Application of next generation sequencing in genetic counseling a case of a couple at risk of cystinosis

Elmonem M.A., Veys K.R., Soliman N.A., van Dyck M., van den Heuvel L.P., Levtchenko E.

Cystinosis is the most common hereditary cause of renal Fanconi syndrome in children. It is an autosomal recessive lysosomal storage disorder caused by mutations in the CTNS gene encoding for the carrier protein cystinosin, transporting cystine out of the lysosomal compartment. Defective cystinosin function leads to intra-lysosomal cystine accumulation in all body cells and organs. The kidneys are initially affected during the first year of life through proximal tubular damage followed by progressive glomerular damage and end stage renal failure during mid-childhood if not treated. Other affected organs include eyes, thyroid, pancreas, gonads, muscles and CNS. Leucocyte cystine assay is the cornerstone for both diagnosis and therapeutic monitoring of the disease. Several lines of treatment are available for cystinosis including the cystine depleting agent cysteamine, renal replacement therapy, hormonal therapy and others; however, no curative treatment is yet available. In the current review we will discuss the most important clinical features of the disease, advantages and disadvantages of the current diagnostic and therapeutic options and the main topics of future research in cystinosis.

Levtchenko E., van den Heuvel L., Emma F., Antignac C.

Nesterova G., Gahl W.A.

Cystinosis is a rare autosomal recessive disorder involving lysosomal storage of the amino acid cystine due to a defect in the membrane transport protein, cystinosin. Since the introduction of kidney transplants and the availability of cystine-depleting medical therapy, this previously fatal disease was transformed into a treatable disorder. Renal allografts and medical therapy targeting the basic metabolic defect have altered the natural hisotry of cystinosis so drastically that patients have a life expectancy extending past 50 years. Consequently, early diagnosis and appropriate therapy are critically important. In this article, we offer a review of the manifestations of cystinosis, including the proximal tubular dysfunction of renal Fanconi syndrome, and discuss the prevention and treatment of the disorder’s systemic complications. We focus on the nephropathic forms of cystinosis, aiming to assist nephrologists and other physicians to develop early recognition and appropriate management of cystinosis patients.

Brodin-Sartorius A., Tête M., Niaudet P., Antignac C., Guest G., Ottolenghi C., Charbit M., Moyse D., Legendre C., Lesavre P., Cochat P., Servais A.

Nephropathic cystinosis is a multisystem autosomal recessive disease caused by cystine accumulation, which is usually treated by oral cysteamine. In order to determine long-term effects of this therapy, we enrolled 86 adult patients (mean age 26.7 years) diagnosed with nephropathic cystinosis, 75 of whom received cysteamine. Therapy was initiated at a mean age of 9.9 years with a mean duration of 17.4 years. By last follow-up, 78 patients had end-stage renal disease (mean age 11.1 years), 62 had hypothyroidism (mean age 13.4), 48 developed diabetes (mean age 17.1 years), and 32 had neuromuscular disorders (mean age 23.3 years). Initiating cysteamine therapy before 5 years of age significantly decreased the incidence and delayed the onset of end-stage renal disease, and significantly delayed the onset of hypothyroidism, diabetes, and neuromuscular disorders. The development of diabetes and hypothyroidism was still significantly delayed, however, in patients in whom therapy was initiated after 5 years of age, compared with untreated patients. The life expectancy was significantly improved in cysteamine-treated versus untreated patients. Thus, cysteamine decreases and delays the onset of complications and improves life expectancy in cystinosis. Hence, cysteamine therapy should be introduced as early as possible during childhood and maintained lifelong.

JAOUAD I.C., ELALAOUI S.C., SBITI A., ELKERH F., BELMAHI L., SEFIANI A.

