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Inherited and Non-Inherited Genome - Coggle Diagram
Inherited and Non-Inherited Genome
NON-INHERITED GENOME
Cancer Incidence varies between tissues : cancer is a group of conditions that share an increase in cell number w/in a particular tissue - benign or malignant - arise anywehre - carcinomas have highest risk as they are in direct contact w/environment - epithelial cells have high mitotic rate and thus higher cancer rate, low cancer chnace in brain and muscle cells as they divide slowly
Cancer caused by mutation in genes that regulate cell division, death, dna repair etc: we evolve from mutations but can increase/decrease protein activity or generate opp function or decrease protein expression - proteins like a bike with some essential and non-essential parts
Mutation types:
-Loss of Function: reduction in function of protein encoded by gene - partial or complete loss of function // - gain of function: activity of protein increased - protein could be more stable or bind to substrate better, resist inhibitor better, expressed at higher level // dominant-interfering mutations: protein opposes function of wild type protein ie by binding to it or its target - block function
P53 Protein: functions as a tetramer if a subunit is mutated the whole tetramer loses function - mutant p53 that can still binds bind to tetramer can act as dominant interfering mutant
it acts as a tumor suppressor by regulating cell division
Ras Protein- commonly mutated where gain of function mutations found - mutations increase liklihood that Ras will be in its active (GTP-Bound) State
correlation between cell damage/infection and cancer incidence: tissues w/highest carcinogen exposure have highest risk of mutation eg epithelial cells // viruses can cause cancer - provoke cancer by insertion of their genome into host - results :1. viral genome may carry gene that enables host to escape controls to restrict cell division or limit lifespan , 2. virus may integrate into genome close to host gene regulating cell division/apoptosis
Cancer is a Rare Occurrence: there is a huge potential for mutation due to the rate of constant cell replacement - uncommon due to several layers of controls that have evolved to limit uncontrolled division
when mutations arise they facilitate further mutation accumulation at an accelrated rate // tumours undergo a Darwinian selection process cells carrying diff combos of mutations are produced - fittest survive
tumours are monoclonal - arise from a single precursor cell that became cancerous eg B cells that become cancerous - lymphomas, myelomas - produce unique antibodies via VDJ recombination - the tumours would also produce this antibody if they arose from a single transformed B cell - can discover clonal origin of tumours by studying antibodies
Cancer = Muiltistep Process: a single mutation doesnt cause cancer due to developed safegaurding events - apoptosis to mutant cells to limit potential for devloping cancers
exception: large scale chromosomal breakage can produce multiple mutataions at once = chromothripsis - result of faulty chromosomal repair
mutations on oncogenes and tumour supressor genes contribute to ancer development ie mutations of host genes that enhance proliferation or other processes that increase cancer liklihood
oncogene = gene, when mutated, promotes cancer - typically gain-of-function
tumour supressor genes oppose cancer development - genes inactiavted in cancers so they cant carry out normal function (loss of function)
genetic mutations may produce dominant interfering muattion - fail to carry out normal function
progressive transformation to more transformed cancerous state - further mutations at each point facilitated by prev. mutations - enables cells to progres sto next stage - long time frame - accelerated if pre-existed mutations inherited = fertile soil for mutations to develop (cancer has an inheritable component)
barriers to transformation
requirement of growth factors: cells need grwoth factors to divide - are provided by other cells (paracrine) - if supply of growth factors is limiting cancers acquire ability to make them or mutating downstream signalling molecules associated w/binding growth factors to their memebrane receptors so receptors appear constantly switched on
