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Balkan endemic nephropathy (BEN) is a disease that affects people that live in the alluvial plains along the tributaries of the Danube River in the Balkan region. BEN is a chronic tubulointerstitial disease with a slow progression to terminal renal failure and has strong association with upper tract urothelial carcinoma (UTUC). There are several hypotheses about the etiology of BEN, but only the toxic effect of aristolochic acid has been confirmed as a risk factor in the occurrence of the disease. Aberrantly expressed miRNAs have been shown to be associated with many types of cancers. A number of studies have investigated the expression of microRNAs in urothelial carcinoma, mainly on urothelial bladder cancer, and only a few have included patients with UTUC. Here we present the first study of microRNA profiling in UTUC tissues from patients with BEN (BEN-UTUC) and patients with UTUC from nonendemic Balkan regions (non-BEN-UTUC) in comparison to normal kidney tissues. We found 10 miRNAs that were differentially expressed in patients with BEN-UTUC and 15 miRNAs in patients with non-BEN-UTUC. miRNA signature determined in BEN-UTUC patients differs from the non-BEN-UTUC patients; only miR-205-5p was mutual in both groups.

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.

New generation genomic platforms enable us to decipher the complex genetic basis of complex diseases and Balkan Endemic Nephropathy (BEN) at a high-throughput basis. They give valuable information about predisposing Single Nucleotide Polymorphisms (SNPs), Copy Number Variations (CNVs) or Loss of Heterozygosity (LOH) (using SNP-array) and about disease-causing mutations along the whole sequence of candidate-genes (using Next Generation Sequencing). This information could be used for screening of individuals in risk families and moving the main medicine stream to the prevention. They also might have an impact on more effective treatment. Here we discuss these genomic platforms and report some applications of SNP-array technology in a case with familial nephrotic syndrome.

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.

Background: Balkan Endemic Nephropathy (BEN) is late-onset kidney disease thought to arise from chronic exposure to aristolochic acid, a phytotoxin that contaminates wheat supplies in rural areas of Eastern Europe. It has recently been demonstrated that humans are capable of perceiving aristolochic acid at concentrations below 40 nM as the result of high-affinity interactions with the TAS2R43 bitter taste receptor. Further, TAS2R43 harbors high-frequency loss-of-function mutations resulting in 50-fold variability in perception. This suggests that genetic variation in TAS2R43 might affect susceptibility to BEN, with individuals carrying functional forms of the receptor being protected by an ability to detect tainted foods., Methods: To determine whether genetic variation in TAS2R43 predicts BEN susceptibility, we examined genotype-phenotype associations in a case-control study. A cohort of 88 affected and 99 control subjects from western Bulgaria were genotyped with respect to two key missense variants and a polymorphic whole-gene deletion of TAS2R43 (W35S, H212R, and wt/Δ), which are known to affect taste sensitivity to aristolochic acid. Tests for association between haplotypes and BEN status were then performed., Results: Three major TAS2R43 haplotypes observed in previous studies (TAS2R43-W35/H212, -S35/R212 and -Δ) were present at high frequencies (0.17, 0.36, and 0.47 respectively) in our sample, and a significant association between genotype and BEN status was present (P = 0.020; odds ratio 1.18). However, contrary to expectation, BEN was positively associated with TAS2R43-W35/H212, a highly responsive allele previously shown to confer elevated bitter sensitivity to aristolochic acid, which should drive aversion but might also affect absorption, altering toxin activation., Conclusions: Our findings are at strong odds with the prediction that carriers of functional alleles of TAS2R43 are protected from BEN by an ability to detect and avoid aristolochic acid exposure. Evidence for a positive association between high-sensitivity alleles and BEN status suggests instead that possession of toxin-responsive receptor variants may paradoxically increase vulnerability, possibly by shifting attractive responses associated with low-intensity bitter sensations. The broad-spectrum tuning of the ~25-member TAS2R family as a whole toward xenobiotics points to a potentially far-reaching relevance of bitter responses to exposure-related disease in both individuals and populations.

(1)H NMR spectroscopy of urine has been applied to exploring metabolomic differences between people diagnosed with Balkan endemic nephropathy (BEN), and treated by haemodialysis, and those without overt renal disease in Romania and Bulgaria. Convenience sampling was made from patients receiving haemodialysis in hospital and healthy controls in their village. Principal component analysis clustered healthy controls from both countries together. Bulgarian BEN patients clustered separately from controls, though in the same space. However, Romanian BEN patients not only also clustered away from controls but also clustered separately from the BEN patients in Bulgaria. Notably, the urinary metabolomic data of two people sampled as Romanian controls clustered within the Romanian BEN group. One of these had been suspected of incipient symptoms of BEN at the time of selection as a 'healthy' control. This implies, at first sight, that metabolomic analysis can be predictive of impending morbidity before conventional criteria can diagnose BEN. Separate clustering of BEN patients from Romania and Bulgaria could indicate difference in aetiology of this particular silent renal atrophy in different geographic foci across the Balkans.

