Hot Topics Flashcards
Name 5 genes associated with FH
APOB LDLR PCSK9 APOE LDLRAP (AR) 70-95% FH - APOB, LDLR, PCSK9 - Going to do MLPA on LDLR
When should FH be suspected?
Total cholesterol >7.5mmol/l
Personal or family history of premature CHD (event before age 60)
What is FH caused by clinically?
1) Decreased number of LDL receptors
2) Poor binding of receptors
3) Increased degradation of receptors
LDL (cholesterol rich lipoprotein) is recycled by the liver - LDL binds to receptor on surface and is internalised
What is incidence of FH
1/200-250
AD Disorder
Treatment for FH
Statins - inhibit cholesterol synthesis
Genes associated with Lynch Syndrome
MLH1, MSH2, MSH6, PMS2, EPCAM
Caused by defects in DNA mismatch repair MutS (MSH) and MutL(MLH) family of proteins
Which 2 main cancer types are associated with Lynch Syndrome?
Colorectal cancer and Endometrial cancer
What is molecular cause of Lynch?
Germline mutn in MLH1, MSH2, MSH6 or PMS2 followed by 2nd somatic loss of remaining copy - in almost all cases
Define anticipation
Signs and symptoms of genetic condition become more severe and appear at earlier age as disorder is passed from one generation to the next
Good eg - triplet expansion disorders
Define age related mosaicism
Accumulation of somatic/germline mutns over the course of someone’s life resulting in mosaicism
Good eg - loss of 1 X or Y to give 45,X cell line (benign) or cancer
Define Variable Expressivity
Phenotype is expressed to a different degree among individuals with the same genotype
Good eg: various neurosusceptibility loci in arrays, Marfan Syndrome
Define Penetrance
Proportion of individuals carrying a variant that expresses and associated trait
Good eg: BRCA1 - 80% lifetime risk of developing breast cancer
What can complicate the calculation of penetrance?
Ascertainment bias, attributable risk (would cancer arise without genotype), polygenic traits, cancer
Define TMB
Tumour mutation burden - number of small non-synonymous variants per Mb
What is DPYD
Pyramidine catabolic enzyme - initial and rate limiting factor in pathway of uracil and the midline catabolism
What is consequence of DPYD path mutn?
Error in pyrimidine metabolism - increased risk of toxicity in cancer patients receiving 5-fluorouracil chemotherapy
What drugs are relevant wrt DPYD testing?
Capecitabine and 5-fluorouracil - widely used treatment for solid tumours including colorectal and metastatic breast cancer
How many variants do we test for DPYD in Liverpool?
4 - 5 day TAT - either high risk of fatal toxicity (half dose/alternate therapy) or risk of severe toxicity (3/4 dose and raise over cycles)
Give some limitations of short read NGS
Affected by GC rush/repetitive regions
Hard to map around regions of structural variation
Mapping to imperfect reference genomes is difficult
Phasing of variants not possible if more than a few hundred bps apart
Give 2 egs of commercially available Long Read Sequencing technologies
Single Molecule Real time (SMRT) sequencing - PacBio
Nanopore sequencing - Oxford Nanopore Technologies
What are some limitations of Long Read Sequencing
For optimal LRS - fresh material or even intact cells are required
DNA isolation pro tools require improvements
Error rate is higher than that seen in short read sequencing
Currently more expensive than SRS
Data analysis pipelines not as mature and need more work
Ranges for Huntington Disease
Normal - 6-25 Intermediate - 27-35 - risk of expansion down paternal line Reduced Penetrance - 36-39 Affected - >39 Juvenile onset >60 rpts
Pathogenesis for HD
CAG rots Translated into polyglutamine tract thought to acquire deleterious function when abnormally expanded, neuronal degradation, accumulation of mutant protein in insoluble aggregates
Risk of expansion through transmission in HD depends on:
Repeat length
Age/sex transmitting parent
Family history
Sequence surrounding repeat - polys eg Glu2645del overrepresented in HD alleles compared to normal
What genes are involved in NTRK rearrangements
NTRK1, NTRK2, NTRK3 - Code for TRKA, TRKB and TRKC - collectively known as TRK
Neutrophil Receptor Tyrosine Kinases
Describe NTRK fusions
Inter-intra chromosomal rearrangements form hybrid genes - 3’ sequence of NTRK1/2/3 that include kinase domain are juxtaposed to 5’ sequences of a different gene
Chimaeric oncoprotein characterised by ligand-independent constitutive activation of the TRK kinase
Treatment for NTRK rearrangement cancers
First generation TRK tyrosine kinase inhibitors - Larotractinib or entrectinib - Immunotherapies - result in histology-agnostic responses in both adult and paediatric patients - generally well tolerated
Why would you mainstream genomics?
