1
Q
What is the gross structure of the human genome?
A
23 pairs of chromosomes
2
Q
What is the molecular structure of the human genome?
A
DNA sequence
3
Q
How big is the human genome?
A
- 3 billion bases (3000Mb)
- ~20,000 genes
- ~2% genome codes for protein
~99.9% DNA same between any 2 people (i.e. ~3million bases different)
Any position in the genome that varies between individuals is considered polymorphic
4
Q
What is a ‘common’ thing in genome variation?
A
• We see lots of these types of variants throughout the genome
• The frequency of the different alleles is relatively high
- Population frequency
- Ie proportion of chromosomes that carry each allele in the population
• polymorphism: minor allele frequency >1%
- rare polymorphism: MAF 1-5%
- common polymorphism: MAF >5%
• all variants start off rare
• evolutionary forces affect whether or not a variant remains rare
• rare variant may be damaging and/or recent
5
Q
What are examples of types of common genetic variation?
A
- single nucleotide polymorphism (SNP) – also Single Nucleotide Variant (SNV)
- microsatellite (short tandem repeat, STR)
- minisatellite (variable number of tandem repeats, VNTR)
- copy number variation (CNV)
6
Q
About single nucleotide polymorphisms (SNPs)…
A
- high frequency: 1 every 300 nucleotides
- ~17 million SNPs identified in human genomes
- generated by mismatch repair during mitosis
7
Q
Where might an SNP be?
A
• Gene:
- No amino acid change (synonymous)
- Amino acid changed (non-synonymous/missense)
- Introduce stop codon (nonsense)
• Promoter protein level changed
• Non-coding region
• Without a deleterious effect or population annihilation, SNPs do not disappear
8
Q
What is an example of an SNP causally associated with a trait?
A
MC1R…
• Melanocortin 1 receptor • Binds alphaMSH eumelanin • alphaMSH does not bind phaeomelanin • some SNPs tend towards lack of binding and therefore red hair, freckling, pale skin • eg c.451C>T; Arg151Cys (R151C) - Frequency = 2%
9
Q
What are microsatellites?
A
- Number of repeats varies between individuals
- Totally length of microsatellite sequence varies between individuals
10
Q
Where might you find a microsatellite?
A
• Part of the 98% of genome not coding for protein:
- Intronic or UTR: may affect gene expression
- Intergenic
• Exonic
- Extra amino acids in protein
11
Q
Where might CNVs be?
A
- intergenic
- but – quite large (>1kb) so typically affect one or more genes (parts of genes)
12
Q
What associations can genome variation have with traits?
A
• Height • Coronary heart disease traits - Plasma concentraitons of triglyceride - Total cholesterol - High-density lipoprotein cholesterol - Low-density lipoprotein cholesterol - Apolipoprotein Al - Apolopoprotein B - Fibrinogen - AMI - Systolic blood pressure - Diastolic blood pressure • Aging
13
Q
What types of variation can there be?
A
¥ Rare
Ð Mutns: MAF
14
Q
What types of variant effects can there be?
A
- Can be beneficial
- Can be pathogenic
- Most are neutral
- Are these of any use?
- Yes, can be used as markers to help find disease-causing genes and mutations
homozygosity mapping and linkage studies (microsatellites, SNPs)
association analysis (SNPs,CNVs)
15
Q
What is disease gene mapping?
A
• A way of identifying which gene causes a disease when not working properly
20,000+ genes in the genome – how do we know which ones cause which disease? – mapping
16
Q
Why is gene mapping useful?
A
- Assume a disease is caused by a single gene, but we do not know what the gene is or where it is located
- If we look at enough markers in a family, some are bound to be near the disease gene while many others are not
- We then simply need to correlate shared chromosomal regions with disease state to find the location of the disease gene
17
Q
About gene mapping…
A
- Neighbouring loci on a chromosome have a tendency to “stick together” when passed on to offspring
- Therefore, a disease is often passed on to offspring with a specific marker allele and it can be concluded that the gene which is responsible for the disease if located close to these markers on the chromosome
18
Q
About learning disability…
A
- Significantly reduced ability to understand new or complex information, to learn new skills
- Reduced ability to cope independently which starts before adulthood with lasting effects on development.
- Incidence 1-2.5% ( ratio M:F 1.3:1)
- Prevelence 2011 - 1.19 million (total pop 63.3 mill)
- Mild: IQ 50-70
- Moderate: IQ 35 – 50
- Severe: IQ 20-35
- Profound: IQ
19
Q
About autistic spectrum disorders…
A
Autism affects ~1% of the UK population
Developmental conditions present from birth.
Characterised by:
- Impaired social interaction
- Impaired social communication
- Impaired imagination
Can occur in isolation or in combination with LD
20
Q
What are possible causes of learning difficulties?
