Mechanisms of acute hypercalcemia in pediatric patients following the interruption of Denosumab.

PURPOSE: Denosumab is a fully human monoclonal anti-RANK-L antibody that is clinically used to counteract the bone loss induced by exacerbated osteoclast activity. Indeed, its binding to RANK-L prevents the interaction RANK-L/receptor RANK that is essential for osteoclastogenesis and bone resorbing activity. Although there are many medications available to treat bone loss diseases, including bisphosphonates, Denosumab is highly effective since it reduces the bone erosion. The use in pediatric patients is safe. However, some concerns are related to the interruption of the treatment. Indeed, in this study, we reported hypercalcemia in two pediatric patients and alterations of circulating osteoclast precursors. METHODS: Peripheral Blood Mononuclear Cells (PBMC) were isolated from two pediatric patients with hypercalcemia after Denosumab interruption and from 10 controls. Cytofluorimetric analysis and in vitro osteoclastogenesis experiments were performed. RESULTS: Increase of CD16-CD14+CD11b+ cells was revealed in PBMC from patients reflecting the enhanced in vitro osteoclastogenesis. CONCLUSION: Our data suggest that precautions must be taken when Denosumab therapy is interrupted and gradual decrease of dose and/or timing of treatment should be performed. To prevent the onset of hypercalcemia that could be in the discontinuation phase, cytofluorimetric analysis of PBMC should be performed to evaluate osteoclast precursors.

Swallowing dysfunction in patients with nephropathic cystinosis.

INTRODUCTION: Nephropathic cystinosis is a rare autosomal recessive lysosomal storage disorder caused by mutations in the CTNS gene. Patients with nephropathic cystinosis suffer not only from renal disease but have also other systemic complications like myopathy and swallowing dysfunction. Dysphagia for solid food is mentioned in patients with cystinosis, but in clinical practice swallowing investigations are only performed when the patient has complaints. The aim of this study was to explore the swallowing function in patients with cystinosis by use of the Test of Mastication and Swallowing Solids (TOMASS), and to compare their performance with patients with myotonic dystrophy type 1 - a neuromuscular disease in which dysphagia for solid food is a known problem. METHODS: Twenty adult patients with cystinosis (11 men and 9 women, range 19-51 years) and 10 patients with myotonic dystrophy type 1 (5 men and 5 women, range 20-60 years) were included. All cystinosis patients were treated with cysteamine. Data of the two groups were compared with normative data using independent-samples t-tests. In case the variables were not normally distributed, the non-parametric Mann-Whitney U test was used. RESULTS: There was a significant difference in the number of bites, masticatory cycles, swallows and total time between the normal values and cystinosis patients. The results of the cystinosis patients were comparable to those of the patients with myotonic dystrophy. DISCUSSION AND CONCLUSION: Adult patients with cystinosis have significant dysphagia for solid food. Clinicians treating these patients should be aware of this fact. The TOMASS can be performed easily in clinical practice to investigate whether patients with cystinosis have swallowing dysfunction. The swallowing dysfunction can now be diagnosed by use of a non-invasive, very simple, non-harmful test. It can be discussed whether this should be added to the regular care scheme of cystinosis patients in order to regularly follow-up swallowing function.

TRPV1 dysfunction in cystinosis patients harboring the homozygous 57 kb deletion.

Cystinosis is a rare autosomal recessive disorder characterized by lysosomal cystine accumulation due to loss of function of the lysosomal cystine transporter (CTNS). The most common mutation in cystinosis patients of Northern Europe consists of a 57-kb deletion. This deletion not only inactivates the CTNS gene but also extends into the non-coding region upstream of the start codon of the TRPV1 gene, encoding the capsaicin- and heat-sensitive ion channel TRPV1. To evaluate the consequences of the 57-kb deletion on functional TRPV1 expression, we compared thermal, mechanical and chemical sensitivity of cystinosis patients with matched healthy controls. Whereas patients heterozygous for the 57-kb deletion showed normal sensory responses, homozygous subjects exhibited a 60% reduction in vasodilation and pain evoked by capsaicin, as well as an increase in heat detection threshold. Responses to cold, mechanical stimuli or cinnamaldehyde, an agonist of the related nociceptor channel TRPA1, were unaltered. We conclude that cystinosis patients homozygous for the 57-kb deletion exhibit a strong reduction of TRPV1 function, leading to sensory deficiencies akin to the phenotype of TRPV1-deficient mice. These deficits may account for the reported sensory alterations and thermoregulatory deficits in these patients, and provide a paradigm for life-long TRPV1 deficiency in humans.

