Exploring the Contribution of the Transporter AGT1/rBAT in Cystinuria Progression: Insights from Mouse Models and a Retrospective Cohort Study.

More than 20 years have passed since the identification of SLC3A1 and SLC7A9 as causative genes for cystinuria. However, cystinuria patients exhibit significant variability in the age of lithiasis onset, recurrence, and response to treatment, suggesting the presence of modulatory factors influencing cystinuria severity. In 2016, a second renal cystine transporter, AGT1, encoded by the SLC7A13 gene, was discovered. Although it was discarded as a causative gene for cystinuria, its possible effect as a modulatory gene remains unexplored. Thus, we analyzed its function in mouse models of cystinuria, screened the SLC7A13 gene in 34 patients with different lithiasic phenotypes, and functionally characterized the identified variants. Mice results showed that AGT1/rBAT may have a protective role against cystine lithiasis. In addition, among the four missense variants detected in patients, two exhibited a 25% impairment in AGT1/rBAT transport. However, no correlation between SLC7A13 genotypes and lithiasis phenotypes was observed in patients, probably because these variants were found in heterozygous states. In conclusion, our results, consistent with a previous study, suggest that AGT1/rBAT does not have a relevant effect on cystinuria patients, although an impact in patients carrying homozygous pathogenic variants cannot be discarded.

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.

CRISPR/Cas9 engineering of albino cystinuria Type A mice.

Cystinuria Type A is a relatively common genetic kidney disease occurring in 1 in 7,000 people worldwide that results from mutation of the cystine transporter rBAT encoded by Slc3a1. We used CRISPR/Cas9 technology to engineer cystinuria Type A mice via genome editing of the C57BL/6NHsd background. These mice are an improvement on currently available models as they are on a coisogenic genetic background and have a single defined mutation. In order to use albinism to track Cas9 activity, we co-injected gRNAs targeting Slc3a1 and tyrosinase (Tyr) with Cas9 expressing plasmid DNA into mouse embryos. Two different Slc3a1 mutational alleles were derived, with homozygous mice of both demonstrating elevated urinary cystine levels, cystine crystals, and bladder stones. We used whole genome sequencing to evaluate for potential off-target editing. No off-target indels were observed for the top 10 predicted off-targets for Slc3a1 or Tyr. Therefore, we used CRISPR/Cas9 to generate coisogenic albino cystinuria Type A mice that could be used for in vivo imaging, further study, or developing new treatments of cystinuria.

Polymorphism in the PBX1 gene is related to cystinuria in Brazilian families.

The aim of our study was to determine regions of loss of heterozygosity, copy number variation analysis, and single nucleotide polymorphisms (SNPs) in Brazilian patients with cystinuria. A linkage study was performed using DNA samples from six patients with cystinuria and six healthy individuals. Genotyping was done with the Genome-Wide Human SNP 6.0 arrays (Affymetrix, Inc., Santa Clara, CA, USA). For validation, SNPs were genotyped using a TaqMan® SNP Genotyping Assay Kit. The homozygote polymorphic genotype of SNP rs17383719 in the gene PBX1 was more frequent (P = 0.015) in cystinuric patients. The presence of the polymorphic allele for this SNP increased the chance of cystinuria by 3.0-fold (P = 0.036). Pre-B-cell leukaemia transcription factor 1 (PBX1) was overexpressed 3.3-fold in patients with cystinuria. However, when we compared the gene expression findings with the genotyping, patients with a polymorphic homozygote genotype had underexpression of PBX1, while patients with a heterozygote or wild-type homozygote genotype had overexpression of PBX1. There is a 3-fold increase in the risk of the development of cystinuria among individuals with this particular SNP in the PBX1 gene. We postulate that the presence of this SNP alters the expression of PBX1, thus affecting the renal absorption of cystine and other amino acids, predisposing to nephrolithiasis.

Stone growth patterns and risk for surgery among children presenting with hypercalciuria, hypocitraturia and cystinuria as underlying metabolic causes of urolithiasis.

