HLA-DRB1 allele impact on pediatric multiple sclerosis in a Hellenic cohort

Abstract

Background:
Pediatric-onset multiple sclerosis (POMS) is considered a complex disease entity with many genetic and environmental factors implicated in its pathogenesis. Linkage studies in Caucasian adult populations consistently demonstrate the major histocompatibility complex and its HLA (human leukocyte antigen) polymorphisms as the genetic locus most strongly linked to MS.

Objective:
To investigate the frequencies and possible clinical and imaging correlations of HLA-DRB1 alleles in a Hellenic POMS sample.

Methods:
Fifty POMS patients fulfilling the IPMSSG (International Pediatric Multiple Sclerosis Study Group) criteria were enrolled using 144 adult-onset MS (AOMS) patients and 246 healthy controls for comparisons. HLA genotyping was performed with standard low-resolution sequence-specific oligonucleotide (SSO) techniques. Clinical and imaging correlations with specific HLA-DRB1 alleles were also examined.

Results:
The HLA-DRB1*03 genotype was significantly higher in POMS patients compared to both the AOMS population (26% vs. 12.5%, p = 0.042) and the general population (26% vs. 12.6%, p = 0.004). HLA-DRB1*03-positive POMS patients had significantly more relapses (6.9 ± 4.9 vs. 4.2 ± 4.4, p = 0.005) and more thoracic spinal cord lesions than HLA-DRB1*03-negative patients (61.5% vs. 27%, p = 0.043).

Conclusion:
In our Hellenic population, HLA-DRB1*03 allele confers increased risk for POMS and it is also correlated with possibly increased disease activity, expanding the existing knowledge on HLA associations and POMS.

Full text

Original Research Paper
HLA-DRB1 allele impact on pediatric multiple
sclerosis in a Hellenic cohort
Maria Gontika, Charalampos Skarlis, Artemios Artemiadis , Roser Pons,
Sotiria Mastroyianni, George Vartzelis, Virginia Theodorou, Konstantinos Kilindireas,
Leonidas Stefanis, Marinos Dalakas, George Chrousos and Maria Anagnostouli
Abstract
Background: Pediatric-onset multiple sclerosis (POMS) is considered a complex disease entity with
many genetic and environmental factors implicated in its pathogenesis. Linkage studies in Caucasian
adult populations consistently demonstrate the major histocompatibility complex and its HLA (human
leukocyte antigen) polymorphisms as the genetic locus most strongly linked to MS.
Objective: To investigate the frequencies and possible clinical and imaging correlations of HLA-DRB1
alleles in a Hellenic POMS sample.
Methods: Fifty POMS patients fulfilling the IPMSSG (International Pediatric Multiple Sclerosis Study
Group) criteria were enrolled using 144 adult-onset MS (AOMS) patients and 246 healthy controls for
comparisons. HLA genotyping was performed with standard low-resolution sequence-specific oligonu-
cleotide (SSO) techniques. Clinical and imaging correlations with specific HLA-DRB1 alleles were also
examined.
Results: The HLA-DRB1*03 genotype was significantly higher in POMS patients compared to both
the AOMS population (26%vs. 12.5%,p¼0.042) and the general population (26%vs. 12.6%,p¼0.004).
HLA-DRB1*03-positive POMS patients had significantly more relapses (6.9 4.9 vs. 4.2 4.4,
p¼0.005) and more thoracic spinal cord lesions than HLA-DRB1*03-negative patients (61.5%vs.
27%,p¼0.043).
Conclusion: In our Hellenic population, HLA-DRB1*03 allele confers increased risk for POMS and it is
also correlated with possibly increased disease activity, expanding the existing knowledge on HLA
associations and POMS.
Keywords: Multiple sclerosis, pediatric-onset multiple sclerosis, human leukocyte antigens, clinical
phenotype, magnetic resonance imaging, therapeutics
Date received: 30 September 2019; revised: 3 January 2020; accepted: 19 January 2020
Introduction
Early-onset (pediatric and adolescent) multiple scle-
rosis (MS) accounts for approximately 3–5%of all
MS cases, with the term pediatric-onset MS (POMS)
being used largely interchangeably in the interna-
tional literature for both age groups.
1
In parallel
with adult-onset MS (AOMS),
2
POMS is considered
a complex multi-factorial disease entity with many
genetic, epigenetic, and environmental factors impli-
cated in its pathogenesis.
Since the 1970s, it has been well established that the
major histocompatibility complex (MHC) and its
polymorphisms have a strong genetic effect on the
risk of AOMS.
3
Carrying the HLA-DRB1*15:01
allele is associated with a threefold greater risk of
developing MS, while HLA-A*02:01 confers protec-
tion against the disease.
4
A series of studies have
confirmed the predisposing role of also HLA-
DRB1*15 in POMS, although its effect is lower
than that for AOMS, suggesting a fundamental
genetic similarity between different age groups.
5–7
However, whether the HLA-DRB1*15:01 allele
is associated with lower age at MS onset by
itself still remains controversial,
8
with one study
indicating significance of epistatic interactions at
Creative Commons Non Commercial CC BY-NC: This article is distributed under the terms of the Creative Commons Attribution-NonCommercial 4.0
License (https://creativecommons.org/licenses/by-nc/4.0/) which permits non-commercial use, reproduction and distribution of the work without further
permission provided the original work is attributed as specified on the SAGE and Open Access pages (https://us.sagepub.com/en-us/nam/open-access-at-sage).
Multiple Sclerosis Journal—
Experimental, Translational
and Clinical
January–March 2020, 1–8
DOI: 10.1177/
2055217320908046
!The Author(s), 2020.
Article reuse guidelines:
sagepub.com/journals-
permissions
Correspondence to:
Maria Anagnostouli,
Demyelinating Diseases Unit
& Director of
Immunogenetics Laboratory,
First Department of
Neurology, Medical School,
National and Kapodistrian
University of Athens,
NKUA, Aeginition Hospital,
Vas. Sophias, 74, 115 28,
Athens, Greece.
managnost@med.uoa.gr
Maria Gontika,
Charalampos Skarlis,
Immunogenetics Laboratory,
First Department of
Neurology, Medical School,

