GPX3 rs8177412 Polymorphism Modifies Risk of Upper Urothelial Tumors in Patients with Balkan Endemic Nephropathy.

Current data suggest that aristolochic acid (AA) exposure is a putative cause of Balkan endemic nephropathy (BEN), a chronic kidney disease strongly associated with upper tract urothelial carcinoma. The cellular metabolism of AA is associated with the production of reactive oxygen species, resulting in oxidative distress. Purpose: Therefore, the aim of this study was to analyze individual, combined and cumulative effect of antioxidant gene polymorphisms (Nrf2 rs6721961, KEAP1 rs1048290, GSTP1AB rs1695, GSTP1CD rs1138272, GPX3 rs8177412 and MDR1 rs1045642), as well as GSTP1ABCD haplotypes with the risk for BEN development and associated urothelial cell carcinoma in 209 BEN patients and 140 controls from endemic areas. Experimental method: Genotyping was performed using polymerase chain reaction (PCR) and PCR with confronting two-pair primers (PCR-CTTP) methods. Results: We found that female patients carrying both variant GPX3 rs8177412 and MDR1 rs1045642 genotypes in combination exhibited significant risk towards BEN (OR 1 = 3.34, 95% CI = 1.16-9.60, p = 0.025; OR 2 = 3.79, 95% CI = 1.27-11.24, p = 0.016). Moreover, significant association was determined between GPX3rs8174412 polymorphism and risk for urothelial carcinoma. Carriers of variant GPX3*TC + CC genotype were at eight-fold increased risk of BEN-associated urothelial tumors development. There was no individual or combined impact on BEN development and BEN-associated tumors among all examined polymorphisms. The haplotype consisting of variant alleles for both polymorphisms G and T was associated with 1.6-fold increased risk although statistically insignificant (OR = 1.64; 95% CI = 0.75-3.58; p = 0.21). Conclusions: Regarding GPX3 rs8177412 polymorphism, the gene variant that confers lower expression is associated with significant increase in upper urothelial carcinoma risk. Therefore, BEN patients carrying variant GPX3 genotype should be more frequently monitored for possible upper tract urothelial carcinoma development.

The role of HLA in Balkan endemic nephropathy.

Balkan endemic nephropathy (BEN), a progressive chronic tubulointerstitial disease, occurs in the endemic focus of Croatia in a population of about 10,000 inhabitants. One of its most peculiar characteristics is a strong association with upper tract urothelial carcinoma (UTUC). Despite a high number of studies, currently there are insufficient data about the association of BEN and HLA genes. The aim of this study was to investigate the polymorphism of HLA-A, -B, and -DRB1 alleles and haplotypes among BEN patients and to determine whether an association between HLA and BEN exists. In this study, we investigated HLA-A, -B, and -DRB1 alleles and haplotypes in a population of patients with BEN (N = 111) and matched healthy controls (N = 190). All individuals were tested by PCR-SSO and PCR-SSP methods to assess the possible contribution of HLA alleles and haplotypes to the development of/protection from BEN. Our results showed a positive association between the presence of HLA-B*35:02 and DRB1*04:02 alleles and BEN (P = 0.0179 and P = 0.0151, respectively) in contrast to the protective effect of HLA-A*01:01, B*27:05 and B*57:01 alleles (P = 0.0111, P = 0.0330 and P = 0.0318, respectively). Moreover, when BEN patients' HLA haplotypes were compared to controls, two haplotypes were associated with BEN susceptibility among Croatians (HLA-A*02:01~B*08:01~DRB1*03:01 and HLA-A*02:01~B*27:02~DRB1*16:01, P = 0.0064 and P = 0.0023, respectively), while haplotypes HLA-A*02:01~B*27:05~DRB1*01:01 and HLA-A*02:01~B*38:01~DRB1*13:01 each showed a possible protective effect (P = 0.0495). Our results point toward genetic susceptibility to BEN and observed differences in both susceptible/protective HLA profiles indicate the necessity of further studies in order to elucidate the pathogenesis of this disease.

