Genetic Analysis of DBA

Gareth Gerrard

Imperial Molecular Pathology / Centre for Haematology

Hammersmith Hospital

Molecular Diagnostics Begins With…

DNA, Codons and the Amino Acid Code

Genes: Exons, Introns & Splicing

DNA, RNA & Proteins (& Cake)

Amino acids

The Ribosome (80S)

40S (S) unit: 18S RNA + 33 proteins

40S (S) unit: 18S RNA + 33 proteins

60S (L) unit: 5S RNA,  28S RNA, 5.8S RNA + ~49 proteins

A cake making machine that uses mRNA as the recipe and amino acids as the ingredients

DBADBA

25-35%RPL5, RPL11,

RPS26, RPS24, RPS17, RPS10, RPL35a,RPS7, RPL26, RPL15

25-35%RPL5, RPL11,

RPS26, RPS24, RPS17, RPS10, RPL35a,RPS7, RPL26, RPL15

25% RPS1925%

RPS19

40-50% ??40-50% ??

• ~80 RP genes in total• 10 are known to be affected in

DBA• GATA1 may also have a role

• ~80 RP genes in total• 10 are known to be affected in

DBA• GATA1 may also have a role

Mutations affecting:

Mutations affecting ribosomal protein

(RP) genes

Heterozygous, autosomal dominant

Leading to RP haploinsufficiency

DBA is a ribosomopathy*

*probably

Types of Mutations in DBA – 1) Missense

Change in recipe – use salt instead of sugar= cake no good!Change in recipe – use salt instead of sugar= cake no good!

Types of Mutations – 2) Nonsense

Change in recipe – leave out half of ingredients= cake no good!Change in recipe – leave out half of ingredients= cake no good!

Types of Mutations – 3) Frameshift

Change in recipe – words become unreadable= cake no good!Change in recipe – words become unreadable= cake no good!

Types of Mutations – 4) Splice Site

Change in recipe – pages left out or go blank= cake no good!Change in recipe – pages left out or go blank= cake no good!

Types of Mutations – 5) Copy Number Variation (CNV)

Change in recipe – pages torn out= cake no good!Change in recipe – pages torn out= cake no good!

Why Screen?

• Accurate diagnosis

• Donor selection for

allogeneic haematopoietic

stem cell transplantation

• Reproductive choices

• Linking genotype to

phenotype

• Accurate diagnosis

• Donor selection for

allogeneic haematopoietic

stem cell transplantation

• Reproductive choices

• Linking genotype to

phenotype

Unknown

RPS19

RPL5

RPS10

RPL11RPS35a

RPS26 RPS24 RPS7 RPS17 RPL26

10 Commonly Identified DBA associated RP Genes

= 7 genes in conventional molecular screen

Mutations are mostly SNVs and indels, but large deletions & insertion are also seen

Mutation Detection Technology – Sanger Sequencing

1 Sample / 1 Gene / day1 Sample / 1 Gene / day

ABI 3130ABI 3130 ABI 3500xlABI 3500xl

5 Samples / 1 Gene / day5 Samples / 1 Gene / day

Peripheral BloodExtract DNA

RPS19

RPL5

RPL11

RPS24

RPS17

RPL35a

RPS7

Standard DBA Screening Pipeline Measure & QC

Sanger Sequence PCR target gene exons

Next Generation Sequencers

Roche 454Roche 454 Illumina MiSeqIllumina MiSeq Ion Torrent PGMIon Torrent PGM

Getting on a bit / Expensive

Getting on a bit / Expensive

Highest throughputHighest throughput Fastest / most flexibleFastest / most flexible

V1 - PilotV1 - Pilot V2 - CurrentV2 - Current

Why Next Generation Seq (NGS)?

• Very high throughput (fast)

• Can look at all 80+ RP genes at once

• Can multiplex many samples at once

• Potential to pick up allele-loss deletions & insertions (CNV)

• Cost effective per-gene / per-sample

• Once identified, family members can be screened by conventional sequencing

• Very high throughput (fast)

• Can look at all 80+ RP genes at once

• Can multiplex many samples at once

• Potential to pick up allele-loss deletions & insertions (CNV)

• Cost effective per-gene / per-sample

• Once identified, family members can be screened by conventional sequencing

Small Location (for capture)SA RPSA chr3:39448180-39453929S2 RPS2 chr16:2012061-2014861S3 RPS3 ch11:75110530-75133324S3A RPS3A chr4:152020725-152025804

RPS4X chrX:71475529-71497150RPS4Y chrY:2709527-2734997

S5 RPS5 chr19:58898636-58906170S6 RPS6 chr9:19375713-19380252S7 RPS7 chr2:3622795-3628509S8 RPS8 chr1:45240923-45244451S9 RPS9 chr19:54704610-54752862S10 RPS10 chr6:34385231-34393902S11 RPS11 chr19:49999634-50002944S12 RPS12 chr6:133135580-133138703S13 RPS13 chr11:17095936-17099334

