identifying disease genes and genetic susceptibility to complex disease

approach toward genetic testing

depends on if single person or population is carrying the problem, depends on if gene is known or unknown

single person

always start w/ pedigree analysis (2 gen above and below)

population

can't draw a pedigree, use population testing/screening technique instead

known gene

mutational analysis: must do pretest counseling FIRST; warn about things like potential to find out about infertility, new diseases etc. -> depending on results of pretest counseling then you can decide on mutational analysis

unknown gene

linkage analysis (starting test): done when you have multigenerational info or multiple ppl in family w/ unknown gene disorder, tells about general area of where disease is occurring on chromosome, tells you area you should focus one -> then you can apply other tests

indications for cytogenic analysis

developmental delay/intellectual disability (esp. w/ anomoalies), ambiguous internal/external genitalia, girls w/ primary amenorrhea (don't get period) and boys w/ delayed puberty, males w/ learning/behavioral disorders who are taller than expected, specific malignant and premalignant diseases, family history of similarly affected children, couples w/ multiple spontaneous abortions of unknown cause, prenatal diagnosis

prenatal diagnosis indication for cytogenetic analysis

advanced maternal age, carrier present, prior affected child, abnormal serum or cell-free DNA screening (non-invasive screening, in maternal blood, tests for chromosomes of baby, tells you gender), fetal anomaly or growth restriction on US (scans to predict appearance of disease after birth and see if you can fix it before birth)

challenges in dx of genetic disease

complex/multifactorial diseases (no obviously predominant disease locus)

monogenic

influenced predominantly by single gene locus

oligogenic

disease phenotype may be dependent on a few genes

polygenic

multiple genetic factors determine phenotype, each making a small contribution, eg. T1 and T2 DM, CAD, stroke, RA, alzheimers

genetic risk factors

don't cause disease, just increase risk

whole genome sequencing

process that determines the DNA sequence of an entire genome, HGP

next generation sequencing (NGS)

takes <24 hrs (quicker), <$1k, getting cheaper still, sequencing by synthesis, resequencing, 99.9% accurate (lots of data given), replaces some older techniques, only makes sense if looking at MULTIPLE genes (15-20), NOT used for single gene mutations, can identify single nucleotide changes, can't identify balanced translocation, preferred over karyotyping

sequencing by synthesis (NGS)

break genomic DNA and add linkers (short stretch of oligonucleotides of known sequence) -> amplify the linkers, put on a chip, then read them -> fluorescent signal (G attached to green, T attached to blue) -> captured by computer and computer organizes/aligns it, millions of rxns

resequencing (NGS)

compare w/ sequence genome, find abnormalities thru comparison

karyotyping

examines chromosomes in sample of cells usually in metaphase or prometaphase when the chromosomes are most condensed, giemsa stain, slow and labor intensive (1-2 weeks), preimplantation genetic testing - IVF, gradually being replaced by NGS, can't identify anything under 5 million bp, easily done anywhere, low resolution of chromosome

karyotyping process

chromosome dense (metaphase/prometaphase) -> apply anti-tubulin drug (colchicine) -> arrests chromosomes by stopping spindle fiber function -> chromosome visible -> stain them (giemsa stain) -> bind in areas w/ A and T, gives banding pattern, can identify translocations, etc.

FISH

fluorescence in situ hybridization

chromosome FISH

less expensive and fast (1-2 days), KNOWN mutation is required (premade probe according to defect), up to 100k bp, less expensive than NGS but varies on how expensive it is vs karyotyping, open up chromosome and apply signal

chromosome FISH process

fix chromosome preparations on microscopic slides, treat slides to denature DNA and hybridize fluorescently labeled probes of interest to denatured DNA, locations of fluorescent signals are recorded against a background stain that binds to all DNA sequences

gene linkage

alleles at closely neighboring loci w/ recomb frequency of

linkage analysis

only tells location on chromosome of mutation -> once identified use NGS to analyze area, req. multiple people to have disease, checks for linked genes with recomb frequency <50%, LOD score

LOD score

statistical estimate of whether 2 loci are likely to lie near each other on a chromosome and are therefore likely to be inherited tog, score of 3 or more indicates they are linked and close to one another

linkage analysis example

SNP B = ppl w/out disease, SNP A = ppl w/ disease (pattern), disease-causing mutation should be somewhere near SNP A -> NGS around this area to find the mutation more closely

exome

collective term for all exons in genome, majority of disease causing mutations occur in exons of protein-coding genes

exome sequencing

cost effective, less laborious, sequencing fraction (just over 1% of genome), is easier and cheaper than genome sequencing, ONLY sequences exomes (exons = <2% of coding genome), 95% of genetic disorders occur in protein coding region, start in pt w/ genetic disorder w/out diagnosis and doesn't know gene and NGS, can find RARER diseases for a single person

exome sequencing process

involves first capturing exons from DNA of affected individuals -> then sequencing the captured DNA

thrifty phenotype

adaptation that maximizes change of surviving in an adverse environment w/ limited calorie intake, fetal nutrient starvation -> thrifty phenotype -> low metabolic rate and reduced pancreatic B cell mass and islet function

low birth rate risks

lead to increased risk of HTN, CVD, stroke, T2DM

thrifty phenotype hypothesis

low birth weight babies/babies in restricted womb environment; after birth can't handle normal amounts of nutrients bcos it got used to having limited amt of nutrients (similar to spice tolerance), decreased metabolic rate -> baby gets used -> normal nutrients feel excessive to baby -> increased risk of disease

CVD, diabetes (can't handle sugar), obesity

phenocopy

similar phenotype arising from a different cause -> often environmental or nongenetic -> can often mimic genetic disorder, makes diagnosis challenging

phenocopy example

alzheimers disease diagnosis vs other dementia disorders (lewy dementia, frontotemporal dementia, etc.), can only diagnose after death and biopsy brain

genome-wide association study (GWAS)

helps identify susceptibility locus in complex diseases, looks into genetic factors of complex disease, useful in finding genetic variations of common, complex diseases like asthma, cance, diabetes, heart disease, mental illness

GWAS process

rapidly scanning markers (SNPs) across complete sets of DNA or genomes of many people to find genetic variations associated w/ a particular disease, compare SNPs from all these ppl w/ and w/OUT disease to figure out which variant is likely pathogenic, new genetic associations -> better strategies to detect, treat, and prevent disease

GWAS in IBD

new insights into biological pathways -> new approaches in treating disease, successful in Crohn's disease and ulcerative colitis (both results from abnormal immune responses to gut microbiota), identified 320 IBD risk loci (2023); most are shared by both diseases, revealed role of autophagy in Crohn's disease, IL23R locus pointed to IL-23 blockers (ustekinumab) -> now approved for IBD

environmental basis of complex disease

case-control studies, prospective cohort studies

case-control studies

most widely used method of investigating genetic and environmental basis of complex disease, HOWEVER cases and controls are investigated retrospectively -> BIAS (eg. bias of selection of subjects to be studied), identifies ppl w/ and w/out disease and what they were exposed to, easier and cheaper but prone to bias

prospective cohort studies

NO BIAS, takes a long time (months-years), ppl can fall out of study over time or come in, effective at finding environmental causes/how disease develops