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1 2 genome structure
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1 2 Genome Structure ____________________________________ Start - - PDF document

Apr 20, 2023 217 likes •779 views

B ASICS OF MOLECULAR DIAGNOSTICS FOR THE EVERYDAY PRACTICING PATHOLOGIST Artur Rangel, MD, PhD Pathology Consultants of South Broward Medical Director, Clinical and Molecular Pathology - Memorial Healthcare System 1 2 Genome Structure

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SLIDE 1

BASICS OF MOLECULAR DIAGNOSTICS FOR THE

EVERYDAY PRACTICING PATHOLOGIST

Artur Rangel, MD, PhD

Medical Director, Clinical and Molecular Pathology - Memorial Healthcare System Pathology Consultants of South Broward

____________________________________ |||||||||||||||||||||||||||||||||||| ATGACGGATCAGCCGCAAGCGGAATTGGCGACATAA TACTGCCTAGTCGGCGTTCGCCTTAACCGCTGTATT ||||||||||||||||||||||||||||||||||||

Start Stop

Genome Structure 1 2 3

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SLIDE 2

Chromosomal GAINS / LOSSES (CNV)

Common Types of Genetic Variants

Complete vs Partial Balanced vs unbalanced Large translocations

4 5 6

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SLIDE 3

1p / 19q Co-deletion Oligodendroglioma 1p 19q 1p / 19q Co-deletion Oligodendroglioma 250,000,000 bp 7 8 9

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SLIDE 4

Technique: Karyotype Resolution Scope Variants

Quantitative

Specimen Limitations GAINS / LOSSES / Rearrangements Chromosome level / Megabases Genome wide Semi ( x / 20 cells) Requires living / dividing cells Specimen / resolution Sub-chromosomal GAINS / LOSSES (CNV)

Common Types of Genetic Variants

Balanced vs unbalanced Small Translocations Several kilobases range

Genome Structure 10 11 12

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SLIDE 5

DNA Complementarity

A <–> T G <–> C

DNA Complementarity -> hybridization DNA Hybridization DNA Complementarity -> hybridization 13 14 15

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SLIDE 6

DNA Complementarity -> hybridization DNA Complementarity -> hybridization 16 17 18

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SLIDE 7

19 20 21

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SLIDE 8

Millions

  • f Bases

Thousands

  • f Bases

22 22

22 23 24

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SLIDE 9

Karyotype FISH Break apart probe Karyotype FISH

Technique: FISH Resolution Scope Variants

Quantitative

Specimen Limitations GAINS / LOSSES / Rearrangements Sub-Chromosome / 100’s Kilobases Targeted – specific probes Yes, % affected cells FFPE or dividing cells (metaphase) Specimen / Decal / resolution

25 26 27

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SLIDE 10

Chromosomal GAINS / LOSSES (CNV)

Common Types of Genetic Variants

Complete vs Partial Single Nucleotide Polymorphisms (SNP) Gene Expression Over, Under Expression profiling Comparative Genomic Hybridization - array CGH

