1. NHS Diagnostics: Healthcare Science Genetics
William Cross

2. Agenda 1) Introduction to healthcare science in the NHS
Genetic Disorders: Aneuploidy diagnosis using Karyotyping
Karyotyping Workshop
Genetic Disorders: Aneuploidy diagnosis using QF PCR (finger printing)
QF PCR workshop
Answers and Questions
William Cross

3. Working as a Healthcare Scientist
Around 50 scientific disciplines covering patient care and research in the NHS.
Occupy 5% of the total NHS workforce (55,000 staff).
Many job roles within each discipline; Clinical Scientists, Scientist Practitioners, Biomedical Scientists, Support Workers.
William Cross

4. Healthcare Genetics
Aims to give patients better quality of life using knowledge of genetics and diagnostic tests.
NHS genetic services are based in UK hospitals, but can also have separate clinical centres.
Co-ordinated by the UKGTN a subsidiary group of the department of health.
Our local laboratory is BGL at Southmead Hospital and our clinical centre is at St.Michaels hospital.
William Cross

5. What does Bristol Genetics Laboratory do day to day?
Receives patient referrals from GPs, Hospital wards and other specialist services such as Paediatrics and Oncology centres.
Arrange appropriate tests to be carried out based on the patients needs/symptoms.
Analyse results and write reports for Doctors and Specialists to interpret.
Offer specialist advice to all healthcare professionals.
William Cross

6. Testing Workflow Blood sample sent to BGL
DNA extracted from blood sample
Doctor examines patient
Report sent to Doctor
Report written
Genetic analysis performed
William Cross

7. Types of Genetic Testing
NHS genetic services cover many areas of patient healthcare.
Diagnostics - When a patient has symptoms of a disorder
Predictive - When a family member has a disorder
Cancer work - diagnosis of cancer types
Prenatal diagnosis - diagnosing from abnormal foetal scans
Neonatal screening - screening newborn infants
William Cross

8. Southmead ‘Super’ Hospital
William Cross

9. See NHS Careers/Jobs websites for further details of scientific careers in the NHS:
William Cross

10. Genetic Disorders: Aneuploidy diagnosis using Karyotyping
William Cross

11. Genetic Disorders: Overview
Mutations within the genes of an individual can cause genetic disorders.
Can be ‘new’ to a family (de-novo mutation) or inherited.
Mutations come in many types from whole genome duplications (triploidy), to a single nucleotide change (point mutation).
Mutations can be detected by a variety of techniques: PCR, sequencing, Southern blotting, Real-time PCR, MLPA.
William Cross

12. Aneuploidy Mutations
Aneuploidy is the condition of having an abnormal number of chromosomes (karyotype).
Can be a chromosome number reduction or increase (monosomy / trisomy)
Often caused by faults in the cell division of the gametes (meiotic non-disjunction)
Chromosome 21 pair in a dividing gamete
Normal division
Abnormal division
William Cross

13. Aneuploidy disorders Edwards syndrome.
Chromosome 18 trisomy (2nd most common autosomal trisomy, 1:7900).
High mortality rate; life expectancy is a few days from birth and less than 1 in 10 live to the first year of life.
Many clinical features, which can be observed from 1st trimester scan and after birth.
William Cross

14. Edwards Syndrome Congenital heart defects Growth retardation
Dysmorphic features (see diagram)
Facial clefts
Spina bifida
Sever developmental delay
William Cross

15. Aneuploidy disorders Patau syndrome Chromosome 13 trisomy.
High mortality rate; median life expectancy is 2.5 days after birth.
Rare; 1:9500.
Many clinical features, which can be observed from 1st trimester scan and after birth.
William Cross

16. Patau Syndrome Growth retardation Cardiac malformations
Kidney malformations
Cultis aplaisa (scalp defects)
Omphalocele
William Cross

17. Aneuploidy disorders Down syndrome
Chromsome 21 trisomy (most common autosomal trisomy).
1:1000 births
Life expectancy around 60 years.
Well established clinical features that can be determined through scans or at birth.
William Cross

18. Down Syndrome Delayed milestones
Mean intelligence quota (IQ) in children and young adults is 45-48
Cardiac defects
Neonatal hypotonia
Dysmorphic features
William Cross

19. Aneuploidy Mutations Can be detected by karyotyping.
Karyotyping involves culturing patient cells then spreading on a microscope slide and staining (G-banding).
Cells in Metaphase of the cell cycle are viewed under a microscope and specialist software image captures chromosomes for analysis.
A karyotype is produced that enables the chromosomes to be counted and analysed.
William Cross

20. Karyotype Example Increasing Size Chromosome numbers Female Patient
Pairs look nearly identical
William Cross

21. Workshop Examples
Each group will be given four anonymous prenatal/infant karyotypes with accompanying referral forms.
Read referral forms for each foetus/infant examine the karyotype and record your answers in the spaces provided.
Keep your results for later.
Please ask questions if unsure.
William Cross

22. Genetic Disorders: Aneuploidy diagnosis using QF PCR (finger printing)
William Cross

23. QF PCR Overview Why is QF PCR required?
Enables high throughput chromosome screening for most common abnormalities (chromosomes 13, 18 and 21).
Increases scientific evidence for abnormal cases.
Allows a cursory result to be obtained far quicker than karyotyping. Very useful in prenatal cases where there are time constraints.
William Cross

