Genetics Flashcards
Next generation sequencing (NGS) aka massively parellel sequencing or deep sequencing
Revolutionised genetic sequencing. Umbrella term many different technologies. Can now sequence an entire human genome in one day (Sanger sequence technique took 30 years). Benefits: cheap, fast, Good for PGT-A (Copy number chromosome counting)
1. Isolate DNA/RNA sample
2. Generate smaller fragments
3. Small fragments get joined to oligonucleotides - one on each end (they are complementary sequences).
4. These allow them to bind to the flow cell (a channel for absorbing the DNA fragments) as single strands.
5. The fragments hybridise to the flow cell by joining to identical oligonucleotides (image)
6. DNA amplification then occurs using PCR (this is done simultaneously for all fragments on the flow cell.
7. The strand not attached to the flow cell is then washed away.
8. Bridge building then occurs where the free end hybridises with the matching oligonucleotide on the flow cell.
9. PCR used for amplification again but as the two strands separate (denature) they both remain joined to the flow cell.
10. Bridge building and amplification then happens over and over to amplify copies. So from 1 DNA fragment can get massive numbers of copies.
11. Forward and reverse facing strands depending on which oligonucleotide is presenting.
12. All the forward facing strands are cleaved and sequencing starts.
13. A primer binds to the oligonucleotide on the DNA strand and nucleotides free floating attach to their base pair.
14. Nucleotides are fluorescently labelled, this is then excited by a laser and the signal obtained. This is done on mass in sequencing with all the amplified DNA fragments being processed together.
15. Bioinformatic tools are used to overlay all the sequenced fragments to create the genome
16. This technique is only possible because of Sanger sequencing allowing the original mapping of the human genome, to use as a reference genome.
Polymerase chain reaction
Provides a way to make more copies of a portion of DNA extra-cellularly.
1. Denaturing DNA (heat ) - separates the two DNA strands
2. Annealing - cooled and bound to the primer
3. Synthesis - DNA polymerase (enzyme) used to build DNA strand off base strand, nucleotides within buffer/fluid needed.
4. This process is done thousands/millions of times
karyomapping
Historically PGT-M done by using PCR to amplify the gene affected and determine if affected or not. Problem with this technique is that ~10% of single cell amplifications are affected by allele drop out where in a heterozygous individual only one of the alleles amplifies. Contamination also a problem with this technique. This causes unacceptable false positive/negative results.
Now use linked polymorphisms. Usually look at 5-10 polymorphisms close to the gene in question. Prevents ADO and contamination causing an issue.
This polymorphism technique doesn’t identify aneuploidy, can’t do PGT-A and PGT-M at the same time.
Karyomapping:
More generic, less customised method
Allows faster feasibility testing
Able to detect common aneuploidies as well as the single gene condition in question
Looks at 300 000 SNPs (single nucleotide polymorphisms), scattered throughout the genome using an illumino bead array. Looks at the DNA fingerprint of each region of the genome using these SNPs.
Can focus the karyomapping in around the SNPs close to the gene of interest.
Karyomapping essential ignores the mutation and focuses on SNPs around the mutation (need them to be above and below the mutation).
Then genome is typed out in region around mutation for mother, father and another family member with the condition - sibling, grandparent etc.
Can then track the mutation within the embryos and also aneuploidy within the embryo.
Sickle cell disease
Autosomal recessive condition involving Hb S gene defect. Substitution.
EIther homozygous for HbSS or HbS defect with another genetic Hb mutation
- HbSS
- HbSC (Hb C mutation)
- HbSBeta (B-thalassaemia, sick cell thalassaemia)
- HbSD-punjab
- HbSE (HbE mutation)
Black/African highest prevalence as carriers and affected individuals
Characterised - sickle cell shape to RBC and chronic anaemia.
HbSS - HbF protective for first 6/12 as this drops symptoms arise.
Anaemia
Vaso-ooculsive diseas - sickle shaped RBC get stuck in capillaries - severe pain, organ damage, stroke, renal disease, retinopathy, lec ulcers, bone necrosis.
Inc risk of infection (splenic funciton destroyed)
Acute chest syndrome
- fat emboli from bone
Sudden death
On average die 25-30 years younger.
Sickle cell crisis can occur during period of infection.
