1 Population genetics: technology driven Which genotyping technique - - PowerPoint PPT Presentation

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1 Population genetics: technology driven Which genotyping technique - - PowerPoint PPT Presentation

Feb 14, 2023 280 likes •396 views

SNP-analysis Linda Broer (l.broer@erasmusmc.nl) Genetic Laboratory Department of Internal Medicine Erasmus MC, Rotterdam Why do we study DNA variants? Population genetics: technology driven Biology: Time required for genotyping 1 SNP

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SNP-analysis

Linda Broer (l.broer@erasmusmc.nl) Genetic Laboratory Department of Internal Medicine Erasmus MC, Rotterdam

Why do we study DNA variants?

Biology: Mechanism: understand cause of disease Treatment: finding new potential drug targets Prediction: (Early) diagnostics with a stable marker Risk of disease (vulnerability): “personalized medicine” Response to treatment (medication, diet): “pharmacogenetics”

Population genetics: technology driven

  • Time required for genotyping 1 SNP in 7.000 DNA samples from “the Rotterdam Study”:
  • 1996

6 months: RFLP, Epp tubes

  • 1999

3 months: RFLP, 96-well plates

  • 2001

1 week: SBE, 384-well plates

  • 2003

1 day: Taqman (manual)

  • 2004

6 hrs: Taqman (automated)

  • 2005

3 hrs: Taqman, Deerac, “Fast” PCR

  • 2008

6 sec: Illumina 1000K array, 1000 DNAs/week 2010 0.00001 sec Illumia Hiseq, next-gen sequencing

  • 2015

0.000001 sec Illumina X10

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Population genetics: technology driven

  • Time required for genotyping 1 SNP in 7.000 DNA samples from “the Rotterdam Study”:
  • 1996

6 months

  • 1999

3 months

  • 2001

1 week

  • 2003

1 day

  • 2004

6 hrs

  • 2005

3 hrs

  • 2008

6 sec 2010 0.00001 sec

  • 2015

0.000001 sec

Association study with 1 DNA variant Association study with all common DNA variants in one gene Genome-wide association study

Sequencing: causal alleles?

Which genotyping technique to use? Array technology

Illumina bead-array Beads have probes of one SNP attached Each bead is spotted in multifold to increase accuracy and redundancy

Address Probe 23 bp 50 bp bead Fragmented gDNA SNP

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Array technology (and GWAS) based on “Linkage Disequilibrium” or “haplotypes”

SNP1 SNP2 SNP3 SNP4 SNP5 SNP6 A C T A C T G A T G C C Maternal chr Paternal chr Haplotype

Haplotypes movie Haplotypes and LD

Recombination is NOT random Hot spots In between recombination hot spots, variants are in LD They are correlated Often measured as r2 R2 = 1 : two variants provide same information

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How to use haplotypes: imputations

Correlation between variants used to ‘guess’ what the genotype of untyped variants is

Genetic architecture of traits Hemophilia in European royalty

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De-novo mutation

A very personal example

This is me

Genetic architecture of traits

Sequencing

Genetic architecture of traits

Arrays

GWAS analysis

Replication Select SNPs Meta-Analysis of all data Combine GWASs Analyzing all SNPs in 1 run Visualizing results in plots Manhattan-plot Each dot represents 1 SNP

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“ERGO”: The Rotterdam Study

A single-centre, prospective population-based cohort study, started 1990

  • Base-line cohort = 7,983 men and women of age ≥ 55 yrs
  • In 2017: 6 follow-up visits
  • ~1500 measurements per subject each time
  • Ethnically homogeneous: 99% Caucasian
  • Computerized GP + pharmacy monitoring
  • Study determinants and prevalence/incidence of chronic and disabling

disease in the elderly: CVD, Neurodegenerative Disease, Endocrine diseases, Locomotor disease (osteoporosis, osteoarthritis), Eye

  • ~12.000 DNA samples available:
  • 1990: ERGO base-line/ RSI : n=7,000
  • 2000: ERGO plus/ RSII : n=3,000 (55+)
  • 2004: ERGO young/ RSIII : 3,500 (45+)

First GWAS performed in Rotterdam Study

5 x 10-8

LUMBAR SPINE BMD

Rivadeneira et al., Nat Genet., 2009

Most GWAS activities occur within large consortia

Rotterdam Study

CHARGE

GEnetic Factors of OSteoporosis

GENETIC INVESTIGATIONS OF ANTHROPOMETRIC TRAITS

EUROPE by prejudice.…….(according to USA)

