Women’s and
Children’s Hospital
ADELAIDE
'Translating massively parallel sequencing
into clinical diagnostics.’
SA Pathology at the Women’s and
Children’s Hospital and
The University of Adelaide
Adelaide, Australia
Women’s and
Children’s Hospital
ADELAIDE
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
Women’s and
Children’s Hospital
ADELAIDE
ESHG 2011
(Amsterdam, The Netherlands)
May 28-31, 2011
H Brunner (Nijmegen, The Netherlands)
Time to be in
genetics!
•
•
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
Women’s and
Children’s Hospital
Child
ADELAIDE
1/2011
10/2009
3/2010
3
/2010
AJHG
A
JH
HG 10/2010
10/2010
0
4/2010
4
/ 20 10
/2
4/2011
4
/2011
11/2010
1
1/2010
12/2010
12/2010
6/2011
Women’s and
Children’s Hospital
ADELAIDE
Non-invasive prenatal assessment of trisomy 21 by
multiplexed maternal plasma DNA sequencing: large scale
validity study.
BMJ 1/2011
753 pregnant women with high risk of trisomy 21
DNA molecules from 2.0–4.8mL of maternal plasm
6 bp ‘barcode’ indexing
86 had fetus with trisomy 21 (full karyotype based)
Illumina GA II and IIx
753 pregnancies
79.1%
98.9%
91.9%
96.9%
sensitivity 0.3 M reads /sample
specificity
positive predictive value
negative predictive value
314 pregnancies
100%
97.9%
96.6%
100%
sensitivity 2.3 M reads /sample
specificity
positive predictive value
negative predictive value
Women’s and
Children’s Hospital
ADELAIDE
Non-invasive prenatal assessment of trisomy 21 by
multiplexed maternal plasma DNA sequencing: large scale
validity study.
BMJ 1/2011
‘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
Women’s and
Children’s Hospital
ADELAIDE
X chromosome
• 5% of the genome
• 3% of the exome
Women’s and
Children’s Hospital
ADELAIDE
pter
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
22.3
22.2
22.1
21.3
21.2
21.1
11.4
11.3
11.23
11.22
11.21
11.1
11
12
13
21.1
21.2
21.3
22.1
22.2
22.3
2
2
2
2
2
2
qter
MID1, HCCS
OFD1, FANCB
CDKL5, NHS,
SMS, PDHA1
GK
DMD
OTC
BCOR, ATP6AP2
CASK, MAOA,
NDP, PORCN,
SYN1, PHF8
SMC1A, HADH2
ARHGEF
9
SLC16A2
MCT8, KIA2022,
PGK1, ATP7A
MED12 PCDH19
SRPX2
PLP
TIMM8A
NXF5
PRPS1
SIZN1A
DCX
LAMP2
UBE2A
NDUFA1
OCRL FMR1
GPC3 IDS
PHF6 ABCD1
HRPT L1CAM
SOX3 AVPR2
FLNA
IKBKG
DKC1
Genetic heterogeneity
of XLID
• ~ 95 genes
• ~ 14 new (unpublished)
• >> families yet to be
resolved
Women’s and
Children’s Hospital
ADELAIDE
X-exome re-sequencing
EURO MRX/AU GOLD
(V Kalscheuer and H Ropers)
• Probands from 248 unsolved families
• Agilet SureSelect; hybridisation based capture
47 657 baits; 7 591 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
Women’s and
Children’s Hospital
ADELAIDE
Alignment
• eResearchSA supercomputer
– “Corvus” (552 processors 4.46 trillion
calculations per second)
• Galaxy tool: http://galaxy.psu.edu/
• Variants reported at depth of 10 reads
• Annotated using SeattleSeq – easy to use
http://gvs.gs.washington.edu/SeattleSeqAnnotation/
Women’s and
Children’s Hospital
ADELAIDE
Sequencing - Coverage
• Illumina 65 bp single end reads
• Two samples per lane ~8 x 106 reads per sample
Women’s and
Children’s Hospital
ADELAIDE
Chromosome-wide variation in coverage
Women’s and
Children’s Hospital
ADELAIDE
Chromosome-wide variation in coverage
Women’s and
Children’s Hospital
ADELAIDE
Example: Five X-chromosome ‘Exomes’
Mosaik Alignment
Sample
I
II
III
IV
V
57.9
82.8
60.9
42.9
91.1
Minimum
1
1
1
1
1
Maximum
2215
4434
2506
1830
3683
54
75
57
40
83
Mean
Median
# bases covered
2826330 2824649 2813718 2778055 2826976
# bases in bait region
3053381 3053381 3053381 3053381 3053381
% 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.
Women’s and
Children’s Hospital
ADELAIDE
Highly Discrepant (HD) site Filtration
Sample
II
III
IV
V
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
210
153
133
149
No overlap with dbSNP130
63
17
26
23
Blat: best or only match to X
36
16
25
23
Missense
4
4
6
4
Predicted functional effect (any 1 of 4 tools)
