Case 16: genetics 2

Question 1

Application of NIPT

Answer 1

• Aneuploidy testing based on DNA molecule counting (abnormal number of chromosomes)
• Technique: single molecule DNA counting
• Analyse and sequence all foetal DNA. Analyse ratio of DNA to chromosomes to assess disease status
• Then confirm positive result with CVS/amniocentesis
• Can look at alleles and see ratio of those that contain a mutation to those that don’t to assess for recessive conditions (Homozygous or Heterozygous)

Question 2

NIPD/T limitations

Answer 2

• Maternal factors: Some women have very low foetal DNA fractions (due to women’s weight or foetal age). Lab techniques may not pick it up. Underlying chromosomal abnormality or tumour may affect results
• Pregnancy factors: Twin pregnancies (especially for NIPT), Resolving second gestational sac, Placental mosaicism (the foetal blood is mostly from the placenta)

Question 3

Testing for cord blood

Answer 3

only done if implications for management. Otherwise wait till they consent at 18

Question 4

Overview of indications for NIPD/T

Answer 4

• NIPD: determining foetal sex, testing for a specific mutation in a single gene disorder
• Aneuploidy testing

Question 5

Presentations of gene abnormality resul example:
- CFTR c.1521_1523delCTT p.F508del
Heterozygous

Answer 5

• CFTR gene- causative gene in Cystic Fibrosis
• Specific change in DNA coding sequence- CTT deleted between position 1521 & 1523
• Effect of DNA change on the protein produced (p.F508del) Phenylalanine missing at position 508 of the amino acid sequence
• Heterozygous: Present on one allele (one copy of the CFTR gene)

Question 6

Autosomal inheritance

Answer 6

• The mutation is present on both copies of the gene in order for the disease to develop
• Has to be on chromosomes 1-22 (the autosomes)

Question 7

Definitions: Heterozygous, Compound Heterozygous and Homozygous

Answer 7

• Heterozygous: Variant present on one allele, the other is normal. In autosomal recessive condition this means they are a carrier
• Compound Heterozygous: Both alleles contain a different variant, two different mutations present. No functioning copy of the gene so is affected by the recessive condition
• Homozygous: both alleles contain the same variant. Means the individual is affected by the recessive condition

Question 8

Cystic fibrosis pathophysiology

Answer 8

• CFTR gene codes for the Cystic Fibrosis Transmembrane conductance Regulator (CFTR) protein, a cell membrane chloride transporter
• The gene is on chromosome 7
• Most common mutation: Delta F508
• Insufficient functioning CFTR protein for normal chloride transport, resulting in the production of thick sticky mucus production
• Multi-system disorder (lungs, intestines, pancreatic ducts, sweat glands, and reproductive organs).

Question 9

Cystic fibrosis inheritance

Answer 9

• Autosomal recessive inheritance
• Two functioning copies of the CFTR gene: Non carrier, not affected
• Mutation present on one copy of the CFTR gene: carrier not affected
• Mutation present on both copies of the CFTR: affected by CF
• Carrier frequency of CF: 1 in 25

Question 10

Risk of a child being affected by CF if one parent is a known carrier

Answer 10

• 1 in 100
• known carrier: probability of being a carrier is 1
• partner: probability of being a carrier is 1 in 25 (population risk)
• 1 in 4 chance of both passing on the mutation
• therefore overall risk of pregnancy being affected is 1 x 1/25 x 1/4 = 1/100

Question 11

Genetic testing: CF

Answer 11

• Partners of carriers are offered testing for the 50 most common CFTR mutation (account for 90% of mutations in Northern Europe)
• If no mutation is identifies it reduces risk of being a carrier from 1 in 25 to 1 in 250 (still some chance)
• Different mutations in different ethnic groups

Question 12

CF: Diagnosis

Answer 12

• Through newborn blood spot screening programme: measure trypsinogen (IRT) in blood samples. Refereed for further testing
• Sweat chloride test: gold standard, diagnosed if >60 (30-60 require further investigations)
• Genetic tests: not all mutations are detectable
• If clinical features are highly suggestable and investigations are negative then can still be diagnosed. If investigations positive but no symptoms then still diagnose
• After diagnoses refer to a cystic fibrosis centre

Question 13

CF: Monitoring

Answer 13

• Regular sputum cultures
• Pulmonary function tests (PFT): Spirometry and lung volume measurements
• CXR and CT: evaluate lung disease severity, identify complications like bronchiectasis
• Oral glucose tolerance test: screen for cystic fibrosis related complications
• DEXA

Question 14

CF presentation: age

Answer 14

• Infant: Meconium ileus
• Older child: chronic cough, recurrent wheezing, chronic respiratory infections, Malabsorption, failure to thrive
• Adult: cystic fibrosis related diabetes, infertility (absence of vas deferens (CBAVD), irregular menstrual cycle)