S14 RPS14 chr5:149822753-149829319S15 RPS15 chr19:1438363-1440492S15A RPS15A chr16:18792617-18801656S16 RPS16 chr19:39923852-39926618S17 RPS17 chr15:82821158-82824972S18 RPS18 chr6:33239787-33244287S19 RPS19 chr19:42363988-42375482S20 RPS20 chr8:56979854-56987069S21 RPS21 chr20:60962105-60963576S23 RPS23 chr5:81569177-81574396S24 RPS24 chr10:79793518-79816570S25 RPS25 chr11:118886422-118889401S26 RPS26 chr12:56435637-56438116S27 RPS27 chr1:153963235-153964626S27A RPS27A chr2:55459039-55462989S28 RPS28 chr19:8386384-8387809S29 RPS29 chr14:50043390-50053094S30 FAU chr11:64888100-64889945

S4

L3 RPL3 chr22:39708887-39716394L4 RPL4 chr15:66790801-66797221L5 RPL5 chr1:93297597-93307481L6 RPL6 chr12:112842994-112856642L7 RPL7 chr8:74202506-74208024L7A RPL7A chr9:136215069-136218281L8 RPL8 chr8:146015150-146017972L9 RPL9 chr4:39455744-39460568L10 RPL10 chrX:153618315-153637504L10A RPL10A chr6:35436185-35438562L11 RPL11 chr1:24018269-24022915L12 RPL12 chr9:130209953-130213684L13 RPL13 chr16:89627056-89630950L13A RPL13A chr19:49990811-49995565L14 RPL14 chr3:40498783-40506549L15 RPL15 chr3:23958036-23965183L17 RPL17 chr18:47014858-47018906L18 RPL18 chr19:49118585-49122793L18A RPL18A chr19:17970730-17974962L19 RPL19 chr17:37356536-37360980L21 RPL21 chr13:27825446-27830828L22 RPL22 chr1:6241329-6269449L23 RPL23 chr17:37004118-37010064L23A RPL23A chr17:27046411-27051377L24 RPL24 chr3:101399935-101405626L26 RPL26 chr17:8280838-8286568L27 RPL27 chr17:41150446-41154956L27A RPL27A chr11:8703958-8736306L28 RPL28 chr19:55897300-55903449L29 RPL29 chr3:52027644-52029958L30 RPL30 chr8:99037079-99058697L31 RPL31 chr2:101618177-101640494L32 RPL32 chr3:12875984-12883087

Large Subunit L34 RPL34 chr4:109541722-109551568L35 RPL35 chr9:127620159-127624260L35A RPL35A chr3:197676858-197683481L36 RPL36 chr19:5690272-5691674L36A RPL36A chrX:100645812-100651105L37 RPL37 chr5:40825364-40835437L37A RPL37A chr2:217362912-217443903L38 RPL38 chr17:72199721-72206676L39 RPL39 chrX:118920467-118925606L40 UBA52 chr19:18682614-18688269L41 RPL41 chr12:56510370-56511727LP0 RPLP0 chr12:120634489-120639038LP1 RPLP1 chr15:69745123-69748172LP2 RPLP2 chr11:809647-812880

RP Gene loci used for V1 Gene CaptureRP Gene loci used for V1 Gene Capture

http://ribosome.med.miyazaki-u.ac.jphttp://ribosome.med.miyazaki-u.ac.jp

Latest Version adds GATA1, but loses RPS17

Latest Version adds GATA1, but loses RPS17

NGS Workflow – v1

3µg Genomic DNA20 probands

Fragment DNA:Covaris e220

Library quant, pool, clean up and cluster generation

High-throughput SequencingData analysis

Sanger seq validation

Hybridise and capture Ribosomal Protein Gene DNA

including exons, introns, & regulatory regions

Target Enrichment

Total Time = 2 weeksTotal Time = 2 weeks

SG= Stop Gain SNV (Nonsense); FSD= Frame-shift Deletion; FSI= Frame-shift Insertion;SL= Start Loss SNV (Missense); SSD= Splice Site Defect

Gene n= (17) % TypeRPL5 5(4) 29.4% 3(2) SG/2 FSDRPS26 3 17.6% SG/FSI/SLRPL11 2 11.8% FSD/FSIRPS17 2(1) 11.8% 2(1) SGRPS7 1 5.9% SSDRPS10 1 5.9% SGRPS24 1 5.9% SGRPS19 0 0.0%Tot Mut 15(13) 88.2%NoMut 2 11.8%

British Journal of Haematology, 2013, 162,530–536

DBA – NGS v1 – Results from Initial 20 Samples

DBA – NGS – v2 Workflow: Days 1 - 3

20ng gDNA20ng gDNA

AmpliSeq Library Prep (1-2 days)

AmpliSeq Library Prep (1-2 days)

Template& EnrichOneTouch2 & ES

Template& EnrichOneTouch2 & ES

PGMSequence2 x 8 barcode

PGMSequence2 x 8 barcode

qPCR quant & poolKAPA Quant Kit

qPCR quant & poolKAPA Quant Kit

Day 1Day 1 Day 2Day 2 Day 3Day 3

Allows screening of 16 samples for 80+ Genes per runAllows screening of 16 samples for 80+ Genes per run

DBA NGS – Day 4: Analysis...

