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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 4 Frederick Sanger Walter Gilbert Published in 1977 1/4 prize each http://nobelprize.org/chemistry/laureates/1980/sanger-autobio.html February 24, 2017 5 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 February 24, 2017 6 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 7 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 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 February 24, 2017 11 Methods Using Capillary Electrophoresis Principle of agarose or acrylamide gel electrophoresis is still used February 24, 2017 12 Principle of Capillary Electrophoresis A laser is used to detect fluorescently labeled amplified product Multiple capillaries can run samples at the same time February 24, 2017 13 ABI Prism 3100 Genetic Analyzer laser capillaries Syringe with polymer solution Injection electrode Outlet buffer Autosampler tray Inlet buffer February 24, 2017 14 ABI 3100 16-capillary array February 24, 2017 15 Frameshift mutation delT Sequence analysis: ABI PRISM 3100 February 24, 2017 16 DNA Sequence Analysis Using Capillary Electrophoresis Applied Biosystems 3730 48-capillary Genetic Analyzer Applied Biosystems 3500 8-capillary Genetic Analyzer February 24, 2017 17 ABI 3730 Genetic Analyzer laser capillaries polymer inlet buffer cathode/anode buffers February 24, 2017 18 Peering into an ABI 3500 3500 XL 24-capillary February 24, 2017 19 February 24, 2017 20 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 21 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 22 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 February 24, 2017 23 Step One : Punch and Extract DNA Hands on: 5-10 min; Hands-off: 45 minutes February 24, 2017 24 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 25 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 February 24, 2017 26 Set Up Plates in Batches • • • • Saves time Batches better for QI Minimize pipetting errors Minimize inconsistencies February 24, 2017 27 Agarose Gel Electrophoresis Product Check • Sizes okay • Amplification • No Contamination Hands on: 15 min; Hands-off: 45 minutes February 24, 2017 28 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 29 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 30 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 31 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 32 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 34 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 35 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 36 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 37 February 24, 2017 38 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 39 Example of De Novo Mutation ~10% Mom Baby Dad February 24, 2017 40 Thank you to Colleen Stevens, Ph.D. and Suzanne Cordovado, Ph.D. for slides and to you for your attention!