GROWTH – DEDICATED CALL – 1/00
TOPIC III.26
Molecular Genetic Testing
1. CONFORMITY WITH THE WORK PROGRAMME
This topic falls under the Competitive and Sustainable Growth Programme, generic
activity Measurement and Testing. Specifically, it is related to Objective GROW-20006.3 support to the development of certified reference materials for which expressions of
interest have been called.
 Links with other Programmes: Thematic Programme 1 (Life, Generic Activities 7,
8, 9, 14)
2. KEYWORDS
Reference materials / Laboratory Diagnostics / Molecular Genetics / Feasibility study /
Production / Certification /Quality Assurance/ Quality control/ Performance testing / In
vitro diagnostics
3. SUMMARY OF OBJECTIVES AND JUSTIFICATION
The past 10 years have witnessed an explosion in the use of molecular genetics tests in
medical diagnosis. Molecular Genetics testing started in research laboratories, was
developed in specialist centres and is now increasingly being carried out in general
pathology laboratories. Hundreds of thousands of molecular genetics tests are now
performed each year in the EU, and the completion of the Human Genome Project in the
next few years is likely to lead to a rapid expansion in the number of tests available.
Testing laboratories use a wide variety of types of equipment and testing technologies.
New tests and new testing technologies are continuously being introduced. However,
many laboratories still manufacture their own testing reagents, and no CRMs are available
to validate these reagents.
With the advent of commercially-available testing kits, the need for CRMs has become
more acute, as the IVD Directive (Directive 98/79/EC of the European Parliament and of
the Council of 27 October 1998 on in vitro diagnostic medical devices) requires CE
marking of all in vitro diagnostic medical devices offered for sale or transferred between
laboratories.
The objective of this study is the development of the ability to produce and certify
reference materials for a range of diagnostic molecular genetic tests, and to determine
the methods and means required to produce CRMs for any given molecular genetic test.
4. BACKGROUND
Diagnostic molecular genetic testing involves the study of DNA from patients and their
families to diagnose inherited disorders, to predict the onset of such disorders and to
determine the likelihood of such disorders being passed on to the next generation.
DC 1/00/Topic III.26/ Pg 2
Molecular genetic testing will in the future be used to determine individuals’ susceptibility
to developing common disorders such as diabetes, heart disease and cancer, and to
determine their suitability for treatment by pharmaceuticals or susceptibility and resistance
to infectious diseases.
Historically, molecular genetic testing developed in the mid-1980s in research laboratories
where disease genes were being identified. Having identified a disease gene, it was a small
step to proceed to test patients for mutations in that gene which might cause the disease.
Although much diagnostic testing for rare disorders is still carried out in research
laboratories, testing for the common genetic disorders such as cystic fibrosis, the muscular
dystrophies and fragile X syndrome is now usually carried out in specialist diagnostic
laboratories, often associated with Clinical Genetics departments and Cytogenetics
laboratories in Medical Genetics Centres. Most major diagnostic molecular genetic
laboratories still carry out research or are closely associated with research laboratories.
However, Data on numbers of molecular genetic tests carried out are sparse, but the fact
that over 30,000 tests per year are carried out in the UK alone (Harris, R, 1997 and UK
Clinical Molecular Genetics Society; www.cmgs.org) suggests that hundreds of thousands
of tests are performed annually across the EU. The number of tests possible, and therefore
the number of tests performed, is likely to rise rapidly with the anticipated determination of
the entire human DNA sequence (the Human Genome Project) in the next few years, and
as molecular genetic tests are increasingly available to determine susceptibility to common
disorders such as diabetes or heart disease. As commercial testing kits become available,
molecular genetics testing for inherited disorders is increasingly being carried out in
general pathology laboratories and some commercial centres.
Molecular genetics tests are extremely specific – they are usually designed to detect a
single mutation in a specific gene. The results are in the most part qualitative (i.e. presence
or absence of a mutation) though quantitative testing (number of copies of a gene sequence
present) also plays an important role.
Some molecular genetic tests have a quantitative aspect, in that the test must measure the
number of repeats in a stretch of short DNA sequence repeats. Expansions in such repeats
underlie the pathogenesis of many neurological disorders, and such repeat stretches are
also used as linked markers to track disease genes through families. It has become clear
from the experience of external quality assessment schemes that the length of such repeat
stretches is difficult to measure using available sizing standards, for technical reasons. The
only valid sizing standards for such assays are DNA samples in which the repeat length
has been determined by accurate DNA sequencing, as evidenced, for example, by the
variability of results observed in a pilot EU external quality assessment scheme for
Huntington disease carried out by the European Molecular Genetics Quality Network
(Losekoot et al, 1999). Again, there is no central resource for such standards, and testing
laboratories must usually develop their own standards.
