Download Current Second Tier and Future Applications of Gene Sequencing in

Current Second Tier and Future
Applications of Gene
Sequencing in NBS:
Sanger Sequencing
Michele Caggana, Sc.D., FACMG
February 16, 2017
February 24, 2017
S. February
Cordovado,
Ph.D.
24, 2017
2
Molecular Analysis in Newborn Screening
A Staged Approach
Genotyping Panel of
Mutations -- Single Gene
Sequencing Single
Gene
Sequencing
Panel of Genes
Ongoing in routine NBS
Sequencing
of NBS
Genes
Genome
Exome
Experimental in NBS
Ongoing in routine NBS
Experimental
in NBS and
Offered
clinically
research
outside
Offered clinically
andNBS
research outside NBS
February 24, 2017
3
Mix deoxynucleotides
with
ddA, ddT, ddC, ddG
1 scan per
person/fragment
~800 readable bases
Mix deoxynucleotides with
ddA, ddT, ddC*, ddG
4 lanes per person/fragment
~200 readable bases
Chop up the human genome
Make a library of fragments
Sequence billions of bases
Multiplexing multiple people
Millions of ‘reads’
February 24, 2017
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Frederick Sanger
Walter Gilbert
Published in
1977
1/4 prize each
http://nobelprize.org/chemistry/laureates/1980/sanger-autobio.html
February 24, 2017
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Sanger Sequencing
• DNA replication requires a free 3’hydroxyl group; uses dideoxyNTPs
• Originally incorporated a single
ddCTP*35S or ddCTP*32P
• Mix acrylamide:bis-acrylamide and
pour thin gel between 2 glass plates
with spacers
• Polymerizes and is assembled on an
apparatus and loaded
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Sanger Sequencing
• Sequencing reactions set up (ea.
sample set up with ddG, ddA, ddT and
ddC); stop buffer; load each well
• Electrophorese for about 4 hours
• Disassemble and dry gel
• Expose to x-ray film overnight to days
• Manual read
February 24, 2017
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Schematic of the “Sanger” Method
“however, with
people like
Francis Crick
around it was
difficult to
ignore nucleic
acids or to fail to
realize the
importance of
sequencing
them”.
February 24, 2017
8
Radioisotopic Sequencing
Sanger Method
Used radiolabelled P32
https://www.gelcompany.com/gibco-brl
Pasternak, 1999
February 24, 2017
9
Advancements – ABI 373A
•
•
•
•
•
•
•
•
•
•
•
•
Early 1990’s
Fluorescence-based; got
away from using
radioactivity
5’-end labeled primers
PCR to amplify
Single tube
Genescan software
Still had to ‘pour’ a gel
Still had to hand load
Eliminated drying
Eliminated manual reading
http://www.labrecyclers.com/product.html?InventoryID=2301
Longer reads
Human Genome Project
February 24, 2017
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Advancements – Capillary
Electrophoresis Detection
• Still based on size separation
• Still use dideoxy chain terminators
• Labeled primers, laser detects specific
fluorescent tags so we can now
combine nucleotides in one tube
A, C, G, T
• Can automate set-up
• Uses PCR to make DNA
February 24, 2017
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Methods Using Capillary Electrophoresis
Principle of
agarose or
acrylamide gel
electrophoresis
is still used
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Principle of Capillary Electrophoresis
A laser is used to
detect fluorescently
labeled amplified
product
Multiple capillaries
can run samples at
the same time
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ABI Prism 3100 Genetic Analyzer
laser
capillaries
Syringe with
polymer solution
Injection
electrode
Outlet
buffer
Autosampler
tray
Inlet buffer
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ABI 3100
16-capillary
array
February 24, 2017
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Frameshift mutation
delT
Sequence analysis:
ABI PRISM 3100
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DNA Sequence Analysis Using Capillary
Electrophoresis
Applied Biosystems 3730
48-capillary Genetic Analyzer
Applied Biosystems 3500
8-capillary Genetic Analyzer
February 24, 2017
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ABI 3730 Genetic Analyzer
laser
capillaries
polymer
inlet buffer
cathode/anode buffers
February 24, 2017
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Peering into an ABI 3500
3500 XL 24-capillary
February 24, 2017
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Why Even Bother ??
