Cystinosis. A clinicopathological conference. ‘From toddlers to twenties and beyond’ Adult-Paediatric Nephrology Interface Meeting, Manchester 2001

Rachel Middleton1, Mark Bradbury2, Nicholas Webb2, Donal O'Donoghue1 and William Van’t Hoff3

1Department of Renal Medicine, Hope Hospital, Salford, 2Department of Paediatric Nephrology, Manchester Children’s Hospital, Manchester and 3Department of Paediatric Nephrology, Great Ormond Street Hospital for Sick Children, London, UK

Correspondence and offprint requests: Dr Rachel Middleton, Specialist Registrar in Nephrology, Hope Hospital, Stott Lane, Salford, Manchester, M6 8HD, UK. Email: rachel_middleton{at}talk21.com

Keywords: cystinosis; Fanconi syndrome; lysosome; paediatric renal failure



   Case 1
 Top
 Case 1
 Case 2
 Case 3
 Cystinosis: a commentary
 Genetics
 Pathogenesis
 Diagnosis
 Clinical course
 Treatment
 Conclusion
 References
 
In November 1994 a 2-year 8-month-old girl presented with failure to thrive. She was the second child; her parents were first cousins. There was no significant family history other than an uncle with coeliac disease. She was below the 3rd centile for height and weight, and had evidence of rickets on clinical examination. Initial investigations found; haemoglobin (Hb) 12.3 g/dl, white blood cells (WBC) 11.4 x 10–9/l, platelets 309 x 10–9/l, Na+ 134 mmol/l, K+ 2.7 mmol/l, urea 3.1 mmol/l, creatinine 36 µmol/l, bicarbonate 14 mmol/l, albumin 35 g/l, ALP 2748 IU/l, calcium 2.36 mmol/l, phosphate 0.43 mmol/l and PTH 5.9 pmol/l (normal range 1.3–8.5). Urine dipstick revealed +glucose. Urine amino acid chromatography showed marked generalized aminoaciduria consistent with Fanconi syndrome. WBC cystine quantification was 2.24 nmol/1/2 cystine/mg protein. X-ray of hands, wrists and knees revealed florid changes of rickets. Ophthalmology slit lamp examination showed corneal cystine crystals.

These results confirm a diagnosis of cystinosis. She was commenced on phosphate and bicarbonate supplementation 10 ml qds, indomethacin 2 mg/kg and alphacalcidol 1 µg od.

In November 1995, aged 3 years and 8 months, she commenced phosphocysteamine 620 mg bd. Aged 5 years and 5 months, she was transferred from phosphocysteamine to cystagon to a maintenance dose of 150 mg qds. At 7 years and 3 months she was found to have significant proteinuria, protein creatinine ratio 1225 mg/mmol, with an albumin creatinine ratio 291.7 mg/mmol and a plasma albumin of 28 g/l. She was also complaining of vomiting and loss of weight. An upper GI endoscopy was performed; this showed evidence of a pre-pyloric ulcer and linear oesophagitis. A duodenal biopsy showed partial villous atrophy with crypt hyperplasia, in keeping with coeliac disease. She was commenced on a gluten-free diet and increasing doses of enalapril.

In December 1999 she started growth hormone replacement due to continued poor growth below the 3rd centile. The results at last follow up showed K+ 3.6 mmol/l, creatinine 103 µmol/l, bicarbonate 13 mmol/l, phosphate 2.03 mmol/l, urine protein– creatinine ratio (PCR) 1168 mg/mmol, WBC cystine 2.24 nmol/1/2 cystine/mg protein. Bicarbonate supplements were subsequently increased.



   Case 2
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 Case 1
 Case 2
 Case 3
 Cystinosis: a commentary
 Genetics
 Pathogenesis
 Diagnosis
 Clinical course
 Treatment
 Conclusion
 References
 
The younger brother of Case 1 presented after diagnosis of his sister with cystinosis. He was eighteen months old and was asymptomatic other than slight bowing of his legs. Investigations at this time included: creatinine 38 µmol/l, bicarbonate 21 mmol/l, K+ 3.3 mmol/l, phosphate 1 mmol/l, PTH 1.1 pmol/l, WBC cystine 6.4 nmol/1/2 cystine/mg protein. Urine amino acid chromatography revealed a generalized aminoaciduria in a pattern typical of Fanconi syndrome. X-rays of hands, wrists and knees showed evidence of rickets. There was no evidence of crystals on slit lamp examination. He was commenced on phosphate and bicarbonate supplements.

