Lecture 2: Human Genetic variation in Health and Disease Flashcards
What is the human genome and what does it contain?
13
1 * ~37 trillion cells in the body
– Many different types of cells & functions
2 * All cells with a nucleus contain DNA
(mature red blood cells; cornified cells in skin, hair, nails; no nucleus)
3 – 99.9% genes in nucleus
4 – mitochondrial DNA (mtDNA) = 37 genes
5 – Which genes are turned on or off, and when, determine cell type.
6.* 3,000,000,000 base pairs of DNA (X 2).
7 – A standard typist would take 34 years of non-stop typing to type this out!
8 – 1.8m long 1 cell nucleus (tightly wound)
9 * ~20,000 protein-coding genes
10 * ~98% of DNA is “junk DNA”
11 * Any 2 people are ~99.6 – 99.9% identical
12 – 0.4% variation = ~ 12 million base pairs
13 * Finding a molecular diagnosis is essentially searching for one or more typographical errors in 34 years of typing…
What is Human Genetic Variation?
Variation in structure or sequence of the human genome
Human Genetic Variation - whom/where is it among?
= 2
1 – Inter-individual (intra-individual)
2 – Inter-population
Multiple mechanisms contributing To Human Genetic Variation? =6
- Meiotic recombination
2 – DNA replication and repair
3 – Population effects
4—- * Random genetic drift
5 —- * Selection (adaptive advantage)
6 —-* Migration
Understanding Geographical Genetic Diversity
= 3
1 * Out of Africa theory
– H.sapiens evolved into modern humans solely in Africa, 100-200,000 years ago
– One African subpopulation of hominins (among several) was ancestral to all human
beings today
– Some members of that subpopulation left Africa by 60,000 yrs ago
2* Supported by study of present-day mitochondrial DNA, and Y
chromosome sequence variation.
3 * Genetic diversity decreases with migratory distance from Africa
– Due to bottlenecks during human migration (temporarily reduce population size)
Generally speaking ..genetic diversity in world…
- General similarities between neighbouring populations.
- Large differences between different parts of the world.
What are the two types of Variation?
- Structural (>1000bp)
- Sequence level (<1000bp)
Types of Variation
Understanding
* Structural (>1000bp) = 3
1 – Copy number (deletions & duplications)
2 – Positional (insertions, translocations)
3 – Orientational (inversion)
Types of Variation
Understanding Sequence level (<1000bp) = 3
1 – Single base substitutions
2 – Small insertions/deletions/duplications
3 – Repetitive sequence
What is Cytogenetics?
Structure, properties and behaviour of chromosomes
What is Molecular Genetics = 4
1 * Studies structure and function of genes at a
molecular level
2 * Uses molecular biology & genetics
3 * Inter-relationship between DNA, RNA and
synthesis of polypeptides
4 * Tests are typically DNA- or RNA-based
Spectrum of Variation in the Human Genome… SMALL VARIANTS = 3
VARIATION…REARRANGEMENT TYPE… SIZE RANGE
SMALL VARIANTS AT SEQUENCES
- MAY BE DIEASE CAUSING
- SINGLE BASE-PAIR CHANGES
- Single nucleotide polymorphisms, point mutations
- 1bp - SMALL INSERTIONS/DELETIONS
- Binary insertion/deletion events of short sequences (majority < 10bp in size)
- 1-50bp - SHORT TANDEM REPEATS
- Microsatellites and other simple repeats
- 1-500 bp
Spectrum of Variation in the Human Genome…LARGE VARIANTS … MAY BE DISEASE CAUSING OR NOT
…LARGE VARIANTS
… MAY BE DISEASE CAUSING OR NOT
- Large-scale structural variation
- deletions, duplications, large tandem repeats, inversions
- 50kb to 5 Mb - CHROMOSOMAL VARIATION
- Euchromatic variants, large cytogenetically visible deletions, duplications, translocations, inversions, and aneuploidy
– aprox 5Mb to entire chromosomes
Spectrum of Variation in the Human Genome…REPETITIVE ELEMENTS OF VARIOUS SIZES. === 8 features + examples
- Repetitive elements of various sizes.
