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Ho-Wan Ip
Division of Haematology
Department of Pathology and Clinical Biochemistry
Queen Mary Hospital
Hong Kong
18th June 2017
Outline
Myeloid panel
Bioinformatics consideration
Myeloid Panel
Panel Design
Panel of 67 genes implicated to be of diagnostic, prognostic and therapeutic significance in myeloid neoplasms
Acute myeloid leukaemia (AML)
Myelodysplastic syndrome (MDS)
Myeloproliferative neoplasms (MPN)
Myelodysplastic/myeloproliferative neoplasms (MDS/MPN)
Clonal haemopoiesis of indeterminate potential (CHIP)
ABL1, ANKRD26, ASXL1, ATM, ATRX, BCOR, BCORL1, BRAF, CALR, CBL, CBLB, CBLC, CDKN2A, CEBPA,
CREBBP, CSF3R, CUX1, CXCR4, DNMT3A, DDX41, ETNK1, ETV6, EZH2, FBXW7, FLT3, GATA1, GATA2, GNAS,
GNB1, HRAS, IDH1, IDH2, IKZF1, JAK2, JAK3, KDM6A, KIT, KMT2A, KRAS, KMT2B, KMT2D, MPL, MYD88,
NF1, NOTCH1, NPM1, NRAS, PDGFRA, PHF6, PPM1D, PTEN, PTPN11, RAD21, RUNX1, SETBP1, SETD2,
SETDB1, SF3B1, SMC1A, SMC3, SRSF2, STAG2, TET2, TP53, U2AF1, WT1, ZRSR2
Human genome is huge
Genome
Focused interrogation of the human genome: Targeted sequencing
Genome
Fragmentation
Adaptor ligation
Hybridization capture
Sequencing
150 bp
sequence read A T
T A
G C
400 bp fragment
C G
Paired-end sequencing
150 bp
sequence read
400 bp
fragment
150 bp sequenced
section
Massively parallel sequencing
Sequence alignment to reference genome
Reference genome
Variant calling
Myeloid Panel
Type of Variants
Indels
FASTQ
fastQC, Trimmomatic
BAM
SNV & Indel CNV (gene)
Picard MarkDuplicates
SnpEff, ANNOVAR
BAM.indel Population: ESP, 1000G, ExAC_nonTCGA,
gnomAD
GATK Base Recalibration Significance: ClinVar, InterVar, COSMIC
Prediction: dbnsfp33a
BAM.final
Variant List
In-house database: Variant classification,
PMID, frequency
Performance of Myeloid Panel
Quality Metrics
Performance of Myeloid Panel
Quality Metrics: Depth of Coverage
Myeloid Panel: Variant Calling
Single Nucleotide Variants
Performance of Myeloid Panel
Analytical Sensitivity: Validation Using Reference Standard
Gene Variant Expected VAF Measured VAF No. of Alt Allele Total Depth
ABL1 p.T315I 2.5% 2.2% 29 1289
NRAS p.Q61H 2.5% 3.7% 27 739
NRAS p.Q61R 2.5% 1.5% 11 739
IDH1 p.R132H 2.5% 2.1% 27 1311
IDH2 p.R172K 2.5% 1.4% 24 1693
FLT3 p.D835Y 2.5% 2.5% 37 1477
KIT p.D816V 2.5% 2.0% 29 1444
KRAS p.A146T 2.5% 2.8% 29 1081
KRAS p.G12C 2.5% 2.5% 29 1165
KRAS p.G12D 2.5% 2.7% 31 1164
KRAS p.G12S 2.5% 3.4% 40 1165
KRAS p.G13D 25.0% 21.2% 251 1185
KRAS p.Q61H 2.5% 4.3% 50 1169
BRAF p.V600E 8.0% 6.7% 73 1088
BRAF p.V600M 2.0% 1.9% 34 1059
BRAF p.V600R 2.0% (1.2%) 13 1088
PDGFRA p.D842V 2.5% 2.9% 41 1393
Myeloid Panel: Variant Calling
Indels
Spencer DH et al. Detection of FLT3 internal tandem duplication in targeted, short-read-length, next-generation sequencing data. J Mol Diagn
2013;15:81-93.
Myeloid Panel: Variant Calling
Copy Number Variant (CNV)
Myeloid Panel: Variant Calling
Copy Number Variant (CNV)
Myeloid Panel: Variant Calling
Copy Number Variant (CNV): Major Challenges in Cancer
Myeloid Panel
Classification of Sequence Variants
Sukhai MA et al. A classification system for clinical relevance of somatic variants identified in molecular profiling of cancer. Genet Med 2016;18:128-136.
