USE OF TURNITIN: unless otherwise specified, all coursework should be submitted in electronic form via

TURNITIN. For each assignment you may upload one draft so that you can check for accidental plagiarism

marks.
Check that your document has uploaded successfully by reviewing a copy downloaded from
TURNITIN.
TURNITIN will log the time that you uploaded your report and will e-mail a receipt for your work - do not
delete this from your e-mail account until your marked work has been returned to you.

IMPORTANT IF THERE ARE ISSUES WITH TURNITIN !!!!!!!!!

If you are in any doubt about the success of TURNITIN upload of your work you should e-mail your
complete document file (using your University email account) to Ailsa Watson and to Frances Cousins
(copied to yourself) before the deadline; the copy to yourself copy is so that you can check that the email
was sent successfully and that the attached coursework document opens up as a file. As long as the file copy
has been received into the email account of Ailsa & Frances before the deadline you will not be penalised for
late submission.

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 1. Introduction
This booklet contains background information for each exercise. You must read through this
information before coming to preparatory sessions and try to answer as far as possible the questions
which are set.
During the preparatory sessions or small group work sessions the staff will be assessing how well
prepared you are if you come to the session without having read carefully through the protocols
and attempting the questions in advance, this will become apparent very quickly. Your overall
performance and preparedness will form a part of any job references written for you by MSc
Programme Coordinators.
Although we will not directly assess the answers you write in this booklet, it is important for your
understanding of the material that you do answer all questions as far as possible, and in many cases
(not all) your answers will be integral to your report.
You will be expected to locate and use appropriate articles from the scientific literature. Additional
resources that may be useful for general background are:
OMIM (Online Mendelian Inheritance in Man) at:
http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=OMIM
Gene Reviews at Gene Tests:
http://www.geneclinics.org/
The Bookshelf of online texts at NCBI:
http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=Books
Various molecular biology and medical genetics textbooks.

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 2. PCR-RFLP detection of variants in HFE gene
Aim
Analyse and interpret data from PCR-restriction fragment length polymorphism (PCR-RFLP) assay
of HFE gene for a series of individuals, and generate a written report that evaluates PCR-RFLP and
compares this to another molecular technique for diagnosis of haemochromatosis.

Ensure that you have read the material in this section, and completed all answers as far as possible
BEFORE coming to the discussion for the report writing. You may need to do a little research on the
web, in journals or in textbooks to find the answers to some of the questions. Note that not all topics
considered in the questions below may be appropriate for inclusion in your report.

Introduction
Diagnostic tests
Many different approaches to diagnostic testing are currently used in genetics laboratories; different
laboratories may use different techniques from each other in order to test for the same diseases (ie
laboratory A might use technique X to test for mutations in gene G, whilst laboratory B might use
technique Y instead).
Q. What do you think are the key attributes of a good diagnostic test?

Q. What factors do you think might govern the choice of technique that is used in a particular
laboratory to detect mutations in a particular gene?

Restriction enzymes
Q. What are restriction enzymes and how do they work?

Q. How may methylation affect the function of restriction enzymes?

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Q. What are the different types of DNA change that may generate restriction fragment length
polymorphism (RFLP)?

An old (and practically obsolete) method of RFLP analysis is by the use of Southern blotting. The
modern approach is to use polymerase chain reaction (PCR) to amplify the relevant piece of DNA
and then carry out restriction digestion on the amplified DNA; this is called PCR-RFLP.
Generate a diagram to illustrate how standard PCR-RFLP analysis works:

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Hereditary haemochromatosis (HH)
HH is an iron overload condition, most frequently as a result of pathogenic variants in the HFE gene.
Most of those affected are homozygous for HFE c.845G>A (p.Cys282Tyr), though other pathogenic
variants have been reported, including c.187C>G (p.His63Asp). A PCR-RFLP assay can assess these
two variants, with fragment lengths predicted as shown in Table 2.1:

Variant Allele with expected fragments Allele with expected fragments

c.845G>A (p.Cys282Tyr) G (Cys): 247, 140 A (Tyr): 247, 111, 29
c.187C>G (p.His63Asp) C (His): 130, 70 G (Asp): 200
Table 2.1: Expected fragments following RFLP analysis of HFE variants

Peripheral blood from seven unrelated individuals with a possible diagnosis of HH has been sent to a
genetic diagnostics laboratory for testing by the above assay (see also notes from class tutorial
session). Each referral was from a different hospital, clinic or GP practice. Referral details are
provided in Table 2.2 and results of the multiplex PCR-RFLP assay are provided in Figure 2.1.

