molecular exam 3 Flashcards
alternative form of a genetic locus
allele
any heritable change in DNA sequence
mutation
genetic variants occurring in >1% of a population
polymorphism
insertion, deletion, duplication, repeating patters of DNA that vary in number
copy number variation
the most common polymorphism
SNP
coding region plus 10kb upstream is
a gene
a set of associated SNP alleles in a region of a chromosome
haplotype
factors effecting gene penetrance
- importance of the function of the protein encoded by the gene
- functional important of the mutation
- interaction with other genes
- onset of somatic mutations
- interaction with the environment
- existence of alternative pathways to substitute for LOF
diseases with high penetrance
SCD, thalassemias, huntingtons, CF, color blindness
most diseases are multifactorial and of low penetrance
true
type 1 and 2 diabetes
RA, chrohn’s, CHD, and asthma are
multifactorial (Low Penetrance)
studies that find correlations of genes with disease by comparing frequency of gene variation with frequency of disease
case control study
a population based study where diseased and non-diseased individuals are unrelated
GWAS
the tendency of genes or other DNA seqences at specific loci to be inherited together as a consequence of their physical proximity to one another on a single chromosome
Linkage disequilibrium
gives strength of association
relative risk
direct causation
epistatic effect
population stratification
linkage disequilibrium
causes of associations
linkage disequilibrium focuses on
two alleles/SNPs
occurs when a set of alleles on one copy of a chromosome stay associated with each other at a higher frequency than would be expected if recombination were completely random
linkage disequilibrium
SNPs are the marker of choice for
Linkage studies, bc they are abundant, have low rate of mutation, and are easy to genotype on a larger scale
association throughout the whole genome of SNPs with diseases or phenotypes
GWAS
provide individuals with info about their risk of developing disease or trait and/or their odds of responding in a particular way to a drug
direct to consumer testing
multiple hypothesis testing
p=0.5 there is a 5% chance for (hypothesis) to occur randomly and a 95% chance the association is real
the failure to detect an allele in a sample or failure to amplify the allele
allele drop out
repeated telomeric breakage and instability of fusion of sister chromatids, as a result they are broken apart during anaphase
breakage fusion bridge cycles
large scale screening of a population for a diseases to ID ppl who probably do and probably do not have a disease
not diagnostic
population screening
population screening for a gene that can cause disease in carrier of offspring
genetic screening
goals of screening
early detection
prevent or reverse disease process
allow informed reproductive decision
principles of screening for a disease
- disease should be serious and common
- disease should be well understood
- there should be effective treatment available
- test should be easy and cheap
- test should be valid/reliable
- sources of dx and treatment should be accessible
the ability of a test to correctly identify those with the disease
sensitivity
the ability of a test to correctly identify those without the disease
specificity
guidelines for heterozygote screening
screen should be voluntary and confidential
informed consent must be given
education and counseling must be available
quality control of test
equal access
genetic testing performed to ID people w/ a disease causing gene before symptoms develop
aid in repro decision
improve health
currently universal pre-symptomatic screening is impractical
limits of genetic testing
not 100% accurate
reveals mutations not disease
may not detect all disease causing mutations
can lead to complex ethical considerations
provides reassurance when normal, provide risk info in pregnancy planning, allows psychological preparation if baby affected, prepares health care team, allows for informed decision making about termination
benefits of prenatal diagnosing
maternal age >35
previous child w/ aneuploidy
family history of genetic defects
increased NT defects
all indicate prenatal screening by amniocentis
preformed 10-11 weeks post LMP
risk of fetal loss approx 1%
mosaicism can confuse Dx
some evidence of increased limb deficiencies
chorionic villus sampling
applications of cordocentisis
for studies when ultrasound shows structural abnormality and rapid Dx is needed