Variant CallerTSv3.6.2Variant CallerTSv3.6.2

VCF FilesVCF Files

VEPEnsembl v72Virtualbox 4.2

VEPEnsembl v72Virtualbox 4.2

Ion Reporterv1.6

Ion Reporterv1.6

CONDEL / Mutation AssessorCONDEL / Mutation Assessor

Human Splicing Finder v2.4.1Human Splicing Finder v2.4.1

MolDiag team for Sanger validation & reporting

MolDiag team for Sanger validation & reporting

NextGene / SeqNext

IGVIGVDON’T PANIC!DON’T PANIC!

SHOW ME THE KITTEHSSHOW ME THE KITTEHS

DBA – NGS - Analysis

DBA Mutation - IGV PileUp showing RPS26 Nonsense

TTC (Phenylalanine) -> TAA (STOP)TTC (Phenylalanine) -> TAA (STOP)

DBA-NGS v2 – Initial Results

DBA-HALO ResultsBarcode Gene Consequence Exon Base Codon LOVD? LOF? dbSNP MAF Sanger Valid?1 RPL15 Stop-Gain 4 3:23960737G>Ap120W>* No Yes(?) n/a n/a Yes2 RPS26 splice donor variant 1 n.30+1G>AINTRON=1/2Yes Yes rs148622862n/a Yes3 RPL13A missense_variant 7 c.481C>A p.Ala161AspNo ? rs150697570 n/a4 RPS7 missense_variant 6 c.562T>C p.133L>S No ??? n/a n/a Yes5 RPL29 inframe_insertion 4 c.386_391dupCCAAGGp.Ala129_Lys130dupn/a ??? rs141201675n/a *6 RPS19 frameshift_insertion 4 c.199-200_insG 67 No ??? n/a n/a *7 RPL7 splice_region_variant 1+8 c.107+8A>GINTRON=1/3n/a ??? rs74460527 0.0096 *8 RPL15 missense_variant 5 c.466T>G p.141S>A n/a ??Splicing n/a n/a *9 -10 -11 -12 -13 -14 RPL17 splice_region_variant,5_prime_UTR_variant1 c.87G>A Exon1/6 (5'UTR)n/a ??? rs140522052<1% *15 RPS10 Stop-Gain c.337C>T p.113R>* Yes Yes rs267607022 Yes - c

3 definite hits (1 novel); 2 very likely; 5 interestingOnly 1 DBA had no mutation (9); 10-13 non-affected family members3 definite hits (1 novel); 2 very likely; 5 interestingOnly 1 DBA had no mutation (9); 10-13 non-affected family members

Summary

• Screening for mutations in DBA is now an established

technology

• We now use NGS technology to screen all 80+

Ribosomal protein genes

• Family members screened by conventional sequencing

(for known mutation)

• Will introduce screening for CNV in near future

• Screening for mutations in DBA is now an established

technology

• We now use NGS technology to screen all 80+

Ribosomal protein genes

• Family members screened by conventional sequencing

(for known mutation)

• Will introduce screening for CNV in near future

IPML Hammersmith

Letizia ForoniKikkeri Naresh

MRDPierre FoskettThet MyintFaisal Abdillah

Mol DiagMikel ValganonAlex FoongNatalie KilleenSarmad Toma

R&DMary AlikianGeorge Nteliopoulos

IPML Hammersmith

Letizia ForoniKikkeri Naresh

MRDPierre FoskettThet MyintFaisal Abdillah

Mol DiagMikel ValganonAlex FoongNatalie KilleenSarmad Toma

R&DMary AlikianGeorge Nteliopoulos

Clinical Team

Josu de la FuenteAnastasios Karadimitris

Jane ApperleyDavid MarinDragana MilojkovicJiri Pavlu

John Goldman

Clinical Team

Josu de la FuenteAnastasios Karadimitris

Jane ApperleyDavid MarinDragana MilojkovicJiri Pavlu

John Goldman

Students

Aysha PatelSakuntala AleRobin Ferrari

Deena Iskander

Students

Aysha PatelSakuntala AleRobin Ferrari

Deena Iskander

ACHS CGL

Tim AitmanMichael MüllerDalia Kasperaviciute

Laurence Game

ACHS CGL

Tim AitmanMichael MüllerDalia Kasperaviciute

Laurence Game

Thank You!