INTRODUCTION: Hypercalciuria, hypocitraturia and cystinuria are the most common underlying metabolic stone abnormalities in children. The present study compared stone growth patterns, stone burden, and the risk of stone-related surgery among these underlying metabolic conditions. METHODS: A retrospective cohort of 356 children with renal stones, followed from 2000 to 2015, was studied. Differences among metabolic groups were determined using Kruskal-Wallis test; the Scheffé-test was used for multiple comparisons to determine differences among single groups. Independent sample t-test was used when adequate, given the sample size, and Chi-squared test was used for categorical variables. Stone growth rates were calculated as differences in diameter divided by time elapsed between U/Ss (mm/year). Logistic regression was performed to assess the effect of initial stone size on the likelihood of surgery. RESULTS: Median stone size at presentation was significantly different among groups, with cystinuria being the group with the largest proportion of stones >10 mm, while patients with stones <5 mm were likely to have a normal metabolic workup (P < 0.05). Stones with a higher growth rate were found in the operative group, while slower growing stones were mostly managed conservatively (3.4 mm/year vs 0.8 mm/year, respectively; P = 0.014). However, stone growth rates were not significantly different among metabolic groups. On the other hand, the rate of new stone formation in cystinuric patients at their first follow-up was 30.4%, which was significantly higher than in patients with hypercalciuria (16.3%) or with a normal metabolic workup (17.2%; P < 0.05). Compared with stones <5 mm, stones measuring 5-10 mm were more than four times more likely to result in surgery, whereas the likelihood of surgery for 10-20 mm or >20 mm stones was almost 16 or 34 times, respectively (P < 0.001). CONCLUSIONS: It is believed that this is the first study to evaluate stone growth patterns, stone burden and surgical risk among children with hypercalciuria, hypocitraturia and cystinuria. Cystinuric patients presented with larger stones at the time of diagnosis, higher new stone formation rates, and were at higher risk of surgery. While no significant difference of growth rate was found among metabolic groups, stones with a higher growth rate were significantly more likely to result in surgical treatment than slower growing stones. Initial stone size, location of largest stone, previous urinary tract infection, and patient's metabolic type significantly influenced the likelihood of a surgical intervention. Better understanding of the natural history ultimately helps surgeons and clinicians defining prognosis, treatment, and prevention plans for pediatric urolithiasis.

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.

Genotype and Phenotype Analysis in Pediatric Patients with Cystinuria.

Cystinuria is an inherited disorder characterized by defective renal reabsorption of cystine and dibasic amino acids leading to nephrolithiasis. This study was conducted to analyze the genotypes and phenotypes of pediatric patients with cystinuria. Eight children from Seoul National University Hospital and Asan Medical Center presenting with cystinuria from January 2003 to June 2016 were retrospectively analyzed. Mutational studies were performed by direct sequencing. Two of the 8 were male and 6 were female. The median ages at onset and diagnosis were 1.5 (range, 0.3-13.6) and 2.6 (range, 0.7-16.7) years, respectively. The median followed up was 7.7 (range, 3.4-14.0) years. Mutational analyses were performed in 7 patients and revealed biallelic SLC3A1 mutations (AA genotype) in 4 patients, a single heterozygous SLC3A1 mutation (A- genotype) in 1 patient, biallelic SLC7A9 mutations (BB genotype) in 1 patient, and a single heterozygous SLC7A9 mutation (B- genotype) in 1 patient. Two of the mutations were novel. No genotype-phenotype correlations were observed, except for earlier onset age in patients with non-AA genotypes than in patients with the AA genotype. All patients suffered from recurrent attacks of symptomatic nephrolithiasis, which lead to urologic interventions. At the last follow-up, 3 patients had a mild-to-moderate degree of renal dysfunction. This is the first study of genotypic and phenotypic analyses of patients with cystinuria in Korea.

PREVALENCE OF CYSTINURIA IN SERVALS ( LEPTAILURUS SERVAL) IN THE UNITED STATES.