(From: Yanko Tsvetkov, alphadesigner.com)

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N=5,000

5 x 10-8

  • Rotterdam Study
  • ERF Study
  • Twins UK
  • deCODE Genetics
  • Framingham Study

LUMBAR SPINE BMD

Rivadeneira et al., Nat Genet., 2009

N=6,200

5 x 10-8

  • Rotterdam Study
  • ERF Study
  • Twins UK
  • deCODE Genetics
  • Framingham Study

LRP5

LUMBAR SPINE BMD

Rivadeneira et al., Nat Genet., 2009

N=8,500

5 x 10-8

  • Rotterdam Study
  • ERF Study
  • Twins UK
  • deCODE Genetics
  • Framingham Study

LRP5

LUMBAR SPINE BMD

Rivadeneira et al., Nat Genet., 2009

N=15,000

  • Rotterdam Study
  • ERF Study
  • Twins UK
  • deCODE Genetics
  • Framingham Study

LRP5 OPG MHC C6ôrf10 1p36 RANK-L

LUMBAR SPINE BMD

Rivadeneira et al., Nat Genet., 2009

5 x 10-8

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N=19,125

  • Rotterdam Study
  • ERF Study
  • Twins UK
  • deCODE Genetics
  • Framingham Study

1p36 C6ôrf10 LRP5 RANK-L OPG

LUMBAR SPINE BMD

SP7

Rivadeneira et al., Nat Genet., 2009

5 x 10-8

Consortia: working together does work

Much larger sample sizes can be achieved Go from competition to cooperation Creates better science! But… Only ‘cosmopolitan’ variants found Trying to set up a call with the US, Europe and Australia is impossible Can slow things down as you are waiting for each other Typical GWAS takes ~3-7 years

The success of consortia Example current GWAS study: age-related hearing loss

Heritability: 35-55% Previous GWAS studies (N~3,500) have not found anything Collected a bigger sample size (N~9,700) Redefined the phenotype HIGH: 4 & 8 kHz LOW: 0.5, 1 & 2 kHz

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Manhattan plot Chr 3 hit for HIGH phenotype ILDR1: further evidence

Knock-out mice are deaf Zebrafish knock-down have impaired auditory responses 11 families known with autosomal recessive mutations in ILDR1 Deafness is more pronounced at higher frequencies

Not always so easy…

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Ethnicity should be kept in mind

Africans have more variants than Europeans/Asians ‘Unique’ variants appeared in those that left Africa Adaptation to new environment Some of these came from already existing hominids outside Africa Frequencies of variants can differ between Ethnic groups

Example: rs776746

SNP in gene CYP3A5 which metabolizes clinical drugs G allele encodes CYP3A5*3 allele Inactivates the gene

What has/will GWAS achieve

E D. Green et al. Nature 470, 204-213 (2011) doi:10.1038/nature09764

Examples of links between GWAS discoveries and drugs

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Price GWAS measurement

100 200 300 400 500 600 700 800 2007 2008 2009 2010 2011 2012 2013 2014 2015 2016

28 euro:

GWAS + clinical research content, HLA typing, pharmacogenetics

Human Genomics facility (HuGE-F) FACILITY & RESEARCH

BIOBANKING

Rotterdam Study, GenR, Parelsnoer, BBMRI, many more

GENOTYPING

Bench marking with top institutes of the world

NEXT GEN SEQUENCING

Collaborations in large consortia

BIOINFORMATICS

GWAS, imputation, methylation analysis, exome and transcriptome analysis

TRANSCRIPTOMICS EPIGENETICS HIGH THROUGPUT ARRAYS MICROBIOMICS

Functional studies in mouse models and cell lines

www.glimdna.org

…..IGNORANCE CAN BE DAUNTING……EDUCATION IS IMPORTANT !!

Annual Courses organized by the Genetic Laboratory: in 2018:

  • “Genetic for Dummies” (MolMed): 15-30 participants
  • “SNPs and human diseases” (MolMed): 40-70 participants
  • “Genomics in Medicine” (ESP57; NIHES): 20-40 participants

www.molmed.nl www.nihes.nl Questions