Delivering the Genomic Revolution in Health Dr Ang Davies 26th February Senior Lecturer

What is a Genome?!

Whole Genome Sequencing

https://www.gov.uk/government/news/matt-hancock-announces-ambition-to-map-5-million-genomes

• “Every genome sequenced moves us a step closer to unlocking lifesaving treatments”, Matt Hancock Secretary for Health and Social Care

Healthy patients can be sequenced

https://www.thetimes.co.uk/article/gene-test-for-sale-on-nhs-rkln85wfp

Healthcare Scientists make a difference to peoples’ lives... and so can you • Only make up 5% of NHS workforce but involved in

80% of all clinical decisions • Eg blood science, audiology, genomic counselling,

bioinformatics • Are developing some of the most amazing clinical and

technological advancements. • Are involved in improving clinical service and

undertaking research

Who are Healthcare Scientists?

Content

• Molecular biology 101

• Sequencing then and now

• Landmark sequencing initiatives and whole genome sequencing

• So how do you interpret a genome?

• Gene therapy

• Healthcare transformation

Human Genome Project

Nature volume409 (15 February 2001) doi:10.1038/35057062

Creating a reference genome Is it representative?

What’s in it? DNA Structure and Function

DNA

• Deoxyribose/phosphate backbone

• 4 base code

• Complementarity

• Sugar backbone difference RNA (ribose)

Antiparallel structure

Photo by OpenStax College [CC BY 3.0]

https://www.ensembl.org/Homo_sapiens/Info/Annotationhttps://creativecommons.org/licenses/by/3.0

DNA – secondary structure

• B form

• Right-handed helix 10 bps per turn

• A (more bp/turn)

• Z left handed helix

Minor

groove

Major

groove

Pasta

• What is a gene? Part of a DNA molecule that serves as a template for making functionally important messenger RNA.

• What is a chromosome? A chromosome is created by tightly coiling a length of DNA around histone molecules and is a way of compacting DNA within the nucleus

• What is a locus? A unique chromosomal location defining the position of a gene.

• What is an allele? Alternative versions of a gene, eg ABO locus.

• What is a genotype? List of all alleles present at a loci/locus.

• What is a phenotype? Observable trait/characteristic – contributed by genotype/epigenetic/environmental factors.

A few definitions!

• What is an exon? Part of the gene that encodes the final mature mRNA

• What is an intron? Part of the gene that can be removed during a process known as splicing to produce the final mature mRNA

• What is a transcript? A length of RNA that has been transcribed respectively from a DNA template.

A few definitions

http://www.ensembl.org/Homo_sapiens/Info/Annotation

Assembly GRCh38.p10 (Genome Reference Consortium Human Build 38), INSDC AssemblyGCA_000001405.25, Dec 2013

Base Pairs 3,554,996,726

Golden Path Length 3,096,649,726

Annotation provider Ensembl

Annotation method Full genebuild

Genebuild started Jan 2014

Genebuild released Jul 2014

Genebuild last updated/patched

Jun 2017

Database version 90.38

Gencode version GENCODE 27

Coding genes 20,338 (incl 562 readthrough)

Non coding genes 22,521

Small non coding genes 5,363

Long non coding genes 14,720 (incl 238 readthrough)

Misc non coding genes 2,222

Pseudogenes 14,638 (incl 6 readthrough)

Gene transcripts 200,310

Human genome

Ensembl release 93 - July 2018 © EMBL-EBI http://www.ensembl.org/Homo_sapiens/Info/Annotation

http://www.ensembl.org/Homo_sapiens/Info/Annotationhttps://www.ebi.ac.uk/ena/data/view/GCA_000001405.25https://www.ensembl.org/Homo_sapiens/Info/Annotationhttp://www.ebi.ac.uk/http://www.ebi.ac.uk/http://www.ebi.ac.uk/http://www.ensembl.org/Homo_sapiens/Info/Annotationhttp://www.ensembl.org/Homo_sapiens/Info/Annotation

• Gene: ABO gene resides on chromosome 9 and contains 7 exons

• Locus: band 9q34.2

• Major Alleles: A, B & O

• Genotype (one from each parent) : AA, AO

• Phenotype: A

Picture By Genetics Home Reference, Genome Decoration Page/NCBI [Public domain], via Wikimedia Commons https:/, at band 9q34.2/commons.wikimedia.org/wiki/File:Human_chromosome_09_ideogram_from_GHR.png

ABO blood group locus

Central Dogma of Molecular Biology

What is a gene?

