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Development of a genomic dna bank of iga nephropathy (igan) patients and family members. new trends in genetics for the early diagnosis of familial igan

Deliverables

IgA nephropathy (IgAN) or Berger’s disease is the most common glomerulonephritis in the world diagnosed in patients undergoing renal biopsy. The involvement of genetic factors on the pathogenesis of the IgAN is evidenced by ethnic and geographic variation in prevalence, familial clustering in isolated populations, familial aggregation and more recently by the identification of a genetic linkage to one locus (IGAN1) mapped on 6q22-23. Although such a study seems to imply a single major locus, the hypothesis of multiple interacting loci or genetic heterogeneity cannot be ruled out. A group of European scientists, specialists in this disease, constituted an IgAN Consortium and organized a DNA Biobank through the recruitment of IgAN patients and family members following a common protocol. The main aim of the project was to constitute a genomic DNA bank of well-characterized IgAN patients and their family members belonging to three different geographic areas of Europe (Germany, Greece and Italy) to be used for genetic studies. A web-site (http://www.igan.net) was organized to collect personal data and clinical findings of all subjects enrolled in the study. Data collected were available by appropriate passwords to all partners respecting the subject’s privacy. DNA samples of IgAN patients and family members belonging to 71 multiplex extended pedigrees were collected. Thirty-five families with <10 members, 21 with 10-20 members and 15 families with >20 members were enrolled. For each family, all the first-degree relatives were enrolled. Moreover, 158 trios (sons /daughters affected and healthy parents), 1028 sporadic IgAN patients, and 1040 healthy subjects were included in the IgAN Consortium Biobank. The strategy used by the IgAN Consortium is to perform genetic studies separately in an homogeneous population belonging to a restricted area and only in case of interesting results to offer the possibility to check for the same data in the others. DNA samples from patients, family members and an adequate number of healthy subjects of different European geographic areas have been collected. This organization could be highly representative of the IgAN genetic background. The focus of the IgAN Biobank is molecular genetics research and identification of disease susceptibility genes. The knowledge gained from the study of Mendelian diseases has shown that the genetic dissection of complex trait is more powerful when combined linkage-based, association-based, and sequence-based approaches are performed. This is the first and unique DNA bank of IgA Nephropathy in the world. The policy of the IgAN Consortium which is responsable of the Biobank is to offer different chances of collaboration as follows: - To be member of the IgAN Consortium by collecting DNA samples of the IgAN patients and families members in according to estabilished protocol which is available in the Consortium website (http://www.igan.net); - To be a researcher of the Consortium after submission of a designed scientific project which will be analysed by the partners of the Consortium; - To collaborate with other scientists in multi-centres genetic or clinical studies. The genomic DNA bank is based on an electronic database (IgAN Registry-database), which contains the images of the familial IgAN pedigrees, clinical findings and laboratory data of patients and family members enrolled in the studies and the continuous updating of clinical patient status.
The IgAN consortium has created a website (www.igan.net) for public dissemination and confidential sharing of the results achieved by the Consortium. The website contains three sections: 1) Information; 2) Registry; 3) News. Section 1 and 3 are available for public vision. They are dedicated to the exploitation and dissemination of data on IgA nephropathy obtained by the IgAN Consortium. Section 1 contains a booklet, which includes the definition of the disease as a sporadic form and a familial or a suspected familial form. In addition, there is the definition of affected, probably affected and unaffected subjects belonging to these families. There are the guidelines for the collection of blood samples, which will be stored in the Biobank. The booklet contains letters of information about the disease and an informed consent to be used for the collection of blood samples. Section 3 is dedicated to the dissemination of data obtained by the IgAN Consortium since the constructed Biobank is the unique DNA bank on IgA nephropathy available in the world. IgA nephropathy is the most frequent worldwide glomerulonephritis and 20-40% of subjects affected by this disease receive renal replacement therapy 20 years after the onset of the disease. The disease involves young subjects and this means that socio-economic aspects represent major points of the disease. The section No.2 contains the Registry which is the core of the website for basic and clinical research. The database is accessible only to the partners of the IgAN Consortium, who have received a valid password.