2 * Tandem (vary in length) or interspersed (vary in copy number)
3 * Small (<5Mb) or cytogenetically visible (>5Mb)
4 * Non disease-causing but may predispose to
rearrangements or expansions/contractions. - FINE-SCALE STRUCTURAL VARIATION
- Deletions, duplications, tandem repeats, inversions
- 50bp to 5 kb - RETROELEMENT INSERTIONS
- SINEs, LINEs, LTRs, ERVs
- 300 bp to 10 kb - INTERMEDIATE-SCALE STRUCTURAL VARIATION
- deletions, duplications, tandem repeats, inversions
- 5-kb to 50kb - Large-scale structural variation
- deletions, duplications, large tandem repeats, inversions
- 50kb to 5 Mb - CHROMOSOMAL VARIATION
- Euchromatic variants, large cytogenetically visible deletions, duplications, translocations, inversions, and aneuploidy
– aprox 5Mb to entire chromosomes
Causes of Genetic variation? = 4
1 * DNA repair mechanisms contending with damage due to mutagens
– Ionising radiation
– UV
– Chemicals
2 * DNA replication errors
– Proof-reading errors
– Fork stalling
3 * Homologous DNA recombination during meiosis
– Allelic and non-allelic
4 * Retrotransposition
– Elements able to amplify themselves throughout the genome.
– Copy themselves to RNA and then back into DNA that integrates back
into the genome.
Causes pf Genetic variation?
Understanding DNA repair mechanisms contending with damage due to mutagens… 3
1 – Ionising radiation
2– UV
3 – Chemicals
Causes pf Genetic variation?
Understanding DNA replication errors = 2
– Proof-reading errors
– Fork stalling
Causes pf Genetic variation?
Understanding Homologous DNA recombination during meiosis
Allelic and non-allelic
Causes pf Genetic variation?
Understanding RETROTRANSPOSITION ..2
- Elements able to amplify themselves throughout the genome.
– Copy themselves to RNA and then back into DNA that integrates back
into the genome.
Defining Variation: Reference Genome
- The human reference genome is: 5
- Maintained by the Genome Reference Consortium
- – A product of multiple international contributions
- – Updated by new full releases and minor patches/fixes
- – Represents “normal”
- However we know that there are differences between “normal” populations
Defining Variation: Reference Genome
* The human reference genome is:
CREATED? WHEN NOT USEFUL? 3
- Created by sampling lots of individuals of varying genetic make-up.
- – Not necessarily appropriate when comparing large range of ethnicities (e.g.
Australia)?
—- 3* Probably less of an issue in Mendelian inheritance, however could be an issue when considering complex inheritance and epistasis (suppression of the effect of a gene by another).
Variation and Phenotype IN GENETIC VARIATION = 3
1 * Any individual will have 10s of thousands of structural and sequence variants when compared to
the reference genome.
2 – Any may contribute to that individual’s phenotype
3 – Phenotype = “set of observable characteristics of an individual resulting from the interaction of it’s genotype with the environment”.
Spectrum of consequences of genetic variation and Phenotype…4
1 – No change in phenotype
2 – Alternative phenotypes of no medical consequence
3 – Disease susceptibility
4 – Pathogenic
Continuous Vs Discontinuous Variation …IN POPULATION =6 GRAPH?
*1 Continuous variation has no limit on the value that
can occur within a population.
2 – Line graph is used to represent.
3 * Height
4 * Weight
5 * Heart rate
6 * Finger length
Continuous Vs Discontinuous Variation IN ORGANISIMS.. GRAPH? =7
1 * Discontinuous variation has distinct groups for
organisms to belong to
2 – A bar graph is used to represent
3 * Tongue rolling
4 * Finger prints
5 * Eye colour
6 * Blood groups
7* Detached ear-lobes
Databases of Variation… TYPES…3
1 * Range of web-based databases containing normal or disease-associated variants.
- – “Normal” variation
* Database of Genomic Variants - Copy number variants (CNVs)
* ExAC, Exome Variant Server, 1000 Genomes, dbSNP (sequence variants). - – Disease-associated variation
* Disease/gene specific databases
* Database of Human Gene Mutation Data (HGMD, sequence variants)
* DECIPHER (CNVs)
Databases of Variation… Generally speaking… 4
1 – Different levels of curation, evidence base
2 – Generally free to access, some require a fee
3 – In-house collection of variants.
….4 * International pressure to contribute local data to
international databases
What is a Reference genome?
normal vs Abnormal …5
1 * Caution needs to be taken when calling genetic variation “normal”
vs “abnormal” using a single reference
2 – Variation occurs between populations.
