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