Genetics of
Hashimoto's Thyroiditis
Evidence for genetic susceptibility to
Hashimoto's thyroiditis
Abundant epidemiologic data (population-based and
family-based studies, twin studies) suggest a strong genetic contribution to
the development of Hashimoto disease. The disease clusters in families [22,73].
Thyroid abnormalities with clinical outcomes were observed in 33% of
offspring of patients with Hashimoto disease or
Graves' Disease [73]. The sibling
risk ratio (λS), that is the ratio of
the prevalence of disease in siblings to the prevalence in the general
population, can be used as a quantitative measure of the genetic
contribution to the disease. Usually, a λS
of more than five indicate a significant genetic contribution to the disease
development. Based on historical data, the λS
for AITD (autoimmune thyroid disease) is estimated to be greater than 10, supporting a strong case of
genetic influence on disease development [74]. Using
Hashimoto disease
prevalence data from the NHAHES III study, an estimated
λS value is about 28 for Hashimoto's Thyroiditis [74].
In Danish twin study, the concordance rates for
Hashimoto's
disease were 38% for monozygotic (MZ) twins and 0 for dizygotic (DZ) twins [75].
For Hashimoto's Thyroiditis, a recent twin study in California confirmed these results, showing
concordance rates of 55% and 0% in MZ and DZ twins, respectively [76].
For thyroid antibodies, the concordance rate in the Danish twin study was
twice high in MZ twins (80%) than that in DZ twins [75].
In a recent twin study in the UK, the concordance rates for Tg-antibodies
were 59% and 23% in in MZ and DZ twins, respectively [77].
In this study, the concordance rates for TPO-autoantibodies were 47% and 29%
in MZ and DZ twins, respectively [77]. These data suggest
that Hashimoto's Thyroiditis and other Autoimmune Throid Disease outcomes such as antibody production against
thyroid-specific antigens have a substantial inherited susceptibility. Hashimoto
disease
seems to be a polygenic disease with a complex mode of inheritance. Immunomodulatory genes are expected to play an important role in
predisposing and modulating the pathogenesis of Hashimoto's thyroiditis.
It is recommended that Transfer
Factor Advanced Formula to be used in autoimmune conditions.
Transfer Factor Plus is generally preferred for
conditions caused by infection. Transfer Factors
suppress over acting immune system
to ease autoimmune conditions.
Animal Models of Autoimmune Thyroiditis
Animal models of AITD
(autoimmune thyroid disease) still hold immense promise for the
discovery of pathways, genes and environmental factors that determine the
development of thyroid autoimmunity. Animals affected by experimental
autoimmune thyroiditis (EAT) provide a unique opportunity to uncover
disease-associated pathways, which are complicated to define in man.
One of the oldest inbred models is the obese strain chicken
(OS), which develops goitrous lympholytic thyroiditis with the subsequent
atrophic lympholytic thyroiditis followed by a rapid onset of
hypothyroidism
[78]. The biobreeding diabetes-prone (BB-DP) rat
expresses a form of focal lympholytic thyroiditis that under normal
conditions does not lead to hypothyroidism [79]. The
nonobese diabetes (NOD) mouse strain NOD-H2h4 spontaneously
develops iodine-induced autoimmune thyroiditis but not
diabetes [26].
In particular, this murine strain has been extensively used to evaluate the
role of iodine in the development of autoimmune thyroiditis [16].
EAT can be induced in mice by injecting with murine or
human Tg, [80] and in normal syngenic recipients it is
induced by the adoptive transfer of in vitro activated T cells from
Tg-immunized mice [81]. The induced disease is
characterized by the production of murine Tg-specific antibodies and
infiltration of the thyroid by lymphocytes and other monocytes, with murine
or human Tg-specific CD4+ T cells as the primary effector cells [80,82].
Clinical features of EAT induced in the animal models
mentioned above are similar to those of human Hashimoto thyroiditis. For example, autoimmune thyroiditis in the NOD-H2h4 mouse is induced by dietary iodine
that supports epidemiologic data on human populations. In addition, the
iodinified mouse represents high levels of IgG2b that is similar to Hashimoto thyroiditis
patients expressing the predominance of IgG2 subclass, the human analog of murine IgG2b [83]. IgM class generally restricts Tg-antibodies
of normal individuals and mice, while Hashimoto thyroiditis individuals and affected mice
commonly produce Tg-antibodies of the IgG isotype [17].
However, anti-TPO antibodies generally detectable in Hashimoto thyroiditis patients could not
be found in NOD-H2h4 mice. Despite some differences between EAT
and Hashimoto thyroiditis, these animal models have greatly contributed to the knowledge
concerning the etiology and the pathogenesis of thyroid autoimmunity, most
notably on the events occurring in the very early prodromal phases.
Major Histocompatibility Complex (MHC) molecules are
thought to play an important role in the initial stages of the development
of Hashimoto thyroiditis and AITD. MHC molecules, or Human Leukocyte Antigen (HLA) homologs,
play a pivotal role in T-cell repertoire selection in the thymus and in
antigen presentation in the periphery. Crystal structures of MHC molecules
show a peptide-binding cleft containing the variable region of these
molecules. Genetic polymorphism of the MHC molecule determines the
specificity and affinity of peptide binding and T-cell recognition.
Therefore, polymorphisms within MHC class I and class II loci can play a
significant role in predisposition to autoimmune disease [84].
A role of selected HLA class II genes susceptible to
Hashimoto disease has
been significantly clarified using transgenic NOD (H2Ag7) class
II-knockout mice with EAT as a model for Hashimoto thyroiditis [85,86].
In mouse genome, the H2 class II locus is homologous to the human HLA class
II region [51]. A role for HLA-DRB1 polymorphism as a
determining factor in Hashimoto's Thyroiditis-susceptibility, with DR3-directed predisposition
and DR2-mediated resistance to the disease, was demonstrated using H2 class
II-negative mice injected with HLA-DRA/DRB1*0301 (DR3) and HLA-DRB1*1502
(DR2) transgenes [85]. A role for HLA-DQ polymorphism was
shown with human thyroglobulin-induced EAT in HLA-DQ*0301/DQB1*0302 (DQ8),
but not HLA-DQ*0103/DQB1*0601 (DQ6), transgenic mice [52].
In summary, DR3 and DQ8 alleles are found to be susceptible, whereas DR2,
DR4 and DQ6 alleles are resistant [30,87].