Aged 2 years and 6 months he was commenced on alphacalcidol 1 µg od, indomethacin 12.5 mg bd and phosphocysteamine 620 mg bd, subsequently changing to cystagon. Height and weight were maintained at the 25th centile. An opthalmology review aged 7 years confirmed the presence of corneal and conjunctival crystal deposition. The results at last follow up showed K+ 3.1 mmol/l, creatinine 64 mmol/l, bicarbonate 18 mmol/l, phosphate 1.08 mmol/l, urine PCR 167 mg/mmol, WBC cystine 1.29 nmol/1/2 cystine/mg protein.



   Case 3
 Top
 Case 1
 Case 2
 Case 3
 Cystinosis: a commentary
 Genetics
 Pathogenesis
 Diagnosis
 Clinical course
 Treatment
 Conclusion
 References
 
The brother of Cases 1 and 2 presented 4 days post natally as the parents had declined antenatal diagnosis. Investigations at that time were Na+ 140 mmol/l, K+ 4.7 mmol/l, urea 5.3 mmol/l, creatinine 54 mmol/l, bicarbonate 25 mmol/l, blood pH 7.35 and PTH 6.1 pmol/l. Urinalysis showed +protein. On urine amino acid chromatography there was no evidence of aminoaciduria. WBC cystine was elevated at 3.9 nmol/1/2 cystine/mg protein. X-ray and slit-lamp examination were normal.

He was commenced on phosphocysteamine 5 mg/kg qds in the postnatal period, increasing by 5 mg/kg increments every 3–5 days. This was subsequently changed to cystagon. Height and weight were maintained at the 50th centile. At 5 years of age, ophthalmology review showed evidence of low-density crystal deposition within the cornea. The results at last follow up showed K+ 3.7 mmol/l, creatinine 69 µmol/l, bicarbonate 21 mmol/l, phosphate 1.21 mmol/l, WBC cystine 1.67 nmol/1/2 cystine/mg protein.



   Cystinosis: a commentary
 Top
 Case 1
 Case 2
 Case 3
 Cystinosis: a commentary
 Genetics
 Pathogenesis
 Diagnosis
 Clinical course
 Treatment
 Conclusion
 References
 
Cystinosis is a rare disorder with 120–150 cases in the UK and accounts for ~5% of childhood chronic renal failure. It is a genetic disorder of autosomal recessive inheritance due to defective lysosomal cystine transport leading to excessive intracellular cystine accumulation. The proximal tubule is very sensitive to cystine storage and patients manifest the features of the Fanconi syndrome. Cystinosis is the commonest cause of Fanconi’s syndrome in a child presenting in infancy, whereas in the newborn period other possible causes for consideration include tyrosinaemia, mitochondrial defects, galactosaemia and Lowe’s syndrome. Another site of cystine storage is the cornea where cystine crystals can be seen on slit-lamp examination.

Cystinosis typically presents as in Case 1 with features of the renal Fanconi’s syndrome with anorexia, vomiting, excessive thirst, polyuria, delayed growth, weakness and rickets. These children progress to end-stage renal failure (ESRF) by the end of the first decade unless appropriate therapy is commenced. The later onset forms of the disease, of which there are two to three families in the UK, tend to present in adolescence or early adult life, with proteinuria or chronic renal failure but the Fanconi syndrome is not a classic feature. A third form of the disease is asymptomatic and only comes to light on detection of corneal cystine crystals by routine slit-lamp examination. Patients with this adult, benign form of cystinosis have no evidence of nephropathy.



   Genetics
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 Case 1
 Case 2
 Case 3
 Cystinosis: a commentary
 Genetics
 Pathogenesis
 Diagnosis
 Clinical course
 Treatment
 Conclusion
 References
 
A linkage study was used to localize the cystinosis gene to the short arm of chromosome 17 and a positional cloning approach led to the isolation of the cystinosis gene, CTNS, a relatively small gene at 12 exons. The CTNS gene encodes a new class of integral membrane protein of 367 amino acids, cystinosin. This is predicted to have seven transmembrane domains typical of an integral membrane protein. It has a GY-XX hydrophobic motif in the C-terminus shared by a number of known lysosomal membrane proteins giving strong evidence for a lysosomal membrane location. The N-terminus is heavily glycosylated; characteristic of lysosomal associated membrane proteins and this is thought to infer protection against hydrolytic enzymes within the lysosomal lumen. A large number of mutations have deletion of the gene. Data exists on almost 300 patients from Europe and North America in whom 55 individual genetic mutations have been described. The commonest mutation is a 57 kb deletion, spanning the 5' end of the gene, found in 40% of patient samples tested, particularly those of North European origin. In non-Caucasian families we have never found this deletion, as is the case of this family presented. This is a mutation of amino acid 270 leading to a deletion of serine at that site. Although on reverse transcriptase there is production of cystinosin, it is non-functional, speculated to be due to loss of serine affecting the conformational binding of the cystinosin molecule. Early-onset patients have deletion or mutations predicted to cause loss of protein whereas milder cases are heterozygous for a severe mutation or are homozygous for a milder mutation in non-conserved or functionally less relevant areas of the protein. The mechanisms whereby CTNS mutations lead to cystinosis remain to be elucidated.