A
• Genetic causes • Problems during pregnancy and birth - Maternal infections - Teratogens - Prematurity - Pre/peri/postnatal trauma • Incidents after birth - Serious illness - Head injury - Poor nutrition - Exposure to toxins
21
Q
What are types of cytogenic abnormalities?
A
• Aneuploidy • Translocations - Robertsonian - Reciprocal • Deletions/duplications
Abnormalities of chromosome number = ANEUPLOIDY
Other potential causes of microscopically visible deletions or aneuploidy
22
Q
About robsertsonian translocations…
A
- Other causes of microscopically visible abnormalities
- Only occurs with certain chromosomes
- 13, 14, 15, 21 or 22
- Two chromosomes stuck end to end
- Carrier has 45 chromosomes, but normal amount of genetic material
- Affected has 46 chromosomes but extra copy of one
- usually 21
- Down syndrome caused by translocation
23
Q
About reciprocal translocations…
A
- Involve any part of any chromosome
- Exchange of material between 2 chromosomes
- Carrier completely normal
- Can result in offspring with unbalanced amount of the two chromosomes
With newer genetic techniques we can now study someone’s genetic make-up at a level that falls between the big picture of the chromosome test and the minute detail of a single gene test.
We can highlight a few books on the shelf to show if those books are present or absent
24
Q
About the 22q11 micro deletion…
A
- Antenatally detected VSD – repaired at birth
- Significant speech and language difficulties
- Moderate LD
Symptoms
- Cleft palate/nasal speech
- Congenital heart disease
- Hypocalcaemia
- Mild to moderate learning difficulties
- Renal abnormalities
Also known as:
- Velocardiofacial
- DiGeorge syndrome
90% de novo
25
About the 15q11.2 micro deletion...
* Often inherited from parent with few problems…
* Variable phenotype
* No reliably recognisable dysmorphology
* Seizures
* Mild-moderate delay
26
What are symptoms of phenylketonuria?
* Developmental delay
* Behavioural or social problems
* Seizures
* Hyperactivity
* Growth retardation
* Eczema
* Microcephaly
* A must odor in the child’s breath, skin or urine
* Fair skin and blue eyes
27
About fragile X syndrome...
* 5% of all males with LD
* high forehead
* long ears
* long face
* prominent jaw
* macro-orchidism
28
How might we test for single genes?
* sanger sequencing is still mainstay
* next generation panels
* whole exome
* whole genome
29
About triplet repeat expansions...
* unstable/dynamic expansions therefore can increase in size in next generation
* instability depends on parent of origin
* general correlation between size of expansion and severity of the disorder
30
What is imprinting?
Disease is present even though there is no apparent cytogenetic or molecular genetic abnormality
- certain parts of the genome are “imprinted”/methylated
- importance of having both parents’ contribution
31
About Prader-Willi syndrome...
* Short stature
* Hypotonia
* Obesity
* Hypogonadism
* Learning disability
Loss of paternally expressed SNRPN and adjacent genes
- 75% due to deletion of paternal 15q11-13
- 25% maternal uniparental disomy 15
- 2% abnormalities at ICR
32
About Angelman Syndrome...
* epilepsy
* severe learning disability
* ataxia
* happy affect
Loss of maternally expressed UBE3A
- 70% maternal microdeletion of 15q11-13
- 5% paternal uniparental disomy
- 10% mutation un UBE3A
- 5% abnormalities of ICT
33
What are examples of environmental/teratogens syndromes?
- Fetal Alcohol Syndrome
- Fetal Valproate Syndrome
o Risks 5-10%
o LD
o Behavioural difficulties
o Characteristic facial features
34
What are methods of linkage analysis?
- Gene mapping
- Using an observed locus (marker) to draw inferences about an unobserved locus (disease gene)
- Family based design (from few large families to many small nuclear or subpairs)
35
What are the principles of genetic linkage?
* The tendency for alleles at neighbouring loci to segregate together at meiosis is the phenomenon of genetic linkage
* Therefore to be linked, two loci must lie very close together
* Alleles at linked loci is called a haplotype. Haplotypes mark chromosomal segments which can be tracked through pedigrees and populations
* Cross-overs are more likely to occur between loci separated by some distance than those close together
36
Basics of recombination...
RF was introduced to describe the proportion of recombinants in the total number of offspring. Therefore RF is how frequently recombination occurs in a family.
When loci are far apart and ~ 0.5 no linkage, ie in equilibrium
When loci are close and ~ 0 linkage, ie in disequilibrium
37
What is linkage and linkage disequilibrium?
