Postnatal outcome of children with antenatal colonic hyperechogenicity.

OBJECTIVE: To evaluate the postnatal outcome of children with antenatal colonic hyperechogenicity, currently considered as a sign of lysinuria-cystinuria, but which may also be a sign of other disorders with a more severe prognosis. METHOD: We carried out a French multi-centric retrospective study via 15 Multidisciplinary Center for Prenatal Diagnosis from January 2011 to January 2021. We included pregnancies for which fetal colonic hyperechogenicity had been demonstrated. We collected the investigations performed during pregnancy and at birth as well as the main clinical features of the mother and the child. We then established the prevalence of pathologies such as lysinuria-cystinuria (LC), hypotonia-cystinuria syndrome (HC), or lysinuric protein intolerance (LPI). RESULTS: Among the 33 cases of colonic hyperechogenicity collected, and after exclusion of those lost to follow-up, we identified 63% of children with lysinuria-cystinuria, 8% with lysinuric rotein intolerance, and 4% with hypotonia-cystinuria syndrome. CONCLUSION: Management of prenatal hyperechoic colon should include a specialized consultation with a clinical geneticist to discuss further investigations, which could include invasive amniotic fluid sampling for molecular diagnosis. A better understanding of diagnoses and prognosis should improve medical counseling and guide parental decision making.

Antenatal Hyperechogenic Colon and Cystinuria.

A male newborn was investigated for history of antenatal hyperechogenic colon (HEC) detected at 32 weeks of gestation. In the first week of life, urinary ultrasonography showed nephrolithiasis. Urinary amino acid analysis expressed increased excretion of dibasic amino acids, and high urinary cystine levels were detected in both spot and 24-hour urine specimens. He was diagnosed as cystinuria, and genetic analysis of the patient revealed a heterozygous mutation in SLC7A9 gene. Antenatal presentation of cystinuria with HEC is rare and reported to be associated with a more severe disease course.

A mouse model of type B cystinuria due to spontaneous mutation in FVB/NJcl mice.

Cystinuria is an autosomal metabolic disorder caused by mutations in the SLC3A1 and SLC7A9 genes, encoding the amino acid transporter proteins rBAT and b0,+AT, respectively. Based on the causative gene, cystinuria is classified into 3 types: type A (SLC3A1), type B (SLC7A9), and type AB (SLC3A1 and SLC7A9). Patients with cystinuria exhibit hyperexcretion of cystine and dibasic amino acids in the urine and develop cystine crystals due to its low solubility in the urine, often resulting in calculus formation. In this study, we present an inbred strain FVB/NJcl mice affected with cystinuria. In the affected mouse kidney, Slc7a9 expression was completely abolished because of a large sequence deletion in the promoter region of the Slc7a9 mutant allele. Slc7a9-deficient mice with FVB/NJcl genetic background developed cystine calculi in the bladder with high penetrance, as compared to the previously reported mouse models of cystinuria. This model may be useful to understand the determinants of crystal aggregation, affecting calculus formation.

Lipidomics characterization of the lipid metabolism profiles in a cystinuria rat model: Precalculus damage in the kidney of cystinuria.

Cystinuria is a genetic disorder of cystine transport, including defective protein b0,+AT (encoded by SLC7A9), and/or rBAT (encoded by SLC3A1). Patients present hyperexcretion of cystine in the urine, recurrent cystine lithiasis, and progressive decline in kidney function. Moreover, heterodimer transport is defective. To date, little omics data are accessible regarding this metabolic disease caused by membrane proteins. Since membrane function is closely related to changes in the lipidome, we decided to explore the changes in kidney tissue of a self-established cystinuria rat model by performing lipidomic analysis by LC-MS/MS. Our results demonstrated that Slc7a9 deficiency changed the lipid profile of the renal cortex and induced vital modifications in the lipidome, including major alterations in ChE, LPA, and PA. Among those alterations, this lipidomic study highlights the lipid changes that participate in inflammatory responses during cystinuria. As a result, lipid research, perhaps has great potential, for it may lead to the identification of novel therapeutic targets for the prevention and treatment of cystinuria.