Question 15

CF: respiratory

Answer 15

• Persistent cough:Productive or non-productive, often exacerbated by mucus accumulation and infections.
• Wheezing and dyspnea:Resulting from airway obstruction and bronchospasm.
• Recurrent respiratory infections: pathogens such asStaphylococcus aureus,Haemophilus influenzae, andPseudomonas aeruginosa.
• Nasal polyps and chronic sinusitis

Question 16

CF: GI

Answer 16

• Meconium ileus:Neonates with CF may present with intestinal obstruction due to thickened meconium.
• Pancreatic insufficiency:Steatorrhea, weight loss, and malabsorption of fat-soluble vitamins due to pancreatic duct obstruction.
• Distal intestinal obstruction syndrome (DIOS):Partial or complete intestinal obstruction due to inspissated faecal material.
• Biliary cirrhosis:Impaired bile flow leading to liver damage, portal hypertension, and potential liver failure.
• Gastroesophageal reflux disease (GERD)

Question 17

CF: other clinical features

Answer 17

• Cystic fibrosis-related diabetes (CFRD):Insulin deficiency and impaired glucose tolerance caused by progressive pancreatic damage.
• Growth failure:Delayed growth and pubertal development due to malabsorption, chronic inflammation, and increased energy expenditure.
• Male infertility:Congenital bilateral absence of the vas deferens (CBAVD)
• Female fertility: irregular menstrual cycles, and decreased fertility
• Clubbing:Chronic hypoxia may lead to digital clubbing, a sign of advanced lung disease.
• Salt loss syndrome:Excessive sodium chloride loss in sweat can lead to hyponatraemic dehydration and metabolic alkalosis, particularly in infants.
• Bone disease:osteoporosis due to malabsorption of calcium and vitamin D

Question 18

CF management: conservative

Answer 18

• Chest physiotherapy:Techniques such as active cycle of breathing, autogenic drainage, and positive expiratory pressure devices help mobilize and clear mucus from the airways.
• High-frequency chest wall oscillation:Devices like the Vest® Airway Clearance System use rapid oscillations to loosen and clear mucus from the airways.
• Exercise
• Patients with CF should try tominimise contact with each otherto prevent cross infection withBurkholderia cepaciacomplex andPseudomonas aeruginosa

Question 19

CF: Medical management

Answer 19

• Mucolytics:dornase alfa and hypertonic saline reduce mucus viscosity
• Bronchodilators: Salbutamol and anticholinergics
• Anti-inflammatories:Beclomethasone Inh and Ibuprofen (NSAIDs) po help improve lung function.
• Antibiotics:Regular use of inhaled antibiotics (e.g., tobramycin, aztreonam) helps manage chronic infections, particularly Pseudomonas aeruginosa.
• CFTR modulators:ivacaftor, lumacaftor, and elexacaftor target specific CFTR mutations, improving protein function and clinical outcomes. Selection depends on the patient’s specific CFTR genotype.

Question 20

CF: surgery and CF modulators

Answer 20

• Lumacaftor/Ivacaftor (Orkambi): in CF who are homozygous for delta F508
• Lung transplant: in advanced lung disease. chronic infection withBurkholderia cepacia is a contraindication

Question 21

CF nutritional support

Answer 21

• Pancreatic enzyme replacement therapy (PERT):given with meals to assist digestion and absorption of nutrients.
• Fat-soluble vitamin supplementation:vitamins A, D, E, and K
• High-energy diet

Question 22

CF prognosis and complications

Answer 22

• Reduced life expectancy: early 40’s
• Dependence on presence of complications like cystic fibrosis related diabetes (CFRD) and liver disease
• Liver disease:Bile duct obstruction can progress to cirrhosis, portal hypertension, and hepatic encephalopathy, requiring liver transplantation in severe cases.
• Respiratory failure: may require supplemental oxygen or lung transplantation.
• Nephrolithiasis:Dehydration, alkaline urine, and hyperoxaluria increase the risk of kidney stones, which may require medical or surgical management.

Question 23

Tay Sachs disease

Answer 23

• HEXA gene encodes part of the beta-hexosaminidase A enzyme which plays a key role in the breakdown of toxic products in the brain and spinal cord.
• Autosomal recessive condition
• Severe neurodegenerative condition

Question 24

Consanguinity

Answer 24

• A consanguineous relationship is one between individuals who are second cousins or closer i.e. when two first cousins marry
• Important to ask- increased risk of recessive disorders. On a family tree is denoted with a double horizontal line between husband and wife (=)
• Doubles the risk of serious congenital and genetic disorders for children of first cousins
• More common in parts of the Middle East, South Asia, certain religious communities and amongst Irish travellers

Question 25

Carrier testing for CF and other autosomal recessive conditions: Familial risk

Answer 25

- Initially offered to first degree relatives - Checks if they are a carrier - Main indication is to find out the chance of having an affected child - Specifically test for the mutation identified in the family - For children testing should be deferred until old enough to consent

