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1 They have 3 things in common Rule breaker #1: telomeres What are - PowerPoint PPT Presentation

Mar 22, 2024 •188 likes •334 views

DNA is stable Stability of DNA main selling point for genetic research Cause and effect is clear Can use DNA from blood to study brain Dynamic DNA: Challenges and Opportunities Linda Broer Disease l.broer@erasmusmc.nl Department of

  1. DNA is stable � Stability of DNA main selling point for genetic research � Cause and effect is clear � Can use DNA from blood to study brain Dynamic DNA: Challenges and Opportunities Linda Broer Disease l.broer@erasmusmc.nl Department of Internal Medicine Human Genetics Facility (HuGe-F) Rules are meant to be broken Meet the rule breakers… 1

  2. They have 3 things in common Rule breaker #1: telomeres What are telomeres? Function of telomeres: hide end of chromosomes 2

  3. Telomeres: driving force behind the Hayflick limit Hayflick limit in action: Dolly � Hayflick limit: number of times a cell can divide � Once limit is reached � senescence � Telomere length (TL) determines this limit Only lived 7 years (instead of 10-12) Telomere length (TL) with age Regulation of TL � Telomerase � Consists of TERT and TERC � Telomerase down-regulated in all long-lived mammals � 90% of carcinomas have active telomerase When cloning TL from somatic cell given to the clone 3

  4. Telomerase regulation How to measure telomere length (TL)? TRF qPCR Q-FISH STELA / TESLA (gold standard) DNA amount +++ + ++ ++ Labor intensity +++ + +++ ++ Skill level +++ + +++ ++ Longest TL + N/A ++ + Average TL +++ +++ ++ N/A Smallest TL + N/A ++ +++ TL per chromosome N/A N/A ++ + Established techniques Kim, W, et al. 2016, PLOS Biology Analysis of TL TL and gender � TL is linear variable � No different in analysis than BMI, age, etc. � Classical epidemiology issues � Confounding � Reverse causality � Usually measured in blood, not known how this links to other tissues � Different cell types in blood � Currently not considered, but should be Lin, J, et al. 2015, J Immuno Research 4

  5. TL itself is genetically determined Largest GWAS to date 70% of variability explained by genetics N ~ 38,000 All loci associated with telomerase Broer, L, et al. 2013, Eur J Hum Genet Codd, V, et al. 2013, Nat Genet Known TL associations Keep an eye on sample size though… � Cardiovascular � Age-related hearing impairment � Cancer � BMI � COPD � Smoking � Alzheimer’s � Leptin � Osteoarthritis � Stress � Rheumatoid Arthritis � Lung function � Osteoporosis � Height � Macular Degeneration � Etc. � Depression 5

  6. TL and mortality Results in Rotterdam Study (1,073 deaths, 674 censored) � Biologically plausible � Long telomeres � higher replicative ability � cellular senescence postponed � live longer Linear TL Short VS average TL Long VS average TL Mortality outcome beta se p-value beta se p-value beta se p-value � Cancer mortality hypothesized to be associated with both short AND All-cause -0.130 0.032 4.18E-05 0.187 0.075 0.012 -0.098 0.077 0.203 long telomeres Cancer 0.046 0.062 0.459 0.014 0.160 0.928 0.156 0.151 0.301 -0.245 0.057 1.82E-05 0.007 -0.235 0.140 CHD 0.339 0.126 0.093 � Only half of studies show association � Performed in oldest-old (85+ at baseline) � selection � Solution � Long follow-up Broer, L et al. Unpublished Telomerase: the future is calling! � Experiments in mice with telomerase gene therapy Worst side-effect: cytopenia (reduction in number of � Tissue degeneration reversed mature blood cells) � No increase in cancer! � Increased lifespan Not surprising as � Not known if this translates to humans myelofibrosis is a chronic � 90% of carcinomas have active telomerase blood cancer � Seen as important step for cancer development “Imagine a drug that’s � First port of call: telomerase inactivation as cancer treatment almost too effective at � Should only affect cancer cells! killing cancer cells” Jaskelioff, M et al., 2011, Nature De Jesus, BB et al., 2012, EMBO Mol Med 6

  7. Rule breaker #2: Sex chromosomes Sex chromosome loss with age � Preferential loss of X-chromosome in women � 0.07% at age <16 � 7.3% at age >65 � Preferential loss of Y-chromosome in men � 0.05% at age <15 � 1.34% at age >75 � Currently primarily shown in lymphocytes � Presence and/or rate of loss in other tissues unclear Guttenbach, M et al., 1995, Am J Hum Genet Russell, LM et al., 2007, Cytogenet Genome Res Inactive X-chromosome gets lost with age Measuring sex chromosome loss � In situ hybridization with chromosome-specific DNA probes � At metaphase of cell cycle � Using array intensities as proxy � Mean intensity value of all SNPs on X or Y chromosome � Negative intensity values indicate loss of chromosome 7