tumour suppressor genes act as a brake on proliferation : certain proteins suppress uncontroled proliferation by monitoring cells for signs of dna damage/aberrant behaviour - p53 TF most important - collabs w/proteins involved in DNA damagae detection and repair - when p53 activated in cells (via stabilisation of it when normally labile) it induces transcription of gene that block entry into mitosis - enables dna repair to be carried put before cell is permitted to re-initiate mitosis - extensive DNA damage results in p53 dependmet expression of genes (Noxa, Puma, bax) that promote apoptosis - p53 loss can accelerate cancer - mutataed p53 gene in 50% of cancers
requirement for oxygen and nutrients: supply is regulated by proximity to blood vessels - as tumours are abnormal cell masses they need blood vessels to grow beyond a certain size - restraint upon growth - tumours need to induce new blood vessels to grow or tumour cells will die of hypoxia and nutrient deprivation / ability to grow new blood vessels linked to prodcution of VEGF (vascular endothelial growth factor)
Cellular Transformation: transformed cells display characteristics not displayed by a normal cell
ability to grow in a lab culture (in vitro) for long time periods: growth factors make it possible to stimulate cells to undergo cell division - serum - source of factors that promote cell division - in presence of serum, untranformed cells undergo a set number of divisions (25-30) until theyre unresponsive - Hayflick Limit - shortening of chromosome-ends (telomeres) during each round of cell division - so much erosion thta further chromosomal duplication is impossible - cells display breakages etc --> thus hallmark of a tranformed cell is ability to divide beyond Hayflick Limit - related to expression of enzyme (telomerase) thatcan repair telomeres and therfore enhnace no. of cell divisions it can undertake - many cancers display reactivation of telomerase gene expression
reduced requirement for growth factors: transformed cells have ability to make their own growth factors (autocrine growth) - achieved by amplifying no. of growth factors receptors expressed by cell - mimicking growth factor receptor engagement - can supplement growth factor requirment by acquiring gene mutations that function in growth factor signalling pathways eg signalling cascade is permanently on (Mutation on Ras found in cancers)
Anchorage Independence : w/exception of RBCs, normal cells wont grow unless attached to a solid support- support provided by lactic cell culture vessel (petri dish) - normal cells stop dividing when a monolayer is formed unlike transformed cells that will continue to divide and pile on top of each other - will grow even when detached from flask - transformed cell can betray their presence in a culture of healthy cells by forming disorganised clumps - piled up in 3D
Altered Morphology: transformed cells have aberrant morphology and normla cells are fully flat in a culture - transformed cells can become round or look like spiindles - disorganised cytoskelton - more flexible morphology - look v diff to their normal counterparts
loss of contact Inhibition: normal cells cease to divide when theyre in contact w/other cells at all sides - formation of ordered monolayers when untransformed cells are grown in culture - transformed cells dont display this and so grow to higher densities
ability to form tumours when introduced into immunocomprimised mice
Introduction of mutant genes into normal cells can result in features of cellular transformation
ways to transform normal cells into cancerous cells - introduce individual genes (isolated from cancer cell) - DNA transfection - formation of complex between -DNA and calcium salt - precipitates the DNA when added to cells - escapes into cell cytosol - can enter nucleus and become expressed
individual mutations may produce one or more features of tranformed cellls but may not fully transform cells
Cellular transformation assays - cells traeted with mutagenic chemicals - DNA isolated from chemically-transformed cell population - cut into small pisces and reintroduced into normals cells using transfection method ← can find and isolate the DNA that transformed normal cells - can sequence to pinpoint the mutation
Oncogenes and Tumour Suppressor Gnenes:
oncogenes = genes that in mutant form promote cancer development, usually promote cell division eg growth factors, their receptors - some are involved in apoptosis regulation - prolong lifespan of cells expressing mutant forms of genes
mutations that turn a gene into a oncogene - gain of function mutations - increase activity of protein encoded by gene- achieved by: 1. enhancing stability of protein, 2. increasing expression rate of gene encoding protein, 3. altering protein active site so it functions at an enhances rate
