Genetic Testing Technologies Flashcards
Uses of Genetic Tests
- diagnostic/confirmatory
- predictive
- presymptomatic
- prognostic
- carrier risk assessment
- newborn screening
- prenatal diagnosis
- preimplantation diagnosis
- pharmacogenetics
Diagnostic/Confirmatory Test
testing designed to confirm or exclude a known or suspected genetic disorder in a symptomatic individual or prenatally in a fetus at risk for a certain genetic condition
Predictive Test
testing offered to asymptomatic individuals with a family history of a genetic disorder and a potential risk of eventually developing disorder
Presymptomatic Test
testing of asymptomatic individual in whom discovery of a gene mutation indicates certain development of findings related to specific diagnosis at some future point; negative result excludes diagnosis
Carrier Risk Assessment
testing used to identify usually asymptomatic individuals who have a gene mutation for an AR or XL disorder
Newborn Screening
testing done within days of birth to identify infants at increased risk for specific genetic disorder so treatment can begin asap; when result is positive, further diagnostic testing usually required to confirm/specify results and counseling offered to educate parents
Prenatal Diagnosis
testing performed during pregnancy to determine whether fetus is affected with particular disorder
Preimplantation Diagnosis
procedure used to decrease chance of particular genetic condition for which fetus specifically at risk by testing one cell removed from early embryos conceived by IVF and transferring to mother’s uterus only those embryos determined not to have inherited mutation in question
Pharmacogenetic Test
testing used to identify potential interactions of individual’s genetic makeup with drug or class of drugs that predict drug effects
Prognostic Test
testing used to predict probable outcome or clinical course of a disease, or chance of recovery
Test Parameters
- accuracy
- precision
- sensitivity
- specificity
- predictive value
- validity
Accuracy
how close the measurement is to the true value
Precision
how close together are each of the measurements
Sensitivity
TP/(TP +FN)
can the test detect the analyte or the disease
Specificity
TN/(TN+FP)
can test detect only analyte of interest or discriminate between who does/doesn’t have disease
Positive Predictive Value
TP/(TP+FP)
if a person tests positive, how likely are they to have disease
Negative Predictive Value
TN/(TN+FN)
if a person tests negative, how likely are they to not have the disease
Validity
ability of test to accurately measure analyte/genotype or predict disease phenotype
Clinical Utility
ability of test to effect positive outcomes
Reasons for False Positives
- lack of perfect analytic specificity
- test is cross-reacting
- genotype result is correct but phenotype not visible
- disease clinically misclassified
Reasons for False Negatives
- insufficient analytic sensitivity
- untested genes responsible for disease
- untested mutations responsible for disease
- disease was misclassified
- genotype not present in cell population tested
- ethnicity-dependent variations in frequency of genotype
Ideal Genetic Test
high sensitivity, high specificity, low false positives, low false negatives, high PPV, high NPV
Possible Technical Issues
- not all genes contributing to pathogenesis are known
- testing may fail to identify mutation
- misinterpretation of results by patients and healthcare providers
- significance of results unclear
- human error/sample mix-up
- wrong diagnosis, testing wrong person, mistaken paternity
Ethical Issues in Testing
- genetic information is familial information; duty to warn?
- non-paternity and disclosure
- testing for fetus or child for adult-onset disorder
- genetic discrimination
Legislative Initiatives for Testing Protection
- Americans with Disabilities Act
- Health Insurance Portability and Accountability Act
- Secretary’s Advisory Committee on Genetics, Health, and Society
- GINA
Screening Test
any testing procedure designed to separate people according to a fixed characteristic or property with intention of detecting early evidence of disease; high v low risk
Diagnostic Test
any kind of medical test performed to aid in diagnosis or detection of disease
Clinical Labs
- must be CLIA certified
- usually provide re-testing for mutations identified in non-CLIA lab
- preferable testing setting
Research Labs
- do not need CLIA certification
- may take long time to obtain results/may never obtain results
- testing usually free
Cytogenetic Technologies
- karyotype
- FISH
- comparative genomic hybridization
Biochemical Technologies
- tandem mass spectrometry
- enzymatic/metabolite assay
Molecular Technologies
- microarray
- NIPS
- NGS
- WES
- WGS
“Classic” Technologies
- blotting
- allele specific oligonucleotide analysis (ASO)
- PCR
- mutiplex ligand-dependent probe amplification (MLPA)
- sequencing
Karyotype
- looks at whole chromosomes
- uses phytohemagluttinin (PHA) to stimulate cell division and colchicine to arrest cells in metaphase
- benefits: can use multiple specimen types, detects balanced translocations, accounts for MCC, can create back up culture for POC
- limitations: long culture time and TAT 10-14 days, limited resolution (5Mb), need live cells, normal result does not rule out single gene disorders or small/subtle gains/losses/rearrangements
- used for aneuploidy, polyploidy, balanced translocations, leukemia
FISH
- probes for known regions on chromosomes
- benefits: fast TAT 24-48hrs, identification of leukemia rearrangements
- limitations: resolution only as good as probe, need to know what specifically looking for
- used for deletion/duplication syndromes, CHARGE, imprinting conditions
Types of FISH Probes
- centromere
- telomere
- whole chromosome
- locus
- break apart
- dual color-dual fusion
Comparative Genomic Hybridization
- modification of FISH in which relative hybridization intensity in a genomic DNA sample is compared to relative intensity of a reference genome to determine copy number of evaluated sequences
