Hot topics Flashcards
Hemophilia B (Christmas diseasefactor IX deficiency)- why is current therapy not always successful? what therapy is in development for haem A and B?
Genetically engineered recombinant factor 9 - patients can develop immune response.
Adeno-associated viruses - A gene and promoter are inserted into the viral genome. doesn’t cause disease but may have immune response.
Haemophilia A - R338L encoded by AAV. gain-of-function mutation that has approximately eight times the specific activity as wild-type factor IX. in phase III trials - only suitable if dont have neutralising antibodies.
Why is genotyping useful for PKU testing?
genotype determines residual PAH activity and may help determine BH4 responsiveness in some instances
2 null variants highly predictive of non-responsiveness – response test not recommended
how is DPYD testing used in 5FU treatment?
5fu used in treatment of solid tumours
5FU toxicity seen in 10% of patients
DPYD metabolises 5FU & deficiency causes cytotoxicity
ARMS, sanger, NGS can be used to detect variants in DPYD that cause poor metabolism and so dose-management is critical
DPD activity values assigned to certain variants - sum up alleles
poor metabolisers = avoid 5FU
intermediate = 50% of dose
normal = full dose
Need short TAT- actionable time frame
how might WGS be used for NBS? what are pros and cons?
Genomic England ongoing research study to assess use of WGS for actionable genetic conditions
Timely diagnosis, access to treatment pathways, and enable better outcomes and quality of life
preventative
lifetime storage
research - identify new conditions
No phenotype in healthy newborns-
carrier status
incidental findings
what should be reported
TAT
consent, data storage, eligibility, reanalysis
discrimination
what is aminoglycoside ototoxicity testing? what is the most common variant? what test can be done to detect the variant and what is the advantage?
MT-RNR1 variants - ototoxicity(hearing loss) when treated with amnioglycosides (eg. Gentamicin given for translational read-through in CF)
- m.1555A>G MT-RNR1 - results in destruction of sensory hair cells in the inner ear
- urgent R65 in TD
- portable GeneDrive kit Detects m.1555A and m.1555G by melt curve analysis (30 mins rapid bedside testing)
- 100% sensitivity and specificity
what are limitations of short reads data?
high GC content regions/ amplification bias
Repetitive regions where short reads will not map uniquely
SVs and STRs
phase
pseudogenes
what are advantages of long read data? eg. SMRT Sequencing: PacBio and Nanopore
de novo assembly - spans repetitive regions for accuracy
better for pseudogenes and phase
structural variants (e.g. segmental duplications, gene loss and fusion events)
STRs
characterization of methylation
what are drawbacks of long read sequencing?
DNA sample must be of high/sufficient quality
low sequencing accuracy compared to short reads
cost
may be lower throughput than short reads
what are NTRK genes
neurotrophic tyrosine kinase (NTRK) gene family responsible for the normal development and function of both the central and peripheral nervous system, regulating neuronal growth, proliferation and survival
what are NTRK gene fusions?
occur in 1% of solid tumours
TRK fusion proteins - Oncogenic drivers of adult and paed cancers eg. thyroid, breast, lung, CRC
NTRK gene fusions are also detected in ALL and AML at a frequency of <5%.
over 80 different partners - 3’ end of NTRK gene (kinase domain) fuses with 5’ end of other gene creating chimeric oncoprotein
how are NTRK gene fusions detected?
- NGS multiplex detection of FFPE samples
- dual colour breakapart FISH BUT unable to identify novel fusion gene partners
- RT-PCR: only able to detect known fusions included in the assay, primer design challenging
- IHC: antibody targeting NTRK C-terminal domain: false positives, not amenable to multiplex
what is the treatment for NTRK fusions?
TKI eg. larotrectinib or entrectinib
second line TKIs if resistance mutation