Patient Sex Age Referral reason(s)

1 F 28 Family history of HH
2 M 48 Enlarged liver, joint problems, elevated serum ferritin
3 M 32 Family history of HH
4 M 57 Enlarged liver, elevated serum ferritin
5 F 54 Fatigue, lethargy, recent diabetes diagnosis
6 M 45 Family history of HH, elevated serum ferritin
7 M 51 Enlarged liver, heart failure, elevated serum ferritin
Table 2.2: Referral reasons as detailed on referral forms

Figure 2.1: PCR-RFLP analysis for HFE variants c.845G>A (p.Cys282Tyr)and c.187C>G
(p.His63Asp) in seven patients (1-7) referred for genetic testing. Expected fragment sizes for
each allele are provided in Table 1. The final lane contains a DNA marker set with sizes shown in
base pairs at the right.

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Data analysis report
Your report should include the following (i) introduction to PCR-RFLP as a diagnostic test for HH;
(ii) a summary of the results of the PCR-RFLP analysis for these samples, with full interpretation;
(iii) evaluation of the PCR-RFLP approach to HH testing and comparison with an alternative
technique of your choice. Throughout your report keep the focus on testing for HH, and in particular
on molecular (ie DNA) testing.
Maximum: 800 words (10% extra allowed up to absolute maximum of 900 words), excluding sub-
headings, tables, figure legends and references (in-text citations and reference list). Figure legends
should be no more than 200 words each. Complete report, including all images, tables, references
should be no more than six A4 pages. Harvard referencing style must be used.
Exceeding the maximum 900 words will result in 1 grade penalty per 100 words (or part thereof).
Exceeding the maximum six pages will result in 1 grade penalty per page (or part thereof).
Please convert file to PDF before submission.
Your name / student ID should not appear in the text of the report, but it would be helpful to staff
who anonymise the reports if your student ID formed part of the filename,
eg 2345678 HFE report.pdf
Note 1: Your name and student ID should NOT appear anywhere within the text of your report file,
since marking is anonymous.
Note 2: General coursework prescriptions for minimum font size and line spacing are provided in
your Course Information Document. Source citation and referencing should be according to
Harvard convention.
Note 3: READ CAREFULLY the advice in the Course Information Document on writing scientific
reports before starting to write!!! Also consult the Feedback Proforma for this report, which will be
available on Moodle this will help you determine what we are looking for when grading.
Note 4 ormat of a
laboratory report; you are analysing data which is provided, rather than data you generated. The
format used for your report should communicate the information in a logical manner, and it would
be sensible to include sub-headings. Your report should not refer to this manual as a source (there is
no need to cite a source for the provided information on the patients or the gel data from the assay).
Note 5: Your focus in the report should be on genetic diagnostic methods for HH.
a echnique to compare PCR-RFLP with, but ideally select a fairly current method
that you consider a good one that is available for use in typical genetic diagnostic laboratories. Note
that the assessment criteria include critical use of literature that demonstrates depth of
understanding.

The grade for this report is formative only, but the individual feedback that you receive should
help with future assessments for your Genetic disease course.

Starter reference
Porto G, Brissot P, Swinkels DW, Zoller H, Kamarainen O, Patton S, Alonso I, Morris M &
Keeney S (2016) EMQN best practice guidelines for the molecular genetic diagnosis of hereditary
hemochromatosis (HH). European Journal of Human Genetics 24: 479 495.

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