- benefits: detects CNVs as small as several hundred bases
- limitations: cannot detected balanced structural rearrangements, VUSs
Microarray
- analyzes multiple regions across genome in parallel
- resolution of 1kb - 5Mb
- detects CNVs (microdels/dups ~400kb, majority benign)
- 15-20% detection rate v 4% with karyotype
- first-line test for multiple anomalies not specific to well-delineated genetic syndrome, apparently non-syndromic DD/ID, ASD
- may be done prenatally when u/s anomalies present, multiple fetal losses, POCs (especially if there’s no cell growth)
- when positive, follow up includes cytogenetic/FISH studies, parental evaluations, and clinical genetic eval + counseling
- may be used in leukemias/lymphomas to direct treatment
Array CGH
- uses targeted probes or probes spread across genome and measures relative intensity of patient and control DNA that binds to probes
- limitations: cannot detect balanced rearrangements or copy neutral gains/losses, does not detect low-level mosaicism, may detect CNVs of unknown significance, no information on structural nature
SNP Array
- patient DNA hybridizes to SNP based array containing SNPs present throughout genome
- benefits: can detect copy neutral gains (triploidy), can detect UPD and LOH (>10% IBD with multiple ROHs 2-5Mb+ suggest 1st or 2nd degree parental relationship), can detect polyploidy, higher resolution than FISH, higher detection than karyotype
- limitations: cannot detect balanced translocations, TAT, may not be able to establish viable culture for POC testing and MCC possible, may not detect low-level mosaicism, VUSs, no information on structural nature
NIPS
- detects fetal anueploidy using cell free DNA in maternal blood from cytrophoblastic cells in placenta
- fetal cfDNA reliably detected after 9+ weeks GA and undetectable within hours postpartum
- MPS approach relies on relative proportion of sequence reads compared to expected proportion
- SNP approach allows deduction of fetal genotype and can detect triploidy
- any patient may opt for cfDNA for aneuploidy screening but biochemical screening most appropriate for low-risk population (8/2020 - cfDNA acceptable for all patients regardless of risk category)
- all positive results should be followed up by invasive testing
NGS
- library of DNA fragments generated, bridge amplification of fragments, and fluorescently labeled nucleotides added and monitored to detect sequencing
- reads aligned to reference sequence to identify differences
- may be used for tumor sequencing
WES
- NGS technology
- coverage of 80x - 200x
- exome comprises approximately 3 million bases (1-2% of genome)
- detection rate of 25-51%
- benefits: identifies atypical presentations of well-described syndromes, best approach for non-specific symptoms, novel gene discovery
- limitations: not all exons targeted, non-coding regions of genome not covered, some regions of exome not covered well (trinucleotide repeats, large CNVs, chromosomal rearrangements, some indels, methylation abnormalities, mitochondrial DNA mutations)
- risks: undisclosed paternity, incidental findings, secondary findings, many VUSs, results my have implications for other family members, identifying novel suspected pathogenic gene with little/no clinical data, future insurance
- most informative when completed as a trio
WGS
- NGS technology
- coverage 20x - 50x
- genome approximately 3,000 million bases (3 billion)
Southern Blot
- restriction enzymes digest DNA, DNA run on gel, pattern transferred to membrane and exposed to x-ray film
- benefits: detects mutations that have significant effect on size of DNA fragment (trinucleotide expansions), may be possible to detect heterozygotes
- limitations: will not see point mutations or small deletions, requires a lot of DNA, time consuming, labor intensive
Northern Blot
- same as Southern blot but looks at RNA to determine gene expression
Allele Specific Oligonucleotide Analysis (ASO)
- one probe matches normal DNA sequence and one matches mutation sequence, hybridized to patient’s DNA
- used for certain genetic diseases where small number of mutations affecting one or small number of bases cause most cases of condition (ex: delF508 in CF)
- normal result does not rule out possibility of mutation elsewhere in gene; just rules out presence of mutations tested
PCR
- denaturation, annealing of primers, and elongation of DNA to generate unlimited amounts of DNA sequence of interest
- multiplex PCR uses multiple different primers in single reaction to determine if specific stretches of DNA present/absent (specific deletions of DMD/BMD, specific mutations in CF)
- benefits: widely used, useful even when small amounts of DNA present, single nucleotide variant detection, low cost
- limitations: limited targets available
Multiplex Ligand-Dependent Probe Amplification (MLPA)
- amplifies many exons at once and amount of amplification is proportional to amount of DNA in original sample
- two probes prepared that hybridize to either end of region of interest, probes ligated together and amplified
- benefits: good for detecting gene dosage, allows analysis of most of gene in one reactions
- limitations: relatively complex
- used for DMD/BMD
Sanger Sequencing
- uses chain-terminating dideoxynucleotides
- used to identify variations in gene sequence like point mutations and small indels
- generally misses full gene deletions or full gene duplications
Capillary Sequencing
- uses thousands of capillary tubes
- benefits: more accurate than NGS, can detect point mutations
- limitations: cannot detect copy number losses, not as much coverage, time, cost
- used for confirmatory sequencing, single gene sequencing
ACMG 59
- list of 59 genes for which specific mutations are known to be causative of disorders with defined phenotypes that are clinically actionable
- variants detected in any of these genes by WES/WGS should be reported (unless patients opt-out)