tumour supressor genes oppose cell proliferation or survival - lose ability to do this in their mutant forms - loss of function mutations / may be mutated into dominant-interfering mutants and oppose function of wild-type protein
discovery of oncogenes: filterable agent derived from chicekn leukaemia cells - transferred to develop disease / peyton rous - grind up sarcoma tissue, filter and trasnfer to other birds - rous sarcoma vrius - first oncogene/ RSV gene called Src - present in genomes of cells - viral Src is a hijacked gene that became part of viral genome due to selective advantgae / viruses arent the only means of initiating cancer
Ras and B-Raf Oncogenes:
growth factor signalling pathways and cancer: growth factors promote cell division by activating signalling cascades - culminates in divsion of duplicated cell contents into two new daughter cells - supply of growth factors must be controlled to ensure constant cell numbers
cells compete for growth factors - must find way of becoming independent of the requirement of receiving GFs to drive mitosis - GF independent by increasing expression of GF receptors (epidermal grwoth factor receptor, erbB) - upregulated and makes cancer cell hyper-responsive to ambient GF levels ; also increaseses rate at which recptors undergo spontaneous activation
other tumours can make their own GF by acquiring mutations in key signalling molecules that act downstream of their GF receptor - this mutation found in Ras and B-Raf proteins
Mutations of Ras in Cancer: commonly mutated - muattions icnraese Ras affinity for GTP - mostly actiavted - mutations cause increase in activity of Ras - gain of function // 3 Ras Genes: H-Ras, K-Ras, N-ras
Mutant Ras can transform cells via increasing proliferation rates and conferring growth factor independence - More efficient transformation of other cells if they also carry mutant p53 gene - synergistic interactions between independent mutations to promote cell transformation
b-Raf Mutations and Malignant Melanomas
malignant melanoma must diff to treat - occurs when pigment producing cells (melanocytes) becomey transformed due to chromosomal lesions that involve B-Raf kinase - common on exposed skin - prone to spread - acute resistance to therapy due to dysregulation of apoptosis
B-Raf is mutataed in most melanomas - correlate w/progression of melanoma - expressed differntially in a variety of tissues
B-Raf mutations pre-dominantly alter a single AA residue - valine altered to glutamic acid - produces active kinase - 550 time more active - B -Raf mutations also increasesgene expression promoting proliferation - active B-raf may suppress activation of apoptosis machinery - state of chemoresistance
B-Raf mutations arent sufficient to generate cancers - need additional genetuc lesions to fully transofrm cells - known as many B-raf mutations found in benign lesions on skin (moles) - further mutations in gene CDKN2a and p16 proteins cooperate w/B-Raf to transform cells.
Raf inhibitors represent targets cancer chemoteherapies: b-raf is an attractive therapeutic target due to its gain of function mutations - search for C-Raf inhibitor eg ASorafenib - ATP inhibitor // potent inhibitor of B-raf = ZelboRaf - selectively inhibits
Ras controls Raf actiavtion but only if GTP-Bound
Ras = GTP-binding protein, normally tethered to plasma membrane via lipid mods - GTP bound state, ras is active - hydrolyses GTP to GDP (phosphate group split off) to return to inactive state
Ras Role : activate downstream protein kinase, Raf (A, B, C)
Ras actiavtes Raf by recruiting it to plasma membrane - Raf can be phosphorylated by membrane-associated kinase - Ras cant bind Raf unless Ras is in GTP bound state - needs GDP/GTP exchange factor (GEF) to assist Ras in letting go of GDP and exchange for GTP
Major GEF for Ras = Sos (son of sevenless) - Sos doest live in plasma membrane and neither does GRB2 that has domain SH3 that Sos can bind to
many growth factor receptors are kinesis - aggregation of kinases (as a result of ligand binding) → results in receptors phosphorylating each other within their cytoplasmic tails (part of receptor facing cytoplasm) → ligand induced activation of the growth factor receptor creates binding sites for GRB2/Sos → activation of Ras through Sos mediated stimulation of GTP exchange on Ras → Ras recruit s Rfa to membrane- becomes phosphorylated and active Raf propagates the signal further by activating the downstream kinase, MEK - followed by ERK.
ERK (extracellular receptor activated kinase) is switched on - activation of ERK, via Ras and Raf, promotes cell division through action of ERK On TFs.
