Slide 1

Thank you Mr Chairman. I will talk about genetic susceptibility and genome aspects in the progression of renal damage in patients with IgAN.

Slide 2

The first slide shows that the human genome project may be considered the most important discovery of human sciences but at the same time it has been the start-up for further projects and molecular medicine as I will show you at the end of my talk.

Slide 3

Moving from the family cases and applying genetic and genomic studies we may have the possibility to know the pathogenesis of the diseases. This is the rational of our European IgAN Consortium in studying the pathogenesis and susceptibility of the disease.

Slide 4

So, it is well known that the first paper published in 1973 by de Werra et al in a Swiss journal demonstrated that there are familial cases. After that time many papers were published and finally Francesco Scolari who is present in this room described that 15-20% of cases may be in familial forms in IgAN.

Slide 5

In fact, we demonstrated many years ago that if we perform renal analysis in family members of patients with IgAN as you can see ¼ of patients, ¼ of parents and family members are affected by urinary abnormalities which means persistent or microscopic hematuria and overt proteinuria. In contrast in more than 8000 students only 4% were affected by renal abnormalities this means that many relatives are affected probably by IgAN.

Slide 6

In conclusion if we have the possibility to perform urine analysis in relatives of at least 3 generations from patients with IgAN, we have the possibility to distinguish two forms of IgAN; sporadic and familial. Sporadic which means that 1 member of the family is affected by biopsy-proven IgAN and the other members are completely normal and familial IgAN in which we have at least 2 members affected by biopsy-proven IgAN and other members may have renal abnormalities or normal urines. Finally, we have suspected familial IgAN in which we have one member affected by biopsy-proven IgAN and other members may have abnormalities in urine but they do not receive a renal biopsy for many reasons.

Slide 7

This slide shows two examples of IgAN families. In the first is present one generation affected by the disease and in the second one we have 3 generations affected by IgAN.

Slide 8

So by the way the phenotype does not segregate as a simple Mendelian trait but rather displays patterns consistent with a complex trait. This means that IgAN is a complex disease and it is very intriguing concerning genetic susceptibility.

Slide 9

Moving to the first part of my talk, genetic susceptibility to this disease.

Slide 10

The first genome-wide scan study was presented in abstract form at the ASN meeting in 1996 and this genome-wide scan study was organised in 16 inbred IgAN patients and you can see that these patients were linked to some chromosomal traits located in chromosome, 1, 2, 6 and 14.

Slide 11

But a very extensive genome-wide scan study was organised by Ali Gheravi and many nephrologists who are present in this room.

Slide 12

And this study demonstrated in 30 families of which 24 were from Italy that 1 chromosomal trait located in chromosome 6 exactly 22-23 linked to IgAN.

Slide 13

And this linkage trait was called IgA1 and the dominant model of transmission was incomplete penetrance, the LOD score was of 5.6.

Slide 14

After a few years there was another genome-wide scan study but this time organised in ddY mice. These mice develop spontaneous IgAN and – et al demonstrated that there was a susceptibility locus on murine chromosome 10 which is a region syntenic to human IgAN1 on chromosome 6q-22-23.

Slide 15

But if we analyse the Ali Gharavi study only 58% of Italian families linked to chromosome 6q.

Slide 16

And for this reason we organised in Europe an IgAN consortium which organises a DNA bank for collecting blood samples from IgAN patients and the family members and partners at that time were from Italy, Germany, Greece and Switzerland.

Slide 17

Now there are in this consortium nephrologists, epidemiologists, geneticists, statisticians and bioinformaticians.

Slide 18

Well, this is our website. There are 3 sections; information, registry, database registry and the news concerning papers that we publish and the results we obtained.

Slide 19

And this is the paper that we published in BMC nephrology for looking at our cases and for the possibility to organise a collaborative study too.

Slide 20

So the last update collection of DNA samples from patients and families is shown in this slide. We have DNA samples from 70 IgAN families from 69 suspected IgAN families, from Trios and from IgAN patients in sporadic form.

