The role of functionally defective rare germline variants of sialic
acid acetylesterase in autoimmune Addison’s disease
Earn H Gan1,2, Katie MacArthur1, Anna L Mitchell1,2and Simon H S Pearce1,2
1Institute of Genetic Medicine, International Centre for Life, Newcastle University, Central Parkway, Newcastle upon Tyne NE1 3BZ, UK and
2Royal Victoria Infirmary, Newcastle upon Tyne, UK
(Correspondence should be addressed to E H Gan; Email: email@example.com)
Background: Autoimmune Addison’s disease (AAD) is a rare condition with a complex genetic basis.
A panel of rare and functionally defective genetic variants in the sialic acid acetylesterase (SIAE) gene
has recentlybeen implicated in several common autoimmune conditions. We performed acase–control
study to determine whether these rare variants are associated with a rarer condition, AAD.
Method: We analysed nine SIAE gene variants (W48X, M89V, C196F, C226G, R230W, T312M,
Y349C, F404S and R479C) in a United Kingdom cohort of 378 AAD subjects and 387 healthy
controls. All samples were genotyped using Sequenom iPlex chemistry to characterise primer
Results: A heterozygous rare allele at codon 312 (312*M) was found in one AAD patient (0.13%) but
was not detected in the healthy controls. The commoner, functionally recessive variant at codon 89
(89*V) was found to be homozygous in two AAD patients but was only found in the heterozygous state
in controls. Taking into account all nine alleles examined, 4/378 (1.06%) AAD patients and 1/387
(0.25%) healthycontrols carried the defective SIAE alleles, with a calculated odds ratio of 4.13 (95% CI
0.44–97.45, two-tailed P value 0.212, NS).
Conclusion: We demonstrated the presence of 89*V homozygotes and the 312*M rare allele in the AAD
cohort, but overall, our analysis does not support a role for rare variants in SIAE in the pathogenesis of
AAD. However, the relatively small collection of AAD patients limits the power to exclude a small effect.
European Journal of Endocrinology 167 825–828
Autoimmune Addison’s disease (AAD) is a rare
autoimmune endocrinopathy, with a prevalence of one
in 8000 people in the United Kingdom (1). It is
commonly associated with autoimmune thyroid disease
(w50%) and/or type 1 diabetes (w10%), constituting
the type 2 polyendocrinopathy syndrome (2). In
common with many other autoimmune conditions,
AAD is believed to have a complex genetic basis, with
the risk to first-degree relatives of about 2% and a lsO
150 (ratio of risk to a sibling vs the unrelated
background population) (3). Candidate gene studies
have identified numerous susceptibility alleles contri-
buting to AAD, such as the MHC locus on chromosome
6p21, which harbours the strongest susceptibility
variant(s) for this disorder, as well as loci PTPN22 (4),
CTLA4 (5), CLEC16A (6), CIITA (6), PDL1 (7),
CYP27B1 (8) and NLRP1 (9, 10). Nevertheless, given
the high genetic load of this rare condition, there are
likely to be many more susceptibility alleles for AAD,
which have yet to be elucidated. A knowledge of these
variants is essential for improving our understanding of
AAD pathogenesis, with the ultimate aim of designing a
small molecule or protein-targeted therapy as a
Recently, a panel of rare and functionally defective
genetic variants in the sialic acid acetylesterase (SIAE)
gene were identified, in a high-profile publication, as
being strongly associated with many autoimmune
conditions, including Crohn’s disease, type 1 diabetes,
systemic lupus erythematosus (SLE), Sjogren’s syn-
drome, juvenile idiopathic arthritis, multiple sclerosis
(MS), mixed connective tissue disease, rheumatoid
arthritis and ulcerative colitis (odds ratio (OR) O8)
(11). However, a subsequent larger study of more
prevalent autoimmune and inflammatory disorders,
including type 1 diabetes, coeliac disease, Crohn’s
disease and autoimmune thyroid disease, failed to
replicate this finding (12). Nevertheless, SIAE represents
one of the very first associations of rare genomic
variants with common autoimmune disorders. Inter-
estingly, loss-of-function rare variants in the TREX1 (the
major mammalian 30–50exonuclease) and CYP27B1
(vitamin D 1a-hydroxylase) genes have been described
in SLE and MS patients respectively, suggesting that in
European Journal of Endocrinology (2012) 167 825–828ISSN 0804-4643
q 2012 European Society of EndocrinologyDOI: 10.1530/EJE-12-0579
Online version via www.eje-online.org
This is an Open Access article distributed under the terms of the European Journal of Endocrinology’s Re-use Licence which permits unrestricted non-
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contrast to the common disease-common variant
hypothesis, there may be a greater role for rare genetic
variants in the susceptibility to less prevalent auto-
immune diseases (13, 14).
