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| Journal of the New Zealand Medical Association,
09-October-2009, Vol 122 No 1304
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Customised molecular diagnosis of primary immune
deficiency disorders in New Zealand: an efficient strategy for a small developed
country
Rohan Ameratunga, See-Tarn
Woon
There have been major
advances in the understanding of primary immune deficiency disorders (PID) over
the last two decades.1,2 The genetic basis of
many of these conditions has been identified. Most are inherited as single gene
defects. Early identification of PIDs can reduce morbidity and mortality, as
specific treatment is available for the majority of these conditions.
Early work in New
Zealand3 confirmed the value of molecular
analysis of these disorders. The mutation responsible for a family with X-linked
hyper-Immunoglobulin M (XHIM) syndrome was identified shortly after the
molecular basis for the disorder was discovered.
As a result of this work, the
diagnosis was confirmed in the proband and his sister was reassured she was not
a carrier. Thus, molecular diagnostic testing can play a vital role in patient
management.
In 2003 the Immune Deficiency
Foundation of New Zealand (IDFNZ) funded a study to explore the feasibility of
establishing a molecular diagnostic service in New Zealand to assist patients
and families with PIDs. Following the report presented by the late Dr Karen
Snow-Bailey, senior management at LabPlus in Auckland City Hospital made a
decision to fund the service. A senior scientist was appointed in 2004 to lead
the programme.
Over 120 genes have been
implicated in the pathogenesis of PIDs. For most of these conditions, commercial
tests are not available. The molecular immunology laboratory offers customised
testing on a fee-for-service basis with rapid turnaround times. Turnaround time
is usually 1 week for established tests. For customised tests, the turnaround
time is approximately 2 to 3 weeks.
Quality assurance is a
critical part of laboratory testing. Currently there are no external quality
assurance programmes for the genetics of PIDs.4
As part of the quality assurance programme, blinded samples with previously
identified mutations were received from European and North American diagnostic
laboratories. These were correctly identified. A sample initially sequenced in
Perth for XLP was subsequently sequenced in Auckland and the results were
confirmed5 (see below).
The service has been
accredited by IANZ, the laboratory-accrediting agency in New Zealand. The
programme has also been discussed with NATA, the Australian laboratory
accreditation agency (Andrew Griffin personal communication Sydney, 14.3.09).
The service follows the guidelines for molecular diagnostic laboratories issued
recently by the Centres for Disease Control.4
In this paper, we review the
results of patients referred to the service from 2005 to 2008. The analysis of
these patients illustrates the power and limitations of molecular diagnosis of
PIDs. This programme is working well for New Zealand’s small population
(pop 4.2 million) and can serve as a model for other PID diagnostic
services.
MethodsMammalian genes are
usually coded by exons with intervening introns. In our laboratory, genomic
(DNA) sequencing is undertaken. All exons are sequenced with primers designed to
anneal to introns, in order to identify potential splice site mutations. Splice
site mutations can alter the sequence of mRNA leading to clinical disease as a
result of absent or dysfunctional proteins.
Wild type gene sequences
are downloaded from public databases such as Genbank™ and Ensembl. Primers
flanking the exon regions are designed using Oligo version 6.44 (Molecular
Biology Insights, Cascade, CO, USA).
Genomic DNA from blood
samples are extracted using PUREGENE DNA Purification Kit (Gentra Systems,
Minneapolis, MN, USA). Genes of interest are amplified using polymerase chain
reaction (PCR).
The amplicons then
undergo BigDye® Terminator sequencing cycle
sequencing and the products are subjected to electrophoresis in an Applied
Biosystems (ABI PRISM®) 3100 Genetic
Analyzer. The DNA sequence files are compared to wild type sequence using SeqMan
v5.01 software (DNASTAR, Madison, WI, USA).
The laboratory creates
immortalised Epstein-Barr virus (EBV) transformed B cell lines, which can be the
source of DNA for genetic studies. Fibroblast cell lines are useful after bone
marrow transplantation, as B cells may be of donor origin. These cell lines
obviate the need for multiple blood tests. Creation and storage of the cell
lines are undertaken with the consent of the patient or family in the case of
children.
The laboratory also
offers analysis of T cell receptor excision circles (TRECs), which is a marker
of T cell production by the thymus.6 This assay
has a variety of uses including confirmation of a Severe Combined Immune
Deficiency (SCID) phenotype, when the mutation is not obvious. It can also be
used to follow T cell maturation following bone marrow transplantation.
Figure 1. Laboratory workflow of genetic
testing
 ResultsThe number of samples
received by the laboratory is such that a rapid turnaround time can be achieved.
If necessary, a repeat sample can be rapidly tested for confirmation. A list of
patients referred to the service is outlined in Table 1.