Microarray Applications

Gene Expression Profiling Single Nucleotide Polymorphism - SNP array

Microarray 28 29 30

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SLIDE 11

Microarray

CTTAACCG CTTAACCG CTTAACCG CTTAACCG CTTAACCG

Microarray s

CTTAACCG CTTAACCG CTTAACCG CTTAACCG CTTAACCG

Patient

ATGACGGATCAGCCGCAAGCGGAATTGGCGACATAA TACTGCCTAGTCGGCGTTCGCCTTAACCGCTGTATT

Control

ATGACGGATCAGCCGCAAGCGGAATTGGCGACATAA TACTGCCTAGTCGGCGTTCGCCTTAACCGCTGTATT

Microarray - CGH 31 32 33

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SLIDE 12

CTTAACCG CTTAACCG CTTAACCG CTTAACCG CTTAACCG

Patient

ATGACGGATCAGCCGCAAGCGGAATTGGCGACATAA TACTGCCTAGTCGGCGTTCGCCTTAACCGCTGTATT

Control

ATGACGGATCAGCCGCAAGCGGAATTGGCGACATAA TACTGCCTAGTCGGCGTTCGCCTTAACCGCTGTATT

Microarray - CGH

CTTAACCG CTTAACCG CTTAACCG CTTAACCG

Normal Patient Patient DNA Control DNA

CTTAACCG CTTAACCG CTTAACCG CTTAACCG

Microarray - Copy Number Variation (CNV) Normal Patient

34 35 36

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SLIDE 13

CNV : GAIN Patient DNA Control DNA

+4 copies / Fold change: 3 / CNV:6

CTTAACCG CTTAACCG CTTAACCG CTTAACCG

CTTAACCG CTTAACCG CTTAACCG CTTAACCG

CNV : GAIN

+4 copies / Fold change: 3 / CNV:6

37 38 39

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SLIDE 14

CNV : LOSS Patient DNA Control DNA

  • 1 copy / Fold change: 0.5 / CNV:1

CTTAACCG CTTAACCG CTTAACCG CTTAACCG CTTAACCG CTTAACCG CTTAACCG CTTAACCG CTTAACCG CTTAACCG CTTAACCG CTTAACCG

CNV : LOSS

  • 1 copy / Fold change: 0.5 / CNV:1

Comparative Genomic Hybridization - array CGH

Microarray Variations

Gene Expression Profiling Single Nucleotide Polymorphism - SNP array

40 41 42

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SLIDE 15

Microarray Applications Expression Profiling

TACTGCCTAGTCGGCGTTCGCCTTAACCGCTGTATT |||||||||||||||||||||||||||||||||||| ____________________________________ |||||||||||||||||||||||||||||||||||| ATGACGGATCAGCCGCAAGCGGAATTGGCGACATAA

Start Stop

DNA Gene Expression

____________________________________ |||||||||||||||||||||||||||||||||||| ATGACGGATCAGCCGCAAGCGGAATTGGCGACATAA

Start Stop

Gene Expression DNA

UACUGC |||||| CUAGUCGG |||||||| CGUUCGCCUU |||||||||| AACCG ||||| CUGUAUU |||||||

mRNA

exon 1 exon 2 exon 3 intron intron

UACUGC |||||| CGUUCGCCUU |||||||||| CUGUAUU |||||||

exon 1 exon 2 exon 3

spliced mRNA cDNA

TACTGC |||||| CGTTCGCCTT |||||||||| CTGTATT |||||||

43 44 45

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SLIDE 16

UACUGC |||||| CGUUCGCCUU |||||||||| CUGUAUU |||||||

mRNA cDNA

TACTGC |||||| CGTTCGCCTT |||||||||| CTGTATT |||||||

cDNA Probes Microarrays Variations Gene Expression Profiling

CTTAACCG CTTAACCG CTTAACCG CTTAACCG CTTAACCG CTTAACCG CTTAACCG CTTAACCG CTTAACCG CTTAACCG CTTAACCG CTTAACCG CTTAACCG CTTAACCG CTTAACCG CTTAACCG CTTAACCG CTTAACCG

Comparative Genomic Hybridization - array CGH

Microarray Variations

Gene Expression Profiling Single Nucleotide Polymorphism - SNP array

46 47 48

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SLIDE 17

Single Nucleotide Polymorphisms

G C A * CGTT AGA

SNP array

GCA*CGTTAGA CGT*GCAATCT

Target SNP *

  • Comp. DNA

CGTTGCAATCT CGTCGCAATCT CGTAGCAATCT

(A,G,T)