Association of SOD2 (rs4880) and GPX1 (rs1050450) Gene Polymorphisms with Risk of Balkan Endemic Nephropathy and its Related Tumors.

Background: Experimental data show that superoxide dismutase 2 (SOD2) is involved in ochratoxin (OTA)-induced nephrotoxicity, whereas clinical data indicate the role of SOD2 rs4880 or glutathione peroxidase 1 (GPX1) rs1050450 polymorphisms in end-stage renal disease and urothelial carcinoma risk, known to be the major complications of Balkan endemic nephropathy (BEN). Therefore, we hypothesized that SOD2 and GPX1 gene polymorphisms would influence the risk of BEN and its associated tumors. Materials and Methods: The study was conducted in 207 BEN patients and 86 controls from endemic areas. Results: Individuals with both copies of variant SOD2 allele, known for lower mitochondrial antioxidant protection, are at a significantly higher BEN risk (OR = 2.6, p = 0.021). No association was observed between GPX1 gene polymorphism and BEN risk. Combining SOD2 and GPX1 genotypes did not alter the risk of BEN development. Regarding the risk of urothelial tumors in BEN patients, none of the polymorphisms studied was significantly associated with the risk of these tumors. Conclusions: Polymorphism in SOD2 rs4880 gene affects the risk of BEN development. Hence, SOD2 genotyping could, together with a panel of other enzymes, be used as a biomarker of susceptibility in BEN areas.

Common Polymorphisms in GSTA1, GSTM1 and GSTT1 Are Associated with Susceptibility to Urinary Bladder Cancer in Individuals from Balkan Endemic Nephropathy Areas of Serbia.

Balkan endemic nephropathy (BEN) is a chronic familial form of interstitial nephritis that might eventually lead to end stage renal disease. This nephropathy affects individuals living along of the Danube River and its tributaries in Serbia, Bosnia, Croatia, Bulgaria and Romania. The increased incidence of urinary tract tumors in the BEN areas is well described, but its specific genetic predisposition is still unclear. Certain nephrocarcinogenic compounds, including those associated with BEN, are metabolized by glutathione S-transferase (GST) superfamily of phase II detoxication enzymes. Importantly, the GST-mediated detoxification may result in formation of more toxic compounds. We examined the association of common GST polymorphisms and bladder cancer (BC) risk in individuals from BEN areas in Serbia. A hospital-based case-control study included 201 BC cases (67 from BEN region) and 122 controls. Each polymorphism was identified by a PCR-based method. Individuals from BEN region with low-expression GSTA1 genotype (AB+BB) exhibited a 2.6-fold higher BC risk compared to those with GSTA1 (AA) genotype who were from non-BEN region (OR = 2.60, p = 0.015). In contrast, carriers of GSTM1-active genotype from BEN region had a 2.9-fold increased BC risk compared to those with GSTM1-active genotype from non-BEN region (OR = 2.90, p = 0.010). Likewise, carriers with GSTT1-active genotype from BEN region exhibited 2.1-fold higher BC risk compared to those from non-BEN region with GSTT1-active genotype (OR = 2.10, p = 0.027). Thus, common polymorphisms in GSTA1, GSTM1 and GSTT1 are associated with susceptibility to BC in individuals from BEN areas of Serbia.

Ochratoxin A: 50 Years of Research.

Since ochratoxin A (OTA) was discovered, it has been ubiquitous as a natural contaminant of moldy food and feed. The multiple toxic effects of OTA are a real threat for human beings and animal health. For example, OTA can cause porcine nephropathy but can also damage poultries. Humans exposed to OTA can develop (notably by inhalation in the development of acute renal failure within 24 h) a range of chronic disorders such as upper urothelial carcinoma. OTA plays the main role in the pathogenesis of some renal diseases including Balkan endemic nephropathy, kidney tumors occurring in certain endemic regions of the Balkan Peninsula, and chronic interstitial nephropathy occurring in Northern African countries and likely in other parts of the world. OTA leads to DNA adduct formation, which is known for its genotoxicity and carcinogenicity. The present article discusses how renal carcinogenicity and nephrotoxicity cause both oxidative stress and direct genotoxicity. Careful analyses of the data show that OTA carcinogenic effects are due to combined direct and indirect mechanisms (e.g., genotoxicity, oxidative stress, epigenetic factors). Altogether this provides strong evidence that OTA carcinogenicity can also occur in humans.

