Description of the bioinformatics side of our paper "Truncating mutations in the last exon of NOTCH2 cause a rare skeletal disorder with osteoporosis". published in Nature Genetics. http://www.nature.com/ng/journal/vaop/ncurrent/full/ng.778.html. doi:10.1038/ng.778.

1. NOTCH2
Backstage
Pierre Lindenbaum PhD
UMR915 – Institut du thorax
Nantes, France
http://plindenbaum.blogspot.com
plindenbaum@yahoo.fr
http://www.flickr.com/photos/constuv/4319650519

2. ... the story begins in
Nantes / France ,
in november 2010...
here

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

4. Dr. Cedric LeCaignec
...we're working
on ...
Hajdu–Cheney
syndrome

5. Hajdu-Cheney syndrome
is a rare skeletal disorder
characterized by
the association of facial anomalies,
radiological findings &
premature loss of permanent teeth

6. Although most
cases are
sporadic, few
familial
transmissions
suggest
autosomal
dominant
inheritance
http://commons.wikimedia.org/wiki/File:Autodominant.jpg

7. We have sequenced
the exomes of 6
unrelated patients with
Hajdu-Cheney
syndrome.

8. Exome
Sequencing
http://en.wikipedia.org/wiki/Exome_sequencing
http://en.wikipedia.org/wiki/File:Exome_Sequencing_Workflow_1a.png

9. We'll soon receive
our
“Exome Sequencing”
data from
Integragen .
Can you help us
to analyze the
data ?

10. By chance,
I worked as a
Bioinformatician
at Integragen
(Evry, France) for
7 years !
I know them all ! :-)

11. People from
Integragen
in my
LinkedIn Network
INTEGRAGEN

12. it made the
conversations
easier when we
called
Integragen to
get some
insights about
the data....
Allo Pierre ? Comment
vas-tu yau'd'poil ?
http://www.flickr.com/photos/sweetlog/1108966791

13. We
received a
DVD
containing
the
genotypes...

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

15. #CHROM POS REF ALT
... the data
chr4
chr19
58703510
4068794 C
A
T
G
for each
chr15 93625392 A G
chr1
chr16
73570071
62799884
A
A
C
G
sample
chr19
chr19
2234072 GG
3316536 gg
G
g look like
this...
chr19 3158826 g gG
chr13 22464550 g gA
chr8 80435887 C T
chr4 130829885 A G
chr12 85102835 C T
chr8 56140941 A G
chr2 179036301 A T
chr16 34927854 C T
chr10 104324871 G A
chr4 81581119 T C

16. Linux
JDK6
Eclipse
Coffee

17. 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

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

20. Google-code was used as a repository
for the source-code.
http://code.google.com/p/code915/source/browse/trunk/tools/src/java/fr/inserm/umr915/tools/Integragen20101022.java

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

22. Did you include
Did we try fix the
the results from
maximum
Polyphen ?
number of SNPs
per gene ?
Can you test with a
minimal DEPTH=20 ?
Did we ever
find this
gene ?
And with minimal
number of affected
samples per SNP = 2 ?
And with minimal
number of affected Can you remove
samples per gene = 4 ? all the known
SNPs ? What time is it ?

24. People want to get their hands dirty
http://www.flickr.com/photos/ohm17/162622755

25. A Graphical
User
Interface
(GUI)
was needed...
http://www.jensroesner.de/wgetgui/#screen

26. A quick access to the data was required
http://www.oracle.com/technetwork/database/berkeleydb/overview/index-093405.html

27. The key/value model

29. Example: filtering on Polyphen2

30. filters...

31. Variations have been filtered.
What are the remaining transcripts ?

32. … too many transcripts ?

33. … winnner !

34. WebStart was used to
deploy the
application
The software is always
up-to-date

35. I want to use
your program
from my home
this week-
end....
Proxy
+
WEBSTART

36. I created an executable JAR and
the program was distributed via
http://www.dropbox.com

37. Filter...
Filter...
Filter... Filter...
Filter...
LOSER...
LOSER Filter...
http://www.flickr.com/photos/hurty/401880903/

38. Meanwhile,
we suspected
that another
team was
working on
the very same
topic ...
http://commons.wikimedia.org/wiki/File:Hourglass.jpg

39. We found 78 genes,
which were affected by
distinct missense
variants in three
unrelated patients

40. In contrast, only 5 genes
ARSD, NOMO3, NOTCH2,
OR2T35, PCTK3
were inactivated by nonsense
mutations in
two unrelated patients

41. “NOTCH2 appeared to be an
outstanding functional
candidate”
http://www.ncbi.nlm.nih.gov/gene/4853

42. By checking
unfiltered
sequencing data for
any missed genetic
variant within
NOTCH2
we identified an
additional
nonsense mutation
in a third patient.