Pituitary adenylate cyclase-activating polypeptide deficiency associated with increased platelet count and aggregability in nephrotic syndrome.

BACKGROUND: Pituitary adenylate cyclase-activating polypeptide (PACAP) was recently identified as an inhibitor of megakaryopoiesis and platelet aggregability. OBJECTIVE: We studied PACAP levels in children with nephrotic syndrome (NS), which is associated with thrombocytosis, platelet hyperaggregability, and an increased risk of thrombosis. PATIENTS/METHODS: In four children with congenital NS (CNS) and 24 children with idiopathic NS (INS), plasma and urine levels of PACAP and ceruloplasmin were measured, as were platelet counts and platelet aggregation responses to collagen. In CNS patients, in vitro megakaryopoiesis and nuclear factor-κB expression in platelet lysates were also measured. All tests were performed during the nephrotic state and the non-nephrotic state. RESULTS: Urinary losses of PACAP and ceruloplasmin were observed during the nephrotic state, and disappeared during the non-nephrotic state. Plasma PACAP deficiency was more pronounced in CNS patients than in INS patients. Thrombocytosis was observed in all CNS patients and in 11 of 29 INS patients during the nephrotic state. During the PACAP-deficient state, in vitro megakaryopoiesis was increased for CNS patients, and this effect could be reversed by the addition of recombinant PACAP. Platelet hyperaggregability was observed during the nephrotic state in both CNS and INS patients. In INS patients, the addition of recombinant PACAP to patients' platelets was studied, and resulted in decreased aggregation during the nephrotic state. Platelet aggregation correlated inversely with plasma PACAP levels, but not with serum albumin levels. CONCLUSIONS: We demonstrate urinary losses of PACAP and plasma PACAP deficiency in children with NS, associated with thrombocytosis and platelet hyperaggregability.

Carnitine Profile and Effect of Suppletion in Children with Renal Fanconi Syndrome due to Cystinosis.

BACKGROUND: Cystinosis is an autosomal recessive disorder marked by intralysosomal cystine accumulation. Patients present with generalized proximal tubular dysfunction called renal Fanconi syndrome. Urinary carnitine loss results in plasma and muscle carnitine deficiency, but no clinical signs of carnitine deficiency have been described. Also, the optimal dose of carnitine supplementation is undefined. This study aimed to determine whether currently recommended carnitine doses result in adequate correction of plasma carnitine. METHODS: Five cystinosis patients with renal Fanconi syndrome, aged 2-18 years, were included. L-carnitine was prescribed 50 mg/kg/day since diagnosis: median 36 (range 18-207) months. Total and free plasma and urine carnitine and carnitine profiles were measured at study onset, after stopping L-carnitine for 3 months and 3 months after reintroducing L-carnitine 50 mg/kg/day. RESULTS: At study onset, plasma free carnitine was normal in all patients, total carnitine (1/5), acetylcarnitine (3/5), and several short- and medium-chain acylcarnitines ≤10 carbons (5/5) were increased indicating carnitine over-supplementation. Three months after cessation, carnitine profiles normalized and 3/5 patients showed plasma carnitine deficiency. Three months after reintroduction, plasma free carnitine normalized in all patients, however, carnitine profiles were disturbed in 4/5 patients. Urine free carnitine, acetylcarnitine, and acylcarnitines ≤10 carbons were increased in all patients independent of carnitine supplementation. CONCLUSION: Administration of recommended doses L-carnitine (50 mg/kg/day) resulted in over-supplementation. Although the drug is considered to be rather safe, long-term effects of over-supplementation remain unknown warranting cautious use of high doses. Plasma carnitine profile might be used as a monitor, to prevent overdosing.

Increased human dermal microvascular endothelial cell survival induced by cysteamine.

