Introduction to Molecular Pathology

Shamelessly adapted from talks by Luis E. Ferrer Torres, MD, and Mark

Sobel, MD, PhD

Outline

• Nucleic Acid Structure and Biochemistry• DNA modifying enzymes• The Central Dogma- Transcription, Translation• Regulation of Gene Expression• Mutations and Polymorphisms• Methods:– Sequencing, Array CGH, Southerns, PCR, qPCR, ISH

Molecular Pathology

• Testing of nucleic acids within a clinical context

• Sub-fields within MP:– Virology (biggest in terms of market share)– Blood screening– CT/NG testing, molecular microbiology– Hereditary disorders/Oncology

Goal in molecular Pathology

• To determine the effect of a mutation on the quantity or function of a gene product, and explain why the change is or is not pathogenic for any particular cell, tissue or stage of development.

NUCLEIC ACIDS

• Genetic material of all known organisms• DNA: deoxyribonucleic acid• RNA: ribonucleic acid (e.g., some viruses)• Consist of chemically linked sequences of nucleotides

• Nitrogenous base• Pentose- 5-carbon sugar (ribose or deoxyribose)• Phosphate group

• The sequence of bases provides the genetic information

Bases

• Two types of bases• Purines are fused five- and six-membered rings

• Adenine A DNA RNA• Guanine G DNA RNA

• Pyrimidines are six-membered rings• Cytosine C DNA RNA• Thymine T DNA• Uracil U RNA

DNA: Helix

5’ 5’

3’

3’

In general, DNA is double-stranded. Double-stranded (ds) DNA takes the form of a right handed helix with approximately 10 base pairs per turn of the helix.

DNA StructureA purine always links with a

pyrimidine base to maintain the structure of DNA.

Adenine ( A ) binds to Thymine ( T ), with two hydrogen bonds between them.

Guanine ( G ) binds to Cytosine ( C ), with three hydrogen bonds between them.

Relevance: High [GC] = High Tm

Hydrogen Bonds

H

H

H H

O

O

H

C

C

C C

N

N

C

ThymineH

N

H

H

N

C C

C

C

N

N H

N

C

Adenine

H

O

N

H C

C C

N

N

C

Cytosine

H

H

H

N

C C

C

C

N

N H

N

C

Guanine

NH

O

H

DNA Antiparallel Structure

• Example

• First strand 5’ – GGGTTTAAACCC – 3’• Second strand 3’- CCCAAATTTGGG – 5’

DNA Replication• DNA replication is semi-

conservative. That means that when it makes a copy, one half of the old strand is always kept in the new strand. This helps reduce the number of copy errors.

DNA Modifying enzymes

• Polymerases (lengthen)

• Exonucleases (end-cutters)

• Endonucleases (internal cutters)

• Ligases (linkers)

Polynucleotide kinases and phosphatases (end modifiers)

Nucleases

Endonuclease

5’ Exonuclease 3’ Exonuclease

Restriction enzymes

• Specific endonucleases• Recognize specific short sequences of DNA and

cleave the DNA at or near the recognition sequence

• Recognition sequences: usually 4 or 6 bases but there are some that are 5, 8, or longer

• Recognition sequences are palindromes• Palindrome: sequence of DNA that is the same

when one strand is read from left to right or the other strand is read from right to left– consists of adjacent inverted repeats

Restriction enzymes (cont’d)

• Example of a palindrome:GAATTCCTTAAG

• Restriction enzymes are isolated from bacteria• Derive names from the bacteria• Genus- first letter capitalized• Species- second and third letters (small case)• Additional letters from “strains”• Roman numeral designates different enzymes from the

same bacterial strain, in numerical order of discovery• Example: EcoRI

– E Escherichia– Co coli– R R strain– I first enzyme discovered from Escherichia

coli R

Central Dogma of Molecular Biology

Schema of Gene Transcription

DNA to RNA creates functional translations

• DNA remains in the nucleus, but in order for it to get its instructions translated into proteins, it must send its message to the ribosome's, where proteins are made. The chemical used to carry this message is Messenger RNA

RNA = Ribonucleic acid.

