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Twins: mirrors of the immune system
Marco Salvetti, Giovanni Ristori, Roberto Bomprezzi, Paolo Pozzilli and
R. David G. Leslie
Twin studies are a powerful tool to
assess genetic and nongenetic
I
n the late 19th century, Francis
In the century following Galton’s publifactors in multifactorial, immuneGalton, Charles Darwin’s cousin,
cation, additional methods were used to
mediated diseases. Here, Marco
first thought of using twins to invesexploit the value of twins, such as comparing
tigate the role of heredity and enviidentical twins with and without a disease.
Salvetti and colleagues review
1. Since then, twin
ronment in human life
Immunological investigations in immuneimportant results from such studies
studies in biology, medicine and psychology
mediated disease are an outstanding examand highlight their potential value.
have had a considerable impact on the scienple of the potential of twin studies. Autotific community; this has generally been for
immune diseases affect up to 5% of the
Future developments that should
the better although, in the case of ‘eugenetic’
population and are a major cause of morbidhelp to realize the potential of twin
politics, it has also been for the worse2.
ity and mortality. The vast majority of twin
studies are discussed.
Galton realized that, because twin pairs
studies in immune-mediated diseases have
(whether identical or nonidentical) share the
been carried out in autoimmune diseases,
same environment, greater concordance for
including insulin-dependent diabetes mellia disease in genetically identical twins compared with nonidentical tus (IDDM), multiple sclerosis (MS), rheumatoid arthritis (RA), systwins probably reflects a role for genetic factors leading to the dis- temic lupus erythematosus (SLE), ankylosing spondylitis (AS) and
ease. By contrast, similar concordance rates for a disease in twin coeliac disease. These diseases cover a spectrum of organ-specific
pairs (whether identical or nonidentical), or differences between and non-organ-specific diseases and will be the focus of this review.
identical twins for a given disease, suggest that the disease is probably In such diseases, the impact of genetic factors is so substantial, and
due to non-genetically determined factors.
the identity of all immune response genes sufficiently uncertain, that
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twin studies are the only way, in an outbred population, to rule out
genetic differences contributing to immunological differences. Yet
how much of our current knowledge of the pathogenesis of immunemediated diseases comes from twin studies? Disappointingly little,
probably due to the difficulty in obtaining sufficient numbers of
twins – only about one in every 250 individuals is an identical twin
– and the fact that most twin studies have been cross-sectional and
few have been prospective (an important point because, on follow-up,
twin pairs could show higher concordance rates).
Twin studies and immune-mediated diseases
The impact of environmental factors on disease
Genetic and environmental factors are both thought to contribute to
the pathogenesis of immune-mediated diseases. The most powerful
evidence that immune-mediated diseases are due to environmental
factors comes from the study of identical twins. The majority of identical twins with an autoimmune disease have an unaffected twin;
that is, they are discordant for the disease (Table 1). While the initially unaffected twin may develop the clinical disease some years
after the index twin, the majority remain unaffected on prospective
study even, as with IDDM, 40 years after the clinical diagnosis of
the index twin7. Nevertheless, even identical twins can differ genetically: X-chromosome inactivation in females can lead to different
patterns of mosaicism; differential methylation of CpG islands can
result in repression of transcription; and novel somatic rearrangements are involved in the development of T-cell receptors (TCRs)
and antibodies. Thus, discordance between identical twins may be
determined by nongenetic (epigenetic) factors operating on genetic
expression.
Having established that environmental factors are important, the
next step would be to attempt their characterization. Studies in MS
have shown that nonidentical twins have a similar concordance
rate to their non-twin siblings. Since nonidentical twins would be
expected to share a closer environment than non-twin siblings, these
observations suggest that clustering of diseases in families (familial
aggregation) is genetic and not environmental. Furthermore, this
introduces the important distinction, confirmed by studies on
adoptees and half-sibs, between environmental factors that determine the familial risk of a disease (most probably with limited influence compared with genetic factors) and environmental factors that
act at the population level (and are strong determinants of disease
risk)3. While disease-discordant identical twins should be the perfect
test-bed to identify such critical environmental factors, no diseasedetermining environmental factor has, as yet, been found using twin
studies.