Balkan endemic nephropathy (BEN) is a chronic tubulointerstitial disease associated with urothelial cancer, which affects people living in the alluvial plains along the tributaries of the Danube River. Challenges of studying BEN using the epidemiological method are multiple. The natural history from exposure to occurrence of the disease may take many years. The early stages of BEN are not easily detectable clinically, as the disease is asymptomatic until a significant decline in function occurs, and even then symptoms are usually non-specific. The natural history of BEN is complex, possibly with multiple risk factors operating both at the stage of initiation of renal damage and in its progression. In BEN, genetic susceptibility is due to multiple genes of small effects, gene-gene interactions, and gene-environment interactions of complex nature that are difficult to assess with current study designs. BEN is now kidney disease of the old people, and many risk factors for disease such as smoking, alcohol consumption, obesity, and diabetes could contribute to the kidney damage. Evidence is presented that environmental rather than genetic factors play a decisive role in the etiopathogenesis of BEN. Aristolochic acid, described as a culprit of BEN in 1959, is confirmed in 2007 by the molecular biology methods. Mycotoxins and polycyclic aromatic hydrocarbons, leached from lignites and found in the vicinity of endemic settlements, deserve further investigation. Despite advances in understanding the epidemiology of BEN, more research is needed on the patterns of BEN over time and between places, and on identifying the contributions of modifiable risk factors in initiating and hastening progression of BEN in order to improve the scope for preventing BEN. Primary prevention is still at the beginning. Knowledge accumulated in the fifty years of BEN research and new data about prevention and treatment of chronic kidney disease reveal several effective methods in secondary and tertiary prevention of BEN. Genetic epidemiology could establish the relative size of the genetic effect in relation to other sources of variation in disease risk (i.e., environmental effects such as intrauterine environment, physical and chemical effects, as well as behavioral and social aspects). Public health authorities in the several countries having aristolochic acid nephropathy should take immediate measures for reducing dietary exposure of residents to Aristolochia.

Balkan endemic nephropathy (BEN) is a familial chronic tubulointerstitial disease with insidious onset and slow progression to terminal renal failure. Diagnostic criteria for BEN have been described more than 40 years ago. Research groups on BEN use one of at least three described lists of criteria. Comparison of studies using such criteria is difficult, and a recent meeting of investigators (Zagreb, October 2006) has suggested that unified criteria have to be elaborated. In this paper, an International Panel of BEN Investigators agreed on criteria appropriate to epidemiologic studies and clinical investigations of BEN. A screening procedure of BEN in endemic settlements is proposed.

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., (Copyright 2006 S. Karger AG, Basel)

Objectives: The concept of multifactorial etiology of BEN anticipates that a combination of polymorphic gene variants and various environmental factors causes an increased risk for the disease. CYP enzymes play a key role in the metabolic activation of environmental chemicals and toxins. CYP3A enzymes are particularly relevant for xenobiotic metabolism because of their broad substrate specificity and abundant expression in the human liver, intestine, and kidney. Previous phenotyping analysis on CYP2D6 enzyme activity in BEN patients proposed a modifying effect of CYP2D6 gene variants on BEN risk, but it was not approved with molecular-genetic methods. The aim of the current case-control study was to compare the frequency of CYP2D6 and CYP3A5 polymorphisms, as well as one CYP3A4 promoter variant in BEN patients and controls in order to investigate a possible association between individual genetic variations in these genes and susceptibility to BEN., Design and Methods: Ninety-six nonrelated Bulgarian BEN patients from endemic villages in the Vratza district and 112 healthy Bulgarians from nonendemic areas (controls) were genotyped. Identification of alleles was done by allele-specific PCR or by rapid-cycle amplification on the LightCycler, followed by sequence-specific detection., Results: The UM, PM, and EM + IM genotype frequencies of CYP2D6 did not differ significantly between the two groups (P > 0.05). The CYP3A4*1B allele was only found in the heterozygous form, with allelic frequencies of 5.21% in the patients and 2.23% in the healthy individuals (P = 0.11). The CYP3A5*1 allele was more prevalent in BEN patients with a frequency of 9.38% compared to 5.36% in the controls and was associated with a higher risk for BEN (OR 2.41, 95% CI 1.09-5.33) (P = 0.02)., Conclusions: Our results demonstrate that the CYP3A5*1 allele, previously reported as a marker for CYP3A5 expression in human kidney, is associated with increased risk for BEN, while CYP3A4*1B and CYP2D6 genotypes do not significantly modify the risk for the disease.

