“Monday 10th May 2004, 7 : 11 am, Saint Charles Station in Marseille, my night train coming from La Rochelle comes into the station. I am Marie-Claude BOITEUX, Chair of the Association « Cutis Laxa Internationale ». Our « taxi-driver » Roger PICARD, Chairman of the Federation « Huntington Hope », is waiting. He arrived yesterday evening from Metz. We just have time for breakfast while we wait for the 8:25 train, coming from Dijon, on which Sylvie CASTELLA is. She is the secretary of Federation « Huntington Hope ». She arrives with her daughter, Maïlys, student and volunteer for « Huntington Hope ». Claudine MICHELLAND, who lives in Marseille, joins us. She is a member of the Board of Huntington Hope South-East . She helps us find our way through the Marseille traffic jams, towards the Parc de Luminy where the « School of DNA » is located. There we meet Jean-Claude DUTRIPON, treasurer of the Association « PXE-France » (PseudoXanthome Elasticum). He also lives in Marseille.
It is 9 :25 am. Everyone is here. Our great adventure in the core of DNA and the mysteries of research is about to start, under the guiding hand of our trainers Marion MATHIEU et David KARLIN.
None of us has any specific knowledge in biology and biochemistry. We have gleaned bits of information in the medical literature we consulted in our research on the disorders we are concerned with, but it is only a smattering of it. Thus we must first familiarise ourselves with the laboratory equipment we will be using during these three days :the laboratory benches on which we will work, the pipettes of different capacity ( to add different needed liquids in infinitesimal quantities), the test tubes, the « Vortex » (electric vibrator used for thoroughly mixing the content of the tubes, which leaves us with pins and needles in our fingers), the dry bath (to heat the content of the tubes up to the required temperature), the centrifuge (which allows the heaviest elements of the content to drop down to the bottom of the tube), etc. David also presented us with an educational tool we will refer to throughout the course : a representation of the double helix of DNA, with all its constituents. Funny how it looks like the plastic pearl necklaces, full of colours, we made and loved when we were kids.
After revising the different elements which compose a cell (nucleus, membrane, etc) and memorised again that DNA is located on the chromosomes at the core of the nucleus of each and every cell of our body, we can start our first real experiment. As if we were some kind of Sherlock Holmes we will reproduce the different steps of a DNA analysis made by police forensic scientists, using our own DNA. (extraction of DNA from cells of the mucous membrane in the mouth then amplification of DNA by PCR and visualisation by electrophorsis in agarose gel).”
EXTRACTION OF DNA FROM CELLS OF MUCOUS MEMBRANE IN THE MOUTH
After a mouthwash, we « spit » in a test tube which we then place in the centrifuge. The biggest and heaviest elements form a deposit at the bottom (the “culot” in French). With a pipette, we remove the liquid ( the « surnageant » in French) in which there are cells coming from our mucous membrane. Then we make a PCR.
AMPLIFICATION OF DNA BY P.C.R. : WHAT IS A P.C.R. ??????
PCR ( Polymerase Chain Reaction ) reproduces, in vitro (in a test tube) the duplication of DNA which naturally happens when cells divide. In human beings, the fertilised ovum (egg) divides about fifty times to give 2, 4, 8,……. 50,000,000,000,000 cells throughout the 9 months of pregnancy. Through this process, each and every one of these cells contains a DNA molecule strictly identical to the one in the initial egg. PCR reproduces 40 successive duplication cycles in ….. 40 minutes……instead of 9 months………. Nowadays, this technology is basic in all aspects of biology (genetic testing, judicial identification, testing for bacteria and viruses, marking and identifying organisms, amplification of ancient DNA, etc).
Of course, according to the researches made, it is out of the question to copy (amplify) the entire DNA, but only one specific sequence as required by the type of research being carried out. Remember that genes represent only 5 % of DNA, the rest is composed of intermediary sequences whose function is not yet well known. So one must know, in advance, which sequence will be amplified. It can be used for genetic testing only if the sequence of the mutated gene is perfectly identified. To make a PCR, you need :
- A polymérase : an enzyme (a protein with a specific action) which is able to make a new piece of DNA from the original DNA.
