1 2 Genome Structure ____________________________________ Start - PDF document

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 ____________________________________ Start Stop |||||||||||||||||||||||||||||||||||| ATGACGGATCAGCCGCAAGCGGAATTGGCGACATAA TACTGCCTAGTCGGCGTTCGCCTTAACCGCTGTATT |||||||||||||||||||||||||||||||||||| 3

Common Types of Genetic Variants Chromosomal GAINS / LOSSES (CNV) Complete vs Partial Large translocations Balanced vs unbalanced 4 5 6

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

Technique: Karyotype Variants GAINS / LOSSES / Rearrangements Resolution Chromosome level / Megabases Scope Genome wide Quantitative Semi ( x / 20 cells) Specimen Requires living / dividing cells Limitations Specimen / resolution 10 Common Types of Genetic Variants Sub-chromosomal GAINS / LOSSES (CNV) Several kilobases range Small Translocations Balanced vs unbalanced 11 Genome Structure 12

DNA Complementarity A <–> T G <–> C 13 DNA Complementarity -> hybridization DNA Hybridization 14 DNA Complementarity -> hybridization 15

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

19 20 21

22 Millions Thousands of Bases of Bases 23 22 22 24

Break apart probe Karyotype FISH 25 Karyotype FISH 26 Technique: FISH Variants GAINS / LOSSES / Rearrangements Resolution Sub-Chromosome / 100’s Kilobases Scope Targeted – specific probes Quantitative Yes, % affected cells Specimen FFPE or dividing cells (metaphase) Limitations Specimen / Decal / resolution 27

Common Types of Genetic Variants Chromosomal GAINS / LOSSES (CNV) Complete vs Partial Single Nucleotide Polymorphisms (SNP) Gene Expression Over, Under Expression profiling 28 Microarray Applications Comparative Genomic Hybridization - array CGH Gene Expression Profiling Single Nucleotide Polymorphism - SNP array 29 Microarray 30

Microarray 31 Microarray s CTTAACCG CTTAACCG CTTAACCG CTTAACCG CTTAACCG 32 Microarray - CGH Control Patient ATGACGGATCAGCCGCAAGCGGAATTGGCGACATAA ATGACGGATCAGCCGCAAGCGGAATTGGCGACATAA TACTGCCTAGTCGGCGTTCGCCTTAACCGCTGTATT TACTGCCTAGTCGGCGTTCGCCTTAACCGCTGTATT CTTAACCG CTTAACCG CTTAACCG CTTAACCG CTTAACCG 33

Microarray - CGH Patient Control ATGACGGATCAGCCGCAAGCGGAATTGGCGACATAA ATGACGGATCAGCCGCAAGCGGAATTGGCGACATAA TACTGCCTAGTCGGCGTTCGCCTTAACCGCTGTATT TACTGCCTAGTCGGCGTTCGCCTTAACCGCTGTATT CTTAACCG CTTAACCG CTTAACCG CTTAACCG CTTAACCG 34 Normal Patient Patient DNA Control DNA CTTAACCG CTTAACCG CTTAACCG CTTAACCG 35 Microarray - Copy Number Variation (CNV) Normal Patient CTTAACCG CTTAACCG CTTAACCG CTTAACCG 36

37 CNV : GAIN +4 copies / Fold change: 3 / CNV:6 Patient DNA Control DNA CTTAACCG CTTAACCG CTTAACCG CTTAACCG 38 CNV : GAIN +4 copies / Fold change: 3 / CNV:6 CTTAACCG CTTAACCG CTTAACCG CTTAACCG 39

CNV : LOSS -1 copy / Fold change: 0.5 / CNV:1 Patient DNA Control DNA CTTAACCG CTTAACCG CTTAACCG CTTAACCG CTTAACCG CTTAACCG 40 CNV : LOSS -1 copy / Fold change: 0.5 / CNV:1 CTTAACCG CTTAACCG CTTAACCG CTTAACCG CTTAACCG C TTAACCG 41 Microarray Variations Comparative Genomic Hybridization - array CGH Gene Expression Profiling Single Nucleotide Polymorphism - SNP array 42