• RNA is similar to DNA except:

It has one strand instead of two strands. Has uracil instead of thymine3.Has Ribose instead of Deoxyribose

Translation

• m-RNA directs protein synthesis.• Occurs in ribosomes (rRNA+proteins)• Codons (three bases) are read by transfer

(tRNA)• There are 64 possible codons, therefore most

of the 21 aminoacids are specified by more than 1 codon

Codon Usage Table

What is a Gene?• The gene, the basic

units of inheritance; it is a segment within a very long strand of DNA with specific instruction for the production of one specific protein. Genes located on chromosome on it's place or locus.

Regulation of Gene Expression

• DNA level expression control– Transcriptional– Post-Transcriptional

• Epigenetics– DNA methylation– Histone modification

Regulation of Gene Expression

• DNA level expression control– Transcriptional• House keeping genes

– Always on

• Transcription factors– Usually lie upstream in the promoter region– Enhancer and silencer elements

Regulation of Gene Expression

– Post transcriptional• Export of mRNA out of nucleus• Alternative splicing• mRNA stabilization• mRNA degradation• RNA interference or silencing

– miRNA and siRNA

11 ways to kill gene expressionDelete:(i) the entire gene Most α-thalassemia mutations (Figure 16.2)(ii) part of the gene 60% of Duchenne muscular dystrophy (

Figure 16.3)Insert a sequence into the gene Insertion of LINE-1 repetitive sequence (see 

Section 9.5.6) into F8C gene in hemophilia ADisrupt the gene structure:(i) by a translocation X-autosome translocations in women with

Duchenne muscular dystrophy (Figure 15.9)(ii) by an inversion Inversion in F8C gene (Figure 9.20)Prevent the promoter working:(i) by mutation β-Globin -29A → G mutation (Table 17.2)(ii) by methylation Fragile-X full mutation (FMR1) (Box 16.8)Destabilize the mRNA:(i) by a polyadenylation site mutation α-globin AATAAA → AATAGA mutation(ii) by nonsense-mediated RNA decay Fibrillin mutations (FBN1)Prevent correct splicing (i) by inactivating donor splice site PAX3 451 + 1G → T mutation (Figure 16.1)(ii) by inactivating acceptor splice site PAX3 452-2A → G mutation (Figure 16.1)(iii) by activating a cryptic splice site β-Globin intron 1 -110G → A mutation (

Figure 9.13)Introduce a frameshift in translation PAX3 874–875insG mutation (Figure 16.1)Convert a codon into a stop codon PAX3 Q254X mutation (Figure 16.1)Replace an essential aminoacid PAX3 R271C mutation (Figure 16.1)Prevent post-transcriptional processing(Section 16.6.1).

Cleavage-resistant collagen N-terminal propeptide in Ehlers Danlos VII syndrome

Prevent correct cellular localization of product

F508del mutation in cystic fibrosis

Modes of inheritance

• Mutations of single genes– Patterns: autosomal dominant, autosomal

recessive, X-linked, mitochondrial• Anticipation– Increased severity of a certain diseases in

successive familiar generations associated with triple repeats

• Mosaicism– At least two cell lines derived from a single zygote

Mutations and Polymorphisms

• Mutation: change in DNA sequence• Polymorphism: non disease causing change in

DNA or a change found at a frequency of ≥ 1% in population

• When evaluating changes in DNA sequence use neutral terms: sequence variant, sequence alteration or allelic variant. There may be:– Missense, nonsense, deletions, insertions, frame

shifts, duplications, amplifications, trinucleatide repeats.

Single Nucleotide Polymorhisms and Haplotypes

• SNPs are single base differences in the DNA of individuals

• There are ~10 million SNPs in the human genome• IMPORTANCE: Pharmacogenetics– Ex. CYP (cP450)

• Alleles of SNPs that are close together tend to be inherited together.