Ca2+-mediated higher-order assembly of heterodimers in amino acid transport system b0,+ biogenesis and cystinuria.

Cystinuria is a genetic disorder characterized by overexcretion of dibasic amino acids and cystine, causing recurrent kidney stones and kidney failure. Mutations of the regulatory glycoprotein rBAT and the amino acid transporter b0,+AT, which constitute system b0,+, are linked to type I and non-type I cystinuria respectively and they exhibit distinct phenotypes due to protein trafficking defects or catalytic inactivation. Here, using electron cryo-microscopy and biochemistry, we discover that Ca2+ mediates higher-order assembly of system b0,+. Ca2+ stabilizes the interface between two rBAT molecules, leading to super-dimerization of b0,+AT-rBAT, which in turn facilitates N-glycan maturation and protein trafficking. A cystinuria mutant T216M and mutations of the Ca2+ site of rBAT cause the loss of higher-order assemblies, resulting in protein trapping at the ER and the loss of function. These results provide the molecular basis of system b0,+ biogenesis and type I cystinuria and serve as a guide to develop new therapeutic strategies against it. More broadly, our findings reveal an unprecedented link between transporter oligomeric assembly and protein-trafficking diseases.

Differences in renal cortex transcriptional profiling of wild-type and novel type B cystinuria model rats.

Cystinuria is a genetic disorder of cystine transport that accounts for 1-2% of all cases of renal lithiasis. It is characterized by hyperexcretion of cystine in urine and recurrent cystine lithiasis. Defective transport of cystine into epithelial cells of renal tubules occurs because of mutations of the transport heterodimer, including protein b0,+AT (encoded by SLC7A9) and rBAT (encoded by SLC3A1) linked through a covalent disulfide bond. Study generated a novel type B cystinuria rat model by artificially deleting 7 bp of Slc7a9 gene exon 3 using the CRISPR-Cas9 system, and those Slc7a9-deficient rats were proved to be similar with cystinuria in terms of genome, transcriptome, translation, and biologic phenotypes with no off-target editing. Subsequent comparisons of renal histopathology indicated model rats gained typical secondary changes as medullary fibrosis with no stone formation. A total of 689 DEGs (383 upregulated and 306 downregulated) were differentially expressed in the renal cortex of cystinuria rats. In accordance with the functional annotation of DEGs, the potential role of glutathione metabolism processes in the kidney of cystinuria rat model was proposed, and KEGG analysis results showed that knock-out of Slc7a9 gene triggered more biological changes which has not been studied. In short, for the first time, a rat model and its transcriptional database that mimics the pathogenesis and clinical consequences of human type B cystinuria were generated.

Disrupting Crystal Growth through Molecular Recognition: Designer Therapies for Kidney Stone Prevention.

