CRISPR, HHGE categories only and NIPT Flashcards
what is the Cas 9 complex?
The Cas 9 complex was discovered in bacterial immune system, and is really good at making a ds break at a specific sequence, then runs away
how dose the CRISPR-Cas 9 system work?
Two components: Cas9 (DNA-cutting protein) and guide RNA form a complex to identify and cut specific DNA sections
Cas9 locates and binds to PAM (stands for Protospacer Adjacent Motif, in DNA, it is a short, specific sequence of nucleotides adjacent to the target sequence that the CRISPR-Cas9 system uses as a recognition site)
Guide RNA unwinds DNA helix and matches specific sequence
Cas9 cuts DNA at correct sequence, creating double strand break
Cell attempts error-prone repair via NHEJ, often introducing mutations
CRISPR is therefore useful for knocking out specific genes
how do we use CRISPR-Cas 9?
what are some problems with the system?
Design the guide RNA sequence depending on what DNA sequence you want to target.
problems -
While the repair process NHEJ (non-homologous end joining) is error prone and often causes insertions/deletions, you cannot guarantee the mutation you get, and it will differ each time, it won’t always just simply knock out the gene
This aspect of the process is therefore not ideal for inside the body, except perhaps isolated areas like the retina (choroideremia for example)
metachromatic leukodystrophy - what is it? how is gene therapy used to cure it?
When you can’t produce a functional enzyme ARSA -a involved in degradation. Without it, fatty chemicals called sulfatides build up. These gradually destroy the protective layer around cells in the brain and nervous system, leading to a devastating deterioration
Hematopoietic progenitor cells are removed and the functional gene introduced using a virus. Before being given the treated cells, patient must undergo chemotherapy to kill the faulty cells in the bone marrow
Good because… not in vivo, so you can check for correct mutation/other unwanted DS breaks, and no risk of immune response to virus used
what did He Jiankui supposedly do? why was he criticised?
Apparently altered CCR5, a protein on immune cells used by HIV to target the immune system.
A 32 base deletion is a naturally occurring variation of this gene. Two copies of this D32 mutation are resistant to many forms of HIV infection. He Jiankui supposedly edited embryos to have this homozygous mutation
Was highly criticised - not all mutations seen actually matched the desired one. Also, in the cases this was applied to, where one of the parents had HIV, it is entirely possible and easy to prevent this being passed to the child, so the procedure was unnecessary and not in the best interest of the patients
what are the six categories of HHGE (in germline cells so is heritable)?
Category A - Cases of serious monogenic diseases which ALL children would inherit the disease genotype (e.g. Huntington’s disease (v rare))
Category B - Serious monogenic diseases with some not not all of a couple’s children would inherit the disease-causing genotype (e.g. Cystic fibrosis)
Category C - Other diseases with less serious impact that those in A or B (familial hypercholesterolemia)
Category D - Polygenic diseases (Type II diabetes/ schizophrenia/ some cancers)
Category E - Other applications (not heritable diseases) (e.g. EPO gene for endurance sports, thousands of genes linked to intelligence, resistance to a certain disease as seen above with HIV)
Category F - Genetic conditions that result in infertility
what are the three parts of the first trimester combined test?
- a blood test for a pregnancy associated plasma protein-A, PAPP-A, and HCG (human chorionic gonadotropin). Abnormal levels of these proteins is associated with down syndrome
- Nuchal translucency test - ultrasound used to measure specific area on the back of a foetus’ neck, down syndrome is indicated by a greater depth of this area due to a higher collection of fluid
- Maternal age and results of these two tests are combined to estimate likelihood of down syndrome, then further testing of the foetal DNA via the amniotic fluid can be done
what is NIPT?
NIPT - non-invasive prenatal testing -
Just uses the mother’s blood, the foetus is not involved
There are little fragments of foetal DNA in the mother’s bloodstream (not in cells)
They can then be isolated and sequenced (works in single babies and twins, but not multiple pregnancies)
Tests for aneuploidies like down syndrome, Edwards and Patau syndrome
how does NIPT work?
For down syndrome, you would look at a specific SNP on chromosome 21 where the foetus has the same genotype as mum (I think)
Cases without down syndrome -
In cases where there is no aneuploidy, regardless of the foetal fraction percentage (how much foetal DNA is present in the blood sample), the ratio of alleles will always be 1.0. As in, if both mother and foetus are genotype GA for a certain SNP, the G/A ratio will always be 1:1
Cases with down syndrome -
In cases of aneuploidy, the foetus will be contributing an extra allele for the SNP from chromosome 21 you are looking at, e.g. the mother is GA while the foetus is GGA. this will change the G/A ratio
How much the GA ratio changes depends on the amount of foetal DNA present - the foetal fraction
In the example given, if the foetal fraction was 20% the G/A ratio will be 1.2
Any deviation from a 1.0 ratio indicates trisomy 21
Nuffield council (UK) on Bioethics has three major concerns on NIPT - what are they (in title only)?
misleading statistics
poor information/ limited and biased information
lack of follow up support
explain in detail Nuffield Council’s concerns about NIPT and misleading statistics
Claim - websites of most private providers state that NIPT is ‘99% accurate’ or has ‘99% sensitivity’
Reality - the conditions affect less than 1% of all children born, so a dummy test that gave everybody a low chance result would be 99% accurate.
A more helpful statistic is the positive predictive value P(positive test | sick)
positive predictive value of NIPT for Downs’ syndrome is 81.6%, for Edwards’ syndrome is 37%, and for Patau’s syndrome is 49%.
The negative predictive value (NPV) tells you how likely it is that a ‘low chance (negative)’ NIPT result is correct.
NIPT has a very high NPV for Down’s, Edwards’ and Patau’s syndromes(99.9%)
REMEMBER - P(healthy | positive result) i.e. false positive rate = 20% for down syndrome, 50% for Patau’s, and 60% for Edward’s
is the 99.9% NPV value from NIPT useful?
it’s more likely that someone won’t have down syndrome so the 99% NPV isn’t very useful as this could also be obtained just by guessing. the 80% PPV is more useful as it shows % probability the test will correctly diagnose someone actually affected (small population), but 20% of the time this is incorrect
Explain why Nuffield Council is concerned about poor information surrounding NIPT
Provide very limited information that is unbiased, on the conditions being tested for, meaning parents cannot make informed decisions
Private companies are also offering NIPT for conditions that are extremely rare, like Turner’s syndrome for example, where positive predictive values aren’t even available most likely due to the limited research that has been done showing low accuracy of NIPT for these conditions
So positives from NIPT for these rare conditions, that are quite likely to be false positives, cause anxiety and unnecessary tests like an amniocentesis which has a small risk of miscarriage
explain Nuffield Council’s concerns on lack of follow up support regarding NIPT
While some places provide both pre and post testing support, many don’t
Stressed mothers with positive results then have to turn to the NHS who aren’t necessarily prepared, especially for the other rare conditions some of these private clinics test for
note - improvements - Care Quality Commission has already started carrying out inspections of clinics in England that offer NIPT
what does a negative NIPT result mean?
actually no such thing as negative, it’s low chance - so she has a lower chance than the general population of having a baby with DS
more specifically, for DS NIPT has super high NPV, so this low chance result has >99% chance of being right, (tho chances of DS are 1% anyway)