- « Amorces » : short sequences of DNA which will identify the section of DNA to be copied, complementary sequences to the first and the last of the portion of DNA that is to be amplified.
- Bases : A (Adenin), T (Thymin), G (Guanin), C (Cytosin) , the necessary « bricks » to create DNA. According to the order in which they are associated, they create a different amino acid :
ATC = Methionin, GGA = Glycin,…….. Each protein is made up of different amino acids. There are 20 amino acids (serin, glycin, cystein, tryptophane,…….). The bases are associated in pairs.
THE EXPERIMENT MADE ON A SMALL SECTION OF CHROMOSOME 8
1st step : Denaturating = heating : the DNA molecule opens up
2nd step : Hybridation = the « amorces » stick to the DNA
3rd step : Elongation = with the polymerase DNA, the 2 strands of DNA are replicated
Each time these 3 steps are reproduced, 2 new strands of DNA are made. They are strictly identical to the initial DNA ( DNA reference). Amplification is exponential. Normally, reproducing the 3 steps 40 times, allows the sequence to be amplified a thousand billions of times.
The piece of DNA we have amplified can exist in two versions (allèles in French) in the population (the presence or the absence of one of these two versions is not associated to any genetic risk) :
- Short version , 100 pairs of bases
- Long version , 400 pairs of bases
As we all have two chromosomes 8 ( one inherited from the father, and one from the mother),the PCR reactions on our DNA can give 3 possible results :
- 2 short segments of DNA if they have the short version on both their chromosomes (homozygotous)
- 2 long segments if they have the long version on both their chromosomes (homozygotous)
- 1 short segment and 1 long segment if there is the short version on 1 chromosome and the long version on the other (heterozygotous)
“Up to that point, everything takes place in test tubes. We added, with the pipettes, the various elements required for the reaction to occur, we « vortexed » the tubes, heated them, centrifuged them ,etc.….. and we also…… waited….. for the reaction to come through at each step…. waited…… for the heating time to be long enough. One must be very attentive, must not make the smallest mistake. It is vital to throw away the tip of the pipette and replace it with a new one for each new use, one must no use the wrong test tube, etc. Each time researchers devise a new experiment, they must, in addition to all that, systematically write down each step in a small exercise book (how many micro-litres have been used, which product, heating time and degrees,etc,…… that’s not all, and not the half of it……..) . This is absolutely necessary to be able to change, even in the smallest way, one or other of the parameters, which will change the result of the experiment. We start understanding that researchers’ time scale is not the same as ours……..”
During one of these waiting times Mrs Constance HAMMON visits us. She is Chair of the Association « The School of DNA » which is welcoming us today. We thank her for that. We touched on one of the many difficulties which associations struggle with : Fundraising. This is something we know too well. Even if, today, the laboratory in which we work is fairly well equipped, there are still a lot of machines to buy to be able to carry out all the experiments needed in genetics (especially microscopes , as well as equipment for cell biology experiments). Their cost is quite high. The laboratory is located in the newly built premises of the INMED ( Mediterranean Institute of Neurobiology). It was partly funded by the Regional Council and the town of Marseille. It stands on the Campus du Parc de Luminy where there is part of the University as well as various scientific institutions and organisations such as the Immunology Centre, the Particle Physics Centre, etc. The INMED houses the School of DNA, but also the Association « Hippocampe » which organises training sessions for secondary school pupils, as well as laboratories for neurobiological research ( working on epilepsy). Some rooms are still waiting for the installation of laboratories for pharmacological research. We deeply appreciate this pleasant and relaxing moment, talking about this and that.
VISUALISATION OF FINGERPRINTS BY ELECTROPHORESE IN AGAROSE GEL
Our test tubes are now ready. We can watch the results with special equipment. First we must put the Agarose gel between two glass plates. This gel will filter the content of the tubes in order to “see”, according to their size, our genetic fingerprints.
Our samples are placed between two control samples. The first one(control +) is David’s DNA (he knows in advance the result that must be obtained), in the second one (control -) DNA was replaced by water in the tube for the PCR. These control samples will help us verify that there is no contamination in the tubes and in the products we used and that the experiment worked well.