Aberrant crystallization within the human body can lead to several disease states or adverse outcomes, yet much remains to be understood about the critical stages leading to these events, which can include crystal nucleation and growth, crystal aggregation, and the adhesion of crystals to cells. Kidney stones, which are aggregates of single crystals with physiological origins, are particularly illustrative of pathological crystallization, with 10% of the U.S. population experiencing at least one stone occurrence in their lifetimes. The human record of kidney stones is more than 2000 years old, as noted by Hippocrates in his renowned oath and much later by Robert Hooke in his treatise Micrographia. William Hyde Wollaston, who was a physician, chemist, physicist, and crystallographer, was fascinated with stones, leading him to discover an unusual stone that he described in 1810 as cystic oxide, later corrected to cystine. Despite this long history, however, a fundamental understanding of the stages of stone formation and the rational design of therapies for stone prevention have remained elusive.This Account reviews discoveries and advances from our laboratories that have unraveled the complex crystal growth mechanisms of l-cystine, which forms l-cystine kidney stones in at least 20 000 individuals in the U.S. alone. Although l-cystine stones affect fewer individuals than common calcium oxalate stones, they are usually larger, recur more frequently, and are more likely to cause chronic kidney disease. Real-time in situ atomic force microscopy (AFM) reveals that the crystal growth of hexagonal l-cystine is characterized by a complex mechanism in which six interlaced anisotropic spirals grow synchronously, emanating from a single screw dislocation to generate a micromorphology with the appearance of stacked hexagonal islands. In contrast, proximal heterochiral dislocations produce features that appear to be spirals but actually are closed loops, akin to a Frank-Read source. These unusual and aesthetic growth patterns can be explained by the coincidence of the dislocation Burgers vector and the crystallographic 61 screw axis. Inhibiting l-cystine crystal growth is key to preventing stone formation. Decades of studies of "tailor-made additives", which are imposter molecules that closely resemble the solute and bind to crystal faces through molecular recognition, have demonstrated their effects on crystal properties such as morphology and polymorphism. The ability to visualize crystal growth in real time by AFM enables quantitative measurements of step velocities and, by extension, the effect of prospective inhibitors on growth rates, which can then be used to deduce inhibition mechanisms. Investigations with a wide range of prospective inhibitors revealed the importance of precise molecular recognition for binding l-cystine imposters to crystal sites, which results in step pinning and the inhibition of step advancement as well as the growth of bulk crystals. Moreover, select inhibitors of crystal growth, measured in vitro, reduce or eliminate stone formation in knockout mouse models of cystinuria, promising a new pathway to l-cystine stone prevention. These observations have wide-ranging implications for the design of therapies based on tailor-made additives for diseases associated with aberrant crystallization, from disease-related stones to "xenostones" that form in vivo because of the crystallization of low-solubility therapeutic agents such as antiretroviral agents.

A case of early onset cystinuria in a 4-month-old girl.

Cystinuria is an autosomal recessive disorder characterized by a decrease in the reabsorption of cystine and dibasic amino acids (lysine, ornithine, and arginine) in the renal proximal tubule. It presents with recurrent urolithiasis. Cystinuria accounts for 6-8% of all pediatric urolithiasis. The age of onset is typically 10-30 years. Here, we report a case of early-onset cystinuria. A 4-month-old girl presented with hematuria. We noticed multiple renal calculi in ultrasonography and abdominal computerized tomography scans. The diagnosis was cystinuria with urinary calculus analysis and urinary amino acid analysis. The patient was treated with urine alkalinization and cystine chelating drugs. Gene analysis showed a P482L heterozygous mutation from her mother, and an A70V heterozygous mutation from her father, in the SLC7A9 gene. This gene encodes a putative subunit of the neutral and basic amino acid transport protein, BAT1. Although cystinuria is an autosomal recessive disease, there have been previous reports of P482L heterozygous mutations greatly suppressing cystine reabsorption and causing cystinuria symptoms. Therefore, the highly influential P482L mutation of the SLC7A9 gene may have contributed to the onset of this autosomal recessive disease at an extremely young age.

Milestones in treatments for inborn errors of metabolism: Reflections on Where chemistry and medicine meet.

From Sir Archibald Garrod's initial description of the tetrad of albinism, alkaptonuria, cystinuria, and pentosuria to today, the field of medicine dedicated to inborn errors of metabolism has evolved from disease identification and mechanistic discovery to the development of therapies designed to subvert biochemical defects. In this review, we highlight major milestones in the treatment and diagnosis of inborn errors of metabolism, starting with dietary therapy for phenylketonuria in the 1950s and 1960s, and ending with current approaches in genetic manipulation.

Metabolic consequences of cystinuria.

