L16 -Genomics for Diagnosis and Disease monitoring: Examples from Haematology (Dr Andrew Mumford) Flashcards
1. Understand the broad applications of clinical genomic testing. 2. Understand that there is a detailed NHS infrastructure to ensure access to genomic testing through next generation sequencing technologies. 3. Be able to illustrate some specific examples of patient benefit from genomic testing.
what is genomic testing used for?
Genomic testing identifies genetic mutations, chromosomal abnormalities and hereditary cancer syndromes to diagnose conditions e.g. Down syndrome, haemophilia and polycystic kidney disease
What is down syndrome?
Trisomy 21 which is a chromosomal disorder where an individual has three copies of chromosome 21 instead of 2
What are the key features of Down Syndrome?
Characteristic facial features, intellectual disability, congenital heart defects, increased risk of leukaemia and early onsest of Alzheimer’s disease
What is haemophilia B
A blood clotting disorder caused by a mutation in the Factor IX gene on the X chromosome leading to excessive bleeding
What is the inheritance pattern of haemophilia B?
X-linked recessive (mostly affects males, while females are typically carriers)
What os Noonan syndrome?
A genetic disorder that affects multiple organs, causing short stature, congenital heart defects, facial abnormalities and developmental delays
what is the inheritance pattern of Noonan syndrome?
Autosomal dominant - a single mutation can cause the disease
What is sickle cell disease?
A genetic blood disorder where a mutation in the HBB gene leads to abnormal sickle-shaped red blood cells that cause blockages, pain and organ damage
What is the inheritance pattern of sickle cell disease?
Autosomal recessive whre both parents need to carry the mutated gene for the disease to manifest
What is polycystic kidney disease? (PKD)
A genetic disorder causing multiple cysts in the kidneys, leading to kidney enlargement and loss of function
What is the inheritance pattern of polycystic kidney disease (PKD)
Autosomal dominant where one mutated gene is enough to cause the disease
How does PKD affect kidney function?
Cysts replace normal kidney tissue reducing function and leading to kidney failure
What is acute myleoid leukaemia (AML)?
A cancer of immature blooc cells in the bone marrow, often linked to chromosomal abnormalities or gene mutations
What is lymphoma?
A cancer of the lymphatic system, affecting lymphocytes (B and T cells) with both sporadic and genetic risk factors
What is melanoma?
A skin cancer arising from melanocytes, which can be caused by UV radiation or genetic mutations (e.g. BRCA, CDKN2A or RB1 mutations)
What is adenocarcinoma of the large bowel?
A colorectal cancer that may develop sporadically (most common) or due to genetic syndromes (e.g. FAP, Lynch syndrome)
What is familial adenomatous polyposis (FAP)?
A genetic disorder caused by a mutation in the APC gene, leading to hundreds of precancerous polyps in the colon, which progress to cancer by early adulthood if untreated/
What is the difference between sporadic and hereditary colorectal cancer?
Sporadic cases occur randomly in older adults, while hereditary cases (E.g. FAP, lynch syndrome… ) occur earlier in life due to germline mutations
What is lynch syndrome?
A hereditary colorectal cancer syndrome caused by mutations in DNA mismatch repair genes (MLH1, MSH2, MSH6 and PMS2) leading to early-onset colorectal and endometrial cancer
What is the role of APC mutations in colorectal cancer?
APC mutations lead to uncontrolled cell growth in the colon, seen in both sporadic and hereditary (FAP) colorectal cancer
What is a germline mutation?
A mutation present in sperm or egg cells, meaning it is heritable and can be passed to offspring
What is a somatic mutation?
A mutation occuring in non-reprodictive cells that cannot be inherited, often leading to sporadic cancers
How does a mutation in the RB1 gene affect cancer risk?
RB1 mutations increase the risk of retinoblastoma and melanoma, as the RB1 protein normally regulates cell division
What is the difference between germline and somatic mutations?
Germline mutations occur in egg or sperm cells and are inherited by all cells in the offspring while somatic mutations occur after birth in specific cells and are not inherited
How do germline mutations affect genetic testing?
Since every cell in the body carries the mutation, it can be detected in any tissue sample, including blood, saliva or placenta cells
Why do somatic mutations lead to cancer?
