Session 4 - Screening and biochemical genetics Flashcards
Define Linkage
A statistical method to determine the link between a region of the genome and a given genotype in a family or group of affected patients.
It is performed on small sets of data, often large pedigrees, but can also be used for sib-pair analysis.
Used to identify genes with large effects, often Mendelian.
Define an Association study
A statistical method of determining association between SNPs and disease in large populations.
Performed on large cohorts in a case-control fashion.
Can identify genes with small effects
What are the advantages and disadvantages of Linkage analysis?
Advantages:
Localisation of disease region on a chromosome.
Multiple markers can be studied at once
Disadvantages:
Need multigenerational cases
Difficult to study complex traits, however data from multiple families can be combined to increase the power of the study
What is used to measure Linkage? What is the cut off?
LOD scores
no linkage -2 < indeterminate < 3 Linkage
Define linkage disequilibrium
The likelihood that two independent markers will be inherited together at a frequency greater than chance. This is different from Linkage.
What can cause linkage disequilibrium?
Natural selection
Chance
How is linkage disequilibrium expressed? How is it calculated?
D
D(ab) = P(ab) - P(a)P(b)
Alleles in complete LD have a score of 1.
What factors affect LD?
Recombination Gene conversion Selection Population structure New mutations Genetic drift Gene flow Population history.
What is the Transmission Disequilibrium Test (TDT)?
A method of linkage analysis that can be performed in trios or between sib-pairs. It involves calculating the likelihood of two affected individuals inheriting the same allele from parents. It is used to study complex disease traits in families. Overtransmission of an allele suggests disease susceptibility
Name a successful Linkage study
Identification of NOD2 in Crohn’s disease
Name a successful GWAS study
Identification of associations between genotypes and age-related macular degeneration
Name the 4 possible explanations for finding a positive association between variant and disease.
- There is a true association
- variant is in LD with the true variant.
- Association by chance
- Case-controls my be selected from different populations
Give some advantages and disadvantages of GWAS
Advantages:
No pre-hypothesis
Good for studying complex disease
Tend to have better genomic resolution that linkage studies.
Disadvantages:
Need large cohort of ethnically matched cases and controls
Cannot test causality
Can be expensive
Doesn’t test for CNVs
Large number of statistical tests means a very low P value is required.
What assumptions are made in order for GWAS results to be valid?
Case-controls are ethnically matched
Case participants reflect disease phenotype
Genomic and clinical data are collected in the same way in cases and controls.
How can study power be defined? What is it determined by?
The ability of a study design to pick up associations accurately.
The MAF of the risk allele, samples size, LD between true risk allele and another, genetic heterogeneity of the sample population, relative risk of the risk allele.
What are the two types of linkage analysis? What information do each of them require?
Parametric: Gene information, MOI, penetrance, allele frequency
Non-parametric: no data.
What type of linkage analysis can be used in consanguineous populations
Autozygosity mapping.
What are the problems encountered during LOD score analysis?
Vulnerable to lots of sampling and experiemntal errors
Computational limitations
Locus heterogeneity
Low resolution
Parameters need to be correctly spec’d
Low penetrance and phenocopies in a family.
What is the principle of non-parametric linkage analysis?
That the region of a genome in which disease-causing variants are located is more likely to be shared by affected individuals than would be expected by chance.
What must be distinguished in non-parametric linkage analysis?
Whether regions are identical by descent (the patients are related and the variant has been inherited from an ancestor), or if they are identical by state (the allele has entered the family twice - not identical due to ancestral inheritance)
The frequency of the allele in the population must be taken into account.
What can Affected Sib Pair analysis be used for?
To identify genes that cause multifactorial disorders in affected siblings.
Explain the process behind ASP analysis
Genotype affected siblings
Look for chromosomal regions where sharing of allele is above the expected mendelian values
Identify regions of the genome that are IBD
Re-examine region to narrow down candidate area
List some population genome studies. State their purpose
1000G - catalogue of human genetic variation - 26 populations ~2600 individuals
100KG - stimulate UK genomics, introduce genomics into healthcare, identify new disease, produce ethical programme based on consent.
COSMIC - Catalogue of Somatic mutations in Cancer
HapMap - Identify linked haplotypes (international)
ExAC - Aggregation of data from multiple large scale genome studies. 60706 individuals from multiple populations. Now GnomAD
EVS - combination of multiple US studies
The Cancer Genome Atlas - somatic mutations identified in different cancers. Tumour profiling and clinical information.