Tumour Suppressor Genes: p53, Guardian of the Genome: p53 is a TF that responds to DNA damage upon activation - stops cell division/promotes apoptosis - cell cycle arrest to permit DNA cell damage to be repaired/kill cell - perfofrmed by switching expression of genes on/off to stop cell cycle (p21) or induce apoptosis (Noxa, Puma, Bax)
discovery: monkey virsu, SV40 - infected mouse with it - strong immune response against SV40 large T antigen - specific antibodies used to look for cellular targets - large T antigen immunoprecipitated from infected cells - contained p53 protein - involved in cancer development // p53 v freq. muatted in cancer - comparison of normal and utant p53 show genes differ by a single point mutation that converts it into a dominant-interfering mutant protein
p53 normally funtions to supress proliferation in response to DNA damage and other cell stress: p53 alleles v common in tumours - most mutant p53 alleles are missense (not nonsense) - tumours show benefit from mutant p53 rather than loss of p53 function
p53 normally functions as a heterotetramer: only 1/16 of a p53 in a cell carrying dominat negative p53 allele will be functional
p53 has a short half-life - stability of p53 is controlled by another protein, Mdm2 - binds to p53 and targets protein for degradation via ubiquitin-protease protein destruction pathways; it also exports p53 out of nucleus thus preventing DNA access to p53 and blocking transcriptional activity // expression of Mdm2 controlled by p52 = p53 controls synthesis of its own behavior !
Mdm2 is degraded as a conseuqnce od DNA damage : Mdnm2 has upper hand in healthy cells and p53 is constantly degraded - upon DNA damage (ie radiation), Mdm2 is phosphorylated by kinase (ATM) - stabilisation of p53 - some reduncy as p53 can also be phosphorylated by ATM and other kinases involved in DNA damage response pathways - phosphorylation of p53 stabilises it - p53 half life enhances in response to DNA damage
DNA damage-induced p53 activation promotes cell cycle arrest/cell death: p21 is the most notable gene controlled by p53 wrt cell division arrest - upon p53 stabilisation, p21 protein is rapidly expressed - halts cell cycle inhibiting kinases crucial to cell division // p53 also induces expression of DNA polymerase beta involved in DNA repair upon detected DNA damage (by ATM, Chk1, Chk2) - p53 is stabilised, halts cell division and induce proteins required for DNA repair
p53 also controls expression of genes that can directly trigger apoptosis: products of these genes: Bax, Puma, Noxa - engage in apoptosis machinery - relate to p53 levels taht accumulate in cell and degree of DNA damage cell suffered - low DNA damage results in cell division arrest and repair while higher levels trigger p53-dependent apoptosis
Driver and Passenger Mutations:
most mutations w/in a tumour are irrelevant - no functional advantage to tumour = passenger mutations - arise due to other mutations that result in increase in mutations rate or decrease rate of DNA repair
key mutations that drive towards cancerous state = driver mutations - gain of function mutations in growth factors, growth factor receptors (oncogenes eg Ras) or tumour suppressor mutations - have a functional impact on cancerous state - driver n bus analog for driver and passenger mutations
Cancer genomics/implications for treatment
most mutagenic events have little consequnce that drive tumour formation // some mutagenic events produce cells w/ properties that make them disobey normal cell behaviour rules ege growth factors - predispose towards acquiring additional muatations by generating DNA insability // diff combos of muations in diff tissues
insights from large scale genomics: reveal sequence mutations / ID differentially expressed genes // chemical mods of DNA can be mapped - a tumour may have 500 unique mutations - majority of mutations have little consequnce for cancer progression (passenger) // early diagnosis if can ID specific mutation signatures - guide treatment (most are p53 mutants)
insights on acquired resistance w/chemotherapy: relapse due to emerging resistance - subclones of mutation escape drug effects that target original tumour - two main novel mechanisms
Comparing sequencing data of pretreated metastatic cancer patients and untreated primary tumour patients
Sequencing paired pretreatment and post treatment biopsy samples obtained at diff times from same patient → mutational profiles compared
genomics and precision medicine: Detection of specific oncogenic mutations may be ‘clinically actionable’ and indicate treatment w/ a targeted therapy - eg known mutations in other cancers - common therapeutic vulnerabilities but not always