A mutation in a single cell can trigger clonal expansion, where abnormal cells multiply uncontrollably, forming tumours
What are the types of prenatal genetic testing?
✅ Preimplantation genetic diagnosis (PGD): Tests embryos created via in vitro fertilization (IVF) before implantation.
✅ Amniocentesis: Extracts amniotic fluid containing fetal cells to check for chromosomal abnormalities.
✅ Chorionic villus sampling (CVS): Takes a small sample of the placenta to analyse fetal DNA.
✅ Non-invasive prenatal testing (NIPT): Detects fetal DNA in maternal blood to screen for genetic conditions.
What are the risks of amniocentesis and chorionic villus sampling (CVS)
Both procedures carry a small risk of miscarriage due to their invasive nature
Why is non-invasive prenatal testing (NIPT) preferred?
Safer and easier than amniocentesis or CVS, as it only requires a blood sample from the mother
What genetic conditions can be detected before birth?
Trisomy 21 (Down syndrome), trisomy 18 (Edwards syndrome), trisomy 13 (Patau syndrome), cystic fibrosis, sickle cell disease, Tay-Sachs disease, and other single-gene disorders.
/how can genetic testing assist in diagnosing rare diseases?
It helps identify disease-causing mutations early, allowing for timely interventions (e.g. enzyme replacement for metabolism disorders e.g. Phenylketonuria)
What is Marfan syndrome and how is it diagnosed?
Marfan syndrome is a genetic disorder (caused by a mutation in FBN1 gene) affecting connective tissue. It usually results in patients having a tall stature, long limbs, hyperflexivility, heart problems (aortic aneurysms) and is diagnosed via clinical features and genetic testing
What is pharmogenomics?
The study of how genetic variations affect an individual’s response to drugs
How does the DPYD gene variant affect chemotherapy treatment?
DPYD mutations can cause severe fluorouracil (5-FU) toxicity, leading to life-threatening side effects. Genetic testings helps identify at risk patients and adjust dosage
How can germline DNA testing assess cancer risk?
Detects inherited mutations like BRACA1/2, which increases breast and overian cancer risk. this allows preventative measures to be taken e.g. mastectomy or ovary removal
why is genetic testing important in breast cancer?
It identifies BRCA mutations (higher lifetime risk of breast and ovarian cancer) and it helps personalise treatment plans (e.g. avoiding certain chemotherapies based on genetic response)
How is tumour DNA testing used in cancer treatment?
It identified molecular amrkers e.g. BCR-ABL in chronic myeloid leukemia, to guide targeted therapies. It also monitors minimal residual disease (MRD) after treatment to detect relapses
How is genetic testing used in infection diagnosis
Detects pathogen DNA/RNA to identify infections faster than traditional cultures
Why is genetic testing important for tuberculosis (TB) diagnosis?
Mycobacterium tuberculosis grows very slowly in culture so genetic testing e.g. PCR provides a rapid and reliable diagnosis
How did COVID-19 change the use of genetic testing?
Highlighted the impirtant of PCR and sequencing in tracking virus mutations and drug resistance
What are some key genetic mutations in lung cancer?
AKT1, ALK, BRAF, CDKN2A, DDR2, EGFR, ERBB2, KRAS, MET, NRAS, PIK3CA, PTEN, TP53, TMB
Which genes are frequently mutated in melanoma?
AKT1, BRAF, CDKN2A, CTNNB1, GNA11, GNAQ, KIT, MAP2K1, NF1, NRAS, PIK3CA, PTEN, TP53
What are common genetic alterations in colorectal cancer?
AKT1, BRAF, FBXW7, KRAS, MAP2K1, MSH2, MSH6, NRAS, PIK3CA, PTEN, TP53
Which genetic mutations are seen in ovarian cancer?
BRAF, BRCA1, BRCA2, KRAS, MAP2K1, PIK3CA, PTEN, TP53
What is next generation sequencing (NGS)
A high-throughput sequencing method that allows simultaneous analysis of multiple genes to identify genetic mutations in cancers and inherited conditions.
What is the TruSight Oncology 500 gene panel used for?
It is used for somatic testing of solid tumours, analysing 50-1000 genes to identify key mutations for diagnosis, prognosis and targeted therapy selection
Why is NGS useful for cancer diagnostics?
