Lecture 2: Human Genetic variation in Health and Disease

Question 1

What is the human genome and what does it contain?

13

Answer 1

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…

Question 2

What is Human Genetic Variation?

Answer 2

Variation in structure or sequence of the human genome

Question 3

Human Genetic Variation - whom/where is it among?

= 2

Answer 3

1 – Inter-individual (intra-individual)

2 – Inter-population

Question 4

Multiple mechanisms contributing To Human Genetic Variation? =6

Answer 4

1. Meiotic recombination

2 – DNA replication and repair

3 – Population effects

4—- * Random genetic drift
5 —- * Selection (adaptive advantage)
6 —-* Migration

Question 5

Understanding Geographical Genetic Diversity
= 3

Answer 5

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)

Question 6

Generally speaking ..genetic diversity in world…

Answer 6

• General similarities between neighbouring populations.
• Large differences between different parts of the world.

Question 7

What are the two types of Variation?

Answer 7

• Structural (>1000bp)
• Sequence level (<1000bp)

Question 8

Types of Variation

Understanding
* Structural (>1000bp) = 3

Answer 8

1 – Copy number (deletions & duplications)

2 – Positional (insertions, translocations)

3 – Orientational (inversion)

Question 9

Types of Variation

Understanding Sequence level (<1000bp) = 3

Answer 9

1 – Single base substitutions

2 – Small insertions/deletions/duplications

3 – Repetitive sequence

Question 10

What is Cytogenetics?

Answer 10

Structure, properties and behaviour of chromosomes

Question 11

What is Molecular Genetics = 4

Answer 11

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

Question 12

Spectrum of Variation in the Human Genome… SMALL VARIANTS = 3

VARIATION…REARRANGEMENT TYPE… SIZE RANGE

Answer 12

SMALL VARIANTS AT SEQUENCES

• MAY BE DIEASE CAUSING

1. SINGLE BASE-PAIR CHANGES
   - Single nucleotide polymorphisms, point mutations
   - 1bp
2. SMALL INSERTIONS/DELETIONS
   - Binary insertion/deletion events of short sequences (majority < 10bp in size)
   - 1-50bp
3. SHORT TANDEM REPEATS
   - Microsatellites and other simple repeats
   - 1-500 bp

Question 13

Spectrum of Variation in the Human Genome…LARGE VARIANTS … MAY BE DISEASE CAUSING OR NOT

Answer 13

…LARGE VARIANTS
… MAY BE DISEASE CAUSING OR NOT

1. Large-scale structural variation
   - deletions, duplications, large tandem repeats, inversions
   - 50kb to 5 Mb
2. CHROMOSOMAL VARIATION
   - Euchromatic variants, large cytogenetically visible deletions, duplications, translocations, inversions, and aneuploidy
   – aprox 5Mb to entire chromosomes

Question 14

Spectrum of Variation in the Human Genome…REPETITIVE ELEMENTS OF VARIOUS SIZES. === 8 features + examples

Answer 14

1. 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.
2. FINE-SCALE STRUCTURAL VARIATION
   - Deletions, duplications, tandem repeats, inversions
   - 50bp to 5 kb
3. RETROELEMENT INSERTIONS
   - SINEs, LINEs, LTRs, ERVs
   - 300 bp to 10 kb
4. INTERMEDIATE-SCALE STRUCTURAL VARIATION
   - deletions, duplications, tandem repeats, inversions
   - 5-kb to 50kb
5. Large-scale structural variation
   - deletions, duplications, large tandem repeats, inversions
   - 50kb to 5 Mb
6. CHROMOSOMAL VARIATION
   - Euchromatic variants, large cytogenetically visible deletions, duplications, translocations, inversions, and aneuploidy
   – aprox 5Mb to entire chromosomes

Question 15

Causes of Genetic variation? = 4

Answer 15

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.

Question 16

Causes pf Genetic variation?

Understanding DNA repair mechanisms contending with damage due to mutagens… 3

Answer 16

1 – Ionising radiation

2– UV

3 – Chemicals

Question 17

Causes pf Genetic variation?

Understanding DNA replication errors = 2

Answer 17

– Proof-reading errors

– Fork stalling

Question 18

Causes pf Genetic variation?

