Neuromyelitis optica (NMo) and multiple sclerosis (MS) are
autoimmune diseases of the central nervous system with
complex pathogeneses. Evidence suggests that genetic and
environment factors may induce these diseases1-3. Neuromyelitis
optica was once considered to be a severe variant of MS. Since
2004, serum anti-aquaporin-4 antibody (AQP4-Ab) has been
suggested to be a reliable biomarker of NMo4.
HLA-DRb1 on chromosome 6p21 was the most remarkable
genetic locus contributing to MS susceptibility. However, a large
number of non-HLA single nucleotide polymorphisms (SNP)
were also confirmed to increase the risk for MS5-7. In NMo,
although HLA-DPb1 might be associated with susceptibility to
NMo3,8,9, the effect on non-HLA SNPs in NMo have been less
CD226 belongs to the immunoglobulin supergene family of
receptors and was widely expressed on the CD4+and CD8+T
cells, natural killer cells, monocytes, b cells and platelets.
CD226 could lead to various biological responses, including
target cell lysis and immune cell activation12. Rs763361/
ABSTRACT: Background: Neuromyelitis optica (NMo) and multiple sclerosis (MS) are autoimmune diseases of the central nervous
system with complex pathogeneses. NMo was once considered to be a severe variant of MS. There has been more evidence that a non-
synonymous exchange (rs763361/Gly307Ser) in the gene for CD226 is linked to several autoimmune diseases including multiple
sclerosis (MS). However, no studies have investigated the role of rs763361 in the pathogenesis of NMo. Objectives: The goal of our
study is to evaluate the role of CD226 Gly307Ser in neuromyelitis optica (NMo) in Southern Han Chinese. Methods: Eight-nine NMo
patients, 93 relapsing-remitting multiple sclerosis (RRMS) patients, and 122 controls (CTLs) were enrolled. The rs763361 alleles of the
subjects were determined by sequencing-based typing. Results: The results strongly support that the TT genotypes are associated with
NMo but are not significantly correlated with susceptibility for MS. Conclusions: CD226 Gly307Ser may correlate with risk of NMo
in Southern Han Chinese.
RÉSUMÉ: Association de la variante Gly307Ser du gène CD226 avec la neuromyélite optique chez les Chinois Han du Sud. Contexte : La
neuromyélite optique (NMo) et la sclérose en plaques (SP) sont des maladies autoimmunes du système nerveux central dont la pathogenèse est
complexe. La NMo était anciennement considérée comme une variante sévère de la SP. Il existe maintenant des données supplémentaires en faveur de
l'hypothèse selon laquelle un échange non-synonyme (rs763361/Gly307Ser) dans le gène CD226 serait lié à plusieurs maladies autoimmunes dont la
SP. Cependant, aucune étude n'a porté sur le rôle de rs763361 dans la pathogenèse de la NMo. Objectifs : Le but de notre étude était d'examiner le rôle
de la variante Gly307Ser du gène CD226 dans la NMo chez les Chinois Han du Sud. Méthode : Quatre-vingt-neuf patients atteints de NMo, 93 patients
atteints de SP récurrente-rémittente et 122 sujets témoins ont participé à l'étude. La présence de l'allèle rs763361 chez les sujets a été déterminée par
séquençage. Résultats : Les résultats sont en faveur d'une association entre le génotype TT et la NMo, mais il n'existe pas de corrélation significative
entre ce génotype et la susceptibilité à la SP. Conclusions : La variante Gly307Ser du gène CD226 pourrait être corrélée au risque de la NMo chez les
Chinois Han du Sud.
Can J Neurol Sci. 2012; 39: 488-490
CD226 Gly307Ser Association With
Neuromyelitis Optica in Southern Han
Chao Liu, Guansan Wang, Hong Liu, Yue Li, Jin Li, Yongqiang Dai,
From the Guangzhou Forensic Science Institution (Guangdong Provincial Key
Laboratory of Forensic Genetic) (CL, GW, HL, YL); Multiple Sclerosis Center,
Department of Neurology (JL, YD, XH), The Third Affiliated Hospital of Sun Yat-Sen
University, Guangzhou, Guangdong Province, China.
RECEIvED NovEMbER 16, 2011. FINAL REvISIoNS SUbMITTED FEbRUARY 15, 2012.
Correspondence to: Xueqiang Hu, Multiple Sclerosis Center, Department of
Neurology, The Third Affiliated Hospital of Sun yat-sen University, No.600 Tianhe
Road, Guangzhou 510630, Guangdong Province, China.
Gly307Ser in the immune response gene CD226 on
chromosome 18q22 may increase the susceptibility of multiple
autoimmune diseases, such as Type 1 diabetes (T1D),
autoimmune thyroid disease (AITD) and rheumatoid arthritis
(RA)13,14. In Chinese Han population, rs763361 was also
associated with systemic lupus erythematosus (SLE)15.
Rs763361 was also found to be associated with MS in Western
and Asian countries12,13,16. However, no studies have
LE JoURNAL CANADIEN DES SCIENCES NEURoLoGIQUES
Volume 39, No. 4 – July 2012
investigated the role of rs763361 in the pathogenesis NMo and
MS in Chinese Han population.
In this study, we investigated the frequency of rs763361 in
NMo and MS patients in Chinese Han population, and analyzed
the susceptibility risk of rs763361 in those patients.