SummaryConsanguineous marriage is traditionally common throughout Arab countries. This leads to an increased birth prevalence of infants with recessive disorders, congenital malformations, morbidity and mortality. The aim of this study was to evaluate the rate of consanguineous marriage in families with autosomal recessive diseases, and to compare it with the average rate of consanguinity in the Moroccan population. The study was conducted in the Department of Medical Genetics in Rabat on 176 families with autosomal recessive diseases diagnosed and confirmed by clinical, radiological, enzymatic or molecular investigations. The rate of consanguinity was also studied in 852 families who had infants with trisomy 21 confirmed by karyotyping. These families were chosen because: (i) there is no association between trisomy 21 and consanguinity, (ii) these cases are referred from different regions of Morocco and (iii) they concern all social statuses. Among 176 families with autosomal recessive disorders, consanguineous marriages comprised 59.09% of all marriages. The prevalence of consanguinity in Morocco was found to be 15.25% with a mean inbreeding coefficient of 0.0065. The differences in the rates of consanguineous marriages were highly significant when comparing the general population and couples with offspring affected by autosomal recessive conditions. These results place Morocco among the countries in the world with high rates of consanguinity. Autosomal recessive disorders are strongly associated with consanguinity. This study better defines the health risks associated with consanguinity for the development of genetic educational guidelines targeted at the public and the health sector.

Drube J., Schiffer E., Mischak H., Kemper M.J., Neuhaus T., Pape L., Lichtinghagen R., Ehrich J.H.

The renal Fanconi syndrome (FS) is characterized by renal glucosuria, loss of electrolytes, bicarbonate and lactate, generalized hyperaminoaciduria and low-molecular-weight proteinuria. We studied the urinary low-molecular-weight proteome to identify excreted peptides indicative of a pathogenetic mechanism leading to tubular dysfunction.We established a urinary proteome pattern using capillary electrophoresis mass spectrometry (CE-MS) of 7 paediatric patients with cystinosis and 6 patients with ifosfamide-induced FS as the study group, and 54 healthy volunteers and 45 patients suffering from other renal diseases such as lupus nephritis (n = 8), focal segmental glomerulosclerosis (n = 27), minimal change disease (n = 7) and membranous glomerulonephritis (n = 3) as controls. Consequently, we conducted a blinded study consisting of 11 FS patients and 9 patients with renal disease other than FS. Additionally, we applied this pattern to 294 previously measured samples of patients with different renal diseases. Amino acid sequences of some marker proteins were obtained.Specificity for detecting FS was 89% and sensitivity was 82%. The marker peptides constituting the proteome pattern are fragments derived from osteopontin, uromodulin and collagen alpha-1.CE-MS can be used to diagnose FS in paediatric patients and might be a future tool for the non-invasive diagnosis of FS. The reduced amount of the marker proteins osteopontin and uromodulin indicates loss of function of tubular excretion in all patients suffering from FS regardless of the underlying cause. In addition, the six different fragments of the collagen alpha-1 (I) chain were either elevated or reduced in the urine. This indicates a change of proteases in collagen degradation as observed in interstitial fibrosis. These changes were prominent irrespectively of the stages of FS. This indicates fibrosis as an early starting pathogenetic reason for the development of renal insufficiency in FS patients.

Gahl W.A., Balog J.Z., Kleta R.