   Pathogenesis
 Top
 Case 1
 Case 2
 Case 3
 Cystinosis: a commentary
 Genetics
 Pathogenesis
 Diagnosis
 Clinical course
 Treatment
 Conclusion
 References
 
Definitive work demonstrating that cystinosis is due to cystine accumulation within the lysosome took place in the early 1980s. Gahl et al. [1] loaded lysosomes with cystine in three groups: normals, parents (obligate heterozygotes) and patients. Normals had increased transport of cystine out of the lysosome given the increased rate of loading but with a saturable maximum. In patients, in contrast, there was no transport of cystine out of the lysosome irrespective of the degree of increased cystine loading, illustrating a complete defect in cystine transportation out of the lysosome. Heterozygotes have a maximum transportation of cystine at 50% of the normal rate, giving a good example of a gene–dose effect in an autosomal recessive disorder. Lysosomal cystine transport is carrier-mediated but it has proved impossible to directly isolate the protein responsible for lysosomal cystine transport. Lysosomes are critical to the understanding of cystinosis but probably also critical to the modern understanding of the generation of the Fanconi’s syndrome in a number of different disorders. There is a proportion of evidence to suggest the reason why the proximal tubule is affected in such a profound way is due to mitochondrial dysfunction.

Normal rabbit proximal tubules loaded with cystine by incubation with the cystine dimethyl ester have intracellular cystine concentrations similar to cystinotic cells and exhibit defective proximal tubular transport, analogous to the human Fanconi phenotype [2,3]. DME-loaded tubules exhibit reduced intracellular ATP, reduction in substrate oxidation and in the oxygen consumption directed to Na-K ATPase [4]. This suggests inhibition of mitochondrial oxidative phosphorylation thereby reducing the energy gradient that drives sodium extrusion at the proximal tubule cell and so reducing the gradient for solute entry at the apical membrane [57]. Data from animal studies indicate decreased ATP production in in vitro models but there are no data from human studies. Knockout (ctns-/-) mice accumulate cystine in the kidney and non-renal tissues but do not show evidence of a tubulopathy. However, the proximal tubules in such mice show mitochondrial enlargement and other features that would suggest cell dysfunction [8]. There are studies indicating elevated pyroglutamate excretion in cystinosis and it is thought that ATP depletion might lead to inhibition of the gamma glutamyl cycle with secondary elevation of pyroglutamate [9]. The gamma glutamyl cycle has a less important role in amino acid uptake in the proximal tubule and is heavily dependent on ATP generation, this in turn causes glutathione depletion, which is known to damage mitochondria at the level of cytochrome oxidase (complex IV).



   Diagnosis
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 Case 1
 Case 2
 Case 3
 Cystinosis: a commentary
 Genetics
 Pathogenesis
 Diagnosis
 Clinical course
 Treatment
 Conclusion
 References
 
A marker of the extent of renal tubulopathy is the quantification of tubular proteinuria and enzymuria. Tubular proteins such as retinol binding protein, albumin, beta-2-microglobulin and enzymes such as the lysosomal enzyme N-acetyl-glucosaminidase are grossly elevated compared with normal age-matched controls reflecting the severity of proximal tubular dysfunction in cystinosis. The diagnosis is confirmed clinically by the presence of cystine crystals on slit-lamp examination and biochemically by measurement of cystine in leucocytes. Measurement of cystine in leucocytes is also useful for monitoring response to therapy. Cystine can be measured in leucocytes or granulocytes. Whilst there are theoretical reasons for using polymorphonuclear leucocytes (cystine will accumulate more in these) compared with mixed leucocyte cell sample, there is sufficient data to recommend the mixed preparation. In addition, there are considerable variations in cystine measurements between different European laboratories. Use of a mixed pellet is technically easier and therefore favoured at present. It is generally accepted that heterozygotes will have a WBC cystine concentration of 0.2–1 nmol/1/2 cystine/mg protein and the normal population will have undetectable levels, at least with our method.