Studies on EAT-developing mice showed the differential effects of class II
molecules on EAT induction. Susceptibility can be determined when class II
molecules from a single locus, H2A or HLA-DQ, are examined in transgenic
mice, but the overall effect may depend upon the presence of both class II
molecules H2A and H2E in mice and HLA-DQ and HLA-DR in humans [88].
Polymorphism within DQ alleles can determine predisposition to Hashimoto's
thyroiditis while DRB1
molecules associated with susceptibility to Hashimoto's thyroiditis may appear to play a
permissive role. The combination of susceptibility-inducing HLA-DQ and
permissive DR alleles is responsible for the association of the HLA class II
region with the disease.
T cells recognize an antigenic peptide via interaction of
their membrane T cell receptors (TcR) with antigen-MHC complexes presented
on the surface of APC. Biased or restricted TcR gene use has been reported
in a variety of human or murine autoimmune diseases [89].
Biased TcR V gene in intrathyroidal T cells was also observed in mice with
spontatenous (NOD strain) or human Tg-induced (CBA/J strain) thyroiditis.
This confirms the primary role played by T cells in initiating EAT and the
phenomenon of oligoclonal expansion of intrathyroidal T lymphocytes in early
thyroiditis [90]. Sequencing of amplified TCR V beta cDNA
showed that within each NOD thyroid sample at least one of the overexpressed
V beta gene families was clonally expanded. For example, in the CBA/J mouse
immunized with human Tg, clonally expressed T cells were shown to primarily
express the murine TcR Vβ1 and Vβ13
sequences [91].
A new murine model that developed destructive thyroiditis
with histological and clinical features comparable with human Hashimoto's thyroiditis has been
recently reported [92]. The transgenic mice express the
TcR of the self-reactive T-cell clone derived from a patient with autoimmune
thyroiditis. The T-cell clone is specific for the autoantigen thyroid
peroxidase (TPO) peptide comprising amino acid residues at positions 535-551
(TPO535-551) of the TPO amino acid sequence. This includes a
cryptic epitope (TPO536-547) preferentially displayed after
endogenous processing during inflammation [93]. These
results underline the pathogenic role of autoreactive human T cells and the
potential significance of recognition of cryptic epitopes in target
molecules such as TPO for inducing thyroid-specific autoimmune response.
The two-signal theory for T cell activation requires TcR
engagement of its cognate antigen-MHC complex and CD28 binding to B7 ligands
(B7-1 and B7-2) on APC. Activation of T cells results in increased
expression of the cytotoxic T cell antigen-4 (CTLA-4) molecule that shares
homology with CD28. Although B7-1 (CD80) and B7-2 (CD86) expressed on APC
can bind to both CD28 and CTLA-4 (CD152), because of higher affinity, they
preferentially bind to CTLA-4 on activated T cells and attenuate the T cell
response [94].
The importance of CTLA-4 in the down-regulation of T cell
responses and in the induction of anergy and tolerance to alloantigens,
tumors and pathogens, has been clearly demonstrated in experiments with
CTLA-4 deficient mice. The mice developed a severe inflammatory disorder due
to up-regulated proliferation of T cells [95,96].
CTLA-4 can down-regulate T cell responses involving binding and sequestering
B7 molecules from CD28, therefore preventing CD28-mediated co-stimulation.
Another possibility is that CTLA-4 through its intracellular domain could
actively transmit a negative signal resulting in down-regulation of
activated T cells [97]. The crucial role of CTLA-4 in
maintaining self-tolerance breakdown of which leads to the initiatition of a
primary autoimmune response has been demonstrated in several murine models
of autoimmune diabetes [98] and autoimmune thyroiditis [32].
It is recommended that Transfer
Factor Advanced Formula to be used in autoimmune conditions.
Transfer Factor Plus is generally preferred for
conditions caused by infection. Transfer Factors
suppress over acting immune system
to ease autoimmune conditions.
Human Leukocyte Antigen Class I and II
Genes
Genes of the human MHC region are clustered on chromosome
6p21 and encode HLA glycoproteins and a number of additional proteins, which
are predominantly related to immune response. The MHC locus itself contains
three groups of genes: class I genes encoding HLA antigens A, B and C, class
II genes encoding HLA-DR, DP and DQ molecules and class III genes [99].
Previous studies in the early 1980s investigated the HLA
locus in relation to the genetics of Hashimoto disease. Associations between HLA and
Hashimoto
disease
have both been analysed by serologic typing of HLA and DNA typing using
sequence-specific oligonucleotide probe analysis or restriction fragment
length polymorphism. In Asians, HLA class I (A2, B16, B35, B46, B51, B54,
C3) and HLA class II (DR2, DR9, DR53, DQ4) genes showed an association with
the disease [31,100-105]. In
Caucasians, Hashimoto's thyroiditis is associated with HLA class II genes such as DR3, DR4, DR5,
DQA1*0301, DQB1*0201 and DQB1*0301 [106-120] but not
with the HLA-DP and HLA class I (HLA-A, HLA-B and HLA-C) genes [113,114,121].
However, some studies could not reveal an association between HLA-DQ and DR
genes and Hashimoto thyroiditis [114,122,123].
Reports of disease-associated alleles are not consistent, but associations
appear to be strongest with alleles in the HLA-DR and -DQ loci. This has
also been suggested by studies in transgenic mouse [30,52,85-87].
Early linkage, non-genome-wide studies of the HLA region
have failed to detect linkage between the HLA locus and Hashimoto's
thyroiditis [124-129].
Using dataset of 56 US Caucasian multigenerational families, genome-wide
scans has revealed a susceptibility locus AITD-1 located on chromosome 6p [130].
The AITD-1 locus is common for both general forms of thyroid autoimmunity,
Hashimoto's thyroiditis and Grave's
disease [130]. This locus was replicated in the
expanded dataset of 102 US Caucasian families but is distinct from the HLA
gene cluster [131]. Whole-genome scans of a large family
with members affected with vitiligo and Hashimoto's thyroiditis mapped a Hashimoto
thyroiditis susceptibility locus
that shared both the MHC region and the non-MHC AITD-1 [132].