Cystinuria is a condition caused by defects in amino acid transport within the kidneys and small intestines. It has been reported in humans, dogs, domestic cats, ferrets, nondomestic canids, and nondomestic felids, including servals ( Leptailurus serval). Genetic mutations have been identified in dogs, humans, and domestic cats. Cystinuria usually follows an autosomal recessive inheritance, although it can be autosomal dominant and sex linked. The primary objective of this study was to screen urine samples dried on filter paper from captive servals in the United States for cystinuria by using the cyanide-nitroprusside screening test. A second objective was to determine whether cystinuria is inheritable in servals. Servals were initially recruited for the study by survey. Owners and institutions interested in participating were sent a second survey and filter paper for collecting urine samples. Samples were collected from 25 servals. One additional serval with confirmed cystine urolithiasis was added for a total sample size of 26 servals. Twenty-seven percent (7/26) were positive, 54% (14/26) were weakly positive, and 19% (5/26) were negative. Sex, reproductive status, and urine collection method had no significant association with test results. This condition is likely underreported in servals and should be ruled out in any serval with nonspecific signs of illness; neurologic signs such as lethargy, ataxia, or seizures; ptyalism; or signs of lower urinary tract disease such as dysuria, hematuria, stranguria, pollakiuria, or urethral obstructions.

Further delineation of genotype-phenotype correlation in homozygous 2p21 deletion syndromes: first description of patients without cystinuria.

Homozygous contiguous gene deletion syndromes are rare. On 2p21, however, several overlapping homozygous gene deletion syndromes have been described, all presenting with cystinuria but otherwise distinct phenotypes. Hypotonia-cystinuria syndrome (HCS, OMIM606407) is characterized by infantile hypotonia, poor feeding, and growth hormone deficiency. Affected individuals carry homozygous deletions including the cystinuria gene SLC3A1 and the adjacent PREPL gene. Larger homozygous deletions in this region encompassing the PPM1B, SLC3A1, PREPL, and C2orf34 (CAMKMT) genes result in a more severe phenotype, the 2p21 deletion syndrome. A phenotype intermediate to HCS and the 2p21 deletion syndrome is termed atypical HCS and is caused by deletion of SLC3A1, PREPL, and C2orf34 (CAMKMT). Using high resolution SNP array molecular karyotyping we identified two siblings with a homozygous deletion of 83 kb partially encompassing the genes PREPL and C2orf34 (CAMKMT), but not the SLC3A1 gene. The affected siblings display a recognizable phenotype which is similar to atypical HCS with regard to growth failure and neuro-muscular features, but is characterized by lack of cystinuria. The patients also exhibit features which have not been reported to date such as cleft palate and genital abnormalities. In conclusion, we report the first patients with a homozygous 2p21 deletion syndrome without cystinuria and further delineate the complex genotype-phenotype correlations of homozygous microdeletion syndromes of this region.

Two novel deletions in hypotonia-cystinuria syndrome.

Hypotonia-cystinuria syndrome (HCS) is an autosomal recessive disorder caused by combined deletions of SLC3A1 and PREPL. Clinical features include cystinuria, neonatal hypotonia with spontaneous improvement, poor feeding in neonates, hyperphagia in childhood, growth hormone deficiency, and variable cognitive problems. Only 14 families with 6 different deletions have been reported. Patients are often initially misdiagnosed, while correct diagnosis enables therapeutic interventions. We report two novel deletions, further characterizing the clinical and molecular genetics spectrum of HCS.

Deletion of C2orf34, PREPL and SLC3A1 causes atypical hypotonia-cystinuria syndrome.

BACKGROUND: Hypotonia-cystinuria syndrome (HCS) and 2p21 deletion syndrome are two recessive contiguous gene deletion syndromes associated with cystinuria type I. The deletions differ in size and the number of genes involved. In HCS patients, only SLC3A1 and PREPL are disrupted. In the 2p21 deletion syndrome, two additional genes (C2orf34 and PPM1B) are lost. OBJECTIVE: Clinical and molecular analysis of two siblings who presented with an atypical HCS phenotype. METHODS: Molecular analysis of the SLC3A1/PREPL locus was performed in the patients using quantitative polymerase chain reaction (PCR) methods. RESULTS: HCS in both siblings was confirmed with the deletion screen of the SLC3A1/PREPL locus. Fine mapping of the breakpoint revealed a deletion of 77.4 kb, including three genes: SLC3A1, PREPL and C2orf34. Features not present in classical HCS were a mild/moderate mental retardation and a respiratory chain complex IV deficiency documented in patient 2. CONCLUSIONS: We report the first patients with a deletion of SLC3A1, PREPL and C2orf34. They present with a phenotype intermediate between HCS and 2p21 deletion syndrome. These patients facilitate the elucidation of the contribution of each gene to the phenotype in the different 2p21 deletion syndromes.

