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Molecular genetic studies on cystinuria
Linköping University, Department of Clinical and Experimental Medicine, Cell Biology. Linköping University, Faculty of Health Sciences.
2007 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Cystinuria is defined as an inherited disorder characterized by increased urinary excretion of cystine and the dibasic amino acids arginine, lysine and ornithine. The only clinical manifestation of cystinuria is renal cystine stone formation due to the low solubility of cystine in the urine. Cystinuria can be attributed to mutations in the SLC3A1 and SLC7A9 genes in the majority of all cases and it has been a common expectation that molecular genetic studies of cystinuria would aid in understanding of the varying clinical outcome seen in the disease. Besides human, the disease has been most extensively studied in the domestic dog.

The present study was undertaken to investigate the molecular genetic basis of cystinuria in patients from Sweden and to correlate genetic findings with phenotypes produced regarding cystine and dibasic amino acid excretion. Further, attempts were made to elucidate the molecular genetics of cystinuria in the dog.

The entire coding sequences of the SLC3A1 and SLC7A9 genes were analysed by means of SSCA and DNA sequencing in 53 cystinuria patients and genetic findings were related to urinary excretion of cystine and dibasic amino acids in a subset of the patient group. We detected a total number of 22 different mutations in the SLC3A1 and SLC7A9 genes, 18 of which were described for the first time. We have found a probable genetic cause of cystinuria in approximately 74 % of our patients and a possible contribution to the disease in another 19 %. Mutations in the SLC3A1 gene is the major cause of cystinuria in our group, with only a minor contribution of SLC7A9 mutations. The group of patients presenting SLC3A1 mutations in a heterozygous state or lacking mutations in both genes had higher values of total urinary cystine and dibasic amino acids compared to patients homozygous for SLC3A1 mutations. The reason for this discrepancy remains unclear, but the possible impact of medical treatment with sulfhydryl compounds on total cystine values was ruled out.

Sequencing of the full-length canine SLC7A9 cDNA was accomplished using the RACE technology and results from mutation analyses of SLC7A9 and SLC3A1 in cystinuric dogs showed that only two out of 13 dogs have mutations with possible impact on protein function in these genes. DNA sequencing was used for all exons of both genes in the dog, and in human cystinuria patients, all samples lacking mutations or showing heterozygosity after SSCA screening were sequenced in both genes as well. This implies that all point mutations present have been detected, but the possibility of mutations escaping PCR based methods as well as mutations in regulatory parts of the SLC3A1 and SLC7A9 genes remains in cases lacking a full molecular genetic explanation of the disease.

Finally, clinical and genetic data from our study of cystinuria both in man and dog exemplifies that manifestation and clinical severity of cystinuria is not determined by genetic alterations in the SLC3A1 and SLC7A9 alone. Environmental factors, congenital malformations and modulating genetic factors are all possible contributors to the clinical outcome of cystinuria.

Place, publisher, year, edition, pages
Institutionen för klinisk och experimentell medicin , 2007. , 50 p.
Series
Linköping University Medical Dissertations, ISSN 0345-0082 ; 1034
Keyword [en]
Amino acid transport systems, Amino acids diamino, Mutation, Sulphydryl compounds, urine, Cystine, Carrier proteins, Cystinuria, DNA mutational analysis, Dog diseases, genetic, Membrane glycoproteins
National Category
Medical Genetics
Identifiers
URN: urn:nbn:se:liu:diva-10465ISBN: 978-91-85895-22-9 (print)OAI: oai:DiVA.org:liu-10465DiVA: diva2:17217
Public defence
2007-12-13, Eken, Campus US, Linköpings universitet, Linköping, 09:00 (English)
Opponent
Supervisors
Available from: 2008-02-13 Created: 2008-02-13 Last updated: 2009-04-29
List of papers
1. Identification of 12 novel mutations in the SLC3A1 gene in Swedish cystinuria patients
Open this publication in new window or tab >>Identification of 12 novel mutations in the SLC3A1 gene in Swedish cystinuria patients
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2001 (English)In: Human Mutation, ISSN 1059-7794, E-ISSN 1098-1004, Vol. 18, no 6, 516-525 p.Article in journal (Refereed) Published
Abstract [en]

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.

Keyword
cystinuria, CSNU, CNSU1, CNSU3, SLC3A1, SLC7A9, transporter, amino acid
National Category
Medical and Health Sciences
Identifiers
urn:nbn:se:liu:diva-12855 (URN)10.1002/humu.1228 (DOI)
Available from: 2008-02-13 Created: 2008-02-13 Last updated: 2017-12-14Bibliographically approved
2. Mutation analysis of SLC7A9 in cystinuria patients in Sweden
Open this publication in new window or tab >>Mutation analysis of SLC7A9 in cystinuria patients in Sweden
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2003 (English)In: Genetic Testing, ISSN 1090-6576, E-ISSN 1557-7473, Vol. 7, no 1, 13-20 p.Article in journal (Refereed) Published
Abstract [en]

