DUMMIES COURSE FOR PEOPLE who want to know more about genetics - - PowerPoint PPT Presentation

GENETICS

FOR

DUMMIES

COURSE

FOR PEOPLE who want to know more about genetics INTRODUCTION to BASIC GENETICS

ANNEMIEKE VERKERK j.verkerk@erasmusmc.nl

First Day, Wednesday 1 November, room OWR-72

Moderators: Annemieke Verkerk & Robert Kraaij Time Speaker Subject 09.15-09.30 Registration and coffee 09.30-10.30 Annemieke Verkerk Basic genetics: history & concepts 10.30-10.45 Coffee break 10.45-11.45 Robert Kraaij Molecular genetics (1) 11.45-12.00 Coffee break 12.00-13.00 Robert Kraaij Molecular genetics (2) 13.00-13.45 Lunch 13.45-14.45 André Uitterlinden Introduction to complex genetics (1) 14.45-15.00 Coffee break 15.00-16.00 André Uitterlinden Introduction to complex genetics (2)

SOME

HISTORY

CHARLES DARWIN (1809-1882) UK

1859 : launched evolution theory _life evolves from one species to another _evolution is driven by natural selection _and characteristics can somehow be past on to next generations and change

1860 CHARACTERISTICS ARE TRANSMITTED

Mendel (Austria) 1822 - 1884

WHAT IS MENDELIAN INHERITANCE ?

BB

X

bb

X

Bb Bb Bb Bb BB bb Bb Bb

1869 Friedrich Miescher (Switserland) 1878 Albrecht Kossel (Germany) 1919 Phoebus Levene (Litouwen, USA) 1928 Frederick Griffith (UK) 1927 Nikolai Koltsow (Russia) 1937 William Astbury (UK) 1943 Oswald Avery (Canada, USA) Colin MacLeod (Canada) Maclyn McCarty (USA) 1951 Linus Pauling (USA) 1952 Alfred Hershey, Martha Chase (USA) 1952 Rosalind Franklin, Maurice Wilkins (UK) 1953 Francis Crick, James Watson (USA)

SEARCH FOR THE “inheritance molecule”

X-RAY CRYSTALLOGRAPHY

1953 London’s King’s College: “DNA is a molecule in which two “strands” form a tightly linked pair”. Rosalind Franklin Maurice Wilkins

Watson and Crick proposed: that the structure of DNA was a winding helix in which pairs of bases Adenine -- Thymine Guanine --- Cytosine held the two strands together.

James Watson (age 25; still alive!) Francis Crick (age 36)

MODEL OF THE DNA DOUBLE HELIX

Publication of Watson and Crick in Nature in April 1953

In 1962 Francis Crick, James Watson and Maurice Wilkins were awarded the Nobel Prize in Physiology or Medicine, for their discovery of the structure of DNA Rosalind Franklin, had passed away before then (1958).

SOME OTHER MILESTONES

1970 Discovery that DNA can be cleaved by Restriction Enzymes and isolation of the first restriction enzyme HindII Hamilton O. Smith (Nobel Prize Physiology/Medicine 1978) 1977 Development of the DNA Sanger sequencing method Fred Sanger (Nobel Prize Chemistry 1958) 1985 DNA can be replicated in vitro by Polymerase Chain Reaction – PCR Kary Mullis (Nobel Prize Chemistry 1993) 1989 Introduction of the term fingerprinting and use of DNA polymorphisms in paternity testing and murder cases…, using PCR Alec Jeffreys (Knighted by Queen Elizabeth in 1994) 1988-1989 Identification of the gene for Cystic Fribrosis on chromosome 7, the first disease gene identified by positional cloning Francis Collins and Lap-Chee Tsui 1990 Start Human Genome Project – sequence total human DNA 1998 first large scale detection of Single Nucleotide Polymorphisms (SNPs) using PCR and Sanger sequencing

SOME OTHER MILESTONES

1999 start of The SNP consortium: aim: to discover 300.000 SNPs in the human genome in two years result: described 1.4 million SNPs 2001-2003 Human Genome Project : from working draft to “finished” Sequence MAP 2002 start International HapMapProject: aim: investigate SNPs in populations 2005 Start publications on Population studies with Genome Wide Association Studies (GWAS) 2007 Start development techniques for Next Generation Sequencing 2010 First paper on gene finding with Next Generation Sequencing 2012 ENCODE project consortium: our junk DNA isn’t junk: many regulatory elements present 2011-2017 Development op Non-invasive prenatal testing = NIPT test 2014-2017 Clinical exome sequencing

