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1 They have 3 things in common Rule breaker #1: telomeres What are - - PowerPoint PPT Presentation
1 They have 3 things in common Rule breaker #1: telomeres What are - - PowerPoint PPT Presentation
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
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Telomeres: driving force behind the Hayflick limit
Hayflick limit: number of times a cell can divide Once limit is reached senescence Telomere length (TL) determines this limit
Hayflick limit in action: Dolly
Only lived 7 years (instead
- f 10-12)
Telomere length (TL) with age
When cloning TL from somatic cell given to the clone
Regulation of TL
Telomerase Consists of TERT and TERC Telomerase down-regulated in all long-lived mammals 90% of carcinomas have active telomerase
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Telomerase regulation
Kim, W, et al. 2016, PLOS Biology
How to measure telomere length (TL)?
TRF (gold standard) qPCR Q-FISH STELA / TESLA 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
Analysis of TL
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
TL and gender
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TL itself is genetically determined
Broer, L, et al. 2013, Eur J Hum Genet
70% of variability explained by genetics
Largest GWAS to date
N ~ 38,000 All loci associated with telomerase
Codd, V, et al. 2013, Nat Genet
Known TL associations Cardiovascular Cancer COPD Alzheimer’s Osteoarthritis Rheumatoid Arthritis Osteoporosis Macular Degeneration Depression Age-related hearing impairment BMI Smoking Leptin Stress Lung function Height Etc. Keep an eye on sample size though…
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TL and mortality
Biologically plausible Long telomeres higher replicative ability cellular senescence postponed live longer Cancer mortality hypothesized to be associated with both short AND long telomeres Only half of studies show association Performed in oldest-old (85+ at baseline) selection Solution Long follow-up
Results in Rotterdam Study (1,073 deaths, 674 censored)
Linear TL Short VS average TL Long VS average TL Mortality outcome beta se p-value beta se p-value beta se p-value All-cause
- 0.130 0.032 4.18E-05
0.187 0.075 0.012 -0.098 0.077 0.203 Cancer 0.046 0.062 0.459 0.014 0.160 0.928 0.156 0.151 0.301 CHD
- 0.245 0.057 1.82E-05
0.339 0.126 0.007 -0.235 0.140 0.093
Broer, L et al. Unpublished
Telomerase: the future is calling!
Experiments in mice with telomerase gene therapy Tissue degeneration reversed No increase in cancer! Increased lifespan Not known if this translates to humans 90% of carcinomas have active telomerase Seen as important step for cancer development First port of call: telomerase inactivation as cancer treatment Should only affect cancer cells!
De Jesus, BB et al., 2012, EMBO Mol Med Jaskelioff, M et al., 2011, Nature
“Imagine a drug that’s almost too effective at killing cancer cells” Worst side-effect: cytopenia (reduction in number of mature blood cells) Not surprising as myelofibrosis is a chronic blood cancer
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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
Russell, LM et al., 2007, Cytogenet Genome Res Guttenbach, M et al., 1995, Am J Hum Genet
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
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Genetics of sex chromosome loss
Heritability 34% for Y loss 26% for X loss GWAS on Y loss 19 loci identified
Wright, DJ et al., 2017, nature Genetics
Known associations
Not much evidence yet, but a few have been described Decreased survival Increased cancer risk Smoking increases sex chromosome loss
Rule breaker #3: Mitochondrial DNA (mtDNA) Main function of mitochondria: ATP production
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Why do mitochondria have DNA? mtDNA
Most mitochondrial genes located in nuclear genome
How mtDNA differs Nuclear DNA Inherited from both parents Linear 3.2 billion base pairs 20,000 genes Varied by recombination Has introns Low mutation rate 2 copies mtDNA Inherited from mother only Circular 16,569 base pairs 37 genes No recombination No introns High mutation rate ~100-1,000 copies mtDNA measurements
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How to measure mtDNA
Copy number qPCR (gold standard) SNP arrays (under investigation) Sequence data (promising) Heteroplasmy Deep sequencing of mtDNA only r = 0.71
mtDNA copy number mtDNA copy number Known associations: mtDNA copy number (1)
60 70 80 90 100 5 10 15 20
Female Male
Age mtDNA copy number
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Known associations: mtDNA copy number (2)
Obesity Cancer CHD Diabetes Physical activity Smoking Frailty BMD Etc.
mtDNA copy number and mortality
Broer, L et al., unpublished
Mortality outcome beta se p-value All-cause
- 0.085 0.027
0.002 Cancer
- 0.120 0.056
0.034 CHD
- 0.137 0.048
0.004 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
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Difficulty with measuring mtDNA heteroplasmy
Nuclear MiTochondrial DNAs: NUMTs Blocks of mtDNA in nuclear genome with high homology Caused by ongoing process of organelle-to-nuclear DNA transfer NUMTs can thus differ between individuals Largest NUMT in humans covers 90% of mtDNA genome New sequencing method claims to have overcome this issue 1% frequency of heteroplasmy detectable
mtDNA heteroplasmy analysis
Unclear how to define for complex traits Overall Per mtDNA site Average burden per mtDNA molecule In healthy people, abundance of any given heteroplasmic site <1% Well below classical phenotypic expression threshold However, if enough heteroplasmic mutations are present they can still 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
25% 75%
At birth
Heteroplasmic Homoplasmic 50% 50%
Adult
Heteroplasmic Homoplasmic
Number of heteroplasmies per individual
Zhang, R et al., 2017, BMC Genomics
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Frequency of observed heteroplasmies
Zhang, R et al., 2017, BMC Genomics
63% at MAF<5%
Location of heteroplasmies
Zhang, R et al., 2017, BMC Genomics
~14% known pathogenic
One example: 3243A>G
Most common pathogenic mtDNA point mutation 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
Heteroplasmies might be curable
Theoretically all that is needed to treat mtDNA disease is to reduce the heteroplasmy level Reducing heteroplasmy is possible…in mice
Bacman, SR et al., 2018, Nat Med Gammage, PA et al., 2018, Nat Med
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