The impact of genetic factors on disease
Twin studies can also be used to estimate the impact of genetic
factors on the cause of a disease. A genetic effect is suggested when
the concordance rate in identical twins exceeds that in nonidentical
twins. This is the case for all the diseases listed in Table 1, where
identical twins are more often concordant for the disease than are
Table 1. Concordance rates in identical and nonidentical twin pairs in population-based studies of
immune-mediated diseasesa
Disease
MS
RA
IDDM
SLE
Identical twin pairs
(%)
Nonidentical twin pairs
(%)
Refs
26.7
12.3
13
33
3.5
3.5
2.5
0
3
4
5
6
Abbreviations: IDDM, insulin-dependent diabetes mellitus; MS, multiple
sclerosis; RA, rheumatoid arthritis; SLE, systemic lupus erythematosus.
a
The concordance rates are consistently less than 100% and are higher in
identical than nonidentical pairs.
nonidentical twins, indicating that genetic factors are important. The
concordance rates in nonidentical twins are less than 50% of those of
identical twins, suggesting a polygenic model for these major
immune-mediated diseases (in the case of a single dominant gene,
the risk for nonidentical twins would be half that of identical twins;
with increasing numbers of genes, the difference between identical
and nonidentical twins would be increasingly higher). However,
even infectious diseases such as polio or tuberculosis show differences in identical and nonidentical twin pair concordance rates (36%
and 6% respectively for polio8, and 51% and 26% respectively for
tuberculosis9). Taken together, these studies are consistent with
genetic susceptibility influencing the appearance of clinical disease
even when environmental factors play a major role in causing the
disease. This conclusion is further strengthened by migrant studies10. Children with a Sardinian heritage maintain the same high incidence of IDDM abroad as the indigenous Sardinian population, even
if born in a region of low disease incidence.
The impact of HLA genes on autoimmune disease
An association between major histocompatibility complex (MHC)
HLA type and disease has been noted in many autoimmune conditions. The relative contribution of the HLA locus to disease can
be estimated by comparing concordance rates in HLA-identical but
dizygotic twins (or siblings) with that in monozygotic twins.
Alternatively, the impact of a gene on the development of a disease
(penetrance) can be estimated by comparing susceptibility genes
in identical twin pairs who are discordant and concordant for the
disease.
AS is strongly associated with HLA-B27. In one study, 6/8 (75%)
HLA-B271 identical twin pairs were concordant for the disease, as
compared with only 4/32 (12.5%) nonidentical twins and 4/15 (27%)
HLA-B271 nonidentical twin pairs11. No nonidentical twin pairs
have been reported to be concordant for AS but discordant for
HLA-B27. These observations suggest that HLA-B27 is almost essential to the development of AS but that other genetic, as well as
environmental, factors determine which HLA-B271 individuals will
manifest the disease.
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A similar suggestion can be drawn from twin studies in RA, as
demonstrated by the importance of the HLA-DR shared epitope for
disease concordance and the gene-dose effect in RA susceptibility12,13. The potential of twin studies to define the role of HLA and
non-HLA genes has not been exploited in other immune-mediated
diseases, the main problem being the limited numbers of twin pairs
available for study. Developments in population-based twin ascertainment allied to the identification of those non-HLA genes that are
probably involved in disease susceptibility could lead to the wider
application of twin studies in genome research.
Limitations of twin studies in assessing genetic impact on disease
There are other problems with assessing the genetic contribution to
disease using the twin study method. First, identical twins may have
a more similar environment both in utero and in childhood, which
would lead to an over-estimate of heritability. Second, in contrast to
nonidentical twins, identical twins are always the same sex. Many
autoimmune diseases have a clear sex bias; for example, RA, MS,
SLE and autoimmune thyroid disease are more common in females.
Third, twin studies are hindered by potential biases in ascertainment. In the traditional ‘clinic-based’ approach, identical twin pairs
concordant for a disease, or with severe disease, are more likely to
be identified. In the ‘population-based’ approach, individuals are
identified first as twins and are then assessed for illness. These
population-based studies must ascertain large numbers of twin pairs
to detect sufficient numbers of affected twins. Finally, most twin
studies have been cross-sectional, but, by following twins for a
longer period, it might be possible to detect a higher concordance
rate3.
Immunological studies
Humoral immune responses
Identical twins show a similar genetic regulation of the production
of antibodies, even when they are reared apart14. A recent study confirmed that such a genetic control remains largely unaltered by chronic
autoimmune stimulation14. Moreover, genetic factors can regulate
the level and extent of antibody production. Autoantibodies are correlated with certain HLA types (e.g. HLA-DR4 and insulin autoantibodies16 or rheumatoid factor17). A study of IgM and IgG rheumatoid
factor isotypes in 70 identical and 84 nonidentical twins discordant
for RA concluded that genetic factors were important in determining
the levels and frequency of these isotypes; for example, IgM and IgG
positivity was higher in healthy identical than healthy nonidentical
twins18. In agreement with such a genetic effect, combinations of
IDDM-associated autoantibodies were more often found in healthy
identical than healthy nonidentical twins of IDDM patients19.