Evaluation of cystine transport in cultured human kidney cells and establishment of cystinuria type I phenotype by antisense technology.

Cystinuria is a rare hereditary disease resulting in recurrent stone formation and the need for repeated invasive interventions. So far, two responsible genes have been identified which encode the two transporters, rBAT and b(0,+)AT forming a heterodimer to transport cystine in proximal tubular cells (PTC) and whose defect results in increased excretion of cystine. A human cell line mimicing the phenotype of cystinuria in vitro is yet to be developed. Human kidney (HK)-2 is a PTC line derived from normal HK. After determining the presence of rBAT gene by RT-PCR and Western blot analysis, radioactively labeled cystine (S(35)) was used to evaluate the functional presence of the amino acid transport in HK-2 cells when cultured in vitro. To achieve a cystinuria type I phenotype in HK-2 cells, the rBAT gene was silenced using antisense oligonucleotides complimentary to human rBAT mRNA. The reduced transport activity of cystine was then determined by radiolabeled cystine uptake measurements. RT-PCR and Western blot confirmed the expression of the rBAT gene in HK-2 cells. Considerable transport of the radio labeled cystine was observed in HK-2 cells and was linearly dependent on the incubation time with the amino acid. The cystine transport in rBAT knockdown cells after incubation with antisense oligonucleotides was significantly lower compared to control (0.76 vs. 0.98%; P=0.0008), proving a transient knock-down of the rBAT gene. This study demonstrates the presence of the b(0,+) amino acid transport system in human proximal tubular HK-2 cells when cultured in vitro. Inhibition of this transport system is possible by using antisense technology. A permanent inhibition of the cystine transport, based on our model, would be useful for the development and evaluation gene therapeutic approaches.

Slc7a9 knockout mouse is a good cystinuria model for antilithiasic pharmacological studies.

Cystinuria is a hereditary disorder caused by a defect in the apical membrane transport system for cystine and dibasic amino acids in renal proximal tubules and intestine, resulting in recurrent urolithiasis. Mutations in SLC3A1 and SLC7A9 genes, that codify for rBAT/b(0,+)AT transporter subunits, cause type A and B cystinuria, respectively. In humans, cystinuria treatment is based on the prevention of calculi formation and its dissolution or breakage. Persistent calculi are treated with thiols [i.e., d-penicillamine (DP) and mercaptopropionylglycine (MPG)] for cystine solubilization. We have developed a new protocol with DP to validate our Slc7a9 knockout mouse model for the study of the therapeutic effect of drugs in the treatment of cystine lithiasis. We performed a 5-wk treatment of individually caged lithiasic mutant mice with a previously tested DP dose. To appraise the evolution of lithiasis throughout the treatment a noninvasive indirect method of calculi quantification was developed: calculi mass was quantified by densitometry of X-ray images from cystinuric mice before and after treatment. Urine was collected in metabolic cage experiments to quantify amino acids in DP-treated and nontreated, nonlithiasic mutant mice. We found significant differences between DP-treated and nontreated knockout mice in calculi size and in urinary cystine excretion. Histopathological analysis showed that globally nontreated mutant mice had more severe and diffuse urinary system damage than DP-treated mice. Our results validate the use of this mouse model for testing the efficacy of potential new drugs against cystinuria.

Renal crystal deposits and histopathology in patients with cystine stones.

We have biopsied the papillae of patients who have cystine stones asking if this stone type is associated with specific tissue changes. We studied seven cystine stone formers (SF) treated with percutaneous nephrolithotomy using digital video imaging of renal papillae for mapping and obtained papillary biopsies. Biopsies were analyzed by routine light and electron microscopy, infrared spectroscopy, electron diffraction, and micro-CT. Many ducts of Bellini (BD) had an enlarged ostium, and all such were plugged with cystine crystals, and had injured or absent lining cells with a surrounding interstitium that was inflamed to fibrotic. Crystal plugs often projected into the urinary space. Many inner medullary collecting ducts (IMCD) were dilated with or without crystal plugging. Apatite crystals were identified in the lumens of loops of Henle and IMCD. Abundance of interstitial Randall's plaque was equivalent in amount to that of non-SF. In the cortex, glomerular obsolescence and interstitial fibrosis exceeded normal. Cystine crystallizes in BD with the probable result of cell injury, interstitial reaction, nephron obstruction, and with the potential of inducing cortical change and loss of IMCD tubular fluid pH regulation, resulting in apatite formation. The pattern of IMCD dilation, and loss of medullary structures is most compatible with such obstruction, either from BD lumen plugs or urinary tract obstruction from stones themselves.