24. PCR: A Quick Overview
Used to amplify specific DNA strands for molecular analysis. The ‘main’ technique of genetics.
Well established technique with many variations e.g. QF PCR, TP PCR, RT PCR.
Utilizes thermostable enzymes (Taq polymerase) and thermocyclers.
Primers can be designed to target most regions in the human genome.
William Cross

25. PCR Reaction 30 sec @ 95ºc Target Loci Primers 45 sec @ 55ºc
Exponential amplification
William Cross

26. Visualizing PCR Products
Gel Electrophoresis
PCR products added to gel well.
Gel contains Ethidium bromide that ‘stains’ DNA as it migrates.
Electrical current applied to the gel.
DNA migrates to positive electrode.
After a predetermined run time DNA can be seen in the gel under a UV light.
Enables fragments to be sized; larger fragments towards the top of the gel (smaller fragments run quicker).
Large to small fragments
Samples
-ve M
William Cross

27. Visualising: Sizing Products
Large to small fragments
Molecular weight ladder allows known size fragments to be seen
Sample bands line up with known size standard band (250bp) as ladder has fragments 50bp apart.
Product sizes can be calculated from the size standard and quantity can also be estimated from band intensity.
William Cross

28. Multiplex PCR
A method of amplifying more than one PCR target simultaneously.
Very useful when designing assays for specific tasks performed routinely e.g. CF mutation screens.
Attention has to be paid to expected PCR product sizes when designing multiplex assays. As overlaps can be difficult to interpret.
William Cross

29. Multiplex Example Why do you think patient 3 has only 4 product bands?
Large to small fragments
There are 5 products in this PCR reaction.
Each product is a distinctly different size otherwise overlap wouldn’t be seen.
Why do you think patient 3 has only 4 product bands? M
William Cross

30. PCR With Variable Number Tandem Repeats
VNTRs are found throughout the human genome in various untranslated regions (UTRs).
They represent regions of DNA where a nucleotide repeat occurs e.g (GCGCGC)n or (CGGTCGGTCGGT)n
VNTRs have varying repeat units thus different alleles present in the general population, e.g repeat units of GC.
A person can be heterozygous for the number of repeats in a given loci so will produce two different sized PCR products.
William Cross

31. PCR With Variable Number Tandem Repeats: Example
Patient 1 is Homozygous for a tandem repeat on chromosome 13.
His/Her genotype therefore is 45/45 repeats, meaning each allele of this loci in both the patients chromosome 13s are 45 repeats long; the same size on a gel.
Large to small fragments
Patient 2 is Heterozygous for this VNTR on chromosome 13 so has two bands.
His/Her genotype therefore is 45/50 repeats, meaning the patients chromosomes have different alleles at this loci. This is very common. M
William Cross

32. Why Are VNTR Useful?
Since most people are heterozygous for some VNTR alleles, this can be used to distinguish between each chromosome within an individual (fingerprinting).
This also allows the amount of each chromosome to be compared thus PCR can be used to test for Aneuploidy by examining the band intensities on a gel.
William Cross

33. Aneuploidy Gel Example
Ratios represent patients genotypes
This gel displays 4 patients, 2 heterozygous and 2 homozygous.
Patient 1 has two bands with equal intensity.
Patient 2 has double the intensity on the upper (48 repeat) band.
Why?
45/ / / / M
48 repeat allele
30 repeat allele
William Cross

34. Aneuploidy Gel Example
Patient 2 has a trisomy at this loci that is detectable by the VNTR
Since VNTR lengths are different in most people, this patient has inherited two different versions from his/her parents.
Hence there is twice as much 48 repeat allele than 30.
30 repeat allele
48 repeat alleles
William Cross

35. Problems With Gel Electrophoresis
Interpretation can be subjective
Bands not always clear.
Intensity can be subjective so quantification not very reliable.
Bands made brighter the longer the fragments are run through the gel.
Gel bed electrophoresis is still used but other techniques are more common.
William Cross

36. Modern Detection Methods: QF PCR
Stands for Quantitative Fluorescence PCR.
Replaces a flat bed gel with a capillary gel inside a genetic analysis machine.
The principles are exactly the same as gel beds, except no Ethidium bromide is needed to stain DNA.
A detector within the machine can measure DNA levels as electrophoresis is performed.
Superior quantification over viewing band intensities.
William Cross

37. Capillary Electrophoresis
45/45 repeats
Gel Bed Version
Capillary Versions
45/50 repeats
Ladder in red
Beckman Coulter CEQ 8000
William Cross

38. Electropherograms in Detail
Numbers on the ‘y’ axis represent amount of PCR product
Fragment sizes are shown at the top of peaks
This kind of analysis is made far easier with genetic analysers!
Patient peaks are any colour other than red
Red peaks always indicate sizing ladder
Numbers on the ‘x’ axis represent fragment size
William Cross

39. Workshop Examples
Each group is given electropherograms corresponding to each patient karyotyped earlier.
These are QF PCR sex chromosome, 13, 18 and 21 Aneuploidy tests.
See if these results match your assessments from earlier.
Please ask for help if unsure.
William Cross

40. Workshop Results Patient A: Normal male foetus William Cross

41. Workshop Results Patient B: Trisomy 18 (Edwards syndrome) foetus
William Cross

42. Workshop Results Patient C: Turners syndrome infant William Cross

43. Workshop Results
Patient D: Translocation 1:22. Undetectable from QF PCR result, may be balanced and display no pathogenicity
QF PCR looks normal
William Cross

44. Thank you for listening. I hope this workshop was helpful
Thank you for listening. I hope this workshop was helpful. Good luck with your final exams.
Questions welcomed
William Cross