'Translating massively parallel sequencing into clinical diagnostics.

Size: px

Start display at page:

Download "'Translating massively parallel sequencing into clinical diagnostics."

Joella Roxanne Lane
5 years ago
Views:

1 'Translating massively parallel sequencing into clinical diagnostics. SA Pathology at the Women s and and The University of Adelaide Adelaide, Australia

2 MPS is revolutionising genetics & biology - >100 genes for various rare human disease discovered - de novo mutations (ID, ASD) identified, more to come? - understanding of the cancer, role of somatic mutation - diagnostic re-sequencing

unclassified variants? Why do diagnostic exome?

3 ESHG 2011 (Amsterdam, The Netherlands) May 28-31, 2011 H Brunner (Nijmegen, The Netherlands) genetics! Time to be in Capture only genes of interest Capture all and select only those of interest Diagnostic problem = too many unclassified variants? Why do diagnostic exome? Cost effective (~ 2000 EURO/exome) Clinical diagnosis not good enough as a guide for specific inquiry Applicable to singletons MPS is becoming a preference to clinical diagnostic testing They aim for ~500 diagnostic exomes in 2011

4 A D E L A I D E 1/ /2009 3/2010 4/2011 AJHG 4/ / / /2010 6/2011

8mL of maternal plasm 6 bp barcode indexing Illumina GA II and IIx 753 pregnancies 314 pregnancies 79.1% sensitivity 0.3 M reads /sample 98.

5 Non-invasive prenatal assessment of trisomy 21 by multiplexed maternal plasma DNA sequencing: large scale validity study. BMJ 1/2011 Women s and 753 pregnant women with high risk of trisomy had fetus with trisomy 21 (full karyotype based) DNA molecules from mL of maternal plasm 6 bp barcode indexing Illumina GA II and IIx 753 pregnancies 314 pregnancies 79.1% sensitivity 0.3 M reads /sample 98.9% specificity 91.9% positive predictive value 96.9% negative predictive value 100% sensitivity 2.3 M reads /sample 97.9% specificity 96.6% positive predictive value 100% negative predictive value

6 Non-invasive prenatal assessment of trisomy 21 by multiplexed maternal plasma DNA sequencing: large scale validity study. BMJ 1/2011 Women s and Our data reveal that the main value of the maternal plasma DNA sequencing test is to rule out fetal trisomy 21. The false positive rates of the current screening programs = 5% Using MPS the trisomy 21 could be ruled out in 98% of those (5%) Leaving only 0.1% of women with referrals for amniocentesis or CVS

7 X chromosome 5% of the genome 3% of the exome Women s and

8 NLGN4 AP1S2 RSK2 ARX IL1RAPL1 TM4SF2 ZNF674 ZNF41 ZNF81 FTSJ1 PQBP1 SYP1 SHROOM4 JARID1C IQSEC2 HUWE1 FGD1 OPHN1 SLC16A2 DLG3 NLGN3 XNP/ATRX MAGT1 BRWD3 ZNF711 ACSL4 PAK3 AGTR2 CUL4B GRIA3 UPF3B ZDHHC9 SLC9A6 ARHGEF6 FMR2 (FRAXE) MECP2 RPL10 GDI1 SLC6A8 RAB39B pter qter MID1, HCCS OFD1, FANCB CDKL5, NHS, SMS, PDHA1 GK DMD OTC BCOR, ATP6AP2 CASK, MAOA, NDP, PORCN, SYN1, PHF8 SMC1A, HADH2 ARHGEF SLC16A2 9 MCT8, KIA2022, PGK1, ATP7A MED12 PCDH19 PLP SRPX2 TIMM8A NXF5 PRPS1 SIZN1A DCX LAMP2 UBE2A NDUFA1 OCRL FMR1 GPC3 IDS PHF6 ABCD1 HRPT L1CAM SOX3 AVPR2 FLNA IKBKG DKC1 Women s and Genetic heterogeneity of XLID ~ 95 genes ~ 14 new (unpublished) >> families yet to be resolved

9 X-exome re-sequencing EURO MRX/AU GOLD (V Kalscheuer and H Ropers) Women s and Probands from 248 unsolved families Agilet SureSelect; hybridisation based capture baits; exons Sequenced using Illumine GA IIx 96.8% regions covered by >3 reads Average 4 novel missense variants/x chromosome 99% sequence variants confirm by Sanger sequencing

Alignment eresearchsa supercomputer Corvus (552 processors 4.46 trillion calculations per second) Galaxy tool: http://galaxy.psu.