* Lack of recombination indicated that loci are close together
* Recombination indicates that loci are not close together
* Linkage = phenomenon of two loci being close together and therefore alleles on the same chromosome being likely to be inherited together
* Two alleles from different loci being co-inherited more than would be expected by chance are said to be in linkage disequilibrium
38
About micro satellites...
• Di-, tri-, tetra- nucleotide sequence with a variable numbers of repeats
- Number of repeats is constant within an individual
- Number of repeats varies between individuals
39
How do we detect microsatellites?
1. Isolate DNA from individuals to be studied
2. Design primers specific to the flanking sequences
3. PCR amplification
4. Gel electrophoresis
5. Use fragment length to determine number of repeats
40
What is micro satellite genotyping?
* PCR amplification of microsatellite region
* Genotype size of fragments on gel-bases system
* Homozygous = single band
* Heterozygous = two bands
* Don’t particularly care about size of repeat for linkage
Scenario 1: disease gene on diff chromosome independent assortment, i.e. expect approx = number of recombinants to non-recombinants
Scenario 2: disease gene on same chromosome but a long way away independent assortment
Scenario 3: disease gene quite close to marker on same chromosome not independent assortment, i.e. expect a smaller proportion of recombinants to non-recombinants
41
Where is the disease gene?
• Null hypothesis: the disease gene is not near the marker
• Possible outcome:
- True: alleles from the marker locus and the disease gene locus will segregate independently
- False: certain marker alleles will co-segregate with disease
42
What is mendel's second law?
Second Law: independent assortment
- Alleles of different genes assort independently of each other during meiosis
- Not true if linked, eg marker and disease gene
43
When is mendel's second law most helpful?
- When you already know the genomic region the disease gene is in but you don’t know that exact gene
- When you already know the disease gene but there are so many different mutations that you cant test for them all
44
What is ADPKD?
* Autosomal dominant polycystic kidney disease
* ~1 in 1000 people affected
* cysts may go unnoticed until early adulthood/family screening
* other problems: hypertension, brain aneurysms, polycystic liver
* incurable
* ESRF: 40-50%
45
What is a centimorgan (cM)?
The frequency of recombination events is related to distance – measured in centimorgans (cM)
1cM = the distance between 2 points where 1% of the products of meiosis are recombinant
1cM ~ 1Mb (1,000,000 base pairs)
2 or 3 recombination events per chromosome
46
Is it possible to somehow combine the statistical and distance measure into one measurement?
• LOD score = logarithm of odds
• LOD = log10[Lθ/L(0.5)]
= log10 (likelihood if linked)/(likelihood if unlinked)
= log10[(1-θ)NR. θR/0.5NR+R]
• Lθ = Likelihood of the observed data if the loci are linked at the given RF (θ)
• L(0.5) = Likelihood of the observed data if the loci are not linked, i.e. RF=0.5
47
What are examples of screening tests in pregnancy?
• Hb electrophoresis in all women (and their parteners if from high risk ethnic background)
- Sickle cell disease
- Beta-thalassaemia
• Routine booking ask about:
- Metabolic disorders – eg MCAD, G6PD
- Cystic fibrosis, Huntington, hypercholesterolemia
- Consanguinity
- Any other genetic disorder in the family
• Ultrasound scans
- Nuchal translucency/combined test at 11-13 weeks
- Detailed anomaly scan 20-23 weeks
48
What is assessed at the 12 week scan?
Nuchal Translucency
Now combined in some hospitals
- PAPA levels
- Beta HCG
- (maternal age of course)
Increase NT associated with:
- downs syndrome
- chromosomal abnormalities
- congenital heart disease
- other genetic (syndromes)
49
What is assessed at the ultrasound scan at 16 to 20 weeks?
- foetal sexing possible (not 100% reliable)
- structural abnormalities seen
- congenital heart disease can be detected
50
What is the prenatal diagnosis of the CVS?
* miscarriage rate 0.5 to 2%
* maternal contamination
* placental mosaicism
* transverse limb defects
51
What is the prenatal diagnosis of amniocentesis?
* >16 weeks
* extraction of amniotic fluid
* biochemical diagnosis possible
* lower miscarriage risk (0.5-1%)
52
What is early dating ultrasound?
- foetal heart seen at about 6 weeks
- gestational age can be accurately assessed
- detection of multiple pregnancy
53
What are the ethical and legal implications of prenatal diagnosis?
* Timing of diagnosis – post 24 week termination for significant risk of severe medical conditions only
* What should we offer diagnosis for – adult onset conditions? Hearing loss?
* Cultural/religious sensitivities
* Technologies are capable of fetal genome sequencing – should we?
54
What is pre symptomatic testing?
Predictive testing
- Prior to a person developing symptoms
- Often gives a likelihood of developing disease
- Often does not indicate disease severity
55
What are examples of genomic technologies?