CHIP - identification of mutations normally associated with haem-onc that don’t have disease
PAGE - 1000 foetus/parent trios with abscan of NT>4mm, looking to develop good exome test for prenatal testing
DGV - database of CNVs and SVs in the normal population
DDD - 12,000 trios with ID
CR-UK stratified medicine programme - aim to combine genetic testing with trial enrollment. 2 stages. First stage looked at 10 genes in multiple tumours to ID markers for disease/therapy/prognosis. Stage 2 now looking at the Lung Cancer Matrix Trial of nsc lung cancers to identify genetic faults driving their disease.
ENCODE
What types of DTC testing are there?
Testing for predisposition to disease Dispositional health tests (warfarin dose, lifetime risk of dementia) Nutrigenomics Compatibility tests Paternity tests Related-ness tests Ancestry testing
What motivates people to chose a DTC test?
Curiosity
Taking part in research
Generate actionable knowledge
What concerns to consumers have?
Insurance Confidentiality Privacy Unwanted information Unreliable test results Not understanding results
What impact do the results of DTC testing have?
Emotional and psychological stress or happiness if results are all low risk
Changing behaviour
Able to carry drug sensitivity card with them
List issues with DTC testing
Inadequate provision of pre and post test support.
People not being prepared for a bad result
False reassurance if found to be negative
Poor understanding of results by GPs
Costs mean there is no equity of access
What ethical considerations are their surrounding DTC testing?
- Autonomy - The patient has the right to know
- Beneficience - With-holding information may prevent an individual taking preventative action
- Non-maleficence - may give false reassurance or unnecessary burden
- Justice - inequity of access due to cost
Which organisation has introduced legislation to protect consumers ordering DTC genetics tests?
ESHG
Give the 9 points summarised in the ESHG policy on DTC testing
- Clinical utility of a test shall be an essential criterion when deciding whether to offer the test
- Labs should comply with accepted quality standards
- Information about the purpose and scope of the test should be given before testing
- Genetic counselling and pyschosocial assessment should be offered before some testing
- Privacy and confidentiality must be secured
- Avoid inappropriate testing of minors
- All advertising claims should be transparent
- Ethical principles, and international treaties/recommendations should be taken into account
- Nationally approved guidelines should be followed
What regulations must companies operating in the UK adhere to?
Data protection act
Human tissue act
Which UK advisory body has also drawn up guidelines to protect consumers?
UK Human Genetics Commission.
What are the WHO criteria for genetic screening programmes? Who were they drawn up by?
Wilson and Junger 1968.
- Clinically and biochemically well defined disorder
- Significant health burden on the population (disease frequency)
- Disease is associated with significant mortality or morbidity
- Effective treatment is available
- Effective testing is available
- Cost effective
- Early treatment is associated with better outcomes.
What are the benefits of screening?
Improve the prognosis
Identify individuals at high risk of disease before onset
Less radical treatment needed
Reassurance for those with negative results
May inform reproductive choices in carriers
What are the disadvantages of screening?
Longer period of morbidity if there is no effective treatment
Overtreatment
Costly
False negative results give false reassurance
Anxiety from positive result
Unmask carriers
Testing minors
Informed consent
May be undue pressure on individuals to be screened
May be undue pressure to abort affected pregnancy
Genetic disclosure to other family members may be hard.
Name the 9 disorders currently screened for under the UK NSC newborn screening programme.
CF MSUD Homocysteinuria PKU MCADD CHT IVA GA1 Sickle Cell
What screening tests may be available, pre-conception, and to what populations?
B-thal in Cypriots (1/7 carrier freq)
AJ screening - 1/5 are carriers for a rare disorder
What are the ethical issues associated with screening programmes?
Autonomy
Beneficience
Non-maleficance
Justice
List some potential future uses of screening in genetic disease.
NIPS - may replace current combined screening
Rapid screening of NICU patients
Begin screening for carriers of diseases with high UK frequency (CF, SMA)
ACMG59 screening
Screening for late-onset adult disease which may be preventable
How common is PKU?
What gene is mutated?
What metabolite accumulates?
How is it tested for?
1/10,000, AR
PAH
Phenylalanine
MS/MS for Tyrosine:Phenylalanine ratio and absolute concentrations on day 2-5 bloodspot. Genetic testing not routine.
Consider DHPR mutations - they provide a similar phenotype, but are sensitive to BH4 supplementation (a restricted deit is not required in these patients)
How common is CHT?
What gene is mutated?
What metabolite accumulates?
How is it tested for?
1/4000, AR. 10% due to a genetic defect
Can be primary (thyroid a-/dys-genesis) or secondary (TSH insensitivity)
Cause by mutations in 8 different genes.
Screen for present of TSH. Treat with Thyroxine to prevent serious disability
How common is MCADD?
What gene is mutated?
What metabolite accumulates?
How is it tested for?
1/10,000
ACADM, common mutation in 88% (p.Lys304Glu) clinically diagnosed cases, but only homozygous in 50% of NBSP cases
Build-up of medium-chain fatty acids (C8) that accumulate in the lysosome.