Comprehensive analysis of multiple genetic alterations which helps when personalising therapy selection and identifying mutations beyond the primary tumour type
How does next generation sequencing help in clinical trials?
It identified genetic profiles that determine eligibility for targeted therapy trialsH
How can NGS stratify cancer risk?
It detects mutations that predict tumour behaviour and response to treatment
what is the advantage of using gene panels instead of single gene testing
It is more cost-effective, it captures multiple genetic alterations at one and it helps when no clear single candidate gene is suspected
What is whole genome sequencing (WGS)
A sequencing method that analyses the entire genome, including coding and non-coding regions to identify both somatic and germline mutations
How does WGS differ from gene panels?
WGS covers:
Coding regions (like gene panels) as well as introns, untranslated, and regulatory regions (unlike gene panels)
What types of samples can be analysed with WGS
somatic samples (like luekemia and tumour biopsies) and germline samples (like skin, blood or other tissues)
What are the advantages of WGS in cancer diagnostics?
Rapid analysis (<4 weeks)
Affordable (<$500)
Detects single nucleotide variants (SNVs), insertions/deletions (indels), and copy number variants (CNVs)
What is circos plot, and how is it used in cancer genetics?
A visual representation of chromosomal changes e.g. copy number variants and translocations, commonly used in multiple myeloma and other cancers
How does WGS add value in haematological malignancies
✅ Comprehensive genotyping for diagnostic variants
✅ Tissue typing & blood group detection
✅ Detection of inherited susceptibility genes
✅ Pharmacogenomic safety insights
✅ Identification of other risk factors or diseases
How is genomic testing organised in England?
Seven NHS genomic laboratory Hubs provude standardised testing for rare diseases and cancers
What is the NHS national genomic test directory?
A database listing test indivations and technologies for 3000 rare disease indications and 18- cancer indications (a database that specifies which genetic tests are available for which conditions (i.e., their indications) and what technologies are used to perform them)
Where is the largest genetic testing center in SouthWest Engalnd
Southmead hospital
Why do major hospitals in London perform extensive genetic testing?
London has tertiary referral centers that recieve complex cases from across the UK due to specialist expertise
What is haemophilia A
a genetic bleeding disorder caused by deficiency in factor VIII (8O, a crucial protein for blood clotting. It is X-linked recessive, meaning it mostly affected males while females can be carriers
what is haemophilia B
an X linked recessive disorder caused by a mutation in the F9 gene, leading to factor IX defiency
Why are males more likely to be affected by haemophilia B than females
Males have only one X chromosome (XY) so if they inherit a faulty F9 gene, they will have the disease. FEmales (XX) can be carriers if they have one normal and one mutated X chromosome
Who is the most famouse historical carrier of haemophilia B?
Queen Victoria of England, who passed the mutation to several royal families in Europe (she was a carrier and passed the mutated X chromosome to her daughters who then transmitted it to Russian, Spanish and German Royal families)
How common is haemophilia A compared to Haemophilia B
Hemophilia A: 1 in 5,000 males (caused by factor VIII deficiency).
Hemophilia B: 1 in 30,000 males (caused by factor IX deficiency).
What happens if a carrier female has children
50% chance of passing the affected X chromosome to sons (who will have hemophilia).
50% chance of passing the affected X chromosome to daughters (who will be carriers).
what is the relationship between the haemophilia bleeding phenotype and genotype?
the bleeding phenotype in haemophilia can be predicted based on the residual clotting factor level and the type of gene defect
what are the clotting factor levels associated with different severities of haemophilia?
🩸 Severe haemophilia: <0.01 IU/dL
🩹 Moderate haemophilia: 0.01-0.05 IU/dL
🔵 Mild haemophilia: 0.05-0.4 IU/dL
What types of genetic variants cause different severities of haemophilia?
🛑 Severe haemophilia: Insertion/deletion, stop gain/loss, splice mutations, some missense mutations.
🟠 Moderate haemophilia: Missense mutations.
🟢 Mild haemophilia: Missense mutations.
why do people with haemophilia experience excessive bleeding and bruising ?
🩸 People with haemophilia have impaired blood coagulation, leading to excessive bleeding, bruising, and accumulation of blood in soft tissues.