IgA nephropathy (IgAN) is a disorder related to an abnormal immune response which is characterized by increased synthesis of deglycosylated IgA1. T helper (h) lymphocytes in pathogenic immune response at mucosal effector site have been shown to play a key role in IgAN. The cytokine levels themselves have the most critical role in Th cell polarization. We considered cytokine gene polymorphisms, which in turn influence the production of the respective cytokines. The following gene polymorphisms were analysed: - Interferon (IFN) gamma intron-1 CA-repeat at position 1349–1373 (Capillary electrophoresis) for Th1-type cytokine; - Interleukin (IL)-13 –1055C/T (SSCP) for Th2; - Transforming growth factor (TGF) beta 915G/C (SSCP) for Th3; IL-10 5’-proximal and distal microsatellites (PAGE) for TR; - Tumor necrosis factor (TNF) alfa -308G/A (RFLP/NcoI), -238G/A (RFLP/BglII) for monocytes/macrophages. We carried out a family-based association study on 50 families, which included 53 IgAN patients, 45 complete trios, 4 incomplete trios and 36 discordant siblings. Statistical analysis was performed using the family-based association test (FBAT) software freely available from: http://www.biostat.harvard.edu/~fbat/default.htlm. It incorporates a set of statistical procedures to accommodate variable pedigree constellations, dichotomous or quantitative phenotypes, phenotype-unknown subjects, bi- or multi-allelic marker data, multiple loci and various models for the mode of inheritance and gene-environment interactions. Moreover, genotyping of IFNg intron-1 microsatellite was carried out in a cohort of 174 IgAN patients to evaluate the influence on disease outcome of the seven alleles identified in our population. Multi-allelic analysis showed the association between IFNg polymorphism and susceptibility to IgAN (p=0.03). No significant association was found between other cytokine gene polymorphisms and the disease. The bi-allelic analysis, to evaluate how the seven different IFN gamma alleles influenced the disease, evidenced that the 13-CA repeat allele was preferentially transmitted to the affected individuals (p=0.006). Association between the presence/absence of risk genotype and gender, age at onset of the disease and at the time of renal biopsy, serum creatinine and renal function, daily proteinuria progressively increasing, and histological lesions did not yield significant results. In addition, 163 out 174 patients of the cohort with a mean follow-up of 5 years (range 1-30 years) were distinguished as progressors (46) and non-progressors (117). Progressor patient was defined as one who reached end stage renal disease (ESRD) or had a serum creatinine (sCr) value, which doubled from the time of renal biopsy. Progression was observed in 42.4% of 13 CA-repeat allele carriers versus 57.6% of non-carriers. No significant difference in IFN gamma 13 CA-repeat allele distribution between these two groups was found (p= 0.72). No association with disease progression was found. A correlation of 12-CA repeat IFN gamma allele and a higher cytokine production has previously been demonstrated by in vitro assays. In our IgAN patient population 12-CA and 13-CA repeat IFNg allele distribution was 43.4% and 44.3%, respectively. We can conclude that the association between 13-CA repeats IFN gamma allele as a lower producer genotype status and IgAN development could be suggestive of a predominance of the Th2 immune response. A relative or absolute increase in Th2 cytokine production is a significant pathogenic factor in human IgAN since it may lead to abnormalities in IgA1 glycosylation and formation of circulating IgA1-IgG (anti IgA1) immunocomplexes. This is the first and largest to date family-based study, which suggests an important role of IFN gamma in the development of the disease.
IgA nephropathy (IgAN [MIM 161950]) is a common form of primary glomerulonephritis characterised by diffuse glomerular mesangial IgA1 deposition that leads to mesangial proliferation and chronic glomerular inflammation. Analysis of serum IgA1 from IgAN patients revealed an abnormally galactosylation of the O-linked carbohydrate moieties of IgA. It may be hypothesised that the abnormal glycosylation of the hinge region glycans could result from a reduced quantity or enzymatic activity of the Core1 beta 1,3- Galactosyltransferase. Recently, C1GALT1 gene coding for this molecule has been characterised. In order to evaluate the association between C1GALT1 and IgAN, we performed a case control study among the Italian population. We sequenced C1GALT1 coding and promoter regions in 196 IgAN patients and 234 healthy controls. We identified 8 single nucleotide polymorphisms (SNPs) two of which were never described: 5 were in the promoter region (-796T/C, -784G/T, -511G/A, -392G/T, -353insC) and 3 in the 3’UTR (1365G/A, 1484T/A, 1513T/A). Comparing allelic frequencies of C1GALT1 SNPs between patients and controls, we evidenced that allele 1365G in 3’UTR was significantly more frequent in IgAN patients than in healthy controls (Chi-square =14.68, p = 0.0001, pc = 0.008, odds ratio 2.47 [95% CI: 1.54-3.97]). For the linkage disequilibrium analysis, and in order to calculate the haplotype frequencies, we processed genotypes data with Genotype Transposer and Arlequin softwares. The promoter CGATW haplotype was significantly less frequent in IgAN patients than in healthy controls (Chi-square =6.76, p = 0.009, pc = 0.05 odds ratio 0.564 [95% CI: 0.365-0.872]). The genotype 1365G/G confers susceptibility to IgAN (Chi-square =12.26, p = 0.0005, pc= 0.004, odds ratio 2.51 [95% CI: 1.48-4.24]) but it is not associated with the progression of the disease. The role of this SNP in the pathogenesis of IgAN has been investigated by Real-time quantitative PCR experiments that have shown reduced expression of C1GALT1 in homozygous 1365 G/G (mean ratio +/-SD: 1,4+/1,46) individuals in respect to those with other genotypes (mean ratio +/- SD: 2,96+/-2,5; unpaired t test p value =0.0295). The results of this study suggest that C1GALT1 plays a possible role as a pathogenetic factor for IgAN.