•
If a laboratory has the technology, the costs are different
than commercial laboratory costs
•
“Pseudodeficiency only” specimens ‘rule – out’ Pompe
•
Some phenotype/genotype correlations
•
Turnaround time ~ a work day with pre-preparation
•
Physicians like the information – urgency, family
interaction; health care disparity concerns
•
Can release premature infants from further work-up or
re-focus efforts; 42% reduction of referrals for Krabbe
disease
February 24, 2017
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GAA Enzyme Activity (MS/MS) (<15%)
Daily Mean Average Percent
DNA Sequencing
2
Pathogenic
Variants
Referral
1 Pathogenic
Variant
c/ or s/
VOUS /
Polys
Referral
VOUS (<1%
in General
Population)
Referral
Only
Pseudodeficiency
Alleles*
No Referral
VOUS = variant of unknown significance
Polys – common variants that are not disease-causing
*p.Gly576Ser; p.Glu589Lys
No
Pathogenic
Variants
No Referral
February 24, 2017
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2nd Tier DNA Assay - Overview
• Obtain two 3-mm punches from dried blood spots and
extract DNA
• Amplify 14 separate gene fragments by PCR;
encompasses exons 2-20 including intron/exon
boundaries
• PCR products used for Sanger sequencing
• Sequences analyzed for mutations or variants
• Referral according to the algorithm
• Parental phasing; sibling testing
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Step One : Punch and Extract DNA
Hands on: 5-10 min;
Hands-off: 45 minutes
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20 Exons Covered in 14 Fragments
Chromosome 17 –952 amino acids
PCR Number
Exons
Covered
Base
Pairs
PCR Number
Exons
Covered
Base
Pairs
1
2
772
8
13-14
679
2
3
384
9
15
486
3
4-5
630
10
16
583
4
6-8
781
11
17
497
5
9
483
12
18
400
6
10-11
890
13
19
342
7
12
465
14
20
460
•
•
•
•
Primer design
Check for SNPs; minor allele frequencies on dbSNP
Need to amplify all with the same PCR program
Can use different master mixes
February 24, 2017
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PCR Plate Map
1
2
3
4
5
6
7
A
E2 –1a
E4/5 –1a
E9 –1a
E12 –1a
E15 –1a
E17 –1a
E19 –1a
B
E2 –1b
E4/5 –1b
E9 –1b
E12 –1b
E15 –1b
E17 –1b
E19 –1b
C
E2 –WTC
E4/5 –WTC
E9 –WTC
E12 –WTC
E15 –WTC
E17 –
WTC
E19 –WTC
D
E 2 –NTC
E4/5 – NTC
E9 –NTC
E12 –NTC
E15 –NTC
E17 –NTC
E 19 –NTC
E
E3 –1a
E6/8 –1a
E10/11 –
1a
E13/14 –
1a
E16 –1a
E18 –1a
E20–1a
F
E3 –1b
E6/8 –1b
E10/11 –
1b
E13/14 –
1b
E16 –1b
E18 –1b
E20–1b
G
E3 –WTC
E6/8 –WTC
E10/11 –
WTC
E13/14 –
WTC
E16 –WTC
E18 –
WTC
E20 –WTC
H
E3 –NTC
E6/8 –NTC
E10/11 –
NTC
E13/14 –
NTC
E16 –NTC
E18 –NTC
E20 –NTC
Hands on: 5 min;
Hands-off: 2 hours
WTC = wild type control; NTC = no template control
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Set Up Plates in Batches
•
•
•
•
Saves time
Batches better for QI
Minimize pipetting errors
Minimize inconsistencies
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Agarose Gel
Electrophoresis
Product Check
• Sizes okay
• Amplification
• No Contamination
Hands on: 15 min;
Hands-off: 45 minutes
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PCR Clean-up Before Sequencing
ExoSap-IT Treatment
1
2
3
4
5
6
A
E2 –1a
E6/8 –1a
E12 –1a
E16 –1a
E20–1a
B
E2 –1b
E6/8 –1b
E12 –1b
E16 –1b
E20–1b
C
E2 –1a
E6/8–1a
E13/14 –1a
E17 –1a
D
E2 – 1b
E6/8–1b
E13/14 –1b
E17 –1b
E
E3 –1a
E9 –1a
E13/14 –1a
E18 –1a
F
E3 –1b
E9 –1b
E13/14 –1b
E18 –1b
G