Understanding Homologous DNA recombination during meiosis

Answer 18

Allelic and non-allelic

Question 19

Causes pf Genetic variation?

Understanding RETROTRANSPOSITION ..2

Answer 19

• Elements able to amplify themselves throughout the genome.

– Copy themselves to RNA and then back into DNA that integrates back
into the genome.

Question 20

Defining Variation: Reference Genome

• The human reference genome is: 5

Answer 20

1. Maintained by the Genome Reference Consortium
2. – A product of multiple international contributions
3. – Updated by new full releases and minor patches/fixes
4. – Represents “normal”
5. However we know that there are differences between “normal” populations

Question 21

Defining Variation: Reference Genome
* The human reference genome is:

CREATED? WHEN NOT USEFUL? 3

Answer 21

1. Created by sampling lots of individuals of varying genetic make-up.
2. – 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).

Question 22

Variation and Phenotype IN GENETIC VARIATION = 3

Answer 22

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”.

Question 23

Spectrum of consequences of genetic variation and Phenotype…4

Answer 23

1 – No change in phenotype

2 – Alternative phenotypes of no medical consequence

3 – Disease susceptibility

4 – Pathogenic

Question 24

Continuous Vs Discontinuous Variation …IN POPULATION =6 GRAPH?

Answer 24

*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

Question 25

Continuous Vs Discontinuous Variation IN ORGANISIMS.. GRAPH? =7

Answer 25

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

Question 26

Databases of Variation... TYPES...3

Answer 26

1 * Range of web-based databases containing normal or disease-associated variants. 2. – “Normal” variation * Database of Genomic Variants - Copy number variants (CNVs) * ExAC, Exome Variant Server, 1000 Genomes, dbSNP (sequence variants). 3. – Disease-associated variation * Disease/gene specific databases * Database of Human Gene Mutation Data (HGMD, sequence variants) * DECIPHER (CNVs)

Question 27

Databases of Variation... Generally speaking... 4

Answer 27

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

Question 28

What is a Reference genome? normal vs Abnormal ...5

Answer 28

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.

Question 29

Genetic vs Complex vs Environmental Diseases

Answer 29

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

Question 30

Genetic diseases = 3

Answer 30

1. Down syndrome 2. Cystic fibrosis 3. Sickle cell disease

Question 31

Complex diseases ...4

Answer 31

1. Diabetes (type 2) 2. Alzheimer disease 3. Cardiovascular disease 4. Obesity

Question 32

Environmental diseases...3

Answer 32

1. Measles 2. Hepatitis 3. Influenza

Question 33

Mendelian Inheritance (monogenic disease).... 8

Answer 33

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

Question 34

Understanding Online Mendelian Inheritance in Man (OMIM https://omim.org) = 5

Answer 34

1. >15,500 genes 2. – >4,900 phenotypes described, molecular basis known 3. – >1,600 phenotype/locus, molecular basis unknown .... 4* Too many genes and diseases to try to learn! 5. * A large number of genetic diseases have the molecular basis known, but there is still a significant proportion that are yet to be solved.

Question 35

What is Epigenetics?

Answer 35

* Heritable changes to gene expression that do not involve changes to the underlying DNA sequence. – Change in phenotype without change in genotype.

Question 36

* Epigenetic mechanisms = 3

Answer 36

– DNA methylation – histone modification – non-coding RNA

Question 37

Epigenetic changes are influenced by; 3

Answer 37

– age – environment/lifestyle – disease state

Question 38

Epigenetic changes associated with; 3

Answer 38

– Cancer – Chromosomal instabilities – Mental retardation

Question 39

Epigenetic tests

Answer 39

Epigenetic tests in most diagnostic labs are limited to detection of DNA methylation

Question 40

Central Dogma of Molecular Biology

Answer 40

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.