Testing kits are currently only available for a few of the most common genetic disorders
(cystic fibrosis [CF], Y-chromosome deletions [AZF], fragile X syndrome [FraX]).
Consequently, most testing reagents are produced in-house by testing laboratories. These
reagents are routinely validated against standards also developed in the same laboratories –
usually patients with mutations detected by DNA sequencing or some alternative method
of detection. In some cases positive controls are obtained from colleagues in other testing
laboratories or from research centres. The implementation of the IVD Directive may
DC 1/00/Topic III.26/ Pg 3
severely limit the transfer of such materials between laboratories. There is no central
source of reference materials, and no standards have been set for the development of such
materials. Lack of reference materials has been recognised to contribute to diagnostic error
in some tests developed in research centres (e.g., Huntington's Disease [HD]; Losekoot et
al, 1999 and Haemochromatosis [HH]; Jeffery et al, 1999).
An added complication is that most molecular genetics tests can be carried out by several
different methodologies, and any CRM will have to be validated for all the methodologies
in use. Many genetic tests rely on amplification of the DNA sequence of interest by the
polymerase chain reaction (PCR). As PCR primers must precisely match DNA sequences
flanking the mutation site to be tested, any variation in these flanking sequences can affect
the outcome of the test (Jeffrey et al, 1999)
The European external QA for Cystic Fibrosis, organised in 1996-97 and 98 by the
European Concerted Action on Cystic Fibrosis (BMH4-CT96-0462) and which will
continue and be extended in the Thematic Network on CF in the FP5 program, has already
shown that from the more than 100 laboratories who participated in these three trials 20%
or more of the laboratories made technical and administrative errors in the testing for
frequent mutations. Moreover, while about 50% of the laboratories used commercially
available kits, errors or weaknesses were uncovered in all of the kits used, confirming that
the validation of these commercial products in the laboratories is still insufficient and
inadequate (Dequeker and Cassiman, 1998).
Genetic tests are unlike other forms of laboratory diagnostic testing in several respects:
Firstly, because an individual’s genetic composition does not alter with time, patients are
usually only tested once, and an incorrect result can stay with them for life. Secondly, the
results of genetic tests can have profound implications for other family members. The
results of prenatal tests to determine the genetic status of the fetus are often used by the
family to decide whether a pregnancy will be terminated or not. Furthermore, the public
and the medical profession have a high degree of confidence in the results of molecular
genetics tests, perceiving them as “state-of-the-art”, and are thus perhaps less likely to
question the validity of the results of these tests. All of these factors contribute to the
importance of maintaining the highest possible quality standards in molecular genetics
testing laboratories, a requirement which is reinforced when one considers the growth in
potential for testing in this discipline and the rate of development of new technologies.
Quality (accuracy, reproducibility) and comparability of test results relies on laboratory
accreditation, adherence to standard protocols, training of personnel, participation in
external quality assessment schemes and the routine use of Reference Materials
(McGovern et al, 1999). The availability of Certified Reference Materials is therefore
of central importance in ensuring and demonstrating quality in all forms of laboratory
testing. All of these quality systems are at some stage of development through national
or EU programmes but the diagnostic community has yet to make a concerted effort to
develop CRM's for molecular genetics.
5. ECONOMIC AND SOCIAL BENEFITS
As outlined above, there are several reasons which make it important that genetic tests, in
particular, must be as accurate as possible. The benefits of the availability of CRMs for
molecular genetic testing are increased accuracy and reliability of testing, and fall into the
category of health benefits, with associated social and economic consequences.
DC 1/00/Topic III.26/ Pg 4
Standardisation of quality standards will help to retain the confidence of end-users (the
public and their clinicians) in the accuracy of genetic tests. The availability of CRMs will
improve the accuracy of tests across the EU, with consequent increases in efficiency of
testing, avoidance of costly repeat testing and avoidance of legal action consequent to
incorrect test results. The operation and usefulness of national and pan-European external
quality assurance schemes would also be greatly enhanced by the availability of molecular
genetic CRMs.
With the introduction of the IVD Directive (Directive 98/79/EC of the European
Parliament and of the Council of 27 October 1998 on in vitro diagnostic medical
devices), CE marking is now required on all diagnostic kits offered for sale in the EU.