Context Nephropathic cystinosis causes the renal Fanconi syndrome in childhood. With renal replacement therapy, affected children are living longer and exhibiting previously unseen manifestations of the disease. Contribution This case series describes 100 adults age 18 to 45 years with cystinosis. Ninety-two persons received a renal allograft. Most persons had multiple complications, such as hypothyroidism, hypergonadotropic hypogonadism, pulmonary insufficiency, myopathy, retinopathy, and diabetes. One third died. A history of long-term cysteamine therapy seemed to be associated with a decreased risk for complications and death. Cautions The study was retrospective. Data were from selected patients attending a national referral center. Implication Nephropathic cystinosis is a multisystemic disease that may be mitigated by cysteamine therapy. The Editors Nephropathic cystinosis, the most common identifiable cause of the renal Fanconi syndrome in childhood, is an autosomal recessive storage disease caused by defective transport of cystine out of lysosomes (13). The renal tubular damage of cystinosis, which begins at 6 to 12 months of age, is associated with polyuria, polydipsia, dehydration, acidosis, hypophosphatemic rickets, hypokalemia, hypocalcemic tetany, hypocarnitinemia, and growth retardation. These disorders are treated with nutritional replacements and, sometimes, growth hormone therapy. Renal glomerular damage generally becomes apparent by 2 to 5 years of age and results in end-stage renal disease by 9 to 10 years of age unless cystine-depleting therapy is initiated early in life (13). Renal replacement therapy has transformed cystinosis from an exclusively pediatric disease to one that affects individuals up to (and potentially beyond) 50 years of age. Nonrenal complications of nephropathic cystinosis were initially thought to be limited to photophobia and hypothyroidism. Once kidney transplantation allowed survival past 10 years of age, the multisystemic nature of cystinosis became apparent (4). Complications include retinal blindness (5), vacuolar myopathy (6, 7), swallowing dysfunction (8, 9), diabetes mellitus (10), pancreatic exocrine insufficiency (11), central nervous system involvement (12, 13), pulmonary dysfunction (14), male hypogonadism (15), benign intracranial hypertension (16), vascular calcifications (17), and nodular regenerating hyperplasia of the liver (18). The basic defect in cystinosis was elucidated in 1982 (1, 2), and the causative gene, CTNS (OMIM 606272), was discovered in 1998 (19). CTNS is located on chromosome 17p (20) and encodes cystinosin, a 367amino acid protein with 7 transmembrane domains (19). Cystinosin transports the disulfide amino acid cystine out of lysosomes and into the cytoplasm of cells, where it is reduced to cysteine. This transport process is defective in cystinosis (2123), causing intralysosomal accumulation and, in most cells, crystal formation (Figure 1). This pathologic process is attenuated in some variants of cystinosis with residual transport activity (24). Specifically, onset of renal disease in adolescence indicates intermediate cystinosis (2, 25), and photophobia due to corneal crystals is the only symptom of ocular cystinosis (also called nonnephropathic cystinosis) (2, 26). Figure 1. Clinical findings in adults with nephropathic cystinosis not treated with oral cysteamine. A. B. C. Targeted therapy for nephropathic cystinosis involves oral administration of the free aminothiol cysteamine (27). This membrane-permeable compound enters lysosomes, where it participates with cystine in a disulfide interchange reaction, forming cysteine and cysteinecysteamine mixed disulfide (28), both of which can exit the cystinotic lysosome by using transporters other than the defective cystinosin (29). Oral cysteamine (Cystagon, Mylan Pharmaceuticals, Morgantown, West Virginia), which was approved by the U.S. Food and Drug Administration in 1994, is given every 6 hours at a dosage of 60 to 90 mg/kg of body weight per day (1.3 to 1.95 g/m2 of height per day). When adherence is consistent, this therapy achieves leukocyte cystine depletion of up to 95% and reduces the cystine content of parenchymal tissues, such as the muscle and liver (30). Oral cysteamine therapy preserves renal glomerular function, enhances growth, and obviates the need for l-thyroxine replacement therapy (3133). It also prevents the development of swallowing difficulties (9), coronary artery calcifications (17), and damage to the posterior segment of the eye (34). In addition, topical cysteamine eye drops dissolve the corneal crystals of cystinosis (3537). We present the clinical characteristics of 100 adults with nephropathic cystinosis who were examined at the National Institutes of Health (NIH) Clinical Center between 1985 and 2006. Untreated cystinosis is a devastating disease, with a 33% mortality rate and nearly universal morbidity in adults. Long-term oral cysteamine administration has substantial beneficial effects on survival and final height and weight and helps prevent diabetes mellitus, myopathy, pulmonary dysfunction, and hypercholesterolemia, which are associated with nephropathic cystinosis. Methods Patients All patients were enrolled in NIH Clinical Center protocol 78-HG-0093, Use of Cysteamine in the Treatment of Cystinosis, and gave written informed consent to participate. The protocol was approved by the National Human Genome Research Institute Institutional Review Board and is consistent with the principles of the Declaration of Helsinki. Patients seen only for ophthalmic evaluation and those with intermediate or ocular cystinosis were excluded. Otherwise, every patient with classic nephropathic cystinosis who was admitted to the NIH Clinical Center between January 1985 and May 2006 and was at least 18 years of age at admission was included. Data from each patient's latest admission were examined. This process resulted in analysis of data from 100 consecutive patients, of whom 35 were described in 1993 (38). Twenty-four of the 35 patients have since had follow-up admissions. The diagnosis of nephropathic cystinosis was based on a typical history, the presence of corneal crystals, and an off-treatment polymorphonuclear leukocyte cystine level greater than 3 nmol half-cystine/mg protein (normal value, 200 mg/dL) or current receipt of a statin drug to treat hypercholesterolemia. Retinopathy was defined by clinical examination, legal blindness in at least 1 eye, or an abnormal electroretinogram. Vascular and cerebral calcifications were identified by computed tomography of the chest and brain, respectively. Diabetes mellitus was diagnosed if patients required insulin therapy. Molecular Diagnostic Techniques Mutation analysis of the CTNS gene was done by using a multiplex method to detect the 57-kb deletion, which is present in approximately 50% of North American patients (4244). Role of the Funding Source The study received no external funding. Results General Characteristics One hundred patients age 18 to 45 years (mean age, 26.2 years [SD, 6.5]) who had cystinosis met our criteria for analysis. The male-to-female ratio was 58:42. Of the 44 patients who were evaluated for sexual development, 1 was Tanner stage I, 2 were stage II, 6 were stage III, 21 were stage IV, and 14 were stage V. Three women each delivered 1 healthy child. The leukocyte cystine level while not receiving cysteamine therapy was available for 32 patients; the mean value was 8.3 nmol half-cystine/mg protein (SD, 3.6). Kidney Transplantation Most patients (92%) had received a renal allograft (Table 1), and many received more than 1. For all 92 patients who received a transplant, the first allograft was done at a mean age of 12.3 years (SD, 4.2). Of the 92 initial allografts, 42 were from living donors and 42 were from cadavers; for 8 allografts, the donor was not specified. Forty-four patients received a second renal transplant (13 from living donors, 29 from cadavers, and 2 from an unspecified source), and 6 patients received a third renal transplant (3 from living donors and 3 from cadavers). Seven of the 8 patients with native kidneys were 18 to 21 years of age, and 1 was 27 years of age. Table 1. Patient Characteristics Of the 92 patients who underwent transplantation, 17 were uremic at the time of their most recent NIH admission. Laboratory data, which were available for 74