SIAE is a negative regulator of B lymphocyte
signalling by acting at inhibitory receptors that
attenuate B-cell receptor signalling. Spontaneous auto-
antibody production has been demonstrated in SIAE-
mutant mice on a C57Bl/6 background, suggesting that
defects in SIAE function conferred by SIAE variants
might contribute to human autoimmunity (15).
Genome-wide studies have not demonstrated any
association or linkage with SNPs at the SIAE locus in
patients with autoimmunity; however, under a multiple
rare variants model, this might not be expected.
Nevertheless, AAD is rarer than all the previously
studied disorders and hence rare variants might have a
significant contribution to its pathogenesis. The purpose
of our study is to explore whether these rare SIAE
variants are associated with AAD, which has not
previously been studied (11, 12, 16).
Materials and methods
Three hundred and seventy-eight Caucasian subjects
with AAD have been recruited since 1996 through
outpatient endocrinology services in the North East of
England and the UK Addison’s disease self-help group.
The diagnosis of AAD was confirmed by either a
subnormal response to the ACTH1–24stimulation test
(using 250 mg of parenteral synthetic ACTH), or a low
basal cortisol with a high ACTH level. Patients with
APS1, primary adrenal failure owing to infiltrative
or infective causes or secondary adrenal failure were
excluded. Three hundred and eighty-seven healthy local
Caucasian controls were used for comparison (including
113 individuals from a 1958 birth cohort). This study
was carried out with approval of the Leeds (East)
Research Ethics Committee (Ref 05/Q1206/144).
We studied nine rare germline variants within the
SIAE gene (chromosome 11), which are among the
12 rare non-synonymous SIAE variants demonstrated
by Surolia et al. to be functionally defective in
esterase activity or enzyme secretion (11). These
rare SNPs comprise 21 of the 24 cases (88%) of
functionally defective rare SIAE variants reported by
Surolia et al. The SNPs selected include C196F, T312M,
C226F, F404S, R230W, R479C, W48X, Y349C and
M89V. These SNPs were selected and genotyped in
Caucasian individuals with AAD and healthy controls
in the UK.
Genomic DNA was extracted from venous blood from
each subject and used for multiplex PCR at a
concentration of 20 ng/ml. PCR was performed in a
10 ml reactionvolume usinga Qiagen PCR kit, using the
following concentrations per reaction: 20 ng/ml
genomic DNA, 1.25! PCR buffer, 25 mM magnesium
chloride, 0.5 mM of each primer, 6.25 mM of each dNTP,
and 5 U/ml HotStar Taq DNA polymerase. Primer
sequences are available from the authors on request.
PCR was then performed by initial denaturation for
15 min at 95 8C, followed by 45 cycles of denaturation
at 95 8C for 20 s, annealing at 56 8C for 30 s and
extension at 72 8C for 1 min, followed by a final
extension at 72 8C for 3 min. Amplified products were
then genotyped using Sequenom iPlex chemistry for
MALDI-TOF characterisation of primer extension
products. Ten to 15% of all samples were re-genotyped
blind for each assay to ensure fidelity of genotyping
(O99% for each SNP). The results were verified by
direct DNA sequencing where appropriate, using
Eurofins MWG Operon’s DNA sequencing service.
Statistical analysis and power
Fisher’s exact test was used for association analysis, by
means of 2!2 contingency tables. Control genotypes
(threshold PO0.05). A power estimation, using the
Table 1 Genotypes for SIAE variants in autoimmune disease cases and controls.