GCAACGTTAGA

Patient Genotype at *: AA Patient DNA

A G T

CGTTGCAATCT CGTCGCAATCT CGTAGCAATCT

GCAACGTTAGA

GCAACGTTAGA GCAACGTTAGA

A G T A/A

49 50 51

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SLIDE 18

GCAACGTTAGA

Patient Genotype at *: AT Patient DNA

A G T

CGTTGCAATCT CGTCGCAATCT CGTAGCAATCT

GCATCGTTAGA

GCAACGTTAGA GCATCGTTAGA

A G A/T T

52 53 54

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SLIDE 19

Technique: Microarrays Resolution Scope Variants

Quantitative

Specimen Limitations CNV, SNP, Gene Expression SNP to 10’s Kilobases Targeted / genome wide Yes, CNV /Expression Blood, Fresh, Frozen, FFPE, cells Balanced alterations / Decal

55 56 57

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SLIDE 20

Insertions / Deletions (InDel)

Common Types of Genetic Variants

CNV Gene Expression Single Nucleotide Polymorphisms (SNP) Fusions (translocations)

TACTGCCTAGTCGGCGTTCGCCTTAACCGCTGTATT |||||||||||||||||||||||||||||||||||| ____________________________________ |||||||||||||||||||||||||||||||||||| ATGACGGATCAGCCGCAAGCGGAATTGGCGACATAA

Start Stop

DNA Polymerase Chain reaction - PCR

____________________________________ |||||||||||||||||||||||||||||||||||| ATGACGGATCAGCCGCAAGCGGAATTGGCGACATAA

Start Stop

AATACAG ||||||| TACTGCCTAGTCGGCGTTCGCCTTAACCGCTGTATT |||||||||||||||||||||||||||||||||||| TACTGC |||||| CTAGTCGG |||||||| CGTTCGCCTT |||||||||| AACCG ||||| CTGTATT ||||||| _____________________________ ||||||||||||||||||||||||||||| ATGACGGATCAGCCGCAAGCGGAATTGGC

for primer rev primer

58 59 60

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SLIDE 21

____________________________________ |||||||||||||||||||||||||||||||||||| ATGACGGATCAGCCGCAAGCGGAATTGGCGACATAA

Start Stop

TACTGCCTAGTCGGCGTTCGCCTTAACCGCTGTATT |||||||||||||||||||||||||||||||||||| CTAGTCGG |||||||| CGTTCGCCTT |||||||||| AACCG ||||| CTGTATT ||||||| _____________________________ ||||||||||||||||||||||||||||| ATGACGGATCAGCCGCAAGCGGAATTGGC

Polymerase Chain reaction - PCR ~ 34 Billion fold amplification Typical 35 cycle PCR => 2^35 PCR - Results

Gel electrophoresis Capillary electrophoresis Quantitative PCR

61 62 63

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SLIDE 22

Gel electrophoresis Capillary Electrophoresis

TACTGCCTAGTCGGCGTTCGCCTTAACCGCTGTATT |||||||||||||||||||||||||||||||||||| ____________________________________ |||||||||||||||||||||||||||||||||||| ATGACGGATCAGCCGCAAGCGGAATTGGCGACATAA

Start Stop

TACTGC ||||||

qPCR – Quantitative PCR

Quantitative PCR

____________________________________ |||||||||||||||||||||||||||||||||||| ATGACGGATCAGCCGCAAGCGGAATTGGCGACATAA

Start Stop

TACTGC |||||| CGTTCGCCTT |||||||||| GCCTT ||||| CGTTC ||||| CTAGTCGG ||||||||

64 65 66

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SLIDE 23

Technique: PCR applications Resolution Scope Variants

Quantitative

Specimen Limitations typing (SNP, InDel), CNV, Expression SNP to 100’s of bases Targeted Yes: qPCR (CNV /Expression) Blood, Fresh, Frozen, FFPE, cells Targeted / Resolution / Decal