BACKGROUND: Cystinosis is an autosomal recessive disease caused by intralysosomal cystine accumulation, treated with cysteamine. Recently, new adverse effects of cysteamine were reported. Skin biopsies showed microvascular proliferation (angioendotheliomatosis). To examine the mechanism of angioendotheliomatosis associated with cysteamine toxicity, we examined the effect of cysteamine on human dermal microvascular endothelial cells (HDMVEC). METHODS: After cysteamine exposure (range 0-3.0 mM) during 24 h, cell viability was measured using water soluble tetrazolium salt-1 (WST-1) in both control HDMVEC and fibroblasts. Cell proliferation and apoptosis rate were measured in HDMVEC by bromodeoxyuridine (BrdU) incorporation and caspase 3 and caspase 7 activity, respectively. Intracellular glutathione (GSH) was measured in HDMVEC after cysteamine exposure of 0, 0.1 or 1.0 mM. Medium and cysteamine were refreshed every 6 h to mimic the in vivo situation. Next, cell viability in HDMVEC was measured after 24 h of GSH exposure (range 0-10.0 mM). RESULTS: HDMVEC viability and proliferation increased after cysteamine exposure 0.03-3.0 mM (p < 0.01) and 0.03-1.0 mM (p = 0.01) respectively; cell viability in fibroblasts was not affected by incubation with cysteamine. Apoptosis remained unaffected by incubation with 0-1.0 mM cysteamine, 3.0 mM caused increased apoptosis. Intracellular GSH was significantly increased after incubation with cysteamine 0.1 mM (p = 0.02) and 1.0 mM (p < 0.01). HDMVEC viability increased after exposure to GSH 1.0-5.0 mM (p < 0.01). CONCLUSION: Cysteamine concentrations, similar to those described in plasma of cystinosis patients, stimulate HDMVEC viability and proliferation and increase intracellular GSH content. We postulate that this mechanism might underlie angioendotheliomatosis induced by cysteamine.

Elevated concentrations of sedoheptulose in bloodspots of patients with cystinosis caused by the 57-kb deletion: implications for diagnostics and neonatal screening.

Cystinosis is an autosomal recessive lysosomal storage disease caused by mutations in CTNS. The most prevalent CTNS mutation is a homozygous 57-kb deletion that also includes an adjacent gene named SHPK (CARKL), encoding sedoheptulokinase. Patients with this deletion have elevated urinary concentrations of sedoheptulose. Using derivatisation with pentafluorobenzyl hydroxylamine and liquid chromatography-tandem mass spectrometry (LC-MS/MS), we developed a new sensitive method for the quantification of sedoheptulose in dried blood spots. This method can be utilized as a quick screening test to detect cystinosis patients homozygous for the 57-kb deletion in CTNS; which is the most common mutation of cystinosis. Sedoheptulose concentrations in the deleted patients were 6 to 23 times above the upper limit for controls. The assessment of sedoheptulose in a bloodspot from a known cystinosis patient homozygous for the 57-kb deletion retrieved from the Dutch neonatal screening program showed that sedoheptulose was already elevated in the neonatal period. There was no overlap in sedoheptulose levels between cystinosis patients homozygous for the 57-kb deletion and cystinosis patients not homozygous for this deletion. Our presented method can be used prior to mutation analysis to detect cystinosis patients homozygous for the 57-kb deletion. We feel that the presented method enables fast (pre)-symptomatic detection of cystinosis patients homozygous for the 57-kb deletion, allowing early treatment.

Neurocognitive functioning in school-aged cystinosis patients.

INTRODUCTION: Cystinosis is an autosomal recessive disorder leading to intralysosomal cystine accumulation in various tissues. It causes renal Fanconi syndrome and end stage renal failure around the age of 10 years if not treated with cysteamine. Children with cystinosis seem to have a normal intelligence but frequently show learning difficulties. These problems may be due to specific neurocognitive deficits rather than impaired renal function. Whether cysteamine treatment can improve cognitive functioning of cystinosis patients is thus far unknown. We aim to analyze neurocognitive functioning of school-aged cystinosis patients treated with cysteamine in order to identify specific deficits that can lead to learning difficulties. PATIENTS AND METHODS: Fourteen Dutch and Belgian school-aged cystinosis patients were included. Glomerular filtration rate was estimated using the Schwartz formula. Children were tested for general intelligence, visual-motor integration, inhibition, interference, sustained attention, accuracy, planning, visual memory, processing speed, motor planning, fluency and speed, and behavioural and emotional functioning using standardized methods. RESULTS: Glomerular filtration rate ranged from 22 to 120 ml min(-1) 1.73 m(-2). Median full-scale intelligence was below the average of a normal population (87, range 60-132), with a discrepancy between verbal (median 95, range 60-125) and performance (median 87, range 65-130) intelligence. Over 50% of the patients scored poorly on visual-motor integration, sustained attention, visual memory, planning, or motor speed. The other tested areas showed no differences between patients' and normal values. CONCLUSION: Neurocognitive diagnostics are indicated in cystinosis patients. Early recognition of specific deficits and supervision from special education services might reduce learning difficulties and improve school careers.

