Genomics Flashcards
How does different patterns present?
Taking a family history can highlight potential patterns of inheritance. While it can be difficult to be sure about this without a genetic diagnosis, there can be important clues in the family history which may point to a specific pattern of inheritance.
In this talk, we will explore some of the common modes of inheritance, and what clues may suggest these modes of inheritance in a family history.
With autosomal dominant inheritance, only one copy of an altered allele is necessary for the condition to be present
There is a 50% risk to children of affected individual
This risk is independent for each sibling
The gene change does not skip generations
Males and females have same risk of inheriting the gene change
Variable penetrance e.g. the likelihood that disease will manifest if the gene change is present
With autosomal recessive inheritance, two copies of the altered allele are necessary for the condition to be present
If a couple are both carriers, there is a 25% chance of each child they have together being affected by the condition
There is a 50% chance the child will be unaffected but will be a carrier of the condition
There is a 25% chance the child will be unaffected and not a carrier
Typically, both parents of an affected child will be carriers
All children of affected individuals will at least be carriers
Unaffected siblings of an affected individual each have a 2 in 3 chance of being a carrier
Males and females have same risk
X-linked inheritance involves a change in a gene that is on the X chromosome
Males with a gene change on their X chromosome will have the condition
Females with a gene change on one of their two X chromosomes are usually called carriers of the condition
Female carriers are not usually affected, although some females may manifest some symptoms (this is disease specific)
Carriers females have the following reproductive risk with each pregnancy:
25% risk of affected male in each pregnancy
25% chance of a carrier female
25% chance of an unaffected male
25% chance of non-carrier female without the condition
50% risk to sons
50% risk of carrier daughters
What genes are tested in Germline testing?
Tumour Suppressor Genes
Protective role in repairing cells during growth
Germline mutations need a ‘second hit’ in the working copy (Knudson’s hypothesis)
In reality often more complex than this
Oncogenes
Promote growth
Often activate during early/embryonic life
If they acquire ‘gain-of-function’ (i.e. ‘activating’) mutations they are termed ‘proto-oncogenes’…..and increase the chance of cancer
E.g. activating mutations in MEN2 as a cause of Multiple Endocrine Neoplasia type 2
What are examples of tumour suppressor gene and how these can result in inherited cancers?
Retinoblastoma
RB1
Cell division, DNA replication, cell death
Li-Fraumeni syndrome (brain tumors, sarcomas, leukemia)
TP53
Cell division, DNA repair, cell death
Familial adenomatous polyposis
APC
Cell division, DNA damage, cell migration, cell adhesion, cell death
Breast and/or ovarian cancer
BRCA1, BRCA2
Repair of double-stranded DNA breaks, cell division, cell death
How is germline testing done?
How is it done? Need ‘germline’ – generally blood - DNA Gene by gene – traditional approach e.g. test BRCA1/2 in breast/ovarian family; test CDH1 in diffuse gastric cancer family Panel approach (often used now) e.g. in family with colorectal cancer/polyps test a panel of 15 relevant genes ‘phenotype agnostic approach’ – large panel to test all known cancer predisposition genes
What is array CGH and how does it work?When is it used?
Array comparative genome hybridisation (aCGH) allows high resolution chromosome analysis and is done using DNA in solution rather than whole chromosome preparations. In principle, DNA from the patient is broken into tiny fragments which are labelled with a fluorescent dye. DNA from a normal control is treated in the same way but labelled with a different coloured dye. Both are mixed together in equal quantities and allowed to hybridise (stick to their matching sequence).
Each fragment then hybridises to a detector array – essentially, known fragments of DNA sequence fixed to a solid support (e.g. a glass slide). If the patient is missing a fragment, more control DNA will bind to the corresponding detector molecule and will give a ‘normal control’ colour signal when exposed to UV light. If the patient sample and control sample are the same, they will give a fusion colour signal; and if the patient has a duplication of a fragment, more patient DNA will bind to the detector and give a ‘patient’ colour signal.
This is a highly automated process that gives reliable results. It has completely changed the way in which we investigate children with learning disability and is changing the world of prenatal diagnosis.
aCGH will in theory detect trisomy, CNVs, microdeletions and smaller ins/dels but it will not detect single nucleotide changes and cannot, at present, detect balanced translocations.
What are the methods of DNA sequencing?
A variety of DNA sequencing technologies has evolved over the past 20 years or so which differ from the original technique (known as Sanger sequencing) and are collectively known as ‘next generation sequencing’ (NGS). The key feature of NGS is the ability to read – and re-read – fragments of DNA in solution, very quickly and reliably. See this paper on DNA sequencing technologies for an overview.