[Endemic nephropathy in Croatia]. / Endemska nefropatija u Hrvatskoj.

Endemic nephropathy (EN) is a chronic tubulointerstitial aristolochic acid nephropathy (AAN) affecting residents of the certain villages in the valleys of the major tributaries of the Danube river in the south-east Europe including Croatia. Patients with EN have a significantly higher incidence of transitional cell carcinoma of the ureter than the general population. A-T transversion of the p53 gene is now considered to be a mutational "signature" of aristolochic acid, which is a cause of endemic nephropathy. Currently used diagnostic criteria for EN are outdated, uneven (three types of criteria) and are not in agreement with proposed new guidelines for kidney diseases. Therefore, based on current knowledge and expertise of a group of scientists and experts from all countries with EN as well as world where AAN has been reported, new diagnostic criteria and the new classification of the population of endemic villages were created at a symposium on EN. EN presents a major public health problem and current knowledge about this disease as well as new diagnostic criteria should help us in its early detection and treatment and maybe in a near future its eradication.

Balkan nephropathy.

Balkan endemic nephropathy (BN), frequently associated to upper urothelial cancer, is a familial chronic tubulointerstitial disease with insidious onset and slow progression to end-stage renal disease. After 60 years of research, its cause remains the major unanswered question. Etiology assumes polygenic susceptibility to the disease in interaction with multiple environmental factors. Chronic intoxication with Aristolochia is the major environmental risk factor for this disease. The mycotoxin hypothesis considers that BN is produced by ochratoxin A. The Pliocene lignite hypothesis assumes that the disease is caused by long-term exposure to organic toxins leached from coal nearby the endemic villages. Exome sequencing of 22,000 genes revealed that mutant genes (CELA1, HSPG2, and KCNK5) in BN patients encode proteins involved in basement membrane/extracellular matrix and vascular tone, which are tightly connected to the process of angiogenesis. SEC61G, IL17RA, and HDAC11 proved to be differently methylated throughout all patient-control pairs. The acetylation of histone lysine residues was detected and found increased at specific sites of H3 and total H4 histones isolated from urothelial cells of patients with BN. The results of molecular biological research will allow the discovery of genetic markers of BN and associated urothelial cancer, permitting early detection of BN-predisposing mutations and identification of susceptible individuals who might be at risk of exposure to environmental agents. The research of gene-gene and gene-environment interactions could lead to further studies to determine the precise risk for BN.

Genetic, Genomic and Epigenomic Studies of Balkan Endemic Nephropathy (Ben).

BEN is a primary, chronic tubulointerstitial nephritis characterized with chronic anemia, absence of edema, xantoderma, normal blood pressure and normal findings on the fundus oculi. The disease is distributed in restricted areas in Bulgaria, Romania, Croatia, Bosnia, Former Yugoslavia. Despite numerous studies on genetic and environmental factors and their possible involvement in BEN, its etiopathogenesis still remains elusive. Our recent study aim to elucidate the possible epigenetic component in BEN development. Whole genome DNA array methylation analysis was applied to compare the methylation profiles of male and female BEN patients from endemic regions in Bulgaria and Serbia and healthy controls. All three most prominent candidate genes with aberrations in the epigenetic profile discovered with this study are involved in the inflammatory/immune processes and oncogenesis. These data are in concordance with the reported pathological alterations in BEN. This research supports the role of epigenetic changes in BEN pathology. Exome sequencing of 22.000 genes with Illumina Nextera Exome Enrichment Kit revealed three mutant genes (CELA1, HSPG2, and KCNK5) in BEN patients which encode proteins involved in basement membrane/extracellular matrix and vascular tone, tightly connected to process of angiogenesis. We suggest that an abnormal process of angiogenesis plays a key role in the molecular pathogenesis of BEN.