With our pipettes we slip a tiny quantity of the content of our tubes between the two glass plates, and we write down the order in which we are putting them. Each one of us will soon know if s/he is Homozygotous or Heterozygotous. The glass plates are placed in an “electrophorese” machine. Thanks to electrical impulses the amplified DNA will move and stop at the level corresponding to its pairs of bases.
“Unfortunately, one moment of inattention and……the content of Jean-Claude’s and Claudine’s tubes are mixed…….. even so we put them between the glass plates, knowing that this result will be skewed….. It only takes very few things to ruin the work you have done. It is so hard to be a researcher ! And now we must wait…..always wait….. The reaction takes time. As it is after 12:30, we decide to go and have lunch. Some of us , remembering our student days, a long time ago, appreciate the gastronomic pleasures of …….. the University Restaurant .”
Our experiment is a success………. We see our genetic fingerprints on the gel.
“I am the only one to be, and proud to be, Homozygotous Long Version…… All the others are Heterozygotous, ………even Jean-Claude’s and Claudine’s mix up…….
If we had carried out this experiment for a police enquiry , the control+ sample would have been the one found in the murder room, and the others, the victim’s and the suspects’. Comparing the results of these samples and superimposing the images would have allowed the identification of the murderer. In the same way, when one knows the deficient or mutated gene of a genetic disorder, one can say if the samples examined carry the same deficiency or mutation. It can be done for Huntington disease.
2 : 30pm, we are back at our benches. Marion is our afternoon teacher. She prepared for us some “recipes” which, as ”research apprentices” we are will have to be successful with……. At least we hope so. We will have to introduce the gene coding for GFP (Green Fluorescent Protein) which renders organisms fluorescent, into bacteria (Echerichia). These bacteria can naturally be found in our intestines and are not dangerous for us…… Phew !!!!. This gene comes from a chinese jellyfish, Aequorea Macrodactyla, which naturally produces GFP.
We are going to make transgenic bacteria………. GMO (Genetically Modified Organisms)…… which, once the gene is introduced, will be able to produce GFP protein themselves. We start “cooking”………”
HOW TO GET BACTERIA TO PRODUCE A PROTEIN OF INTEREST ?
DNA is a universal code. The information it contains can be understood by any living organism (living on earth !!!!!! of course !!!!!!!……..). So our little bacteria won’t have any trouble reading the instructions of the jellyfish gene. In the same way one can use micro-organisms (bacteria, yeasts, etc) to produce therapeutic proteins ( hormones, vaccines, coagulating agents, etc). It bypasses the problems linked to the difficulties of purifying these proteins when they come from their natural producers (human beings for instance). It also ensures that there is no infection (viruses or prions for instance) and, enables a manufacturer to control completely the production line. That is why this method was one of the first considered in genetics.
After having heated, then quickly cooled down, our bacteria, thus creating a thermal shock, their membrane is weakened and allows the foreign DNA to enter. The segment of DNA is also associated with an antibiotic-resistant gene to allow us to control our work, throughout the experiment, and to select only the bacteria which accepted this « stranger » inside them.
To complete the process the boxes are placed for 24 hours, upside down, in a 37°incubator.
“Pipettes, dry bath, vortex, test tubes, Petri boxes, are…… our dishes, our spoons, our oven, our mixers and our cake tins …..
Bacteria, CaCL², Segment of DNA, H²O, culture environment are…… our flour, our butter, our eggs, our yeast………
All afternoon long we cook, we make « pastry »………
We take some break while waiting for the mixture to fit the recipe……. Tiny apportionments…….Special precautions………….. All this is much more precise and complicated than to cook a « home made stew » ……. But, most of all the last cooking time is so long : 24 hours…….. It is only tomorrow morning that we will see if our « cakes » are successful……
It is 5 : 30 pm. We are quite tired after this intense day. We have already learned a lot. Tomorrow the pieces of equipment will no longer hold any secrets for us, which is great, compared to what we knew this morning….. Sylvie, Maïlys, Roger and I decide to go straight back to the Hotel for dinner. All the inhabitants of Marseille are also going back home……The joys of traffic jams……. We arrive at 6 : 30 pm and decide to meet for dinner at 7 : 30. As soon as dinner is finished we go back to our bedrooms, exhausted but happy, ready to start our second training day,….after a well deserved night of rest…..