However, evidence for linkage between the HLA locus and Hashimoto disease (or autoimmune
thyroid disease) has not been confirmed by further whole-genome scans of
other affected families [133,134],
sibling pairs [135], or within HLA-DR3 positive families
[120]. The lack of linkage means, for instance, the DR3
gene did not cause the familial segregation of Hashimoto's disease while a
relatively strong and consistent association showed that HLA-DR3 conferred a
generalized increased risk of Hashimoto's thyroiditis in the general population. These data did
not support a major role for the HLA region in the susceptibility to Hashimoto
disease and
may imply that the DR3 gene modulates the effect of other non-HLA
susceptibility gene.
However, a linkage between the HLA region and
Hashimoto's thyroiditis was
recently shown in the data set of 40 US multiplex families affected with AITD
(autoimmune thyroid disease) and type 1 diabetes [136]. The linkage to Hashimoto
disease was
found to be weaker than to diabetes, suggesting that additional, non-HLA
loci were contributing to the joint susceptibility to AITD and T1D. Among
HLA-DR alleles, HLA-DR3 was detected as the only associated gene for
Hashimoto's thyroiditis and diabetes [136]. Indeed, DR3
seems to represent the major HLA allele, which contributes to the shared
susceptibility to T1D and AITD. These findings, however, need to be
replicated in larger data sets because early family [137,138]
and case-control [139,140] studies
have not shown the unique role for HLA-DR3 allele in conferring shared
susceptibility to T1D and thyroid autoimmunity.
The HLA region has been established to be involved in
multiple autoimmune disorders [141]. The mechanisms by
which HLA molecules influence the susceptibility to autoimmune disorders
become more and more clear. Different HLA alleles could have different
affinities to autoantigenic peptides. Therefore, certain alleles can bind
the autoantigenic peptide, with the subsequent recognition by T cells that
have escaped self-tolerance, whereas others may not [142].
The possibility of certain class II alleles to bind and present
thyroid-specific antigens such as TSHR or Tg peptides has been shown in
vitro [143] and in mice with EAT [144].
Thyroid autoantigens need to occur in the thyroid or its
draining lymph nodes in order for them to be presented by HLA molecules. It
has been suggested that an aberrant intrathyroidal expression of MHC class
II molecules by thyrocytes is necessary to initiate thyroid autoimmunity [145,146].
This hypothesis is supported by detection of the expression of HLA class II
molecules by thyroid epithelial cells in Hashimoto thyroiditis and
Graves' disease patients [147,148]
and in studies on animal models with experimentally induced thyroid
autoimmunity [85,145,149,150].
The aberrant expression of HLA class II antigen by thyrocytes can initiate
autoimmune responses through direct thyroid self-antigen presentation or a
secondary event following on from cytokine secretion by infiltrated T
lymphocytes [148,151].
Genetic contribution of HLA varies depending on the
disease. HLA involvement in T1D, rheumatoid arthritis or
multiple sclerosis
is large and can constitute more than 50% of the genetic risk [84,152].
Contributions of HLA alleles as genetic risk factors to Hashimoto disease are much weaker [118,153].
HLA class I and II genes appear to contribute to the autoimmunity in general
but not to organ specificity. Their role in the predisposition to Hashimoto's thyroiditis is
rather non-specific [62,117]. The HLA
class I and II genes appear not to be the primary Hashimoto's thyroiditis genes, and are likely
to be modulating genes that increase the risk for AITD contribution by other
genes. HLA class III and other non-HLA genes, located in the HLA region, are
also critical to the immune response. It is possible that HLA associations
as seen in thyroid autoimmunity are due partially to genetic variation in
these closely linked immune regulatory genes and their linkage
disequilibrium with class I and II genes [154].
It is recommended that Transfer
Factor Advanced Formula to be used in autoimmune conditions.
Transfer Factor Plus is generally preferred for
conditions caused by infection. Transfer Factors
suppress over acting immune system
to ease autoimmune conditions.
HLA class III genes and non-HLA genes of
the HLA region
The HLA class III region lies between class I and II genes
and encodes important immunoregulatory proteins such as cytokines [tumour
necrosis factor (TNF), lymphotoxin alpha (LT-α)
and beta (LT-β)], complement components (C2, C4,
properdin factor B) and heat shock proteins (HSP) [155].
Both TNF and LT-α mediate B-cell proliferation
and humoral immune responses [154]. TNF has been found
to enhance cellular expression of HLA class I and II antigens, and enhances
adhesion and complement regulatory molecules in the thyroid gland of Hashimoto
disease
patients. Alterations to the above could promote the autoimmune process [156].
However, case-control studies showed no association between polymorphisms
within the TNF and LT-α genes and Hashimoto
disease in Germans
[112], UK Caucasians [118] and
Koreans [157].
HSP70 gene cluster consists of three genes encoding
HSP70-1, HSP70-2 and HSP-Hom proteins. They are expressed in response to
heat shock and a variety of other stress stimuli (e.g. oxidative free
radicals, toxic metal ions and metabolic stress). HSPs are also important
for antigen processing and presentation [158]. Genetic
variations within all three HSP70 genes were tested in British patients with
Hashimoto disease and no associations were found [118]. Polymorphisms
of complement component-encoding genes have not yet been evaluated in
relation to Hashimoto's thyroiditis. Meanwhile, finding a link between frequency disturbances in
BI and C4A allotypes and one of the forms of thyroid autoimmunity,
postpartum thyroiditis [159], may be an intriguing
future study in Hashimoto disease patients.
Other genes crucial to the
immune response, including TAP
(transporters associated with antigen processing), LMP (large
multifunctional protease), DMA and DMB genes are located within the HLA
class II region [155]. Protein products of TAP (TAP1 and
TAP2) and LMP2 (LMP2 and LMP7) genes participate in the proteolysis of
endogenous cytoplasmic proteins into small fragments and subsequent
transportation of these self-peptides from the cytoplasm into the
endoplasmic reticulum, the site of HLA class I assembly [160].
To date, one investigation has been concerned with the association between
TAP1 and TAP2 genes and Hashimoto's thyroiditis. No significant association
was observed in the British population [118]. The
genetic role of LMP in Hashimoto disease has not yet been examined. An association between
the R60 allele of the LMP2 gene and
Graves' disease was observed [161].
Additionally, quantitative defects in the amount of transcription products
of TAP1, TAP2, LMP2 and LMP7 genes were found in lymphocytes of patients
with AITD (autoimmune thyroid disease) [160]. These findings suggest that defective
transcription of HLA class I-processing genes could contribute to the
quantitative defect in cell-surface expression in autoimmune lymphocytes in
Hashimoto disease. Further evaluation of the role of such class I-processing genes as TAP
and LMP is necessary.