NGS nominated CELA1, HSPG2, and KCNK5 as candidate genes for predisposition to Balkan endemic nephropathy.

Balkan endemic nephropathy (BEN) is a familial chronic tubulointerstitial disease with insidious onset and slow progression leading to terminal renal failure. The results of molecular biological investigations propose that BEN is a multifactorial disease with genetic predisposition to environmental risk agents. Exome sequencing of 22 000 genes with Illumina Nextera Exome Enrichment Kit was performed on 22 DNA samples (11 Bulgarian patients and 11 Serbian patients). Software analysis was performed via NextGene, Provean, and PolyPhen. The frequency of all annotated genetic variants with deleterious/damaging effect was compared with those of European populations. Then we focused on nonannotated variants (with no data available about them and not found in healthy Bulgarian controls). There is no statistically significant difference between annotated variants in BEN patients and European populations. From nonannotated variants with more than 40% frequency in both patients' groups, we nominated 3 genes with possible deleterious/damaging variants--CELA1, HSPG2, and KCNK5. Mutant genes (CELA1, HSPG2, and KCNK5) in BEN patients encode proteins involved in basement membrane/extracellular matrix and vascular tone, tightly connected to process of angiogenesis. We suggest that an abnormal process of angiogenesis plays a key role in the molecular pathogenesis of BEN.

Endemic (Balkan) nephropathy is aristolochic acid nephropathy.

Endemic nephropathy is a syndrome that comprises two entities: chronic interstitial nephropathy and urothelial cell cancers predominantly of the upper urinary tract. The etiological agent for the disease is aristolochic acid, a compound found in the plants of Aristolochia spp. The development of urothelial cancers is characterized by the formation of aristolactam DNA adducts leading to mutations, predominantly A: T->T: A transversions. In order to comprehensively understand the gene regulation programs in upper urothelial cancers we performed integrated miRNA and mRNA expression profiling of paired tumours and unaffected urothelium samples. The obtained data will help us to understand the carcinogenesis caused by aristolochic acid and might be the source for the design of a diagnostic biomarker.

Whole genome methylation array analysis reveals new aspects in Balkan endemic nephropathy etiology.

BACKGROUND: Balkan endemic nephropathy (BEN) represents a chronic progressive interstitial nephritis in striking correlation with uroepithelial tumours of the upper urinary tract. The disease has endemic distribution in the Danube river regions in several Balkan countries.DNA methylation is a primary epigenetic modification that is involved in major processes such as cancer, genomic imprinting, gene silencing, etc. The significance of CpG island methylation status in normal development, cell differentiation and gene expression is widely recognized, although still stays poorly understood. METHODS: We performed whole genome DNA methylation array analysis on DNA pool samples from peripheral blood from 159 affected individuals and 170 healthy individuals. This technique allowed us to determine the methylation status of 27 627 CpG islands throughout the whole genome in healthy controls and BEN patients. Thus we obtained the methylation profile of BEN patients from Bulgarian and Serbian endemic regions. RESULTS: Using specifically developed software we compared the methylation profiles of BEN patients and corresponding controls and revealed the differently methylated regions. We then compared the DMRs between all patient-control pairs to determine common changes in the epigenetic profiles.SEC61G, IL17RA, HDAC11 proved to be differently methylated throughout all patient-control pairs. The CpG islands of all 3 genes were hypomethylated compared to controls. This suggests that dysregulation of these genes involved in immunological response could be a common mechanism in BEN pathogenesis in both endemic regions and in both genders. CONCLUSION: Our data propose a new hypothesis that immunologic dysregulation has a place in BEN etiopathogenesis.