Tuesday 11th May , 8 : 00 am we meet for breakfast and even if Sylvie has overslept a little we almost leave the Hotel at the scheduled time. Unfortunately, as soon as we leave the hotel, we are caught in traffic jams. Thanks to « Miss GPS » in Roger’s car we try another route, but it is no better…….. roadworks, dustcarts, drivers in a hurry,……..it doesn’t matter, we arrive safely, but a little late. Everyone is waiting for us….quick….let’s go back to work….. but the morning will be a little « perturbed » by the presence of journalists (M6TV and « la Provence » newspaper. In fact, it is the first time the School of DNA welcomes patients’ associations. It is worth a scoop. We willingly lend ourselves to interviews, photos and filmed sequences. It is very important, for us too, that the School of DNA is better known. It deserves advertising. There is no other place where one can learn what we learned here. Our members, our sufferers need the knowledge, the information we will share with them, allowing a better understanding of researchers’ work.
We go and watch our little “cakes” of yesterday. They “cooked” all night long and are ready. In each of our petri boxes we find the expected results.”
We will use the box containing the transgenic bacteria for our morning’s work :
GFP PURIFICATION BY HYDROPHOBIC CHROMATOGRAPHY
Why and how to purify a protein are this morning’s lessons.
As we learned yesterday, proteins have a primordial role in our system. They induce the different functions of our organs. There are 30,000 genes in the human genome. Each gene contains an average of 1,200 nucleotides (group of proteins) with each protein composed on average of 400 Amino-Acids ( 3,500 in the Huntingtin !!!!). The human genome is composed of 3,000,000,000 nucleotides. Studying proteins is thus primordial in all genetic research. To complete these studies, it is necessary to have a great quantity of the protein to be studied. As was the case in our experiments where we had GFP produced by bacteria. These bacteria, placed in a culture, develop. We must now extract (purify) the protein to be able to study it. In the experiment we lead, we want to purify GFP which is hydrophobic. It does not like water and tends to « stuck » to non aqueous supports (lipids (fats), non charged carbon polymers, etc) which are also hydrophobic : two hydrophobic substances tend to attract each other. So we will use plastic balls (very tiny) covered with a hydrophobic substance (resin) to catch and purify GFP. The principle is quite simple : bring together GFP and the other hydrophobic substance in an aqueous solution with added salt (salt helps to link the two), then “disconnect” (éluer in French) the GFP by adding …….. clear or almost clear water (without salt). But………..
The method is quite complex and consists of many steps :
- Breaking the membrane of the bacteria (« Lyser » in French ) thanks to a solution ( “tampon” in French) which contains a detergent, putting it in the Vortex in order to mix it all well and incubating it for 15 minutes . The resulting mixture is called « Lysat »
- Getting rid of the big debris and the DNA through 10 minutes in the centrifuge then picking up the « surnageant » ( surface liquid).
- Adding to GFP a fixation « tampon » which has a high salt content.
- Adding the balls and incubating for 15 minutes
- “Washing” the whole mixture with a washing « tampon » of an intermediate saltiness to eliminate the slightly linked contaminants
- Adding a disconnecting « tampon » ( in fact it is just water).
- Putting in the vortex
- Putting in the centrifuge
- Removing the surface liquid (called « éluat » in French, in this experiment)
Steps 6 to 9 must be done again and again, until the balls are not fluorescent any more, which means that there is no more GFP stuck on them. We must be careful not to mix up the resulting “éluats” ! Each time they must be controlled on an ultraviolets table to « reveal » the fluorescence.
There are other methods used to purify proteins, according to their size, their electrical charge, their attraction for a substance : antibody, DNA, RNA, etc.
“Oh my God !………. And you take the pipette, and you add a little bit of this, and you add a little bit of that, and you place in the vortex, and you pick up the surface liquid……….in short…….. this morning is flying by ……..we are passionate and studious, concentrated on our benches and our tubes, lying in wait for fluorescence …… It’s a hunt…..That’s putting it mildly !….. It’s a desperate struggle… we are going to capture this GFP !!!!!! ………Victory!… It had just struck twelve when we showed, very proud of ourselves, our tiny test tubes containing a few micrograms of GFP. The winner is Claudine. Her tube is a lot brighter than ours on the U.V. table.