HUMAN

GENETICS

BASICS

body

some NUMBERS

Cells in your body: Cell nucleus: contains DNA consisting of chromosome pairs Length of DNA in one nucleus: m Genome: base pairs

codes for genes

100.000.000.000.000 cells = 1014

23

22.000 3.000.000.000 = 3 x 109

2

THE GENOME

 

= the entire genetic information of an organism stored in all chromosomes together, all genes (coding) + non-coding sequences , all basepairs

chromosomes provided by Diane van Opstal and Laura van Zutven Clinical Genetics EMC

BUILD UP of a CHROMOSOME

The complex of DNA and attached proteins in the cell nucleus = Chromatin = very tightly packed 2 sorts of chromatin (after G-banding coloring): Dark bands : Heterochromatin: very compact and not very active Light bands :Euchromatin: less compact and contains most genes and is active 1. 2. 3. 4. 5. DNA consist of 4 bases: A : adenine T : thymine G : guanine C : cytosine bases form pairs (G-C and A-T)

• n a backbone of

_dexoyribose sugar _phosphate groups

KARYOGRAM

All chromosomes in somatic cells are in pairs = 2n = diploid

TYPES OF

CHROMOSOMES

telomere telomere centromere Acro-centric 13 14 15 21 22 Telo-centric not in humans mouse Meta-centric arms equal length 1 2 3 6 Sub-metacentric arms unequal length 4 5 8 9 10 11

short arm -p long arm -q

12 16 17 18 X Y

p-arm q-arm

7 19 20

REPLICATION

1 pair sister chromatids homologous chromosomes

MITOSIS DIVISION OF SOMATIC CELLS

transcription occurs in “interphase” DNA less condensed = chromatin chromosomes have doubled (replication) genes are not transcribed (S phase = synthesis DNA is condense (prophase) Chromosomes line up in the middle (metaphase) Sister chromatids are separated, are again independent chromosomes (anaphase) 2 new cells with normal chromosome content (telophase)

https://www.khanacademy.org/science/biology/cellular-molecular-biology/mitosis/v/mitosis

MITOSIS DIVISION OF SOMATIC CELLS

http://www.youtube.com/watch?v=AhgRhXl7w_g als wmv

MEIOSIS

GERM CELL DIVISION

https://www.khanacademy.org/science/biology/cellular-molecular-biology/meiosis/v/comparing-mitosis-and-meiosis

Meiosis I: recombination between homologous chromosomes and separation of homologous chromosomes Meiosis II: separatio of the sisterchromatids

MEIOSIS

GERM CELL DIVISION

during production of germ cells chromosomes exchange pieces of DNA = recombination or crossing over to create genetic diversity

• 1. recombination

between homologous chromosomes

• 2. pairs of chromosomes

are separated

• 3. sister chromatids are separated

MEIOSIS DIVISION OF GERM CELLS

https://www.youtube.com/watch?v=D1_-mQS_FZ0

STRUCTURE OF A GENE

a promoter region: binding of proteins for regulation 5’ UTR region : for stability of mRNA Exons: together code for a protein Introns: separate exons, are non coding 3’ UTR region: for stability of mRNA splice sites: on the exon-intron boundaries, sequense necessary for correct splicing out of introns

Exon1 Exon2 Exon3 Intron1 Intron2 Promoter

DNA

3’UTR 5’UTR

GT………….….AG GT………….….AG AG AG G-T/A G-T/A

all exons from all 22.000 genes together are called the EXOME

Consists of:

GT………….….AG GT………….….AG

Exon1 Exon2 Exon3 Intron1 Intron2 Promoter

DNA

Splicing Transcription Translation

3’UTR

FROM GENE TO PROTEIN

5’UTR

pre-mRNA Protein

amino acids

mRNA

DNA versus RNA

Deoxyribonucleic - Ribonucleic acid double strand - single strand stable - unstable