Growth retardation in children with cystinosis.

Cystinosis is a rare autosomal recessive disorder characterized by the intralysosomal accumulation of cystine in all tissues due to mutations in the CTNS gene (17p13.3). Infantile nephropatic cystinosis is the most severe and the most frequent form of the disease. It causes renal Fanconi syndrome, leading to end stage renal failure around the age of 10 years if left untreated. Cystine accumulation also affects the eyes, muscles, central nervous system and various endocrine organs. Children with cystinosis often suffer from growth retardation, which is multifactorial in origin. It is not only caused by a decreased renal function, but is aggravated by a poor metabolic status due to renal Fanconi syndrome, pronounced feeding difficulties, often requiring tube feeding and possibly cystine accumulation in the bone. Longitudinal growth can be improved by the correction of metabolic and nutritional deficits and by the treatment of hypothyroidism. The cystine depleting drug cysteamine slows down the progression of renal disease, protects extra-renal organs, accelerates growth and therefore should be administered as early as possible. Despite these treatment strategies, growth retardation remains a severe complication in cystinosis patients and frequently requires the administration of recombinant human growth hormone.

Molecular genetic analysis of podocyte genes in focal segmental glomerulosclerosis--a review.

This review deals with podocyte proteins that play a significant role in the structure and function of the glomerular filter. Genetic linkage studies has identified several genes involved in the development of nephrotic syndrome and contributed to the understanding of the pathophysiology of glomerular proteinuria and/or focal segmental glomerulosclerosis. Here, we describe already well-characterized genetic diseases due to mutations in nephrin, podocin, CD2AP, alpha-actinin-4, WT1, and laminin beta2 chain, as well as more recently identified genetic abnormalities in TRPC6, phospholipase C epsilon, and the proteins encoded by the mitochondrial genome. In addition, the role of the proteins which have shown to be important for the structure and functions by gene knockout studies in mice, are also discussed. Furthermore, some rare syndromes with glomerular involvement, in which molecular defects have been recently identified, are briefly described. In summary, this review updates the current knowledge of genetic causes of congenital and childhood nephrotic syndrome and provides new insights into mechanisms of glomerular dysfunction.

P-glycoprotein-deficient mice have proximal tubule dysfunction but are protected against ischemic renal injury.

The multidrug resistance gene 1 product, P-glycoprotein (P-gp), is expressed in several excretory organs, including the apical membrane of proximal tubules. After inducing acute renal failure, P-gp expression is upregulated and this might be a protective function by pumping out toxicants and harmful products of oxidative stress. We characterized renal function of P-gp knockout mice and studied its consequences in renal ischemic damage. Compared with wild-type mice, knockout mice have a lower glomerular filtration rate and renal plasma flow. An augmented urinary excretion of sodium, numerous amino acids, calcium, glucose, and low molecular weight proteins was observed along with an increased diuresis. A higher lithium plasma clearance in the knockout mice suggested proximal tubular dysfunction. Electron microscopy showed mitochondrial abnormalities in proximal tubular cells that could account for decreased adenosine triphosphate levels in the cortex. After inducing ischemia, wild-type mice showed a decrease in creatinine clearance and severe proximal tubular necrosis. In contrast, knockout mice had no signs of tubular damage. Our data indicate that P-gp knockout mice have impaired renal function but are protected against ischemic renal injury.

Focal segmental glomerulosclerosis in a patient homozygous for a CD2AP mutation.

Focal segmental glomerulosclerosis (FSGS) is a histologic diagnosis in several kidney diseases characterized by proteinuria and a severe decrease in kidney function. Mutations in several genes were found in patients with primary FSGS, one of which is a CD2-associated protein CD2AP (originally referred to as CMS). This gene encodes an adaptor protein that plays a role in endocytosis, cell motility, and cell survival. Mice deficient in Cd2ap (the mouse homolog) die due to kidney failure, while heterozygous mice develop lesions similar to those of FSGS patients. In the kidney, CD2AP regulates the actin cytoskeleton. The only previously described patient with CD2AP mutation had a severely truncated protein. In this study, we describe a patient with a novel mutation resulting in a premature stop codon yielding a protein truncated by only 4%. This shortened CD2AP protein displays a significantly decreased F-actin binding efficiency in vitro with no expression of the mutated allele in the patient's lymphocytes. Heterozygous expression of the CD2AP mutation in both parents did not lead to any kidney pathology, as both have normal glomerular filtration rates and no proteinuria.