What is a dangerous location for bleeding in haemophilia and why
⚠️ Neck bleeding is an emergency because it can cause airway compromise, leading to suffocation. It requires treatment with a major dose of clotting factor.
how does joint bleeding (haemarthrosis) manifest in haemophilia?
🦵 Joint bleeding can cause swelling and pain, particularly in the elbows and knees, and may lead to long-term joint damage.
How does iliopsoas bleeding present in haemophilia, and what is the treatment?
🦠 Iliopsoas bleeding presents with a flexed hip and pain when trying to extend the leg on the affected side. It requires a major dose of clotting factor if compartment syndrome is suspected.
what is iliopsoas bleeding?
Iliopsoas bleeding refers to bleeding into the iliopsoas muscle, a deep muscle in the pelvis that plays a key role in hip flexion. It is a serious complication of haemophilia and can lead to compartment syndrome, which can cause nerve compression and long-term complications.
Symptoms of Iliopsoas Bleeding
🦠 Hip pain – Often on one side, worsening over time.
🚶♂️ Flexed hip posture – The patient may hold their hip in a flexed position to relieve pain.
🦵 Pain with leg extension – Difficulty or pain when trying to extend the leg on the affected side.
🛑 Nerve compression signs – Tingling, numbness, or weakness in the thigh (due to pressure on the femoral nerve).
What are the symtoms of soft tissue bleeding and bruising in haemophilia
💙 Soft tissue bleeding causes bruising and tenderness but usually does not require clotting factor treatment unless severe.
how should deltoid/forearm bleeding be treated in haemophilia?
Routine factor dose is recommended, but a major dose is required if compartment syndrome is suspected
what is compartment syndome
🛑 Compartment syndrome is a serious condition where increased pressure within a muscle compartment restricts blood flow and can cause tissue and nerve damage. It is a medical emergency if left untreated (muscle death (necrosis), nerve damage or amputation)
What are the symtoms and management of buttock bleeds in haemophilia?
A: 🍑 Buttock bleeds can cause pain with or without swelling. If accompanied by tingling or swelling in the leg, it may indicate nerve compression, requiring clotting factor treatment.
How does haemophilia severity vary within families?
🏡 Severity can differ among family members, as genetic mutations do not always result in the same degree of disease. Some individuals may have mild symptoms and live relatively normal lives.
What are the main phenotypes of haemophilia?
🩸 Bleeding phenotype – Severity depends on residual clotting factor and gene defect.
🦵 Haemophilic arthropathy – Joint damage due to recurrent bleeding.
🦠 Chronic synovitis – Persistent joint inflammation from repeated bleeds.
🦠 Treatment-acquired HIV & HCV – Historical complications from contaminated blood products.
What is haemophilic arthropathy?
🦵🔥Joint damage due to recurrent bleeding into the synovial lining. this causes inflammation which destroys the cartilage.
What are x-ray findings and symptoms for haemophilia arthropathy
🦴 X-ray findings: Joint space narrowing, bone erosion.
🚶 Symptoms:
Pain and stiffness in joints (knees, elbows, ankles).
Muscle wasting due to pain-induced immobility.
Early-onset joint replacements in severe cases.
How does chronic synovitis develop in haemophilia?
Frequent joint bleeds irritate the synovial lining, causing inflammation.
Inflammation leads to thickened synovium, increasing the risk of further bleeding which eventually results in cartilage damage, worsening haemophilia arthropathy
How was haemophilia treated in the past?
🩸Factor replacement therapy from blood donors ( 1980s-90s)
What was the risk of haemophilia treatment using blood donors in the past
Blood donors used to be payed so it was typically those desperate for money who would donate (e.g. prisoners, drug users…..) so plasma-derived products were contaminated with blood born infections e.g. HIV and HCV (hepatitis C). this meant that many haemophiliacs contracted life threatening diseases
Why was treatment acquired HIV and HCV a major issue
🏥 High mortality – Many haemophiliacs were infected before screening improved.
📰 UK Blood Scandal: Ongoing discussions about compensation for affected families.
What is Kaposi’s sarcoma and how is it linked to haemophilia?
🦠 Kaposi’s sarcoma (KS) is a tumour of blood vessels caused by HHV-8 (human herpesvirus 8). which lives in two forms :
- Classic KS – Found in elderly Mediterranean men, slow-growing.
- HIV-associated KS – Aggressive, widespread in immunocompromised patients.