Variation in presentation and presence of DNA adducts and p53 mutations in patients with endemic nephropathy--an environmental form of the aristolochic acid nephropathy.

BACKGROUND: Endemic nephropathy (EN) and associated urothelial cell cancers (UUC) are an environmental form of aristolochic acid nephropathy where the most probable rout of ingestion of aristolochic acid (AA) was made by bread contaminated with AA, leading to chronic dietary intoxication. Clinical courses of three members of the same family, similarly exposed to toxin, who exhibited different clinical courses of the disease are presented. METHODS: Questionnaires on AA exposure were taken. Tissue samples were obtained during therapeutic nephrouretectomies. Histopathology, immunohistochemical detection of p53, p53 mutation screening in tumor DNA and analysis on the presence of aristolactam (AL)-DNA adducts were performed. RESULTS: Case 1 had UUC with typical EN histopathological signs, whereas Case 2 had bilateral UUCs with typical EN histopathological signs. In contrast, the patient in Case 3 initially showed renal insufficiency, complicated afterwards by right UUC, and later on by left UUC with histopathological end-stage chronic changes but without typical EN changes. AA-DNA adducts and specific p53 mutational spectra (A:T→ T:A transversion) were found in tissues of cases 1 and 2. CONCLUSION: Diverse clinical courses seem to be related not to differences in exposure but to differences in metabolic activation or detoxification of AA and/or DNA repair resulting from different genetic polymorphisms.

Analysis of TP53 mutation spectra reveals the fingerprint of the potent environmental carcinogen, aristolochic acid.

Genetic alterations in cancer tissues may reflect the mutational fingerprint of environmental carcinogens. Here we review the pieces of evidence that support the role of aristolochic acid (AA) in inducing a mutational fingerprint in the tumor suppressor gene TP53 in urothelial carcinomas of the upper urinary tract (UUT). Exposure to AA, a nitrophenathrene carboxylic acid present in certain herbal remedies and in flour prepared from wheat grain contaminated with seeds of Aristolochia clematitis, has been linked to chronic nephropathy and UUT. TP53 mutations in UUT of individuals exposed to AA reveal a unique pattern of mutations characterized by A to T transversions on the non-transcribed strand, which cluster at hotspots rarely mutated in other cancers. This unusual pattern, originally discovered in UUTs from two different populations, one in Taiwan, and one in the Balkans, has been reproduced experimentally by treating mouse cells that harbor human TP53 sequences with AA. The convergence of molecular epidemiological and experimental data establishes a clear causal association between exposure to the human carcinogen AA and UUT. Despite bans on the sale of herbs containing AA, their use continues, raising global public health concern and an urgent need to identify populations at risk.

NAD(P)H:quinone oxidoreductase expression in Cyp1a-knockout and CYP1A-humanized mouse lines and its effect on bioactivation of the carcinogen aristolochic acid I.

Aristolochic acid causes a specific nephropathy (AAN), Balkan endemic nephropathy, and urothelial malignancies. Using Western blotting suitable to determine protein expression, we investigated in several transgenic mouse lines expression of NAD(P)H:quinone oxidoreductase (NQO1)-the most efficient cytosolic enzyme that reductively activates aristolochic acid I (AAI). The mouse tissues used were from previous studies [Arlt et al., Chem. Res. Toxicol. 24 (2011) 1710; Stiborova et al., Toxicol. Sci. 125 (2012) 345], in which the role of microsomal cytochrome P450 (CYP) enzymes in AAI metabolism in vivo had been determined. We found that NQO1 levels in liver, kidney and lung of Cyp1a1⁻/⁻, Cyp1a2⁻/⁻ and Cyp1a1/1a2⁻/⁻ knockout mouse lines, as well as in two CYP1A-humanized mouse lines harboring functional human CYP1A1 and CYP1A2 and lacking the mouse Cyp1a1/1a2 orthologs, differed from NQO1 levels in wild-type mice. NQO1 protein and enzymic activity were induced in hepatic and renal cytosolic fractions isolated from AAI-pretreated mice, compared with those in untreated mice. Furthermore, this increase in hepatic NQO1 enzyme activity was associated with bioactivation of AAI and elevated AAI-DNA adduct levels in ex vivo incubations of cytosolic fractions with DNA and AAI. In conclusion, AAI appears to increase its own metabolic activation by inducing NQO1, thereby enhancing its own genotoxic potential.