The availability of CRMs for the validation of such kits would be of substantial
competitive economic benefit to EU enterprises producing such kits, and would help
safeguard the public against the introduction and use of inaccurate testing kits. As
detailed above, there is considerable cause for concern about the validation of currentlyavailable testing kits, some of which may not produce the correct result in all testing
situations.
The increasing emphasis on EU-wide quality standards and the traffic in samples for
testing between EU member states combine to create a need for certified reference
materials developed and made available at the EU level.
6. SCIENTIFIC AND TECHNOLOGICAL OBJECTIVES
The final objective of the work to be performed will be development of the ability to
produce and certify reference materials related to diagnostic molecular genetic testing.
The study would necessarily consist of several phases, from the identification and
evaluation of possible sources of RMs, through forming an inventory or database of
materials available, through selection of a representative series of “model” molecular
genetic tests to validation of these tests on the chosen RMs and study of the stability,
consistency and presentation of the CRMs.
The co-ordinator of the study would need to demonstrate broad knowledge and experience
of diagnostic molecular genetic testing and of quality control and quality assessment in
molecular genetics in particular. They would have to demonstrate familiarity with the
principles of the validation and production of RMs.
The objectives of the study will be
1. To identify and evaluate possible sources of RMs, including those currently available
in public and private collections, and including the development of novel sources.
Sources would have to be infinitely renewable to ensure ongoing availability and to
justify the input of effort in certifying the source as a CRM.
2. To develop an inventory or database of potentially suitable sources of RMs
This database would be essential for the feasibility study, and would be a useful
reference source in the future if the study demonstrates the it is feasible to produce
CRMs for molecular genetic testing.
3. To evaluate these various possible sources as to their suitability, using pre-determined
criteria.
Such criteria would include availability, cost, renewability (see point 1 above), ethical
approval and legal ownership status of the RMs and their sources.
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4. To select a limited representative series of “model” molecular genetic tests and
genetic markers on which to assess the feasibility of CRM production.
The criteria for the selection of such model tests would include:
Economic and social justification:
– Test is widely applied in molecular genetics laboratories across the EU
– Test is for a serious inherited disorder
Technical justification
– The tests chosen cover a range of possible testing methodologies
– The tests chosen cover a range of types of inheritance
1.
2.
3.
4.
5.
6.
7.
8.
9.
Prioritized list of diseases and mutations*:
Disorder
Gene Symbols(s)
Cystic Fibrosis
CFTR
Haemochromatosis
HFE
Fragile X syndrome
FMR1
Sickle cell anaemia
HBB
Alpha thalassaemia
HBA
Beta thalassaemia
HBB
Factor V Deficiency
FV
Huntington Disease
HD
Hereditary breast cancer
BRCA1, BRCA2
10. Hereditary non-polyposis colon
cancer
11. Spinocerebellar ataxias
12. Friedreich ataxia
13. Haemophilia A
14. Duchenne Muscular Dystrophy
15. Hereditary Motor & sensory
neuropathy type 1a
16. Myotonic dystrophy
17. Male Infertility
SCA1- SCA7
FRDA
F8
DMD
PMP22
Important Mutations
F508, many others
C282Y, H63D
CGG expansion
"HbS"
Numerous
Numerous
R506Q "Factor V Leiden"
CAG Expansion
185delAG; 5382insC;
6174delT
Representative nonsense
mutations
CAG Expansion
GAA Expansion
Common inversion
Deletions
Common duplication
DM
Y Chromosome
CTG Expansion
Consensus STS markers
HMLH1, HMSH2
*Other diseases and mutations may be added to this list, or the priorities altered, only if
full justification for such addition or alteration is provided.
5. To apply the selected tests to the candidate RMs under the highest quality conditions.
6. To organise a limited collaborative trial of the candidate RMs presented in various
forms, involving laboratories using a range of different testing technologies, including
both Health Service laboratories and private enterprises where applicable.
7. To assess the consistency, stability and homogeneity of the RMs under a range of
preparation,
presentation,
storage
and
handling
conditions.
8. Using the experience gained in the study, to develop general guidelines, protocols
and procedures for the production of CRMs for molecular genetic testing.
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7. TIME SCALE
There is no specific timescale within which such CRMs must be developed, except that
there exists a pressing need to develop them as soon as possible. The “grace period” for
the implementation of the IVD Directive expires shortly, which further emphasises the
urgency of the need.