10 Alignment eresearchsa supercomputer Corvus (552 processors 4.46 trillion calculations per second) Galaxy tool: Variants reported at depth of 10 reads Annotated using SeattleSeq easy to use

11 Sequencing - Coverage Illumina 65 bp single end reads Two samples per lane ~8 x 10 6 reads per sample

12 Chromosome-wide variation in coverage Women s and

13 Chromosome-wide variation in coverage Women s and

Example: Five X-chromosome Exomes Mosaik Alignment Sample I II III IV V Mean 57.9 82.8 60.9 42.9 91.

14 Example: Five X-chromosome Exomes Mosaik Alignment Sample I II III IV V Mean Minimum Maximum Median # bases covered # bases in bait region % bases in bait covered 92.6% 92.5% 92.2% 91.0% 92.6% % bases in bait covered ( 10 fold) 86.2% 87.4% 85.2% 79.8% 86.9% S. Willis-Owen, unpublished.

Highly Discrepant (HD) site Filtration Sample II III IV V All HD sites 103,574 41,989 28,333 65,170 Within bait regions 92,190 36,893 25,130 59,366 Present in only 1 sample 54,599 9,677 5,819 24,315

15 Highly Discrepant (HD) site Filtration Sample II III IV V All HD sites 103,574 41,989 28,333 65,170 Within bait regions 92,190 36,893 25,130 59,366 Present in only 1 sample 54,599 9,677 5,819 24,315 Proportion of reads matching ref allele <=15% No overlap with dbsnp Blat: best or only match to X Missense Predicted functional effect (any 1 of 4 tools) S. Willis-Owen, unpublished.

16 Zero coverage: Known ID Genes 22/90 known (Gecz et al. TIGs 2009) XLID genes affected Gene N bases ACSL4 129 AFF2 154 AP1S2 140 AR 3 ARX 277 BRWD3 59 DLG3 155 FGD1 402 G6PD 115 HUWE1 99 IDS 126 IQSEC2 424 OCRL 83 PAK3 20 PDHA1 26 PGK1 35 SLC16A2 27 SLC6A8 78 SLC9A6 106 SOX3 99 SYN1 328 ZNF

17 ARX gene Women s and

18 X-exome re-sequencing - Results EURO MRX/AU GOLD (V Kalscheuer and H Ropers) 14 novel XLID genes identified non-recurrent (novel) missense/stop variants 366 non-recurrent (novel) silent variants ~ 90/248 families resolved ~37% 61 families (25%) mutations in known XLID genes 29 families (12%) mutations in new XLID genes 38 families (15%) carry changes in candidate XLID genes 120 families (48%) unresolved

19 X-exome re-sequencing - Results EURO MRX/AU GOLD (V Kalscheuer and H Ropers) 14 novel XLID genes identified non-recurrent (novel) missense/stop variants 366 non-recurrent (novel) silent variants ~ 90/248 families resolved ~37% 61 families (25%) mutations in known XLID genes 29 families (12%) mutations in new XLID genes 38 families (15%) carry changes in candidate XLID genes 120 families (48%) unresolved

20 Example 1: A small in/del detected by MPS (Family with X-linked Joubert syndrome with M Field and I Scheffer) I 1 2 II * III 1 2 * * 9 10 * IV V Wt 2 1 * 3 * * 8 * 9 * 2 ASD

24,315 149 23 23 4 All HD sites 100% Within bait

3% Proportion of reads matching ref allele <=15% 0.