mutation signatures may alter treatment choice :
INHERITED GENOME
each person carries 1-2 mutations -> cause genetic disorder/prenatal death when two copies of the same mutation inherited
Mendelian Disorders (=disease due to mutation in a single gene i.e. monogenic)
genetic info can alter treatment e.g. hemophilia(A/B) = x-linked recessive disease --> males affected
Human genome - 46 chromosomes , 22 pairs (autosomes) numbered accord. to size, two sex chromosomes determine whether its a male or female --> 2 m of DNA packaged into chromatin to ft in nucleus
nucleosomes = structural building blocks of packing DNA in chromosome - can pack as it super coils the DNA around spherical histone proteins (nucleosomes)
in chromatin, the dna is bound to histone and non-histone proteins (bind dna as nucleosomes) --> aino and hydroxy terminal tails of hsitones are subject to postranslational modifications (PTMs) on multiple residues, incl. methylation, acetylation, phosphorylation etc
PTMs regulate activity of underlying genomic regions by altering how nucleosomes interact w/each other and the DNA - thus can control acces to DNA seuqnces by recruiting effector proteins that bind PTMs diractly - interpret whether a region should be active or not
thus chromatin considered as regulatory unit of genomes --> further packing within the 3d nuclear space have a direct impact on its activity
Euchromatin = less densely packed form of chromatin found in active regions of genome (93%)
Heterochromatin = v densely packed regions, genetically inactive (7%)
DNA = neg charge, histones= pos charge
Structural Variation in 3D genome
// structural variation = regiom of DNA that can include inversions and balances translocations or genomic imbalances (insertions/deletions) = copy number variants
affect gene dosage but also modulate mechanisms of gene regulation --> can alter number of regulatory elements/modify the 3d genome by disrupting higher-order chromatin organisation such as topological associated domains
result: SVs influence expression of genes distant from SV breakpoints --> cause disease
length, weight and Guanine-cytosine content of human genome -- variation from average can determine disease
anomaly - chromosomes numbered accord to size but Ch22 has more DNA content than Ch21 and thus is bigger (1 biggest)
Human Genome overall structure - only a small amount of human nuclear genome encodes proteins (1%) /
many genes code for RNA (rna doesnt get translated into proteins) but not protein
= non-coding / more non-coding genes than protein coding genes // pseudogenes = non-coding DNA sequences that are defunct (not in use) relatives of their protein-coding parent genes, thus retain high sequence similarity
certain long non-coding RNAs (lncRNAs) expressed from pseudogene loci regulate the protein-coding parents genes of these pseudogenes due to the sequence complementarity
some of the functioning non-coding RNAs regulate gene expression e.g. a class of noncoding RNAs, microRNAs (miRNAs) regulate gene expression
more non-coding DNA --> higher complexity in organism
Types of Non-coding RNAs:
Transfer RNAs, ribosomal RNAs - translation / small nuclear RNAs (snRNAs) - splicing events / small nuckeolar RNAs (snoRNAs) - modifcation of other small RNAs
Short regulatory non-coding RNAs: piwi-associayed RNAs, endogenous short-interfering RNAs, microRNAs - regulators of gene expression
long non-protein codunf RNAs (lncRNA) : Roles in epigenetic control of chromatin, promoter-specific gene regulation, mRNA stability, X-chromosome inactivation and imprinting
lncRNA loci (location) can be : intergenic lncRNAs, lncRNAs expressed from enhancers or promoters, lncRNAs overlapping other genes
lncRNA can have regulatory functions in nucelus
lncRNA transcripts that positvely or negatively regulate the expression of enighbouring (cis) or dstnat (trans) genes by modifying chrmatin states of targeed gene loci
long non-coding transcripts that organize nuclear architecture by forming, maintaining or regulating nuclear structure and domains
lncRNA can act in cytoplasm - the transcripts that bind to and regulate proteins or other rna molecules in cytoplasm
alternative RNA splicing, alternative transcription initiation and alternative transcription termination
occurred more frequent than thought - splicing errors - can effect diversity of non coding gene products and protein coding gene products
RNA processing (eukaryotic genes): a eukaryotic gene is made of two types of DNA sequence : 1. coding sequences = exons, 2. non-coding sequences =introns // after transcription (RNA generated from DNA), introns are removed by RNA splicing to make mature RNA (mRNA)