Aristolactam-DNA adducts are a biomarker of environmental exposure to aristolochic acid.

Endemic (Balkan) nephropathy is a chronic tubulointerstitial disease frequently accompanied by urothelial cell carcinomas of the upper urinary tract. This disorder has recently been linked to exposure to aristolochic acid, a powerful nephrotoxin and human carcinogen. Following metabolic activation, aristolochic acid reacts with genomic DNA to form aristolactam-DNA adducts that generate a unique TP53 mutational spectrum in the urothelium. The aristolactam-DNA adducts are concentrated in the renal cortex, thus serving as biomarkers of internal exposure to aristolochic acid. Here, we present molecular epidemiologic evidence relating carcinomas of the upper urinary tract to dietary exposure to aristolochic acid. DNA was extracted from the renal cortex and urothelial tumor tissue of 67 patients that underwent nephroureterectomy for carcinomas of the upper urinary tract and resided in regions of known endemic nephropathy. Ten patients from nonendemic regions with carcinomas of the upper urinary tract served as controls. Aristolactam-DNA adducts were quantified by (32)P-postlabeling, the adduct was confirmed by mass spectrometry, and TP53 mutations in tumor tissues were identified by chip sequencing. Adducts were present in 70% of the endemic cohort and in 94% of patients with specific A:T to T:A mutations in TP53. In contrast, neither aristolactam-DNA adducts nor specific mutations were detected in tissues of patients residing in nonendemic regions. Thus, in genetically susceptible individuals, dietary exposure to aristolochic acid is causally related to endemic nephropathy and carcinomas of the upper urinary tract.

[Structural and functional characteristics of urinary tract in offspring of Balkan endemic nephropathy patients].

INTRODUCTION: Balkan endemic nephropathy (BEN) is a familial chronic progressive tubulointerstitial disease of unknown aetiology that occurs with high prevalence in endemic rural environments of Serbia, Bosnia and Herzegovina, Croatia, Bulgaria and Romania. It has been documented only in adults. OBJECTIVE: The aim of this study was to examine clinical markers of BEN in children and adolescent offspring of BEN patients. METHODS: Prospective clinical trial involved two groups of children and adolescents: I consisted of 30 offspring of BEN patients and II of 29 offspring of non-BEN dialysis patients, both of them living in the same South Morava region of Serbia. All of them were healthy at the time of the investigation, not receiving any drugs. The study included personal and family history, physical examination, comprehensive laboratory analyses and renal ultrasound. Blood pressure (BP) was determined by using casual BP and 24 h ABPM in subjects older than 5 years. Urinary proteins were investigated by analysing microalbumin, alfa 1 microglobulin, beta 2 microglobulin and SDS-PAGE electrophoresis. GFR was measured by estimated creatinine clearance and by serum Cystatin C concentrations. RESULTS: There were no statistically significant differences in age, gender, history of urinary tract infections or functional voiding disorders between these two groups. All of the studied subjects had normal BP and GFR. Renal ultrasound was abnormal only in BEN offspring (6.66%) as well as increased urine concentrations of microalbumin (3.3%), alpha 1 microglobulin (10%) and beta 2 microglobulin (13.3%) while low molecular protein (<66,000 D) was prevalent in BEN compared with non-BEN offspring (21.43% vs. 3.7%). CONCLUSION: Renal abnormalities in offspring of BEN patients may be an early marker of BEN.This has to be confirmed in long term follow-up of a greater number of BEN paediatric offspring.

p53 mutations as fingerprints for aristolochic acid: an environmental carcinogen in endemic (Balkan) nephropathy.