Strategy/policy - eg 100k
Equity of access
Clinical utility - improve diagnosis and better healthcare - reduce diagnostic odyssey
Increase knowledge
Workload - clinical genetics can deal with numbers
Technology - advances, cheaper etc
What are barriers to mainstreaming genomics?
Education - complicated reports, access support Appointment times Focus on own specialism Informed consent Correct cascading/onward referral Misinterpretation of results Finance IT Routing samples
How could you educate clinicians as part of mainstreaming genomics?
Embed in school/uni curriculum Involved professional bodies Engagement/training events MDTs Staffing - specialist educators Apps/website/social media National level - genomics England courses etc Different formats that can be accessed at any time
Why is consent needed?
Person understands the nature and purpose of the procedure or intervention thereby asserting a right to self determination
What are 3 criteria required before consent is deemed legally valid?
1) person giving consent must have sufficient, appropriate information to be able to make a decision
2) They must be competent to make decision
3) Consent must be voluntarily given
What consent issues should be considered when families undergo trio analysis?
VUS IFs Family implications of results Non-paternity Implications of negative result
What is the criteria for undertaking population screening?
Benefits should outweigh harm
What are potential benefits of whole genome sequencing of newborn screening blood spots?
Detect highly penetrant diseases/treatable diseases
Family choices - cascade testing etc
Additional findings - early monitoring/intervention
Data useful for later life - eg risk, prevention etc
Could reduce/stop diagnostic odyssey in some cases
What are the potential problems of whole genome sequencing of newborn blood spots?
Consent and autonomy of child No clinical context Incidental findings Technical issues Data storage and security How can data be accessed throughout lifetime - IT infrastructure Is it cost effective? Do benefits outweigh harms?
What are the advantages of NIPT over current screening method for common aneuploidies?
High sensitivity and specificity
Reduced need for invasive testing
If strategy right should reduce costs
When can’t NIPT be used?
Maternal malignancy Blood transfusion in last 3 months Transplant Vanished/demised twin Too early More than twin preg Also affected by maternal weight
What are 5 steps in Root cause analysis?
1) Gather info and define problem
2) Fill in the gaps
3) Analyse/identify root cause
4) Develop action plan - SMART
5) Recommend and implement solutions - PDSA cycles
What considerations can you make to reduce costs in lab?
Staff costs
Non staff costs - eg renegotiate contracts, eliminate waste, stationary costs, estate costs, rationalise/improve tests
Income generation
What are 4 tiers of CNVs and CN-LOH in neoplasticism disorders?
1 - Variants with strong clinical significance
1a - specific entity in WHO classification/professional guidelines or germline path variant with cancer predisposition
1b - Assoc with specific neoplasm/prog/treatment response as shown by evidence with expert consensus
2 - Variants with some clinical significance
3 - Clonal variants with no documented neoplastic disorder associated
4 - Benign/likely benign variants
WHO recommendations for Lynch testing
1) Immunohistochemistry 4-panel test for MLH1, MSH2, MSH6 and PMS2 OR do macrosatellite instability test
2) If MLH1 immunohistochem test or MSI test abn do BRAF V600E
3) If BRAF -ve do MLH1 promotor hypermethylation
4) If Hypermethylation -ve -confirm Lynch by genetic testing of germline DNA
Name 7 classes of Myeloproliferative Neoplams
1) CML
2) Polycythaemia Vera
3) Essential Thrombocytopenia
4) Primary Myelofibrosis
5) Chronic neutrophilic leukaemia
6) Chronic eosinophilic leukaemia
7) MPN - Unclassifiable
What variant causes majority of Polycythemia Vera?