Table 1. Genetic
testing results of the patients referred to the molecular immunology diagnostic
service
Abbreviations:
ALPS: autoimmune lymphoproliferative syndrome, AR-HIM: autosomal recessive hyper
immunoglobulin M syndrome, HAE: hereditary angioedema, SCID: severe combined
immune deficiency, WAS: Wiskott-Aldrich syndrome, XLA: X-linked
agammaglobulinemia, XHIM: X-linked hyper immunoglobulin M syndrome, XLP:
X-linked lymphoproliferative syndrome, X-SCID: X-linked severe combined immune
deficiency.
‡
Single nucleotide polymorphism (SNP) is a small genetic change, or
variation, that can occur within the DNA sequence of an individual; * Currently
DNA from patients with suspected type 3 HAE are sent to Sonic laboratories in
Sydney, which offers a quick and cost effective service. Patients are made aware
of the need to send samples overseas; † The original mutation analysis of
the proband was undertaken in Perth, during the time the service was being
established. The mutation was subsequently confirmed in New Zealand.
DiscussionFunding and the role
of genetic services—Genetics services in New Zealand are
centrally funded. Testing is thus free to New Zealand citizens and permanent
residents. Diagnostic studies are undertaken after patients undergo genetic
counselling. The benefits and limitations of testing are explained to patients.
The laboratory offers a
fee-for-service testing programme. The referring clinical service is invoiced
for the testing. The cost depends on the size of the gene. The
recombination-activating gene 2 (RAG2) gene, implicated in some cases of SCID,
attracts a higher fee due to its larger size in comparison with genes such as
the CD40 ligand. Once the mutation is identified, only the abnormal exon is
sequenced in other family members and therefore the cost is proportionately
less. Government funding is available for testing family members.
The exact cost of testing
also fluctuates based on the value of the NZ dollar, as reagents have to be
imported. In general the cost is considerably less than overseas diagnostic
laboratories based in Europe or the United States. Furthermore, many overseas
diagnostic laboratories do not sequence the full gene. Mutations in less
commonly affected areas of the gene may thus be missed.
The programme employs one
full time molecular biologist (S.-T.W.) and a part time immunopathologist
(R.A.). The use of shared molecular diagnostic resources at Lab Plus has
minimised costs. Currently the service at Auckland City Hospital is financially
self-sufficient with revenue covering costs. Samples have been received from
around New Zealand and also Australia.
Advantages of genetic
testing—The ability to identify a disorder at the genetic level
in most cases eliminates any uncertainty about the underlying diagnosis. Genetic
diagnosis may allow treatment decisions to be made with more confidence. The
identification of a PID has profound implications for other family members. This
is illustrated by family 1 with X-linked lymphoproliferative syndrome (XLP).
Currently no males in the immediate family are at risk of
disease.5
The two brothers (family 20)
identified with Wiskott-Aldrich syndrome (WAS) have undergone bone marrow
transplantation. The decision to undertake bone marrow transplantation in these
children was based on the results of mutation analysis by the molecular
immunology diagnostic service. In WAS, the phenotypic severity of the disorder
can be predicted in many instances, based on the nature of the
mutation.7 These two patients were predicted to
have severe disease based on their mutation
(E133K).8
If the situation is urgent,
such as a baby with a Severe Combined Immune Deficiency (SCID) phenotype needing
bone marrow transplantation, testing can be undertaken immediately to confirm
the genotype. Confirmation of the diagnosis may assist with the decision to
undertake bone marrow transplantation. The type of SCID may influence treatment
decisions such as whether to offer conditioning prior to bone marrow
transplantation.9,10 A detailed analysis of
advantages of PID genetic testing, based on our experience, will be the subject
of a future review. (Ameratunga R, Woon S-T and Neas KN submitted)
Limitations of
genetic testing—Despite the advantages of genetic testing, the
limitations of the technology must be made clear to the patients. This
underscores the importance of genetic counselling. The testing strategy
described here has some disadvantages. Promoter mutations and complex DNA
rearrangements for example, may not be identified by DNA sequencing of the
coding region of a gene. Some mutated genes such the C1 inhibitor have a higher
probability of complex mutations.11
Identification may require Southern blotting and/or analysis of cDNA. These
additional tests are available through the laboratory.
Mutation analysis can be
problematic. The significance of an identified mutation may be uncertain (e.g.
patient 7). A mutation may be non-pathogenic and therefore does not alter
cellular function. Several mutations (e.g. patients 7 & 8) may represent
single nucleotide polymorphisms (SNPs). Furthermore, some patients may be
compound heterozygotes, where the second mutation has not been identified. This
was seen in one of the SCID patients, who may have had Artemis deficiency
(patient 22). The second mutation has not been identified.
Another baby with SCID was
identified as having JAK3 deficiency as a result of compound heterozygosity. The
second mutation was not obvious in the patient and required careful evaluation
of both parents. Both babies had very low numbers of T Cell Receptor Excision
Circles (TRECs), confirming the SCID phenotype. A protein or functional assay
may be able to determine deleterious effects of the mutation but could not be
performed as the two patients are deceased. Creating cell lines may allow
functional assays, such as cell signalling and phosphorylation studies, with
greater ease.
