NOTCH2 backstage


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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. doi:10.1038/ng.778.

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NOTCH2 backstage

  1. 1. NOTCH2Backstage Pierre Lindenbaum PhD UMR915 – Institut du thorax Nantes, France
  2. 2. ... the story begins in Nantes / France , in november 2010... here
  3. 3. ... in the “Institut du Thorax” (INSERM/UMR915)
  4. 4. Dr. Cedric LeCaignec ...were working on ... Hajdu–Cheney syndrome
  5. 5. Hajdu-Cheney syndrome is a rare skeletal disorder characterized bythe association of facial anomalies, radiological findings &premature loss of permanent teeth
  6. 6. Although most cases are sporadic, few familial transmissions suggest autosomal dominant inheritance
  7. 7. We have sequenced the exomes of 6unrelated patients with Hajdu-Cheney syndrome.
  8. 8. Exome Sequencing
  9. 9. Well soon receive our “Exome Sequencing” data from Integragen .Can you help us to analyze the data ?
  10. 10. By chance, I worked as a Bioinformatician at Integragen(Evry, France) for 7 years !I know them all ! :-)
  11. 11. People from Integragen in my LinkedIn NetworkINTEGRAGEN
  12. 12. it made the conversations easier when we called Integragen to get some insights about the data.... Allo Pierre ? Comment vas-tu yaudpoil ?
  13. 13. Wereceived aDVDcontainingthegenotypes...
  14. 14. ... and ahard-drivecontainingthe rawdata.
  15. 15. #CHROM POS REF ALT ... the datachr4chr19 58703510 4068794 C A T G for eachchr15 93625392 A Gchr1chr16 73570071 62799884 A A C G samplechr19chr19 2234072 GG 3316536 gg G g look like this...chr19 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
  16. 16. Linux JDK6EclipseCoffee
  17. 17. The first program I wrote was a command -line tool...
  18. 18. ..basically, theprogram is aset of filtersto select theinterestingmutations.
  19. 19. Google-code was used as a repository for the source-code.
  20. 20. … and the program generated a wikicode that we put on our internalwiki...
  21. 21. Did you includeDid 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 removesamples per gene = 4 ? all the known SNPs ? What time is it ?
  22. 22. People want to get their hands dirty
  23. 23. A Graphical User Interface (GUI) was needed...
  24. 24. A quick access to the data was required
  25. 25. The key/value model
  26. 26. Example: filtering on Polyphen2
  27. 27. filters...
  28. 28. Variations have been filtered.What are the remaining transcripts ?
  29. 29. … too many transcripts ?
  30. 30. … winnner !
  31. 31. WebStart was used to deploy the applicationThe software is always up-to-date
  32. 32. I want to use your programfrom my home this week- end.... Proxy + WEBSTART
  33. 33. I created an executable JAR andthe program was distributed via
  34. 34. Filter... Filter... Filter... Filter... Filter... LOSER... LOSER Filter...
  35. 35. Meanwhile, we suspected that another team was working on the very same topic ...
  36. 36. We found 78 genes,which were affected by distinct missense variants in three unrelated patients
  37. 37. In contrast, only 5 genes ARSD, NOMO3, NOTCH2,  OR2T35, PCTK3were inactivated by nonsense mutations in two unrelated patients
  38. 38. “NOTCH2 appeared to be an outstanding functional candidate”
  39. 39. By checking unfilteredsequencing data for any missed genetic variant within NOTCH2 we identified an additional nonsense mutation in a third patient.
  40. 40. Next, we searched for frameshift mutations in NOTCH2 and identified small indels in two additional patients.
  41. 41. ? In the remaining patient, no NOTCH2 mutation could be identified
  42. 42. Sanger sequencing confirmed the heterozygous NOTCH2 mutations
  43. 43. We tested inheritance in two families: both mutations occurred de novo. +/+ +/+ +/m
  44. 44. November 5th 2010 : NOTCH2
  45. 45. Where are themutations inNOTCH2 ?.... Are they locatedbefore or after the “PEST” domain ?
  46. 46.
  47. 47.
  48. 48. mutation mutation D K NR TM M LS AN STEGF repeats (n=36) L RA N NLS PE NOTCH2 mutation mutation mutation
  49. 49. Again, our internal wiki wasused to store the informations about NOTCH2
  50. 50. 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. Im 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
  51. 51. 10th December 2010Dear 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 satisfactionbefore we can proceed further.
  52. 52. Date:    Ven 4 février 2011 19:23To:      Cedric Lecaignec­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­In reply please quote: NG­xxxxx  Le CaignecI 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.
  53. 53.
  54. 54. … just on time ! Our challengers
  55. 55. 2011-03-07. our paper was available on “Nature Genetics” ....
  56. 56. … side-by-side to ourchallengers ....
  57. 57.!/yokofakun/status/44767830905856000
  58. 58. Whats next ?
  59. 59. Whats next ? Wikipedia
  60. 60. Whats next ? Gene RIF
  61. 61. Whats next ? WikiGenes.
  62. 62. Whats 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 LindenbaumTo: NLM/NCBI InfoSubject: OMIMHi OMIM,here are two suggestions for OMIM ID. 102500 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. 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.
  63. 63. Whats next ? : Uniprot
  64. 64. Nantes:Bertrand IsidorOlivier Pichon,Stéphane Bézieau,Christian Dina,Sébastien Jacquemont,Dominique Martin-Coignard,Christel Thauvin-Robinet,Martine Le Merrer Integragen (Evry):Jean-Louis MandelAlbert David Melanie LetexierLaurence Faivre Berengere GeninValérie Cormier-Daire Jean-Paul SaraivaRichard Redon Charles MarcaillouHadja Eldjouzi Francis RousseauCédric Le Caignec Emmanuel Martin
  65. 65. Thank You