Diagnostic laboratories use a range of specific chemical processes and analytical instruments to sequence DNA and the trend is towards larger ‘reads’ all the way up to whole exome (about 1% of the genome, ‘clinical exome’ testing typically covers around 18,000 genes known to be associated with human disease) and whole genome (6 billion nucleotides per diploid genome). Datasets are enormous and require highly specialised ‘bioinformaticians’ to compare the data with ‘normal’ and to identify variants of likely significance. Analysis is usually restricted to ‘panels’ of genes; indeed, most analysis is now done in silico using ‘virtual panels’. It is also possible to undertake ‘agnostic’ analysis which effectively asks the question “is there anything of interest in this exome/genome?” – this is more complex and more likely to generate variants of uncertain significance (see below).
Within the NHS, whole exome and whole genome sequencing, followed by focussed analysis of sequence data, will become the standard ‘genetic test’ for most types of inherited disorder. Some unusual types of mutation – triplet repeat expansions, for example – can also be detected using such technologies, with careful bioinformatic analysis. Such approaches will also become standard for tumour DNA analysis to guide cancer therapy. You’ll learn more about that later in your CDM course.
However, Sanger sequencing still has its uses. It is often used – at present, at least – to verify changes detected using NGS, especially when the clinician intends to offer testing to unaffected members of the family.
What are the pro’s and cons of genetic testing?
Pros: such testing may clarify a genetic diagnosis, the cause of Samantha’s cancer and other diseases risks for Samantha and her family
Cons: a lot to take in at a difficult time, worry about other cancer risks, worry about family
Some patients would genuinely not decide to go ahead, although this is unusual. The usual reasons cited are that the patients have enough on their plate with their current treatment; others don’t wish to ‘open Pandora’s box’.
What are the pro’s and con’s for whole genome sequencing?
We are increasingly living in a world in which people want information. A whole genome sequence offers that possibility:
People may have a previously undetected but significant risk of other health problems for which there may be a preventative or therapeutic intervention.
People who carry recessive disease may wish to understand the reproductive implications if they are thinking about having children.
Pharmacogenomic information may help guide current of future drug treatments.
Such comprehensive analysis can reveal unexpected problems and genomic variants of uncertain significance which can complicate an already challenging health problem. This is certainly an area where informed consent is vital. Always remember that germline information generated in one person is likely to have implications for their close relatives.
What are the types of genetic variants?
Whole chromosomes
Aneuploidy: too many or too few copies of chromosomes
Common anneuploidies:
Trisomy 21
Trisomy 13
Trisomy 18
Sex chromosome anneuploidies eg: 45 X0 (Turner’s)
Translocation: one chromosome stuck onto another chromosome
There are no missing or extra parts of the chromsome so this is a balanced translocation
This would not be expected to have a phenotypic effect but their offspring may have an unbalanced translocation
Only a karyotype (like this image) will detect a balanced translocation currently
Copy number variant: large chunks of DNA that are either duplicated or deleted
Identified by SNP array currently
The structure of the genome predisposes some chunks of DNA to be deleted or duplicated
extra or missing copies or genes within that chunk of chromosome
dose effect means that this causes a genetic disease
Both deletions and duplications can be pathogenic
Why do we need to classify gene variants?
The Human Genome Project catalysed massive improvements in sequencing genes, resulting in a paradigm shift in genetics
Before we use to look at the phenotype and select genes for sequencing(Sanger sequencing) based on that now we use Genome sequencing (or large panel of genes, or exome) Huge numbers of variants to classify
The huge increase in the number of known genetic variants exposed the fact that
Many variants we thought were pathogenic were too common in the general population – they had been incorrectly assumed to be pathogenic
Many variants are extremely rare but we do not have sufficient evidence to say if they cause disease or not
Overall there is more ambiguity about the disease causing status of variants
A robust and structured approach to classifying variants was introduced to improve consistency in interpretation of genetic variants
Single nucleotide variant: a change in a single nucleotide
Synonymous less likely to be pathogenic
Missense ?might be pathogenic
Nonsense
Frameshift high likelihood of being pathogenic
Splice site
Other variants-Insertions or deletions of more than one nucleic acid
Can be any number of nucleic acids inserted or deleted
Triplet repeat
Repeat of an amino acid motif at a particular locus
Can be unstable (i.e. get bigger) on transmission:
Premutation full mutation
Anticipation
Example: Myotonic dystrophy type 1 (DM1)
(CTG)n repeat in a non-coding part of the DMPK gene
Number of repeats corresponds
with phenotype:
Whats the significance of a genetic diagnosis?
What difference does a genetic diagnosis make for a family?
– Diagnostic label is helpful – ending the diagnostic odyssey, psychological impact
– May alter clinical management
• access to specific treatments or screening
• Stop unnecessary screening/treatment/investigations
• May guide withdrawal of care decisions particularly in paediatric/neonates
– Enrolment in clinical trials or disease registries
– Implications for the wider family
• Offer predictive testing to other family members, or counselling about reproductive options
– Reproductive options
• Selection of embryos or fetuses with or without a specific genetic diagnosis as in prenatal testing or preimplantation genetic diagnosis i.e. used as the basis of decisions about making another human being!