BACKGROUND: Cystinuria is an inherited disorder of renal amino acid transport that causes recurrent nephrolithiasis and significant morbidity in humans. It has an incidence of 1 in 7000 worldwide making it one of the most common genetic disorders in man. We phenotypically characterized a mouse model of cystinuria type A resultant from knockout of Slc3a1. METHODS: Knockout of Slc3a1 at RNA and protein levels was evaluated using real-time quantitative PCR and immunofluorescence. Slc3a1 knockout mice were placed on normal or breeder chow diets and evaluated for cystine stone formation over time suing x-ray analysis, and the development of kidney injury by measuring injury biomarkers. Kidney injury was also evaluated via histologic analysis. Amino acid levels were measured in the blood of mice using high performance liquid chromatography. Liver glutathione levels were measured using a luminescent-based assay. RESULTS: We confirmed knockout of Slc3a1 at the RNA level, while Slc7a9 RNA representing the co-transporter was preserved. As expected, we observed bladder stone formation in Slc3a1-/- mice. Male Slc3a1-/- mice exhibited lower weights compared to Slc3a1+/+. Slc3a1-/- mice on a regular diet demonstrated elevated blood urea nitrogen (BUN) without elevation of serum creatinine. However, placing the knockout animals on a breeder chow diet, containing a higher cystine concentration, resulted in the development of elevation of both BUN and creatinine indicative of more severe chronic kidney disease. Histological examination revealed that these dietary effects resulted in worsened kidney tubular obstruction and interstitial inflammation as well as worsened bladder inflammation. Cystine is a precursor for the antioxidant molecule glutathione, so we evaluated glutathione levels in the livers of Slc3a1-/- mice. We found significantly lowered levels of both reduced and total glutathione in the knockout animals. CONCLUSIONS: Our results suggest that that diet can affect the development and progression of chronic kidney disease in an animal model of cystinuria, which may have important implications for patients with this disease. Additionally, reduced glutathione may predispose those with cystinuria to injury caused by oxidative stress. Word count: 327.

Effect of increasing doses of cystine-binding thiol drugs on cystine capacity in patients with cystinuria.

Appropriate dosing of cystine-binding thiol drugs in the management of cystinuria has been based on clinical stone activity. When new stones form, the dose is increased. Currently, there is no method of measuring urinary drug levels to guide the titration of therapy. Increasing cystine capacity, a measure of cystine solubility, has been promoted as a method of judging the effects of therapy. In this study, we gave increasing doses of tiopronin or D-penicillamine, depending on the patients' own prescriptions, to ten patients with cystinuria and measured cystine excretion and cystine capacity. The doses were 0, 1, 2, 3 g per day, given in two divided doses, and administered in a random order. Going from 0 to 1 g/day led to an increase in cystine capacity from - 39.1 to 130.4 mg/L (P < 0.009) and decreased 24 h cystine excretion from 1003.9 to 834.8 mg/day (P = 0.039). Increasing the doses from 1 to 2 to 3 g/day had no consistent or significant effect to further increase cystine capacity or decrease cystine excretion. Whether doses higher than 1 g/day have additional clinical benefit is not clear from this study. Limiting doses might be associated with fewer adverse effects without sacrificing the benefit of higher doses if higher doses do not offer clinical importance. However, trials with stone activity as an outcome would be desirable.

Journey of a cystinuric patient with a long-term follow-up from a medical stone clinic: necessity to be SaFER (stone and fragments entirely removed).

There is a lack of studies looking at the longitudinal follow-up of patients with cystine stones. We wanted to assess the journey of cystinuric patients through our specialist metabolic stone clinic to improve the understanding of episodes, interventions and current outcomes in this patient cohort. After ethical approval, all patients who attended our metabolic stone clinic from 1994 to 2014 with at least one cystine stone episode were included in our study. Data were retrospectively analysed for patient demographics, stone episodes or intervention, clinical parameters and patient compliance. Over a period of 21 years, 16 patients with a median age of 15.5 years underwent a mean follow-up of 8.6 years (1-21 years). The mean number of surgical interventions was 3.1 (1-8/patient), but patients who were stone free after their first treatment had lower recurrences (p = 0.91) and lower number of interventions during their follow-up (2.7/patient, compared to those who were not stone free at 4/patient). During their follow-up period, patients with < 3 interventions had a significantly better renal function than those with ≥ 3 surgical interventions (p = 0.04). Additionally, linear regression analysis showed that eGFR was demonstrated to decline with increasing numbers of stone episodes (r2 = 0.169). It was also noted that patients who began early medical management remained stone free during follow-up compared to those who had medical management after ≥ 2 stone episodes, of whom all had a recurrent episode. Our long-term longitudinal study of cystine stone formers highlights that patients who are stone free and receive early metabolic stone screening and medical management after their initial presentation have the lowest recurrence rates and tend to preserve their renal function. Hence, prompt referral for metabolic assessment, and the stone and fragments entirely removed (SaFER) principles are key to preventing stone episodes and improving long-term function.