Chromosomes
- Karyotype
- FISH
- Qf-PCR
- Array CGH
DNA sequencing
- Single gene sequencing
- Gene panel sequencing
- Exome sequencing
- Whole genome sequencing
- 100,000 genomes project
56
What are ethical and legal implications of genomic technologies?
* variants of uncertain significance
* incidental findings
* non paternity
* predictive testing in childhood
* monozygotic twins
* insurance
57
What are the cancer statistics?
Cancer is a common disease in humans
- There is a 1 in 3 lifetime risk of developing cancer
- Most cancers are caused by a combination of genetic, environmental and lifestyle factors – multifactorial/sporadic
- Only ~5% of cancers are due to the inheritance of a single cancer susceptibility gene
58
When do the mutations occur in multifactorial/sporadic cancers?
```
• Somatic mutations
o Occurs after division of the fertilised egg
o Only present in a subset of cells
o Not inherited from a parent
o Occasionally passed to offspring
```
59
When do the mutations occur in hereditary cancers?
```
• Germline mutations
o Present in the fertilised egg
o Present in every cell in the body
o Can be inherited from parent
o Can be passed to offspring
```
60
What are the differences between sporadic and hereditary cancers?
Sporadic cancers
- No increased risk of other cancers
- Usually small increased risk to relatives
- No genetic testing indicated
- Normal clinical management for affected individuals
Hereditary cancers
- High risks of recurrence/other associated cancers
- High cancer risks in relatives
- We can offer testing to at risk individuals
- We can offer screening and preventative management to gene carriers
- May alter treatment of affected individuals
61
What are cancer susceptibility genes?
- oncogenes
- tumour suppressor genes
- DNA repair genes
62
What is Knudson's 'two hit hypothesis' in sporadic cancer?
* Mutations in a tumour suppressor gene
* Regulates cell growth
* Both mutations are SOMATIC
63
What is Knudson's 'two hit hypothesis' in hereditary cancer?
* One mutation is GERMLINE
| * One mutation is SOMATIC
64
What is penetrance?
• Not every person with a germline mutation develops the disease
• Known as reduced penetrance
• We can give risks of developing disease for a given genotype
- Based on family/population studies
- Unknown modifying factors
65
What is important in taking a family history?
Clues to an underlying genetic susceptibility come from the family history.
In genetics
- 3 generation family history
- ask about consanguinity
- ethnic background – Ashkenazi jewish, other founder populations
- types and ages of all cancers
66
What are the decisions to be made when taking a family history?
Is genetic testing indicated?
- Other investigations required first?
- Confirmation of cancer diagnoses
- Testing of tumour samples (eg IHC)
Is increased screening indicated?
- For affected individual
- For unaffected relatives
67
What needs to be considered when undertaking genetic testing?
Implications for individual
- Recurrence risks
- Risks of other cancers
Implications for relatives
- How to share information
- Concerns about children
- Predictive testing
Insurance implications
- Current moratorium for predictive testing
Family planning options (eg prenatal, PGD)
68
About BRCA1 and BTCA2...
* BRCA1 mutations - 0.11% population
* BRCA2 mutations - 0.12% population
```
• Responsible for
- ~ 16% familial breast cancers
- ~ 5% breast cancer
- ~ 10% ovarian cancer
• Also prostate, pancreatic, fallopian tube and peritoneal cancers
```
Breast cancer risk in BRCA1/BRCA2
Ovarian cancer risk in BRCA1/BRCA2
69
About screening for BRCA carriers...
Breast screening
- 30-50y annual MRI screening
- 30-50y annual mammograms
- - >50 annual mammogram
Ovarian screening
- unproved efficacy
- not currently recommended
70
What are the surgical options for those at risk of cancer?
Risk reducing mastectomy
- most effective way of reducing risk - >5% over lifetime
- avoids need for cancer treatment
- can help women with anxiety
- breast reconstruction available
Risk reducing bilateral salpingo-oophorectomy
- offered at age 40 or after completed family
- can give HRT under specialist guidance
- only proven way to reduce ovarian cancer risk
71
What is the altered clinical management of those at risk of cancer?
- PARP inhibitors
| - sensitivity to platinum chemotherapies
72
What is HNPCC?
• Lynch syndrome
- Germline mutations in DNA mismatch repair genes (MLH1, MSH2, MSH6, PMS2)
• Hereditary predisposition to:
- Colorectal cancer
- Ovarian cancer
- Endometrial cancer
- Plus gastric, pancreatic hepatobiliary tract, urothelial and small intestine cancers
73
How do we screen for HNPCC?
* Colonoscopy every 18-24 months, from age 25
* Discuss endometrial screening from age 35 (but not proven to be effective)
* Discuss option of risk-reducing TAH/BSO from early 40s
Semester 4 Revision
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