Enhancer site

Promoter site

Start Exon

Intron

Stop

3’ 5’

Open Reading Frame

How much DNA in each of us?

Images cc1.0 & 4.0 Annunziato, A. (2008) DNA Packaging: Nucleosomes and Chromatin. Nature Education 1(1):26

To the sun and back more than 300 times

(100 trillion metres)

Rope

Image cc 0

DNA compaction in Eukaryotic Cells

DNA compaction is very complex DNA isn’t just crammed into the nucleus, but organised from the smallest unit – the nucleosome, (via loops and bands) – to the entire chromosome.

Photo by Magnus Manske / CC BY-SA 3.0

https://commons.m.wikimedia.org/wiki/File:Chromatin_chromosome.pnghttps://creativecommons.org/licenses/by-sa/3.0/deed.enhttps://creativecommons.org/licenses/by-sa/3.0/deed.enhttps://creativecommons.org/licenses/by-sa/3.0/deed.enhttps://creativecommons.org/licenses/by-sa/3.0/deed.en

Human variation

• Sequence of nuclear DNA ~ 99.9% identical between any two humans - 0.1% sequence is responsible for genetically determined variability among humans.

• Differences in hair, eye colour, blood group

• Healthcare: distinguishing normal variation from pathogenic variation

Sequencing James Watson

• Sequenced with next generation sequencing

• 3.3 million SNPs

• 10525 positions SNPs different from those in dbSNP & caused amino acid changes

• Did not want to know his APOE status By Cold Spring Harbor Laboratory (National Human Genome Research Institute) [Public domain], via Wikimedia Commons

Wheeler et al., (2008)

The complete genome of an individual by massively parallel DNA sequencing. Nature 452:872-877

WHY?

http://www.nature.com/nature/journal/v452/n7189/full/nature06884.htmlhttp://www.nature.com/nature/journal/v452/n7189/full/nature06884.htmlhttp://www.nature.com/nature/journal/v452/n7189/full/nature06884.htmlhttp://www.nature.com/nature/journal/v452/n7189/full/nature06884.htmlhttp://www.nature.com/nature/journal/v452/n7189/full/nature06884.htmlhttp://www.nature.com/nature/journal/v452/n7189/full/nature06884.htmlhttp://www.nature.com/nature/journal/v452/n7189/full/nature06884.htmlhttp://www.nature.com/nature/journal/v452/n7189/full/nature06884.htmlhttp://www.nature.com/nature/journal/v452/n7189/full/nature06884.htmlhttp://www.nature.com/nature/journal/v452/n7189/full/nature06884.htmlhttp://www.nature.com/nature/journal/v452/n7189/full/nature06884.htmlhttp://www.nature.com/nature/journal/v452/n7189/full/nature06884.htmlhttp://www.nature.com/nature/journal/v452/n7189/full/nature06884.htmlhttp://www.nature.com/nature/journal/v452/n7189/full/nature06884.htmlhttp://www.nature.com/nature/journal/v452/n7189/full/nature06884.htmlhttp://www.nature.com/nature/journal/v452/n7189/full/nature06884.html

• Rare disease, but most common genetic disease in UK (1 in 2500)

• Recessive disorder - 2 copies to be affected

• Tested for in newborn bloodspot test

• Most common form F508

• Deletion of an amino acid

• Caused by deletion of 3 bases

Cystic Fibrosis

Technology Transformation

What is Next Generation Sequencing?