JAK2 p.V617F - 95%
If negative can do JAK2 exon 12 granulocyte DNA sequencing - need fresh sample
What would your testing pathway be for Essential Thrombocytopenia/Primary Myelofibrosis?
JAK2 p.V617F
CALR mutn testing
MPL mutn testing
Can see triple negative so can’t exclude diagnosis
CALR - 80% type 1 (52bp del) or Type 2 (5bp insertion)
MPL - Commonly W515L, also see W515A and W515R
What 3 rare cancers are NTRK rearrangements seen in at over 90%
Infantile fibrosarcoma - ETV6-NTRK3 most common
Mammary analogue secretory carcinoma
Breast secretory carcinoma
Who will get NTRK testing/treatment as it stands?
Metastatic refractory relapse patients
What is WHO classification of AML?
1 - AML with recurrent genetic rearrangement/abnormality
2 - AML with myelodysplasia related changes
3 - AML related to previous chemo/radiation
4 - AML NOS
5 - AML Myeloid Sarcoma
6 - Myeloid proliferations related to Down Syndrome
Name 3 favourable abnormalities in AML
T(8;21)(q22;q22) - RUNX1/RUNX1T1
T/inv(16)(p13.1q22) - CBFB/MYH11
T(15;17)(q24;q21) - PML/RARA
Name 3 unfavourable abnormalities in AML
Deletion of part of chrm 5 or 7 T/inv(3)(q21q26.2) RPN1-EVI1 T(6;9)(p23;q34) DEK-NUP214 T(9;22)(q34;q11) BRC-ABL1 Abn of 11q23 - KMT2A Monosomy Complex karyotype
Name 2 treatments for AML
High dose cytarabine - t(8;21) or t/inv(16)
ATRA - t(15;17)
Name 5 partners to IGH in Multiple Myeloma
FGFR3 - Poor prog - 4p16.3 CCND1 - Good prog - 11q13 MAF - Poor prog - 16q23 CCND3 - Good prog - 6p21 MAFB - Poor/very poor prog - 20q11 (IGH 14q32)
What are the European Myeloma Recommendations for FISH
1) IGH-MAF + IGH/FGFR3, TP53, 1p/1q del’s
2) IGH-CCND1, IGH-MAFB, Ploidy, chrm 12 and 13 abn’s
Karyotype doesn’t work well for Myelomas - enrich for FISH with CD138+ to reduce false -ves
What must your ALL testing strategy encompass?
- Ploidy levels
- KMT2A rearr’s
- ETV6-RUNX1
- BCR-ABL1, TCF3-PBX1, TCF3-HLF
- CNVs
- iAMP21
- ABL like rearrangements
- IGH
What is the common translocation in Ewing Sarcoma?
t(11;22)(q24;q12) EWS-FLI1
What FISH would you do for a Rhabdomyosarcoma?