• Haplotype: a set of associated SNPs alleles in a region of a chromosome

Introduction to Molecular Pathology, Part Deux

Shamelessly adapted from talks by Luis E. Ferrer Torres, MD, and Mark

Sobel, MD, PhD

Outline, Part II

• Methods:– DNA Sequencing (Sanger, Pyro, Next-Gen)– Southern Blotting– Array Comparative Genomic Hybridization– PCR (RT-PCR, Real time PCR)– In Situ Hybridization

Clinical Source Materials for Molecular Pathology

• Peripheral Blood: 1-4 ml in purple top (EDTA) tube preferred, though heparin (green top), Acid Citrate Dextrose (ACD, yellow top), and Sodium Citrate (blue top) are ok

• FFPE: 10 unstained 2 um thick sections

•

Nucleic acid isolation (DNA or RNA)

• Manual vs. automated (Maxwell 16)• Cell lysis

– Dependent of specimen type, nucleic acid being isolated for, desired purity and application to be used in

– FFPE yields ~200 pairs• Purification

– Organic: phenol-chloroform– Non organic: silica, anion exchange chromatography and

magnetic particles• DNA or RNA Isolation

– RNA rapidly degrades…

Methods

• DNA sequencing• Southern Blot• PCR

– RT-PCR– Real Time PCR

• Methylation-Specific PCR

• In-situ PCR• Protein Truncation Test

• Transcription-Mediated Amplification

• Strand Displacement Amplification

• Nucleic Acid Sequence-Based Amplification

• Signal amplification– Branching DNA– Hybrid Capture– Invader– FISH– DNA arrays and chips

DNA sequencing• Determining the exact sequence of the four

bases in a given DNA template• Maxam-Gilbert (1977)- Chemical degradation, uses g-

32P ATP, toxic chemicals

• Sanger: (1977) Chain termination, using dideoxynucleotides Radiolabeled, Dye-prime or Dye-terminator (cycle sequencing)

Fluorescence based Chain Termination Sequencing

• Start with a single stranded template. Add a mixture of all four dNTPs, much lower concentrations of ddNTPs (each with a different fluorescent tag), and DNA polymerase. Chain elongation proceeds until (by random chance) a ddNTP is added. New chains are separated by size using gel electrophoresis. As each labeled DNA fragment passes a detector at the bottom of the gel, the color is recorded and the sequence reconstructed from the pattern of colors.

Gel Electrophoresis vs. High Performance Capillary Electrophoresis

Limitations of Dye Terminator Based sequencing

• Lousy base calls in first 25 bases of sequence• Limited to 700-800 bases per sample (can’t

differentiate 1kb DNA’s that only differ by 1 nt in length….)

Pyrosequencing

• Principle: Start with immobilized ssDNA template, add dNTP’s one at a time.

• Addition of the ‘right’ base releases PPi, regenerated to ATP that activates luciferase.

• Good for 400-500 bp• Can be parallelized into

array-based sequencing, 400 MB of sequence in 10 hours for $5K

Next-Gen or High Throughput Sequencing

Utilize variations on dye terminators:• Illumina/Solexa: Reversible dye terminators• Ion Semiconductor: Detects H+ release

Applications of Direct DNA sequences

Clinical condition Gene

HIV drug resistance HIV-protease, RT

Cystic fibrosis CFTR gene

Beta thalassemia Beta globin

Cancer predisposition

• breast BRCA1

• Hereditary non polyposis colon cancer

TP53

• MEN PTEN Ret proto-oncogene

Congenital hearing loss Connexin 26

HCV genotyping 5’UTR

Methods

• DNA sequencing• Southern Blot• PCR

– RT-PCR– Real Time PCR

• Methylation-Specific PCR

• In-situ PCR• Protein Truncation Test

• Transcription-Mediated Amplification

• Strand Displacement Amplification

• Nucleic Acid Sequence-Based Amplification

• Signal amplification– Branching DNA– Hybrid Capture– Invader– FISH– DNA arrays and chips