Slide 21

Just recently we published a second genome-wide scan study in the American Journal of Human Genetics and we found suggestive linkage for two novel IgAN loci.

Slide 22

22 new families not linked to chromosome 6 were analysed.

Slide 23

And genome-wide scan study demonstrated that there was the possibility that some families linked to chromosome 4, as you can see in the slide with a LOD score of 1.83 and the chromosomal trait was between 26 and 31.

Slide 24

The second chromosome was chromosome 17, the trait was between 12 and 22.

Slide 25

So in conclusion this genome-wide scan study demonstrated that there are some families linked to chromosome 4 others to chromosome 6, others to chromosome 17.

Slide 26

This means that family studies demonstrate that genetic plays an important role in the development of IgAN as many genes maybe implicated in the pathogenesis of this disease.

Slide 27

Moving now to the case-control association studies. Many papers have been published during these years that you can see in this slide.

Slide 28

But if we analyse carefully these studies, many of these studies have some inconsistencies which consist in inadequate sample size. I would like to draw you attention to the possibility that if 1SNP has an allele frequency of 7% at least 240 cases of IgAN and 240 cases of controls should be analysed but many times these papers include a low number of patients and controls. In addition some papers do not have functional genetics, do not have replication and these points are very important for studying, for looking if there is efficacy of genetic studies.
Finally, there is the most important bias in case control studies, population stratification.

Slide 29

So I would like to draw your attention just during the discussion and ask Rosanna the role of interferon gamma because moving from a paper published by Japanese investigators in 2003 they demonstrated that there was a special gene polymorphism, exactly the number of repeats was 13 was significantly higher in the patients developing IgAN and these are Japanese patients with the disease.

Slide 30

We move on from this data for organising a new study. Exactly this was a family-based association study in which there is a no-bias concerning the stratification of the population and we studied the role of interferon gamma gene polymorphisms in our patients.

Slide 31

You can see here that the CA-repeat 13 was involved in patients with IgAN, was highly significantly present, 0.006 and this 13 CA-repeat was preferentially transmitted to affected individuals.

Slide 32

In addition, we also studied the local production of peripheral blood mononuclear cells from patients genotyped. Exactly we have two types of genotype 13 and 12. 13 is responsible for low production of interferon gamma and 12 is responsible for production of high levels of interferon gamma. This slide shows clearly there were patients with low production, low amount of interferon gamma and others with high levels.

Slide 33

When we move on to functional genetics this is a very important point and we transfected HK2 cells with a different allele I mean 12 CA-repeat and 13 CA-repeat we see that after stimulation with soluble CD14 ligand there was a reduced production when the 13 CA-repeat was transfected in HK2 cells.

Slide 34

Well, these two studies are very important because they were carried out in different populations, in Caucasians in our study, in Asians in the Japanese study and this study evidenced the significant higher incidence of this polymorphism in patients with IgAN and this allele is responsible for low production interferon gamma which may influence the development of disease.

Slide 35

This cartoon shows clearly that IgAN may be a TH2 disease in the sense that we have low production of interferon gamma, low production of TNF-α but we have a high production of IL-10 and so there is a prevalence of humoral immunity in our patients with IgAN.

Slide 36

In conclusion many approaches have been used with partial success to determine the genetic determinants of IgAN including analysis based on candidate genes, genome wide-linkage analysis and family-based classical linkage studies. Genomic studies have shown additional candidate genes and all this evidence point to the existence to a complex system in which multiple genes may participate in the determination of IgAN predisposition.

Slide 37

Moving to the second part of my talk, genetic risk factors for progression of renal damage.

Slide 38

We just recently published in the American Journal of Kidney Disease our cases of more than 400 IgAN patients. We analysed the survival of these patients and you can see that the data are approximately the same as other studies described. 60% of patients after 20 years from the time of renal biopsy have normal function and 40 are in end-stage kidney disease or doubling serum creatinine.