We have a well deserved lunch at the University Restaurant like yesterday, but we are hungry and we need to recharge our batteries for the afternoon.
After a coffee-break at the university cafeteria we climb back to the INMED. Yes indeed !!! The scientific Park of Luminy is on a hillside. The INMED is right on top and…. The restaurant and the cafeteria are…….. ..right at the bottom !!!!
The slope is a bit hard to climb after lunch, but never mind, our benches are waiting for us………….”
HOW TO ANALYS THE STRUCTURE OF A PROTEIN ? : THE LIMITED PROTEOLYSE
Taking Huntington disease as an example, we will follow this new experiment step by step.
Proteins are composed of Amino-Acids which are organised in regions with a compact structure (domains) and in regions which are flexible.
Huntington disease is due to an abnormality in the huntingtin protein. In sufferers there is an abnormally high number of glutamin amino-acids following each other which leads to its aggregation through an unknown mechanism. In order to understand this mechanism it is necessary to study the structure of the protein in its normal shape, then in its aggregate shape, and compare them. Huntingtin is a giant protein( over 3,000 Amino-Acids) composed of different parts, each one with a different structure, linked together by flexible parts. One of the methods used to study and understand the way the body of a big protein is designed is to cut it in smaller bits (“proteolyse”) that can be studied separately.
As the analysis of huntingtin by « proteolyse » (or any other method) is very difficult and is the subject of current research on this disorder, we will study a simpler protein : the nucleoprotein of the measles virus .
One needs “tools” to cut a protein : it is the “proteases”. For our experiment, we will use Trypsin, from bovine pancreas.
To see the results we will engage in manipulations similar to those we learned about the electrophorese on Monday morning. But before that, we must prepare our samples.
Research often proceeds by trial and error. We do not know which quantity of trypsin, and in which proportions, we will need to best “cut” our protein. So we must “try” different proportions and keep only the best one. It must “cut” the flexible region enough but without completely destroy the domains. Besides, the solution of trypsin we have has a fixed concentration. So we must first dilute the solution of trypsin to obtain various concentrations.
We have 18 test tubes before us : 6 for each one of us ( we have been working in twos since the beginning of the training session) and 6 which we will use to make trypsin in the various concentrations.
Proteins are first denatured, which means completely “unrolled”. Then they are negatively charged with a strong detergent , SDS. Then, thanks to a magnetic field, they migrate to a polyacrylamide gel which separates the smallest proteins (they move quicker) from the biggest.
This is called “electrophorese in polyacrylamid-SDS gel”.
After the proteins have migrated, the gel is steeped in a Coomassie Blue Solution which colours the proteins . After a few hours of tinting, the excess colouring is eliminated in a discolouration solution.
In Huntington disease, huntingtin, wether normal or mutated, is naturally « cut » in the cell by proteases. It is supposed that this encourages the aggregation of the mutated form. So it is crucial to understand which proteases intervene in the process (maybe to stop their action?) and what bits they generate. As for the structure of huntingtin, it is unknown…for the moment. In fact, huntingtin is very hard to purify from cells. It has not yet been possible to get micro-organisms to produce it. And, apparently, it can have many different structures in the cell.
“What about this then ?…………. Sometimes a kind of panic wind blew over the laboratory……..
All these test tubes on the benches…….. and we must not make any mistake. First dilute the trypsin : knowing that we have a solution of trypsin with a proportion of 2,000mg per litre and that we want to have various solutions with different proportions, how should we go about it?…. Rule of three, calculation, …that’s it ….no that’s wrong….. let’s start again….that’s it, we’ve got it…… It merely brings us back to the leaking taps and crossing trains problems which haunted our childhood’s homework!
