Lecture 11 Flashcards
Models of Disease Etiology
Genetic
Environmental
Multifactorial
Genetic- inherited- cystic fibrous
Environmental - somatic - change in DNA to cells that isnt germ or egg cell- skin cancer
Multifactorial - polygenic ± somatic -
higher risk of heart disease with certain genes
Pedigree
-Family history of phenotype is illustrated on a pedigree diagram
-reveal transmission patterns
autosomal dominate - men and women 50% for children to express
autosomal recessive -
Transmission Patterns
Gain-of-function
Loss-of-function
Dominant negative
-autosomal dominant, autosomal recessive and sex linked recessive -observed in single-gene disorders by one genetic mutation
-prediction of a pattern is called Mendelian inheritance
-observable traits
what is a Autosomal recessive transmission
-most observed
-mutant phenotype is not observed in the heterozygous (normal/mutant) state -must be homozygous (mutant/mutant) to show the abnormal phenotype
Gain-of-function -mutations usually display a dominant phenotype
Loss-of-function -mutations usually display a recessive phenotype
Dominant negative -observed with loss of function in multimeric proteins expressed on both chromosomes
Loss of Heterozygosity
- it is the Loss of the normal allele, revealing the mutant allele caused by somatic mutations or deletions (environmental and not inherited)
-abnormal phenotype a hemizygous (mutant/deletion) state caused by Autosomal recessive mutations
Hemochromatosis Type I
molecular detection of single gene
disorders:
-* Overabsorption of iron from food caused by mutations in the gene for a membrane iron transporter protein
-directs iron stores to overload them
-autosomal recessive
-HFE C282Y Detection -detected by polymerase chain reaction restriction fragment length polymorphism (PCR-RFLP)
Thrombophilic state
molecular detection of single gene
disorders:
- Caused by the Leiden mutation in the gene for coagulation factor 5 (F5) and mutations in the gene for coagulation factor 2 (F2)
-detection by PCR-RFLP,(SSP-PCR) - 2 primers
Few diseases have simple transmission patterns due to
Variable expressivity
Genetic heterogeneity
Incomplete penetrance
Variable expressivity—range of phenotypes from the same genetic mutation
Genetic heterogeneity—different mutations causing the same phenotype observed in diseases with multiple genetic components. Alzheimer’s
Incomplete penetrance—presence of mutation but no abnormal phenotype
Non-Mendelian Transmission Patterns
Gonadal mosaicism:
Genomic imprinting:
Nucleotide-repeat expansion:
Mitochondrial inheritance:
Single-gene disorders or disorders with multiple genetic components with non-classical patterns of transmission:
Gonadal mosaicism: somatic mutation in germline cells (gonads) egg or sperm only offspring has the phenotype and the 1st gen does not
Genomic imprinting: nucleotide or histone modifications that do not change the DNA sequence
Nucleotide-repeat expansion: increased allele sizes disrupt gene function.
Fragile X -(FMR1) - CGG mutations detected by PCR and Southern Blot . Seen on the telomere on the x chromosome . Repeats larger than 200 get methylated on the 5’ end and turns off the gene causing fragile x
- methylation creates the bands at the bottom on the gel
- Huntingtin Gene CAG Expansion Mutations by PCR - presents at 30-40. repeats predict the disease severity
Mitochondrial inheritance: maternal inheritance of mitochondrial genes
Human Disorders Due to Mitochondrial Mutations
- Kearns–Sayre syndrome (KSS) (mitochondrial deletion) detected by southern blot
- Pigmentary retinopathy, chronic progressive external ophthalmoplegia (CPEO)
- Leber hereditary optic neuropathy (LHON)
- Mitochondrial myopathy, encephalopathy, lactic acidosis, and stroke-like episodes (MELAS)
- Myoclonic epilepsy with ragged red fibers (MERFF)
- Deafness
- Neuropathy, ataxia, retinitis pigmentosa (NARP) detected by PCR RFLP
- Subacute necrotizing encephalomyelopathy with neurogenic muscle weakness, ataxia, retinitis pigmentosa (Leigh with NARP)
usually SNP
Mitochondrial Mutations
Homoplasmy
Heteroplasmy
Homoplasmy—all mitochondria in a cell are the same
Heteroplasmy—some mitochondria are normal, and others have mutations
* The severity of the disease phenotype depends on the amount of mutant and normal mitochondria present
* the earlier it occurs the more severe
NARP - PCR RFLP
mitochondrial DNA is digested by restriction enzymes. if the patient has a mutation creating a binding site for the RE creating two fragments. In affected Mito DNA will appear as one large product
KSS - southern blot
in normal patient when DNA is digested only one band of 16.6 is created if there is a mutation there is 5 base deletion - 2 bands seen
Next-Generation Sequencing & Genetic
Testing
Germline mutations
Targeted sequencing panels
Exome sequencing
Genome sequencing
Germline mutations—50% to 100% allele frequency. Sprem or egg - future generations not you
* Mutations discovered by NGS are confirmed by Sanger sequencing
Targeted sequencing panels cover multiple known disease-causing gene mutations
Exome sequencing covers 85% of disease-causing mutations - protein coating of DNA which is where most of the disease is found
* Target/exome enrichment is performed by probe hybridization
* Sensitivity may be lower than targeted gene panel
Genome sequencing covers coding and noncoding regions
Genetic Testing Limitations
- Therapeutic targets (except for gene therapy) are phenotypic
- Non-symptomatic diagnosis where disease phenotype is not (yet) expressed may raise ethical concerns
