William Cross [email protected] NHS Diagnostics: Healthcare Science Genetics.

William [email protected]

NHS Diagnostics:Healthcare Science

Genetics


Agenda1) 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


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.


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.


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.


Testing Workflow

Doctor examines patient

Blood sample sent to BGL DNA extracted from

blood sample

Genetic analysis performedGenetic analysis performed

Report written

Report sent to Doctor


Types of Genetic TestingNHS 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


Southmead ‘Super’ Hospital http://www.superhospitalforbristol.nhs.uk/


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

http://www.nhscareers.nhs.uk/

http://www.jobs.nhs.uk/


Genetic Disorders:Aneuploidy diagnosis

using Karyotyping


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.


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


Aneuploidy disordersEdwards 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.


Edwards Syndrome

•Congenital heart defects

•Growth retardation

•Dysmorphic features (see diagram)

•Facial clefts

•Spina bifida

•Sever developmental delay


Aneuploidy disordersPatau 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.


Patau Syndrome

•Growth retardation

•Cardiac malformations

•Kidney malformations

•Cultis aplaisa (scalp defects)

•Omphalocele


Aneuploidy disordersDown 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.


Down Syndrome

•Delayed milestones

•Mean intelligence quota (IQ) in children and young adults is 45-48

•Cardiac defects

•Neonatal hypotonia

•Dysmorphic features


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.


Karyotype Example

Female Patient

Increasing Size

Chromosome numbers

Pairs look nearly identical


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.


Genetic Disorders: Aneuploidy diagnosis using

QF PCR (finger printing)


QF PCR OverviewWhy 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.


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.


PCR Reaction

Target Loci30 sec @ 95ºc

Exponential amplification

45 sec @ 55ºc

Primers


Visualizing PCR ProductsGel 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


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.



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.


Multiplex Example

• There are 5 products in this PCR reaction.

• Each product is a distinctly different size otherwise overlap wouldn’t be seen.

M 1 2 3 4 5

Why do you think patient 3 has only 4 product bands?



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.


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.



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.


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

1 2 3 4

48 repeat allele

30 repeat allele


Aneuploidy Gel ExamplePatient 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.

48 repeat alleles

30 repeat allele


Problems With Gel ElectrophoresisInterpretation 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.


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.


Capillary Electrophoresis

Beckman Coulter CEQ 8000

Gel Bed Version

Capillary Versions

45/50 repeats

45/45 repeats

Ladder in red


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


Workshop ExamplesEach 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.


Workshop ResultsPatient A: Normal male foetus


Workshop ResultsPatient B: Trisomy 18 (Edwards syndrome) foetus


Workshop ResultsPatient C: Turners syndrome infant


Workshop ResultsPatient D: Translocation 1:22. Undetectable from QF PCR

result, may be balanced and display no pathogenicity

QF PCR looks normal


Thank you for listening.I hope this workshop was helpful.Good luck with your final exams.

Questions welcomed

William Cross [email protected] NHS Diagnostics: Healthcare Science Genetics.

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Transcript of William Cross [email protected] NHS Diagnostics: Healthcare Science Genetics.