Differential (alternative) splicing of RNA can lead to generation of multiple proteins and multiple rna transcripts
sources of protein Variation
(make protein isoforms): many genes transcribed with splice variants // many human proteins undergo PTMs - can strongly influence their activity/function ; incl. phosphorylation, glycosylation, acteylation -> protein variants // selected genes for proteins eg immunoglobulins, t cell receptors undergo somatic recombination to increase no. of potential protein variants
isoforms arise from alternative splicing of RNA and from use of diff promoters or transnational start sites
isoform variation combines w/site-specific changes to generate human proteoforms ; such site-specific changes incl. single-nucleotide polymorphisms (SNPs) and co/post-translational mods eg phosphorylation
Repetitive Sequences
- duplicated genes retain sequence identity - many of these duplications are primate specific - one or more regions of a gene may be repeated or a whole gene may be duplicated // repetitive DNA sequences renature at lower CoT values than single-copy sequences
Multi-gene Families
: clustered gene families arise by tandem duplication events eg the beta and alpha globin gene families on chromosomes 16 and 11 // gene families are distributed over diff chromosomes - arose from duplicated genes separated by rearrangement or form cDNA copies of the mRNA produces by a gene inserted into the genome = such insertions were often w/out regulatory sequences and so result in some nonfunctional pseudogenes
Tandem Gene Duplication: 1/2 or 2/3 of genome is various repetitive sequences - repetitive sequences mat be tandem orientation and/or dispersed throughout genome - can be classfied by function, dispersal patterns and sequence relatednes // eg eukaryotic genome has many transposable elements (TEs)
the human genome has interspersed repeat sequences that have amplified in copy number by movement through genome = transposable elements // almost all transposition happens via an RNA intermediate yielding classes of sequences referred to as retrotransposons/retroposons (Class 1 TEs) / there are TEs that never use RNA intermediates - DNA transposons - always move on their own, inserting or excising themselves form genome - 'cut-and-paste' mechanism
Reptitive Sequences: LINEs (long interspersed nuclear elements)- 41%, SINEs (short interspersed nuclear elements - 29%, LTRs (long terminal repeats) - 18%, DNA transposons -6%, satellite repeats (ie on telomeres, centromeres) - 6%
Common Transposable elements (L1, S49) - confusing!!!!!!! // more than 50% of genome is derived from transposable elements
most common mutagenesis of TEs - disruption of gene regulation - resulting from insertion of new element copies // L1, Alu and SVA cause a broad range of diseases // TEs insertions provide raw material for emergence of protein-coding genes and non-coding RNAs - can take on cellular function eg Rag1, Rag2 - catalyse somatic recombination on the vertebrate immune system
success and diversity of TEs are shaped by evolutionary forces
highly conserved : coding sequnces, non-coding sequneces
poorly conserved: heterochromatin DNA, transposon repeats, other ie unique sequnces
Mitochondria - ETC (electron transport chain) Complexes embedded in inner mitochondrial membrane are protein complexes involved in making 90% of cellular energy
Oxidative phosphorylation (OXPHOS) Complexes - generate cellular energy - ATP --> flow of electrons fro donors eg NADh to electron acptors eg oxygen = exergonic process releasing energy, synthesis of ATP is an endergonic process ie needs energy inout
both ETC and ATP synthase are embedded in inner mitochondrial membrane and energy is transferred from ETC to the ATP synthase by movements of protons across membrane = process called chemiosmosis
the Mitochondrial Genome: mtDNA: 37 genes encoding 13 proteins, 22 tRNAs, and 2 rRNAs --> the 13 mitochondrial gene-encodoes proteins instruct cells to produce protein subunits of enzyme complexes involved in oxidative phosphorylation - enables mitochondria to act a powerhouse of cells
transcription of human mt genome : Heavy (G-rich) & Light (C-rich) DNA strands / D-loop control region contains divergent promoters for the H and L strands / Heavy strand transcript: 2 rRNAs;12 proteins; 14 tRNAs / light strand transcript encodes a single protein ND6 and 8 tRNAs / major 'leaky' bi-directional termination (TERM) site loacted in tRNA (L) - uses DNA -binding protein, MTERF1 / rRNA genes are transcribed 15-60 times more than mRNAs
mtDNA is inherited from a single lineage ie through mothers (maternal inheritance)
mitochondrial disorders