🩸 Seen in haemophiliacs who contracted HIV from contaminated blood products.
How do you assess the risk of a baby inheriting haemophilia?
🤰 If a pregnant woman has a family history of haemophilia she can undergo genetic testing to determine if she is a carrier or not … If she is a carrier, she has a 50% chance of passing the affected X to her baby.
what are some key genetic tests to determine if a mother is a carrier for haemophilia
- Maternal genetic testing – Confirms carrier status.
- Foetal testing – Determines if the baby inherited the mutated X.
Case Study: What is the risk of a baby boy having haemophilia?
👵 Grandmother was a carrier.
👩 Mother may also be a carrier.
👶 If the mother is a confirmed carrier, the baby boy has a 50% risk of haemophilia (inherits either a healthy X or the affected X).
📊 Final probability calculation:
If the mother is confirmed NOT a carrier → 0% risk for baby.
If the mother is a carrier → 50% chance the baby has haemophilia.
what genetic tests can confirm haemophilia risks?
🧬 Carrier screening in the mother – Identifies Factor VIII or IX gene mutations.
🩸 Foetal genetic testing (if needed) – Confirms mutation in the baby’s DNA.
What genetic mutation is associated with severe Haemophilia A?
Inversion 22 (INV 22) in the F8 gene - this helps to predict severe phenotype of Haemophilia A
How does genetic testing help in haemophilia diagnosis and management?
✅ 1. Confirms diagnosis – Differentiates between Haemophilia A (F8 mutation) and Haemophilia B (F9 mutation).
✅ 2. Identifies carriers in family members – Helps with genetic counseling.
✅ 3. Allows for antenatal diagnosis – Options include pre-implantation genetic testing, chorionic villus sampling (CVS), amniocentesis, or foetal DNA testing in maternal blood.
✅ 4. Improves prognosis and management – Predicts:
🩸 Severity of bleeding risk
🚼 Delivery planning (minimizing trauma at birth)
💉 Risk of developing inhibitors to treatment
what non-invasive prenatal genetic tests are available
🩸Foetal DNA in maternal blood (Less risky but not always conclusive).
What invasive prenatal tests are available
Chorionic Villus Sampling (CVS) (10-13 weeks).
Amniocentesis (15-20 weeks).
Foetal blood sampling (Rarely used but an option).
what primplantation genetic diagnosis (PGD) prenatal tests are available?
Used in IVF to select embryos without the mutation.
What is chronic myeloid leukaemia (CML) and genetic abnormalities?
🧬 CML is a type of leukaemia characterized by the clonal proliferation of late myeloid cells.
📊 Accounts for ~15% of all leukaemia cases.
👥 Peak incidence: 40-60 years old.
what are the symtoms for CML
🩸 Early symptoms may be subtle, but later, patients may develop:
✅ Splenomegaly – Enlarged spleen due to excessive white cell production.
✅ Increased white cell count – High number of circulating myeloid cells.
✅ Anaemia – Reduced red blood cells leading to fatigue and weakness.
✅ Thrombocytopenia (sometimes) – Low platelet count, increasing bleeding risk.
What chromosomal abnormalities are associated with CML?
🧬 Philadelphia Chromosome (t(9;22))
A translocation between chromosomes 9 and 22.
Part of chromosome 22 is lost and fuses with chromosome 9.
This creates the BCR-ABL fusion gene, which drives uncontrolled cell division.
🔬 This was the first tumour associated with a chromosomal abnormalit
How is CML diagnosed?
🩺 Cytogenetic Analysis (Karyotyping)
🧪 Technique:
Cells are cultured and arrested in metaphase using colchicine (prevents spindle formation).
Chromosomes are then spread on a slide, fixed, and stained.
Microscopy is used to identify chromosomal abnormalities.
🎨 Staining Method:
Giemsa staining (G-banding) is used to create distinct light and dark bands on chromosomes.
The Philadelphia chromosome (shortened chromosome 22) is identified based on banding patterns.
🧪 Molecular testing (FISH, PCR)
🔬 Fluorescence In Situ Hybridization (FISH):
Uses fluorescently labeled DNA probes to detect the BCR-ABL translocation.
More sensitive and specific than karyotyping.
🧬 Polymerase Chain Reaction (PCR):
Detects the BCR-ABL fusion gene at a molecular level.