H H H H CH3

Uracil

OH OH OH OH

GENE REGULATION

methylation from a distance histon modification

e p i g e n e t i c s

http://www.roadmapepigenomics.org/

http://www.nature.com/news/epigenome-the-symphony-in-your-cells-1.16955 GENE METHYLATION AND EXPRESSION EXPLAINED WITH BEETHOVEN

miRNA

22 nt

lncRNA

> 200 nt

http://www.nature.com/nmeth/journal/v8/n5/pdf/nmeth0511-379.pdf

pseudo genes

http://www.pseudogene.org/

HUMAN

GENETICS

diseases

Phenotype : all physical and mental properties of an organism Genotype: the order and composition of your base pairs Your genotype largely determines your phenotype

PHENOTYPE-GENOTYPE

HUMAN DISEASES CAUSED BY MISTAKES IN THE DNA

   

INHERITED MUTATION



new mutation

• ccurs in germ cell

parent

• r

arises in the early embryo

NEWLY ARISEN

  

DE NOVO MUTATION

BASIC AIM IN HUMAN GENETICS

TO FIND THE GENETIC CAUSE OF A HEREDITARY DISORDER ASSIGN A DEFINITE DIAGNOSIS ! connecting the phenotype with a genotype STUDY AND UNDERSTAND THE CAUSE OF DISEASE DEVELOP MEDICINE and/or TREATMENT CURE?? PREVENTION?? MUTATION IN THE DNA

TYPES OF DISEASES I

Mendelian disorders - monogenic one disease – one genedefect Autosomal or X-linked -- dominant or recessive Complex disorders Interactions between (many) genes and environment Chromosomal disorders Translocations, inversions, deletions, aneuploidy Somatic genetic disorders Predisposition to cancer Mitochondrial diseases (mutation in mitochondrial DNA)

TYPES OF DISEASES II

“Complex” diseases more than one gene involved

• genetic background plays a role
• environment plays a role

thousands of people needed

combinations of Variations in DNA

are risk factors small effect

• ne or a few families needed

caused by Mutation in DNA large effect “Simple” diseases monogenetic

• ne disease – one genedefect

more severe phenotype

• ften early onset

relatively rare Mendelian inheritance e.g. cystic fibrosis,

• steogenesis imperfecta,

intellectual disability, Duchennes muscular dystrophy mild phenotype late onset common complex inheritance e.g. osteoporosis, diabetes, asthma, heart disease, stroke, psychiatric disorders

1992 1998 1966 1994 1968 1971 1975 1978 1983 1985 1988 1990

1966- 1998

Around 6600 monogenetic DISEASES 5108 - phenotype descriptions, molecular basis known 1596 - phenotype descriptions, molecular basis unknown

http://www.omim.org Update from October 2017

= database of human disorders; phenotype descriptions gene descriptions

OMIM

• nline mendelian inheritance

in man

1985

MODELS OF INHERITANCE

autosomal dominant: describes any trait that is expressed in a heterozygote (one mutant allele, one normal allele) autosomal recessive: manifests only in a homozygote (two mutant alleles) X-linked: males are hemizygous (XY) recessive: males are affected; females are unaffected carriers dominant: males are affected; females are “affected”, but often compensate by inactivation of the affected copy not or mildly affected

SYMBOLS USED IN PEDIGREES

healthy carrier

http://haplopainter.sourceforge.net/

50% chance on a healthy child 50% chance on an affected child Affected person has to have an affected parent

Examples: _Osteogenesis imperfecta type I: mutations in the COL1A1/2 genes chr. 17 and 7 _Polycystic kidney disease: mutation in PKD1 gene on chr. 16p13

AUTOSOMAL DOMINANT

• ne parent has the disease

carries a mutation

25% chance on a healthy child 25% chance on an affected child 50% chance on healthy carrier child

Example: _cystic fibrosis (1/30 persons carrier of a mutation in the CFTR gene on chr. 7q31 70% of carriers have the same mutation)

AUTOSOMAL RECESSIVE

both parents healthy carriers

AUTOSOMAL RECESSIVE WITH CONSANGUINITY

Often in first or second cousin marriages _parents have a common ancestor who carries a mutation on one allele _parents share the same chromosome(s)