Sanger Sequencing

____________________________________ |||||||||||||||||||||||||||||||||||| ATGACGGATCAGCCGCAAGCGGAATTGGCGACATAA

Start Stop

TACTGCC* ||||||| TACTGCCTA* ||||||||| TACTGCCTAGT* ||||||||||| TACTGCCTAG* |||||||||| TACTGCCT* ||||||||

Technique: Sanger Sequencing Resolution Scope Variants

Quantitative

Specimen Limitations Novel (SNP, InDel) SNP to 100’s of bases Limited Targeted No, LOD ~20% VAF Blood, Fresh, Frozen, FFPE, cells

Targeted / low throughput/ LOD / Decal

67 68 69

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SLIDE 24

Next Generation Sequencing - NGS

70 71 72

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SLIDE 25

73 74 75

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SLIDE 26

76 77 78

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SLIDE 27

79 80 81

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SLIDE 28

82 83 84

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SLIDE 29

85 86 87

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SLIDE 30

88 89 90

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SLIDE 31

91 92 93

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SLIDE 32

94 95 96

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SLIDE 33

97 98 99

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SLIDE 34

100 101 102

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SLIDE 35

103 104 105

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SLIDE 36

106 107 108

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SLIDE 37

109 110 111

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SLIDE 38

112 113 114

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SLIDE 39

115 116 117

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SLIDE 40

118 119 120

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SLIDE 41

121 122 123

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SLIDE 42

124 125 126

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SLIDE 43

Not meant for humans

127 128 129

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SLIDE 44

130 131 132

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SLIDE 45

Control Patient CNV - Gain

133 134 135

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SLIDE 46

Control Patient CNV - Loss CNV by NGS

EWSR1 (22q12.2) WT1 (11p13)

Fusions by NGS

136 137 138

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SLIDE 47

22 22

FISH Break Apart

Technique: NGS Sequencing Resolution Scope Variants

Quantitative

Specimen Limitations Novel (SNP, InDel, CNV, Fusions) SNP to 100’s of bases Targeted to genome wide

Yes: SNP, CNV, InDel / LOD 1-5% VAF

Blood, Fresh, Frozen, FFPE, cells

CNV sensitivity / Decal / Data Intense

Pathologist’s Roles CLIA validation Tissue assessment Test / Panel selection Data analysis Clinical report

Molec AP + Molec

AP + Molec + onc

Molec Molec

139 140 141

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SLIDE 48

Tertiary analysis - Accurate variant inclusion

Read depth Variant Allele Fraction (VAF) Somatic (tumor) mutations Interpretation Limit of detection LOD (analytical sensitivity) Read depth

20 12

  • Measure of consensus accuracy
  • Acceptable ~>200
  • Usually depth of coverage is in the

thousands for a standard run

Variant Allele Fraction (VAF)

6/20 = 30% VAF in sample Reference Human genome .............................. ..............................

142 143 144

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SLIDE 49

Gene

VAF 50% Pure Tumor

Variant allele fraction (VAF)

Gene

VAF 25%

Gene

50% Tumor VAF 10% 20% Tumor VAF 5% 10% Tumor

145 146 147

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SLIDE 50
  • Variant allele fraction at which 95%
  • f samples would reliably be

detected

  • Usually 1-5% allele frequency
  • "Noise" vs. true allele

○ Polymerase errors ○ Deamination ○ DNA oxidative damage

LOD analytic sensitivity

5% LOD 5%

148 149 150

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SLIDE 51

55% overcall It’s All About Nuclei

Pitfalls...

151 152 153

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SLIDE 52

Pitfalls: Necrosis Pitfalls: Necrosis Pitfalls: Mucinous CA

154 155 156

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SLIDE 53

Pitfalls: Mucinous CA Pitfalls: Mucinous CA

http://www.pathologyoutlines.com

Pitfalls: Lymph nodes

157 158 159

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SLIDE 54

Pitfalls: Melanin

https://en.wikipedia.org/wiki/Melanoma

Tumor Enrichment

4x

Low Power Field 160 161 162

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SLIDE 55

Tissue Selection: Size

Thank You

163 164