• NGS or Massively Parallel Sequencing is any technology that utilises a high-throughput, parallel approach to sequencing

• Molecular barcoding means DNA can be sequenced from many genes from many patients simultaneously

Next Generation Sequencing

Image C/O National Genome Research Institute

Genomics & Precision Medicine

https://www.flickr.com/photos/ dodnewsfeatures/24963709400

By Lsadnh [CC BY-SA 4.0 (https://creativecommons.org/licenses/by-sa/4.0)], from Wikimedia Commons

https://www.flickr.com/photos/https://www.flickr.com/photos/

Copy number variation

Why sequence the whole genome?

• Whole genome = big data challenge (Tera bytes)

• NHS data infrastructure!

• Sequencing is the easy part – interpreting 100s-1000’s of variants (mutations) per person

• Ethics – which data do we look at – how does this affect other family members

• Training & education

Challenges

Clinical Scientist: Bioinformatician

Cli

nic

al

Scie

nti

st:

Ge

ne

ticis

t

Clinician

Genome Technologist

Patient

Genetic Counsellor Treatment?

Workforce Transformation

Treatment of Genetic Disorders

• “concept is to treat a disease by modifying the genetics of the cell itself”

• Bishop was diagnosed with choroideremia – prognosis was blindness – affected gene CHM affects 1 in 50,000

• Replacement genetic material delivered by modified adeno-associated virus which is stripped of its own DNA

• Injected into cells behind the retina

• FDA approved June 2018

Prof. Robert Maclaren and Michael Bishop https://www.theguardian.com/science/2019/jan/19/they-said-i-would-go-blind-gene-therapy-has-changed-that

• Age related macular dystrophy affects 1 in 600, 000 in UK

• Genetic age related decline in macula affecting central vision

• Immune system in the eye starts to attach photoreceptor cells

• Trial to inject modified virus into back of the retina

• Aim is to prevent further decline and vision loss

Janet Osbourne https://www.bbc.co.uk/news/health-47226987

• Build more relevant experimental systems

• Research applications • Drug discovery pipeline

Life science research

• Engineer industrial microorganisms

• Crop improvements

Biotech and agriculture

• Diagnostics • Treat genetic diseases • Target infections • Combine with other

therapy

Medicine and health

CRISPR-Cas9 Gene Editing

Slide courtesy of Antony Adamson

CRISPR Gene Editing

The Clinical Bioinformatician

The MOOC Model

Mass Education

FREE!!!!!! www.futurelearn.com/ courses/bioinformatics

Key Messages

• Whole genome sequencing is entering the NHS

• Allows us to personalise treatment, make prognoses and diagnoses

• Creating a big data challenge for NHS infrastructure

• Transformation of the workforce

• Engagement with patients and public

Homework!

• www.futurelearn.com/courses/bioinformatics

• https://www.genomicseducation.hee.nhs.uk/courses/

• https://www.genomicsengland.co.uk/the-100000-genomes-project/

• https://www.yourgenome.org/

• https://www.gov.uk/government/publications/chief-medical-officer-annual-report-2016-generation-genome

• Angela.davies@manchester.ac.uk

• @MSCclinbioinf

• Thank you!

