Seminar on hemochromatosis the 18^th of September

On the 18^th of September, Evry iGEM team organized a seminar on hemochromatosis and iron related diseases. The aim of this seminar was to present the latest scientific advances on those diseases and futur treatments. It was also an opportunity to present synthetic biology and our project in iGEM competition to both scientists and patients. We also took time to discuss with patients and the presidents of patients' associations about the state of our research and the aim of our project, in order to eliminate all possibly remaining ambiguities.

Day's agenda

Hours	Speaker	Subject
10:00-10:15	Gabriel Guillocheau	Opening speech
10:15-11:00	PHD-DR Gaël Nicolas	Iron homeostasis in mammals and its deregulation
11:00-11:45	PU-PH Pierre Brissot	Hemochromatosis: its diagnostic and its treatment
12:00-12:45	PHD-DR Marie-Paule Roth	Link between HFE, hepcidin and BMP6
14:15-15:00	Result survey with patients participation
15:00-15:45	PHD Nicolas Pollet	Microbiom studies
16:00-16:45	iGEM EVRY team	A bacterial treatment for hemochromatosis
16:45-17:00	Gabriel Guillocheau	Closing speech

Report of the day

Gaël Nicolas: "Iron homeostasis in mammals and its deregulation"

Summary

Iron is necessary to living beings but potentially toxic, that is why homeostasis is required. In a human body there are 4 g to 5 g of iron, that is about a rusted nail. Iron is present as Fe²⁺ and Fe³⁺; as Fe³⁺ it is insoluble and thus must be degraded in Fe²⁺, but as Fe²⁺ it becomes highly toxic because it is a strong oxidant. Much of iron is located in the red blood cells. This explains why we associate iron with oxygen transport, but many other reactions imply iron.
In the 1930's, an experiment showed that iron was not excreted by the body: iron remains stocked in the body. Everyday we absorb 1 mg to 2 mg of iron in order to compensate the 1 mg to 2 mg of iron lost through the death of cells. The production of red blood cells daily uses 20 mg of iron, but these 20 mg exclusively come from the recycling process of old red blood cells by macrophages.
Hepcidin is a hormone that makes the level of iron in the serum decrease. Hepcidin is thus to iron approximatively what insulin is to glucose. Its deficiency is implied in almost all the forms of hemochromatosis. This hormone has four sulphur bridges, that makes it very difficult to synthesize.

PDF of Gaël Nicolas presentation (in french)

Question/Answer

Q1: What are the consequences of iron overload on red blood cells? Are they not biconcave anymore?
A1: No, they remain biconcave because this feature has been naturally selected and it is necessary for the red blood cells to go through the capillaries. But there may be more of them, or they may be bigger.
Q2: Given the number of persons who have this disease, how is it still possible that doctors do not know it, or can not diagnose it properly?
How can you explain the different forms of the disease (particularly regarding the seriousness) between siblings?
A2: Other genes have effects on iron regulation, so they can increase or decrease the gravity of the disease. Food have consequences too, and for example the red wine contains much iron. Moreover alcohol reduces the production of hepcidin.
You mentioned doctors. I want to put things in perspective: in France there are around 200 000 doctors, and there are around 200 000 patients, thus a doctor will have on average one case in all his/her career.
Q3: According to you, will the synthetic mini-hepcidin induce an immune response?
A3: The mini-hepcidin is produced with major modifications, so yes.
Q4: Once iron is stored in organs, how can it be eliminated?
A4: It can not, unless by bloodletting.

Pierre Brissot: "Hemochromatosis: its diagnostic and its treatment"

Summary

Hemochromatosis is a frequent genetical disease: in France one person over 300 is predisposed to develop it. Indeed, it is a disease with an incomplete penetrance, which means that all the people who are homozygote for the mutation will not develop the disease. Hemochromatosis is also a delayed disease because the symptoms are long to appear: hemochromatosis is long silent.
The first symptoms are chronic tiredness and rheumatism. As they are not very specific symptoms, 5 to 10 years often pass between their appearance and the diagnosis. The excessive accumulation of iron has serious consequences on the organs, mainly on the liver. The liver is indeed the first barrier of the body against iron. The over absorption of iron in the liver leads to its cirrhosis, and when the liver can no more stock iron, iron goes to the pancreas and the heart.
The most usual genetic mutation causing hemochromatosis is a mutation of the “iron gene” on the chromosome 6, called C282Y homozygosity. This mutation drastically decrease the production of hepcidin. The genetic test should be done only after a blood test determining iron level. It is also possible to use MRI to see if the liver stocks iron.
Bloodletting forces the organism to use the iron stocks to renew the red blood cells. Bloodletting gives really good results, almost without any side effect, but it is not perfect: for example rheumatism remains. Moreover some patients can not get used to bloodletting. Iron chelators (in oral treatment) can thus be an alternative to bloodletting, or at least complete bloodletting.

