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William Cross NHS Diagnostics: Healthcare Science Genetics.

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Presentation on theme: "William Cross NHS Diagnostics: Healthcare Science Genetics."— Presentation transcript:

1 William Cross NHS Diagnostics: Healthcare Science Genetics

2 William Cross Agenda 1)Introduction to healthcare science in the NHS 2) Genetic Disorders: Aneuploidy diagnosis using Karyotyping 3) Karyotyping Workshop 4) Genetic Disorders: Aneuploidy diagnosis using QF PCR (finger printing) 5) QF PCR workshop 6) Answers and Questions

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

7 William Cross 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 Southmead Super Hospital

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

10 William Cross Genetic Disorders: Aneuploidy diagnosis using Karyotyping

11 William Cross 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 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 divisionAbnormal division

13 William Cross 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 1 st trimester scan and after birth.

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

15 William Cross 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 1 st trimester scan and after birth.

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

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

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

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

23 William Cross 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 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 PCR Reaction Target Loci 30 95ºc Exponential amplification 45 55ºc Primers

26 William Cross 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). M-veSamples Large to small fragments

27 William Cross Visualising: Sizing Products 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. Large to small fragments

28 William Cross 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 Multiplex Example There are 5 products in this PCR reaction. Each product is a distinctly different size otherwise overlap wouldnt be seen. M Why do you think patient 3 has only 4 product bands? Large to small fragments

30 William Cross 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.

31 William Cross PCR With Variable Number Tandem Repeats: Example M 1 2 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. 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. Large to small fragments

32 William Cross 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 Aneuploidy Gel Example 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 Ratios represent patients genotypes repeat allele 30 repeat allele

34 William Cross 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 repeat alleles 30 repeat allele

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

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

39 William Cross 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 Workshop Results Patient A: Normal male foetus

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

42 William Cross Workshop Results Patient C: Turners syndrome infant

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

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