Cystinuria is an autosomal recessive disorder characterized by increased urinary excretion of cystine and dibasic amino acids, which cause recurrent stone formation in affected individuals. Three subtypes of cystinuria have been described (type I, II, and III): type I is caused by mutations in the SLC3A1 gene, whereas non-type I (II and III) has been associated with SLC7A9 mutations. Of the 53 patients reported in our previous work, patients that showed SLC7A9 mutations in single-strand conformation polymorphism (SSCP) screening and/or either lacked or showed heterozygosity for SLC3A1 mutations were included in the present study. The entire coding region and the exon/intron boundaries of the SLC7A9 gene were analyzed by means of both SSCP and DNA sequencing in 16 patients, all but one of which were clinically diagnosed as homozygous cystinurics. Three novel SLC7A9 mutations were identified in the patient group: two missense mutations (P261L and V330M), and one single base-pair deletion (1009 delA). We also detected the previously reported A182T and nine novel polymorphisms in the patients. Mutations V330M and 1009delA occurred on different alleles in one individual, and we suggest that these mutations cause cystinuria in this patient. One patient that was homozygously mutated in the SLC3A1 gene carried the third novel mutation (P261L). We conclude that SLC3A1 is still the major disease gene among Swedish cystinuria patients, with only a minor contribution of SLC7A9 mutations as the genetic basis of cystinuria. The absence of SLC3A1 and SLC7A9 mutations in a substantial proportion of the patients implies that mutations in parts of the genes that were not analyzed may be present, as well as large deletions that escape detection by the methods used. However, our results raise the question of whether other, as yet unknown genes, may also be involved in cystinuria.

National Category
Medical and Health Sciences
Identifiers
urn:nbn:se:liu:diva-12856 (URN)10.1089/109065703321560886 (DOI)
Available from: 2008-02-13 Created: 2008-02-13 Last updated: 2017-12-14Bibliographically approved
3. Urinary excretion of total cystine and the dibasic amino acids arginine, lysine and ornithine in relation to genetic findings in patients with cystinuria treated with sulfhydryl compounds
Open this publication in new window or tab >>Urinary excretion of total cystine and the dibasic amino acids arginine, lysine and ornithine in relation to genetic findings in patients with cystinuria treated with sulfhydryl compounds
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2003 (English)In: Urological research, ISSN 0300-5623, E-ISSN 1434-0879, Vol. 31, no 6, 417-425 p.Article in journal (Refereed) Published
Abstract [en]

Advances in molecular genetics have brought a deeper understanding of cystinuria. This autosomal recessive disease, which is caused by a defective tubular reabsorption of cystine and the three dibasic amino acids arginine, lysine and ornithine, results in a lifelong risk of renal stone formation because of the low solubility of cystine in urine. Mutations detected within the two genes known to be associated with cystinuria, SLC3A1 (related to type I) and SLC7A9 (related to non-type I), cannot, however, in all cases explain the disease. Inasmuch as a high urinary concentration of cystine is the basis of stone formation in these patients, our aim was to measure urinary total cystine, arginine, lysine and ornithine, in patients currently lacking a full genetic explanation for their disease. Thirty-three patients with cystinuria who were on long-term treatment with tiopronin or D-penicillamine were divided into two groups. Group 1 comprised eight patients who carried mutation in one of the SLC3A1 alleles and two patients who completely lacked mutations both in the SLC3A1 and the SLC7A9 genes, that is genetic findings discordant with the increased urinary excretion of cystine and the dibasic amino acids in these patients. Group 2 comprised 23 patients homozygous for mutations within SLC3A1, that is genetic findings in accordance with the excretion pattern of classic type I cystinuria. When the two groups were compared, Group 1 had a significantly higher total urinary excretion of cystine (p<0.01) as well as of arginine, lysine and ornithine (p<0.05) than Group 2. Also, when the two patients without mutations were excluded from the calculations, there still was a significant difference in the urinary excretion of total cystine (p<0.05). This suggests that the two patients without any detected mutations in the two known cystine transport genes also contributed to the difference. These unexpected findings indicate that an additional gene or genes participate in the urinary cystine reabsorption in the cystinuric patients who currently are without a full genetic explanation for their disease.

Keyword
Cystinuria, Urinary cystine, Amino acid transport, SLC3A1, SLC7A9, Inherited disease
National Category
Medical and Health Sciences
Identifiers
urn:nbn:se:liu:diva-12857 (URN)10.1007/s00240-003-0366-6 (DOI)
Available from: 2008-02-13 Created: 2008-02-13 Last updated: 2017-12-14Bibliographically approved
4. SLC7A9 cDNA clonng and mutational analysis of SLC3A1 and SLC7A9 in canine cystinuria
Open this publication in new window or tab >>SLC7A9 cDNA clonng and mutational analysis of SLC3A1 and SLC7A9 in canine cystinuria
2006 (English)In: Mammalian Genome, ISSN 0938-8990, Vol. 17, no 7, 769-776 p.Article in journal (Refereed) Published
Abstract [en]

Cystinuria is a genetic disorder in the domestic dog that leads to recurrent urolith formation. The genetic basis of the disorder is best characterized in humans and is caused by mutations in one of the amino acid transporter genes SLC3A1 or SLC7A9, which results in hyperexcretion of cystine and the dibasic amino acids in the urine and subsequent precipitation of cystine due to its low solubility in urine. In this study we describe the cloning of the canine SLC7A9 cDNA and present a thorough mutation analysis of the coding SLC3A1 and SLC7A9 regions in cystinuric dogs of different breeds. Mutation analysis of the two cystinuria disease genes revealed one SLC7A9 mutation (A217T) and two SLC3A1 mutations (I192V and S698G) in French and English Bulldogs that affect nonconserved amino acid residues, arguing against functional impact on the proteins. The absence of deleterious mutations linked to cystinuria in the remainder of our panel of cystinuric dogs is surprising because SLC3A1 or SLC7A9 mutations explain approximately 70% of all human cystinuria cases studied. The present study, along with previous investigations of canine and human cystinuria, implies that regulatory parts of the SLC3A1 and SLC7A9 genes as well as other unknown genes may harbor mutations causing cystinuria.

National Category
Medical and Health Sciences
Identifiers
urn:nbn:se:liu:diva-12858 (URN)10.1007/s00335-005-0146-4 (DOI)
Available from: 2008-02-13 Created: 2008-02-13

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