Microarray Applications Expression Profiling 43 Gene Expression ____________________________________ Start Stop DNA |||||||||||||||||||||||||||||||||||| ATGACGGATCAGCCGCAAGCGGAATTGGCGACATAA TACTGCCTAGTCGGCGTTCGCCTTAACCGCTGTATT |||||||||||||||||||||||||||||||||||| 44 Gene Expression ____________________________________ Start Stop DNA |||||||||||||||||||||||||||||||||||| ATGACGGATCAGCCGCAAGCGGAATTGGCGACATAA UACUGC CUAGUCGG CGUUCGCCUU AACCG CUGUAUU mRNA |||||| |||||||| |||||||||| ||||| ||||||| exon 1 intron exon 2 intron exon 3 spliced UACUGC CGUUCGCCUU CUGUAUU |||||| |||||||||| ||||||| mRNA exon 1 exon 2 exon 3 cDNA TACTGC CGTTCGCCTT CTGTATT |||||| |||||||||| ||||||| 45

mRNA UACUGC CGUUCGCCUU CUGUAUU |||||| |||||||||| ||||||| cDNA TACTGC CGTTCGCCTT CTGTATT |||||| |||||||||| ||||||| cDNA Probes 46 Microarrays Variations Gene Expression Profiling CTTAACCG CTTAACCG CTTAACCG CTTAACCG CTTAACCG CTTAACCG CTTAACCG CTTAACCG CTTAACCG CTTAACCG CTTAACCG CTTAACCG CTTAACCG CTTAACCG CTTAACCG CTTAACCG CTTAACCG CTTAACCG 47 Microarray Variations Comparative Genomic Hybridization - array CGH Gene Expression Profiling Single Nucleotide Polymorphism - SNP array 48

Single Nucleotide Polymorphisms G C A * CGTT AGA 49 GCA * CGTTAGA SNP array Target SNP * (A,G,T) CGT * GCAATCT Comp. DNA CGT T GCAATCT CGT C GCAATCT CGT A GCAATCT 50 Patient Genotype at * : AA GCA A CGTTAGA Patient DNA GCA A CGTTAGA CGT T GCAATCT GCA A CGTTAGA CGT C GCAATCT GCA A CGTTAGA CGT A GCAATCT A G T A/A A G T 51

Patient Genotype at * : AT GCA A CGTTAGA Patient DNA GCA T CGTTAGA CGT T GCAATCT GCA A CGTTAGA CGT C GCAATCT CGT A GCAATCT GCA T CGTTAGA A G T A/T A G T 52 53 54

55 56 Technique: Microarrays Variants CNV, SNP, Gene Expression Resolution SNP to 10’s Kilobases Scope Targeted / genome wide Quantitative Yes, CNV /Expression Specimen Blood, Fresh, Frozen, FFPE, cells Limitations Balanced alterations / Decal 57

Common Types of Genetic Variants Single Nucleotide Polymorphisms (SNP) Insertions / Deletions (InDel) Fusions (translocations) CNV Gene Expression 58 Polymerase Chain reaction - PCR ____________________________________ Start Stop DNA |||||||||||||||||||||||||||||||||||| ATGACGGATCAGCCGCAAGCGGAATTGGCGACATAA TACTGCCTAGTCGGCGTTCGCCTTAACCGCTGTATT |||||||||||||||||||||||||||||||||||| 59 ____________________________________ Start Stop |||||||||||||||||||||||||||||||||||| ATGACGGATCAGCCGCAAGCGGAATTGGCGACATAA TACTGC CTAGTCGG CGTTCGCCTT AACCG CTGTATT for primer |||||| |||||||| |||||||||| ||||| ||||||| _____________________________ ||||||| ||||||||||||||||||||||||||||| rev primer ATGACGGATCAGCCGCAAGCGGAATTGGC AATACAG TACTGCCTAGTCGGCGTTCGCCTTAACCGCTGTATT |||||||||||||||||||||||||||||||||||| 60