PDF of Pierre Brissot presentation (in french)

Question/Answer

Q1: 98% of the patients have the mutation C282Y/C282Y. We know that this mutation is Celtic, but how can we explain the 2% who have another mutation?
A1: When we notice a major iron overload and a heterozygosity, we look for another mutation: sometimes it is indeed another mutation, but sometimes it is a heterozygosity with two different mutations of HFE gene (but it is very very rare).
We notice a great variety of symptoms, but the personal particularities are still uninterpretable.
Q2: What do we do with H63D patients?
A2: Nothing.
Q2': Even if their serum ferritin values reach 1200?
A2': The most frequent causes of high serum ferritin value are metabolic (alcohol, excess weight, etc.). The H63D patients are rarely iron overloaded.
Q3: Was the HFE mutation C282Y once an advantage?
A3: It was probably once a selective advantage against postpartum haemorrhage for example. But today it is no longer the case. A high amount of heterozygosity is often due to the fact that it was once a selective advantage.
Q4: Does the immune system age more quickly with frequent bloodletting? Has it been researched?
A4: No. But the life expectancy of an early diagnosed patient is average, even slightly above because hemochromatosis patients have a better medical follow-up. And iron overload is far more toxic than immune system aging.

Marie-Paule Roth: "Link between HFE, hepcidin and BMP6"

Summary

What are the pathways of Hepcidin regulation? So far, we know that a large number of proteins are involved in hepcidin synthesis, mainly BMP6, Hemojuvenile, ALK3 and SMAD4. The inactivation of one of these molecules leads to iron overload (this has been demonstrated in knockout mice). Studies on mice has revealed an important role of testosterone on the activation of EGF receptors, which are present in the liver and which is a repressor of hepcidin. In the biosynthesis cascade of hepcidin, the exact role of the HFE protein is still unclear but according to the current research hypotheses of Marie-Paul Roth's team, it seems it is involved in 2 pathways: One with BMP6 and one with BMP2.

PDF of Marie-Paule Roth presentation (in french)

Question/Answer

Q1: How can we integrate the EGF way in the final model?
A1: I could have represented it, but we still do not know very well how it works. We need crossed mice to experiment.
Q2: Did you experiment on mice with KO Bmp6 type 2 receptors?
A2: No, because you can not invalidate those receptors without consequences, unless you only target the liver.

Result survey with patients participation

Summary

We learned three important informations with our survey.
Most patients are satisfied with bloodletting, and the more satisfied they are, the less they want another treatment. Despite this, they remain interested in a complementary treatment.
The most interested patients are the patients who would benefit the less from the bacterian treatment. Indeed this treatment would be most appropriate after the first phase of treatment, to help regulating iron absorption and diminishing the frequence of bloodletting.
Last but not least, our survey shows that a bacterian treatment would be a real help for working patients, who often have difficulties to leave their job to go to bloodletting.

PDF to the result survey (in french)

Nicolas Pollet: "Microbiom studies"

Summary

All the micro-organisms that live in (and on) the human body constitute our microbiome. There are 10 times more of them than our own cells, and the microbiome as an all has 1000 times more genes than us. The microbiome is a necessary part of our organism, so there is a large number of beneficial bacterias.
The microbiome is still relatively unknown. We know that it is very complex. The number of micro-organisms increases exponentially along the digestive system; the microbiome varies depending on the place of the body, but also depending on the topology. Our microbiome changes during our live too: for example during pregnancy. Last but not least, we all have different microbiome, but it seems that there are several bacterial patterns. As the microbiome plays a role in some pathologies, these patterns may be a major research field.
Several probiotics are already marketed, and many more are tested.

PDF of Nicolas Pollet presentation (in french)

Question/Answer

Q1: Concerning the probiotics, how can we avoid colonization?
A1: We remove all the plasmids involved in colonization.
Q2: Is there a dissemination risk, when the bacterias are evacuated in stools?
A2: No, they are anaerobic and without colonization power.
Q3: What about the risk of genetic information sharing?
A3: Bacterias share genetic informations continuously, so it is very unlikely that we increase the risks significantly.

iGEM EVRY team: "A bacterial treatment for hemochromatosis"

Summary

We ended the day by a presentation of our project. We first explain what synthetic biology and iGEM competition are, and described some famous achievements realized in this competition ( Austin 2004 Hello Word project, Edimburgh 2006 Arsenic Biosensor, Groningen 2012 Food Warden…).
We then presented our project, the reasons we chose it and how we designed our different systems: sensor, inverter and chelator systems. After that, we finished our presentation with some results we had obtained with the characterization of our iron sensing system and with our model.

PDF to our presentation (in french)

Impressions of some speakers

Pr. Pierre Brissot, Hospital practitioner and university professor in the Rennes Pontchaillou hospital

When Gabriel Guillocheau contacted me at the beginning of the summer to take part in a seminar about hemochromatosis, I perceived well both the originality and the pertinence of the ongoing approach.