Glomerular involution in children with frequently relapsing minimal change nephrotic syndrome: an unrecognized form of glomerulosclerosis?

Global glomerulosclerosis can be divided in the vascular (obsolescent) type and the glomerulopathic (solidified) type. In biopsies from children with recurrent nephrotic syndrome owing to minimal change nephropathy (MCN), we noticed small, globally sclerosed glomeruli that appeared to be distinct from global glomerulosclerosis. These small sclerosed glomeruli are best described as involuted glomeruli. We have characterized these involuted glomeruli in detail. We studied biopsies of 18 children (11 male, 7 female) with frequently relapsing MCN and evaluated possible explanatory variables. The involuted glomeruli can be differentiated from the other types of global glomerulosclerosis. Most notable is the presence of vital podocytes and parietal epithelial cells, which have retained their staining characteristics, in between the matrix, and the absence of periglomerular and tubulo-interstitial fibrosis. We observed involuted glomeruli in 12 out of 18 biopsies; the median percentage of involuted glomeruli was 6% (range 0-33%). The percentage of involuted glomeruli correlated with age at renal biopsy and the interval between onset of disease and time of renal biopsy, but not with gender, age at onset of disease, or prednisone dose. Multivariate analysis revealed that the interval between onset of disease and time of renal biopsy was the only independent predictor. In conclusion, glomerular involution is a special form of global glomerulosclerosis. The absence of periglomerular and tubulo-interstitial fibrosis suggests a different pathogenesis. Glomerular involution is a slow process. The clinical data suggest that involution is related to the duration of the disease process.

Successful treatment of respiratory dysfunction in cystinosis by nocturnal non-invasive positive pressure ventilation.

Cystinosis is a rare metabolic disorder characterized by lysosomal cystine accumulation leading to multi-organ damage, with kidneys being clinically first affected. Longer survival of cystinosis patients due to successful renal replacement therapy, revealed previously unknown extra-renal symptoms of cystinosis, generally appearing after the first decade. Respiratory insufficiency caused by overall respiratory muscle myopathy is a severely invalidating and sometimes a life-threatening complication of cystinosis. We report a successful treatment of hypoventilation, due to diaphragm myopathy in a cystinosis patient, by nocturnal non-invasive positive pressure ventilation (NIPPV). After initiation of NIPPV the clinical condition of the patient improved and blood-gasses normalized, indicating that this treatment modality should be considered in cystinosis patients with severe respiratory insufficiency.

[A genetic childhood disease with consequences in adult life: the Denys-Drash syndrome]. / Een genetische kinderziekte met gevolgen op de volwassen leeftijd: Denys-Drash-syndroom.

In a 17-year-old woman with absent sexual development and a congenital nephrotic syndrome leading to renal failure, the Denys-Drash syndrome was diagnosed after development of an ovarian dysgerminoma. The Denys-Drash syndrome is characterised by the triad: progressive nephropathy due to diffuse mesangial sclerosis, male pseudo-hermaphroditism (XY karyotype with ambiguous or female genital organs) and an increased risk of developing Wilms' tumour and gonadoblastoma. The syndrome is generally caused by a genetic defect in the Wilms' tumour suppressor 1 gene (WT1 gene). A WT1 mutation and XY karyotype were also found in this patient. The WT1 gene encodes a transcription factor playing an important role in renal and genital development. The diagnosis of Denys-Drash syndrome had important consequences for the follow-up and treatment of the patient. The second gonad and the native kidneys were removed due to the increased risk of malignancy. Moreover, the finding of a XY karyotype could result in serious psychic problems. Physicians responsible for the health of adults are confronted more and more often with the consequences of childhood diseases. This case illustrates the necessity to inform such physicians about previously untreatable genetic diseases of childhood so that the adequate medical management of these patients can be guaranteed.

[From gene to disease: cystinosis]. / Van gen naar ziekte; cystinose.

