what about all these mutations? an introduction to ... · what about all these mutations? an...
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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
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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
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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
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Central Dogma of Molecular Biology
http://genius.com/Biology-genius-the-central-dogma-annotated
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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
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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)
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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
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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
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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
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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
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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
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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
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Drug Resistance in Mtb
http://www.thelancet.com/journals/lancet/article/PIIS0140-6736(10)60410-2/fulltext?&elsca1=TL-210510&elsca2=email&elsca3=segment
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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
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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
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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
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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
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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
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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
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Cellular Targets for First-line Drugs
http://www3.niaid.nih.gov/topics/tuberculosis/Understanding/WhatIsTB/ScientificIllustrations/firstLineIllustration.htm
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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
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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
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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
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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
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MUTATIONS AND EXAMPLES OF PLATFORM SPECIFIC OUTPUT
Mechanisms of drug resistance
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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)
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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
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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%
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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
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Example of MDDR Report
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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
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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: [email protected] 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