DMA and DMB genes are involved in the assembly of HLA class
II peptides. These genes encode subunits of a functional heterodimer that is
critical for class II antigen presentation [160,162].
Based on nucleotide variation within exon 3, three rare DMB alleles (DMB*01kv1,
DMB*01kv2 and DMB*01kv3) have been detected in Korean Hashimoto thyroiditis patients while
these DMB variants have not been found in healthy subjects [163].
However, these DMB alleles have not yet been functionally characterized. In
summary, there is a significant dearth of information on how HLA class III
genes and non-HLA genes, located in the HLA region, contribute to the
pathogenesis of Hashimoto's thyroiditis. Further studies are required to clarify the involvement
of these genes in Hashimoto diseadse susceptibility.
CTLA-4 gene
The CTLA-4 gene is the most frequently studied of the
immune modulatory genes located outside the HLA region, in relation to the
genetics of Hashimoto disease. This gene encodes a costimulatory molecule, which suppresses
T-mediated immune response and is crucial in the maintenance of peripheral
immunological self-tolerance [164]. An inventory of
case-control studies based on the association between three polymorphic
markers within the CTLA-4 gene [A49G dimorphism in the leader peptide, C
(-318) T substitution in the promoter region and a dinucleotide repeat
polymorphism at the 3'-untranslated region (3'-UTR)] and Hashimoto's
thyroiditis is reviewed in [165].
Results of these studies, except for those for the C (-318) T single
nucleotide polymorphism, suggest that polymorphisms within the CTLA-4 gene
are associated with the development of Hashimoto's thyroiditis.
Family studies showed linkage between CTLA-4 and
Graves' disease [166],
thyroid antibody production [167] and autoimmune thyroid
disease [12,62] but not specifically
to Hashimoto's thyroiditis, probably due to lack of their power [129,130,135].
Classical linkage analysis is suitable for detecting susceptibility loci
with major genetic effects. CTLA-4 demonstrates a modest but significant
effect in the genetics of Hashimoto disease. To detect a locus with a modest genetic
effect, a large number (at least 400) of affected families should be tested
[168].
This investigation has recently been performed involving
about 600 AITD (autoimmune thyroid disease) families and more than 1300 affected patients [169].
The CTLA-4 gene has been found to play a critical role in the pathogenesis
of autoimmune diseases such as
Graves' disease, Hashimoto disease and T1D [62,153,169].
Disease susceptibility was mapped in the 6.1-kb 3' untranslating region of
CTLA-4. Allelic variation was correlated to altered mRNA levels of soluble
form of CTLA-4 [169]. This alternative splice form of
CTLA-4 lacks exon 3 encoding the transmembrane domain but maintains exon 2
encoding the ligand-binding domain [170]. The short form
of CTLA-4 can bind CD80/86 and inhibit T-cell proliferation [171].
The soluble CTLA-4 (sCTLA-4) is expressed constitutively by T regulatory
cells suppressing the effector T-cell response [172].
Its role in autoimmune disease is not exactly clear, but sCTLA-4 was
observed significantly more often in patients with AITD [173]
and myasthenia gravis [174] in comparison with
non-affected subjects. Patients with AITD and myasthenia gravis had an
aberrant expression of the CTLA-4 products, with high levels of sCTLA-4 and
low levels of the intracellular form [175]. Soluble
CTLA-4 might play an important role in immune regulation by binding with the
B7 molecules, thus interfering with the binding of CD28 and/or full-length
CTLA-4. Interference of sCTLA-4 with B7/CTLA-4 interactions could block
suppressive signals transferred via surface-bound CTLA-4. Therefore, high
concentrations of sCTLA-4 in serum might contribute to disease
manifestations through interference of sCTLA-4 with B7/CTLA-4 interaction.
It may be that the amino acid change at codon 17 of the
signal peptide could alter the function of the signal peptide to direct
intracellulat trafficking of CTLA-4. In in vitro expreriments, the
Ala17 (G49) allele was found to represent a translation product, which was
not glycosylated in one of two N-linked glycosylation sites [176].
This aberrantly glycosylated product was shown to be further translocated
from the endoplasmic reticulum back to cytoplasm and, probably, to become a
target for proteolytic degradation. In addition, the distribution of Ala17
CTLA-4 variant on the surface of COS1 cells is significantly less density
than the Thr17 variant of CTLA-4 [176]. These fundings
suggest that the Ala17 allele is linked to the inefficient glycosylation of
CTLA-4, which subsequently could affect suppressing effects of the CTLA-4
molecule. This could also explain observations showing that the G49 allele
of the CTLA-4 signal peptide is associated with accelerated proliferation of
T lymphocytes in human subjects homozygous for this allele, and with
suppression of the downregulation of T-cell activation in response to IL-2 [177,178].
The codon 17 single nucleotide polymorphism (SNP) is shown
to be in tight linkage disequilibrium with another SNP situated at position
(-318) of the CTLA-4 promoter region and with the (AT)n repeat
polymorphism at the 3'-UTR of the CTLA-4 gene [176,179-181].
For the C (-318) T SNP, the protective T (-318) allele demonstrated higher
promoter activity than the alternative C allele in a luciferase expression
assay [182]. Since the (-318) dimorphism occurs in a
potential regulatory region, this suggets that this nucleotide substitution
may influence the expression of CTLA-4. However, this possibility remains to
be explored.
The (AT)n repeat polymorphism at the 3'-UTR of
the CTLA-4 gene has been shown to affect the expression of this
costimulatory molecule [174]. Adenylate- and uridylate-rich
elements (AUREs) presented in the 3'-UTRs can regulate stability of
eukaryotic mRNAs, and their presence correlates with rapid RNA turnover and
translational and posttranslational control [183,184].
The AT repeats in the 3'-UTR of CTLA-4 might represent a special type of of
AUREs. CTLA-4 mRNA with longer (AT)n alleles have shorter
half-lives and, hence, are more unstable [174,185].
Indeed, the (AT)n microsatellite in the 3'-UTR influences the
mRNA stability. Additionally, the CTLA-4 AT-repeat polymorphism was recently
shown to alter the inhibitory function of CTLA-4. The long AT-repeat allele
is associated with reduced control of T-cell proliferation and thus
contributes to the pathogenesis of Graves' disease [186].