Hybridization

• Nucleic acid hybridization is the formation of a duplex between two complementary sequences

• Intermolecular hybridization: between two polynucleotide chains which have complementary bases– DNA-DNA– DNA-RNA– RNA-RNA

• Annealing is another term used to describe the hybridization of two complementary molecules

Probes

• Probe is a nucleic acid that– can be labeled with a marker which allows

identification and quantitation– will hybridize to another nucleic acid on the basis of

base complementarity

• Types of labels– Radioactive (32P, 35S, 14C, 3H)– Fluorescent

• FISH: fluorescent in situ hybridization– chromosomes

– Biotinylated (avidin-streptavidin)

Southern Blot

• Edwin M Southern, 1974• DNA extracted• DNA cut into pieces (Restriction

Endonucleases)• Electrophoresis and size separated• Blot (transferred) to a membrane• Anealed with labeled (radioactive,

fluorescence, chemiluminescent) probe

Southern Blot working protocol

Methods

• DNA sequencing• Southern Blot• PCR

– RT-PCR– Real Time PCR

• Methylation-Specific PCR

• In-situ PCR• Protein Truncation Test

• Transcription-Mediated Amplification

• Strand Displacement Amplification

• Nucleic Acid Sequence-Based Amplification

• Signal amplification– Branching DNA– Hybrid Capture– Invader– FISH– DNA arrays and chips

Array-based Comparative Genomic Hybridization

• CGH: Detection of genomic copy number changes – Limited to detection of unbalanced chromosomal

changes, can’t ‘see’ balanced translocations or inversions

• Oligonucleotide-CGH– Can detect Single Nucleotide Pleomorphisms (SNPs) – Used for IUFD/POC testing at Genzyme (Reveal SNP

array)

Array CGH Technique

• DNA isolated from sample and a WT control, labelled red or green

• Hybridized to array of 1000’s of immobilized probes

• Differential red or green signal indicates loss of other DNA at that locus.

• Current spatial resolution on oligonucleotide array CGH: 20-50 nucleotides

• Limitations: quantitation as array complexity increases

• Cellular heterogeneity of sample

PCR

• Kary B. Mullis 1983• Target amplification– Single oligonucletide– Multiplexed

• Mimics the natural process of DNA replication, therefore, requires:– DNA template, DNA polymerase, dNTPs, buffer, Mg++, two

primers to flag the target sequence– Thermal cycler

– Denaturation ~95°C– Annealing ~45-60°C– Extension ~72°C

PCR

• Denaturation• Anealing– Binding to oligonucleotide sequence (probe)

• Extension– DNA polymerase (heat stable, Taq [Thermophilus

aquaticus]) replicates the selected DNA sequence

PCR• Within each cycle:– Denature: (break H-

bonds between ds-DNA base pairs, at 95oC)

– Anneal (primer binding to target sequence, 50-60oC

– Extension (Thermostable DNA polymerase from Thermophilus aquaticus replicates the selected DNA sequence, 72oC

RT-PCR

• To detect or quantify RNA transcripts or viral RNA

• RNA is converted to DNA• Reverse transcriptase (Avian Myeloblastosis

Virus and Moloney Murine Leukemia virus)• Isothermal reaction with primers: oligo dT,

random hexamer primers, or target specific primers

• One step vs. two steps

Real Time - PCR

• Amplifies and detects PCR product fluorescently in each well of PCR plate– Don’t have to run gel afterwards– Use for endpoint detection

• Examples – Fast PCR screening without gels • Locate clone or mutant of interest

– Genotyping SNPs• Genotype individuals using allele specific primers

Real Time - PCR• Regular PCR, but

measure amount of dsDNA at end of each cycle of PCR

• The cycle threshold (Ct) is the amplification cycle number at which fluorescence is detectable