FOXO1 B/A and if positive do PAX3 and PAX7 to determine partner - positive consistent with Alveolar Rhabdomyosarcoma. Don’t see FOXO1 rearrangements in Embryonal Rhabdomyosarcoma
What proportion of PWS results from del/UPD
75-80% PATERNAL del
20-25% MATERNAL UPD
What proportion of AS is due to del/UPD/UBE3A mutn
70-75% MATERNAL del
3-7% PATERNAL UPD
10% UBE3A mutation
Around 10% NAD
Translocation seen in CML
t(9;22)(q34;q11) - BCR-ABL1
5’ end of BCR fused with 3’ end of ABL1 - fusion oncogene on dear (22) has elevated and disregulated tyrosine kinase activity
Treatments of CML
Imatinib - Tyrosine Kinase inhibitor
Second generation TKIs - Nilotinib and Dasatinib
Fanconi Anaemia - gene and test
Whole range of FA complement genes - mutns in FANCA account for nearly 2/3 cases Mytomycin C (or DEB) - breakage testing - set up different conc’s and see a proportional response - need control
Bloom Syndrome - gene and test
BLM - 15q26.1
Harlequin staining - sister chromatid exchange - 6-10/cell in normal, >50 in Bloom Syndrome
Pathogenic mutation in Fragile X Syndrome
Hypermethylation of FMR1 - no FMRP protein product
Over 200 CGG repeats
More likely to expand down maternal line
What are the mutn ranges of FMR1
Normal - 5-44 repeats
Intermediate - 45-54 rpts - Risk of expansion (esp maternal)
Premutation - 55-200 rpts - FXTAS/POI risk
Full mutation - >200 rpts - FRAX
Double paternal contribution triploidy?
Diandry - Cystic villi that have trophoblastic hyperplasia -> Partial hydatidiform mole - most common form of triploidy
Double maternal contribution triploidy?
Dygyny - Non hydronic villi, placenta non molar and generally small
What is a complete hydatidiform mole?
Diploid androgenetic pregnancy where both sets paternally derived
No fetal development, extensive hydrops, placenta have swollen villi and marked widespread hyperplasia of trophoblast
What is risk of invasive mole following molar pregnancy?
15% following complete mole
0.5% following partial mole
Invasion of myometrium which can lead to perforation of uterus - indicated by raised hCG so monitoring recommended
What are technical challenges associated with cffDNA isolation
1) The concentration of cfDNA is low
2) cffDNA is outnumbered by cfmDNA
3) Half the fetal genetic material is maternal in origin and therefore indistinguishable
Some benefits for NIPT/NIPD
- Reduce number of invasive tests - safer, cheaper and less expertise to take blood
- Earlier diagnosis
- Reduced trauma for parents
- Improved quality of care
Some limitations of NIPT/NIPD
- In multiple pregnancies can’t distinguish fetuses
- Proportion of cfDNA in mother with high BMI is low - increases false -ves and inconclusives
- Source of cffDNA is placental - brings around usual limitations
- False +ves/-ves - ff too low, contamination, maternal abnormalities, vanishing twin, triploidy
What is the most common 46,XY DSD
Androgen Insensitivity Syndrome
What is the most common 46,XX DSD
Congenital Adrenal Hyperplasia
Define validation
Testing performed as part of QA programme to determine performance metrics and document evidence that the assay fulfils the requirements for its intended purpose prior to implementation
Are you doing he right test?
Define verification
Testing performed as part of a QA programme to confirm that an assay performs as expected according to the performance metrics as defined during previous test validation
Are you doing the test correctly?
Discuss testing strategies for BRCA1/2
Sequence analysis of all genes - NGS
Targeted analysis for certain ethnic groups
Dosage analysis
Tumour DNA can be examined for LOH
- Co-segregation complicated by phenocopies and incomplete penetrance
What are some ethical considerations of predictive BRCA1/2 testing
Need genetic counselling - age on onset highly variable
Treatment may be mastectomy or increased screening - need to be sure about pathogenicity
Prenatal testing and testing of minors not usually offered
Mutn carrier may not disclose mutn to other family members - then they can’t access testing
Individual might not want testing but other family members might inadvertently determine their result
What pan-cancer markers are there in the TSO500 panel
TMB
NTRK1,2,3
MSI
What cancers are covered as part of the TSO500 Panel?
Lung, bladder, melanoma, breast, colon, ovarian, gastric, myeloid, sarcoma
What is synthetic lethality?