The AT-repeat may also affect splicing of one or more of
the alternative CTLA-4 transcripts but this should be clarified. Ueda et
al. [169] showed that another polymorphism (A6230G,
or CT60 SNP) located in the first position of the 3'-UTR correlates with
higher expression of a soluble CTLA-4. In this study, the highest power of
linkage with Graves' disease was found for this SNP and three other SNPs (JO27, JO30 and
JO31) within a 6.1-kb segment of the 3'-UTR, but not for the (AT)n
repeat polymorphism [169]. However, no T-cell function
data were presented. Thus, further investigations are necessary to evaluate
functional significance of these SNPs. Due to the linkage disequilibrium, it
is currently not possible to determine whether one, or both, are of
physiological importance. It can not be excluded that allele combination of
several closely linked CTLA-4 polymorphisms might form a functionally
significant haplotype that is directly involved in the susceptibility to
autoimmune disease [187,188].
It should be noted that the genomic region 2q33 linked to
autoimmune disease contains cluster of three genes encoding costimulatory
molecules CTLA-4, CD28 and inducible costimulator (ICOS) [189].
However, genetic studies showed that the AITD gene in the 2q33 locus is the
CTLA-4 gene and not the CD28 or ICOS genes [167,169,181].
The CTLA4 gene should be recognised as the first major
known non-HLA locus of human autoimmunity and that its role in the
pathogenesis of Hashimoto disease is rather general and non-specific [74,153].
Association of CTLA-4 with the production of thyroid antibodies [167,190],
an event that often represents the subclinical stage of AITD [1],
can explain non-specific mechanism of CTLA-4-mediated susceptibility to the
development of thyroid autoimmunity. The association of the CTLA-4 gene with
several autoimmune diseases such as T1D [153,169],
Addison's disease [191,192],
multiple sclerosis [193,194],
myasthenia gravis [175] and all clinical outcomes of
AITD [74], can also explain the general contribution of
CTLA-4 to autoimmunity. Interestingly, AITD, Addison's disease and
autoimmune diabetes frequently coexist in patients with the autoimmune
polyendocrine syndrome type II as mentioned above. The above disorders seem
to share a genetic background, and CTLA-4 could represent a common
susceptibility focus for them [195]
It is recommended that Transfer
Factor Advanced Formula to be used in autoimmune conditions.
Transfer Factor Plus is generally preferred for
conditions caused by infection. Transfer Factors
suppress over acting immune system
to ease autoimmune conditions.
Other
Immune Regulatory Genes
In initial phases of AITD
(autoimmune thyroid disease), oligoclonal expansion of T
lymphocytes occurs in the thyroid gland. These T cells are restricted by
their T cell receptor V gene use [89,90].
Therefore, the TcR may be considered a likely candidate gene for AITD and
Hashimoto disease. Early case-control investigations showed a lack of association between
Hashimoto's thyroiditis and the T-cell receptor-α gene in the US
white population [111] but not the T-cell receptor-β
gene in the Japanese [102]. Linkage analysis using a US
Caucasian AITD family dataset [129] and Tunisian
affected pedigree [196] has eliminated the T-cell
receptor V alpha and V beta gene complexes, located on 14q11 and 7q35,
respectively, as candidate genes for susceptibility to thyroid autoimmunity.
Therefore, the TcR genes are not major susceptibility genes for Hashimoto
thyroiditis and AITD.
Another likely candidate among immune-related genes was the
IGH gene because Hashimoto disaease individuals commonly produce Tg-autoantibodies
restricted by IgG class [50]. Early investigations found
an association between IgH Gm allotypes and AITD in the Japanese [197,198].
However, these findings have not been confirmed in Caucasians [129,196].
Cytokines are crucial in the regulation of immune and
inflammatory responses. Multiple investigations showed the important role of
these regulatory molecules in directing autoimmune and apoptotic pathogenic
processes, of particular, in central and late stages of the development of
Hashimoto's thyroiditis [72,80,199].
Therefore, cytokine genes might be good candidates for Hashimoto disease. Intrathyroidal
inflammatory cells and thyroid follicular cells produce a variety of
cytokines, including interleukin-1α (IL-1α),
IL-1β, IL-2, IL-4, IL-6, IL-8, IL-10, IL-12,
IL-13, IL-14, tumor necrosis factor-α, and
interferon-γ [200]. Hunt
et al. [201] evaluated 15 polymorphisms within nine
cytokine genes for IL-1α, IL-1β,
IL-1 receptor antagonist (IL1RN), IL-1 receptor 1, IL-4, IL-4 receptor,
IL-6, IL-10, and transforming growth factor-β in
British patients with AITD. They only found a significant association for
one of those. The T-allele of the IL-4 promoter [T (-590) C] polymorphism
was associated with lower risk of Graves'
disease and AITD but not Hashimoto disease [201].
Blakemore et al. [202] failed to find an association
between a polymorphic minisatellite in the IL1RN gene and Hashimoto's
thyroiditis in another
group of affected patients from UK. Thus, it may be concluded that these
genes are not major susceptibility genes for thyroid autoimmunity but need
to be further studied.
The autoimmune regulator (AIRE1) gene is known to
contribute to the pathogenesis of autoimmune
polyendocrinopathy-candidiasis-ectodermal dystrophy (APECED), a rare
monogenic autoimmune disease with endocrine components including T1D,
adrenal failure, and thyroid dysfunction, with major autoantibodies directed
against adrenal, pancreas, and thyroid tissue [203].
However, studies in UK patients showed no relation between a 13-bp deletion
at nucleotide 964 in exon 8 (964del13) of the AIRE1 gene, a common
disease-associated marker for APECED in British population, and Hashimoto's
thyroiditis [204].
The vitamin D-mediated endocrine system plays a role in the
regulation of calcium homeostasis, cell proliferation and (auto) immunity.
1,25-Dihydroxi-vitamin D3 (1,25(OH)2D3) is
the most active natural vitamin D metabolite that effectively prevents the
development of autoimmune thyroiditis in an animal model [205]
and inhibits HLA class II expression on endocrine cells [206].
C/T polymorphism located at intron 6 of the vitamin D 1α-hydroxylase
(CYP1α) gene failed to show association with Hashimoto's
thyroiditis
in Germans [207]. Two polymorphic markers within the
vitamin D-binding protein gene encoding another member of the vitamin D
metabolic pathway also showed no association with Hashimoto disease in the German
population [208]. However, among two polymorphic sites
tested at the vitamin D receptor (VDR) gene, the Fok I(+) allele of the FokI/restriction
fragment length polymorphism was found to be associated with higher risk Hashimoto
disease
in Japanese females [209]. Meanwhile, the VDR gene
remains to be a likely candidate for the common autoimmune susceptibility
gene because it has been found to be associated with
autoimmune disorders
such as Graves' disease [210], Addison's disease [211],
multiple sclerosis [212] and T1D [213].