Other studies in immune-mediated diseases highlight differences
between healthy and diseased co-twins, implying post-zygotic,
epigenetic or environmental factors influencing the induction of
autoantibodies. In HLA-DR41 identical twin pairs discordant for
RA, the twins with RA had higher titres of antibodies towards
proteins containing QKRAA, an amino acid sequence in the third
hypervariable region common to the HLA-DRb1 alleles associated
with the disease20. In IDDM, disease-discordant identical twins are
often also discordant for disease-associated autoantibodies. Moreover, autoantibodies are as often detected in healthy identical as nonidentical twins of patients with an autoimmune disease, consistent
with a common environmental effect; for example, IDDM-associated
autoantibodies were detected in 6/18 identical twins and 6/18
nonidentical twins of IDDM patients19.
Cellular immune responses
Cellular immune responses are under strong genetic influence.
However, the peripheral TCR repertoire is shaped not only by the
availability of germline receptor elements, but also by their
rearrangement, thymic selection and clonal stimulation. Studies in
identical twins may help define the relative importance of genetic
and epigenetic factors on the shaping of the healthy (Table 2) and
diseased (Table 3) immune repertoire.
Concerning the shaping of the healthy TCR repertoire, shared
complementarity-determining region 3 (CDR3) motifs could be
identified between healthy identical twins in the response to epitopes of myelin basic protein (MBP) and of Mycobacterium bovis
65 kDa heat shock protein21. Similarly, the Jb repertoires of identical
twins were found to be more similar than those of unrelated individuals22. Although shared epigenetic factors cannot be ruled out, these
results may be interpreted as suggesting a predominant genetic
effect. However, they contrast with a later report, again in healthy
identical twins, where a common TCRVa and/or TCRVb gene usage
for shared epitope recognition by MBP-specific T-cell lines was infrequent and there was no significant intrapair concordance, stressing
the importance of epigenetic events23. In accordance with this view,
epitope recognition of MBP-specific T-cell lines in healthy identical
Table 2. Influence of epigenetic factors on the immune repertoire of healthy individuals
Condition
Influence on immune repertoire
No influence on immune repertoire
Healthy identical twins
Diverse TCRVa and/or TCRVb gene usage in the response
to shared T-cell epitopes23
Diverse patterns of epitope recognition24
Shared CDR3 in the response to T-cell epitopes21
Similar Jb repertoires22
Same Va usage in response to antigens28
Abbreviations: CDR, complementarity-determining region; J, joining; TCR, T-cell receptor; V, variable.
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twins24 was distinct in 6/8 pairs, and there was a complete absence
of concordant epitope recognition within two other pairs.
In immune-mediated diseases, the resting peripheral T-cell Vb
repertoire appears to be virtually identical in identical twins irrespective of discordance for IDDM (Ref. 25) or RA (Ref. 26). Similarly,
analysis of six identical twin pairs discordant for RA showed that
their Jb gene repertoires were more similar than those of unrelated
individuals, and Jb expression was not influenced by RA (Ref. 22).
Other studies showed differences in immune repertoire between
twin pairs discordant for RA: one suggested that there may be differences in the TCR Vg repertoire26; another showed RA-associated
alterations of the TCRBV-BJ combination repertoire in the affected
co-twin27. Evidence for disease-associated skewing of the TCR repertoire has also come from studies on the putative antigen in MS
(MBP): identical twins can show differences in TCR Va usage after
repeated stimulation either with MBP or tetanus toxoid28.
Overall, the above data suggest that the gene expression of TCRs
by peripheral lymphocytes is largely regulated by genetic factors.
However, alterations in the TCR repertoire may occur both in
healthy identical twins and in the affected co-twin of pairs discordant for immune-mediated disease. Post-zygotic DNA changes,
epigenetic events or environmental factors may account for such
discordance in the TCR gene usage.
Disappointingly, twin studies have failed to identify diseaserelated epitopes. Characterization of proliferative and cytotoxic
responses, as well as the epitope specificity and HLA restriction of a
large number (.600) of MBP-specific T-cell lines from six twin pairs,
three discordant and three concordant for MS, failed to detect any
difference that could be attributed to the disease29. Those peptide
epitopes of MBP recognized most frequently by the T-cell lines were
already known from previous studies of MS to be immunodominant.
