2024 BIOL 116 pathology lectures.pdf

Molecular pathology: connecting genotypes to phenotypes Mel Bird [email protected] Learning objectives u Define molecular pathology – what is it, and why is it important? u Describe the differences between a loss of funct...

Molecular pathology: connecting genotypes to phenotypes Mel Bird [email protected] Learning objectives u Define molecular pathology – what is it, and why is it important? u Describe the differences between a loss of function (LOF) and gain of function (GOF) mutation. u Describe the importance of protein structure and the consequences of disruption to this u Describe the genomic aberrations that can cause loss of function (LOF) phenotypes (gene deletion, chromosomal rearrangement, promoter deletion) and give examples of diseases caused by LOF mutations. u Describe the genome aberrations that can cause gain of function (GOF) phenotypes (chromosomal rearrangements, missense mutations, toxic RNA/protein products) and give examples of diseases caused by GOF mutations. Molecular pathology: the study and diagnosis of disease on a molecular level u In our context, it’s looking for how genetics affect human phenotypes u Older methods: clinical subjects (families, patients) painstaking search for the chromosome region, possibly a candidate gene with a mutation are 20,000 of them…. Molecular pathology u Now: whole exome sequence u Identifies all variants in the protein coding regions are 20,000 of them…. What is an exome? What is an exome? Molecular pathology BUT – there are 20,000 of them…. Loss vs. gain of function LOSS OF FUNCTION MUTATIONS GAIN OF FUNCTION MUTATIONS  Loss vs. gain of function LOSS OF FUNCTION MUTATIONS GAIN OF FUNCTION MUTATIONS u Gene has lost its function u Gene has taken on a new function loss of function = no gene product respond to incorrect signals Does my variant cause a loss or gain of function? Loss vs. gain of function LOSS OF FUNCTION MUTATIONS GAIN OF FUNCTION MUTATIONS u Gene has lost its function u Gene has taken on a new function u Can be total or partial respond to incorrect signals u Can affect all functions or just one u Total loss of function = no gene product Does my variant cause a loss or gain of function? Loss vs. gain of function LOSS OF FUNCTION MUTATIONS GAIN OF FUNCTION MUTATIONS u Gene has lost its function u Gene has taken on a new function u Can be total or partial u Not usually a completely new function u Can affect all functions or just one u Failure of gene regulation (product expressed in wrong place at wrong u Total loss of function = no gene time) product u May respond to incorrect signals Does my variant cause a loss or gain of function?  What does the variant do? u What the variant does to the gene, AND what the variant does to the person u Healthy people have variants that may ablate gene function u These don’t have to be pathogenic. u Identifying a variant alone isn’t enough – you need to know what it does to the person. How do we get loss of function mutations? u Gene deletion or disruption u Promoter deletion or alteration Gene deletion and loss of function Complete gene deletion leads to protein products absent Gene deletion and loss of function Deletion of first exon prevents accurate translation Gene deletion and loss of function Deletion of last exon (which is a stabilizer of mRNA) may result in no protein product. Gene deletion and loss of function Deletion (or duplication) of an internal exon might lead to frameshift mutation, stops functional protein being produced OR may alter 3D structure of protein, also affecting function Toxic proteins u Proteins need their shape u Stable (non-soluble) proteins need to have their hydrophobic (water-hating) residues on the inside u Hydrophobic residues bind and give the protein stability and shape u Chaperone proteins - recognise misfolded proteins and help them refold Aggregate proteins u Misfolded proteins can form aggregates u Common cause of neurodegenerative disease u Alzheimer’s disease – mutation leads to abnormally folded protein Aggregate proteins u Misfolded proteins can form aggregates u Common cause of neurodegenerative disease u Alzheimer’s disease – mutation leads to abnormally folded protein u Amyloid plaques/fibrils u 70% of dementia is due to Alzheimer’s disease – affects 30 million people. Promoter alteration and loss of function u Promoters live upstream from genes u Bound by transcription factors and RNA polymerase u Absolutely critical for gene regulation Promoter alteration and loss of function e.g. Pyruvate kinase deficiency Metabolic disorder affecting red blood cells Promoter alteration and loss of function e.g. Pyruvate kinase deficiency Mutations in promoter of PKLR gene u G > C mutation u Disrupts PKR-RE1 regulatory element u A > G mutation u Disrupts GATA1 binding site u Reduces PKLR mRNA levels Promoter alteration and loss of function e.g. Pyruvate kinase deficiency Promoter alteration and loss of function e.g. Pyruvate kinase deficiency u Final enzyme in glycolysis (cell turning glucose into energy) u RBC can’t make energy – dies u In the patient, this results in anaemia.. and a bunch of other things Gain of function mutations u GOF mutations are RARE Gain of function mutations u GOF mutations are RARE u The mutation still needs a product to be produced – most mutations prevent a product being made Gain of function mutations u GOF mutations are RARE u The mutation still needs a product to be produced – most mutations prevent a product being made u GOF usually arise from missense changes to a protein, or a change in regulatory sequences Gain of function mutations u GOF mutations don’t usually result in a novel function u The protein does it’s normal thing, but in an abnormal way u A receptor might signal, in the absence of its ligand.  