Cystinosis is an autosomal recessive disorder caused by an impaired transport of cystine out of lysosomes. The most severe infantile form of cystinosis starts with Fanconi syndrome at the age of 3-6 months. Untreated patients develop renal failure before the age of 10. The cystinosis gene (CTNS) maps to chromosome 17p13, spans 23 kb and is composed of 12 exons. CTNS encodes a 367 amino acid protein, cystinosin, which is a H(+)-driven lysosomal cystine transporter. It is enigmatic how lysosomal cystine accumulation induces the clinical symptoms. ATP depletion was demonstrated in an experimental model consisting of loading lysosomes with cystine dimethylester. The amino-thiol cysteamine depletes lysosomal cystine content by a disulfide-exchange reaction with cystine. Therapy with cysteamine should be administered as early as possible and continued after a renal transplantation as the extra renal damage still progresses. Improved life expectancy of cystinotic patients requires the attention of internists with a special interest for this rare disorder.

ACE inhibitorenalapril diminishes albuminuria in patients with cystinosis.

BACKGROUND/AIMS: Cystinosis, a rare autosomal recessive disease, manifests with renal Fanconi syndrome during the first year of life. Interstitial damage is a major cause of renal failure in patients with cystinosis. We presume that albuminuria contributes to the development of renal failure in these patients. The aim of this study was to examine whether the administration of ACE inhibitor enalapril diminishes albuminuria in patients with cystinosis. METHODS: Five patients with cystinosis aged 4 - 9 years were studied. All patients had Fanconi syndrome and were treated with cysteamine. Median creatinine clearance was 48 ml/min/1.73 m2 (range 21 - 61). The excretion of albumin and alpha1 microglobulin as well as arterial blood pressure and serum creatinine were evaluated before and at 3 months on oral administration of enalapril (0.15 mg/kg once daily). RESULTS: At 3 months on enalapril, albuminuria decreased in all patients (1,042 vs 629 mg per 24 h, p < 0.05). The median reduction of albuminuria was 43% (range: 4 - 72%, p < 0.05). Urinary excretion of alpha1 microglobulin remained constant. Systolic blood pressure decreased from median 110 - 100 mmHg (p < 0.05), while diastolic blood pressure remained stable (median 60 mmHg). Creatinine clearance decreased from median 48 - 45 ml/min/1.73 m2 (p < 0.05) and returned to previous values after discontinuation of enalapril. CONCLUSION: ACE inhibitor enalapril diminishes albuminuria in patients with cystinosis and might be used in these patients in order to slow the progression of renal insufficiency attributed to proteinuria.

Negligible urinary cysteamine loss in cystinosis patients with Fanconi syndrome.

Cystinosis is an inborn error of lysosomal cystine transporter, resulting in cystine accumulation in lysosomes of all cells. Renal Fanconi syndrome is an early sign of kidney involvement in cystinosis patients. Cysteamine, a small amino-thiol, depletes intralysosomal cystine content and reduces organ damage. However, it does not reverse renal Fanconi syndrome and only postpones the progression to renal failure. We examined whether cysteamine could be lost in the urine of cystinosis patients with Fanconi syndrome, which may explain the inefficiency of treatment. Urinary cysteamine loss was studied in 6 cystinosis patients with and without Fanconi syndrome and was less than I% of ingested dose in all patients.

The molecular basis of Dutch infantile nephropathic cystinosis.

Infantile nephropathic cystinosis, an inborn error of metabolism with an autosomal recessive inheritance pattern, is characterized by lysosomal storage of the amino acid cystine due to an impaired transport of cystine out of the lysosomes. Initial clinical features consist of the renal Fanconi syndrome and crystals in the cornea. Oral therapy with cysteamine lowers the intracellular cystine content. Recently, the gene coding for the integral membrane protein cystinosin, which is responsible for membrane transport of cystine (CTNS), was cloned. Mutation analysis of the CTNS gene of Caucasian patients revealed a common 57-kb deletion, and several other mutations spread throughout the entire gene. In the present study, we developed an improved screening method for the detection of the common 57-kb deletion. By use of this method we detected the 57-kb deletion in 59% of the examined Dutch alleles. The remaining alleles were screened for other mutations by genomic sequencing of the different exons, revealing three previously described mutations. Furthermore, we studied a possible genotype-phenotype relation of the homozygous deleted patients, which could not be demonstrated in our study population. Next to biochemical determination of cystine in leukocytes or fibroblasts, molecular genetic analysis enables prenatal diagnosis and facilitates identification of carriers.