Renal biopsies are no longer routinely performed in these patients; however, the characteristic appearances would be of proximal tubular lesions and formation of multinucleate giant cells in glomerular podocytes and the presence of birefringent crystals particularly within interstitial cells.



   Clinical course
 Top
 Case 1
 Case 2
 Case 3
 Cystinosis: a commentary
 Genetics
 Pathogenesis
 Diagnosis
 Clinical course
 Treatment
 Conclusion
 References
 
As well as the severe tubulopathy there is progressive glomerular damage leading to ESRF at ~10 years. In addition, patients have severe growth failure and progressive bony deformity secondary to rickets. Renal transplantation is successful and cystinosis does not recur in the graft. Cystine crystals can be observed in the grafted kidney but they occur in the interstitium and not in tubular epithelium. Renal transplant recipients do not develop a Fanconi-like syndrome, at least due to cystinosis. However, the transplanted kidney does not correct the metabolic disorder elsewhere and cystine accumulation progresses in non-renal tissue causing multi-system dysfunction.

Long-term complications are particularly relevant to adult nephrologists. The whole body can be affected. One of the commonest problems is biochemical hypothyroidism occurring in ~50% of 8–10-year-old children and ~80% of patients by the age of 18 years [10]. Primary hypogonadism with delay in pubertal onset and progression is more marked in males. Diabetes mellitus is an important long-term complication exacerbated by the use of prednisolone after transplantation. The incidence of this is between 50 and 80% in early adult life [11]. Many patients have a distal myopathy, which if not clinically evident can be demonstrated on electrophysiological testing. Psychometric defects are rare and seem to be localized to visio-spatial processing. Many adult patients in their 20s suffer memory loss and have an increased risk of seizures. Stroke-like episodes can occur and major vessel damage and arteritis have been described in a few families. The most worrying complication is progressive cerebral degeneration. It is uncommon but is well described.

In the eye there is progressive crystal deposition leading to gradual loss of visual acuity and uncomfortable grittiness. Retinopathy progression also contributes to visual impairment. Recently, crystal deposition has been described in the pupillary body leading to narrow-angle glaucoma. Swallowing problems are a major problem in young children. The gastrointestinal complications in Case 1 are unusually severe for children with cystinosis. Whilst feeding problems and vomiting are almost universal and can progress due to neurological involvement and myopathy, frank ulceration is uncommon. Cysteamine is an extremely potent gastroduodenal ulcerogen in rats but ulceration is rarely seen in cystinosis despite considerable dosage. However, cysteamine is known to increase gastric acid and serum gastrin. In addition, in one small series, two of four children showed visual and histological evidence of inflammation [12]. In another series, administration of omeprazole led to significant attenuation of the cysteamine-mediated increase in acid and gastric production. Pancreatic exocrine insufficiency is rare. A restrictive lung defect has recently been described probably again related to the myopathy.



   Treatment
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 Case 1
 Case 2
 Case 3
 Cystinosis: a commentary
 Genetics
 Pathogenesis
 Diagnosis
 Clinical course
 Treatment
 Conclusion
 References
 
General treatments are critical and were highlighted in the case presentation. Of particular note is nutrition, most of our patients are on enteral or supplementary nutrition many with naso-gastric tube or a gastrostomy in place. Indomethacin has been widely used in Europe since the 1980s reducing renal plasma flow and enhancing tubular sodium reabsorption. It reduces polyuria by ~30%, patients feel better, calorie intake and growth both improve but it has a major, although fortunately rare, side effect of peptic ulceration. It is of value in a selected number of patients for whom biochemical homeostasis and growth is not optimized after 1 year of follow-up.

Cysteamine, or the pro-drug phosphocysteamine, are specific agents that reduce leucocyte and lysosomal cystine. Cysteamine has a biochemical structure similar to cysteine, which is the amino acid that in combination forms cystine with a sulphur–sulphur bond. The fundamental defect in cystinosis consists of failure of the disulphide amino acid cystine to exit the lysosome due to an aberrant cystine carrier in the lysosomal membrane. Cysteamine forms a mixed disulphide bond with cysteine to produce a compound that has a charge and structure similar to lysine and this compound exits the lysosome on the lysine porter, which is intact and fully functional in cystinosis. Cysteamine was the original form given but is unpalatable, phosphocysteamine and cysteamine bitartrate (Cystagon) are pro-drugs of cysteamine. An arbitrary therapeutic target for leucocyte cystine of 1 nmol/1/2 cystine/mg protein is set, as this is the upper limit of cystine seen in unaffected heterozygotes who have slightly elevated cystine levels but no nephropathy.