____________________________________ Start Stop |||||||||||||||||||||||||||||||||||| ATGACGGATCAGCCGCAAGCGGAATTGGCGACATAA CTAGTCGG CGTTCGCCTT AACCG CTGTATT |||||||| |||||||||| ||||| ||||||| _____________________________ ||||||||||||||||||||||||||||| ATGACGGATCAGCCGCAAGCGGAATTGGC TACTGCCTAGTCGGCGTTCGCCTTAACCGCTGTATT |||||||||||||||||||||||||||||||||||| 61 Polymerase Chain reaction - PCR Typical 35 cycle PCR => 2^35 ~ 34 Billion fold amplification 62 PCR - Results Gel electrophoresis Capillary electrophoresis Quantitative PCR 63

Gel electrophoresis 64 Capillary Electrophoresis ____________________________________ Start Stop |||||||||||||||||||||||||||||||||||| ATGACGGATCAGCCGCAAGCGGAATTGGCGACATAA TACTGC |||||| TACTGCCTAGTCGGCGTTCGCCTTAACCGCTGTATT |||||||||||||||||||||||||||||||||||| 65 qPCR – Quantitative PCR Quantitative PCR ____________________________________ Start Stop |||||||||||||||||||||||||||||||||||| ATGACGGATCAGCCGCAAGCGGAATTGGCGACATAA CGTTC TACTGC CTAGTCGG CGTTCGCCTT GCCTT ||||| |||||||||| |||||| |||||||| ||||| 66

Technique: PCR applications Variants typing (SNP, InDel), CNV , Ex pression Resolution SNP to 100’s of bases Scope Targeted Quantitative Yes: qPCR (CNV /Expression) Specimen Blood, Fresh, Frozen, FFPE, cells Limitations Targeted / Resolution / Decal 67 ____________________________________ Start Stop Sanger Sequencing |||||||||||||||||||||||||||||||||||| ATGACGGATCAGCCGCAAGCGGAATTGGCGACATAA TACTGC C* ||||||| TACTGCC T* |||||||| TACTGCCT A* ||||||||| TACTGCCTA G* |||||||||| TACTGCCTAG T* ||||||||||| 68 Technique: Sanger Sequencing Variants Novel (SNP, InDel) Resolution SNP to 100’s of bases Scope Limited Targeted Quantitative No, LOD ~20% VAF Specimen Blood, Fresh, Frozen, FFPE, cells Limitations Targeted / low throughput/ LOD / Decal 69

Next Generation Sequencing - NGS 70 71 72

73 74 75

76 77 78

79 80 81

82 83 84

85 86 87

88 89 90

91 92 93

94 95 96

97 98 99

100 101 102

103 104 105

106 107 108

109 110 111

112 113 114

115 116 117

118 119 120

121 122 123

124 125 126

127 Not meant for humans 128 129

130 131 132

133 134 CNV - Gain Control Patient 135

CNV - Loss Control Patient 136 CNV by NGS 137 Fusions by NGS EWSR1 (22q12.2) WT1 (11p13) 138

FISH Break Apart 22 22 139 Technique: NGS Sequencing Variants Novel (SNP, InDel, CNV, Fusions) Resolution SNP to 100’s of bases Scope Targeted to genome wide Quantitative Yes: SNP, CNV, InDel / LOD 1-5% VAF Specimen Blood, Fresh, Frozen, FFPE, cells Limitations CNV sensitivity / Decal / Data Intense 140 Pathologist’s Roles CLIA validation Molec Tissue assessment AP + Molec Test / Panel selection AP + Molec + onc Data analysis Molec Clinical report Molec 141