User:Etchevers/Notebook/Conference notes/2009/05/04

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Notes for group meeting 4 May 2009

Patrick says meeting room will be preserved and the research office will be moved to the current qPCR/centrifuge room, kept in a “lab” configuration but with appropriate chairs. The “cafeteria” will be converted into an office and may well incumb to our group; Maria Martinez will move into the former office; tables and qPCR and other common equipment into the small L2 which will be moved into the empty space.

Matthias is in charge of “feasibility” study – will see what will be dismounted to bring into the former culture room among other things.

Administration for the university for team renewal – as well as for the INSERM at the end of the year (AERES in October). The dossier is supposed to be for June 15th. The “prospective projects” will be the one that was submitted for the trial Scientific Advisory Board but somewhat revised esp with the patient collection. Four former evaluations – two of them were very positive and two were not because their credo (not scientifically based) was that you need an arbitrarily large number of patients. This document for June 15th to be prepared, is provisory but it needs to be of course as good as possible, especially for 15th of October when it will go in front of the AERES commission. Need to be ready a bit ahead of time because will be sent at the same time as the rest of the INSERM unit and everything needs to be put into the same format etc.

10 pages for team’s past, 10 pages for the future (excluding the references). Anyone who has participated in the team’s work at any point in the last four years should put their publications. They are likely to be more quantitative than qualitative.

  • Nicolas, Heather and Patrick will rewrite the developmental biology part;
  • Matthias and Weihua to look at the “statistical models/genetics of refractive disorders” part – not just GWAS for frequent myopia, but also the approaches will be used for study of rare keratoconus (“recruitment” population collection, controls, any other common methodology, separate sibpair from case-control but otherwise emphasize common themes/strategies to be able to put it all under hat of “genetics of refractive disorders” – why separate the approaches when they are both complex (“probably oligogenic”) disorders with epigenetic/environmental effects as well as multigenic – however to defend that we need fewer cases than a truly “complex” disease such as diabetes);

Weihua says there are papers to support the observation of a fuzzy boundary between what is a multigenic/complex/simple disease.

Mechanical keratoconus after Leber congenital amaurosis; Down syndrome also associated with keratoconus. LA in keratoconus families yielded many loci but never a gene. Why? Not necessarily particularly “complex” but…? Also, ethnic background seems to change the LA loci. No gene or definitive locus, same approach as for myopia. But KC has advantage of true human samples after surgery which are not possible to obtain for myopia.

  • Stephane (using SAB document as a basis) for the keratoconus/scarring/gene therapy part. About a page or two per theme;
  • Francois does part about training opportunities offered in the past: what has become of the people that we have trained in the earlier years?

All of this is to make a provisional file. Invited talks are important, but just the number of posters, not the precise conferences and titles for each, for each researcher part of team.

Documents in progress first meeting ready for the 14th or 15th of May, send them around by e-mail. But Patrick will first send the SAB draft around as a working document from which to start, and Matthias will remind him to do this.

L. Vezinet can help Patrick unblock 40K euros. Matthias explains that Wolfson in London would have been paid in full (purchase order emitted), instead the badly directed payment to Affymetrix will be deducted from Wolfson’s bill. Everyone concurs that we need a written document to make sure this is set. PHRC money was given to Emmanuelle to be administered by the INSERM and Matthias can get a previous purchase order copy from Pauline.

40K euros were for all costs included. Matthias says 338 euros per sample, all costs included. Divided now into two. Approx 250 euros per chip and the rest is consumables. Made a purchase order for 44K euros to Affy London for 200 myopia samples. Now need to make up the rest. Have a kind of credit because have a “bon de commande” for 400 chips for KC and they have only put through 200 chips. So that could be applied to the myopia samples if that makes things go faster? (says Matthias).

S. Tuft told Matthias that for the remaining 120 samples, Affymetrix supplied defective chips and so they are getting replacements and there is a new delay. How many sibpairs COULD BE exploitable? 280 individuals – some trios, somewhat fewer than 140 sibpairs. Only 80 of them have already been genotyped, 120 need to be rehybridized, and another 80 have not yet been sent as they have been coming in piecemeal since November. J. Ravi in charge, Matthias says that Steve has already made his protest. Patrick suggests he and Francois call Fernando Domingues (St Jacques de Compostelle). Or Marie Curie center. Warn the ADR not to honor the purchase order with a bill until we have the service provided.

Try to get the NIH dossier together with Matthias “in Patrick’s name” for what will he will submit on eRA commons to have access to control data.