43. 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

44. ?
In the
remaining
patient, no
NOTCH2
mutation could
be identified

45. Sanger sequencing confirmed the
heterozygous
NOTCH2
mutations
http://commons.wikimedia.org/wiki/File:Sanger_sequencing_read_display.gif

46. We tested inheritance in two families:
both mutations occurred de novo.
+/+
+/+
+/m
http://commons.wikimedia.org/wiki/File:OldFamily.JPG

47. November 5th 2010 : NOTCH2

48. Where are the
mutations in
NOTCH2 ?....
Are they
located
before or after
the “PEST”
domain ?

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

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

51. mutation mutation
D K
NR TM M LS AN ST
EGF repeats (n=36) L
RA N NLS PE
NOTCH2 mutation mutation
mutation

52. Again, our internal wiki was
used to store the informations
about NOTCH2

53. Subject: Notification about reviewers of NG-xxxxxxx Le Caignec from Nature Genetics
De: xxx@boston.nature.com
Date: Mar 30 novembre 2010 19:49
To: Cedric Lecaignec
--------------------------------------------------------------------------
Dear Dr. Le Caignec,
Thank you for submitting your paper, NG-xxxxx Le Caignec, to Nature
Genetics. I'm writing to let you know that we have decided to send your
manuscript for peer review. We usually ask our referees to respond within
two to three weeks, and we will make every attempt to encourage them to
meet this deadline. Once we have received the reviews and made a decision
about your paper, we will contact you by email.
Sincerely,
XX
Senior Editor
Nature Genetics

54. 10th December 2010
Dear Dr. Le Caignec,
Your manuscript "Truncating mutations in the last exon of NOTCH2 cause a
rare skeletal disease with osteoporosis" has now been seen by two
referees, and you will see from the accompanying comments that, whereas
they find your work interesting (as do we), they have raised points that
must be addressed through appropriate revisions and to their satisfaction
before we can proceed further.

55. Date: Ven 4 février 2011 19:23
To: Cedric Lecaignec
­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­
In reply please quote: NG­xxxxx Le Caignec
I am delighted to say that your manuscript "Truncating mutations in the
last exon of NOTCH2 cause a rare skeletal disorder with osteoporosis" has
been accepted for publication in an upcoming issue of Nature Genetics.

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

57. … just on time !
Our challengers

58. http://www.nature.com/ng/journal/vaop/ncurrent/full/ng.778.html
2011-03-07.
our paper was
available on
“Nature Genetics”
....

59. … side-by-side
to our
challengers
....

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

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

62. What's next ? Wikipedia

63. What's next ? Gene RIF

64. What's next ? WikiGenes.
http://www.wikigenes.org/e/gene/e/4853.html

65. What's next ?
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 Lindenbaum
To: NLM/NCBI Info
Subject: 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=20
disorder with osteoporosis."
Isidor B, Lindenbaum P, Pichon O, B=E9zieau S, Dina C, Jacquemont S,=20
Martin-Coignard D, Thauvin-Robinet C, Le Merrer M, Mandel JL, David A,=20
Faivre 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=20
and progressive bone loss."
Simpson MA, Irving MD, Asilmaz E, Gray MJ, Dafou D, Elmslie FV, Mansour=20
S, Holder SE, Brain CE, Burton BK, Kim KH, Pauli RM, Aftimos S, Stewart=20
H, Kim CA, Holder-Espinasse M, Robertson SP, Drake WM, Trembath RC.

66. What's next ? : Uniprot

67. Nantes:
Bertrand Isidor
Olivier Pichon,
Stéphane Bézieau,
Christian Dina,
Sébastien Jacquemont,
Dominique Martin-Coignard,
Christel Thauvin-Robinet,
Martine Le Merrer Integragen (Evry):
Jean-Louis Mandel
Albert David Melanie Letexier
Laurence Faivre Berengere Genin
Valérie Cormier-Daire Jean-Paul Saraiva
Richard Redon Charles Marcaillou
Hadja Eldjouzi Francis Rousseau
Cédric Le Caignec Emmanuel Martin
http://www.flickr.com/photos/constuv/4319650519

68. Thank
You
http://www.flickr.com/photos/constuv/4319650519