Thus, a wide variety of non-HLA immune regulatory genes
located outside the HLA region showed no significant linkage or association
with Hashimoto disease and AITD except for the CTLA4 gene. However, we still cannot
estimate whether or not these genes significantly contribute to Hashimoto
disease
susceptibility due to a serious shortfall in information about their role in
this disorder. It cannot be excluded that other genes in linkage
disequilibrium with these genes are the susceptibility genes at these loci.
It is recommended that Transfer
Factor Advanced Formula to be used in autoimmune conditions.
Transfer Factor Plus is generally preferred for
conditions caused by infection. Transfer Factors
suppress over acting immune system
to ease autoimmune conditions.
Thyroid-specific Genes
Antibodies against thyroid peroxidase are one of the most
specific features of Hashimoto's thyroiditis [214]. Therefore, the TPO gene
is expected to be a putative candidate responsible not only for
susceptibility to Hashimoto disease but also for specific determination between two common
outcomes of AITD (autoimmune thyroid disease), such as Hashimoto's
thyroiditis and Graves' disease. Genetic transmission of the recognition
by antibody of the TPO immunodominant region and the TPO B domain has been
described in families affected with Hashimoto's thyroiditis [215]. This
transmission could be explained by genetic variations within the TPO gene.
However, case-control studies showed lack of association between the TPO
gene polymorphisms and AITD [113,216].
These data suggest that the thyroid peroxidase gene does not play an
important role in predisposition to Hashimoto's thyroiditis. Subsequent studies are necessary to
clarify exactly whether this gene is a true susceptibility gene for AITD.
Within the other thyroid-specific gene, the TSHR gene, the
T52P amino acid substitute was examined in US white and Thai populations but
no association with Hashimoto disease was found [217,218].
Various genome-wide scans have failed to detect linkage between the
thyrotropin receptor gene and Hashimoto disease or AITD [130,133-135,219,220].
However, two microsatellites, an (AT)n marker at intron 2 of the
TSHR gene and a (CA)n marker that was mapped to approximately 600
kb of the TSHR gene, have been shown to be strongly associated with Hashimoto
disease in
Japanese patients [221,222]. The
TSHR gene, therefore, does not seem to be a major susceptibility gene for
Hashimoto disease, although a minor role cannot be excluded.
Tg-specific autoantibodies are common in AITD. The
thyroglobulin gene makes a significant contribution to Hashimoto's thyroiditis and AITD.
Whole-genome scans in Japanese-affected sibling pairs have detected a Hashimoto
disease
susceptibility locus on chromosome 8q24, with a maximum linkage to marker
D8S272 [135]. This marker is separated by 4.6 megabases
(Mb) from the Tg gene. Subsequent studies of the mixed US and European
Caucasian family dataset has confirmed the susceptibility locus to be on
chromosome 8q24, with the maximum linkage to markers D8S514 and D8S284 [74,131,223].
These markers border a large region of chromosome 8 spanning about 15 Mb.
The thyroglobulin gene is located within this region. Moreover, a new
microsatellite marker Tgms2 inside intron 27 of the Tg gene showed strong
evidence of linkage and association with AITD [223,224].
Two new microsatellites have recently been described in introns 29 and 30 of
the thyroglobulin gene that can be useful for further linkage studies in
families with autoimmune thyroid diseases [225]. Using a
high-density panel of SNPs within the human and murine Tg genes, Ban et
al. [226] identified a unique SNP haplotype,
consisting of an exon 10-12 SNP cluster in both genes and, additionally,
exon 33 SNP in the human gene, associated with AITD in humans and with EAT
in mice. Taken together, these data strongly suggest that the thyroglobulin
gene could represent the susceptibility gene for Hashimoto disease and AITD on 8q24 [74,227]
and, therefore, be characterized as the first thyroid-specific
susceptibility gene for thyroid autoimmunity [228].
The Tg gene spanning over 300 kilobases long is expected to
harbour more than one haplotype block associated with AITD since the length
of a linkage disequilibrium block of SNPs is shown to be less than 100
kilobases [229]. It seems that this gene is AITD-specific
but is not a Hashimoto's Thyroiditis-specific susceptibility gene. The manner in which the Tg
gene can be a predisposition to AITD remains unclear. It could be that amino
acid variations within the Tg gene can affect the immunogenicity of Tg. The
evidence that iodination of thyroglobulin affects its immunogenicity favours
this suggestion [230,231]. However,
additional studies are required to evaluate that.
Recent investigation in Tunisians showed significant
association of two polymorphic microsatellites (D7S496 and D7S2459) close to
the PDS gene (7q31) with Graves' disease and Hashimoto
disease, and one of them, D7S496, was linked to Graves' disease only [232]. The PDS gene encodes a transmembrane
protein known as pendrin. Pendrin is a chloride/iodide transporting protein
identified in the apical membrane of the thyroid gland [233].
Data of Kacem et al. [232] suggest that the PDS
gene might be considered a new susceptibility gene to autoimmune thyroid
diseases, having a different involvement with different diseases. However,
studies in other populations are necessary to support a role for the PDS
gene in thyroid autoimmunity and Hashimoto disease.
Finally, a role for other genes specifically expressed in
the thyroid gland, has yet to be defined. These genes include those encoding
thyrotropin-β, thyroid-specific factor-1, sodium
iodide (Na+/I) symporter and paired box transcription factor-8,
among others. They also need to be evaluated for any putative impact on Hashimoto
disease.
Apoptotosis-related Genes
Two polymorphic sites within the FasL gene were recently
tested in Hashimoto's Thyroiditis Caucasian patients from Italy and Germany. No association
between these polymorphisms and the disorder was shown [234].
Assuming a lack of association of the naturally occurring FasL gene
polymorphisms with multiple other autoimmune diseases tested, we conclude
that genetic variation within this gene does not contribute to autoimmunity.
Inactivating mutations within the Fas and FasL genes are associated with
carcinogenesis [235,236]. This
situation is common among apoptotic-related genes encoding caspases, death
receptors, decoy receptors and death ligands as well as for genes that
encode other types of signalling molecules [237].