A novel missense mutation of SLC7A9 frequent in Japanese cystinuria cases affecting the C-terminus of the transporter.

Cystinuria is caused by the inherited defect of apical membrane transport systems for cystine and dibasic amino acids in renal proximal tubules. Mutations in either SLC7A9 or SLC3A1 gene result in cystinuria. The mutations of SLC7A9 gene have been identified mainly from Italian, Libyan Jewish, North American, and Spanish patients. In the present study, we have analyzed cystinuria cases from oriental population (mostly Japanese). Mutation analyses of SLC7A9 and SLC3A1 genes were performed on 41 cystinuria patients. The uptake of 14C-labeled cystine in COS-7 cells was measured to determine the functional properties of mutants. The protein expression and localization were examined by Western blot and confocal laser-scanning microscopy. Among 41 patients analyzed, 35 were found to possess mutations in SLC7A9. The most frequent one was a novel missense mutation P482L that affects a residue near the C-terminus end of the protein and causes severe loss of function. In MDCK II and HEK293 cells, we found that P482L protein was expressed and sorted to the plasma membrane as well as wild type. The alteration of Pro482 with amino acids with bulky side chains reduced the transport function of b(0,+)AT/BAT1. Interestingly, the mutations of SLC7A9 for Japanese cystinuria patients are different from those reported for European and American population. The results of the present study contribute toward understanding the distribution and frequency of cystinuria-related mutations of SLC7A9.

Identification of novel SLC3A1 gene mutations in Spanish cystinuria families and association with clinical phenotypes.

Cystinuria is an inherited metabolic disease characterized by an abnormal urinary excretion of cystine and dibasic amino acids, leading to kidney stone formation. Incidence of cystinuria in the Mediterranean Spanish population is one of the highest in the world. In view of the low prevalence of previously reported mutations in the SLC3A1 gene, analyses to identify novel variants were carried out on 20 cystinuria families. Additionally, we investigated the possible association between these molecular variants and clinical phenotypes. Genomic DNA from 48 cystinuria patients, 44 healthy relatives and 81 unrelated controls from the East Mediterranean coast of Spain was screened by conformation sensitive gel electrophoresis. Abnormal patterns were confirmed by nucleotide sequence determination and by further restriction fragment-length polymorphism. We only found 11 genetic variants within the SLC3A1 gene: five known polymorphisms (114C > A, 231T > A, 1136 + 3delT, 1332 + 7T > C and 1338G > A), four point mutations (M467T, R452W, I105R and Y461X), one single base pair deletion (1767delA) and one 2-bp insertion (1670insAT). Two of these genetic variants (I105R and 1670insAT) were described for the first time. All mutations but one were detected in families classified as Type I cystinuria due to the transmission pattern of the disease. Association analyses revealed that 231T > A (M467T), 1136 + 3delT and 1332 + 7T > C genetic variants were statistically related with urinary amino acid excretion in cystinuria patients. Although some molecular variants within the SLC3A1 gene were associated with clinical traits in cystinuria patients, the low detection rate of mutations in this gene strongly suggests that variation of the SLC3A1 is not the major genetic factor contributing to cystinuria in this Mediterranean population.

A mouse model for cystinuria type I.

Cystinuria, one of the most common inborn errors of metabolism in humans, accounts for 1-2% of all cases of renal lithiasis. It is caused by defects in the heterodimeric transporter system rBAT/b0,+AT, which lead to reduced reabsorption of cystine and dibasic amino acids through the epithelial cells of the renal tubules and the intestine. In an N-ethyl-N-nitrosourea mutagenesis screen for recessive mutations we identified a mutant mouse with elevated concentrations of lysine, arginine and ornithine in urine, displaying the clinical syndrome of urolithiasis and its complications. Positional cloning of the causative mutation identified a missense mutation in the solute carrier family 3 member 1 gene (Slc3a1) leading to an amino acid exchange D140G in the extracellular domain of the rBAT protein. The mouse model mimics the aetiology and clinical manifestations of human cystinuria type I, and is suitable for the study of its pathophysiology as well as the evaluation of therapeutic and metaphylactic approaches.