https://www.genomicseducation.hee.nhs.uk/courses/https://www.genomicseducation.hee.nhs.uk/courses/https://www.genomicseducation.hee.nhs.uk/courses/https://www.khanacademy.org/science/biology/dna-as-the-genetic-materialhttps://www.khanacademy.org/science/biology/dna-as-the-genetic-materialhttps://www.khanacademy.org/science/biology/dna-as-the-genetic-materialhttps://www.khanacademy.org/science/biology/dna-as-the-genetic-materialhttps://www.khanacademy.org/science/biology/dna-as-the-genetic-materialhttps://www.khanacademy.org/science/biology/dna-as-the-genetic-materialhttps://www.khanacademy.org/science/biology/dna-as-the-genetic-materialhttps://www.khanacademy.org/science/biology/dna-as-the-genetic-materialhttps://www.khanacademy.org/science/biology/dna-as-the-genetic-materialhttps://www.khanacademy.org/science/biology/dna-as-the-genetic-materialhttps://www.khanacademy.org/science/biology/dna-as-the-genetic-materialhttps://www.khanacademy.org/science/biology/dna-as-the-genetic-materialhttps://www.khanacademy.org/science/biology/dna-as-the-genetic-materialhttps://www.khanacademy.org/science/biology/dna-as-the-genetic-materialhttps://www.khanacademy.org/science/biology/dna-as-the-genetic-materialhttps://www.khanacademy.org/science/biology/dna-as-the-genetic-materialhttps://www.khanacademy.org/science/biology/dna-as-the-genetic-materialhttps://www.khanacademy.org/science/biology/dna-as-the-genetic-materialhttps://www.khanacademy.org/science/biology/dna-as-the-genetic-materialhttps://www.khanacademy.org/science/biology/dna-as-the-genetic-materialhttps://www.khanacademy.org/science/biology/dna-as-the-genetic-materialhttps://www.khanacademy.org/science/biology/dna-as-the-genetic-materialhttps://www.khanacademy.org/science/biology/dna-as-the-genetic-materialhttps://www.khanacademy.org/science/biology/dna-as-the-genetic-materialhttps://www.khanacademy.org/science/biology/dna-as-the-genetic-materialhttps://www.khanacademy.org/science/biology/dna-as-the-genetic-materialhttps://www.khanacademy.org/science/biology/dna-as-the-genetic-materialhttps://www.khanacademy.org/science/biology/dna-as-the-genetic-materialhttps://www.khanacademy.org/science/biology/dna-as-the-genetic-material

Transcription

Transcription is the DNA directed synthesis of RNA

Like DNA polymerase RNA polymerase can only synthesise

nucleic acid in the 5’-3’ direction while” reading” a DNA template

in the 3’-5’ direction

Direction of Transcription

RNA Splicing

Alternative Splicing

Exon skipping / inclusion

Alternative 3’ splice sites

Alternative 5’ splice sites

Mutually exclusive exons

Intron retention

Constitutive exon Alternatively spliced exon

Splicing and Disease

• 90% of human genes are spliced – variants in canonical splice sites (GT and AG) can lead to disease

• snoRNAs can regulate splice site selection – absence thought to cause Prada WIlli syndrome

• Exon skipping, leading to non-sense mediated decay (eg medium-chain acyl-CoA dehydrogenase deficiency)

• Autosomal dominant retinitis pigmentosa caused by variants in the spliceosome machinery