Then add the various solutions of trypsin to the test tubes containing the nucleoprotein. Identify the tubes according to the proportion of trypsin we have added, place them in the vortex, not forgetting to change the tips of the pipettes after each use (it’s amazing how many we used during these three days !!!) and most of all ………. Don’t forget the tiny little detail which could ruin the whole experiment…….. to heat the tubes. Afterwards, with a micropipette, we must, without shaking, pour the content of the tubes onto the gel stuck between the two glass plates…….. Phew……. We made it !!!!! but….. be patient…. All go things come to those who wait…. We will only have the results tomorrow ….. Oh yes, that’s how it works in research, you sweat, you shake, you go to great lengths, you do your best and then you’re told that the results will be ready……tomorrow……Wait…always wait. And if it fails ????…….. well you’ll have to start all over again from the very beginning, a day’s work lost !!!!!! ………. Claudine sighs…… it’s been a tough day.
We decide it deserves a compensation : We’ll have dinner in a restaurant on the Vieux Port and eat a « Bouillabaisse » (a tasty local fish soup) . We also plan, weather permitting, which is not at all certain, to have a pick-nick for lunch tomorrow…… We are fed up with the University Restaurant…… We will also have a look at the newspapers because the article by the journalist of « La Provence » should be published in the Marseille issue tomorrow. As to M6TV they didn’t give the date of the broadcast. We leave our teachers and are happy to find again the cherished traffic jam. While Roger helps Claudine master a new piece of software on her computer, Sylvie and I go shopping for the pick-nick.
At about 8 : 00 pm we are happily tucking in a delicious Bouillabaisse on the Vieux Port.
We are back at the hotel at midnight for a good sleep. Tomorrow will bring new things and surprises to discover.
Wednesday 11th May, Jean-Claude has bought several issues of « La Provence ». The article is on the second page. Sylvie, Maïlys, Claudine et Jean-Claude have the place of honour in the photo. David and Marion are very pleased, even if, according to them, some issues deserved more accuracy. We wait for Claudine who went to the station to pick up Karen FINSTERLE, a nurse who volunteers for Huntington Hope, who will share with us this last training day. Then we hurry to the laboratory …… we hope our yesterday’s experiment has succeeded, we are impatient to see the results. David is waiting for us with the gel plate, how beautiful it is…”
According to the various proportions of Trypsin, the nucleoprotein (525 Amino-Acids) is progressively cut down to a tough fragment of about 400 Amino-Acids. There is no other tough fragment which means that the remaining 125 Amino-Acids have been totally « digested ». So the nucleoprotein is composed of one very dense fragment (domain) of 400 Amino-Acids and one very flexible region.
This morning we will not work in the laboratory. We go downstairs to the microscope room of the association “ Hippocampe ». Some of Marion’s colleagues from another laboratory have lent her genetically modified worms which are fluorescent. We admire them. It looks like the Milky Way.
A little less « cute », but very interesting, the little fly (drosophile) is also fluorescent. Jean-Claude, in a masterly gesture, picked it up with the tip of a brush from the bottom of its jar to put it on the plate.
We also examine cells, each in turn.
“A « quieter » morning, but what a pleasure for the eyes. Such beauty in the optics of the microscope….. incredible…… Unfortunately, as for the sky, it is not beautiful and our pick-nick is a wash out……. Never mind we settle down in the INMED rest room : fresh bread, ham, melon, crisps, cherry tomatoes, little cheeses, strawberries and « navettes » (small aniseed biscuits of Provence) brought by Marion. What a feast !
2 : 00 pm, this afternoon’s trainer cannot arrive before 4 :00pm as she is a University teacher. So we have some time on our hand. Marion proposes to go for a walk on the (small) mountain above the Scientific Parc of Luminy. After some hesitation due to the threatening colour of the sky, we decide to go….. A little exercise will be very good for us. It’s a bit steep, we do not all have the appropriate shoes, but the path is good and we quietly climb, despite a little « fatigue » for Sylvie.
Marvellous !!!!!! when we arrive at the top, we discover we are just above the Calanques. How beautiful it is !!! Even if the sky is grey and a little rainy, the sea is blue, slightly turquoise. We make the best of this wonderful view. Then we quietly walk down carefully, paying attention to the rocks the rain has made slippery. We are in Spring and there are flowers everywhere : brooms, wild lemon-thyme, sarsaparilla, etc….. the opportunity for a little botany……….”