MATERIALS AND METHODS
Patients and controls
Eight-nine AQP4-Ab positive NMo patients (68 women, 21
men), whose mean of onset age was 28.73 years, were selected
based on the 2006 Wingerchuk criteria17. Indirect
immunofluorescence test systems for human AQP4-Ab detection
Labordiagnostika, Lübeck, Germany) were used. Ninety-three
(57 women, 36 men) relapsing-remitting multiple sclerosis
(RRMS) patients fulfilling the 2010 McDonald criteria18were
enrolled and the mean of onset age was 33.15. These patients
were all enrolled from the MS database of the Third Affiliated
Hospital of Sun Yat-Sen University. Patients with only recurrent
myelitis, recurrent optic neuritis, or recurrent brainstem
symptoms were excluded from this study. one hundred twenty-
two consenting volunteers (84 women, 38 men) were recruited
as controls (CTLs). The selected CTLs had no first-degree
relative with autoimmune diseases. All the subjects were
Southern Han Chinese and were born in Southern China. This
study was approved by the Ethics Committee of the Third
Affiliated Hospital of Sun Yat-Sen University.
EURoIMMUN (EURoIMMUN Medizinische
Peripheral blood samples were Collected and stored at
−20°C. Genome DNA was extracted by using the Tianamp N96
DNA blood Kit (Tiangen, beijing, China). The target DNA
sequence was amplified by polymerase chain reaction (PCR)
method with proper primer as followed: forward primer 5’-
GCACT CATGTCAAGAATAAG-3’ and reverse primer 5-
‘AAGTTCAGACACTGTGTTAG-3’. PCR amplification was
performed with 50 ng of genomic DNA in 20μl PCR reaction
mixture containing 20 mM Tris–HCl pH 8.0, 50 mM EDTA, 0.2
mM dNTPs, 1.5 mM MgCl2, forward and reverse primers (0.5
μmole each), 2.5 units of Taq polymerase. The PCR cycle
consisted of a sequence of denaturation at 95 °C for five minutes
(min), denaturation at 94 °C for 1 min, annealing at 56 °C for
one min, extension at 72 °C for one min. After 35 cycles reaction
was terminated using a final extension at 72 °C for seven min.
The PCR products were 480 bp. After standardization of the
PCR conditions, sequencing was carried out using an automated
DNA sequencer AbI Prism 3700 (Applied biosystems, Foster
City, California, USA). Genotyping was deemed successful if
the concordance rate between duplicates was ≥95%. For samples
not showing a clear genotype, the PCR and sequencing was
repeated until the results were unequivocal.
The Hardy–Weinberg equilibrium (HWE) was initially
determined. Statistical analysis was then performed using SPSS
16.0 (SPSS Inc, Chicago, IL, USA) for Windows. Pearson chi-
square test was used to compare genotypes frequencies and
alleles of rs763361 between NMo, MS, and CTLs. The relative
risk (estimated as the odds ratios, oRs) and 95% confidence
intervals (95% CIs) were calculated. The p values (uncorrected
p, puncorr) were corrected by bonferroni–Dunn's correction to
calculate corrected p values (pcorr). Statistical significance was
set at P<0.05.
As shown in the Table, the frequency of the TT genotype was
higher in NMo patients than in CTLs (pcorr=0.021; oR: 2.668,
95% CI: 1.293–5.587). The frequency of T allele was
significantly higher in NMo patients than in CTLs
(pcorr=0.030; oR: 1.591, 95% CI: 1.071–2.363). There was no
difference in the frequency of the T allele and TT genotype
between NMo and MS. The frequency of T allele and TT
genotype were also not increased in MS patients compare to the
aPuncorr<0.05 and Pcorr>0.05; bPuncorr<0.01 and Pcorr<0.05; cPuncorr<0.05 and Pcorr<0.05
NMo: Neuromyelitis optica; MS: Multiple sclerosis; CTLs: Controls
Table: Genotype and allele distribution of the rs763361 SNP in patients and controls
THE CANADIAN JoURNAL oF NEURoLoGICAL SCIENCES
In this case-control study of rs763361 allele distribution in
Southern Han Chinese NMo and MS patients, we found that
rs763361T was increased in NMo patients. Despite of the fact
that the HLA gene is generally recognized as the strongest risk
factor for NMo in Asian population, non-HLA SNPs in NMo
have been less studied. The leukocyte adhesion molecule
DNAM-1 (CD226) is expressed on the majority of T
lymphocytes, NK cells, and monocytes19, involving both
adaptive and innate immune responses. Lymphocyte function-
associated antigen-1 (LFA-1) is a co-stimulatory molecule
playing an important role in T cell signaling, and CD226 assists
in co-localization with this molecule20. Rs763361 in exon 7 of
the CD226 gene was shown to increase the susceptibility to
multiple autoimmune diseases, such as rheumatoid arthritis21,
systemic sclerosis22, and SLE15. In this study, we proved that the
T allele and TT genotypes in rs763361 may increase the risk of
NMo in Southern Han Chinese. However, the underlying
mechanism is still unclear. Hafler et al hypothesized the
rs763361 variant could alter the expression or signaling of
CD226 as it occurs in the molecular cytoplasmic tail and
Gly307Ser was the causal variant which shared the risk locus for
autoimmune disease13. They also hypothesized rs763361 may
alter mRNA splicing, and result in either CD226 isoform acting
as a non-functional (non-signaling) protein, or with a novel
function, while Gly307Ser could alter the signaling cascade by
affecting the two known phosphorylation sites at positions 322
and 329, which play a critical role in CD226 immune response13.
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