTNS gene and have a phenotypic overlap (Wilmer et al., 2011).

Infantile cystinosis usually starts toward the end of the first 6 months of life; then, the kidneys progressively lose their proximal tubular function, resulting in urinary loss of water, sodium, potassium, bicarbonate, calcium, magnesium, phosphates, amino acids, glucose, proteins, and many other solutes (Levtchenko and Monnens, 2006; Wilmer et al., 2011).

Extrarenal symptoms of cystinosis usually develop in untreated teenagers in the form of painful corneal erosions, peripheral corneal neovascularization, punctate, filamentous or band keratopathy, iris crystals, and retinal degeneration (Tsilou et al., 2002). Also, impairment in endocrinal glands is reported including hypothyroidism, insulin-dependent diabetes mellitus, and hypogonadism. Cystinosis may be accompanied by encephalopathy, stroke-like episodes, benign intracranial hypertension, and myopathy (Wilmer et al., 2011).

The diagnosis of cystinosis should be suspected in all patients with failure to thrive and signs of renal Fanconi syndrome. The index of suspicion is usually increased at the age of 1 year with the observation of cystine crystals in the cornea, a pathognomonic sign. The absence of these crystals after age of 2 years excludes the diagnosis of cystinosis (Wilmer et al., 2011). The definitive diagnosis of cystinosis can be established by the detection of an elevated intracellular cystine content using high-performance liquid chromatography (HPLC) (De Graaf Hess et al., 1999) or tandem mass spectrometry (MS/MS) (Chabli et al., 2007).

Molecular analysis of the CTNS gene facilitates an early presymptomatic diagnosis and could be used for the prenatal diagnosis of the disease. The CTNS gene consists of 12 exons, of which only 10 (from 3rd to 12th) are coding; its mutations mainly result in either complete abolition or reduction of cystine transport (Kalatzis and Antignac, 2003). Many different mutations have been identified in European and American patients, the most common being a large 57-kb deletion (Mason et al., 2003). The association of the severe infantile form with truncating mutations and the intermediate or adult forms with mutations that allow a residual CTNS gene function provides some phenotype–genotype correlates (Kalatzis et al., 2004).

Here, we report a novel mutation of the CTNS gene in an Egyptian family that had members with infantile nephropathic cystinosis in association with major cardiac malformations of different nature. This association, to the best of our knowledge, had not been described before and may add knowledge to the clinical spectrum of the disorder.

Back to Top | Article Outline

Participants and methods


An Egyptian family from the 25 families with documented cases of renal tubular acidosis followed up regularly at the Nephrology Clinic, Mansoura University Children’s Hospital (MUCH), had been enrolled in the current study by November 2010. This family included two suspected cases of infantile cystinosis. The diagnosis of cystinosis was suspected because of the ocular involvement in the form of intense photophobia in early life, indicating corneal irritation, and the evidence for proximal renal tubular involvement. A clinical-laboratory work up was performed for each family member after taking informed written consent and included complete urinalysis including chromatography for amino acids, renal function tests, estimation of serum electrolytes, arterial blood gases, alkaline phosphatase and parathormone levels, skeletal radiographs, renal ultrasound, and slit lamp examination. Because of the accidental discovery of a systolic murmur in the young proband, echocardiography was performed for the two cases.

Back to Top | Article Outline
Molecular analysis

Molecular analysis of the CTNS gene in all members was arranged in collaboration with Bambino Gesù Children’s Hospital and Research Institute, Rome, Italy, to confirm the diagnosis.

Using the DNA purification Capture Column Kit (Gentra kit, Quiagen, Duesseldorf, Germany), genomic DNA was extracted from peripheral blood leukocyte samples withdrawn from all available affected and healthy family members. All coding sequences including flanking introns in the CTNS gene were amplified using a PCR. Direct sequencing for the PCR products was performed using a Big Dye Primer Cycle Sequencing kit and an ABI 310 Genetic Analyzer (PE Applied Biosystems, Foster City, California, USA). The primers and PCR conditions used for sequencing of the target DNA stretch are detailed in Table 1.

Back to Top | Article Outline


A pedigree was constructed (Fig. 1), showing a remote consanguinity; the grandparents (4001, maternal grandfather of the father and 4002, paternal grandfather of the mother) were cousins. Disease had never been suspected in any member of the higher generations. Probable carriage and segregation of the mutant allele had been proposed up to the common ancestor. The two probands had an older sib who developed end-stage renal disease at 10 years of age, was maintained on hemodialysis for 7 months, and died at 11 years because of pulmonary edema.

The clinical parameters of the two living probands (Table 2) were more severe in the older proband because of the relative delay in the diagnosis and the longer disease process. However, a congenital heart lesion was silent in the older patient, only detectable on echocardiography. The initial laboratory parameters in blood and urine were almost indistinguishable among the two cases (Table 3) and included (a) metabolic acidosis with a normal anion gap (hyperchloremic metabolic acidosis), (b) markedly elevated fractional excretion of sodium, potassium, phosphates, and urates, (c) polyuria because of a high solute load in urine, and (d) excessive urinary excretion of amino acids. The diagnosis of cystinosis was established by the slit lamp examination, which showed corneal cystine deposits.