Perhaps a similar study, performed serially over a two- or three-year
period, will be able to clarify whether responses that remain stable
over time differ between affected and non-affected twins.
It is possible that the critical difference between identical twins
discordant for an autoimmune disease lies not in the T-cell epitope
recognition but in the functional response of the cell. The frequency
and functional characteristics of CD42CD82Va24JaQ2 T cells [which
might be important in regulating the T helper 1 (Th1)-cell-mediated
tissue damage in IDDM] were studied in siblings and identical twins
with IDDM. Clones from diabetic patients secreted only interferon g
(IFN-g) on stimulation30. By contrast, nearly all the clones from
normal subjects, and even the nondiabetic identical twins of IDDM
patients, secreted both interleukin 4 (IL-4) and IFN-g. Thus, it was the
loss of the capacity of these Va24JaQ T cells to secrete IL-4 that was
associated with the presence of IDDM. Since identical twins were
discordant for this feature, it is unlikely that the change is genetically
determined. It remains to be established whether the Th1 bias of
these T cells is a consequence of the disease or predisposes to the
condition. As with T-cell studies of epitope recognition, the potential
of twins in the study of T-cell function is yet to be realized.
Predicting disease
Since the healthy identical twin of patients with an autoimmune disease is at high risk of developing the same disease, it has been possible to study twins before the onset of clinical disease. Evidence that
IDDM results from an environmental event operating in early childhood derives in part from such twin studies31. Indeed, twin studies
first revealed the chronic nature of the autoimmune process in the
prediabetic period32. These studies showed immune changes,
including disease-associated antibodies in peripheral blood, months
and even years before the clinical onset of diabetes. The immune
changes were accompanied by a progressive decline in insulin secretory capacity, consistent with a gradual and progressive destruction
of the insulin-secreting islet cells. A recent study suggested that the
variation in the age at clinical onset of IDDM and, by implication, the
disease incubation period following the early induction of autoimmunity, is strongly genetically influenced7. The age of onset in 116
identical twin pairs concordant for IDDM was strikingly correlated
(correlation coefficient 0.94) and the correlation for age at diagnosis
was higher in identical than nonidentical twins.
Extensive twin studies of the co-twins of patients with IDDM
have revealed numerous disease-related immune changes with variable predictive power. Changes that increase the risk of developing
IDDM in such twins include autoantibodies to islet antigens, activation of T cells (particularly CD81 T cells), increased expression of
CD45RA (a marker of naive T cells), increased serum levels of macrophage-derived cytokines, impaired glucose tolerance and decreased
insulin response to intravenous glucose challenge33–36. The positive
predictive value of combinations of disease-related autoantibodies is
as high as 100% (Ref. 18). In broad terms, the predictive power of
immune changes in identical twins has been higher than that in
siblings, giving us an indicator of the potential of combining genetic
Table 3. Influence of epigenetic factors on the immune repertoire of individuals with immune-mediated
diseases
Condition
IDDM-discordant identical twins
RA-discordant identical twins
MS-discordant identical twins
Influence on immune repertoire
repertoire26
Different T-cell Vg
Different TCRBV-BJ repertoire27
Different Va usage in response to antigens28
No influence on immune repertoire
Identical T-cell Vb repertoire25
Identical T-cell Vb repertoire26
Jb expression not influenced by RA22
Abbreviations: IDDM, insulin-dependent diabetes mellitus; MS, multiple sclerosis; RA, rheumatoid arthritis; for other abbreviations, see footnote to Table 2.
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and immune markers in predicting IDDM in both family and population studies. However, in twins of IDDM patients, not all immune,
and even metabolic, changes inevitably result in diabetes. Thus,
increased levels of activated T cells had a positive predictive value of
only 60%, a decrease in the first-phase insulin response to intravenous
glucose gave a positive predictive value of 58% and impaired glucose
tolerance only 33% (Refs 33–36).
Owing to the small numbers of twins available for study, it
has been difficult to extend these observations in twins of IDDM
patients to other autoimmune diseases. In MS, cerebrospinal fluid
immunoglobulin abnormalities37 (oligoclonal bands), magnetic resonance38,39 and visual evoked response alterations39 (i.e. subclinical
defects of the optic pathways that are detectable through electrophysiological techniques) are frequently detected in the healthy cotwin. While the predictive value of such abnormalities is still uncertain, these studies, taken together, suggest that the pathological
process associated with autoimmune conditions encompasses a
spectrum of immune changes and target organ damage that do not
inevitably lead to clinical disease. Moreover, the identification of a
long prodrome before the onset of clinical symptoms in IDDM raises
the possibility that we can predict, and possibly prevent, many of
these diseases.