Slide 39

Multivariate analysis has demonstrated that these 4 points were risk factors responsible for the progression of renal damage. I mean microscopic hematuria at the onset of the disease, renal failure which consists in creatinine more than 1 mg/dL, daily proteinuria more than 1 g/day and finally, grading of renal lesions.

Slide 40

We adapted a new classification in which patients were divided into 2-3 groups G1 patients with mild renal lesions, G2 with moderate and G3 with severe renal lesions and you can see that COX proportional hazard survival curves demonstrated that cumulative failure was present in 93% of patients with severe renal damage, 36% in patients with moderate renal damage.

Slide 41

Well, as I told you at the beginning of my talk, human genome project was the start up of a new project concerning gene. There is now in progress some projects there’s HAPMAP, ENCODE project and VARIOME project and the protein concerning protein we have PROTEOME and METABOLOME project which consist in creating a new medicine which is a molecular medicine.

Slide 42

This is a very important point because we will move in the next years from biochemical medicine to molecular medicine.

Slide 43

For this reason the European IgAN Consortium will organise a new study in which peripheral blood mononuclear cells will be analysed by microarrays and renal tissue will also be analysed by microarrays.

Slide 44

We only have preliminary studies about these situations because we want to study some biochemical points that I just mentioned a few minutes ago by microarrays on peripheral blood mononuclear cells and by microarrays on renal biopsy concerning renal lesions.

Slide 45

Just recently we finished, these are preliminary data, this short paper in which we have 10 patients with IgAN and they were analysed and compared with the patients with polycystic kidney disease and impaired renal function and normals and this slide shows clearly that there is a different distribution of gene expression concerning PKD, IgAN and normals.

Slide 46

And here you can see there are some genes that are upregulating and these genes are included in those chromosomal areas that I demonstrated at the beginning of my talk. I mean chromosome 6, 4 and 17.

Slide 47

Another study was published a few years ago by Preston. They analysed gene expression profiles in circulating leukocytes and they compared the expression of these genes in PBMC or patients with IgAN and other renal diseases which are listed here.

Slide 48

And they found that 14 upregulated genes were present in patients with IgAN, there are a lot of genes.

Slide 49

And they concluded 7 genes correlated with serum creatinine levels. This is the translation from biochemical medicine to molecular medicine. 5 genes correlated with a higher creatinine clearance and so on. In conclusion some genes appear to be specific with the renal impairment in IgAN and not in other diseases.

Slide 50

Now we are moving to another study. We want to study the gene expression in patients with different phenotypes because it is very well known that patients at onset may have frequent episodes on intrainfectious macroscopic hematuria or patients that have sometimes only one episode of gross hematuria followed by persistent microscopic hematuria or patients that have permanent microscopic hematuria.

Slide 51

Finally, we are moving to the microdissection for renal biopsy.

Slide 52

We don’t have data not yet but I would like to present data from Waga et al published a few years ago in Kidney International. They studied 12 patients with IgAN, 4 with mild and 4 with moderate and 4 with severe renal damage.

Slide 53

And they found 13 genes upregulating IgAN in IgA renal biopsies and 7 genes were included in the first cluster, 12 in the second and 1 in the third cluster.

Slide 54

So this is the list of genes, of course, this is not sufficient but we are moving in that direction for transferring biochemical medicine to molecular medicine.

Slide 55

What is the rational of our European IgAN Consortium? We are moving from the linkage analysis after we have demonstrated that some chromosomal traits linked to patients with IgAN, familial IgAN, we are designing RNA interference agents against selective genes that we will show by microarrays and after we will move on to functional genomic analysis for looking at if these genes are responsible for the progression of renal damage and for the development of the disease.

Slide 56

Before concluding my talk I would like to acknowledge the members of the European IgAN Consortium who participated in the 50 framework projects and the members of the Italian IgAN Consortium who participated for other projects that were granted from the Minister of Health at the University in Italy. Thank you for your attention.