• r part of a chromosome and are healthy

carriers of the same gene mutation affected children are homozygous for that area = homozygosity common ancestor

AUTOSOMAL RECESSIVE

WITH COMPOUND HETEROZYGOSITY 25% chance on a healthy child 25% chance on an affected child 50% chance on healthy carrier Example: cystic fibrosis (1/30 persons carrier of mutation in the CFTR gene on chr. 17, but not everybody has the same mutation)

both parents are healthy carriers but have a different mutation in the same gene

X - LINKED

sons of carriers have a 50% chance to be affected daughters of carriers have a 50% chance to be a carrier Example: _colourblindness (Xq28, more genes) _Duchenne muscular dystrophy, DMD gene on Xp21

women are healthy carriers

• f a mutation on
• ne X-chromosome

SOME GENETIC TERMS

locus: a spot on a (pair of) chromosome and consists of 2 alleles locus 1 allele 1 allele 2 locus 2 allele 1 allele 2 locus 3 allele 1 allele 2 haplotype 1 haplotype 2 homologous chromosomes = pair of chromosomes

REPEATS / VARIATIONS

Besides genes DNA contains repeats and variations: Repeats Satellite repeats: in centromeres and telomeres Alu repeats: > 1 million copies, average 300 bp long, (10% of the genome) (is recognized by an enzyme derived from the bacterium Arthrobacter luteus) Low copy number repeats (LCRs) 10-500 kb with > 95% sequence identity ( 5% of the genome) Trinucleotide repeats: CGG, CAG (14 involved with diseases) Dinucleotide repeats (3% of the genome consists of di (CAn) and tetranucleotide repeats) Variations SNPs = single nucleotide polymorphisms (1 in 200-300 base pairs per genome -- freq > 1%) now > 170 million SNPs are in dbSNP CNV’s = copy number variations _deletions _amplifications (12 % of the genome, size: 1 – few 1000 of kb) Other things: insertions, inversions Combinations of these variations make every person unique

EXAMPLE OF CA repeat

TAGAACAAAATAGCGGTATTTTGGGGAGTCTGACTGACCATTGG ACTAGGGGATTGACCAGTAGGCTGCGATTCGGATGCGGATTGAC GATTAAAAAGGCTGACCAGAACCATGTTATAAAGAATGCTGGGC GGCACACACTGTTCNCCCACCTACTCTGGAGACTGAGGCTCAAG GATTGCTTGAGCCCAGGAATTCGGGGCTGCAGTGAGCCATGATT GTGTCACTGTATTCCAGCCTGGATGACAGAGTAAGACCCTGTCC TTCTCTCTCTCTCTTCCTCTTTGGTCTCTCTCGCTCTGTTTCTC TCTCTCTCTCTTATA CTACTGGGAAAGTGAATGTTT GTTTTCCTCGCCANTAGTGGAAGCTATTACGATTAGCTGTGACG TGCAGGATGCTGCGATGCTGGACTGAACGCCCCCCGGGCTTCTT TATTAGCTGCTGACGTGCCAGATGCTGACGTGCAGTGAGGAGTC TGACTGACCATTGGACTAGGGGATTGACCAGTAGGCTGCGATTC GGATGCGGATTGACGATTAAAAAGGATTACGATTAGCTGTGACG TGCAGGATGCTGCGATGCTGGACTGAACGCCCCCCGGGCTTCTT TATTAGCTGCTGACGTGCCAGATGCTGACGTGCAGTGCGGCTGA CGGTGCTTACCTGGATCGGATGCTACCAGTCGATCGATCGATCG TAGCGTAGCGTATGCTAGCTAGTGATCGATGCTAGTAGCTAGCT TAGAACAAAATAGCGGTATTTTGGGGAGTCTGACTGACCATTGG ACTAGGGGATTGACCAGTAGGCTGCGATTCGGATGCGGATTGAC GATTAAAAAGGCTGACCAGAACCATGTTATAAAGAATGCTGGGC GGCACACACTGTTCNCCCACCTACTCTGGAGACTGAGGCTCAAG GATTGCTTGAGCCCAGGAATTCGGGGCTGCAGTGAGCCATGATT GTGTCACTGTATTCCAGCCTGGATGACAGAGTAAGACCCTGTCC TTCTCTCTCTCTCTTCCTCTTTGGTCTCTCTCGCTCTGTTTCTC TCTCTCTCTCTTATA CTACTGGGAAAGTGAATGTTTGTTTTCCTCG CCANTAGTGGAAGCTATTACGATTAGCTGTGACGTGCAGGATGC TGCGATGCTGGACTGAACGCCCCCCGGGCTTCTTTATTAGCTGC TGACGTGCCAGATGCTGACGTGCAGTGAGGAGTCTGACTGACCA TTGGACTAGGGGATTGACCAGTAGGCTGCGATTCGGATGCGGAT TGACGATTAAAAAGGATTACGATTAGCTGTGACGTGCAGGATGC TGCGATGCTGGACTGAACGCCCCCCGGGCTTCTTTATTAGCTGC TGACGTGCCAGATGCTGACGTGCAGTGCGGCTGACGGTGCTTAC CTGGATCGGATGCTACCAGTCGATCGATCGATCGTAGCGTAGCG TATGCTAGCTAGTGATCGATGCTAGTAGCTAGCT