Patients had been provided adequate supportive treatment in the form of polycitra that contained bicarbonate component as potassium citrate as well as phosphates to correct the metabolic acidosis (large amounts of alkali 10–15 mEq/kg/day), an oral calcium supplement, and active vitamin D (Alpha calcitriol, at a dose of 0.04 μg/kg/day), in addition to cysteamine eye drops and bitartrate capsules (Cystagon, Milan Pharmaceuticals, West Virginia, USA), 50, 150 mg, at a dose of 1.30 g/m2/day of the free base, 1/4 to 1/6 dose commenced and then gradually increased over a period of 4–6 weeks to prevent intolerance. Growth curves were linear during the last 2 years of life; weight was 25th–50th percentile and height was 50th percentile despite the presence of clinical, laboratory, and radiological evidence of rickets in the two probands. As the treatment was started at an earlier age in patient 4202, control of eye symptoms and preservation of renal function was observed compared with patient 4201; therefore, more dense cystine crystals in the cornea were observed in the older patient (Fig. 2).

Figure 3 shows the sequencing pherograms for the CTNS gene. A novel G>A substitution in exon 10 at the position 734 (c.734 G>A) is shown in the middle and lower panels; the parents (4101 and 4102) are heterozygous whereas probands (4201 and 4202) are homozygous for the mutation. The nonsense truncating mutation results from the substitution of tryptophan by a stop codon at the position 245 of the cystinosin protein (W245X), leading to truncating loss of 122 amino acid residues and possibly causing a nonfunctioning protein.

Back to Top | Article Outline


Cystinosin is a novel integral lysosomal membrane protein; on analysis of the predicted effects of its mutations among all the clinical forms of cystinosis, Attard et al. (1999) had identified 23 different mutations in the CTNS gene, 14 of which were novel. Twelve out of 25 patients with infantile nephropathic cystinosis (48%) had two severe truncating mutations that caused complete loss of the functional protein; however, the rest (13 patients) had missense or in-frame deletions that resulted in the disruption of transmembrane domains and loss of protein function.

A novel nonsense mutation (W245X) causing the loss of 122 amino acid residues had been detected in our cases. The mutation forms associated with the loss of the functional protein have been linked to the early onset of disease, whereas those reported with later juvenile forms usually affect the functionally unimportant regions of the protein. However, some overlap in the age of onset of disease and severity has been observed (Attard et al., 1999). They found some patients whose disease onset was early had a milder course, thus suggesting the presence of missense mutations that allow the production of a functional protein, in other words, mutations that affect functionally unimportant regions of cystinosin. The novel nonsense mutation (W245X) reported in our Egyptian family could explain the observations of the severe clinical course ending in the death of the first offspring (nontested), the early onset of renal failure in case 4201, and the eye involvement despite the early regular use of cystine chelation in case 4202.

The cardiac involvement of the two alive probands (4201, 4202) in the form of left-sided valvular incompetence in case 4201 and septal defects in case 4202 is uniquely present among reported cystinosis cases. Moreover, we may speculate that pulmonary edema that was the cause of death in the eldest nontested sib could be a sign of hidden left-sided congenital heart disease. Cardiovascular complications associated with cystinosis in the literature have been related to the relative longevity of patients with conditions ranging from arterial stiffness and vascular atheroma formation because of cystine accumulation in the wall of blood vessels to different forms of cardiomyopathy. Adequate cysteamine treatment reduces the risk of vascular involvement even lower than the risk of chronic kidney disease because of other causes (Besouw et al., 2011). Two cases of cardiomyopathy have been diagnosed in young adult females with cystinosis: one involving dilated cardiomyopathy in a Caucasian pregnant female with nephropathic cystinosis and maintained on hemodialysis. Although the patient had stopped cysteamine during pregnancy, the authors argued against cystinosis as the cause but rather suggested the case to be a simple pregnancy-associated cardiomyopathy (Ramappa and Pyatt, 2010). The second woman was a 24 year old who had been diagnosed with an isolated left ventricular noncompaction; she presented with reduced exercise tolerance because of left ventricular dysfunction (Ahmed et al., 2009).

In developing rats, Rosa et al. (2004) studied the toxic effects of the accumulated cystine in myocardium on pyruvate kinase (PK) enzyme activity; cystine caused oxidation of the sulfhydryl groups of PK enzyme by its disulfide moiety, leading to enzymatic inhibition of PK. This theory was confirmed by the protective effects of GSH and cysteamine, which reversed the oxidative inhibition caused by cystine. Cardiac accumulation of cystine was proved in a cadaveric biopsy from a cystinosis patient who died at the age of 33 years because of restrictive cardiomyopathy; cystine crystals were found in interstitial cardiac histiocytes and myocardial cells at a very high concentration, reaching 1000-fold higher than normal (Dixit and Greifer, 2002).

