NOTCH2 backstage

NOTCH2Backstage

http://www.flickr.com/photos/constuv/4319650519

Pierre Lindenbaum PhDUMR915 – Institut du thorax

Nantes, Francehttp://plindenbaum.blogspot.com

plindenbaum@yahoo.fr

... the story begins inNantes / France ,

in november 2010...

here

... in the “Institut du Thorax”(INSERM/UMR915)

...we're workingon ...

Hajdu–Cheney syndrome

Dr. Cedric LeCaignec

Hajdu-Cheney syndromeis a rare skeletal disorder

characterized bythe association of facial anomalies,

radiological findings &premature loss of permanent teeth

http://commons.wikimedia.org/wiki/File:Autodominant.jpg

Although most cases are sporadic, few familial transmissions suggest autosomal dominant inheritance

We have sequenced

the exomes of 6 unrelated patients with

Hajdu-Cheney syndrome.

http://en.wikipedia.org/wiki/File:Exome_Sequencing_Workflow_1a.png

http://en.wikipedia.org/wiki/Exome_sequencing

Exome Sequencing

We'll soon receiveour

“Exome Sequencing”data from

Integragen .

Can you help usto analyze the

data ?

By chance,I worked as a

Bioinformaticianat Integragen

(Evry, France) for 7 years !

I know them all ! :-)

People fromIntegragen

in myLinkedIn Network

INTEGRAGEN

http://www.flickr.com/photos/sweetlog/1108966791

it made the conversations easier when we called Integragen to get some insights about the data....

Allo Pierre ? Comment vas-tu yau'd'poil ?

We received a DVD containing the genotypes...

... and a hard-drive containing the raw data.

... the data for each sample look like this...

#CHROM POS REF ALTchr4 58703510 A Gchr19 4068794 C Tchr15 93625392 A Gchr1 73570071 A Cchr16 62799884 A Gchr19 2234072 GG Gchr19 3316536 gg gchr19 3158826 g gGchr13 22464550 g gAchr8 80435887 C Tchr4 130829885 A Gchr12 85102835 C Tchr8 56140941 A Gchr2 179036301 A Tchr16 34927854 C Tchr10 104324871 G Achr4 81581119 T C

LinuxJDK6

Eclipse

Coffee

The first program I wrote was a command-line tool...

http://code.google.com/p/code915/source/browse/trunk/tools/src/java/fr/inserm/umr915/tools/Integragen20101022.java

..basically, the program is a set of filters to select the interesting mutations.

http://code.google.com/p/code915/source/browse/trunk/tools/src/java/fr/inserm/umr915/tools/Integragen20101022.java

Google-code was used as a repository for the source-code.

… and the program generated a wiki code that we put on our internal wiki...

Can you test with a minimal DEPTH=20 ?

And with minimal number of affected

samples per SNP = 2 ?

And with minimal number of affected

samples per gene = 4 ?Can you remove

all the known SNPs ?

Did we try fix the maximum

number of SNPs per gene ?

Did we ever find this gene ?

Did you include the results from

Polyphen ?

What time is it ?

http://www.flickr.com/photos/ohm17/162622755

People want to get their hands dirty

http://www.jensroesner.de/wgetgui/#screen

A Graphical User

Interface (GUI)

was needed...

A quick access to the data was required

http://www.oracle.com/technetwork/database/berkeleydb/overview/index-093405.html

The key/value model

Example: filtering on Polyphen2

filters...

Variations have been filtered.What are the remaining transcripts ?

… too many transcripts ?

… winnner !

The software is alwaysup-to-date

WebStart was used to deploy the application

I want to use your program from my home

this week-end....

Proxy+

WEBSTART

I created an executable JAR and the program was distributed via

http://www.dropbox.com

http://www.flickr.com/photos/hurty/401880903/

Filter...

Filter...

Filter...

LOSER

LOSER...

Filter...

Filter...

Filter...

http://commons.wikimedia.org/wiki/File:Hourglass.jpg

Meanwhile, we suspected that another team was working on the very same topic ...

We found 78 genes, which were affected by

distinct missense variants in three

unrelated patients

In contrast, only 5 genes

ARSD, NOMO3, NOTCH2, 

OR2T35, PCTK3

were inactivated by nonsense mutations in

two unrelated patients

http://www.ncbi.nlm.nih.gov/gene/4853

“NOTCH2 appeared to be an outstanding functional

candidate”

By checking unfiltered

sequencing data for any missed genetic

variant within NOTCH2

we identified an additional

nonsense mutationin a third patient.

Next, we searched for frameshift mutations in NOTCH2 and

identified small indels in two

additional patients.

http://commons.wikimedia.org/wiki/File:Linen_tester.jpg

?In the remaining patient, no NOTCH2 mutation could be identified

Sanger sequencing confirmed the heterozygous

NOTCH2mutations

http://commons.wikimedia.org/wiki/File:Sanger_sequencing_read_display.gif

We tested inheritance in two families:both mutations occurred de novo.