However, since apoptotic mechanisms play a critical role in pathogenesis and
progression of Hashimoto disease, genes associated with programming cell death should be
evaluated whether or not they confer susceptibility to Hashimoto's thyroiditis.
It is recommended that Transfer
Factor Advanced Formula to be used in autoimmune conditions.
Transfer Factor Plus is generally preferred for
conditions caused by infection. Transfer Factors
suppress over acting immune system
to ease autoimmune conditions.
Other Genes
Due to the prevalence of thyroid autoimmunity in females,
gender-related genes could also be considered as putative candidates for Hashimoto
disease
susceptibility. Some of these genes, such as the CYP19 gene encoding aromatase that participates in estrogen synthesis, and genes for estrogen
receptor-α (ESR1) and -β
(ESR2), were examined but showed no linkage with Hashimoto disease [238].
The ESR1 and ESR2 genes demonstrated no association with AITD (autoimmune
thyroid disease) in the
Japanese [239,240]. It seems that
the CYP19 and both estrogen receptor genes do not predispose to Hashimoto
disease and AITD.
Other gender-specific genes could contribute to AITD. A possible involvement
of such genes has been shown for Graves' disease with the discovery of a susceptible
locus on chromosome X [238].
The SEL1L gene, encoding a novel transcription factor, was
recently described [241]. The gene is located on
chromosome 14q24.3-q31 close to the GD-1 susceptibility locus [128,130,219]
and considered a likely candidate for thyroid autoimmunity. However, a
case-control study in the Japanese population detected no association of a
dinucleotide (CA)n repeat polymorphism in the intron 20 of the
SEL1L gene with AITD [242]. This gene may be a
potentially predisposing gene to T1D because it is specifically expressed in
adult pancreas and islets of Langerhans [241]. It lies
in the vicinity to IDDM11, a susceptibility locus to this autoimmune
disease, on chromosome 14q24.3-q31 [243].
A new zink-finger gene designated ZFAT (a novel zink-finger
gene in AITD susceptibility region) has been recently found on chromosome
8q24 [244]. The T allele of the Ex9b-SNP10 dimorphism
representing an adenine-to-thymidine substitution within intron 9 of this
gene was shown to be associated with high risk for AITD in Japanese
patients. Functional studies showed that the Ex9b-SNP10 significantly
affects the expression of the small antisense transcipt of ZFAT (SAS-ZFAT)
in vitro and this expression results in the decreasing expression of
the truncated form of ZFAT (TR-ZFAT) [244]. This SNP is
located in the 3'-UTR of TR-ZFAT and in the promoter region of SAS-ZFAT.
Full-length ZFAT and TR-ZFAT encode a protein with unknown function, which
has eighteen and eleven repeats of zink-finger domains, respectively. Both
molecular variants of ZFAT are expressed in different tissues including
peripheral blood lymphocytes, while SAS-ZFAT is exclusively expressed in
peripheral blood B cells and represents a non-coding RNA with putative
regulatory function [245]. The disease-associated
polymorphism can play a significant role in B cell function by enfluencing
the expression level of TR-ZFAT through regulation of transcription of
SAS-ZFAT. Interestingly, Shirasawa et al. found no association of the
thyroglobulin gene with AITD when studying different ethnic groups [244].
These results suggest that the ZFAT gene could implicate the susceptibilty
to AITD on chromosome 8q24 but that it needs to be strongly replicated in
other populations. Additionally, the ZFAT gene should be functionally
studied to clarify whether the ZFAT or thyroglobulin gene are true
contributors of genetic susceptibility to AITD and Hashimoto disease on 8q24.
Non-defined Susceptibility
Loci for
Hashimoto's Thyroiditis and Autoimmune Thyroid Disease
At present, the CTLA-4 (chromosome 2q33), thyroglobulin (or
ZFAT) (8q24) and likely HLA genes (6p21.3) are the only susceptibility loci
for Hashimoto's thyroiditis and thyroid autoimmunity to be mapped. Two
Hashimoto's Thyroiditis-specific susceptibility
loci that have been detected in mixed Caucasian families from USA and
Europe, HT-1 (13q) near marker D13S173 and HT-2 on chromosome 12q in the
vicinity of marker D12S351, are still not defined [130].
HT-2 locus has been subsequently replicated in the extended dataset, with a
peak linkage close to marker D12S346, which HT-1 does not have [223].
Possible candidate genes for susceptibility to Hashimoto disease positioned within the HT-2
locus may include the BTG1 and CRADD genes. The BTG1 gene encodes B-cell
translocation protein-1, which play an immune regulatory role as a negative
regulator of the proliferation of B cells [246]. The
GRADD gene encodes CASP-2 and RIPK-domain-containing adaptor with death
domain, that represents apoptotic function, inducing cell apoptosis via
recruiting caspase 2/ICH1, TNF receptor 1, RIPK-RIP kinase and other
proteins [247].
AITD-1 locus located on chromosome 6p is very close yet
distinct from the HLA region [120,130].
It has been shown that the AITD-1 is positioned in the same location as
susceptibility loci for T1D (locus IDDM15) [248] and
systemic lupus erythematosus [249]. This may imply that
a general autoimmunity susceptibility gene is located in this region. The
AITD-1 locus contains an interesting positional candidate gene such the
SOX-4 gene, encoding a transcription factor that modulates differentiation
of lymphocytes [250].
A whole-genome scan in Japanese showed evidence for linkage
with AITD (autoimmune thyroid disease) on chromosome 5q31-q33 [135,251].
The 5q31 locus was replicated by recent genome-wide scan in Caucasian
population, the Old Order Amish of Lancaster County, from Pennsylvania [220].
This locus harbours a cluster of cytokine genes and, therefore, several
positional candidate genes occur in this region and need to be evaluated.
In the Chinese, a whole-genome screening for AITD
susceptibility found two chromosome regions (9q13 and 11q12) linked to AITD
[134]. Susceptibility genes have yet to be defined
within these regions. However, the 9q13 locus harbours a putative candidate
gene such as the ANXA1 gene, whose product annexin A1 prevents the
production of inflammatory mediators [252]. The 11q12
locus contains several interesting candidate genes encoding immune
modulators (CD5 and CD6) and possible components of antigenic peptide
processing (PSMC3) and transport (PTH2).