Background: Polymorphisms in NAD[P]H:quinone oxidoreductase (NQO1) and glutathione S-transferases (GSTs) have been reported to be associated with an increased risk for environmentally and/or occupationally induced renal and bladder cancers. Genetic factors related to chronic nephropathy and to urinary bladder or renal cancer development in Balkan endemic nephropathy (BEN) is unknown. In order to evaluate their possible role in BEN susceptibility, we determined the frequencies of NQO1 alleles *1, *2 and *3, as well as the GSTT1 and GSTM1 null genotypes in BEN patients and healthy subjects from a non-endemic region of Bulgaria., Methods: The respective genotypes of 95 unrelated Bulgarian BEN patients and of 112 healthy individuals (control group) were determined by rapid cycle polymerase chain reaction (PCR) and detected with either SYBR green I fluorescent dye or melting curve analysis using allele specific probes., Results: NQO1 genotyping showed a higher NQO1*2 allele frequency (23.68%) in BEN patients compared to controls (18.75%; p=0.219), while NQO1*3 allele frequencies were similar in both groups (2.63% in BEN patients vs. 2.23% in controls; p=0.791). The GSTT1 deficiencywas observed in 20% of BEN patients vs. 16.1% of controls (p=0.613). The GSTM1 null genotype was found in 45.3% of BEN patients vs. 51.8% of controls (p=0.674). There was no influence of NQO1 and GSTs genotypes found on BEN risk., Conclusions: Our results established that alleles NQO1*2 and NQO1*3, as well as lack of GSTT1 and GSTM1 did not influence the BEN risk. These findings provide novel information on the genetic heterogeneity in the healthy Bulgarian population.

Background: Balkan endemic nephropathy (BEN) is of great clinical importance in restricted areas of Bulgaria, Serbia, Croatia, Bosnia, Herzegovina, and Romania, since the etiology of BEN is still unknown., Methods: In urine samples from 48 patients (41 females and 7 males, aged 65.6 +/- 6.87 years) with BEN living in an endemic area of Vratza district, Bulgaria, neopterin concentrations were measured by high-pressure liquid chromatography (HPLC) and compared with other clinical and laboratory investigations, including creatinine, hemoglobin, and erythrocyte sedimentation rates (ESRs)., Results: Urinary neopterin concentrations were 263 +/- 128 (mean +/- SD; range, 78 to 786 micromol/mol creatinine), 24 (50%) of BEN patients presented with increased concentrations as compared to the established normal ranges. Average ESRs were increased (1 hour, 29.0 +/- 14.7 mm/hour) and hemoglobin was decreased (109.3 +/- 16.4 g/L). Hemoglobin correlated inversely with ESRs (rs = -0.787 and -0.780) and creatinine concentrations (r = -0.690, all P < 0.001), but not with neopterin concentrations. Neopterin concentrations also did not correlate with serum creatinine levels. There existed an age relationship of ESR, creatinine, and hemoglobin, but not of neopterin. Neopterin concentrations were slightly lower in five females with low titers of antibodies against local B1 hantavirus strain (P < 0.05)., Conclusion: The findings can support an immune-mediated inflammatory process in the pathogenesis of BEN only in a subgroup of patients.

New generation genomic platforms enable us to decipher the complex genetic basis of complex diseases and Balkan Endemic Nephropathy (BEN) at a high-throughput basis. They give valuable information about predisposing Single Nucleotide Polymorphisms (SNPs), Copy Number Variations (CNVs) or Loss of Heterozygosity (LOH) (using SNP-array) and about disease-causing mutations along the whole sequence of candidate-genes (using Next Generation Sequencing). This information could be used for screening of individuals in risk families and moving the main medicine stream to the prevention. They also might have an impact on more effective treatment. Here we discuss these genomic platforms and report some applications of SNP-array technology in a case with familial nephrotic syndrome. [ABSTRACT FROM AUTHOR]

¹H NMR spectroscopy of urine has been applied to exploring metabolomic differences between people diagnosed with Balkan endemic nephropathy (BEN), and treated by haemodialysis, and those without overt renal disease in Romania and Bulgaria. Convenience sampling was made from patients receiving haemodialysis in hospital and healthy controls in their village. Principal component analysis clustered healthy controls from both countries together. Bulgarian BEN patients clustered separately from controls, though in the same space. However, Romanian BEN patients not only also clustered away from controls but also clustered separately from the BEN patients in Bulgaria. Notably, the urinary metabolomic data of two people sampled as Romanian controls clustered within the Romanian BEN group. One of these had been suspected of incipient symptoms of BEN at the time of selection as a =healthy' control. This implies, at first sight, that metabolomic analysis can be predictive of impending morbidity before conventional criteria can diagnose BEN. Separate clustering of BEN patients from Romania and Bulgaria could indicate difference in aetiology of this particular silent renal atrophy in different geographic foci across the Balkans. [ABSTRACT FROM AUTHOR]