if hematological issues arise
helps make distinction between true and false mosaicism
lower sensitivity than using amniotic methods
non-invasive
helps detect NTDs and trisomies
maternal serum screening
1 in 10^3 – 10^7 nucleated cells in maternal blood are
fetal, proportion decreases with increasing maternal BMI
fetal cell analysis
FISH, chromosome specific DNA probes
uses cell sorting
less invasive
can be done as early as 7 wks
accurate detection of increased gene dosage due to trisomies can be done with
digital PCR (relative chromosome dosage)
curves that delimit decision boundaries in accepting or denying child is aneuploid
sequential probability ratio test
analysis of fetal-specific differentially methylated regions using methylated DNA immunoprecipitation
MeDiP, also uses qPCR to assess fetal chromosome dosing assessment
higher sensitivity compared to RNA-SNP method
cffDNA in health adults comes from
hematopoietic cells undergoing apoptosis
increased cffDNA can indicate several pregnancy-related disorders
non-invasive prenatal testing methodologies
WGS, SNP, target capture, microarray based
relies on identifying and counting DNA fragments in maternal serum
only first 25-36 BP are sequenced (aka a Tag)
count and compare tags at a specific chromosome and compare to reference chromosome number
WGS
a panorama simultaneously targets 19,000 SNPs
SNP based targeted sequencing
T21 detection by F-S* ratio
euploid is higher ratio
SNP based methods for microdeletions can Dx
digeorge
angelman
prader-willi
cri-du-chat
microdeletion risk is reported using
Odds of being Affected given a Positive Result
OAPR takes into account
clinical incidence of disease
% cases caused by deletion of entire covered region
Digital Analysis of Selected Regions
selected analysis of cell-free DNA in maternal blood for evaluation of fetal trisomy
factors that affect NIPT results
fetal fraction
maternal BMI
fetal fraction increases
by 0.1% in the first 21 weeks
by 1% in weeks 22-40
twins show
increased ff but not doubled
steps for visualizing metaphase chromosomes
- culture patient cells
- add PHA to stimulate division
- colcemid stops cells in metaphase
- methanol and acetic acid fixes chromosomes
dye that binds to AT rich DNA and G band segments
quinacrince
how are R bands visualized
87 Celsius for 10 mins then add Giemsa stain
how to visualize centromeres
alkali treatment
Euchromatin rich areas high in GC content are
R bands
types of fish probes
chromosome painting (whole chromosome) centromere probes, locus specific probes,
locus specific probes detect
gene rearrangements, microdeletions, and amplifications
used to detect aneuploidies and tumors
types of structural chromosome abnormalities
translocation, inversion, deletion, insertion, ring, isochromosome
chromosome 22q11.2 deletion
DiGeorge
chromosome 7q11.23 deletion
williams syndrome
deletion on chromosome 4 4p16
wolf-hirschhorn
amplification copy number variation is the most common change seen in malignancies
true
what does comparative genomic hybridization do
CGH analysis software measures fluorescence intensity values along the length of the chromosomes and translates the ratios into chromosome profiles.
The ratio of green to red fluorescence values is used to quantitate genetic imbalances in tumor samples.
limits of CGH
cannot detect less than 5-10 Mb changes
cannot differentiate between diploid, triploid, and tetraploid
cannot ID balanced structural xsome translocations
cannot distinguish mosaicism from trisomy
applications of micro CGH
classify tumors, tumor progression, genomic changes at various stages
applications of CGH reproductive genetic diagnosis
evaluation of fetal abnormalities/stillbirths, can ID chromosome abnormalities smaller than seen karyotyping, submircroscopic deletions detected,
Benefits of pre-natal CGH
no tissue culture needed
automated
better resolution/detects small rearrangements
first tier test when structural malformations are seen on an ultrasound
chromosomal micro-arrays
cell free fetal DNA is derived from
trophoblast
140-180 bp
massively parallel genomic sequencing is used to
detect aneuploid fetus, gives higher Z-score
who should be considered for whole exome sequencing
patients w/ genetic disease that hasn’t been ID’d or diagnosis is unclear
if testing many genes is cost prohibitive
chromosome structure abnormalities
translocation, deletion, inversion, isochromosome, derivative chromosome, insertion, ring chromosome