- Most disease and normal traits are multicomponent systems
Advantages of Molecular Genetic Testing
- Rapid definitive
- DNA analysis has predictive value for disease risk, diagnosis and therapeutic intervention
- Gene-variant databases for referral
- Gene discovery for inherited diseases has moved from research to clinical applications
Target Microorganisms for Molecular-based
Testing
Those that are difficult or time-consuming to isolate safer and shorter
* Mycobacteria
Hazardous organisms
* Histoplasma, Coccidioides
Those without reliable testing methods
* HIV, hepatitis C virus
High-volume tests- urine is less invasive and pcr is faster
* N. gonorrhoeae, C. trachomatis
Applications of Molecular-Based Testing
- Rapid or high-throughput identification of microorganisms
- Detection and analysis of resistance genes
- Genotyping
- Classification
- Discovery of new microorganisms
Specimen Collection and preparation
- Preserve viability/nucleic acid integrity of target microorganisms
- Avoid contamination
- Appropriate time and site of collection (blood, urine, other)
- Use proper equipment (coagulant, wood or plastic swab shafts)
-depending on the specimen type, the amount, the more rigorous lysis procedures are needed to penetrate the cell wall aka capsule
-Inactivate inhibitors (acidic polysaccharides in sputum or polymerase inhibitors in CSF) and RNases
PCR Detection of Microorganisms:
Quality Control
- Positive control: positive template known org
- Negative control: negative template known org
- Amplification control: omnipresent
Template unrelated to target.
* Reagent blank: no template present to ensure there is contamination
Internal controls would be:
Homologous extrinsic- Controls for amplification- added after extraction before amplication. amplification with primers does not account for NA degradation
Heterologous extrinsic-Controls for extraction and amplification. Non target derived controls added to the sample before extraction other primers need to be added.
Heterologous intrinsic-Human gene control. Normally present to ensure extraction and amplification control happened can use a multiplex pcr
Quality Control: False Positives : False Negatives
False Positives
* Contamination: check reagent blank
* Dead or dying organisms: retest 3 to 6 weeks after antimicrobial therapy. Do a culture will not grow on antibiotic plates
* Detection of less than clinically significant levels
*PCR detects everything - sensitive
False Negatives
* Improper collection, specimen handling
* Extraction/amplification failure: check internal controls
* Technical difficulties with chemistry or instrumentation: check method and calibrations
Viruses methods for detection
-AB , Ag detection- immune responses past or current infection or culture
-target, probe, signal amplificatoin
-tests to determine viral load (measured by qPCR, dPCR) and genotyping by sequence analysis
viral load is number of viruses per millimeter of fluid
Test Performance Features for Viral Load Measurement
Sensitivity
Accuracy
Precision
Specificity
Linearity
Flexibility
Sensitivity- Lowest level detected at least 95% of the time
Accuracy- Ability to determine true value
Precision- Reproducibility of independently determined test results
Specificity- Negative samples are always negative, and positive results are true positives
Linearity-A serial dilution of standard curve closely approximates a straight line
Flexibility- Accuracy of measurement of virus regardless of sequence variations
Antimicrobial agents are classified by:
- -static- inhibit growth/-cidal - kills
- Mode of action
- Chemical structure
sites of action of antimicrobial agents- metabolism, protein synthesis, cell wall integrity, membrane integrity, NA metabolism
Mechanisms for Development of Resistance
to Antimicrobial Agents
- Enzymatic inactivation of agent
- Altered target
- Altered transport of agent in or out
- Acquisition of genetic factors from other resistant organisms
Viral Genotyping
- Viral genes mutate to overcome antiviral agents
- Gene mutations are detected by sequencing
- Primary resistance mutations affect drug sensitivity but may slow viral growth. Original mutation to the first treatment increased virulence
- Secondary resistance mutations compensate for the primary resistance growth defects. fitness of the org increased. worse infection
Advantages of Molecular Detection of
Resistance to Antimicrobial Agents
- Mutated genes are strong evidence of resistance
- Rapid detection without culturing
- Direct comparison of multiple isolates in epidemiological investigations
Epidemiology
collection and analysis of environmental, microbiological, and clinical data
Molecular epidemiology genotypic analysis - plasmid DNA
Species-, strain-, or type-specific DNA sequences are the sources of genotype information
Criteria for PFGE Pattern Interpretation:
Rule of Three
Pulsed-Field Gel Electrophoresis (PFGE) to view coagulase negative staph outbreak - most common
-voltage in 3 directions instead of one
if the isolates is different than less than 3 bands they may be related = same outbreak
Arbitrarily Primed PCR: Random Amplification of Polymorphic DNA (RAPD)
compare test strains to outbreak strains
uses short 10 bp of random sequences to prime sequences the pattern will be another organism
so if two org show the same sequence then they are the same
Interspersed Repetitive Elements
-copies of conserved sequences
- location of these are based on species type
compare the pattern made by your test and known