What clues could you use to determine the clinical significance of a genetic variant?
Has it been seen before in association with disease?
What effect is it predicted to have on the gene/protein?
Is it the same kind of genetic change that is usually pathogenic in this gene/disease?
Has anyone done experiments modelling this genetic variant?
Does the phenotype match my patient?
How common or rare is it?
Is it present in other family members?
What are the Criteria for classifying variants?
Phenotype
Do variants in this gene cause the same pattern of problems / family history that my patient has?
Population data
Is this variant common or rare, or totally novel?
In silico or computational data
What do computer programs predict will be the effect of the protein?
Family studies
Testing other family members
Reported in a disease database
Functional data
Experiments to model this gene change e.g. in cells or in animal models (mice, zebrafish etc)
What needs to be considered in in Silico?Computational data?
What type of variant is it?
Is the variant expected to affect an important part of the protein (‘domain’)?
Does the variant affect an evolutionarily conserved region
How big is the biochemical difference between the substituted amino acids? (only relevant for missense variants)
Computer prediction programs amalgamate all of the above data
What computer predictive programmes can be used and hoe accurate are they?
PolyPhen and SIFT are commonly used prediction programmes
These are not as robust as we would like, and can be conflicting and even wrong
Use as part of a comprehensive assessment of a variant, not in isolation
What are the benefits and harms of population screening?
benefits
Early detection of disease allows for earlier more effective treatment
Identifies at risk individuals so preventive measures can be put in place
Identification of carriers of heritable conditions allows for informed family planning
Increased awareness of own health allowing for lifestyle changes
Control of disease at a population level
Cost-effective
Potential harms
False positive/negative results
Test may be invasive
Insurance implications
Psychological implications
What is the national breast screening program and when is it done?
Most common cancer in the UK
A woman’s lifetime risk – 1 in 8
Around 55,000 people are diagnosed each year (including 400 men)
Just over 80% occur in women over 50 years
National Breast screening programme
Most cases of breast cancer are sporadic
Less than 10% is caused by a germline mutation that increases risk of developing breast cancer
Population screening programme
Offered to women from the age of 50 to 71st birthday every 3 years
May be eligible before the age of 50 if individuals are at a higher risk of developing breast cancer
Women who are 71 or over are still eligible for screening but must request
What is the national bowel screening program and when is it done?
Third most common cancer in the UK
1 in 14 men and 1 in 19 women will get bowel cancer during their lifetime
Mostly sporadic
1 in 4 individuals will have a family history
5-6% inherited e.g. Familial adenomatous polyposis (FAP), Lynch syndrome
Population screening programme
Offered to individuals aged 55 and over
One-off bowel scope screening test offered at age 55 (if available in the individual’s area)
Individuals aged 60-74 offered home testing kit (faecal occult blood test) every 2 years
Individuals 75 or over can request a home testing kit every 2 years
- Whats the diagnostic criteria for Lynch syndrome?
Unaffected carriers
BRCA1
Breast cancer: Lifetime risks (to 80 yrs): 60-90% Ovarian cancer: Lifetime risk 40-60% Majority of lifetime risk conferred after age of 40 Male breast cancer: Lifetime risk ~0.1-1% Prostate cancer: Lifetime risk similar to population risk ~8%
BRACA2
Breast cancer: Lifetime risks (to 80 yrs): 45-85% Ovarian cancer: Lifetime risk 10-30% Majority of lifetime risk conferred after age of 50 Male breast cancer: Lifetime risk ~5-10% Prostate cancer: Lifetime risk significantly increased to ~25%
Affected carriers
BRCA1
Breast cancer:
Lifetime risk contralateral breast cancer ~50%
Overall 5 year risk ~10%
BRCA2
Breast cancer:
Lifetime risk contralateral breast cancer ~50%
Overall 5 year risk ~5-10%
When Should BRACA testing be offered?