a repetition of CA sequences 1 per few thousand base pairs mostly in non-coding sequence length can vary per person and per chromosome, from 3 – 100 CA’s this is a polymorphic repeat, it varies in length (mostly when > 10) a CA repeat can have more than 2 alleles

POLYMORPHIC CA repeats

can be used to distinguish chromosomes and follow them in a pedigree locus 1 CA10 CA15 locus 2 CA12 CA16 locus 3 CA6 CA8 haplotype 1 haplotype 2

HOMOZYGOTE / HETEROZYGOTE

locus 1 CA15 CA15 locus 2 CA12 CA16 locus 3 CA6 CA8 homozygous = two identical alleles on 1 locus heterozygous = two different alleles on 1 locus

SEGREGATION

ca10 ca15 ca14 ca18 ca10 ca14 ca15 ca14 ca18 ca10 ca15 ca15 ca15 ca10 ca15 ca14 ca14 ca15 ca16 ca12 ca16 ca16 ca16

This chromosome segregates with the disease in this family and contains a gene mutation = principle of a linkage study

ca10 ca16 ca12 ca10 ca10 ca16 ca16 ca16 ca16 ca16

a single nucleotide polymorphism, a 1 base variant most frequent variant in the genome, on average 1 every 200-300 bp (common SNPs) can occur in coding and non-coding sequence distributed over the whole genome, exons, introns regulatory regions etc. length is only 1 base, can vary per chromosome and between persons a SNP is also polymorphic, but it has only 2 alleles (less informative than a CA repeat) all SNPs are in a database: dbSNP: http://www.ncbi.nlm.nih.gov/projects/SNP/ every SNP is defined by an rs……. number

