The aminoglycoside geneticin permits translational readthrough of the CTNS W138X nonsense mutation in fibroblasts from patients with nephropathic cystinosis.

BACKGROUND: Cystinosis is an ultrarare disorder caused by mutations of the cystinosin (CTNS) gene, encoding a cystine-selective efflux channel in the lysosomes of all cells of the body. Oral therapy with cysteamine reduces intralysosomal cystine accumulation and slows organ deterioration but cannot reverse renal Fanconi syndrome nor prevent the eventual need for renal transplantation. A definitive therapeutic remains elusive. About 15% of cystinosis patients worldwide carry one or more nonsense mutations that halt translation of the CTNS protein. Aminoglycosides such as geneticin (G418) can bind to the mammalian ribosome, relax translational fidelity, and permit readthrough of premature termination codons to produce full-length protein. METHODS: To ascertain whether aminoglycosides permit readthrough of the most common CTNS nonsense mutation, W138X, we studied the effect of G418 on patient fibroblasts. RESULTS: G418 treatment induced translational readthrough of CTNSW138X constructs transfected into HEK293 cells and expression of full-length endogenous CTNS protein in homozygous W138X fibroblasts. CONCLUSIONS: Reduction in intracellular cystine indicates that the CTNS protein produced is functional as a cystine transporter. Interestingly, similar effects were seen even in W138X compound heterozygotes. These studies establish proof-of-principle for the potential of aminoglycosides to treat cystinosis and possibly other monogenic diseases caused by nonsense mutations.

Stem cell microvesicles transfer cystinosin to human cystinotic cells and reduce cystine accumulation in vitro.

Cystinosis is a rare disease caused by homozygous mutations of the CTNS gene, encoding a cystine efflux channel in the lysosomal membrane. In Ctns knockout mice, the pathologic intralysosomal accumulation of cystine that drives progressive organ damage can be reversed by infusion of wildtype bone marrow-derived stem cells, but the mechanism involved is unclear since the exogeneous stem cells are rarely integrated into renal tubules. Here we show that human mesenchymal stem cells, from amniotic fluid or bone marrow, reduce pathologic cystine accumulation in co-cultured CTNS mutant fibroblasts or proximal tubular cells from cystinosis patients. This paracrine effect is associated with release into the culture medium of stem cell microvesicles (100-400 nm diameter) containing wildtype cystinosin protein and CTNS mRNA. Isolated stem cell microvesicles reduce target cell cystine accumulation in a dose-dependent, Annexin V-sensitive manner. Microvesicles from stem cells expressing CTNS(Red) transfer tagged CTNS protein to the lysosome/endosome compartment of cystinotic fibroblasts. Our observations suggest that exogenous stem cells may reprogram the biology of mutant tissues by direct microvesicle transfer of membrane-associated wildtype molecules.

A variant OSR1 allele which disturbs OSR1 mRNA expression in renal progenitor cells is associated with reduction of newborn kidney size and function.

Human nephrons are formed during fetal life through an interaction between the branching ureteric bud and progenitor cells. The wide variation in final nephron number has been attributed to allelic variants of genes regulating ureteric bud arborization. Here, we hypothesize that dysfunctional variants of the Odd-Skipped Related 1 (OSR1) gene which compromise the renal progenitor cell pool might also limit newborn kidney size and function. We show that OSR1 is expressed in human mesenchymal stem cells, the blastemal component of Wilms tumors and CD24+/CD133+ progenitor cells isolated from the mature kidney. We identified an OSR1(rs12329305(T)) allele in 6% of normal Caucasians which alters an exon2 splice enhancer. This variant is predicted to reduce spliceosome-binding affinity and stability of the OSR1 mRNA. In cultured cells, the OSR1(rs12329305)(T) allele produced no identifiable transcript. Normal Caucasian newborns from Montreal with the OSR1(rs12329305)(T) allele had kidney volume 11.8% smaller (P= 0.006) and cord blood cystatin C levels 12.6% higher (P = 0.005) than those with wild-type genotype. Effects of the OSR1(rs12329305)(T) allele are additive with genes that alter ureteric bud branching. Kidney volume was reduced more in newborns bearing both RET(rs1800860)(A) and OSR1(rs12329305)(T) alleles (22%, P= 0.0008) and cystatin C was increased by 17% (P= 0.006) versus newborns with wild-type alleles. Although only two subjects had PAX2(rs11599825)(A) and OSR1(rs12329305)(T) alleles, kidney size was reduced by 27% and cystatin C was increased by 14% versus wild-types (P= NS).

Natural history of adolescent-onset cystinosis.

Cystinosis is a rare autosomal recessive disease caused by mutations of the CTNS gene in which cystine accumulates throughout the body as a result of a defective efflux of cystine from lysosomes. Three phenotypic forms have been described according to the age of onset and the severity of the clinical symptoms: infantile, intermediate, and ocular non-nephropathic cystinosis. Here we report the natural history of cystinosis in a 55-year-old man with intermediate nephropathic cystinosis diagnosed at 9 years of age. Although tubulopathy was unnoticed in the early years, he required transplantation at age 16. Sequencing analysis of all the CTNS exons revealed that the proband is homozygous for a 21-bp in-frame deletion in exon 5 (c. 198_218del21), resulting in an in-frame deletion of 7 amino acids from the N-terminal domain of the cystinosin protein. Our patient has had relatively mild extra-renal disease despite lack of early cysteamine therapy. He has been able to attend university and pursue a professional career into the 6th decade.