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Immunoglobulin A Nephropathy (IgAN), also known as Berger's Disease, is the most frequent primary glomerulonephritis worldwide and one of the leading cause of end-stage kidney disease (ESKD) requiring dialysis or transplantation. It occurs in early adulthood in sporadic or familial form. The onset is characterized by gross hematuria concomitant with upper respiratory tract infections or persistent microscopic hematuria, usually with a normal renal function associated or not with proteinuria. Such insidious clinical onset has to be supported by a renal biopsy showing a specific histological pattern. A slow progression (10-20 years) toward ESKD is the common course. This project aimed to develop a DNA bio-bank of IgAN patients and family members in order to identify potential genetic markers contributing to the onset and progression of this complex disease. The IgAN Consortium, gathering the leading European scientists in the field, was set up. So far, DNA samples from more than 70 IgAN families, 240 trios, 1300 IgAN patients and 2400 healthy controls have been collected throughout Europe in the first organized bio-bank in the world. A related website (www.igan.net) is available to share demographic, genetic and clinical data within the Consortium and it also contains open sections with general information about the disease and project achievements with published papers. The investigators found that familial IgAN has an increased risk of ESKD and a linkage-based approach to study this form has identified significant loci on chromosomes 6q22-23, 4q26-31 and 17q12-22. Studies are currently under way in order to restrict the linked regions. Familial and case-control association studies with candidate gene polymorphisms have been performed obtaining interesting results. A family based association study evidenced an association of IgAN with 13-CA repeat intron 1 IFN gamma allele while no association was found with MBL2 gene polymorphisms. No difference in the ACE I/D and AGT M235T gene polymorphisms distribution between IgAN patients and controls and between patients with stable and those with deteriorating renal function was shown. A genetic analysis of single nucleotide polymorphisms (SNPs) within the PDGF-B gene evidenced no association with development and progression of IgAN while it has been shown that IgAN patients exhibit high systemic level of PDGF-DD. The haplotype -116C/-51G of the podocin NPHS2 promoter was found more frequent in IgAN patients than in controls. A case-control association study was performed to investigate the role of galactosyltransferase core-1 beta3-galactosyltransferase-1 (C1GalT1) in the pathogenesis of IgAN. The genotype 1365G/G was associated with the onset but not with the progression. Moreover, a reduced expression of C1GalT1 in homozygous subjects for 1365 G/G genotype was observed. The results of this study suggest that C1GalT1 plays a possible role as a pathogenetic factor for IgAN. Direct sequencing of C1GalT1 enabled us to identify, for the first time, two new SNPs -796T and -784G/T. No evidence for its chaperon 'cosmc' has been found. Microarray analysis carried out on peripheral blood leukocytes and renal tissue samples is ongoing. Differences in gene expression between IgAN patients and healthy controls as well as in the same patients during gross hematuria episodes and microhematuria will be highlighted. A study of the regulatory microRNA of IgAN patients has also been undertaken. The robust piece of evidence provided by this project has not only contributed to define the genetic and genomic aspect of this glomerulonephritis but also prompted future directions. A genome-wide association scan, as the next step to cover the genetic spectrum of such complex disease, would benefit from a systems biology approach in order to maximize the impact of the large amount of data generated by the applied high-throughput technology.