Measure using MS/MS to get ratio of C10:C8 and quantification of C8.
Treat with diet, make sure they’re fed regularly
How common is SCD?
What gene is mutated?
What metabolite accumulates?
How is it tested for?
1/2000 UK babies, AR
HBB mutation p.Glu6Val
No accumulation, two mutant b-chains produce HbS and change shape of cells.
Tested by HPLC - will also detect multiple other Hb mutations, and can detect carriers
How common is CF?
What gene is mutated?
What metabolite accumulates?
How is it tested for?
1/2500, AR
CFTR
Abnormal Chloride channel function
IRT test using AutoDELFIA (flurescent Ab-based assay) if high, CF4 kit - if 1 mutation found - second spot, if none found - repeat IRT
How common is MSUD?
What gene is mutated?
What metabolite accumulates?
How is it tested for?
1/120,000, AR
BCKAD complex
Leucine, isoleucine, valine, alloisoleucine - elevated levels of all are suggestive, but not conclusive - second tier biochemical tests required.
Classical form tested using MS/MS, but rarer milder forms may not be detected.
Treat with low protein diet
How common is Homocysteinuria?
What gene is mutated?
What metabolite accumulates?
How is it tested for?
1/100,000, AR
Defects in catabolism of homocysteine to methionine
Homocysteine accumulates
Don’t test within first 24hr of life as methionine levels fluctuate. MS/MS test, quantify homocys and meth
50% of patients in UK are responsive to Vitamin B6
How common is Glutaric acidaemia type 1?
What gene is mutated?
What metabolite accumulates?
How is it tested for?
1/100,000, AR - Glutaric acid, lysine and tryptophan MS/MS to quantify C5DC in blood spot Treat with low protein diet
How common is Isovaleric acidaemia?
What gene is mutated?
What metabolite accumulates?
How is it tested for?
Isovaleric acid
MS/MS for C5 in blood spot - if increased, a full acylcarnitine scan is carried out to rule out GA2
What three methods are responsible for inborn errors of metabolism?
Accumulation of toxic metabolite
Activation of alternative metabolic pathway > toxic
Loss of product.
Why is a genetic diagnosis important for IEMs?
Reproductive choice
Determine carrier status and cascade screening
An exact diagnosis is offered; genotype/phenotype correlations may exist
No invasive procedures needed
Can offer a quicker diagnosis
Promotes use of specific, targeted therapies
Name some IEMs
Pompe disease MCADD Wilson disease Zelwegger syndrome Mucopolysaccaridosis PKU OTC deficiency SLO
What is Pompe disease caused by? What sort of IEM is it? What are the associated clinical features? How is it diagnosed? How is it treated?
Mutations in GAA - lead to build up of GAA
Glycogen storage disorder
Three types: classical: Present in first 2 weeks of life with muscle weakness, hypotonia, cardiomegaly, HCM, feeding difficulties, deafness and failure to thrive.
Death in first year of life.
Non-classical Pompe is less severe. Less cardiac problems and present in first year of life.
Late-onset Pompe shows progressive muscle weakness, but cardiac failure is rare.
Diagnosed by reduced GAA activity in the blood
Treat with high protein diets and enzyme replacement therapy.
Two common founder mutations.
Name the three major haemoglobinopathies
a-thal
b-thal
SCD
What is Hb normally comprised of?
2a2b chains. Though HbF has gamma-chains instead of beta chains and HbA2 has delta chains instead of beta chains.
What is a-thal caused by?
Where is it common?
Loss of multiple copies of the HBA (HBA1 and HBA2) genes on chromosome 16. Deletions are most common, but SNPS and small mutations are responsible for ~10%
Common in Saudi, mediterranean
What causes the following variants of a-thal? a-thal silent carrier a-thal minor HbH disease Barts hydrops foetalis
loss of one copy - asymptomatic
loss of two copies - mild microcytic anaemia
loss of three copies - microcytic anaemia, hepatosplenomegaly, jaundice. Transfusion may be required
loss of all copies - hydrops, not compatible with postnatal life
What is visible in the cells of patients with HbH disease? How are they formed?
Heinz bodies. They are precipitations of Hb beta tetramers (these tetramers form in the absence of Hb alpha).
What are some other alpha thal syndromes?
ATR-16 - a gene deletion syndrome of the region of chromosome 16 containing HBA1 and HBA2 genes.
What is beta thal caused by?
Where is it common?
Loss of HBB expression on chromsome 11. Results in reduced Hb-beta, reduced red cell production and subsequent anaemia.
Caused by ~280 mutations.
Common in Cyprus, tropics and sub-tropics.