8. IMPORTANT ADDITIONAL INFORMATION
As the RMs for this study will of necessity consist of, or be derived from, human
biological materials, the co-ordinator will have to satisfy the Commission that all
necessary ethical approvals and patient consents have been obtained for the use of these
materials in the study
References
Dequeker E, Cassiman JJ Evaluation of CFTR gene mutation testing methods in
136 diagnostic laboratories: report of a large European external quality
assessment. Eur J Hum Genet 1998 Mar-Apr;6(2):165-75.
Harris, R Genetic Services in Europe Eur J Hum Genet 1997 5(suppl. 2): 196-220.
Jeffrey GP, Chakrabarti S, Hegele RA, Adams PC Polymorphism in intron 4 of HFE
may cause overestimation of C282Y homozygote prevalence in haemochromatosis.
Nat Genet 1999 Aug;22(4):325-6.
Losekoot M, Bakker B, Laccone F, Stenhouse S, Elles R A European pilot quality
assessment scheme for molecular diagnosis of Huntington's disease. Eur J Hum Genet
1999 Feb-Mar 7(2): 217-22.
McGovern M, Benach MO, Wallenstein S, Desnick RJ, Keenlyside R Quality
Assurance in Molecular Genetic Testing Laboratories JAMA 1999 Mar 281(9): 835-840.
DC 1/00/Topic III.26/ Pg 7
Addendum to the supporting document for all topics related to
the development of Certified Reference Materials (CRMs)
Introduction
Research in support of the development of CRMs is an objective of the Growth
Programme. The modality for this research is a shared cost action on RTD (cf work
programme Competitive and Sustainable Growth). The guidelines for the submission of
the proposal are laid down in the Guides for Proposers.
In addition to the requirements for research on a specific topic that are laid down in the
supporting document*, there are general requirements for the development of CRMs.
These requirements are described below and are in addition to the general requirements
for shared cost actions, as described in the guides for proposers and the model contract.
The requirements have been divided in 2 major parts:
I) Description of the work
This part concentrates on the technical requirements of the research as well as
the reporting requirements in view of the future certification.
II) Implementation of the objectives of the research
The research on CRMs consists of the development of the ability to produce and
certify reference materials that are fit for purpose. The planned exploitation of
the results has to aim at the production and certification of the reference
material(s) in accordance with prevailing international quality standards.
*
The dedicated call system enables the Commission to identify priority topics that have
been forwarded under the call for the expressions of interest. The supporting document
that has been used to evaluate the topic now serves as a reference document to the
proposers for the preparation of the proposal.
DC 1/00/Topic III.26/ Pg 8
I) Description of the work
Research on CRMs consists of the development of the ability to produce and certify
reference materials. The actual production of the CRM is not part of the research but is
part of the exploitation of the result(s).
In the first phase of the research project the certification strategy should be elaborated
(development of the materials and methods for characterisation), in the second phase of
the research project the ability to produce and certify should be demonstrated and
finally the specifications for the final CRM should be set.
The development of a CRM normally consists of 4 major parts:
1) development of the know-how required to produce (and package) the future CRM
2) development of the methods needed for future certification of the material
3) demonstration of the feasibility of the CRM production (production of a small batch
+ mimicking its certification)
4) reporting of detailed and realistic specifications for the future CRM
1. Development of the know-how required to produce (and package) the future
CRM






specify the sort of material that is most likely to meet both the targets laid down in
the supporting document and be suitably homogeneous, stable, safe to handle, and
acceptable in cost
identify the options available for selecting / preparing such a material
take into account special storage and / or transport requirements existing for certain
types of materials
develop of methods for production / selection and preparation, homogenisation,
stabilisation, protection and packaging of material as required, to enable the
production of a CRM which will be fit for purpose
if a material needs to be altered substantially (e.g. freeze drying), or when it is
artificially spiked with the substance of interest, it is necessary to check if the material
behaves in the same manner as the routine samples when applying the relevant
methods of measurement.
Ensure the availability of a suitable reconstitution procedure if a material will need
reconstitution
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2. Development of the methods needed for future certification of the material
Certification can be performed according to different criteria. The research will
define which certification route, following internationally recognised quality
requirements and standards, will be followed.
Certification on the basis of pure substances

If the CRM is a pure substance the preparation methods should be such that the
presence of impurities are reduced to a minimum. Additionally the substance should
be tested for impurities by carefully selected methods at least in two laboratories.