3 – Common variant = “normal”
* BRCA2, c.865A>C (p.Asn289His)
* ExAC frequency all people = 0.052 (e.g. not rare)
4 – Rare variant = “abnormal”
* DMD, c.10412T>A (p.Leu3471*)
– * = X or stop
5 * Does not exist in any benign variant databases
– most likely disease-causing
– also gene fits with phenotype, and type of mutation fits with previous
knowledge for this gene.
Genetic vs Complex vs Environmental Diseases
1 * Genetic diseases
– Down syndrome
– Cystic fibrosis
– Sickle cell disease
2 * Complex diseases
– Diabetes (type 2)
– Alzheimer disease
– Cardiovascular disease
– Obesity
3 * Environmental diseases
– Measles
– Hepatitis
– Influenza
Genetic diseases = 3
- Down syndrome
- Cystic fibrosis
- Sickle cell disease
Complex diseases …4
- Diabetes (type 2)
- Alzheimer disease
- Cardiovascular disease
- Obesity
Environmental diseases…3
- Measles
- Hepatitis
- Influenza
Mendelian Inheritance
(monogenic disease)…. 8
1 * Gregor Mendel (1822 - 1884) - Austrian Augustinian monk
2 – “father” of genetics.
3 * Autosomal dominant
4 * Autosomal recessive
5 * X-linked dominant
6 * X-linked recessive
7 * Mitochondrial
8 * De novo
Understanding Online Mendelian Inheritance in Man (OMIM https://omim.org) = 5
- > 15,500 genes
- – >4,900 phenotypes described, molecular basis known
- – >1,600 phenotype/locus, molecular basis unknown
…. 4* Too many genes and diseases to try to learn!
- A large number of genetic diseases have the molecular basis known, but there is still a significant proportion that are yet to be solved.
What is Epigenetics?
- Heritable changes to gene expression that do not involve changes to the underlying DNA sequence.
– Change in phenotype without change in genotype.
- Epigenetic mechanisms = 3
– DNA methylation
– histone modification
– non-coding RNA
Epigenetic changes are influenced by; 3
– age
– environment/lifestyle
– disease state
Epigenetic changes associated with; 3
– Cancer
– Chromosomal instabilities
– Mental retardation
Epigenetic tests
Epigenetic tests in most diagnostic labs are limited to detection of DNA methylation
Central Dogma of Molecular Biology
The central dogma of molecular biology is a theory stating that genetic information flows only in one direction, from DNA, to RNA, to protein, or RNA directly to protein.
Locus vs Allele
- Locus:
– location of a gene/marker on the chromosome - Allele:
– one variant form of a marker at a particular locus
Draw locus vs allele
slide 39
Polymorphism vs Mutation = 8
- Polymorphism
1 – Variations in DNA sequence (e.g. deletions, insertions etc)
2 * >1% in a population.
3 – Ancient & common
4 – Weak or no effect - Mutation
5 – Variants in DNA sequences (substitutions, deletions etc)
6 * <1% in a population.
7 – Recent & rare
8 – Can produce a loss or gain of function
What is Monogenic Disease = 8
1 * Monogenic: change/s in one gene sufficient for disease.
2 – One gene —> one disorder
- – One gene —> multiple disorders
- – Multiple genes —> individually cause same single disorder
- – Comparatively rare
- ~3000 males with Fragile X (AUS)
- ~3000 people with cystic fibrosis (AUS)
- ~3.5M people with cardiovascular disease (AUS)
Polygenic & Complex Disease = 3
- Polygenic:
1 – multiple genes contribute to phenotype, each exerting
a small effect.
2 – e.g. eye colour. - Complex/multifactorial
3 – gene(s) & environment.
Germline Vs Somatic = 5
1 * Germ line is the cell line from which gametes are derived.
2 * Cells of the germline are called germ cells.
3 * Germ cells are deliberately set aside to later form gametes.
….4 – Originate in the primitive streak and migrate to the developing gonads
where they undergo cell division & differentiation sperm, ova.
5 * Somatic cells arise from the germline, but are not part of it
What happens in Germline Mutation …each stages….4
- GAMETES… germ-line mutation in SPERM ONLY
- EMBRYO: ….