Question 41

Locus vs Allele

Answer 41

* Locus: – location of a gene/marker on the chromosome * Allele: – one variant form of a marker at a particular locus

Question 42

Draw locus vs allele

Answer 42

slide 39

Question 43

Polymorphism vs Mutation = 8

Answer 43

* 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

Question 44

What is Monogenic Disease = 8

Answer 44

1 * Monogenic: change/s in one gene sufficient for disease. 2 – One gene ---> one disorder 3. – One gene ---> multiple disorders 4. – Multiple genes ---> individually cause same single disorder 5. – Comparatively rare 6. * ~3000 males with Fragile X (AUS) 7. * ~3000 people with cystic fibrosis (AUS) 8. * ~3.5M people with cardiovascular disease (AUS)

Question 45

Polygenic & Complex Disease = 3

Answer 45

* Polygenic: 1 – multiple genes contribute to phenotype, each exerting a small effect. 2 – e.g. eye colour. * Complex/multifactorial 3 – gene(s) & environment.

Question 46

Germline Vs Somatic = 5

Answer 46

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

Question 47

What happens in Germline Mutation ...each stages....4

Answer 47

1. GAMETES... germ-line mutation in SPERM ONLY 2. EMBRYO: .... 3. ORGANISM.... Entire organism carries the mutation 4. GAMETES OF THE ORGANISM: Half of the gametes carry the mutation

Question 48

What happens in SOMATIC Mutation ...each stages....4

Answer 48

1. GAMETE - ...zygote 2. EMBRYO: SOMATIC MUTATION OCCURS 3. ORGANISM: Patch of affected area 4. GAMETES: None of the gametes carry the mutation

Question 49

Germline vs Somatic Mutations = 4

Answer 49

* 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.

Question 50

DNA Variant “Jargon” * Variant

Answer 50

= any change

Question 51

DNA Variant “Jargon” * Pathogenic

Answer 51

= capable of causing disease

Question 52

DNA Variant “Jargon” * Mutation

Answer 52

= change that causes disease – e.g. a pathogenic variant

Question 53

DNA Variant “Jargon” * Polymorphisim

Answer 53

= variant is common in normal population, therefore presumed benign. – e.g. a benign variant

Question 54

DNA Variant “Jargon” * Benign

Answer 54

not harmful in effect

Question 55

Types of mutations : 4

Answer 55

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

Question 56

Mutation: Mode of inheritance...4

Answer 56

Dominant, recessive, autosomal, X-linked

Question 57

Genomic location... mutation type..3

Answer 57

Coding, promoter/regulatory, splice-site

Question 58

mutation: MOLECULAR CHANGE ...3

Answer 58

1. Structural or sequence level 2. Substitution (synonymous, non-synonymous, missense, nonsense), deletion/insertion, expansion/contraction 3 – Homozygous, heterozygous, compound heterozygous, hemizygous

Question 59

MUTATION TYPE: Functional effect... 3

Answer 59

– Loss or gain of function, haploinsufficiency dominant negative

Question 60

Sequence level mutations: 5

Answer 60

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)

Question 61

Missense mutations

Answer 61

– altered function due to changes in protein structure (functional domains and binding sites)

Question 61

Nonsense and frame-shifting mutations:

Answer 61

– nonsense mediated decay or truncated protein

Question 62

Splicing mutations

Answer 62

– exon skipping or inclusion of novel exons

Question 63

Other mutations that affect gene expression by ...3

Answer 63

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

Question 64

Dynamic mutations

Answer 64

Dynamic mutations (triplet repeats)

Question 65

Copy number mutations

Answer 65

* Single gene, contiguous gene and multiple gene syndromes

Question 66

Copy number mutations...Molecular mechanisms: 5

Answer 66

1. Gene dosage 2. Gene interruption 3. Gene fusion 4. Position effects 5. “Unmasking” of recessive alleles

Question 67

Cytogenetics & Molecular Genetics * Line between both cytogenetics & molecular genetics specialties is blurring:

Answer 67

* Line between both cytogenetics & molecular genetics specialties IS BLURRING: Molecular methods can detect cytogenetic abnormalities, e.g. microarray for submicroscopic deletions & duplications.

Question 68

Molecular Diagnosis – significance? 6

Answer 68

1. Accurate Diagnosis 2. Possible Prognosis 3. Better Informed Genetic Counseling (cascade screening) 4.Prenatal, preimplantation, presymptomatic diagnosis 5. Appropriate trial enrolment, treatment 6. Drug interactions/ pharmacogenomics

Question 69

Genotype-phenotype correlation.... 5

Answer 69

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