ChIP discussion – Pax6 overexpression like KO in mice leads to corneal clouding; Patrick wants to imagine a precise delivery with precise gene dosage to the corneas of PAX6-mutated aniridia patients to improve what vision they have left? (This being because Adeline seems to have succeeded in cloning at least one of the two common isoforms of hPAX6.)

KC – mass spec results forthcoming as all five pairs have been purified and should all have been passed on the mass spectrometer by early June. The analysis takes two to four months! Manual annotations. The relevant person takes vacation all the month of August. Map theoretical peptides with trypsin digest to the real peptides that are generated and provide statist. probability that these real peptides actually correspond to such or such a protein. Preliminary results for the first three couples around desmoglein, IL16 perhaps… for the on/off most extreme results.

12 KC and 8 control corneas – all extracted and of good quality. There are 10 more to extract – the idea was to hybridize 20 and 20 in an ideal world. Suggestion to put at least one aliquot of each RNA extraction into another -80 freezer. Matthias sends the first 20 DNAs to the INSA Affymetrix platform for hybridization this week. Next week he will bring the ten next ones. Normalization question from Patrick. On the chips there is an internal control. I have suggested to put off this week’s group until next and combine with the 10 additionals to label with the same kit all at the same time, and hybridize all at the same time as well (keeping buffers, temperatures the same).

Francois asks if bring all of them Monday, when done? Answer – one week, ten days (really four days). 22K euros for 30 chips= 733 euros a chip?! The person over there (Nathalie Marsaud) at INSA will help out with normalization and initial analyses.

Scarring – Stephane. AAV 1/250 injections with enzyme seems to be quite helpful in reducing the trepanation-induced corneal opacity within 14 days. Some nearly entirely transparent. Also less oedema. Thinner corneas though. Alpha-smooth muscle actin, no obvious difference with the numbers of myofibroblasts. Isabelle at the Ecole Veterinaire is doing the histology for these mice. This week 15 treated/15 controls, joining a pool of nearly 100 animals. Pierre and Emilie are assisting in the evaluation of the severity of the lesions. Now looking how partners of this protease are changing transcriptionally – col I, collagen III, VEGF, col XVIII, sir-61, MMP2, alpha-SMA. This article will be descriptive including histology as well as the PCRs. Maybe add keratocan but Stephane thinks not necessary. Collagen III slowly upregulated as far as transcription, but also immunofluorescence works. If there is a difference between RT results and protein there are other hypotheses possible.

qPCR – Angelique has extracted corneas, will do them next week.

Two M1 students – Anabelle has ID’d 3 reporter genes that are stable in all states of the cornea (healing, normal) for qRT-PCR. Anais was doing immuno with Jackie – also looking for qRT-PCR primers for stem cell markers. TEP1 (hTERT1 partner) seemed to work alright and got upregulation over time in the healing corneas. Will check if telomerase itself is also following the same dynamics and if so, it may well be a better immuno antibody as we have no luck with the polyclonal antibodies against Pax6 etc. Monoclonal antibody against alpha-SMA works very nicely. Jackie said today that the rabbit IgG negative control stuck to everything.

Francois wants Stephane to organize a meeting with collaborator “Vergniolles” and Dawiyat included as well. But not necessarily, if it is too time-consuming. Quid the collab with Lyon? Steph waiting for PCPE1/PCPE2 KO mouse eyes for immunostaining with PCPE1 and BMP1, from American collab of Lyon group. Steph will do month after trepanation at different time points qRT-PCR for a number of markers. Francois wants to make sure that we can easily justify the interest of the study given that the results may not all be published before the evaluation committee.

Pierre-Julien new intern from ophthalmology in clinical genetics. May well propose something else in case he wants to do an M2 degree – Patrick proposes can do some quantitative analysis of genes – deletions/duplications of Rieger genes in micro.anophthalmias – OTX2 and RAX to add to the “Quantitative Multiplex PCR of Short Fragments” analysis that Adeline has already done. And also help out for the collection of controls for myopia. Patrick wants more efficiency in recruitment! Jackie has 40-odd controls, only 15 or so more from the last time we asked ophthalmology for them. So PJ may help with that recruitment. Bérangère is also getting blood from controls at the Ecole d’Aides-Soignantes with new students arriving in June (Patrick warns against sex bias!).

Lille cohort from diabetes study possibly available but less info because older study and Illumina chips, less useful than Wellcome and NIH control data as these are genotyped with higher density (Wellcome is the same SNP chip version).

8 aniridias born in France per year; 100-120 analysis of PAX6 per year. Axenfeld-Rieger is less frequent. Complete maybe 3/year.

Tired secretary signing off.

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