No evidence for point mutations in the novel renal cystine transporter AGT1/SLC7A13 contributing to the etiology of cystinuria.

BACKGROUND: Cystinuria is caused by the defective renal reabsorption of cystine and dibasic amino acids, and results in cystine stone formation. So far, mutations in two genes have been identified as causative. The SLC3A1/rBAT gene encodes the heavy subunit of the heterodimeric rBAT-b0,+AT transporter, whereas the light chain is encoded by the SLC7A9/ b0,+AT gene. In nearly 85% of patients mutations in both genes are detectable, but a significant number of patients currently remains without a molecular diagnosis. Thus, the existence of a further cystinuria gene had been suggested, and the recently identified AGT1/SLC7A13 represents the long-postulated partner of rBAT and third cystinuria candidate gene. METHODS: We screened a cohort of 17 cystinuria patients for SLC7A13 variants which were negative for SLC3A1 and SLC7A9 mutations. RESULTS: Despite strong evidences for an involvement of SLC7A13 mutations in cystinuria, we could not confirm a relevant role of SLC7A13 for the disease. CONCLUSION: With the exclusion of SLC7A13/AGT1 as the third cystinuria gene accounting for the SLC3A1 and SLC7A9 mutation negative cases, it becomes obvious that other genetic factors should be responsible for the cystinuria phenotype in nearly 15% of patients.

Clinical, Biochemical, and Genetic Findings of Cystinuria in Chinese Children.

BACKGROUND: Cystinuria is a rare inherited renal stone disease caused by mutations in the SLC3A1 and SLC7A9 genes. The Chinese cystinuria phenotype and genotype have rarely been reported in the literature. METHODS: For this research, the clinical features and genetic etiology were analyzed in seven children, and the clinical characteristics were summarized. The blood and urine amino acids and acylcarnitine were analyzed. Additionally, the whole coding sequence and exon-intron junctions of the SLC3A1 and SLC7A9 genes were analyzed. RESULTS: These seven patients with cystinuria were from seven unrelated Chinese families, and they were diagnosed between the ages of 1 month and 16 years old. The urinary amino acids, including ornithine, arginine, and threonine, were elevated in these patients. A homozygous c.325G>A mutation in SLC7A9 was identified in two patients, and six SLC3A1 mutations were found in five patients. CONCLUSIONS: The core pedigree analysis showed that most of the parents carried mutations; however, there was no association between the clinical course and the genotype.

Associating mutations causing cystinuria with disease severity with the aim of providing precision medicine.

BACKGROUND: Cystinuria is an inherited disease that results in the formation of cystine stones in the kidney, which can have serious health complications. Two genes (SLC7A9 and SLC3A1) that form an amino acid transporter are known to be responsible for the disease. Variants that cause the disease disrupt amino acid transport across the cell membrane, leading to the build-up of relatively insoluble cystine, resulting in formation of stones. Assessing the effects of each mutation is critical in order to provide tailored treatment options for patients. We used various computational methods to assess the effects of cystinuria associated mutations, utilising information on protein function, evolutionary conservation and natural population variation of the two genes. We also analysed the ability of some methods to predict the phenotypes of individuals with cystinuria, based on their genotypes, and compared this to clinical data. RESULTS: Using a literature search, we collated a set of 94 SLC3A1 and 58 SLC7A9 point mutations known to be associated with cystinuria. There are differences in sequence location, evolutionary conservation, allele frequency, and predicted effect on protein function between these mutations and other genetic variants of the same genes that occur in a large population. Structural analysis considered how these mutations might lead to cystinuria. For SLC7A9, many mutations swap hydrophobic amino acids for charged amino acids or vice versa, while others affect known functional sites. For SLC3A1, functional information is currently insufficient to make confident predictions but mutations often result in the loss of hydrogen bonds and largely appear to affect protein stability. Finally, we showed that computational predictions of mutation severity were significantly correlated with the disease phenotypes of patients from a clinical study, despite different methods disagreeing for some of their predictions. CONCLUSIONS: The results of this study are promising and highlight the areas of research which must now be pursued to better understand how mutations in SLC3A1 and SLC7A9 cause cystinuria. The application of our approach to a larger data set is essential, but we have shown that computational methods could play an important role in designing more effective personalised treatment options for patients with cystinuria.