TGCGATTCGGATGCGGATTGACGATTAAAAAGGATTACGATTAGCTG TGACGTGCAGGATGCTGCGATGCTGGACTGAACGCCCCCCGGGCTTC TTTATTAGCTGCTGACGTGCCAGATGCTGACGTGCAGTGCGGCTGAC GGTGCTTACCTGGATCGGATGCTACCAGTCGATCGATCGATCGTAGC GTAGCGTATGCTAGCTAGTGATCGATGCTAGTAGCTAGCTAGCTGAT CGATCATCGATCGTAGCTAGCTAGCTAGCTAGCTGATCGATCGATGC TAGCTAGCTAGCTAGTCATCTGTGGTGGGGGGTTAAATGCGATTGCC GCTAGCTAGAACAAAATAGCGGTATTTTGGGGAGTCTGACTGACCAT TGGACTAGGGGATTGACCAGTAGGCTGCGATTCGGATGCGGATTGAC GATTAAAAAGGATTACGATTAGCTGTGACGTGCAGGATGCTGCGATG CTGGACTGAACGCCCCCCGGGCTTCTTTATTAGCTGCTGACGTGCCA GATGCTGACGTGCAGTGAGGAGTCTGACTGACCATTGGACTAGGGGA TTGACCAGTAGGCTGCGATTCGGATGCGGATTGACGATTAAAAAGGA TTACGATTAGCTGTGACGTGCAGGATGCTGCGATGCTGGACTGAACG TGCGATTCGGATGCGGATTGACAATTAAAAAGGATTACGATTAGCTG TGACGTGCAGGATGCTGCGATGCTGGACTGAACGCCCCCCGGGCTTC TTTATTAGCTGCTGACGTGCCAGATGCTGACGTGCAGTGCGGCTGAC GGTGCTTACCTGGATCGGATGCTACCAGTCGATCGATCGATCGTAGC GTAGCGTATGCTAGCTAGTGATCGATGCTAGTAGCTAGCTAGCTGAT CGATCATCGATCGTAGCTAGCTAGCTAGCTAGCTGATCGATCGATGC TAGCTAGCTAGCTAGTCATCTGTGGTGGGGGGTTAAATGCGATTGCC GCTAGCTAGAACAAAATAGCGGTATTTTGGGGAGTCTGACTGACCAT TGGACTAGGGGATTGACCAGTGGGCTGCGATTCGGATGCGGATTGAC GATTAAAAAGGATTACGATTAGCTGTGACGTGCAGGATGCTGCGATG CTGGACTGAACGCCCCCCGGGCTTCTTTATTAACTTCTGACGTGCCA GATGCTGACGTGCAGTGAGGAGTCTGACTGACCATTGGACTAGGGGA TTGACCAGTAGGCTGCGATTCGGATGCGGATTGACGATTAAAAAGGA TTACGATTAGCTGTGACGTGCAGGATGCTGCGATGCTGGACTGAACG

EXAMPLE OF a SNP

SOME FACTS ABOUT SNPs

_every individual has about 10 million common SNPs in their genome _ a SNP has a Minor Allele Frequency = MAF that is the frequency at which the less common allele occurs in a given population example SNP: rs1042725 variant located on chromosome 12 (at chromosomal position 66358347 (build 37) CTCAATACTACCTCTGAATGTTACAA[C/T]GAATTTACAGTCTAGTACTTATTAC the ancestral allele = C has allele frequency of 0.6 = major allele the variant allele = T MAF of 0.4 = minor allele 60% of the chromosomes in the population carry a C at this position 40% of the chromosomes in the population carry a T at this position

DIFFERENT TYPES OF SNPs IN CODING REGIONS

synonymous SNP variation on DNA level no change in amino acid TTT or TTC both code for Phenylalanine no change in protein non-synonymous SNP variation on DNA level gives change in amino acid TGG or TGC Tryptofane Cysteine change in protein

SNPs IN NON-CODING REGIONS

EXAMPLE: SNP in promoter region of a gene _can have effect on binding of i.e. transcription factors _ influence transcription of the gene

GT………….….AG

Exon1 Exon2 Intron1 promoter

DNA

5’UTR

TAGCTTTGACTAAGCTTA

GT………….….AG

Exon1 Exon2 Intron1 promoter

DNA

5’UTR

TAGCTTCGACTAAGCTTA

high level transcription low level transcription

db SNP

dbSNP database was set up in 1998 (by NCBI and NHGRI) = public database of genetic variation (human + other species) _it contains SNPs (99.7%) short insertion/deletion polymorphisms (0.2%) and short tandem repeats and other things (0.1%) _neutral variants as well as disease-causing clinical mutations ! _Academic research laboratories as well as private research companies can contribute variations to dbSNP _consortia contribute: HapMap Project, 1000 Genomes Project, Wash_UV ESP dbSNP progressed from Build 1 with 11 SNPs in 1998 Build 138 with 62.676.337 SNPs in 2013 Build 142 with 112.743.739 SNPs in 2014 Build 144 with 149.735.377 SNPs in 2015 Build 150 with 171.000.000 SNPs in 2017

With the collection of SNPs growing, emphasis shifted to studying SNPs in populations (2002) _How are allele frequencies of SNPs distributed between different populations _How are these variants in individuals associated with a - complex - disease HapMap Project (2002) started: genotyping of 270 samples from different populations (Africa, USA, Japan, China) and produced allele frequencies for millions of SNPs can be used for Genome Wide Association Studies (GWAS)