In a large Tunisian family affected with AITD, a
susceptibility locus was mapped on 2p24 [133] This locus
harbors two possible candidate genes such as the FKBP1B gene, product of
which demonstrates immune modulating activity [253], and
the TP53I3 gene encoding p53-inducible protein 3 that is involved in
p53-mediated apoptotic pathway [254].
These data suggest that both
Hashimoto
disease and AITD show genetic
heterogeneity in different populations. Susceptibility loci differ in their
chromosome location depending on the population being tested. The
contributory value of these genes to the disease pathology varies
significantly depending on the ethnic background. A gene, that has a major
effect on the susceptibility to Hashimoto disease in one population, may contribute weakly
in other population. To date, several regions of linkage to Hashimoto
disease and AITD have
been defined. Further studies are required to find a true susceptibility
gene in these genomic regions to reveal the functional significance of
disease-associated polymorphisms within these genes.
It is recommended that Transfer
Factor Advanced Formula to be used in autoimmune conditions.
Transfer Factor Plus is generally preferred for
conditions caused by infection. Transfer Factors
suppress over acting immune system
to ease autoimmune conditions.
Conclusion
AITD
(autoimmune thyroid disease)
can be
initiated in individuals genetically predisposed to AITD by non-genetic
(environmental) triggers such as dietary iodine, infection, pregnancy,
cytokine therapy (Fig.
1). This interaction leads to different clinical phenotypes of thyroid
autoimmunity such as Graves' disease,
Hashimoto's thyroiditis or production
of thyroid antibodies. Hashimoto disease and Graves' disease are two distinct but related clinical
outcomes of AITD. It seems that both thyroid diseases have common pathogenic
mechanisms as their initial steps including breakdown of the immune
tolerance and accumulation of T lymphocytes in the thyroid gland.
Sequence variants of CTLA-4, associated with increased
levels of the soluble form of this immune costimulator and with stability of
CTLA-4 mRNA, could play a crucial role in the earliest stages of AITD (i. e.
breakdown of self-tolerance and surviving autoreactive T lymphocytes). This
role might be sufficient to regulate subsequent steps in the development of
autoimmune responses to the production of thyroid autoantobodies [167].
Environmental factors (particularly, iodine intake and
infection) could cause insult of the thyrocyte followed by abnormal
expression of MHC class I and class II molecules, as well as changes to
genes or gene products (such as MHC class III and costimulatory molecules)
needed for the thyrocyte to become an APC [255]. In this
stage, a modulating role of sequence variants of HLA class II molecules
could become pivotal in binding and presenting thyroid antigenic peptides
derived from Tg, TPO and TSHR. Genetic variations in Tg, and probably in
TSHR and other thyroid-specific genes, might be responsible for generating
an autoimmune response.
In later stages, thyroid autoimmunity could be switched
towards Graves' disease or Hashimoto's disease. Graves' disease is characterized by TH2-mediated switching
of thyroid-infiltrating T cells. These induce the production of stimulating
anti-TSHR antibodies by B cells and anti-apoptotic mechanisms that lead to
clinical hyperthyroidism. In Hashimoto's disease, preferential TH1 response
initiates apoptosis of thyroid cells and results in clinical hypothyroidism
[22].
It is clear that a number of loci and genes determine
genetic predisposition to Hashimoto's thyroiditis, with varying effects. These loci could be
unique to Hashimoto disease or general for both Hashimoto disease and
Graves' disease. Several whole-genome scans
showed results suggesting that there is significant shared susceptibility to
Hashimoto's disease and Graves' disease [130,131,134,135].
This is also supported by the frequent coexistance of both diseases in
affected families [74,133].
Preliminary data suggest that shared genetic susceptibility involves both
immune regulatory (i. e. CTLA-4 and HLA) and thyroid-specific genes (i.e. Tg).
These genes are not responsible for the determination of pathogenic
mechanisms of thyroid autoimmunity distinct for Hashimoto disease and
Graves' disease. It remains
unclear which susceptibility genes are specifically involved in the Hashimoto's
thyroiditis
pathogenesis.
The CD40 gene, an important immune modulator, appears to
act as a GD-specific susceptibility gene. The gene is located within the
20q11 locus and shows significant linkage to Graves' disease, but not to Hashimoto
disease, in UK
Caucasians [74,130,131,256,257].
Subsequent analysis found the CD40 gene to be associated with Graves'
disease [258].
However, this finding needs to be independently confirmed in other
population samples.
A probable susceptibility gene that could direct switching
towards Graves' disease or
Hashimoto disease is thought to be located within the 5q31 locus, which is
linked to AITD and contains a cytokine gene cluster. Different sets of
cytokines are known to regulate switching to TH1 or TH2
type mechanisms [58]. There may be two susceptibility
genes, each of which uniquely contributes to the development of Hashimoto's thyroiditis- or Graves' disease-specific
pathogenesis. The IL-4 promoter [T(-590) C] polymorphism also appears to be
associated with Graves' disease, but not with Hashimoto's thyroiditis [199]. IL-4 mediates
TH2 type mechanism, which can lead to hyperthyroidism [259].
Another Hashimoto's thyroiditis-specific susceptibility gene(s) may be an
apoptotic gene. Apoptosis of thyroid follicular cells is the hallmark of Hashimoto
disease
and might be the primary cause of death of thyrocytes compared to T
cell-mediated cytotoxity [69].
Thus, it is necessary to identify additional susceptibility
genes and disease-associated polymorphisms in apoptotic genes in AITD- and
Hashimoto's Thyroiditis-linked loci by using a fine mapping approach and high-density panels of SNPs. Further functional analysis and search for correlations between
genotype and phenotype will help to evaluate the role of these genes in the
development of autoimmune thyroid disease. Susceptibility genes interact
with thyroid autoimmunity [62,130],
and the level of these interactions could affect disease severity and
clinical expressions.
The molecular mechanisms of these interactions is
unknown. However, significant progress has been made in identifying
susceptibility genes to Hashimoto disease and AITD along with intriguing findings regarding
the functional characterization of disease-associated polymorphisms. These
should stimulate further studies towards the in-depth understanding of the
mechanisms by which these genes contribute to thyroid autoimmunity.
It is recommended that Transfer
Factor Advanced Formula to be used in autoimmune conditions.
Transfer Factor Plus is generally preferred for
conditions caused by infection. Transfer Factors
suppress over acting immune system
to ease autoimmune conditions.
Competing
interests
The author(s)
declare that they have no competing interests.
Authors'
contributions
ABFG
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