2
1
2
3
All HD sites
Proportion of reads matching ref allele <=15%
S. Willis-Owen, unpublished.
Women’s and
Children’s Hospital
ADELAIDE
Zero coverage: Known ID Genes
• 22/90 known (Gecz et al. TIGs 2009)
XLID genes affected
Gene
ACSL4
AFF2
AP1S2
AR
ARX
BRWD3
DLG3
FGD1
G6PD
HUWE1
IDS
IQSEC2
OCRL
PAK3
PDHA1
PGK1
SLC16A2
SLC6A8
SLC9A6
SOX3
SYN1
ZNF674
N bases
129
154
140
3
277
59
155
402
115
99
126
424
83
20
26
35
27
78
106
99
328
190
Women’s and
Children’s Hospital
ADELAIDE
ARX gene
Women’s and
Children’s Hospital
ADELAIDE
X-exome re-sequencing - Results
EURO MRX/AU GOLD
(V Kalscheuer and H Ropers)
14 novel XLID genes identified
1 108 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
Women’s and
Children’s Hospital
ADELAIDE
X-exome re-sequencing - Results
EURO MRX/AU GOLD
(V Kalscheuer and H Ropers)
14 novel XLID genes identified
1 108 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
Women’s and
Children’s Hospital
ADELAIDE
Example 1: A small in/del detected by MPS
(Family with X-linked Joubert syndrome
with M Field and I Scheffer)
I
1
1
II
1 *
2
2
2
3
4
3
5
6
7
8
9
III
1
2 *
3
4
5
6
7
*
8
IV
10
*
Wt
1 *
2
3 *
4
5
6
7
2
ASD
V
9
* 8
* 9
*
Women’s and
Children’s Hospital
ADELAIDE
pter
qter
Xp22.2 – Xp21.3 (hg18, chrX: 10,052,226- 27,452,338)
Original New
Mutation
Codo A Codo A
class
n
A
n
A
• All HD sites
65,170 • 100%
N
Position
% reads % reads Allele
Chr read
(bp)
ref
alt
change
s
• Within bait regions
59,366 • 91.1%
27750326 chrX 48
2.10%
97.90%
G/A
48006166 chrX 84
4.80%
95.20%
T/G
48637316 chrX 22
15236403
9 chrX 18
4.50%
95.50%
G/A
TIMM17B Missense CGG R TGG W best X
5.60%
94.40%
A/T
TREX2 Missense CTG L CAG Q best X
• Present in only 1 read
24,315 • 37.3%
149
23
23
4
Gene
MAGEB1
0
Missense GTT V ATT
SSX1
Blat
I only X
Missense TTC F TGC C best X
• 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%
p.230-235del IKMEAK of the OFD1 gene
OFD1 NP_003602
200 NEYKREIEEQLRAEMCQKLKFFKDTEIAKIKMEAKKKYEKELTMFQNDFEKACQAKSEAL 260
OFD1 NP_003602 Del 200 NEYKREIEEQLRAEMCQKLKFFKDTEIAK------KKYEKELTMFQNDFEKACQAKSEAL 260
Women’s and
Children’s Hospital
ADELAIDE
Example 2: mutation missed by large scale
Sanger sequencing
(with M Field and A Hackett, GOLD Newcastle)
Women’s and
Children’s Hospital
ADELAIDE
Sanger family #423
#423
ACSL4 Sanger sequencing
Missed exon 13 (15) of ACSL4 and
that is where the R654X mutation is!
Women’s and
Children’s Hospital
ADELAIDE
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
Women’s and
Children’s Hospital
ADELAIDE
Example 4: NO mutations found by Sanger or
MPS (exomes)
(with M Field and A Hackett, GOLD Newcastle)
MRX3 studied since 1989
DXS304-Xq28; LODmax = 2.89
Women’s
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Children’s
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ADELAIDE
Women’s and
Children’s Hospital
ADELAIDE
Example 5: MRX12
22.3
22.2
22.1
21.3
21.2
21.1
OLD
11.4
11.3
11.23
11.22
11.21
11.1
(with M Field and A Hackett, GOLD Newcastle)
LODmax = 3.01
11
12
13
21.1
21.2
21.3
22.1
22.2
22.3
23
24
NEW
25
26
27
28
Mutation in a novel XLID identified in the NEW linkage interval
Women’s and
Children’s Hospital
ADELAIDE
Example 6: IGOLD#586 family
(with Julie McGaughran, Brisbane, QLD)
Innocuous DNA variant found my MPS (1/4)
4
2
2
4
3
3
2
2
2
4
2
Expression profiling
Pointed towards
CCDC22 gene
Women’s and
Children’s Hospital
ADELAIDE
Summary
• 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
Women’s and
Children’s Hospital
ADELAIDE
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).
Women’s and
Children’s Hospital
ADELAIDE
XLID
NS-ARID
>60 genes identified
- 110 genes identified
-
- ~10-15% of ID
- ~85-90% of ID
~ 500 genes causing ID
Women’s and
Children’s Hospital
ADELAIDE
How much rare variation?
Women’s and
Children’s Hospital
ADELAIDE
How much rare variation?
Women’s and
Children’s Hospital
ADELAIDE
How much rare variation?
“The total somatic mutational load must be enormous. For example, the
intestinal epithelium contains approximately 106 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 >109
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.”
Women’s and
Children’s Hospital
ADELAIDE
M. Shaw, L. Hobson
M. Corbett, L. Nguyen
C. Shoubridge, A. Gardner
L. Huang, S. Rujirabanjerd
S. Willis-Owen, E. Douglas
L. Jolly
A. Hackett, M. Field, G. Turner
I. Scheffer et al.
H. Ropers, V. Kalscheuer
Funding:
Neurogenetics Research
Program, Adelaide
GOLD, Newcastle, AU
Melbourne, AU
Max Planck, Berlin
NH&MRC
Women’s and
Children’s Hospital
ADELAIDE
Thank you for your attention.