+/m

+/+

+/+

http://commons.wikimedia.org/wiki/File:OldFamily.JPG

November 5th 2010 : NOTCH2

Where are the mutations in NOTCH2 ?....

Are they located

before or after the “PEST” domain ?

http://biostar.stackexchange.com/questions/3662

http://plindenbaum.blogspot.com/2010/11/blastxmlannotations.html

EGF repeats (n=36) TMDANK

NLSPEST

LNR

NLSRAM

NOTCH2

mutation mutation

mutation

mutation

mutation

Again, our internal wiki was used to store the informations

about NOTCH2

Subject: Notification about reviewers of NG-xxxxxxx Le Caignec from Nature GeneticsDe: xxx@boston.nature.comDate: Mar 30 novembre 2010 19:49To: Cedric Lecaignec--------------------------------------------------------------------------

Dear Dr. Le Caignec,Thank you for submitting your paper, NG-xxxxx Le Caignec, to NatureGenetics. I'm writing to let you know that we have decided to send yourmanuscript for peer review. We usually ask our referees to respond withintwo to three weeks, and we will make every attempt to encourage them tomeet this deadline. Once we have received the reviews and made a decisionabout your paper, we will contact you by email.

Sincerely,XXSenior EditorNature Genetics

10th December 2010

Dear Dr. Le Caignec,Your manuscript "Truncating mutations in the last exon of NOTCH2 cause arare skeletal disease with osteoporosis" has now been seen by tworeferees, and you will see from the accompanying comments that, whereasthey find your work interesting (as do we), they have raised points thatmust be addressed through appropriate revisions and to their satisfaction before we can proceed further.

Date:    Ven 4 février 2011 19:23To:      Cedric Lecaignec­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­In reply please quote: NG­xxxxx  Le Caignec

I am delighted to say that your manuscript "Truncating mutations in thelast exon of NOTCH2 cause a rare skeletal disorder with osteoporosis" hasbeen accepted for publication in an upcoming issue of Nature Genetics.

http://friendfeed.com/yokofakun/85dfa44b

… just on time !

Our challengers

http://www.nature.com/ng/journal/vaop/ncurrent/full/ng.778.html

2011-03-07.our paper was

available on“Nature Genetics”

....

… side-by-sideto our

challengers ....

http://twitter.com/#!/yokofakun/status/44767830905856000

http://biostar.stackexchange.com/questions/6215/we-found-a-gene-involved-in-a-genetic-disease-now-what-is-the-todo-list

What's next ?

What's next ? Wikipedia

What's next ? Gene RIF

http://www.wikigenes.org/e/gene/e/4853.html

What's next ? WikiGenes.

Dear Colleague,I have forwarded your message to the OMIM editors for their consideration.Note that the editors of OMIM attempt to include the essential references f=or a particular topic, not necessarily everything that has been published. = In addition, a neighboring feature has been applied to OMIM that links add=itional, relevant articles from MEDLINE to each entry, thereby ensuring tha=t all relevant articles are easily retrievable.The OMIM editors will contact you directly should they require any further =information.Best regards,

NCBI User Services-----Original Message-----From: Pierre LindenbaumTo: NLM/NCBI InfoSubject: OMIM

Hi OMIM,here are two suggestions for OMIM ID. 102500

http://www.ncbi.nlm.nih.gov/pubmed/21378989

Nat Genet. 2011 Mar 6."Truncating mutations in the last exon of NOTCH2 cause a rare skeletal=20disorder with osteoporosis."Isidor B, Lindenbaum P, Pichon O, B=E9zieau S, Dina C, Jacquemont S,=20Martin-Coignard D, Thauvin-Robinet C, Le Merrer M, Mandel JL, David A,=20Faivre L, Cormier-Daire V, Redon R, Le Caignec C.

http://www.ncbi.nlm.nih.gov/pubmed/21378985

Nat Genet. 2011 Mar 6"Mutations in NOTCH2 cause Hajdu-Cheney syndrome, a disorder of severe=20and progressive bone loss."Simpson MA, Irving MD, Asilmaz E, Gray MJ, Dafou D, Elmslie FV, Mansour=20S, Holder SE, Brain CE, Burton BK, Kim KH, Pauli RM, Aftimos S, Stewart=20H, Kim CA, Holder-Espinasse M, Robertson SP, Drake WM, Trembath RC.

What's next ?

What's next ? : Uniprot

http://www.flickr.com/photos/constuv/4319650519

Nantes:

Bertrand Isidor Olivier Pichon,Stéphane Bézieau,Christian Dina,Sébastien Jacquemont,Dominique Martin-Coignard,Christel Thauvin-Robinet,Martine Le MerrerJean-Louis MandelAlbert DavidLaurence FaivreValérie Cormier-DaireRichard RedonHadja EldjouziCédric Le Caignec

Integragen (Evry):

Melanie LetexierBerengere GeninJean-Paul SaraivaCharles MarcaillouFrancis RousseauEmmanuel Martin

ThankYou

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