Synthetic lethality arises when a combination of deficiencies in the expression of two or more genes leads to cell death, whereas a deficiency in only one of these genes does not. Good example is use of PARP inhibitors in BRCA cancer
How do PARP Inhibitors work?
BRCA mutated cells lack homologous recombination repair and therefore rely on base excision repair. PARP enzymes involved in BER therefore if you inhibit them, BRCA mutant cells die
What risk do BRCA1/2 mutns confer?
BRCA1 - 60-85% BC, 40-60% OC, <1% male BC
BRCA2 - 40-85% BC, 30% OC, 10% Male BC (prostate too)
When should you consider BRCA test?
Early onset BC, 2+ primaries, BC and OC in same person, OC/BC in family, FHx of BRCA mutn, Ashkenazi Jewish, Male BC, personal Hx OC, Bilateral BC
NICE suggests testing if 10% risk of BRCA mutn
What are some advantages of data sharing?
Making accurate/safe diagnosis
More effective disease management and precision medicine
Accurate advice for family members
Improved understanding of genetic disease
What are some barriers to data sharing
Time/resources Nomenclature Transcripts/genomes build Technology/confidence range Inheritance Phenotype
Repeat ranges for Myotonic Dystrophy
5-34 repeats - Normal
35-49 repeats - Mutable normal
>50 repeats - Affected
Increased CTG repeat expansion in DMPK
What is a polygenic risk score?
Mathematical aggregate of risk conferred by many DNA variants to estimate the likelihood of a specific outcome such as disease onset in an individual
How can polygenic risk score models differ from each other?
Number of genetic variants considers
Specific type of statistical model used to combine risks
Ability to score to generalise to entire population
No universally agreed upon standards for developing polygenic risk scores
Give some egs of disorders where polygenic risk scores may be in use in the near future
FH
Breast Cancer
What issues are associated with DNA extracted from FFPE tissues
Hydrolysis of phosphodiester bonds - varying degrees of DNA fragmentation
Cross linking - artificial nucleotide mutations
Limited yield of DNA at extractions
What are limitations of direct tumour sampling?
Accessibility of tumour
Frequency of sampling
Existence of clinical overt disease
Cost
What can affect ctDNA testing
Anatomical site of disease Heterogeneity Field defects TMB Blood/brain barrier Age related clonal haematopoesis
Where could ctDNA testing be useful
Pre cancer diagnosis and monitoring
MRD
Recurrent/metastatic disease
What are some benefits to pharmacogenomic testing
Safer dosing options Avoid drug toxicity and adverse side effects Ensure maximum efficacy Improve drug development Explain variable response to drugs Reduce healthcare costs
What are pharmacokinetics and pharmacodynamics?
Pharmacokinetics: What the body does to the drug - absorption, distribution, metabolism, elimination
Pharmacodynamics: What the drug does to the body - Target proteins, downstream messengers
Give some cancer pharmacogenomic drug egs
Breast - Herception (overexpression of HER2) or Olaparib (BRCA mutn, PARP inhibitor)
Lung - Gefitinib (EGFR activating mutations, is an EGFR tyrosine kinase inhibitor)
Pan cancer - Entractinib/Larotractinib (NTRK rearrangements)
Give some examples of adverse drug reactions due to genetics
Gentamicin - m.1555A>G - mit deafness
Sodium valproate - anti-retroviral therapy in HIV - HLA-B*57:01 allele - liver toxicity
5FU and Capecitabine - Chemo - DPYD variants - risk of toxicity
Give some egs of targeted germline therapies
Ivacaftor/Lumacaftor (orkambi) - CF p.Phe508del
Ataluren - DMD - stop codon read through
EXONDYS51 - DMD - exon skipping
Spiranza - SMA
What is BSGM statement on DTC testing
Analytical validity, sensitivity and clinical utility is low
High chance of false positive and false negative results
Patients should be offered NHS care that they would otherwise receive regardless of DTC result