Plasma from a case of recurrent idiopathic FSGS perturbs non-muscle myosin IIA (MYH9 protein) in human podocytes.

The MYH9 gene encodes a non-muscle myosin IIA heavy chain (NMMHC-IIA) expressed in podocytes. Heterozygous MYH9 mutations cause a set of overlapping syndromes characterized by variable degrees of deafness, morphologic abnormalities of platelets and focal segmental glomerulosclerosis (FSGS) with progressive renal dysfunction. Similar glomerular lesions are seen in a variety of nephropathies, including an idiopathic form of FSGS in children which recurs in renal allografts, implying a circulating factor that affects glomerular podocyte biology. It is unknown whether NMMHC-IIA is perturbed in the idiopathic form of FSGS. We describe a pediatric patient with typical idiopathic FSGS, in whom proteinuria recurred within hours of deceased donor renal transplantation but who responded to plasmapheresis. We demonstrate in vitro that plasmapheresis effluent from our patient rapidly decreased cultured podocyte levels of the phosphorylated myosin light chain (MLC) that mediates NMMHC-IIA binding to actin and induced dispersion of NMMHC-IIA from its usual position along actin stress fibers. FSGS plasma also caused dispersion of slit diaphragm proteins (nephrin and podocin) and vinculin-positive focal adhesion complexes. Our observations suggest that the putative circulating factor in idiopathic FSGS disrupts normal NMMHC-IIA function in podocytes and might contribute to the pathogenesis of recurrent FSGS in other children.

A human ALDH1A2 gene variant is associated with increased newborn kidney size and serum retinoic acid.

Nephron number varies widely between 0.3 and 1.3 million per kidney in humans. During fetal life, the rate of nephrogenesis is influenced by local retinoic acid (RA) level such that even moderate maternal vitamin A deficiency limits the final nephron number in rodents. Inactivation of genes in the RA pathway causes renal agenesis in mice; however, the impact of retinoids on human kidney development is unknown. To resolve this, we tested for associations between variants of genes involved in RA metabolism (ALDH1A2, CYP26A1, and CYP26B1) and kidney size among normal newborns. Homozygosity for a common (1 in 5) variant, rs7169289(G), within an Sp1 transcription factor motif of the ALDH1A2 gene, showed a significant 22% increase in newborn kidney volume when adjusted for body surface area. Infants bearing this allele had higher umbilical cord blood RA levels compared to those with homozygous wild-type ALDH1A2 rs7169289(A) alleles. Furthermore, the effect of the rs7169289(G) variant was evident in subgroups with or without a previously reported hypomorphic RET 1476(A) proto-oncogene allele that is critical in determining final nephron number. As maternal vitamin A deficiency is widespread in developing countries and may compromise availability of retinol for fetal RA synthesis, our study suggests that the ALDH1A2 rs7169289(G) variant might be protective for such individuals.

Newborn serum retinoic acid level is associated with variants of genes in the retinol metabolism pathway.

Retinoic acid (RA) is a critical regulator of gene expression during embryonic development. In rodents, moderate maternal vitamin A deficiency leads to subtle morphogenetic defects and inactivation of RA pathway genes causes major disturbances of embryogenesis. In this study, we quantified RA in umbilical cord blood of 145 healthy full-term Caucasian infants from Montreal. Sixty seven percent of values were <10 nmol/L (84 were <0.07 nmol/L) and 33% had moderate or high levels. Variation in RA could not be explained by parallel variation in its precursor, retinol (ROL). However, we found that the (A) allele of the rs12591551 single nucleotide polymorphism (SNP) in the ALDH1A2 gene (ALDH1A2rs12591551(A)), occurring in 19% of newborns, was associated with 2.5-fold higher serum RA levels. ALDH1A2 encodes retinaldehyde dehydrogenase (RALDH) 2, which synthesizes RA in fetal tissues. We also found that homozygosity for the (A) allele of the rs12724719 SNP in the CRABP2 gene (CRABP2rs12724719(A/A)) was associated with 4.4-fold increase in umbilical cord serum RA. CRABP2 facilitates RA binding to its cognate receptor complex and transfer to the nucleus. We hypothesize that individual variation in RA pathway genes may account for subtle variations in RA-dependent human embryogenesis.

Wilms tumor arising in a child with X-linked nephrogenic diabetes insipidus.

We report on a child with X-linked nephrogenic diabetes insipidus (NDI) who developed Wilms tumor (WT). Nephrogenic diabetes insipidus is caused by mutations of the arginine vasopressin receptor (AVPR2) or aquaporin-II (AQP2) genes. Wilms tumor is also genetically heterogeneous and is associated with mutations of WT1 (15-20%), WTX (20-30%) and other loci. The boy presented at 5 months with failure to thrive, polyuria, hypernatremia and abdominal mass. Analysis of leukocyte DNA showed a novel missense mutation (Q174H) of the AVPR2 gene, which was not present in his mother. In cells (WitS) isolated from the tumor, WTX mRNA expression and coding sequence were intact. However, we identified a 44-kb homozygous deletion of the WT1 gene spanning exons 4 to 10. The WT1 deletion was not present in leukocyte DNA from the patient or his mother. We also noted strong beta-catenin (CTNNB1) expression in the tumor cells and identified a heterozygote missense Ser45Cys mutation of exon 3 of CTNNB1. However, the mutation was absent both in the constitutional DNA of the patient and his mother. The concurrence of WT and NDI has not been previously reported and may be unrelated. Nevertheless, this case nicely illustrates the sequence of events leading to sporadic Wilms tumor.