Back at the laboratory, we meet Mrs Nicole PHILIP, hospital practionner at the medical genetics department of Marseille. She is a specialist in Huntington disease.
Here and there around the benches we start talking about this and that. Roger and Karen would like her to answer all their questions, but research is on its way and many answers have yet to be found. It is not always enough to identify the gene responsible to be able to consider therapies. We do not know enough about Huntingtin . So far, no one knows its real role inside our organism and if one could live without it. Even if it is already possible to introduce the mutated gene in the DNA of the fertilised ovum of a mouse, it is impossible to get rid of the huntingtin completely in an adult mouse to study the consequences of the total lack of huntingtin. In fact, the huntingtin is everywhere, in each and every cell of our organism. How could we reach every single cell ?
Jean-Claude and I, concerned with other genetic disorders, are nevertheless interested by this discussion. Mrs PHILIP asks us about the disorders our children are suffering from : Cutis Laxa and PseudoXanthome Elasticum. She knows these diseases. Our statements turn the conversation to patient care. Mrs PHILIP is convinced that it is absolutely necessary that each patient, whatever the pathology, has a local referral doctor in hospitals. This doctor must be able, in an emergency, to set up all the necessary protocols to avoid useless, dangerous, and even at times life-threatening treatments or operations. The local town specialists do not answer phone calls on Saturdays and Sundays . Hospital specialised consultations are often too far from the place of residence to allow emergency care.
“It is 5 : 30 pm. Our training has come to an end. Our brain is full and enriched with everything we have learnt during the past three days. Time has passed by quickly, very quickly. We fill in our training feedback forms. It is time to exchange our impressions with Marion and David. It is very important for them to hear of any criticism we may have (they are very few and concern the form more than the content). It is the first time they work with patients’ associations (normally they work with secondary school pupils) and it will help them improve the training .
As for us……. What can we say?……. Just Thank You for everything : Thank You for sharing their knowledge, Thank You for the friendly welcome, Thank You for the walk to the Calanques………
We inaugurate the “Visitors’ book”………. It is time to go……Long drawn-out goodbyes ……
We’ll come back to see you……It’s a promise………
6 : 30 pm the car moves off, Roger is driving us back. We drop Karen in Lyon, Sylvie and Maïlys in Dijon. At 2 : 00 am he drops me near Paris and is back home, in Metz, at 4 : 00 am.
As for me, I just want to add :
Thanks to Roger for offering me to share this training organised by Huntington Hope. I would have missed so many things if you had not done it. Thanks also for being an unequalled driver……. Right to very end……
Thanks to Sylvie for preparing the logistics for these three days : Nothing to say, it was perfect… ;
Thanks to Claudine, Karen, Maïlys and Jean-Claude for being such good « school friends », working seriously, but also with a lot of good humour ;
Thanks to Marion and David for being great teachers .
And just simply Thanks to “the School of DNA” for existing.
P.S. M6TV report was broadcasted on Friday 14th May during « 6 Minutes » News. It could be watched on the internet for 5 days after that.”
Names and Addresses
School of DNA
INMED – Parc Scientifique de Luminy – 13273 Marseille Cédex 09
email : email@example.com – Tel : 04 91 92 81 45 – Fax : 04 91 82 81 01
Docteur Nicole PHILIP
Hôpital d’Enfant de la Timone – 264 rue Saint Pierre – 13385 Marseille Cédex 05
Email : firstname.lastname@example.org – Tel : 04 91 38 66 30 ou 04 91 38 67 34 –
Fax : 04 91 38 46 04 – Mobile : 06 19 57 82 40
Fédération Huntington Espoir
20, le Mas au Lièvre – BP 26 – 57645 Noisseville
Tel & Fax : 03 87 76 61 65 – Email : email@example.com
4, impasse des Closeries – 35170 Bruz – Tel : 02 99 57 16 75
Email : firstname.lastname@example.org
Cutis laxa Internationale
35 route des Chaîgnes – 17740 Sainte Marie de Ré – Tel & Fax : 05 46 55 00 59
Email : email@example.com