Myeloid Panel
Interpretation of Sequence Variants
Population frequency: Variant with >1% frequency in normal population can be assumed to have no clinical
significance (Benign)
1000 Genomes Project, Exome Sequencing Project, Exome Aggregation Consortium, gnomAD
Literature and database review (Class 1 and 2)
ClinVar: Database of clinical significance
COSMIC: Cancer database
PubMed, Google
Determine mechanism of implicated gene in causing disease phenotype
Gain of function
Loss of function
Function prediction software (Class 3 and 4)
Somatic variant
Myeloid Panel
Result Reporting
Myeloid Panel: NGS Study in Myelofibrosis in Hong Kong
Patient Demographics
Myeloid panel
Bioinformatics consideration
Targeted RNA-seq for Haematological Malignancies
Panel Design and Application
Hybridisation capture
FASTQ
Trimmomatic
FASTQ.trim
TopHat-Fusion STAR-Fusion
TopHat-Fusion-Post
FNDC3B RARA
Cheng CK et al. FNDC3B is another novel partner fused to RARA in the t(3;17)(q26;q21) variant of acute promyelocytic leukemia. Blood 2017;129:2705-2709.
NGS Panel for Inherited Haematological Diseases
Panel Design
Hybridization capture of all exons, with some genes including promoter regions
Curation process
Literature review
OMIM: Search of disease phenotype and associated genes
NGS Panel for Inherited Haematological Diseases
Disease Phenotypes
FASTQ
fastQC, Trimmomatic
BWA
BAM
SNV & Indel CNV (gene)
Picard MarkDuplicates
BAM.uniq
SnpEff, ANNOVAR
BAM.indel
Myeloid panel
Bioinformatics consideration
Bioinformatics Considerations
Personnel
For targeted sequencing, personal computers can handle most, if not all, the data analysis
i5 or i7 CPU, or equivalent
8 Gb memory
Installation of software and first use of software may need some troubleshooting efforts
Clinical Implementation of NGS: Experience in Hong Kong
Summary
30% of AML
Insertion of 15 to 300 bp
Spencer DH et al. Detection of FLT3 internal tandem duplication in targeted, short-read-length, next-generation sequencing data. J Mol Diagn
2013;15:81-93.
Myeloid Panel: Variant Calling
FLT3 Internal Tandem Duplication
Reference genome
Reference genome
Soft-clipped read
Soft-clipped read
Myeloid Panel: Variant Calling
FLT3 Internal Tandem Duplication
Myeloid Panel: Variant Calling
Surveying the Copy Number of the Whole Genome Using Targeted Data
Genome
Myeloid Panel: Variant Calling
Surveying the Copy Number of the Whole Genome Using Targeted Data
Myeloid Panel: Variant Calling
Surveying the Copy Number of the Whole Genome Using Targeted Data
Myeloid Panel: NGS Study in Myelofibrosis in Hong Kong
Survival Analyses
Case Study: Patient TCA
History and Physical Examination
M/86d
Born in private hospital. 1st child. Parents not consanguineous.
Admitted from OPD for haemolysis after birth
Anaemia, severe jaundice
Require red cell transfusions and exchange transfusion
Case Study: Patient TCA
Investigation: Complete blood count
Case Study: Patient TCA
Investigation: Peripheral blood smear
Case Study: Patient TCA
Investigation: PK study & Sanger sequencing in family trio
Anaemia
Phenotypically PK deficiency
Appear to be homozygous PKLR:p.R510Q
Is this due to hemizygous mutation due to inheritance of a deletion from the mother?
Is this due to uniparental disomy from a paternally inherited mutation?
Case Study: Patient TCA
Investigation by NGS: PKLR(NM_000298.5):c.1529G>A p.R510Q
Case Study: Patient TCA
Investigation by NGS: PKLR deletion
Case Study: Patient TCA
Summary of genetic findings
Costa C et al. Severe hemolytic anemia in a Vietnamese family, associated with novel mutations in the gene encoding for pyruvate kinase. Haematologica
2005;90:25-30.
So CC et al. First reported case of prenatal diagnosis for pyruvate kinase deficiency in a Chinese family. Hematology 2011;16:377-379.
Case Study: Patient TCA
Summary of genetic findings
PKLR(NM_000298.5):c.1529G>A p.R510Q
Frequently occurring mutation, found primarily in north Europeans and Caucasian Americans
Baronciani L et al. Analysis of pyruvate kinase-deficiency mutations that produce nonspherocytic hemolytic anemia. Proc Natl Acad Sci U S A
1993;90:4324-4327.
Wang C et al. Human erythrocyte pyruvate kinase: characterization of the recombinant enzyme and a mutant form (R510Q) causing nonspherocytic
hemolytic anemia. Blood 2001;98:3113-3120.
Bioinformatics Considerations
Possible Options for Analysis Platform
Windows
Windows 10: Bash on Ubuntu on Windows
Dual boot
Virtual machine (not preferred)
Note: Windows-based text files are not compatible with Linux-based text files
MacOS is Unix-based
Chang J. Rewarding bioinformaticians. Nature 2015;520:151-152.