Can quantify minimal residual disease (MRD) to monitor treatment response.
how does BCR-ABL fusion gene cause CML
🔥 BCR-ABL produces a continuously active tyrosine kinase, leading to:
Uncontrolled myeloid cell proliferation.
Reduced apoptosis (cell death).
Genomic instability, increasing mutation risk.
🛑 Targeted therapy with tyrosine kinase inhibitors (e.g., Imatinib/Gleevec) has revolutionized treatment!
What is the somatic t(9;22) translocation
The somatic t(9;22)(q34:q11) translocation involves the fusion of the BCR gene on chromosome 22 and the ABL gene on chromosome 9.
What is the somatic t(9;22) translocation result in
This translocation results in the creation of a chimeric BCR-ABL protein that has constitutive tyrosine kinase activity, which plays a critical role in the pathogenesis of chronic myeloid leukaemia (CML). 🧬
How does the BCR-ABL fusion protein contribute to CML
The BCR-ABL fusion protein has strong tyrosine kinase activity, which leads to abnormal cell proliferation and contributes to CML.
how does Imatinib target CML
Imatinib is a synthetic, specific inhibitor of the BCR-ABL fusion protein. It targets the tyrosine kinase activity, reducing the uncontrolled cell growth associated with CML. 💉
What are the clinical signs and laboratory findings of a 55-year-old woman diagnosed with CML?
The patient presented with 4 weeks of unexplained weight loss, fever, and abdominal discomfort. Physical examination revealed an enlarged spleen (15 cm below the costal margin). Laboratory results showed:
WBC: 89 x10^9/L (Normal range: 4-11)
Platelets (PLT): 564 x10^9/L (Normal range: 150-400)
Hemoglobin (HB): 10.2 g/L (Normal range: 12-15)
These findings are indicative of CML, with leukocytosis and splenomegaly. 🩸📊
How was the patient treated, and what were the results?
The patient was started on Imatinib 400 mg daily.
At 3 months, the WBC count reduced to 5.2 x10^9/L, and the BCR/ABL transcript ratio dropped from 71 to 5.2, showing a positive haematological and cytogenetic response.
At 12 months, however, the WBC count rose to 35.1 x10^9/L, and the BCR/ABL transcript ratio increased to 23.4, indicating acquired resistance to Imatinib.
This illustrates the initial success of Imatinib therapy followed by the development of resistance. 💊
What mechanisms lead to resistance to Imatinib in CML patients?
Resistance to Imatinib often arises due to somatic single nucleotide variants (SNVs) in the ABL gene. Missense mutations in ABL that confer Imatinib resistance undergo positive selection. These mutations reduce Imatinib’s effectiveness. New ABL inhibitors, such as Ponatinib, Dasatinib, Nilotinib, and Bosutinib, have been developed to overcome this resistance. 🔬⚔️
How has genetic testing helped in managing CML?
Genetic testing plays several key roles in CML management:
Confirmation of Diagnosis: Detecting the BCR-ABL translocation confirms the diagnosis of CML.
Informed Treatment Decisions: It guides the choice of first-line treatment (e.g., Imatinib).
Monitoring Disease Activity: Monitoring the BCR-ABL transcript ratio helps track treatment efficacy and disease progression.
Personalized Therapy: Genetic testing enables selection of alternative therapies for patients with relapsed or resistant disease. 🧬🧪
What is the significance of the Philadelphia chromosome in CML?
Imatinib targets the BCR-ABL tyrosine kinase, inhibiting its activity. This prevents the overactivation of cell proliferation pathways that are linked to the Philadelphia chromosome. Unlike traditional chemotherapy, which indiscriminately targets rapidly dividing cells, Imatinib specifically targets cells with the BCR-ABL fusion protein, making it more selective and effective with fewer side effects. 💊🛑
How does monitoring help in managing CML?
In the case of the 55-year-old patient, genetic testing and disease monitoring were crucial:
The reduction in BCR-ABL transcript ratio from 71 to 5.2 at 3 months indicated a positive response to Imatinib.
At 12 months, the increase in the BCR-ABL ratio to 23.4 suggested acquired resistance, prompting a shift in therapy.
Monitoring allows clinicians to adjust treatment based on the disease’s response and progression. 📅🔍