Individual affected with breast cancer:
In general BRCA1/2 is indicated in a person affected with breast cancer where the Manchester Score is 15 or above
The Manchester Scoring System (MSS) allows the calculation of the probability for the presence of mutations in the BRCA1/2 genes in families suspected of having hereditary breast and ovarian cancer
The MSS, is used alongside the family history:
Scores are added for each cancer
A Manchester score of 15 is equivalent to a 10% threshold for finding a BRCA variant
Individuals with a grade 3 triple negative breast cancer <50 should also be offered BRCA testing
Testing for the 3 Ashkenazi founder mutations offered to women with breast cancer and Ashkenazi ancestry
Full analysis offered when Manchester Scoring is 15 and above
Newer genes such as PALB2 can also be tested
P53, Stk11, CDH1, PTEN can also be considered if appropriate
Individual unaffected with breast or ovarian cancer:
BRCA1/2 testing is indicated in an unaffected person where there is no living affected person available for testing. The Manchester score should be 20 or above
The individual should have a 1st degree relative affected with a relevant cancer
Ideally testing should be offered to an affected individual where there is a greater chance of identifying a mutation
Testing for the 3 Ashkenazi founder mutations offered to women with breast cancer and Ashkenazi ancestry. Full analysis of offered when Manchester Scoring is 20 and above
Testing of other genes e.g. PALB2, P53, Stk11, CDH1, PTEN can also be considered if appropriate
What is Lynch syndrome?
Lynch syndrome, also known as hereditary non-polyposis colorectal cancer (HNPCC), is caused by mutations in one of several DNA mismatch repair (MMR) genes; MLH1, MSH2, MSH6 and PMS2.
Carriers*:
Men – up to 80% risk of colorectal cancer
Women – up to 70% risk of colorectal cancer and 60% endometrial cancer
* Exact risk will depend on the gene
What is predictive testing and when is it done?
Predictive testing for known pathogenic variants should be offered to all families where a pathogenic variant has been identified. This is testing in individuals who are not yet affected by the condition.
In some conditions, risk management options are available to individuals who test positive.
For adult-onset conditions, testing is typically offered from the age of 18. Therefore in cancer predisposition syndromes, testing in children is not usually considered as onset is typically in adulthood and surveillance is not normally recommended before the age of 25/30.
Exceptions to this include FAP and TP53 where earlier surveillance may be possible.
Typically predictive testing is offered to first degree relatives in the first instance. If they test positive, then their first degree relatives would then be eligible for predictive testing. If they test positive, their first degree relatives would be eligible for testing, and so on.
It is important that individuals understand the implications of predictive testing. While it is different in some ways to diagnostic testing, it is equally important that patients are fully informed, and are aware of potential medical, psychological, familial and financial implications of genetic testing.
Who is eligible for predictive testing
Typically, all first degree relatives of individuals who have a gene change identified in them will be offered predictive testing.
Therefore, if a gene change is identified in Samantha, both of her parents, and her brother and sister would be eligible for predictive testing.
If they test positive, then their first degree relatives would be eligible for testing. For example, if her mum tested positive, her maternal aunt would then be eligible for testing.
Individuals who test positive may then be eligible for risk management options themselves.
Individuals who test negative are usually considered to be at population risk. Their descendants are also ‘off the hook’.
How does result of genetic tests affect insurance?
The ‘Code on Genetic Testing and Insurance’ is an agreement between the Government and the Association of British Insurers (ABI)
Outlines what an insurance company needs to know about testing, and how they should act:
To not require or pressure you into a predictive or diagnostic genetic test
To not ask for or take into account the result of a predictive genetic test if you are applying for insurance (the only exception being if you are applying for life insurance over £500,000 and you have had a predictive genetic test for Huntington’s Disease)
Avoids patients with positive result facing discrimination from insurance companies
However, insurance companies can put two and two together by asking, ‘do you have family history of cancer’, ‘have you had an outpatient’s appointment’ and increase premiums
Advise patients before undergoing genetic testing to consider getting life insurance/mortgage insurance in place, as answering questions with knowledge they had at that time
What are the factors contributing tio increased breast cancer risk?How can patients risk of developing can classified?
Risk factors
Family history Smoking Obesity Alcohol Lack of exercise Nulliparity or first pregnancy >30 Early menarche (<12) and/or late menopause (>55) Current or previous HRT use Previous radiotherapy Previous breast cancer (Prolonged breast feeding is protective)
Risk stratification
can be done based on genotypes and pheotype
The genetics service aims to provide risk assessments for family members and further screening recommendations. An initial family tree is compiled and diagnoses are confirmed via cancer registries or medical records. Genetic testing may also be performed. From this information, we can classify cases into different types of risk categories from very high to low and determine appropriate further management.
What genetic testing is done in breast cancer?
Current options
Single mutation (predictive) test – if known mutation in the family
Standard panel test – BRCA1/BRCA2/PALB2 – if fhx breast/ovarian cancer meeting testing criteria of 10% chance of finding a mutation
Bespoke panel test – by adding other relevant genes to above panel
Future options
Large panel testing
Polygenic risk scores
What are some high pentrance breast cancer genes
BRCA1 and BRCA2
PALB2
TP53 Li Fraumeni(Brain tumors,leukemias,sarcomas)
PTEN Cowden(microcephaly,autism,endometrial,kidney,thyroid ca,bengin skin lesions)
CDH1 Hereditary Diffuse Gastric Cancer(lobular breast cancer)
STK11 Peutz Jeghers