Future developments
If the potential of twin studies is to be realized, a spirit of close collaboration must exist between clinicians and scientists, including
epidemiologists, immunologists and geneticists. To date, twin studies have provided a patchy outline of immune responses associated
with autoimmune diseases, usually in limited numbers of twin pairs
with little or no prospective analysis. Important issues that twin
studies could resolve, such as the genetic impact on disease severity
(to explain the variation between patients for arthritis, neurological
deficit or vascular damage), have not been addressed. To improve
epidemiological and immunological twin research it will be necessary to increase the numbers of pairs under study. This can be attained
through the construction of population-based twin registries. Scandinavian countries have a long-standing tradition in this respect and
similar registries are now being assembled in other countries40. The
creation of centralized facilities for the storage of large amounts of
frozen peripheral blood mononuclear cells and sera should be an
early goal in establishing these registries, considering also that this
would increase the chances of sampling the material from subjects
free of any therapy.
Recent technological progress highlights the potential of twin
studies: a prelude to these developments has been the successful
application of techniques such as the differential display of thousands of genes to quantify gene expression; for example, in the study
of identical twins discordant for MS (Ref. 41). This analysis has led
to the detection of a deficient expression of the inhibitory transcription factor Sp3 in mononuclear blood cells of the affected co-twin; the
defect was confirmed by comparing a series of MS patients with
control subjects. This study anticipates what might be attained in
the near future by employing new and more powerful ‘functional
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genomics’ techniques such as microarray analysis. As this technology allows the simultaneous monitoring of hundreds and thousands
of genes at their expression level, through the quantitative measurement of the amount of mRNA transcripts, it is well suited for
investigating complex diseases. Early applications of the microarray-based approach to the study of human autoimmune disorders,
namely RA and inflammatory bowel disease, have been particularly
promising. Not only has it allowed identification of novel genes
associated with the disease but, more importantly, it has identified
distinct patterns of gene expression42. This recognition of distinct
gene expression profiles is of particular interest; for instance, using
DNA microarrays, Alizadeh and colleagues were able to identify
two molecularly distinct forms of diffuse large B-cell lymphoma that
had gene expression patterns indicative of different stages of B-cell
differentiation43. Cluster analysis of gene expression patterns thus
provides a molecular classification of tumours, revealing previously
undetected subtypes of cancer that are clinically significant44,45. The
recognition of specific features of histological and physiological variation in tumour samples using microarrays suggests that this will
be a potent tool to dissect the heterogeneity and complexity of
multifactorial disorders.
It may be an arduous task to discern differences in mRNA expression that are pathogenetically relevant and distinct from those resulting simply from the many different genetic backgrounds in an
outbred population. Performing such studies on identical twins discordant and concordant for a disease could overcome this problem.
Since microarray analysis is a relative of differential display in the
functional genomics family of techniques, its application in twins
should be possible – the more so with the imminent availability of
gene sequences.
Twin studies might have other potential applications in the
assessment of aspects of potential relevance in the pathogenesis of
autoimmune diseases. Possible precipitating factors of autoimmune
conditions that await definition include epigenetic effects due to
DNA or protein modifications (methylation patterns or acetylation
of histones), the accumulation of somatic mitochondrial DNA mutations, X chromosome inactivation, and the presence of transcriptionally active endogenous retroviral sequences.
Research has come a long way since Francis Galton conceived the
potential of twin studies to detect genetic traits. Today, it is possible
to define genetic and epigenetic features of a disease using twin studies. New technologies are knocking at the door, and once we have
overcome the limited number of twins available for study, the full
power of twin studies should be realized.
The authors are supported by the British Diabetic Association, the Diabetic
Twin Research Trust, the Istituto Superiore di Sanità and the Fondazione Italiana
Sclerosi Multipla. We thank R. Tosi for his critical review of the manuscript.
Marco Salvetti, Giovanni Ristori and Roberto Bomprezzi are at
the Dept of Neurosciences of the University of Rome ‘La Sapienza’, 00185
Rome, Italy; Paolo Pozzilli ([email protected]) is at the University
Campus Biomedico, 00100 Rome, Italy and at the Dept of Diabetes
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and Metabolism, St Bartholomew’s Hospital and Medical College, London,
UK EC1A 7BE; R. David G. Leslie is at the Dept of Diabetes and
Metabolism, St Bartholomew’s Hospital and Medical College, London, UK
EC1A 7BE.
22
23
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