Introduction to Genomic MedicineExeter Expert Series: Respiratory

12-13 October, 2017

Session I. Genes and Genomes

Presented by

Júlia Baptista PhD

Clinical Scientist and Honorary Lecturer

Developed with Sawsan Khuri PhD

What will be covered

• Structure of a gene

• Genomes and the birth of genomics

• Types of genomic variants and clinical interpretation

DNA

• DNA = deoxyribonucleic acid

• Stable double-stranded polymer of nucleotides: – Deoxyribose sugar, Phosphate, Base (A, T, C & G)

• Function: storage of genetic code

Central dogma of biology

RNA

• RNA = ribonucleic acid • Unstable single-stranded polymer of nucleotides: –

Ribose sugar, Phosphate, Base (A, U, C & G)

• Different types with different functions:– Messenger RNA (mRNA): coding intermediary between DNA and proteins– Non-coding RNA (ncRNA): various roles• Protein synthesis: ribosomal (rRNA), transfer RNA (tRNA).• Gene regulation: microRNA (miRNA), small interfering or

silencing RNA(siRNA), long non-coding RNA (lncRNA)• Catalysis: ribozymes

Proteins

• Polymers of amino acids

• Many different functions: transport, structural, catalytic, signalling, etc.

What is a gene?

• A gene is the basic physical and functional unit of heredity.

• Genes, which are made up of DNA, act as instructions to make proteins.

Structure of a gene

Coding Exons

Un-Translated Regions at 5’ and 3’

as well as

• sequence variations (SNPs etc)

• Regulatory elements, in promoter region and throughout

Gerstein et al, Genome Research, 2007

Non protein-coding Introns

alternative splicingone transcript has this exon the other doesn't

Exon 1 Exon 2

TSSTranscriptional Start Site

5’UTR

Translation initiation codonATG

Promoter region

5’ 3’

Exon 1 Exon 2

Promoters have transcription factor binding sites (regulatory elements)

The 5’ structure

Barrett et al, Cell Mol Life Sci, 2012

The 3’ end structure

Exon nExon n-15’ 3’

Usually contains:• PolyA Tail addition signal• microRNA binding sites• Silencer regions• AU-rich elements& other regulatory elements

Stop codonTAG/TAA/TGA

3’UTR

Barrett et al, Cell Mol Life Sci, 2012

Genes, genomes, and genomics

• A genome is the total genetic content of an organism, whether it is coding or not.

• Genomic Medicine is the use of genomic information and its emerging technologies for disease diagnosis, prognosis, and intervention.

The Human Genome Project

ENCODE: identify all functional elements in the human genome

from the ENCODE project

Mapping genomic variation

Retired in 2016

Completed in 2015

UK now on 100,000 Genomes ProjectAfrican, Japan, Singapore, India... genome projects currently ongoing

Next generation sequencingNew technologies helped reduce the cost of sequencing human genomes

Data handling is now the bottleneck. It costs more to analyse a genome than to sequence it.

– Gene panel sequencing

• Targeted sequencing that will detect changes in a selected panel of genes known to cause the phenotype of interest.

– Clinical exome sequencing

• Targeted sequencing of exons of “all” known disease genes (~6,000 genes).

– Whole exome sequencing (WES)

• Targeted sequencing of “all” exons of all known genes (~23,000 genes)

– Whole genome sequencing (WGS) (entire genome sequencing) in the NHS in 2018

Next generation sequencing: genomic medicine

Large scale genetic variation

• Mainly chromosomal changes. These can be changes to number or structure. Structural: deletions, duplications, translocations, inversions and insertions

• Copy Number Variants

– Insertions or deletions of >1000 nucleotides, causing gains or losses of genomic regions

Relevant in various lung cancers and a decreased risk of chronic obstructive pulmonary disease (e.g. Qiu et al., 2017; Wang and Lemos, 2017; Jamal-Hanjani et al., 2017; Chen et al., 2017)

Cytogenetics/Cytogenomics detects large scale variants

DNA loss DNA gainG-banded karytotypeArray-CGH

Small scale: Single nucleotide variants (SNVs)

Types of SNVs• Substitutions in non-coding sequences

– Splice sites, regulatory elements binding sites

• Substitutions in coding sequences

– Synonymous (do not change the amino acid), Non-synonymous (missense or nonsense)

• Insertions/deletions (indels)

– In coding sequences may lead to malformed proteins

– In non-coding sequences may delete or insert regulatory signals

Synonymous variants: no change to protein sequence

Normal sequence

GGTCTCCTCACGCCA

↓

Pro-Glu-Glu-Cys-Gly

Substitution : C>T

GGTCTTCTCACGCCA

↓

Pro-Glu-Glu-Cys-Gly

Missense variants: amino acid substitution

Normal sequence

AGC TAC GGG GTG GGC

↓

Ser Tyr Gly Val Gly

Substitution

AGC TAC AGG GTG GGC

↓

Ser Tyr Arg Val Gly

Some amino acids are similar to each otherNot all amino acids are important for protein folding/function

Nonsense variants

Normal sequence

GGTCTCCTCACGCCA

↓

Pro-Glu-Glu-Cys-Gly

Often lead to premature termination of the protein.The truncated protein can be targeted for degradation by the nonsense-mediated pathway.

Substitution

GGTCTCCTCACTCCA

↓

Pro-Glu-Glu-STOP

Insertions/Deletions

Normal sequence

GGTCTCCTCACGCCA

↓

Pro-Glu-Glu-Cys-Gly

Insertion of G

GGTGCTCCTCACGCCA

↓

Pro-Arg-Gly-Val-Arg

Insertions and deletions can be in-frame or out-of-frame

(frame shift)

Insertions or deletions of a nucleotide number that disrupt the reading frame will lead to frameshifts, often a premature stop codon is formed/

Sources of variation

• Exogenous factors (chemical agents, UV radiation)

• Endogenous factors

– -Replication slippage/slipped strand mis-pairing

– -Recombination (large scale variants)

– -Segregation (Aneuploidies)

• Variants that are present in the germ cells (cells that give rise to the gametes) can be passed onto the next generation when that cell participates in fertilization. These variants are important in evolution.

• Variants that arise in a cell lineage other than the germline are somatic. These are postzygotic events and cannot be inherited. Somatic changes are mosaic.

Somatic and germline variation

From ThinkLink.com

Meiosis

• Starts with replication of genetic material (where SNPs can happen)

• Chromosomes are intertwined and crossing over can occur (where CNVs can happen)

• Results in unique haploid cells called gametes which could be quite different from parent cell, each containing a single copy of the genome

• Gametes come together during mating to produce a diploid embryo

• Hence, any SNVs and CNVs are passed down to the next generation

Evolution is driven by natural selection of the variants brought about during meiosis.

Mitosis

• Starts with replication of genetic material (where SNPs can happen)

• Chromosomes are lined up in middle ready to split into two cells, but they are not intertwined

• Results in diploid cells with usually only slight differences from parent cell

• Repeats to heal a wound, or to renew or grow a tissue, organ, etc

• Mitotic variants are not passed down to the next generation

Evolution is not affected by variants brought about during mitosis. Genetic changes during cancer are mitotic.

Interpreting variants in the clinic

• ~ 3-4 million SNVs per genome

• Mostly “common variants” not connected with a disease phenotype

• SNVs associated with disease tend to be rare.

• Important evidence:• Population data

• Disease databases

• De novo data

• Segregation

• Functional data

• Computational predictions

Nature website: Human Genome Variation

Reading a gene