heteroplasmic mtDNA mutations - show a 'Threshold effect' - below a certain level of mutant mt DNA there may be no obvious phenotypic effect, but just above this level there are full phenotypic consequneces
mitochondrial replacement therapy: 3 parent baby born using spindle nuclear transfer
two approaches: pronuclear transfer and spindle transfer - allow nuclear genome to be transferred to a donor zygote (fertilised egg)//oocyte containing mitochondria w/out mtDNA mutation
Mendelian Disease: Autosomal Recessive (25% affected, 50% carrier, 25% normal) and Autosomal Dominant (50% normal, 50% affected) // X-Linked (sex linked) - all female children of an affected male are carriers, 50% of male children from a carrier female have disease and 50% female children will be carriers if both parents are affected
Types of Disease related genetic variants: chromosomal abnormalities, subchromosomal abnormalities, small scale DNA changes - one or more single base changes in nuclear or mitochondrial genomes
Effects of variants:non-functional protein or RNA, protein or RNA w/aberrant effects, over or under expression of one or more proteins or RNAs, splicing defects, regulatory mutations, chromatin remodeling
chromosomal abnormalities - aneuploidy detection using quantitative fluorescence PCR: use of multiple short tandem repeat markers from chromosomes 13(Patau syndrome),18 (edwards syndrome), 21 (down syndrome) - pairs of fluorescently labelled primers are used for a multiplex PCR, products seperated by polyacrylamide capillary electrophoresis
Patau Syndrome - chromosomal abnormality where some or all cells in body contain extra genetic material from chromosome 13 - disrupts normal development - multiple organ defects
edwards syndrome: extra chromosome at chromosome 18 - more common in girls - usually dont survive past first week of life, small head, small jaw, unusual shape chest
genetic varients may have no effects (silent) but can influence traits ie skin colour etc or cause a specofic mendelian disease eg haemophilia
variants can involve single bases of DNA or involve multiple bases ie can be large deletions or insertions of DNA - can have large copy number variants (CNVs) and structural variants (SVs)
Missense mutation = AA specifying codon is replaced by a codon for a diff AA
Stop-gain (nonsense) mutation = AA specifying codon is replaced by a premature stop codon
Stop-Loss mutation = natural stop codon is replaced by an AA specifying codon
Consequences of Point Mutations in DNA - some are silent and some cause big changes - can cause partial or total loss of function of encoded protein (soem reccessive) - may produce a protein thats toxic to the cell eg if the protein function s in blood clotting - mutation may cause Haemophilia A --> uncontrolled bleeding
sequence of bases in DNA determine the properties of the protein encoded by the gene - insertion or deletion of one base can shift the reading frame --> nonsense or missense mutation
base-pair substitution --> missense or nonsense mutation
splicing process and splice site sequnces - pathogenix splicing mutations can cause exon skipping or intron retention / non obvious mutations still have effects on splicing eg deep intron mutation
assessin gnovel variants : searching allele freq. in databases, evaluate conservation of AA between species, in silico modeling of effects of variant on protein structure or of variant on splicing
Rhodopsin (RHO) variants causing retinal degenration
autosomal dominant RP (rhodopsin) - mutations in rhodopsin cause dominantly inherited eye disorder Retinitis Pigmentosa - encoded protein w/ a chnage structure - photoreceptor cells die - lead to blindness
eg Autosomal recessive EB (skin blistering disorder) - caused by mutations in genes whose encoded proteins function in strengthening skin - a result of a point mutation (single base pair change)
common cystic fibrosis (CF) mutation - delta F508 deletion
genesis of genetic variation
provenance of mutations:
single nucleotide substitutions can arise due to chemical degredation of DNA that hasnt been repaired (methylated cytosine can be deaminated to thymine) - small insertions/deletions occur by replication slippage / templates w/tandem repeats are vulnerable to replication slippage
mutation rates
: not all the same in all parts of genome - mutational hotspots - some more likely to be mutated - mt gene has a higher mutation rate compared w/nuclear genome
somatic cells
: to maintain tissues being replaced daily - many cells dividing -> DNA damage due to enviornmental factors and normal metabolic processes occurs to cells each day - mistakes in DNA need to be fixed to maintain integrity of our genomes