Retrospective analysis of a 30-year experience by the National Institute of Health [13] provides the best available data on efficacy of cysteamine therapy. Patients are divided into three groups; Group 1, who never received cysteamine, followed up at ~8 years old had a mean creatinine clearance of 8 ml/min. Group 2 having received cysteamine treatment after the age of 2 years, had a marginally improved creatinine clearance. Group 3 who received cysteamine before 2 years of age and consistently had low levels of cystine, followed up at 8 years had a much improved creatinine clearance and better cystine depletion. Growth is also improved with cysteamine and the cases in point illustrate this. Cysteamine can be of value for the extra-renal complications of cystinosis so this must be continued after transplantation.

Systemic cysteamine does not penetrate the cornea and topical eye drops need to be given intensively especially in the early phase and are very effective in clearing cystine crystals.



   Conclusion
 Top
 Case 1
 Case 2
 Case 3
 Cystinosis: a commentary
 Genetics
 Pathogenesis
 Diagnosis
 Clinical course
 Treatment
 Conclusion
 References
 
Cystinosis is a metabolic disorder of lysosomal storage causing a profound Fanconi’s syndrome but also multi-system dysfunction. It is amenable to cysteamine therapy. The defect is due to mutations in CTNS leading to defective cystinosin function and the most common mutation is a large deletion.

The authors recognise that there has been further progress in our understanding of the molecular basis of cystinosis since this case discussion.

Conflict of interest statement. None declared.



   References
 Top
 Case 1
 Case 2
 Case 3
 Cystinosis: a commentary
 Genetics
 Pathogenesis
 Diagnosis
 Clinical course
 Treatment
 Conclusion
 References
 

  1. Gahl WA, Tietze F, Bashan N, Bernadini I, Raiford D, Schulman JD. Characteristics of cystine counter-transport in normal and cystinotic lysosome-rich leucocyte granular fractions. Biochem J 1983; 216: 393–400[ISI][Medline]
  2. Sakarcan A, Timmons C, Baum M. Intracellular distribution of cystine in cystine-loaded proximal tubules. Paediatr Res 1990; 35: 447–450
  3. Salmon RF, Baum M. Intracellular cystine loading inhibits transport in the rabbit proximal convoluted tubule. J Clin Invest 1990; 85: 340–344[ISI][Medline]
  4. Coor C, Salmon RF, Quigley R, Marver D, Baum M. Role of adenosine triphosphate (ATP) and NaKATPase in the inhibition of proximal tubule transport with intracellular cystine loading. J Clin Invest 1991; 87: 955–961[ISI][Medline]
  5. Foreman JW, Benson JL. Effect of cystine loading and cystine dimethyl ester on renal brush border membrane transport. Paediatr Nephrol 1990; 4: 236–239[ISI][Medline]
  6. Coor C, Salmon RF, Quigley R, Marver D, Baum D. Role of adenosine triphosphate and NAK ATPase in the inhibition of proximal tubule transport with intracellular cystine loading. J Clin Invest 1991; 87: 955–961[ISI][Medline]
  7. Sakarcan A, Arichetta R, Baum M. Intracellular cystine loading causes proximal tubule respiratory dysfunction; effect of glycine. Paediatr Res 1992; 32: 710–713[Abstract]
  8. Haq S, van’t Hoff W. Cellular dysfunction in cystinosis. In: Broyer M, ed. Cystinosis. Elsevier, 1999; 14–19
  9. Rizzo C, Ribes A, Pastore A et al. Pyroglutamic aciduria and nephropathic cystinosis. J Inher Metab Dis 1999; 22: 224–226[CrossRef][ISI][Medline]
  10. Broyer M, Tête MJ. Complications tardives de la cysinose. A propos de 33 cas ayant dépassé 18 ans. Ann Paediatr 1995; 42: 635–642
  11. Broyer M, Tête MJ, Gubler MC. Late symptoms in infantile cystinosis. Paediatr Nephrol 1987; 1: 519–524[ISI][Medline]
  12. Wenner WJ, Murphy JL. The effects of cysteamine on the upper gastrointestinal tract of children with cystinosis. Pediatr Nephrol 1997; 11: 600–603[CrossRef][ISI][Medline]
  13. Markello TC, Bernadini IM, Gahl WA. Improved renal function in children with cystinosis treated with cysteamine. N Engl J Med 1993; 328: 1157–1162[Abstract/Free Full Text]