As illustrated by several
patients in this series, a causative mutation may not be identified in spite of
the patient having the classical phenotype. This is seen in several patients
with suspected XLP (patients 3–6) and suspected XLA (patients 12 &
13). One possibility is genocopy, where mutations in unrelated genes can cause a
similar phenotype. A good example of genocopy is a defect of BLNK, where the
phenotype produced is very similar to X-linked agammaglobulinaemia.
12
The results of testing are
only meaningful if they are interpreted in the appropriate clinical context. The
laboratory offers an extensive panel of tests for PIDs including flow cytometry,
lymphocyte proliferation and vaccine antigen responses. Weekly meetings are held
to discuss results and progress with clinical staff. The results of other tests
including flow cytometry are also discussed at the same time.
Diagnostic tests in
research laboratories—Some research laboratories offer free
testing as part of their research programme. These laboratories are often run by
leading authorities in the field with extensive clinical knowledge of these
disorders. While the main advantage is free testing, potential disadvantages
need to be carefully evaluated. The cost of the DNA extraction and sample
transportation need to be considered. Some overseas research programmes require
patients to travel to their institutions before being offered free testing. The
cost of travel needs to be balanced with a free service.
Another concern is a long
turnaround time. Samples may be batched in research laboratories until a
sufficient number have been received. Sometimes results may not be available for
months, which could impact on the ability to offer a family prenatal diagnosis,
where time is of the essence. Genetics services in many centres including
Auckland require the results from research laboratories be confirmed in a
diagnostic laboratory before being used for prenatal diagnosis.
Molecular studies are
expensive and labour intensive. Because of cost constraints, testing may be
performed by a junior staff member or a student in a research laboratory.
Testing could be discontinued abruptly if a research grant is not renewed or the
senior investigator moves to another institution. We also had the experience of
free testing for a limited time after the discovery of a novel disease-causing
gene. Free testing is not offered after some time, presumably because new
mutations cannot be published in high-impact journals.
Research laboratories may not
be obliged to participate in external quality assurance programmes, which can be
an expensive process. Sample mix ups and PCR contamination can occur in any
laboratory. Without close clinical communication, there may not be an easy way
to identify an error in a remote laboratory. Repeating a test may also take a
considerable amount of time, especially in a distant country.
Cultural issues need to be
considered. Tikanga is the traditional system of beliefs, values and
spirituality of Māori. For Māori there is concern about sending tissue
and DNA samples abroad. For some Māori, there may be added concerns about
long-term storage of DNA samples in overseas laboratories. Culturally
appropriate disposal of tissue and DNA samples is important. The Auckland City
Hospital follows Tikanga-recommended best practice policy. If safe to
do so, there is the option of returning specimens to patients if requested.
Samples are stored long term only with the consent of the patient.
If a mutation cannot be
identified in a diagnostic laboratory, the condition being investigated may not
have been previously described. In such cases, a candidate gene approach may
need to be considered. Genes that are likely to be mutated, based on the known
physiology of the suspected gene can be analysed with ethics approval and
patient consent. Alternatively samples could be sent to a research laboratory
once ethics and cultural issues have been addressed. In this specific situation,
research laboratories can play a complementary role to diagnostic
laboratories.
ConclusionsThe model presented here is
an efficient and cost-effective solution for a small developed country. It
allows self-sufficiency in PID diagnosis. We have shown here that a dedicated
PID programme allows rapid diagnosis, leading to early treatment and improved
patient outcomes with reduced mortality and morbidity. A similar model may be
feasible for other specialties in New Zealand, where genetic diagnosis plays a
critical role. More importantly, close involvement of referring clinicians is
likely to improve the quality of the results.
Competing
interests: None declared.
Author
information: Rohan Ameratunga, Adult and Paediatric Immunologist;
See-Tarn Woon, Senior Scientist; LabPlus, Auckland City Hospital, Auckland Acknowledgements:
We are very grateful to IDFNZ for its assistance in creating and supporting this
programme. We would like to thank the late Dr Karen Snow-Bailey for her support
in the early stages of this programme, Dr Kitty Croxson and senior management at
Auckland City Hospital for their ongoing support, and Dr Maia Brewerton for
advice on cultural issues relating to Maori. We thank colleagues Drs Don Love,
Miriam Hurst, Salim Aftimos, and Lochie Teague for helpful suggestions. The
authors can be contacted for more information on specific tests.
Correspondence:
Associate Professor Rohan Ameratunga. LabPlus, Auckland Hospital, Park Rd,
Grafton, Auckland, New Zealand. Fax: + 64 (0)9 3072826; email: rohana@adhb.govt.nz
References:
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