Question 26

Pharmacogenetics

Answer 26

explores the ways in which the persons genome contributes to a persons response to drugs

Question 27

What does Pharmacogenetics affect

Answer 27

- Pharmacokinetics: influences what the body does to the drug - Pharmacodynamics: what the drug does to the body

Question 28

When is it indicated to use Pharmacogenetics in medicine

Answer 28

- Drugs with a narrow therapeutic index and a serious adverse drug reaction i.e. chemotherapy - Genes can encode for liver enzymes which metabolise the drug, if there is a mutation this can cause the enzyme to be inactive - The P450 (CYP) liver enzyme encodes for genes which metabolise warfarin, Clopidogrel, antidepressants and analgesics - The plasma concentration of the drug varies signicantly depending on the activity of the enzymes

Question 29

The 5 drug gene pairs in which testing occurs relatively routinely

Answer 29

- azathioprine: TPMT and NUDT15; - abacavir: HLA-B; - fluoropyrimidines: DPYD; - carbamazepine: HLA-A and HLA-B; and - aminoglycoside antibiotics: MT-RNR1.

Question 30

Variants in which genes can affect warfarin sensitivity

Answer 30

- CYP2C9: codes for an enzyme primarily responsible for warfarin metabolism - VKOR1: codes for the warfarin drug target - Variants cause warfarin dosing issues/longer time to stabilise INR and increased risk of life threatening bleeding events

Question 31

DPYP gene

Answer 31

- Tested prior to prescribing fluorouracil based chemo - Inherited variants in DPYD can result in deficiency of the enzyme encoded by this gene which can result in severe toxicity to drugs like 5-fluorouracil

Question 32

Direct to consumer genetic testing

Answer 32

- Tests are marketed via a range of web- and print-based media, including social media platforms; they can even be bought over-the-counter - DNA sample are typically collected using saliva kits and sent directly to the testing laboratory by the consumer - Results are uploaded to a secure website or provided in a written report format. - Dont need to speak to doctor/clinician - Can have access to raw data allowing other people to reanalyse the data to include other disorders/trait

Question 33

What can direct to consumer genetic testing offer

Answer 33

- Disease risk prediction - Carrier status: for common autosomal conditions i.e. cystic fibrosis - Mendelian disease i.e. BRCA. - Lifestyle testing - Ancestry - Privacy: data stored safely - Reanalysis

Question 34

What do direct to consumer genetic tests offer: disease risk prediction

Answer 34

- Risk of developing a number of common condition: - Dementia / Alzheimer’s disease, IBD, T2D, Coeliac disease, Autoimmune disease, Parkinson’s disease - Looks at genes which are present with individuals who have these which may not be relevant to that patient. Based in control of people who bought the test which causes a bias as it doesn’t represent the whole population - Many of the diseases are multifactorial i.e. diet, smoking and family history which are not considered in direct to consumer genetic tests

Question 35

Direct to consumer genetic tests: carrier status

Answer 35

- Doesn’t report on all autosomal recessive conditions - Doesn’t look for all pathogenic variations (only a small number of mutations)

Question 36

Direct to consumer genetic tests: Mendelian disease

Answer 36

- Normally only done after the patient has undergone genetic counselling - Variations may be ethnic specific and when excluded have no clinical significance - Only look at a few mutations

Question 37

Direct consumer genetic tests: Lifestyle testing

Answer 37

- Fitness and exercise capacity - Nutrition, metabolism and weight - Sleep preferences - Skin care - Taste i.e. wine - The link between genomic variation and trait may be indirect or even unknown

Question 38

Direct to consumer genetic tests: Ancestry

Answer 38

- Compares your data to standards across the world so based on current population distribution rather than bone/teeth samples - These traits are based on conjecture and observation- other people filling in questionnaires and comparing their genes

Question 39

Direct to consumer genetic tests: privacy

Answer 39

- What does the company do with the consumer’s DNA sample once the analysis is complete? Will it be stored, shared, sold, or destroyed? - Who owns the consumer’s genetic data? Is it the company? Do have a right to decide how the data are used? - Can the consumer delete their data? - What are the company’s data security measures? How are the data stored? Can the data be accessed for unauthorised use?

Question 40

Direct to consumer genetic tests: Reanalysis

Answer 40

- When you download the raw data and ask a third party to look at it - The original DNA analysis often includes false positive findings, which are carried through to re-analysis. - 40% of reported findings were not confirmed in a clinical laboratory. - Once the consumer has downloaded their raw data file, it is no longer protected by the original service’s privacy policy. - Third-party re-interpretation services are usually unregulated.

Question 41

Pro's and cons of direct to consumer genetic tests

Answer 41

- Pro’s: sample collection is easy, doesn’t require blood. Broad selection of tests, Quick results. Can use data in research and product development - Cons: expensive, risk of false positives and negatives. Results can give false reassurance. Little evidence with some traits. Largely unregulated and privacy might not be guaranteed