The activation of protooncogenes and inactivation of tumor suppressor genes are considered to be the main molecular events in the multistep process of carcinogenesis. Mutations of the TP53 tumor suppressor gene have been found in nearly all tumor types and are estimated to contribute to more than 50% of all cancers. Most mutations lead to the synthesis of highly stable, inactive proteins that accumulate in the nucleus of cancer cells. Among the 393 codons of the human p53 gene, 222 are targets of 698 different types of mutations. Alterations of codons 175, 248, 273 and 282 correspond to 19% of all mutations and are considered general hot spot mutations. Dietary exposure to aristolochic acid (AA), an established nephrotoxin and human carcinogen found in all Aristolochia species was shown to be the causative agent of aristolochic acid nephropathy (previously called Chinese herbs nephropathy). This syndrome is characterized by proximal tubular damage, renal interstitial fibrosis, slow progression to the end stage renal disease and a high prevalence of upper urinary tract urothelial carcinoma (otherwise a highly unusual location). AA preferentially binds to purines in DNA and is associated with a high frequency of A-->T transversions in the p53 gene. Rats treated with AA develop A:T-->T:A mutations in codon 61. The pathological and clinical features of endemic (Balkan) nephropathy closely resemble those associated with aristolochic acid nephropathy except for the slower progression to end stage renal disease and longer cumulative period before the appearance of urothelial cancer. Recently, we reported the presence of AA-DNA adducts in renal cortex and A-->T p53 mutations in tumor tissue of patients from Croatia and Bosnia with endemic nephropathy. These data support the hypothesis that dietary exposure to AA is a major risk factor for endemic (Balkan) nephropathy.

Germline p53 single-base changes associated with Balkan endemic nephropathy.

The Balkan endemic nephropathy (BEN) is a significant clinical and scientific problem in need of novel effective therapies. Though many genetic and environmental factors have been investigated the basis, cause, and predisposition to BEN are still unclear. In this study, based on the hypothesis that the genetic pathways leading to BEN might be associated with p53 dysfunction, we screened for p53 gene mutations 90 Bulgarian BEN patients using optimized PCR-SSCP-sequencing analysis. Germline p53 single-base changes were found in blood samples in 10% of BEN cases. Three of them caused amino acid substitutions (p.Arg283Cys, p.Gln317His, and p.Lys321Glu); the other six were either synonymous amino acid substitutions (p.Arg213Arg) or intron polymorphisms (T14766C). To the best of our knowledge, these are the first data investigating tumor suppressor gene mutations in patients with BEN. The obtained results are in support of our hypothesis that p53 gene alterations are possibly involved in BEN genetic pathways.

Etiology of Balkan endemic nephropathy and associated urothelial cancer.

Balkan endemic nephropathy (BEN) is a familial chronic tubulointerstitial disease with insidious onset and slow progression to terminal renal failure. Evidence has accumulated that BEN is an environmentally induced disease. There are three actual theories attempting to explain the environmental cause of this disease: (1) the aristolochic acid hypothesis, which considers that the disease is produced by chronic intoxication with Aristolochia, (2) the mycotoxin hypothesis, which considers that BEN is produced by ochratoxin A, and (3) the Pliocene lignite hypothesis, which proposes that the disease is caused by long-term exposure to polycyclic aromatic hydrocarbons and other toxic organic compounds leaching into the well drinking water from low-rank coals in the vicinity to the endemic settlements. Moreover, it was suggested that BEN risk is influenced by inherited susceptibility. Therefore, it has been expected that molecular biological investigations will discover genetic markers of BEN and associated urothelial cancer, permitting early identification of susceptible individuals who may be at risk of exposure to the environmental agents. Since kidney pathophysiology is complex, gene expression analysis and highly throughput proteomic technology can identify candidate genes, proteins and molecule networks that eventually could play a role in BEN development. Investigation of gene-gene and gene-environment interactions could be the content of further studies determining the precise risk for BEN.