Slc7a9-deficient mice develop cystinuria non-I and cystine urolithiasis.

Cystinuria is a common recessive disorder of renal reabsorption of cystine and dibasic amino acids that results in urolithiasis of cystine. Cystinuria is caused by defects in the amino acid transport system b0,+ (i.e. the rBAT/b0,+AT heteromeric complex). Mutations in SLC3A1, encoding rBAT, cause cystinuria type A, characterized by a silent phenotype in heterozygotes (phenotype I). Mutations in SLC7A9, encoding b0,+AT, cause cystinuria type B, in which heterozygotes in most cases hyperexcrete cystine and dibasic amino acids (phenotype non-I). To facilitate in vivo investigation of b0,+AT in cystinuria, Slc7a9 knockout mice have been generated. Expression of b0,+AT protein is completely abolished in the kidney of Slc7a9-/- mice ('Stones'). In contrast, Stones expressed significant amounts of rBAT protein, which is covalently linked to unidentified light subunit(s). Stones mice present a dramatic hyperexcretion of cystine and dibasic amino acids, while Slc7a9+/- mice show moderate but significant hyperexcretion of these amino acids (phenotype non-I). Forty-two per cent of Stones mice develop cystine calculi in the urinary system. Calculi develop during the first month of life and grow throughout the life span of the animals. Histopathology in kidney reveals typical changes for urolithiasis (tubular and pelvic dilatation, tubular necrosis, tubular hyaline droplets and chronic interstitial nephritis). The fact that some Stones mice, generated in a mixed genetic background, develop cystine calculi from an early age, while others do not develop them in their first year of life, suggests the involvement of modifier genes in the lithiasis phenotype. Thus, Stones provide a valid model of cystinuria which can be used in the study of genetic, pharmacological and environmental factors involved in cystine urolithiasis.

Identification of 12 novel mutations in the SLC3A1 gene in Swedish cystinuria patients.

Cystinuria is an autosomal recessive disorder that affects luminal transport of cystine and dibasic amino acids in the kidneys and the small intestine. Three subtypes of cystinuria can be defined biochemically, and the classical form (type I) has been associated with mutations in the amino acid transporter gene SLC3A1. The mutations detected in SLC3A1 tend to be population specific and have not been previously investigated in Sweden. We have screened the entire coding sequence and the intron/exon boundaries of the SLC3A1 gene in 53 cystinuria patients by means of single strand conformation polymorphism (SSCP) and DNA sequencing. We identified 12 novel mutations (a 2 bp deletion, one splice site mutation, and 10 missense mutations) and detected another three mutations that were previously reported. Five polymorphisms were also identified, four of which were formerly described. The most frequent mutation in this study was the previously reported M467T and it was also detected in the normal population with an allelic frequency of 0.5%. Thirty-seven patients were homozygous for mutations in the SLC3A1 gene and another seven were heterozygous which implies that other genes may be involved in cystinuria. Future investigation of the non-type I cystinuria gene SLC7A9 may complement our results but recent studies also suggest the presence of other potential disease genes.

Spectroscopic and ultrastructural comparative study of cystine calculi in humans and dogs.

The careful analysis of cystine calculi may be important to detect the presence of other urinary alterations (such as hyperuricosuria, hypercalciuria, hyperoxaluria or bacterial infections) that coexist with cystinuria in many patients. For this reason, in the present study, 14 human and 17 canine cystine uroliths have been studied by infrared spectroscopy (IR), scanning electron microscopy (SEM), and energy dispersive X-ray analysis (EDX). According to the infrared analysis, most of the human and canine stones were composed of nearly pure cystine. However, in these calculi of apparently pure cystine, the study by SEM and EDX showed in many cases the presence of small amounts of calcium apatite. The infrared study of several samples heated at 750 degrees C confirmed the presence of phosphate, when it was difficult to detect this component in the spectra of the original samples owing to band overlapping. Other components detected in varying proportions in cystine calculi were magnesium ammonium phosphate hexahydrate (struvite), calcium hydrogen phosphate dihydrate (brushite), calcium oxalate (mono and/or dihydrate) and, in one case, a drug (oxolinic acid).