new insights: can be born w/mutations that occured during gametogenesis and postzygotically - de novo muattions can cause sisorders such as autsim - de novo mutations are mostly paternal origin and their no. increases w/ paternal age - sperm cells undergo many more mitoses than oocytes - errors in replication/ repair of DNA higher ; oogenesis has a fixed no. of mitosees but mutations can accumulate over time due to failed DNA repair
consequnces of crossing over during meiosis - recombination - crossing over results in random shufflling of genetic material during proocess of gamete formation --> crossing over at Chiasmata during meiosis - genome mapping
recombinant events during meiosis mapped by genome wide DNP genotyping or by NGS - pattern of meiotic recombination in chromosome passed form mother to daughter
males have lower recombination rates , opp is true near the telomeres
indirect association:
genotyped single nucleotide polymorphisms (SNPs) lie in region of high linkage disequilibrium w/an influential allele - genotyped SNP will be statistically associated w/the disease and can act as a surrogate for teh disease SNP through indirect association
DNA from family members can be tested for the association between disease and a 'marker' locus using DNA SNP microarrays through genome
in Mendelian disorders, one region of genome is causative hence are hunting for one gene but modifier genes can influence disease phenotype
can use DNA SNP microarrays, NGS(next generation sequencing)-based sequencing eg whole exome/whole genome sequencing (WES, WGS) to search for disease genes for mednelian or complex disorders
Complex/Polygenic/Multifactorial Disorders - diseases that are influenced by a combo of multiple genes and environmental factors
Genome Wide Asssociation Studies (GWAS) to track complex traits/diseases - DNA microarrays can test single nucleotide polymorphisms (SNPs) - can decipher genetics of complex traits / GWAS study can be influences by study sample size, minore-allele freq., strength of linkage disequilibrium, effect sizes of alleles - GWAS has a case-control format - two large groups of indiviuals compared (one group = control ie healthy, the other group is diseased) - all genotyped for common SNPs - see if allele freq. is altered between case and control - allele count of each evaluated (use chi squared test to identify variants associated in trait tested) ; mechanism for reporting effect sizes/quantifying strength of association is odds ratio - measures association between a genotype and being diseased
null hypothesis of chi squared test is that there is no association between given marker and disease labels ; a low p value mean sthe observed data is unlikely under the null hypothesis thus can reject null hypothesis and declare an association between SNP and disease state
Manhattan plots - statistical summary for GWAS , y-axis : neg log of p values obtained for association tests, x-axis : chromosome 1-22, X, Y etc // each dot on plot represents a SNP // strongest associations have smallest p values thus their negative logarithms will be the greatest
genome wide significance = specific threshold for determining the statistical significance of a reported association between a single-nucleotide polymorphism (SNP) and given trait // most common accepted threshold: 5e-8 , if p value is lower than this the null hypothesis of no association is rejected
Heritability = measure of the contribution of genetics to phenotype - values between 0-1 (1= excusively due to genetic factors)
Vp = Varience due to genes (VG) + variance due to organisms environment : VP = VG + VE
H2b = broad sense heritability: H2b = VG/VP
h2 = narrow sense heritability : h2 = VA/VP
Twin studies and Hertability: higher proportion of genetic disease in monozygotic twins than dizygotic twins
ACE Model: A = additive genetic variation ie genetic similarities and differences between twins, C = common environment -> =1, E = unique/non-shared variance ie if twins go to diff schools
falconers formula underlies the ACE model : rMZ = A+C, as monozygotic twins share two of the three components, rDZ = A/2 + C
can calculate difference bwteen MZ and DZ correlations: A = 2 (rMZ -r DZ), where A = additive genetic variance
polygenic, complex traitsinvolving variants in multiple genes may appear continuous, quantitative eg height // some traits that are determine dby multiple genes can appear dichotomous (binary - you have or dont have the trait) - are also polygenic but pehnotyoe only evident when liability threshold reached
if youre related to someone w/a complex disorder involving multiple genes you have a greater susceptibility due to the greatr proportion of shared genes