Spectrum of mutations in cystinuria patients presenting with prenatal hyperechoic colon.

Cystinuria is a heterogeneous, rare but important cause of inherited kidney stone disease due to mutations in 2 genes: SLC3A1 and SLC7A9. Antenatal hyperechoic colon (HEC) has been reported in some patients as a non-pathological consequence of the intestinal transport defect. We report 83 patients affected by cystinuria: 44 presented prenatally with a HEC (HEC group) and 39 with a classical postnatal form (CC group). SLC3A1 and SLC7A9 were sequenced. All patients were fully genotyped, and the relationship between the genotype and clinical features was analyzed. We identified mutations in SLC3A1 in 80% of the HEC group and in only 49% of the CC group. The SLC3A1 p.Thr216Met mutation was found in 21% of the alleles in the HEC group but was never found in the CC group. Most of the mutations found in the HEC group were considered severe mutations in contrast with the CC group. Twenty-five novel mutations were reported. This study shows a relationship between genotype and the clinical form of cystinuria, suggesting that only the patients with the most severe mutations presented with an HEC. These results emphasized the need for prenatal cystinuria screening using classical third-trimester ultrasound scan and the early management of suspected newborns.

α-Lipoic acid treatment prevents cystine urolithiasis in a mouse model of cystinuria.

Cystinuria is an incompletely dominant disorder characterized by defective urinary cystine reabsorption that results in the formation of cystine-based urinary stones. Current treatment options are limited in their effectiveness at preventing stone recurrence and are often poorly tolerated. We report that the nutritional supplement α-lipoic acid inhibits cystine stone formation in the Slc3a1-/- mouse model of cystinuria by increasing the solubility of urinary cystine. These findings identify a novel therapeutic strategy for the clinical treatment of cystinuria.

Novel cystine transporter in renal proximal tubule identified as a missing partner of cystinuria-related plasma membrane protein rBAT/SLC3A1.

Heterodimeric amino acid transporters play crucial roles in epithelial transport, as well as in cellular nutrition. Among them, the heterodimer of a membrane protein b(0,+)AT/SLC7A9 and its auxiliary subunit rBAT/SLC3A1 is responsible for cystine reabsorption in renal proximal tubules. The mutations in either subunit cause cystinuria, an inherited amino aciduria with impaired renal reabsorption of cystine and dibasic amino acids. However, an unsolved paradox is that rBAT is highly expressed in the S3 segment, the late proximal tubules, whereas b(0,+)AT expression is highest in the S1 segment, the early proximal tubules, so that the presence of an unknown partner of rBAT in the S3 segment has been proposed. In this study, by means of coimmunoprecipitation followed by mass spectrometry, we have found that a membrane protein AGT1/SLC7A13 is the second partner of rBAT. AGT1 is localized in the apical membrane of the S3 segment, where it forms a heterodimer with rBAT. Depletion of rBAT in mice eliminates the expression of AGT1 in the renal apical membrane. We have reconstituted the purified AGT1-rBAT heterodimer into proteoliposomes and showed that AGT1 transports cystine, aspartate, and glutamate. In the apical membrane of the S3 segment, AGT1 is suggested to locate itself in close proximity to sodium-dependent acidic amino acid transporter EAAC1 for efficient functional coupling. EAAC1 is proposed to take up aspartate and glutamate released into luminal fluid by AGT1 due to its countertransport so that preventing the urinary loss of aspartate and glutamate. Taken all together, AGT1 is the long-postulated second cystine transporter in the S3 segment of proximal tubules and a possible candidate to be involved in isolated cystinuria.