As the process of lysosomal cystine accumulation starts in the early intrauterine period (Levtchenko and Monnens, 2006; Wilmer et al., 2011), it could be speculated that the left-sided valvular dysfunction as well as the structural discontinuity of cardiac septa might be logical consequences of myocardial cystine accumulation. However, the suggestion of a causal relationship between this novel mutation and the cardiac involvement in the reported Egyptian family would be immature only on the basis of the current report, considering the relatively high incidence of congenital heart diseases. However, this might provide the basis for more surveys in other similar cases.

In view of the previously unreported disease association and the novel truncation mutation of the CTNS gene, an echocardiographic screening of cases with infant cystinosis, especially those who have a severe course or a documented truncating mutation that possibly leads to nonfunctioning protein, could be suggested.

Back to Top | Article Outline

The authors acknowledge the efforts of Dr. Mona Hafez, Assistant Professor of Pediatric Cardiology, Mansoura University Children's Hospital, Egypt, for the Echocardiographic evaluation, and Dr. Iman Azmi, Lecturer of Ophthalmology, Mansoura Ophthalmology Center, Egypt, for her kind slit lamp examination and fundus evaluation of the patients.

Back to Top | Article Outline
Conflicts of interest

There are no conflicts of interest.

Back to Top | Article Outline


Ahmed I, Phan TT, Lipkin GW, Frenneaux M. Ventricular noncompaction in a female patient with nephropathic cystinosis: a case report. J Med Case Rep. 2009;3:31
Attard M, Jean G, Forestier L, Cherqui S, Van’t Hoff W, Broyer M, et al. Severity of phenotype in cystinosis varies with mutations in the CTNSgene: predicted effect on the model of cystinosin. Hum Mol Genet. 1999;8:2507–2514
Besouw MTP, Holewijn S, Levtchenko EN, Janssen MCH. Non-invasive measurements of atherosclerosis in adult cystinosis patients. J Inherit Metab Dis. 2011;34:811–818
Chabli A, Aupetit J, Raehm M, Ricquier D, Chadefaux Vekemans B. Measurement of cystine in granulocytes using liquid chromatography-tandem mass spectrometry. Clin Biochem. 2007;40:692–698
De Graaf Hess A, Trijbels F, Blom H. New method for determining cystine in leukocytes and fibroblasts. Clin Chem. 1999;45:2224–2228
Dixit MP, Greifer I. Nephropathic cystinosis associated with cardiomyopathy: a 27-year clinical follow-up. BMC Nephrology. 2002;3:1–4
Gahl WA, Thoene JG, Schneider JA. Medical progress: cystinosis. N Engl J Med. 2002;347:111–121
Kalatzis V, Antignac C. New aspects of the pathogenesis of cystinosis. Pediatr Nephrol. 2003;18:207–215
Kalatzis V, Nevo N, Cherqui S, Gasnier B, Antignac C. Molecular pathogenesis of cystinosis: effect of CTNS mutations on the transport activity and subcellular localization of cystinosin. Hum Mol Genet. 2004;13:1361–1371
Levtchenko E, Monnens L. Development of Fanconi syndrome during infancy in a patient with cystinosis. Acta Paediatr. 2006;95:379–380
Mason S, Pepe G, Dall’Amico R, Tartaglia S, Casciani S, Greco M, et al. Mutational spectrum of the CTNS gene in Italy. Eur J Hum Genet. 2003;11:503–508
Middleton R, Bradbury M, Webb N, O’Donoghue D, Van’t Hoff W. Cystinosis. A clinicopathological conference. ‘From toddlers to twenties and beyond’ Adult-Paediatric Nephrology Interface Meeting, Manchester 2001. Nephrol Dial Transplant. 2003;18:2492–2495
Ramappa AJ, Pyatt JR. Pregnancy-associated cardiomyopathy occurring in a young patient with nephropathic cystinosis. Cardiol Young. 2010;20:220–222
Rosa TG, Wyse ATDS, Wajner M, Wannmacher CMD. Cysteamine prevents and reverses the inhibition of pyruvate kinase activity caused by cystine in rat heart. Biochim Biophys Acta. 2004;1689:114–119
Town M, Jean G, Cherqui S, Attard M, Forestier L, Whitmore SA, et al. A novel gene encoding an integral membrane protein is mutated in nephropathic cystinosis. Nat Genet. 1998;18:319–324
Tsilou ET, Rubin BI, Reed GF, Iwata F, Gahl W, Kaiser Kupfer MI. Age-related prevalence of anterior segment complications in patients with infantile nephropathic cystinosis. Cornea. 2002;21:173–176
Wilmer MJ, Schoeber JP, Van Den Heuvel LP, Levtchenko EN. Cystinosis: practical tools for diagnosis and treatment. Pediatr Nephrol. 2011;26:205–215

cardiac manifestation; codon 245; CTNS; cystinosin; cystinosis; novel truncating mutation

© 2012 Middle East Journal of Medical Genetics