NHS Diagnostics: Healthcare Science Genetics

1. NHS Diagnostics:Healthcare ScienceGenetics William Cross william.cross@nbt.nhs.uk

2. William Cross william.cross@nbt.nhs.uk 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

3. William Cross william.cross@nbt.nhs.uk 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.

4. William Cross william.cross@nbt.nhs.uk 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.

5. William Cross william.cross@nbt.nhs.uk 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.

6. William Cross william.cross@nbt.nhs.uk Testing Workflow Blood sample sent to BGL DNA extracted from blood sample Doctor examines patient Report sent to Doctor Report written Genetic analysis performed

7. William Cross william.cross@nbt.nhs.uk 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

8. William Cross william.cross@nbt.nhs.uk Southmead ‘Super’ Hospital http://www.superhospitalforbristol.nhs.uk/

9. See NHS Careers/Jobs websites for further details of scientific careers in the NHS: http://www.nhscareers.nhs.uk/http://www.jobs.nhs.uk/ William Cross william.cross@nbt.nhs.uk

10. William Cross william.cross@nbt.nhs.uk Genetic Disorders:Aneuploidy diagnosis using Karyotyping

11. William Cross william.cross@nbt.nhs.uk 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.

12. William Cross william.cross@nbt.nhs.uk 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

13. William Cross william.cross@nbt.nhs.uk 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.

14. William Cross william.cross@nbt.nhs.uk Edwards Syndrome • Congenital heart defects • Growth retardation • Dysmorphic features (see diagram) • Facial clefts • Spina bifida • Sever developmental delay

15. William Cross william.cross@nbt.nhs.uk 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.

16. William Cross william.cross@nbt.nhs.uk Patau Syndrome • Growth retardation • Cardiac malformations • Kidney malformations • Cultis aplaisa (scalp defects) • Omphalocele

17. William Cross william.cross@nbt.nhs.uk 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.

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

19. William Cross william.cross@nbt.nhs.uk 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.

20. William Cross william.cross@nbt.nhs.uk Karyotype Example Increasing Size Chromosome numbers Female Patient Pairs look nearly identical

21. William Cross william.cross@nbt.nhs.uk 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.

22. William Cross william.cross@nbt.nhs.uk Genetic Disorders: Aneuploidy diagnosis using QF PCR (finger printing)

23. William Cross william.cross@nbt.nhs.uk 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.

24. William Cross william.cross@nbt.nhs.uk 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.

25. William Cross william.cross@nbt.nhs.uk PCR Reaction 30 sec @ 95ºc Target Loci Primers 45 sec @ 55ºc Exponential amplification

26. William Cross william.cross@nbt.nhs.uk 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

27. William Cross william.cross@nbt.nhs.uk 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.

28. William Cross william.cross@nbt.nhs.uk 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.

29. William Cross william.cross@nbt.nhs.uk Multiplex Example 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 1 2 3 4 5

30. William Cross william.cross@nbt.nhs.uk 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 20-30 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.

31. William Cross william.cross@nbt.nhs.uk 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 1 2

32. William Cross william.cross@nbt.nhs.uk 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.

33. William Cross william.cross@nbt.nhs.uk 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? 45/30 48/30 51/51 51/51 M 1 2 3 4 48 repeat allele 30 repeat allele

34. William Cross william.cross@nbt.nhs.uk 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

35. William Cross william.cross@nbt.nhs.uk 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.

36. William Cross william.cross@nbt.nhs.uk 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.

37. William Cross william.cross@nbt.nhs.uk Capillary Electrophoresis 45/45 repeats Gel Bed Version Capillary Versions 45/50 repeats Ladder in red Beckman Coulter CEQ 8000

38. William Cross william.cross@nbt.nhs.uk 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

39. William Cross william.cross@nbt.nhs.uk 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.

40. William Cross william.cross@nbt.nhs.uk Workshop Results Patient A:Normal male foetus

41. William Cross william.cross@nbt.nhs.uk Workshop Results Patient B:Trisomy 18 (Edwards syndrome) foetus

42. William Cross william.cross@nbt.nhs.uk Workshop Results Patient C:Turners syndrome infant

43. William Cross william.cross@nbt.nhs.uk Workshop Results • Patient D:Translocation 1:22. Undetectable from QF PCR result, may be balanced and display no pathogenicity QF PCR looks normal

44. William Cross william.cross@nbt.nhs.uk Thank you for listening.I hope this workshop was helpful.Good luck with your final exams. Questions welcomed