Gain of function mutations e.g. Alpha-1-antitrypsin (AAT) u Protects lungs from elastin molecules u Elastin can harm body tissues u AAT degrades it tion = bleeding disorder Gain of function mutations e.g. Alpha-1-antitrypsin (AAT) u Protects lungs from elastin molecules u Elastin can harm body tissues u AAT degrades it u P.Met358Arg mutation – AAT binds thrombin, not elastin u Thrombin = key in blood clotting u Leads to bleeding disorder AND elastin damage GOF mutations in cancer – how do they work? 1) Extra copies of active gene – increases amount of gene 2) Chromosomal rearrangements Oncogene under control of enhancer (increase expression) Create novel chimeric genes (combine to exons) 3) Missense changes alter protein properties Summary u Molecular pathology is about connecting genotypes to phenotypes, looking at disease on a molecular level u Mutations are either loss of function (LOF) or gain of function (GOF) u Toxic proteins and aggregates can be either LOF or GOF, but are another consequence of gene mutation u LOF caused by gene or promoter deletions, chromosomal rearrangements & missense changes and make up the vast majority of mutations e.g. Pyruvate kinase deficiency u GOF are rare, caused by missense changes to a protein, or changse in regulatory sequences e.g. Alpha-1-antitrypsin (AAT) up next: Cancer and the cell cycle Learning objectives u Understand the cell cycle. u Explain the three internal control checkpoints u Describe how cancer is caused by uncontrolled cell growth u Understand how proto-oncogenes are normal genes that, when mutated, become oncogenes u Describe how tumour suppressor genes function u Explain how mutant tumour suppressors cause cancer Cancer is many different diseases u Uncontrolled cell growth is a hallmark of all cancers, regardless of their cause u To understand cancer, we need to understand the cell cycle. Cell cycle refresher u Growth and cell division = daughter cells Cell cycle refresher u Two regulared phases: mitosis and interphase Cell cycle refresher u G1 phase – accumulating building blocks of DNA, energy stores Cell cycle refresher u S phase – DNA is replicating Cell cycle refresher u G2 phase – synthesises proteins for mitosis Control of the cell cycle – why do we need checkpoints and regulators? u To maintain a stable genome, daughter cells need to be exact duplicates of parental cells regulators, to make sure the cell cycle is completed properly. Control of the cell cycle – why do we need checkpoints and regulators? u To maintain a stable genome, daughter cells need to be exact duplicates of parental cells u There are DNA repair mechanisms, but these don’t always work with 100% accuracy properly. Control of the cell cycle – why do we need checkpoints and regulators? u To maintain a stable genome, daughter cells need to be exact duplicates of parental cells u There are DNA repair mechanisms, but these don’t always work with 100% accuracy u There are internal checkpoints, and external regulators, to make sure the cell cycle is completed properly. Internal checkpoints u Three internal cell cycle checkpoints can’t proceed to S phase….. Internal checkpoints u Three internal cell cycle checkpoints u G1 – are the conditions favourable to proceed? Is the cell the right size? Does it have enough energy? Is there any DNA damage? u No? You can’t proceed to S phase….. Internal checkpoints u Three internal cell cycle checkpoints u G2 – Is the cell the correct size? Does it have all the proteins it needs for mitosis? Have all the chromosomes been replicated? Are they intact? u No? You can’t move on to mitosis yet…. Internal checkpoints u Three internal cell cycle checkpoints u M – Are the sister chromatids attached to the microtubules? u No? The cell cycle will stop! Control of the cell cycle - molecules u Control molecules: u Move the cell to the next phase of the cycle (positive, proto-oncogenes) u Halt the cycle (negative, tumour suppressors) Control of the cell cycle - molecules u Control molecules: u Move the cell to the next phase of the cycle (positive, proto-oncogenes) u Halt the cycle (negative, tumour suppressors) u They may act themselves, or work with other proteins to do their job. u Sometimes, more than one mechanism controls the same event, so a failure of a single regulator might not impact the cell cycle. Control of the cell cycle - molecules u Control molecules: u Move the cell to the next phase of the cycle (positive, proto-oncogenes) u Halt the cycle (negative, tumour suppressors) u They may act themselves, or work with other