What about all these mutat ions?An Introduct ion to Molecular Biology of TB

Drug Resistance

Division of Tuberculosis EliminationNational Center for HIV/AIDS, Viral Hepatitis, STD & TB Prevention

Angela M. Starks, PhD Tracy L. Dalton, PhDChief, Laboratory Branch Deputy Chief, Laboratory Branch

Outline of Presentat ion Review the basic principles of molecular biology

Summarize the language used and major concepts for understanding molecular aspects of drug resistance

Discuss the molecular basis of drug resistance in Mycobacterium tuberculosis

Examine different types of mutat ions

Generated using wordle.net

What is Molecular Biology? Branch of science concerned with the study of

biological act ivity at the molecular level Focuses on formation, structure, and funct ion of nucleic

acids (i.e., DNA/RNA) and proteins Techniques used in molecular biology are unique and

different from those used for classical microbiology

https://simple.wikipedia.org/wiki/Molecular_biology#/media/File:Schematic_relationship_between_biochemistry,_genetics_and_molecular_biology.svg

Central Dogma of Molecular Biology

http://genius.com/Biology-genius-the-central-dogma-annotated

Double Helix: http://ghr.nlm.nih.gov/handbook/illustrations/dnastructure.jpg

Understanding the Basics of DNA Deoxyribonucleic acid (DNA)

Serves as the carrier of genetic information

Consists of two strands arranged in a double helix

Polynucleotide chains consisting of 4 bases

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In double-stranded DNA

A always bonds to T

C always bonds to G

Organizat ion of Genetic Material

DNA is packaged into individual chromosomes Human cells contain 23 chromosomal pairs Mycobacterium tuberculosis (Mtb) has a single circular chromosome

Genome is the total of all genet ic content of an organism Genome of Mtb ~4 million base pairs

Generally, a gene is a nucleic acid sequence that carries the information required for synthesis of a specific protein (i.e., encodes a protein) Genome of Mtb contains ~4,000 genes For some genes, end product is RNA and not a protein (e.g., tRNAs

or rRNA)

APHL Essentials of the Mycobacteriology Laboratory : Promoting Quality Practices, Molecular Biology 101 Module

AAACGCGTTAGC

Gene

Locus

AAATGCGTTAGC

A distinct sequence of nucleotides along a segment of DNA that provide the coded instructions for synthesizing a protein or RNA molecule

A region of interest in a gene

Codon A combination of three consecutive nucleotides within a gene that specifies a particular amino acid

Important Definit ions

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Central Dogma of Molecular Biology

http://genius.com/Biology-genius-the-central-dogma-annotated

http://cronodon.com/BioTech/Ribosomes.html

Ribonucleic Acids (RNA)

Polynucleotide formed by 4 bases Adenine, Cytosine, Guanine, Uracil

Single-stranded

Transcribed from DNA by an enzyme called RNA polymerase

RNA transcribed from a gene is messenger RNA (mRNA)

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Adapted from APHL Essentials of the Mycobacteriology Laboratory: Promoting Quality Practices, Molecular Biology 101 modulehttp://www.nature.com/scitable/topicpage/protein-structure-14122136

Amino Acids

Building blocks of protein DNA is read in sets of

three nucleotides, called codons within the open reading frame*

Each codon encodes one amino acid in the protein

*regions of sequences that code for proteins

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http://bio1152.nicerweb.com/Locked/media/ch17/central_dogma.html

Translat ion for Protein Synthesis

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www.vce.bioninja.com.au

Genetic Code

Codons and Reading Frame

64 codons for 20 amino acids (redundancy)Reading frame determined by start codon

(usually first AUG in mRNA)

http://www.nature.com/scitable/content/if-the-fourth-nucleotide-in-the-sequence-6927613

Generated using wordle.net

General Concepts for Understanding Resistance

Genotype Set of genetic determinants of an organism Understanding these genetic determinants through genotyping

(i.e., examining the DNA/molecular test) and not observation Mtb example is genotyping for identifying potential transmission

chains (i.e., MIRU and spoligotyping) but also examining DNA to identify mutations associated with resistance (e.g., DNA sequencing)

Phenotype Observed or expressed characteristics Depends on genotype but environment can influence Mtb example is growth-based (i.e., phenotypic) drug susceptibility

testing

Putt ing the Concepts Together

Phenotypic suscept ibility or resistance to a part icular ant ituberculosis drug is observed by drug suscept ibility test ing

Observed resistance is result of various factors that could include intrinsic resistance (e.g., waxy cell wall), changes at the genet ic level (i.e., mutat ions) or expression of efflux pumps