006% Position (bp) Chr N read s % reads ref % reads

90% G/A Gene Mutation class Original Codo A n A New

21 pter qter Xp22.2 Xp21.3 (hg18, chrx: 10,052,226-27,452,338) 65,170 59,366 24, All HD sites 100% Within bait regions 91.1% Present in only 1 read 37.3% Proportion of reads matching ref allele <=15% 0.23% No overlap with dbsnp130 (1/2 bp features) 0.035% Best or only match to X (Blat) 0.035% Missense only 0.006% Position (bp) Chr N read s % reads ref % reads alt Allele change chrx % 97.90% G/A Gene Mutation class Original Codo A n A New Codo n A A Blat MAGEB1 0 Missense GTT V ATT I only X chrx % 95.20% T/G SSX1 Missense TTC F TGC C best X chrx % 95.50% G/A TIMM17B Missense CGG R TGG W best X chrx % 94.40% A/T TREX2 Missense CTG L CAG Q best X p del IKMEAK of the OFD1 gene OFD1 NP_ NEYKREIEEQLRAEMCQKLKFFKDTEIAKIKMEAKKKYEKELTMFQNDFEKACQAKSEAL 260 OFD1 NP_ Del 200 NEYKREIEEQLRAEMCQKLKFFKDTEIAK------KKYEKELTMFQNDFEKACQAKSEAL 260

22 Example 2: mutation missed by large scale Sanger sequencing (with M Field and A Hackett, GOLD Newcastle)

23 Sanger family #423 Women s and #423 ACSL4 Sanger sequencing Missed exon 13 (15) of ACSL4 and that is where the R654X mutation is!

24 Example 3: Sanger and BAC acgh negative (with M Field and GOLD NSW) 2 missense changes found of unknown significance A duplication involving DLG3 identified based on read depth

25 Example 4: NO mutations found by Sanger or MPS (exomes) (with M Field and A Hackett, GOLD Newcastle) MRX3 studied since 1989 DXS304-Xq28; LOD max = 2.89

26 Women s and AD EL AI DE

27 Example 5: MRX12 Women s and OLD (with M Field and A Hackett, GOLD Newcastle) LOD max = NEW Mutation in a novel XLID identified in the NEW linkage interval

28 Example 6: IGOLD#586 family Women s and (with Julie McGaughran, Brisbane, QLD) Innocuous DNA variant found my MPS (1/4) Expression profiling Pointed towards CCDC22 gene

29 Summary Women s and The MPS technology on its own will not address all your questions (find all variants) Important to know what MPS can do and what it can not do ALL or a PART (Whole Genome Sequencing or Targeted Enrichment and MPS)? DNA variant annotation or functional effect interpretation Coding variants vs non-coding variants Missense variants vs silent variants Unique variants vs recurrent variants De novo variants vs inherited SNVs vs small in/dels vs large CNVs

30 Thoughts. MPS will not be the only technology in molecular diagnostic testing MPS is likely going to be the first test (WG >> WE >> Targeted) MPS (WGS) done only once & revised according to advancing knowledge (& annotation and tools for data mining) Even if ~50% of MPS tests deliver the causal DNA variant $$ and effort well spent! Currently 1 exome ($1900) = Sanger sequencing of 1 gene (e.g. ARX).

31 XLID genes identified - ~10-15% of ID NS-ARID - >60 genes identified - ~85-90% of ID ~ 500 genes causing ID

32 How much rare variation? Women s and

33 How much rare variation? Women s and

34 How much rare variation? The total somatic mutational load must be enormous. For example, the intestinal epithelium contains approximately 10 6 independent stem cells, each of which generates transient daughter cells every week or two. Thus, the intestinal epithelium of a 60-y-old is expected to harbor >10 9 independent mutations. This implies that, not far beyond the age of 60y, nearly every genomic site is likely to have acquired a mutation in at least one cell in this single organ.

35 M. Shaw, L. Hobson M. Corbett, L. Nguyen Neurogenetics Research C. Shoubridge, A. Gardner Program, Adelaide L. Huang, S. Rujirabanjerd S. Willis-Owen, E. Douglas L. Jolly A. Hackett, M. Field, G. Turner GOLD, Newcastle, AU I. Scheffer et al. Melbourne, AU H. Ropers, V. Kalscheuer Max Planck, Berlin Funding: NH&MRC

36 Thank you for your attention. Women s and

The Japanese collaborated study of Fragile X syndrome and its related disorders

The Japanese collaborated study of Fragile X syndrome and its related disorders Yuji Nakayama, PhD Research Center for Bioscience and Technology, Tottori University, Japan TOTTORI Tsukuba Kyoto TOKYO Okayama