proteins to do their job. u Sometimes, more than one mechanism controls the same event, so a failure of a single regulator might not impact the cell cycle u BUT! Can be fatal to the cell if multiple processes affected. Cancer cells u Key characteristics of cancer cells Cancer cells u Key characteristics of cancer cells u They need to bypass or inactivate the anti-cancer control mechanisms u Multiple mutations are usually involved in the formation of a cancer cell. Mechanisms that cause cancer u Two main mechanisms that cause cancer u 1) Growth advantage u Daughter cells of mutant cell with a growth advantage are one step closer to becoming cancerous Mechanisms that cause cancer u Two main mechanisms that cause cancer u 1) Growth advantage u Daughter cells of mutant cell with a growth advantage are one step closer to becoming cancerous u 2) Genome destabilisation u A mutation may increase mutation rate and make the genome unstable (more likely to suffer structural abnormalities) Passenger and driver mutations u Cancers depend on mutations that increase growth rate AND stabilisation u Cancers develop in stages u Tumours are heterogeneous – cancer cells in a tumour are all different u How do you know which cell had the first mutation? The ‘driver’ mutation? u Which mutations are just background ‘passenger’ mutations? Passenger and driver mutations u Cancers depend on mutations that increase growth rate AND stabilisation u Cancers develop in stages u Tumours are heterogeneous – cancer cells in a tumour are all different u How do you know which cell had the first mutation? The ‘driver’ mutation? u Which mutations are just background ‘passenger’ mutations? Passenger and driver mutations u Cancers depend on mutations that increase growth rate AND stabilisation u Cancers develop in stages u Tumours are heterogeneous – cancer cells in a tumour are all different u How do you know which cell had the first mutation? The ‘driver’ mutation? u Which mutations are just background ‘passenger’ mutations? Passenger and driver mutations u Cancers depend on mutations that increase growth rate AND stabilisation u Cancers develop in stages u Tumours are heterogeneous – cancer cells in a tumour are all different u How do you know which cell had the first mutation? The ‘driver’ mutation? u Which mutations are just background ‘passenger’ mutations? Passenger and driver mutations u Cancers depend on mutations that increase growth rate AND stabilisation u Cancers develop in stages u Tumours are heterogeneous – cancer cells in a tumour are all different u How do you know which cell had the first mutation? The ‘driver’ mutation? u Which mutations are just background ‘passenger’ mutations? Protooncogenes u Protooncogenes are positive cell cycle regulators u When mutated, they are called oncogenes u Normal mutations lead to non- functional proteins, the cell will probably die (can’t complete cell cycle) u What about when a mutation increases the activity of a positive regulator?? u Subsequent generations of cells may accumulate more mutations in cell cycle regulators u Oncogene = a gene that increases rate of cell cycle progression when it’s mutated. Protooncogenes u Protooncogenes are positive cell cycle regulators u When mutated, they are called oncogenes u Normal mutations lead to non- functional proteins, the cell will probably die (can’t complete cell cycle) u What about when a mutation increases the activity of a positive regulator?? u Subsequent generations of cells may accumulate more mutations in cell cycle regulators u Oncogene = a gene that increases rate of cell cycle progression when it’s mutated. Protooncogenes u Protooncogenes are positive cell cycle regulators u When mutated, they are called oncogenes u Normal mutations lead to non- functional proteins, the cell will probably die (can’t complete cell cycle) u What about when a mutation increases the activity of a positive regulator?? u Subsequent generations of cells may accumulate more mutations in cell cycle regulators u Oncogene = a gene that increases rate of cell cycle progression when it’s mutated. Protooncogenes u Protooncogenes are positive cell cycle regulators u When mutated, they are called oncogenes u Normal mutations lead to non- functional proteins, the cell will probably die (can’t complete cell cycle) u What about when a mutation increases the activity of a positive regulator?? u Subsequent generations of cells may accumulate more mutations in cell cycle regulators u Oncogene = a gene that increases rate of cell cycle progression when it’s mutated. Protooncogenes u Protooncogenes are positive cell cycle regulators u When mutated, they are called oncogenes u Normal mutations lead to non- functional proteins, the cell will probably die (can’t complete cell cycle) u What about when a mutation increases the activity of a positive regulator?? u Subsequent generations of cells may accumulate more mutations in cell cycle regulators u Oncogene = a gene that increases rate of cell cycle progression when it’s mutated. Oncogenes u Natural role – promote cell proliferation u Human cancer can be caused by abnormal activation of oncogenes u Gain of function mutation – cells grow out of control u Can be activated in numerous ways. Oncogenes u Amplification – breast, ovarian, gastric, colon = multiple copies of ERBB2 (HER2) u Mutation/deletion – EGFR mutation in non-small- cell lung cancer u Chromosome translocation to form chimeric genes – chronic myelogeous leukaemia Oncogenes u Amplification – breast, ovarian, gastric, colon = multiple copies of ERBB2 (HER2) u Mutation/deletion – EGFR mutation in non-small- cell lung cancer u Chromosome translocation to form chimeric genes – chronic myelogeous leukaemia By Nephron - Own work, CC BY-SA 3.0, https://commons.wikimedia.org/w/index.php?curid=9584461 Oncogenes u Amplification – breast, ovarian, gastric, colon = multiple copies of ERBB2 (HER2) u Mutation/deletion – EGFR mutation in non-small- cell lung cancer u Chromosome translocation to form chimeric genes – chronic myelogeous leukaemia Tumour suppressor genes u Segments of DNA that code for negative regulator proteins u Roadblocks for cell cycle progression u Mutations mean the cell cycle can’t be halted u 50% of human cancers have mutated p53 – errors in DNA not detected, or may be unable to recruit the repair enzymes, or be unable to trigger apoptosis Tumour suppressor genes u Segments of DNA that code for negative regulator proteins u Roadblocks for cell cycle progression u Mutations mean the cell cycle can’t be halted u 50% of human cancers have mutated p53 – errors in DNA not detected, or may be unable to recruit the repair enzymes, or be unable to trigger apoptosis Tumour suppressor genes u Segments of DNA that code for negative regulator proteins u Roadblocks for cell cycle progression u Mutations mean the cell cycle can’t be halted u 50% of human cancers have mutated p53 – errors in DNA not detected, or may be unable to recruit the repair enzymes, or be unable to trigger apoptosis Tumour suppressor genes u Segments of DNA that code for negative regulator proteins u Roadblocks for cell cycle progression u Mutations mean the cell cycle can’t be halted u 50% of human cancers have mutated p53 – errors in DNA not detected, or may be unable to recruit the repair enzymes, or be unable to trigger apoptosis Tumour supressor genes u TS genes work to keep the cell under control p53 u They work by restraining or suppressing out of control cell division, or sentence broken cells to death u p53 is the most well known TS gene GENOME  P53 – the guardian of the genome u Transcription factor that regulates the cell cycle u It initiates the repair of e.g. double strand breaks u p53 gets phosphorylated (activated) and can transcribe genes such as: u P21 (CDKN1A) – inhibitor of cell cycling (pauses cell cycle so DNA can be repaired) u PUMA and NOXA – pro-apoptotic proteins (stimulate apoptosis to kill cell if DNA can’t be repaired)  P53 – the guardian of the genome u Transcription factor that regulates the cell cycle u It initiates the repair of e.g. double strand breaks u p53 gets phosphorylated (activated) and can transcribe genes such as: u P21 (CDKN1A) – inhibitor of cell cycling (pauses cell cycle so DNA can be repaired) u PUMA and NOXA – pro-apoptotic proteins (stimulate apoptosis to kill cell if DNA can’t be repaired)  P53 – the guardian of the genome u Transcription factor that regulates the cell cycle u It initiates the repair of e.g. double strand breaks u p53 gets phosphorylated (activated) and can transcribe genes such as: u P21 (CDKN1A) – inhibitor of cell cycling (pauses cell cycle so DNA can be repaired) u PUMA and NOXA – pro-apoptotic proteins (stimulate apoptosis to kill cell if DNA can’t be repaired)  P53 – the guardian of the genome u Transcription factor that regulates the cell cycle u It initiates the repair of e.g. double strand breaks u p53 gets phosphorylated (activated) and can transcribe genes such as: u P21 (CDKN1A) – inhibitor of cell cycling (pauses cell cycle so DNA can be repaired) u PUMA and NOXA – pro-apoptotic proteins (stimulate apoptosis to kill cell if DNA can’t be repaired)  P53 – the guardian of the genome u Transcription factor that regulates the cell cycle u It initiates the repair of e.g. double strand breaks u p53 gets phosphorylated (activated) and can transcribe genes such as: u P21 (CDKN1A) – inhibitor of cell cycling (pauses cell cycle so DNA can be repaired) u PUMA and NOXA – pro-apoptotic proteins (stimulate apoptosis to kill cell if DNA can’t be repaired) P53 can be lost my mutation or deletion Most common single genetic change in cancer  Summary u Cancer is a heterogeneous disease, with multiple causes u Cancer forms when there are problems with the genetic control of cell proliferation and cell death u Multiple mutations are needed to enable a cancer cell to form, and these mutations need to confer a growth advantage AND destabilise the genome u Activation of oncogenes (via gene amplification, mutation/deletion or chromosome abnormalities) enable the formation of cancer cells u Tumour suppressor genes guard the genome against potential cancer-causing changes, by initiating DNA repair or signaling destruction of an unrepairable cell u Colorectal cancer is the result of a series of cumulative mutations in tumour suppressor and oncogenes