Certification on the basis of preparation data

When dealing with a synthetic CRM and a quantity which is directly related to (or
depends in a well-established way on) composition, examine if preparation under
metrological conditions and certification on that basis (e.g. using gravimetric data) is
possible
 Identify nevertheless a method of measurement that can be used as an independent
check for large errors
Certification on the basis of measurements - method-dependent quantities

If the quantity to be certified is defined on the basis of a specific (standard) sample
treatment or method of measurement, certification will be based on statistical
processing of results produced by a sufficient number of laboratories (e.g. 12 - 15),
which follow strictly the standard protocol but include all variability which is
allowed by the protocol (do not use common sources of calibrants, reagents, ...
unless these will be available to the future CRM users and prescribed in the
protocol). Such protocol may have to be developed
 Work may be necessary to achieve an acceptable level of reproducibility
Certification on the basis of measurements - method-independent quantities

As error-free, matrix-independent, calibration-independent methods are exceptional,
the basis for certification will usually be an agreement between different methods and
different laboratories, applied under conditions of independence which rule out the
risk of a common systematic error. Methods with a high potential for matrixindependence and/or calibration-independence (e.g. IDMS for determining
polyisotopic elements) should be included if available
 it may be necessary to perform a substantial collaborative method improvement (to
achieve the required level of agreement between methods and between laboratories),
or even research (method development) to achieve the required ability to measure.
Guidance for collaborative method improvement is given in the BCR Guidelines*
*
Guidelines for the production and certification of BCR reference materials, available from the
Commission on request (E. Michel, tel: 32/2-295.11.33, fax: 32/2-295.80.72, email:
ellen.michel@cec.eu.int )
DC 1/00/Topic III.26/ Pg 10
3. Demonstration of the feasibility of the CRM production

prepare a small batch of material, similar to that intended to be prepared as a CRM,
using the method intended to be used to prepare the actual reference material
 Check the fitness for purpose:
 form, shape or texture of the material; if applicable, contamination (air, moisture,
vapours, dust and dirt, micro-organisms etc.)
 homogeneity
 stability (the analyte / measurand as well as the matrix)
 safety of packaging when exposed to shocks, extreme temperatures and other possible
causes of damage during storage and transport
 ease of opening and use
 lyophilisation and reconstitution procedures
Guidance for homogeneity and stability testing is given in the BCR Guidelines.

Use the test batch to mimic the certification of a future CRM in a measurement
campaign, designed as a certification exercise (see BCR Guidelines)

If the results show that a CRM prepared and certified as in the test round is not good
enough, then the development work (1. or 2. above) must be recommended
4. Reporting of detailed and realistic specifications for the future CRM
The reporting requirements are set in the guidelines for reporting. In addition to the
Final Technical Report the coordinator should prepare an overview of all the results,
as foreseen in the Technical Implementation Plan. In this report the outcome of the
research that sets the boundaries for the future CRM production should be given
preferably in the form of a technical data sheet. It should contain the following
information:
 form / type of samples (with preference for individually packed portions for single
use)
 lyophilisation and reconstitution procedures
 specify bottling, labelling, packing, storage and transport requirements
 identify any legal restrictions on the transport of such material and need for safety
labelling, collect data for material safety data sheets
 magnitude of the quantity of interest (acceptable range)
 acceptable uncertainty on the certified value for an individual sample (this includes
at least: acceptable uncertainty on the mean value for the entire batch, and
acceptable level of inhomogeneity of the batch)
 method requirements (if the CRM is method dependent)
 acceptable magnitude of other quantities
 an estimate of the quantity of CRM that should be produced as well as an estimate
of the production costs.
DC 1/00/Topic III.26/ Pg 11
II) Implementation of the objectives of the research
As for all shared cost contracts the contractors are obliged to exploit the knowledge
arising from the project. In the case of research on CRMs the contractors are, among
others, obliged to assure that the CRM is produced and certified within a reasonable
time limit (article 10 of Annex II General Conditions of model contract).
The "Technological Implementation Plan" (TIP), which has to be prepared in any
RTD project of the 5th Framework Programme, shall explain the actions planned to
assure that results of the project will be exploited. In case of CRMs this TIP has to
outline the strategy for the CRM production, certification and marketing within a
reasonable time frame, at a reasonable cost and according to internationally
recognised quality requirements. A draft TIP has to be available at the project midterm. Options, ranging from production by the consortium to production by a third
party or the Commission, will be explored in consultation with the Commission
Services.