E4/5 –1a
E10/11 –1a
E15 –1a
E19 –1a
H
E4/5 – 1b
E10/11 –1b
E15 –1b
E19 –1b
Hands on: 5 min;
Hands-off: 30 minutes
7
8
February 24, 2017
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Cycle Sequencing
1
2
3
4
5
6
7
8
9
10
A
E2 F–
1a
E 6/8 F–
1a
E12 F–
1a
E16 F–
1a
E 20
F–1a
E2 R–
1a
E 6/8
R–1a
E12
R–1a
E16
R–1a
E20
R– 1a
B
E2 F–
1b
E 6/8 F–
1b
E12 F–
1b
E16 F–
1b
E 20
F–1b
E2 R–
1b
E 6/8
R–1b
E12
R–1b
E16
R–1b
E20
R– 1b
C
E2
SEQF
1a
E6/8
SEQF1a
E13/14
F–1a
E17 F–
1a
E2
SEQR –
1a
E6/8–
SEQR1a
E13/14
R–1a
E17
R–1a
D
E2
SEQF1
b
E6/8
SEQF1b
E13/14
F–1b
E17 F–
1b
E2
SEQR1
b
E6/8
SEQR1b
E13/14
R–1b
E17
R–1b
E
E3 F–
1a
E9
F–1a
E13/14
SEQF
1a
E18 F–
1a
E3 R–
1a
E 9 R–
1a
E13/14
SEQR1a
E18
R–1a
F
E3
F–1b
E9
F–1 b
E13/14
SEQF
1b
E18 F–
1b
E3 R–
1b
E9
R–1a
E13/14
SEQR1b
E18
R–1b
G
E4/5
F–1a
E10/11F
–1a
E15 F–
1a
E19 F–
1a
E4/5
R–1a
E10/11
R–1a
E15 R–
1a
E19
R–1a
H
E 4/5
F–1b
E10/11
F–1b
E15 F–
1b
E 19 F–
1b
E4/5
R– 1b
E10/11
R–1b
E15 R–
1b
E19
R–1b
Hands on: 5 min;
Hands-off: 2.5 hours
SEQ primers are internal sequencing primers
February 24, 2017
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The Rest of the Story
• Centri-sep columns to remove dye terminators
•
Hands-on: 5 minutes; Hands-off: 10 minutes
• Load Sequencer and run
•
Hands-on: 5 minutes; Hands-off: 2 hours
February 24, 2017
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The Rest of the Story 2
• Analyze results on SeqScape
• Map new mutations
• Search databases, literature for detected mutations
• Run new variants / VOUS through programs to
determine if pathogenic
• Make referral with best interpretation possible in
absence of any clinical information
• Results available the next morning
• Total time: 10-12 hours
Hands on: 1 - 2 hours
February 24, 2017
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Exon 18 Deletion Analysis
p.Gly828_Asn882del; c.2482_2646del
PCR # 15
GAP-PCR
•
902 bp only = no deletion
•
902 bp + 536 bp =
heterozygous deletion
•
536 bp product only –
homozygous deletion
•
Present in 5-7% of Dutch
alleles; 1 in NY so far
February 24, 2017
NYS Newborn Pathogenic Mutations
Mutation
(Nucleotide)
Mutation
(Protein)
c.-32-13T>G
33
(Erasmus & Emory Databases)
Classification
# Cases
with
Mutation
# Homozygous
# Heterozygous
Common Late Onset, Splicing
11
3
8
c.2560C>T
p.R854X
Very Severe
3
0
3
c.752C>T
p.S251L
Homozygosity with p.S254L has been
seen in Late Onset Pompe
3
0
3
c.761C>T
p.S254L
Homozygosity with p.S251L has been
seen in Late Onset Pompe
3
0
3
c.2238G>C
p.W746C
Potentially Mild
3
0
3
c.1979G>A
p.R660H
Potentially Less Severe
2
0
2
Less Severe
1
0
1
Potentially Less Severe
1
0
1
Previously Reported Mutation
1
0
1
c.692+5G>T
c.1099T>C
p.W367R
c.1754+2T>A
c.1843G>A
p.G615R
Potentially Less Severe
1
0
1
c.2219_2220delTG
p.Val740GfsX55
Very Severe
1
0
1
c.2646+2T>A
Very Severe
1
0
1
c.2647-7G>A
Potentially Mild
1
1
0
Very Severe
1
0
1
c.2662G>T
p.E888X
February 24, 2017
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NYS Newborn Screening VOUS
Variant of
Unknown
Significance
Variant of
Unknown
Significance
Information Found in
Literature
and
Other Notes
# Cases
with
Mutation
#
Homozygous
#
Heterozygous
c.664G>A
p.V222M
Reported as likely nonpathogenic based on in vitro
studies, however this mutation
was reported in several infants
identified by the Hungarian
NBS program as having low
GAA enzyme activity.