CRISPR-Cas-9 gene editing technology approved by FDA Dc 23 in USA for tx of sicklecell. Casgevy . Targets patients blood stem cells - increases production of HbF
Tay-Sach disease
Autosomal recessive condition.
Common in Ashkenazi Jewish population
Lysosomal storage disorder, caused by gene mutation on Chrosomone 15 which codes for a lysosomal enzyme B-hexosaminidase (HEX-A).
Normal breaksdown lipids in neurons - accumulates
Progressive symptoms of CNS degeneration - decreased muscel tone, weakness, visual difficulites, seizures.
Either have total deficiency of HEX-A or variable production (occurs for later onset and slower progression).
Huntington disease
Rare Autosomal Dominant triplet repeat disorder
Causes neurodegenerative disease (abnormal movements and cognitive problems). Average age of onset ~40 yo. (chorea, abnormal eye movements, dementia, personality change, depression).
Chrosomome 4 - CAG repeat. Normal 10-36, Huntington >/= 36.
Codes for glutamine - excess glutamine.
Likely causes cell death.
Higher number of repeats the earlier the patient starts to have symptoms -often earlier symptom onset in each generation.
Pre-mutation alleles 27-35 repeats. 36-39 repeats = variable penetrance. 40 repeats = definite disease.
Repeat expansion happens more in male line. Anticipation and new disease alleles more common in males line.
Examples of triplet repeat disorders
Huntingtons (CAG) (autosomal dominant)
Fragile X (CGG) (X-linked dominant)
Myotonic dystrophy (CTG) (autosomal dominant)
Friedreich Ataxia (GAA) (autosomal recessive condition)
X linked recessive condition
Haemophilia A and B
Red-green colour blindness
Duchenne muscular dystrophy
Becker’s muscular dystrophy
G6P dehydrogenase deficiency
X linked dominant conditions
Rett syndrome (95% sporadic mutation) in 5% germline - X linked dominant and most males die in utero or after birth. Females - NDD - imparied language and coordination. Seizures.
Fragile X syndrome
Alport syndrome (glomerulonephritis, CKD, hearing loss)
Autosomal dominant conditions
Huntington disease
Polycystic kidney disease
Neurofibromatosis
Marfan syndrome
What causes increasing aneuploidy rates in older women
Chromosome segregation error is the underlying cause.
-Meiotic recombination failure – exchange of genetic material between homologous chromosomes during prophase 1 doesn’t occur properly.
-Cohesin deterioration – cohesion holds sister chromatids together and is important to maintain proper alignment required to achieve correct chromosome segregation
-Spindle assembly checkpoint (SAC) dysregulation – SAC usually - monitors proper kinetochore attachment to the spindle and prevents chromosome missegregation by delaying anaphase until accurate attachment to the spindle apparatus occurs.
-Mitochondrial dysfunction – increases with age and negatively affect occyte – ROS production and oxidative stress increases, can lead to meiotic spindle damage, chromosome misalignment, aneuploidy and oocyte death.
Why are normal infants born from mosaic embryos?
Mosaicism in trophectoderm doesn’t mean mosaicism within the inner cell mass – confined placental mosaicism.
Embryo self-corrected by aneuploidy cell apoptosis.
False positive result from biopsy and actually euploid embryo – test artifacts, laboratory error
Overall level of mosaicism within individual may be low enough to not cause any pathogenic impact.
Natural conception unrelated to embryo transfer.
array CGH
comparative genomic hybridisation
Form of chromosomal microarray analysis.
BAsed on use of differentially labelled test and reference genomic DNA samples.
These are simultaneously hybridised to DNA targets arrayed on a glass slide or other solid platform.
Essentially scans the entire genome providing additional information such as CNVs.
Avoid the limitations of convention karyotyping and FISH, such as culture failure, limited chromosomal examination
SNP microarray
SNP array is a form of chromosomal microarray analysis similar to array CGH.
Fragments of DNA from the human genome where we know that there are multiple known alleles of that region of the DNA (SNPs). Each position on an array corresponds to a specific location on a chromosome.
Array platforms can be built to examine particular areas of interest, i.e. areas we know are associated with pathogenic SNPs. Patients DNA is then amplified – labelled with probes and hybridised onto that array platform. Results are then computed and deletions and duplications (CNVs) can be identified.
It can additional detect the parental source of chromosomes and anomalies and can therefore be used to rule out maternal cell contamination.