- ORGANISM…. Entire organism carries the mutation
- GAMETES OF THE ORGANISM: Half of the gametes carry the mutation
What happens in SOMATIC Mutation …each stages….4
- GAMETE - …zygote
- EMBRYO: SOMATIC MUTATION OCCURS
- ORGANISM: Patch of affected area
- GAMETES: None of the gametes carry the mutation
Germline vs Somatic Mutations = 4
- Germline mutation
1 – Any tissue from that patient can be sampled for genetic testing.
2 – Can give genetic predisposition to certain cancers (e.g. BRCA1,
BRCA2) - Somatic mutation
3 – Mutation will only be in affected tissues, not all tissues.
4– Cancer can occur due to the accumulation of somatic mutations in
DNA over time.
DNA Variant “Jargon”
* Variant
= any change
DNA Variant “Jargon”
* Pathogenic
= capable of causing disease
DNA Variant “Jargon”
* Mutation
= change that causes disease
– e.g. a pathogenic variant
DNA Variant “Jargon”
* Polymorphisim
= variant is common in normal population, therefore presumed benign.
– e.g. a benign variant
DNA Variant “Jargon”
* Benign
not harmful in effect
Types of mutations : 4
1 * Mode of inheritance
– Dominant, recessive, autosomal, X-linked
2 * Genomic location
– Coding, promoter/regulatory, splice-site
3 * Molecular change
– Structural or sequence level
– Substitution (synonymous, non-synonymous, missense, nonsense),
deletion/insertion, expansion/contraction
– Homozygous, heterozygous, compound heterozygous, hemizygous
4 * Functional effect
– Loss or gain of function, haploinsufficiency and dominant negative
Mutation: Mode of inheritance…4
Dominant,
recessive,
autosomal,
X-linked
Genomic location… mutation type..3
Coding,
promoter/regulatory,
splice-site
mutation: MOLECULAR CHANGE …3
- Structural or sequence level
- Substitution (synonymous, non-synonymous, missense, nonsense), deletion/insertion, expansion/contraction
3 – Homozygous, heterozygous, compound heterozygous, hemizygous
MUTATION TYPE: Functional effect… 3
– Loss or gain of function,
haploinsufficiency
dominant negative
Sequence level mutations: 5
1 * Missense mutations
– altered function due to changes in protein structure
(functional domains and binding sites)
2 * Nonsense and frame-shifting mutations
– nonsense mediated decay or truncated protein
3 * Splicing mutations
– exon skipping or inclusion of novel exons
4 * Other mutations that affect gene expression by
– affecting interactions of the promoter with transcription
enhancers/suppressors,
– resulting in unstable mRNA that is rapidly degraded
– affecting splicing efficiency or translation
5 * Dynamic mutations (triplet repeats)
Missense mutations
– altered function due to changes in protein structure
(functional domains and binding sites)
Nonsense and frame-shifting mutations:
– nonsense mediated decay or truncated protein
Splicing mutations
– exon skipping or inclusion of novel exons
Other mutations that affect gene expression by …3
1 – affecting interactions of the promoter with transcription enhancers/suppressors,
2 – resulting in unstable mRNA that is rapidly degraded
3 – affecting splicing efficiency or translation
Dynamic mutations
Dynamic mutations (triplet repeats)
Copy number mutations
- Single gene, contiguous gene and multiple gene syndromes
Copy number mutations…Molecular mechanisms: 5
- Gene dosage
- Gene interruption
- Gene fusion
- Position effects
- “Unmasking” of recessive alleles
Cytogenetics & Molecular Genetics
- Line between both cytogenetics & molecular genetics specialties is blurring:
- Line between both cytogenetics & molecular genetics specialties IS BLURRING:
Molecular methods can detect cytogenetic abnormalities,
e.g. microarray for submicroscopic deletions & duplications.
Molecular Diagnosis – significance? 6
- Accurate Diagnosis
- Possible Prognosis
- Better Informed Genetic Counseling (cascade screening)
4.Prenatal, preimplantation,
presymptomatic diagnosis
- Appropriate trial enrolment, treatment
- Drug interactions/ pharmacogenomics
Genotype-phenotype correlation…. 5
1 * The link between a specific genetic mutation (genotype) and disease characteristics (phenotype)
2 * Observed genotype-phenotype correlations for loss of function mutations can sometimes be explained by considering residual protein function
3 * Other important mechanisms that might explain the presence or
absence of a genotype-phenotype correlation include:
4 – Mosaicism
5 – Modifier genes, chance effects and environment