Autoimmune Addison’s diseaseUK healthy controls
aData presented as number of rare homozygote/heterozygote/common homozygote genotypes.
bHomozygous 89*V genotype.
E H Gan and others
EUROPEAN JOURNAL OF ENDOCRINOLOGY (2012) 167
pooled case and control allele frequencies found by
Surolia et al. (2.6 and 0.3% respectively) (11), showed
that our study design had 78% power to detect a
similar-sized effect (aZ0.05).
Among the 761 subjects (378 cases and 387 controls),
we did not find any rare allele variants for the SIAE
germline variants encoding W48X, C226G, R230W,
Y349C, F404S and R479C (Table 1). One AAD patient
was a heterozygous carrier of the codon 312*M
(T312M) variant, but all controls were found to have
the wild-type allele. A single heterozygous carrier of the
SIAE 196*F (C196F) allele was found amongst both the
case and the control cohorts. As previously found (11),
the codon 89*V allele was present heterozygously in
12.1 and 12.0% of the patient and control cohorts
respectively. However, no codon 89*V homozygotes
were found amongst control patients, whereas two AAD
patients were 89*V homozygotes (PZ0.242).
All AAD patients who are either heterozygous
carriers of the rare codon 312*M (T312M) and 196*F
(C196F) variants, or the homozygous carriers of
the codon 89*V (M89V), have other associated
autoimmune diseases in the spectrum of type 2
autoimmune polyendocrinopathy syndrome (APS2).
Among them, three had pernicious anaemia, two
autoimmune hypothyroidism and one premature ovar-
ian failure (Table 2).
In summary, taking into account all nine alleles
examined, 4/378 (1.06%) AAD patients and 1/387
(0.25%) healthy controls inherited SIAE genotypes that
would be expected to lead to functionally detrimental
consequences; OR of 4.13 (95% CI 0.44–97.45;
two-tailed P value 0.212, NS).
The association between rare SIAE genetic variants and
disease susceptibility has recently been explored in some
common autoimmune diseases, with conflicting results
among various research groups (11, 12, 16, 17).
Nevertheless, the loss-of-function rare variants in the
TREX1 and CYP27B1 genes have been significantly
implicated in SLE and MS patients respectively (13, 14),
suggesting the possibility that rare genetic variants may
have a role in risk susceptibility for some of the less
prevalent autoimmune diseases. We explored the
hypothesis that rare SIAE variants would be associated
with AAD, one of the least common autoimmune
conditions. We demonstrated the presence of two codon
89*V homozygotes and one heterozygous carrier of the
312*M allele in the AAD cohort, which was not
significantly different from healthy control genotypes.
Thus, our findings extend the negative study of Hunt
et al. (12) who showed no differences for nine of the
SIAE gene rare variants (including M89V but not
T312M) in cohorts of the commoner autoimmune and
inflammatoryconditions, such as type 1 diabetes, atopic
eczema, celiac disease, Graves’ disease and Hashimoto
thyroiditis. However, this large cohort of nearly 67 000
subjects did not include patients with the rarer
condition of AAD.
While our study had good power to replicate findings
of a similar magnitude to those previously seen in other
autoimmune conditions (OR 8.6) (11), our analysis is
underpowered to detect a more subtle genetic effect,
such as is more frequently seen for commoner
autoimmune disease susceptibility alleles. We cannot
exclude that a future analysis of an enlarged AAD
cohort or a family-based genetic association study may
cast further light on this question. However, the
parameters for declaring significance in studies of rare
genetic variants are not yet well defined (18), and the
key attribute of reproducibility has not been fulfilled for
SIAE variants in autoimmune diseases to date.
Declaration of interest
The authors declare that there is no conflict of interest that could be
perceived as prejudicing the impartiality of the research reported.
This study has been funded by the EU-Framework-7 grant 201167 to
the Euradrenal Consortium.
The authors are grateful to Katherine White and the members of the
UK Addison’s Disease Self-Help Group (http://www.addisons.org.uk)
for their help in organising and donating blood samples.
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Received 4 July 2012
Revised version received 4 September 2012
Accepted 25 September 2012
E H Gan and others
EUROPEAN JOURNAL OF ENDOCRINOLOGY (2012) 167