The day of the seminar indeed confirmed completely this first impression. I am pleased to underline the two points that seemed to me the most remarkable:

• The originality of the scientific project aiming to chelate the intestinal iron with genetically oriented intestinal bacterias, in order to inhibit the duodenal iron absorption, and so helping to thwart the digestive hyperabsorption responsible for the development of iron overload, in particular in case of genetic hemochromatosis.
• The performance, and the daring showed by the organizers, coordinated by Gabriel. In only a few weeks of summer, they set up an entire day of exchange with not only the main national scientific experts of hemochromatosis, but also with the presidents of the main hemochromatosis patients' associations. This was a particular dimension of the day, and a special attention.

Thus, I congratulate warmly the entire team – students, advisors and instructors – for this successful meeting.

Dr Gaël Nicolas, PHD specialist of iron metabolism at Cochin institut

This event was an opportunity to bring together hemochromatosis patients, presidents of patients' associations, expert researchers and clinicians, and the 2013 Evry iGEM team in an extremely warm atmosphere.

During the day, very varied themes were discussed: the fundamental research on iron homeostasis, the clinical aspects of hemochromatosis, the ethical and philosophical aspects of the creation and use of a probiotics to decrease iron overload, and finally the state of the art of a promising research field, the microbiome.
On the remarkable (and rare!) initiative of the 2013 Evry iGEM team, the patients had the possibility to express their opinion about their disease by answering a survey: 135 women and 135 men answered it, which constitutes an absolutely remarkable cohort. One session, presented by the 2013 Evry iGEM team, was dedicated to analysing some of the datas.
Finally, the 2013 Evry iGEM team presented their project. They could present the beginning of a proof of concept: they had obtained a modified bacteria capable of modulating GFP expression according to iron concentration.
The day ended with a visit of the laboratory given to the 2013 Evry iGEM team to elaborate their project.

During this kind of day, it is not so easy to use a vocabulary such as all the participants could reach a common base of knowledge, in order to have exchanges as fruitful as possible. Nevertheless I feel that all the speakers managed it, and thus it enabled each time an enriching exchange of questions and answers.

From a personal point of view, it is always very enriching to meet patients, to hear their difficulties, to understand how they live, accept or refuse some aspects of their disease. It is during these exchange that I hear their expectation for new medicines, and that, in return, I have the possibility to explain that it is always very long between a proof of concept and a new treatment. 10 to 15 years usually pass between the two.

I repeat my congratulations to all the members of the 2013 Evry iGEM team for the organisation of this day, for their dynamism, for their scientific daring, and most of all for the way they have developed their project scientifically as well as philosophically... starting from scratch... and in the record time of a few months!

Brigitte Pineau, President of the French Federation of Hemochromatosis Patients' Associations:

We were very interested in the project of the iGEM Evry-Genopole team. So, during summer, we enthusiastically spread a survey to around 500 contacts that our regional associations have. Indeed, how could we not respond to the solicitation of these students, when their project aims to improve the life quality of hemochromatosis patients, who have still no pharmaceutical treatment, and are bled like in the time of Molière? What a fortunate initiative!

The 4th of September 2013, I had the pleasure, as President of the FFAMH (French Federation of Hemochromatosis Patients' Associations), to be invited to come and meet with this very nice multidisciplinary team. They were very motivated and willing to go as far as the iGEM final in Boston. They were asking for advice, and they presented their project to us.

The seminar of the 18th of September was a great success. The most renowned researchers in the field of iron metabolism were present: Dr. Gaël Nicolas from Cochin Institut, Pr. Pierre Brissot - coordinator of the Center of reference of rare iron iron overloads -, Dr. Marie-Paule Roth from Toulouse Inserm. The specialists were given the floor before the project was presented by the team to the physicians, researchers and patients invited to this exceptional day.

If some of the aspects of the presentations were sometimes too hard to understand for the patients, particularly during the presentation of Dr. Marie-Paule Roth about the link between HFE gene and hepcidin, the presentations enable us to understand that despite the discovery of hepcidin - key regulator of iron homeostasis - no pharmaceutical firm tried to produce this complex molecule yet. The presentation of the microbiome by Dr. Nicolas Pollet, introduce us to an unknown world. We were far from imagining that our digestion system contained between 500 and 1000 different bacteria, and that they impact our health. We ignored also that the bacteria love iron very much…

So, thinking of a bacterial treatment in substitution of bloodletting, this was to us a very innovative perspective.

As, in the FFAMH, we never thought about bloodletting in the perspective of a new treatment, we learned something important through the survey: the more the patients are experienced with their disease, the less they are ready to change their treatment.

The FFAMH thanks these students very much for their interest in hemochromatosis, and wishes them the success they earned in Boston.

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