• Ct is inversely proportional to the amount of starting template in the sample

PCR

Advantages– Sensitivity– Specificity– Speed– Versatility– Automated– No need for intact DNA/RNA

Disadvantages• Target sequence needs to be

known• Target needs to be conserved

among individuals (polymorphisms)

• Oligonucleotide length• Can fail in the detection of

chromosomal abnormalities like translocations, inversions, large addition or deletions

• Contamination (F+)

Methods

• DNA sequencing• Southern Blot• PCR

– RT-PCR– Real Time PCR

• Methylation-Specific PCR

• In-situ PCR• Protein Truncation Test• In Situ Hybridization

• Transcription-Mediated Amplification

• Strand Displacement Amplification

• Nucleic Acid Sequence-Based Amplification

• Signal amplification– Branching DNA– Hybrid Capture– Invader– FISH– DNA arrays and chips

In Situ Hybridization

• Probe types:– Centromeric or CEP (chromosome enumeration probe)– Whole chromosome probes or paints [metaphase only]– Locus specific probe or identifier (LSI)

• Section pretreatment.• The labeled probe is first denatured (by heating or under

alkaline conditions) into single DNA strands • Hybridized to the target DNA (~Southern blotting) or RNA

(~northern blotting) immobilized on a membrane (blotting) or in situ.

• Metaphase and Interphase cells

In Situ Hybridization• More in use Chromosomal translocations• Useful in CMV, HSV, VZV• Sub types of papilloma virus• Useful in Mycobacteria, fungi and parasites• Helicobacter pylori from gastric biopsies• Legionella pneumophila • Pneumocystis jiroveci• Tests done on paraffin embedded specimen• Need applications in Infectious diseases• Doctortvrao’s ‘e’ learning series

ISH - Examples

• Genotyping of Neoplasms– Polysomy and other gains• Trisomy 12 in B-CLL

– Losses• del 1p / del 19q in oligodendroglioma

– Amplification• HER2/neu

– Translocations• t(9,22)(q34;11) BCR/ABL in CML

1p / 19q - Oligodendroglioma

ISH – Solid Tumors

HER2/CEP17 Identifies the subset of breast carcinoma patients eligible for Herceptin™ (trastuzumab) therapy.

MDM-2/SE12 Well-differentiated liposarcoma, dedifferentiated liposarcoma, atypical lipomatous tumor, and pleomorphic lipoma

SS18 (SYT) translocations (Breakapart) Synovial Sarcoma

TOP2A / CEP17 A predictive biomarker in a subset of breast carcinomas. TOP2A gene amplification may predict response to anthracycline-containing breast chemotherapy.

ISH - Lymphomas

MALT1 (18q21) translocations (Breakapart)

Translocations involving the MALT1 gene have been detected in approximately 20-30% of patients with extranodal low grade marginal zone B-cell lymphomas of MALT type (i.e., MALT lymphomas). Patients with t(11;18)(q21;q21)-positive gastric MALT lymphomas do not respond to Helicobacter pylori eradication therapy, are associated with more advance stage disease, and usually do not show transformation to large cell lymphoma.

t(14;18) IGH/MALT1 Subset of MALT lymphomas (Marginal zone B cell lymphoma)

t(11;18), MALT1/API2 Subset of MALT lymphomas (Marginal zone B cell lymphoma)

ISH - Lymphomas

MYC (8q24) translocations (Breakapart) Burkitt lymphoma; MYC translocations (MYC/IGH, MYC/kappa, MYC/lambda); t(8;14), t(2;8), t(8;22)

t(11;14) CCND1/IGH Identifies mantle cell lymphoma and subset of plasma cell neoplasms. Patients with multiple myeloma that have a t(11;14)(q13;q32) have been reported to have a neutral to slightly improved clinical course. FISH-based assays provide the most sensitive and specific methodology for detecting the t(11;14)(q13;q32).