Molecular tests focus on detect ing mutat ions Mutat ions are ident ified by comparison with (or lack of

consistency with) what is the typical or wild-type sequence

Development of Drug Resistance

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Spontaneous mutations occur in the DNA of all cells Mutations can change the structure of a protein that is a drug

target Protein still functions, but is no longer inactivated by the drug and

Mtb can grow

Resistance is linked to large bacterial populations Mutants resistant to any drug naturally occur on average once in

every 108 cells Pulmonary TB — cavities often contain 107 – 109 organisms By using two antibiotics, chances for both targets to be mutated

and resistant to both drugs is extremely small (10-8 x 10-8 = 10-16) This is the rationale for treatment regimens with more than one

drug

Drug Resistance in Mtb

http://www.thelancet.com/journals/lancet/article/PIIS0140-6736(10)60410-2/fulltext?&elsca1=TL-210510&elsca2=email&elsca3=segment

Mutat ionsA mutation is a change in the DNA sequence resulting

in variation from previous generations that may be transmitted to subsequent generations

Range in size from a single base to a large segment of DNA

Possible causes of mutations Mutagens (UV-light) Selective pressure (antibiotics) Spontaneous, due to replication errors

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Adapted from APHL Essentials of the Mycobacteriology Laboratory : Promoting Quality Practices

Types of Mutat ions

Point mutat ion—a single base (i.e., A,T, C, or G) in the sequence is changed.

Delet ion—a single base or set of bases is deleted from the sequence

Insert ion—a new base or set of bases is inserted into the sequence

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APHL Essentials of the Mycobacteriology Laboratory : Promoting Quality Practices

Another important term: SNP

Single nucleotide polymorphism A variation in a single base pair in a DNA

sequence (i.e., point mutation) Multiple SNPs can occur in a locus Can be naturally occurring polymorphism

useful for strain differentiation (e.g., S95T gyrA) Could be mutational event

• Can result in phenotypic change but not always

Types of Mutat ions Effect of a mutat ion on resistance phenotype depends on the

locat ion and the type of mutat ion

Types of Mutat ions

Missense/non-synonymous mutat ion—a nucleotide change that results in a change in the amino acid sequence

Silent/synonymous mutat ion—a nucleotide change that does not affect the resulting amino acid sequence

Nonsense mutat ion—a mutation that results in a stop codon.

Frame Shift—a mutation that affects the reading frame of the gene (insertion or deletion not divisible by 3)

Point mutat ion in a promoter or regulatory region—nucleotide change only

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Reading Frame and Types of Mutat ions

• http://www.brooklyn.cuny.edu/bc/ahp/BioInfo/MUT/Mut.Types.html

What do mutat ions look like in a DNA sequence?

http://www.ucl.ac.uk/~ucbhjow/bmsi/bmsi_6.html

Synonymous mutat ion

Non-synonymous mutat ion

Influence of Mutat ions Could have no effect on drug resistance

Change in protein structure inhibit ing drug act ivity (e.g., no act ivat ion of prodrug)

Change in protein structure such that drug cannot bind to target

Change in promoter region leading to changes in expression level to overcome the effects of drug

Change in rRNA or modifying enzyme (change in affinity for drug)

Can result in different levels of resistance depending on the part icular mutat ion

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MYCOBACTERIUM TUBERCULOSIS

Mechanisms of drug resistance

Cellular Targets for First-line Drugs

http://www3.niaid.nih.gov/topics/tuberculosis/Understanding/WhatIsTB/ScientificIllustrations/firstLineIllustration.htm

Rifampin Resistance (1)Up to 98% of rifampin (RIF) resistant strains

contain mutations within the 81 base pair “hot spot” (RRDR) of a gene called rpoB

Figure 3. Single amino acid subst itut ions in the 81 bp core-region of the rpoB gene responsible for conferring rifampicin (RIF) resistance (Insert ions and delet ions that confer the RIF-resistance phenotype are not depicted). Amino acids are represented with single let ter abbreviat ions. Changes in codon Ser531 and His526 account for more than 70% of the mutat ions with RIF resistance (depicted in shaded ellipses).

http://www.cdc.gov/ncidod/EID/vol4no2/rattang.htm

Rifampin Resistance (2)

RIF targets the β subunit of the RNA polymerase

RIF binds and physically blocks the progress of the RNA polymerase (no mRNA = no protein = cell death)

Mutat ions in rpoB alter the structure of the β subunit so it no longer binds to RIF and retains funct ion

Isoniazid Resistance Isoniazid (INH) affects mycolic acid biosynthesis INH is a prodrug that must be activated by catalase peroxidase

encoded by katGMutations in resistant isolates commonly detected in katG

Prodrug not activated High level resistance

When activated INH targets InhA-NADH complexMutations commonly detected in promoter region of inhA