Shotelersuk V., Larson D., Anikster Y., McDowell G., Lemons R., Bernardini I., Guo J., Thoene J., Gahl W.A.

Nephropathic cystinosis is an autosomal recessive lysosomal storage disease characterized by renal failure at 10 years of age and other systemic complications. The gene for cystinosis, CTNS, has 12 exons. Its 2.6-kb mRNA codes for a 367-amino-acid putative cystine transporter with seven transmembrane domains. Previously reported mutations include a 65-kb "European" deletion involving marker D17S829 and 11 small mutations. Mutation analysis of 108 American-based nephropathic cystinosis patients revealed that 48 patients (44%) were homozygous for the 65-kb deletion, 2 had a smaller major deletion, 11 were homozygous and 3 were heterozygous for 753G-->A (W138X), and 24 had 21 other mutations. In 20 patients (19%), no mutations were found. Of 82 alleles bearing the 65-kb deletion, 38 derived from Germany, 28 from the British Isles, and 4 from Iceland. Eighteen new mutations were identified, including the first reported missense mutations, two in-frame deletions, and mutations in patients of African American, Mexican, and Indian ancestry. CTNS mutations are spread throughout the leader sequence, transmembrane, and nontransmembrane regions. According to a cystinosis clinical severity score, homozygotes for the 65-kb deletion and for W138X have average disease, whereas mutations involving the first amino acids prior to transmembrane domains are associated with mild disease. By northern blot analysis, CTNS was not expressed in patients homozygous for the 65-kb deletion but was expressed in all 15 other patients tested. These data demonstrate the origins of CTNS mutations in America and provide a basis for possible molecular diagnosis in this population.