INTEREST IN

SNPs IN POPULATIONS

WHAT IS A GENOME WIDE ASSOCIATION STUDY - GWAS

_study designed to identify possible variants in the human genome that contribute to complex diseases _compare the DNA of thousands of people with a specific medical condition (or trait) the DNA of thousands of similar people without that condition searche for differences between their genomes _ = association study because certain variants / SNPs will occur more often in the disease group than in the non-disease group and are "associated" with the condition _these variants are not the direct cause of the condition they contribute to the disease and have a small effect

... ATGCCGATCGCT ... ... ATGCCGATCGCT ... ... ATGCCGATCGCT ... ... ATGCCGATCGCT ... ... ATGCCGATCACT ... ... ATGCCGATCACT ...

a variation/ SNP with MAF of 33%

                                                                       

SNP| G SNP| A

some individuals have

• ne version the the

SNP, some the other healthy people people with a disease in a normal population a certain percentage will have one allele, the rest the other in the disease group a higher than normal incidence of one allele suggests that SNP |A is associated with the disease

_the idea was that association to common genetic variations (MAF >5% in the population) would explain the cause of complex disease but they contribute only a modest risk for the diseases. _ the next idea was that the genetic burden of common diseases must be carried by large numbers of rare variants (MAF 0.05 – 1% of the population) but these also only contribute modestly to disease risk _many loci that have been discovered to be associated to a disease do not map to exons of genes: these loci might contain regulatory or functional sequences is being investigated by the ENCODE project : ENCyclOpedia of DNA Elements https://genome.ucsc.edu/encode goal: build a comprehensive map of functional elements in the human genome

THE HUMAN GENOME PROJECT

National Human Genome Research Institute, NIH

Started in October 1990 goal: _determine the sequence of all 3 x 109 base pairs _identify all the human genes (20.000-25.000) _store information in databases _improve tools for data analysis _ coordinated and funded by US department of Energy and the National Institutes of Health (NIH) – director Francis Collins _ performed by a consortium of scientists from diff. countries (USA, UK, France, Australia, Japan and others) _public project _was expected to take 15 years

In May 1998 Craig Venter, started the company Celera Genomics and announced that he would sequence the human genome in

• nly two years (privately funded project)

_ also he wanted to patent human genes, which is what Watson wanted and was of great concern to Francis Collins _the race and competition started between Collins and Venter

June 26, 2000 It was decided that the competitors announce the completion of the first working draft of the entire human genome together

Craig Venter (Celera) Bill Clinton Francis Collins (NIH)

Febr 2001: first draft of the genome published in separate journals April 2003: announcement of the essentially complete genome May 2006: sequence of the last human chromosome was published

SHOTGUN SEQUENCING

The human genome project was performed using shotgun sequencing – elaborate and time consuming

_the whole genome is cut randomly into small fragments (500-600 basepairs) _these fragments are inserted into vectors = cloning _each fragment of DNA is amplified by a PCR reaction _and sequenced by Sanger sequencing _overlapping sequences are then aligned into a whole again by computer analysis

NEXT GENERATION SEQUENCING

FROM SHOTGUN TO NEXT GENERATION SEQUENCING

because of the human genome project more modern sequencing techniques were developed which are less elaborate and also faster (lectures from Robert Kraaij tomorrow) this started the 1000 genomes project in 2008 with the aim to sequence DNA of 1000 participants uncover genetic variation

1000 GENOMES PROJECT

www.1000genomes.org

MANY NGS SEQUENCING PROJECTS

1000 genomes project 2008 2500 Exome Variant Project (ESP) 2010 6503 National Heart, Lung and Blood Institute (NHLBI) http://evs.gs.washington.edu/EVS/ ErasmusMC, genetic lab, internal medicine 2010 3000 healthy Rotterdam citizens UK10K 2010 10.000 6000 extreme phenotypes http://www.uk10k.org 4000 whole genome Genomics England 2016 100.000 rare disease + cancer funded by National Health Service ENCODE project 2007 1% of the genome (44 regions) https://www.encodeproject.org/ in 147 cell types http://www.nature.com/encode/#/threads Exome Aggregation Consortium (ExAC) 2014 variant frequency of 60,706 individuals http://exac.broadinstitute.org/ combined in one database Million Veteran Program 2016 1.000.000 participants

https://www.research.va.gov/mvp/

start # individuals/samples