Mutations can also occur in inhAstructural gene

Results in overexpression of InhA Overcome effects of drug Low level resistance

Limitat ion and Considerat ions Not all mechanisms of resistance are known and the lack of a mutation ≠

susceptibility

Limited genes and sites are targeted

Emerging resistance (mixed populations) may not be detected; limit of detection

Not all mutations are associated with phenotypic resistance Silent (synonymous) mutations—no change in protein Neutral polymorphisms (e.g., gyrA codon 95 may be Ser or Thr ) Output is platform dependent

MUTATIONS AND EXAMPLES OF PLATFORM SPECIFIC OUTPUT

Mechanisms of drug resistance

Molecular-based Assays

APHL Essentials of the Mycobacteriology Laboratory : Promoting Quality Practices

MethodGeneXpert®

MTB/RIFHAIN Genotype®

MTBDRplusSanger

SequencingPyrosequencing

Company Cepheid HAIN Lifescience Laboratory developed test

Laboratory developed test

Genet ic loci rpoB rpoB, katG, and inhA

First and second-line drugs

Varies but can include rpoB, inhA, katG, aphC, gyrA,

and rrsFormat Semi-automated

real-time PCRLine probe assay DNA sequencing DNA sequencing

FDA approved Market authorization

No N/A N/A

Expected turn-around t ime from specimen receipt in laboratory

1-2 working days 1-2 working days (depends on how often performed

in lab)

1-2 working days(depends on how often performed

in lab)

1-2 working days (depends on how often performed

in lab)

GeneXpert instrument generated result

Interpretat ion of XpertMTB/RIF result

Recommended “minimum report ing language”

MTB detected, RIF resistance detected

MTB detected within sample, mutation in rpoB detected

MTBC detected. A mutation in rpoBhas been detected, indicating possible RIF resistance. Confirmatory test ing should follow

MTB detected, RIF resistance not detected

MTBdetected, but no mutation in rpoBdetected

MTBC detected. No rpoBmutation suggests probably RIF susceptible

MTB detected, RIF resistance indeterminate

MTB detected,unable to determine if there is an rpoBmutation

MTBC detected, presence of rpoBgene mutations cannot be accurately determined

MTB not detected MTB target is not detected within the sample

MTBC not detected

Recommended Report ing Language for GeneXpert® MTB/RIF

MMWR 62(41):821-4. October 18, 2013

CDC Molecular Detect ion of Drug Resistance (MDDR) Panel

Drug Loci Sensit ivityRifampin rpoB(81 bp region) 97%Isoniazid inhA, katG 86%Ethambutol embB 79%Pyrazinamide pncA 86%Fluoroquinolones gyrA 80%Amikacin rrs 91%Kanamycin rrs, eis 87%Capreomycin rrs, tlyA 55%

How are mutat ions reported?Mutations may be reported by describing the DNA

change and, when applicable, the amino acid change that occurs at the protein level

Changes in DNA sequence can be indicated by the base position in combination with the specific change that has occurred or by the wild-type codon followed by the mutated codon Substitution: 76A>C Substitution TCG>TTG Insertion: 76_77insT Deletion: 76_78del

If the mutation occurs before the start of the open reading frame, a negative number is used to indicate the position relative to the start codon C(-15)T

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How are mutat ions reported? (2)Changes in the protein sequence will be indicated

by the original (i.e., wild-type) amino acid followed by corresponding codon number and the resulting amino acid due to changes in nucleotide sequence His526Asp or H526D for a missense mutation Phe514Phe or F514F for a silent mutation

Report could include 3-letter abbreviation or single letter amino acid code

Multiple substitutions could occur within a single locus Ser315Thr, Ile335Val

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Example of MDDR Report

Example of MDDR Report

Acknowledgements• Division of Tuberculosis Elimination, Surveillance, Epidemiology, and

Outbreak Investigations Branch

• Division of Tuberculosis Elimination, Laboratory Branch

• Association of Public Health Laboratories

• State and Local Public Health Laboratories

For more information please contact Centers for Disease Control and Prevention

1600 Clifton Road NE, Atlanta, GA 30333Telephone, 1-800-CDC-INFO (232-4636)/TTY: 1-888-232-6348E-mail: cdcinfo@cdc.gov Web: www.cdc.gov

The findings and conclusions in this report are those of the authors and do not necessarily represent the official position of the Centers for Disease Control and Prevention.

Thank You

National Center for HIV/AIDS, Viral Hepatitis, STD & TB PreventionDivision of Tuberculosis Elimination