Diagnostic info was not
published.
3
2
1
Likely benign due to its
location in the intron
3
0
3
3
2
1
2*
2
0
2
0
2
(Nucleotide)
(Protein)
c.858+17_858+2
3
delCGGGCGG
c.1194+37G>A
c.316TC>T
p.R106C
c.2051C>T
p.P684L
*Twins
Seen once by Duke; only mut
detected in a 21 y.o. with
progressive scoliosis and
diminished GAA activity.
Predictive algorithms
conflicting as to whether its
pathogenic. Reported in
dbSNP and ExAC, but rare.
February 24, 2017
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The IDUA Gene – MPS 1
•
•
•
•
•
The IDUA gene codes for the alpha-L-iduronidase
enzyme
Cytogenetic Location: 4p16.3
Gene spans approximately 19 kb and has 14 exons;
653 amino acids; GC-rich gene – tricky
Approximately 100 mutations have been reported
Majority seem to be private mutations but a few have
been seen with some frequency
•
In North America two of the most common mutations are
p.W402* (45-60%) and p.Q70* (17%)
February 24, 2017
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Possible Pseudodeficiency Alleles
Molecular analysis:
c.235G>A (p.A79T); c.246C>G (p.H82Q); c.755G>A (p.D223N);
c.965T>A (p.V322E);
• 3 Black infants are p.A79T / p.A79T [MAF=2.8%; Blacks only]
• 2 Black infants are p.A79T / p.V322E [MAF<1% Blacks & Eur. Am.]
• 1 Black infant is p.A79T / p.D223N [MAF <1%; Blacks only]
• p.H82Q (MAF<1% in both European and Blacks) was detected in 2
infants: homozygous in a Caucasian infant, and compound
heterozygous with p.V322E in a biracial infant.
• Too early to rule out MPS 1 in these patients based on clinical
features; however six of the eight have had urine studies performed
and had normal chromatography/electrophoresis.
• Previously described pseudodeficiency allele c.898G>A (p.A300T)
Three apparent pseudo-deficiency alleles in the IDUA gene identified by newborn screening. L. M.
Pollard1, S. R. Braddock2, K. M. Christensen2, D. J. Boylan2, L. D. Smith3, B. A. Heese3, A. M. Atherton3, C. E.
Lawson3, M. E. Strenk3, M. Willing4, L. Manwaring4, T. C. Wood1
February 24, 2017
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February 24, 2017
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Third Tier: DNA Sequencing – ALD
• Full sequencing of ABCD1 gene
• Not intended to reduce referrals
• Helps to determine
o if females are ALD carriers (ALD females)
o if males have mutation
o if no mutation, consider other PBD
• Neither marker concentration nor
genotype correlates with phenotype!!
February 24, 2017
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Example of De Novo Mutation
~10%
Mom
Baby
Dad
February 24, 2017
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Thank you to Colleen Stevens, Ph.D.
and Suzanne Cordovado, Ph.D. for slides
and to you for your attention!