Town M., Jean G., Cherqui S., Attard M., Forestier L., Whitmore S.A., Callen D.F., Gribouval O., Broyer M., Bates G.P., Hoff W.V., Antignac C.

Nephropathic cystinosis, an autosomal recessive disorder resulting from defective lysosomal transport of cystine, is the most common inherited cause of renal Fanconi syndrome. The cystinosis gene has been mapped to chromosome 17p13. We found that the locus D17S829 was homozygously deleted in 23 out of 70 patients, and identified a novel gene, CTNS, which mapped to the deletion interval. CTNS encodes an integral membrane protein, cystinosin, with features of a lysosomal membrane protein. Eleven different mutations, all predicted to cause loss of function of the protein, were found to segregate with the disorder.

Bois E., Feingold J., Frenay P., Briard M.L.

A national distribution of 66 French patients, from 49 sibships, has been studied. Segregation analysis, using the maximum likelihood method, was found to agree with the theoretical values expected in recessive autosomal inheritance. The birthplaces of these patients show an unequal geographic distribution of cystinosis, the incidence being higher in Western France. Compared with the total number of live births during the period 1959 to 1972, the minimum incidence of the condition in the province of Brittany is 1 per 25 909, and the gene frequency 0.0062. In the rest of France, the minimum incidence is 1 per 326,440 and the gene frequency 0.0018. Application of Dahlberg9s formula gives a similar result. The mean inbreeding coefficient is 530 X 10(-5), a figure 23 times higher than the mean coefficient of France. An indirect test of inbreeding, the distance between parental birthplaces, was studied, first using the French administrative boundaries, second by using kilometers. This distance was constantly smaller for the parents of patients than for the parents of controls. Analysis of two erythrocyte polymorphisms (ABO and Rh) showed a large excess of group A patients when compared with overall French data. These findings are difficult to interpret on genetic grounds. The genetic reasons for the unequal geographic distribution of cystinosis in France are discussed.

Abderhalden E.

El Younsi M., Trabelsi M., Ben Youssef S., Ouertani I., Hammi Y., Achour A., Maazoul F., Kharrat M., Gargah T., M’rad R.

Nephropathic cystinosis is an autosomal recessive disease caused by a mutation in the CTNS gene which encodes cystinosin, a lysosomal cystine transporter. The spectrum of mutations in the CTNS gene is not well defined in the North African population. Here, we investigated twelve patients with nephropathic cystinosis belonging to eight Tunisian families in order to analyze the clinical and genetic characteristics of Tunisian children with infantile nephropathic cystinosis. Clinical data were collected retrospectively. Molecular analysis of the CTNS gene was performed by Sanger sequencing. We describe a new splicing mutation c.971-1G > C in the homozygous state in 6/12 patients which seems to be a founder mutation. The reported deletion of 23nt c.771_793 Del (p.Gly258Serfs*30) was detected in a homozygous state in one patient and in a heterozygous compound state with the c.971-1G > C mutation in 3/12 patients. Two of 12 patients have a deletion of exons 4 and 5 of the CTNS gene. None of our patients had the most common 57-kb deletion. The mutational spectrum in the Tunisian population is different from previously described populations. Thus, a molecular diagnostic strategy must be implemented in Tunisia, by targeting as a priority the common mutations described in this country. Such a strategy will allow a cost-effective diagnosis confirmation as well as early administration of treatment with oral cysteamine. A higher resolution version of the Graphical abstract is available as Supplementary information