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February 24, 2017

Current Second Tier and Future Applications of Gene Sequencing in NBS: Sanger Sequencing

Michele Caggana, Sc.D., FACMGFebruary 16, 2017

February 24, 2017 2

Molecular Analysis in Newborn ScreeningA Staged Approach

Ongoing in routine NBS

Experimental in NBS

Offered clinically and research outside NBS

Genotyping Panel of Mutations -- Single Gene

Sequencing Single Gene

Sequencing Panel of Genes

Sequencing of NBS Genes

GenomeExome

Ongoing in routine NBS

Experimental in NBS

Offered clinically and research outside NBS

S. Cordovado, Ph.D.

February 24, 2017 3

Mix deoxynucleotides with ddA, ddT, ddC*, ddG4 lanes per person/fragment~200 readable bases

Mix deoxynucleotideswith ddA, ddT, ddC, ddG1 scan per person/fragment~800 readable bases

Chop up the human genomeMake a library of fragmentsSequence billions of basesMultiplexing multiple peopleMillions of ‘reads’

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http://nobelprize.org/chemistry/laureates/1980/sanger-autobio.html

Frederick Sanger Walter Gilbert

1/4 prize each

Published in 1977

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• 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

Sanger Sequencing

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• 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

Sanger Sequencing

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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”.

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Radioisotopic SequencingSanger MethodUsed radiolabelled P32

Pasternak, 1999

https://www.gelcompany.com/gibco-brl

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• 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 • Longer reads• Human Genome Project

Advancements – ABI 373A

http://www.labrecyclers.com/product.html?InventoryID=2301

February 24, 2017 10

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


Methods Using Capillary Electrophoresis

Principle of agarose or

acrylamide gel electrophoresis

is still used


Principle of Capillary Electrophoresis

Multiple capillaries can run samples at the same time

A laser is used to detect fluorescently labeled amplified product


ABI Prism 3100 Genetic Analyzer

capillaries

Syringe with polymer solution

Autosamplertray

Outlet buffer

Injection electrode

Inlet buffer

laser


ABI 310016-capillary

array


Sequence analysis:ABI PRISM 3100

Frameshift mutationdelT


Applied Biosystems 3730 48-capillary Genetic Analyzer

Applied Biosystems 3500 8-capillary Genetic Analyzer

DNA Sequence Analysis Using Capillary Electrophoresis


ABI 3730 Genetic Analyzer

capillaries

cathode/anode bufferspolymer

laser

inlet buffer


Peering into an ABI 35003500 XL 24-capillary


• 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

Why Even Bother ??


GAA Enzyme Activity (MS/MS) (<15%) Daily Mean Average Percent

DNA Sequencing

2 Pathogenic

Variants

Referral

No Pathogenic

Variants

Referral

VOUS (<1% in General Population)

No Referral

OnlyPseudodeficiency

Alleles*

No ReferralReferral

1 Pathogenic Variantc/ or s/VOUS /Polys

VOUS = variant of unknown significance Polys – common variants that are not disease-causing*p.Gly576Ser; p.Glu589Lys


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


Step One : Punch and Extract DNA

Hands on: 5-10 min; Hands-off: 45 minutes


PCR Number Exons Covered

Base Pairs

1 2 772

2 3 384

3 4-5 630

4 6-8 781

5 9 483

6 10-11 890

7 12 465

PCR Number Exons Covered

BasePairs

8 13-14 679

9 15 486

10 16 583

11 17 497

12 18 400

13 19 342

14 20 460

20 Exons Covered in 14 FragmentsChromosome 17 –952 amino acids

• Primer design• Check for SNPs; minor allele frequencies on dbSNP• Need to amplify all with the same PCR program• Can use different master mixes


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

PCR Plate Map

Hands on: 5 min; Hands-off: 2 hoursWTC = wild type control; NTC = no template control


Set Up Plates in Batches

• Saves time• Batches better for QI• Minimize pipetting errors• Minimize inconsistencies


Agarose Gel Electrophoresis

Product Check

• Sizes okay• Amplification• No Contamination

Hands on: 15 min; Hands-off: 45 minutes


PCR Clean-up Before SequencingExoSap-IT Treatment

1 2 3 4 5 6 7 8

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


Cycle Sequencing1 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

E6/8SEQF1a

E13/14 F–1a

E17 F–1a

E2 SEQR –

1a

E6/8–SEQR1a

E13/14 R–1a

E17 R–1a

D E2 SEQF1

b

E6/8SEQF1b

E13/14 F–1b

E17 F–1b

E2 SEQR1

b

E6/8 SEQR1b

E13/14 R–1b

E17 R–1b

E E3 F–1a

E9F–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

E9R–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 hoursSEQ primers are internal sequencing primers


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


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


Exon 18 Deletion Analysisp.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


Mutation(Nucleotide)

Mutation(Protein)

Classification(Erasmus & Emory Databases)

# Cases with

Mutation

# Homozygous # Heterozygous

c.-32-13T>G 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

c.692+5G>T Less Severe 1 0 1

c.1099T>C p.W367R Potentially Less Severe 1 0 1

c.1754+2T>A Previously Reported Mutation 1 0 1

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

c.2662G>T p.E888X Very Severe 1 0 1

NYS Newborn Pathogenic Mutations


Variant of Unknown

Significance(Nucleotide)

Variant of Unknown

Significance(Protein)

Information Found in Literature

andOther Notes

# Cases with

Mutation

# Homozygous

# Heterozygous

c.664G>A p.V222M Reported as likely non-pathogenic 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

c.858+17_858+23

delCGGGCGG

Likely benign due to its location in the intron

3 0 3

c.1194+37G>A 3 2 1

c.316TC>T p.R106C 2* 2 0

c.2051C>T p.P684L 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.

2 0 2

*Twins

NYS Newborn Screening VOUS


• The IDUA gene codes for the alpha-L-iduronidaseenzyme

• 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%)

The IDUA Gene – MPS 1


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

Possible Pseudodeficiency Alleles


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 mutationo if no mutation, consider other PBD

• Neither marker concentration nor genotype correlates with phenotype!!


Dad

Mom

Baby

~10%

Example of De Novo Mutation


Thank you to Colleen Stevens, Ph.D. and Suzanne Cordovado, Ph.D. for slides

and to you for your attention!

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