TRANSLATION

The Genetic Code

DNA RNA SECOND BASE

T C A G U C A G

T

TTT Phe TCT Ser TAT Tyr TGT Cys T

U

UUU Phe UCU Ser UAU Tyr UGU Cys U

TTC Phe TCC Ser TAC Tyr TGC Cys C UUC Phe UCC Ser UAC Tyr UGC Cys C

TTA Leu TCA Ser TAA Stop TGA Stop A UUA Leu UCA Ser UAA Stop UGA Stop A

TTG Leu TCG Ser TAG Stop TGG Trp G UUG Leu UCG Ser UAG Stop UGG Trp G

FIR

ST B

ASE

C

CTT Leu CCT Pro CAT His CGT Arg T

C

CUU Leu CCU Pro CAU His CGU Arg U

THIR

D B

ASE

CTC Leu CCC Pro CAC His CGC Arg C CUC Leu CCC Pro CAC His CGC Arg C

CTA Leu CCA Pro CAA Gin CGA Arg A CUA Leu CCA Pro CAA Gin CGA Arg A

CTG Leu CCG Pro CAG Gin CGG Arg G CUG Leu CCG Pro CAG Gin CGG Arg G

A

ATT Ile ACT Thr AAT Asn AGT Ser T

A

AUU Ile ACU Thr AAU Asn AGU Ser U

ATC Ile ACC Thr AAC Asn AGC Ser C AUC Ile ACC Thr AAC Asn AGC Ser C

ATA Ile ACA Thr AAA Lys AGA Arg A AUA Ile ACA Thr AAA Lys AGA Arg A

ATG Met ACG Thr AAG Lys AGG Arg G AUG Met ACG Thr AAG Lys AGG Arg G

G

GTT Val GCT Ala GAT Asp GGT Gly T

G

GUU Val GCU Ala GAU Asp GGU Gly U

GTC Val GCC Ala GAC Asp GCG Gly C GUC Val GCC Ala GAC Asp GGC Gly C

GTA Val GCA Ala GAA Glu GGA Gly A GUA Val GCA Ala GAA Glu GGA Gly A

GTG Val GCG Ala GAG Glu GGG Gly G GUG Val GCG Ala GAG Glu GGG Gly G

Remember Biochemistry!

Translation

NON-CODING RNA

• 1-3% of genomes encodes polypeptides

• Junk DNA – BUT intergenic sequence actually transcribed

• ENCODE project - https://www.encodeproject.org/

• The great majority of all the genome is transcribed, at least at some times and in some types of cell.

• Some sort of function (coding, protein-binding…) can be assigned to 80.4% of nucleotides in the genome = Pervasive transcription

Non-coding RNA

https://www.encodeproject.org/

RNA is not just a messenger

lincRNAs

Image created by Prof Sam Griffiths-Jones, The University of Manchester

Morris and Mattick,

2014

miRNA 122 a biomarker in liver disease

microRNAs (miRNA)

https://commons.wikimedia.org/wiki/File:MiRNA-biogenesis.jpg CC Attribution share alike 3.0

http://www.sciencedirect.com/science/article/pii/S0168827814007314http://www.sciencedirect.com/science/article/pii/S0168827814007314http://www.sciencedirect.com/science/article/pii/S0168827814007314https://commons.wikimedia.org/wiki/File:MiRNA-biogenesis.jpghttps://commons.wikimedia.org/wiki/File:MiRNA-biogenesis.jpghttps://commons.wikimedia.org/wiki/File:MiRNA-biogenesis.jpghttps://commons.wikimedia.org/wiki/File:MiRNA-biogenesis.jpghttps://commons.wikimedia.org/wiki/File:MiRNA-biogenesis.jpg

MEIOSIS, MITOSIS & CELL DIVISION

• A karyotype is the complete set of all chromosomes of a cell of any living organism.

• The chromosomes are arranged and displayed (often on a photo) in a standard format: in pairs, ordered by size.

• Upper right is a typical karyotype of a human male somatic cell.

Diploid somatic cell (2n) Karyotype

Chromosome bands

By Geer, R.C. & Messersmith, D.J [Public domain]

https://commons.wikimedia.org/wiki/File:Cytogenetic_Banding_Nomenclature.png

Cell cycle has two parts:

(I) Growth preparation

Interphase- 75% of cell life cycle

G1: rapid growth

S: DNA replicates; centrioles replicate.

G2: cell prepares for cell division; microtubular structures form.

(II) Cell Division

Mitosis (nuclear division)

Cytokinesis (cytoplasm division)

CellCycle

DNA Replication

Stages in mitosis

Photo by OpenStax College [CC BY 3.0]

[CC BY 3.0]

https://www.ensembl.org/Homo_sapiens/Info/Annotationhttps://creativecommons.org/licenses/by/3.0https://www.ensembl.org/Homo_sapiens/Info/Annotationhttps://creativecommons.org/licenses/by/3.0

• Sex cells (gametes ) chromosomes divide by meiosis

• Sex cells are haploid (n)

• After cell division the chromosome number is halved

• Results in genetic variation by shuffling of maternal and paternal chromosomes.

Meiosis