  8. Genetics of sex chromosome loss Known associations � Heritability � Not much evidence yet, but a few have been described � 34% for Y loss � Decreased survival � 26% for X loss � Increased cancer risk � GWAS on Y loss � 19 loci identified � Smoking increases sex chromosome loss Wright, DJ et al., 2017, nature Genetics Rule breaker #3: Mitochondrial DNA (mtDNA) Main function of mitochondria: ATP production 8

  9. Why do mitochondria have DNA? mtDNA Most mitochondrial genes located in nuclear genome How mtDNA differs mtDNA measurements Nuclear DNA mtDNA � Inherited from both � Inherited from mother only parents � Linear � Circular � 3.2 billion base pairs � 16,569 base pairs � 37 genes � 20,000 genes � No recombination � Varied by recombination � Has introns � No introns � Low mutation rate � High mutation rate � ~100-1,000 copies � 2 copies 9

  10. How to measure mtDNA mtDNA copy number � Copy number � qPCR (gold standard) � SNP arrays (under investigation) � Sequence data (promising) r = 0.71 � Heteroplasmy � Deep sequencing of mtDNA only mtDNA copy number Known associations: mtDNA copy number (1) 20 Female Male mtDNA copy number 15 10 5 0 60 70 80 90 100 Age 10

  11. Known associations: mtDNA copy number (2) mtDNA copy number and mortality � Obesity � Cancer � CHD � Diabetes Mortality outcome beta se p-value � Physical activity All-cause -0.085 0.027 0.002 � Smoking Cancer -0.120 0.056 0.034 � Frailty CHD -0.137 0.048 0.004 � BMD � Etc. Broer, L et al., unpublished mtDNA heteroplasmy mtDNA heteroplasmy � Due to high mutation rate (5-15x nuclear genome) � not all mtDNA molecules are identical � Heteroplasmy: mixture of two or more mtDNA genotypes in the cell 11

  12. Difficulty with measuring mtDNA heteroplasmy mtDNA heteroplasmy analysis � Nuclear MiTochondrial DNAs: NUMTs � Unclear how to define for complex traits � Blocks of mtDNA in nuclear genome with high homology � Overall � Caused by ongoing process of organelle-to-nuclear DNA transfer � Per mtDNA site � NUMTs can thus differ between individuals � Average burden per mtDNA molecule � Largest NUMT in humans covers 90% of mtDNA genome � In healthy people, abundance of any given heteroplasmic site <1% � Well below classical phenotypic expression threshold � New sequencing method claims to have overcome this issue � However, if enough heteroplasmic mutations are present they can still � 1% frequency of heteroplasmy detectable be deleterious � Overall burden of heteroplasmy most likely of interest � Burden in specific regions of mtDNA should not be ignored Percentage of individuals harboring at least 1 heteroplasmic site Number of heteroplasmies per individual At birth Heteroplasmic Homoplasmic 25% Adult Heteroplasmic Homoplasmic 75% 50% 50% Zhang, R et al., 2017, BMC Genomics 12

  13. Frequency of observed heteroplasmies Location of heteroplasmies 63% at MAF<5% ~14% known pathogenic Zhang, R et al., 2017, BMC Genomics Zhang, R et al., 2017, BMC Genomics One example: 3243A>G Heteroplasmies might be curable � Most common pathogenic mtDNA point mutation � Theoretically all that is needed to treat mtDNA disease is to reduce the heteroplasmy level � Reducing heteroplasmy is possible…in mice � Heteroplasmy of 50-90% MELAS syndrome � Weakness of muscles throughout the body (M) � Encephalopathy - central nervous system (E) � Abnormal build-up of lactic acid (LA) � Stroke (S) � Lower levels of heteroplasmy associated with � Autism � Diabetes Gammage, PA et al., 2018, Nat Med Bacman, SR et al., 2018, Nat Med 13

  14. The fourth rule breaker … somatic mutations Take-home messages � DNA is stable, but not everyone has received the memo � Telomere length, sex chromosomes and mtDNA don’t follow our rules � Neither do somatic mutations � Studies have been small and/or incomplete � Cell-type specificity often ignored � Cause/effect often ignored � Replication often not performed � Telomere length and mtDNA copy number/heteroplasmy might be modifiable Questions 14

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