the HLA-DRB1 locus, specifically between the
HLA-DRB1*04:01 and HLA- DRB1*15:01 alleles.
9
In 2014, a putative predisposing role of HLA-
DRB1*03 in POMS was proposed in the Hellenic
population.
5
This allele has been correlated with a
presumably better MS prognosis in Caucasian pop-
ulations, but also with neuromyelitis optica (NMO),
a mainly humoral demyelinating disease entity,
3,5
A protective role of the HLA-DRB1*16 allele also
emerged, expanding our knowledge in the field.
5
Previous studies in AOMS correlated HLA genotype
with a series of clinical and paraclinical character-
istics. Besides age of disease onset, proposed
correlations suggested sex predominance,
10,11
spe-
cific phenotype and disease course,
12–14
as well as
magnetic resonance imaging (MRI) findings and
response to treatment.
15,16
Similar studies are largely
missing in POMS,
17–19
creating space for further
research toward a more personalized prognosis and
therapeutic decision-making.
This study investigated the influence of HLA-DRB1
alleles on disease risk and/or protection in a Hellenic
sample of 50 POMS patients, using 144 AOMS
patients and 246 healthy controls (HCs) for compar-
isons. Clinical and imaging correlations with
specific HLA genotypes were also examined in the
Hellenic population for the first time.
Materials and methods
Patients
The First Department of Neurology of National
and Kapodistrian University of Athens (NKUA)
is the reference center for a wide spectrum of neu-
rological disorders, including MS and other demye-
linating diseases. MS patients are admitted
in our Demyelinating Diseases Unit and are fre-
quently followed up in the outpatient clinic. POMS
patients were enrolled in our inpatient/outpatient
Demyelinating Diseases Unit, as well as in other
pediatric neurology reference centers. Fifty POMS
patients (32 females, 18 males, mean age 26.7 
10.4 years old) fulfilling the IPMSSG
(International Pediatric Multiple Sclerosis Study
Group) criteria for POMS diagnosis were selected.
20
All patients and legal guardians provided written
informed consent. The study received ethical
approval by the Hospital Ethics Committees, consis-
tent with the Declaration of Helsinki.
At this time, no patients were identified with primary
progressive MS (PPMS), while 11 (22%) patients
had the secondary progressive type (SPMS). The
mean age of disease onset was 15.3 2.8 years
old; the mean disease duration was 138.6 114.1
months and the median Expanded Disability Status
Scale (EDSS) was 2.8 1.5.
21
We used two differ-
ent control groups of Hellenic origin; an AOMS
group with 144 AOMS patients from our center
and a general population group of 246 HCs who
were genotyped in the past, from centers all over
the country, in the biggest genotyping research
study in the Hellenic population, contacted by the
reference Immunogenetics and Histocompatibility
Center in Greece.
22
AOMS patients fulfilled the
McDonald criteria of 2010.
23
Valid MRI results of
the brain, cervical and spinal cord were available for
all patients, while cerebrospinal fluid (CSF) (i.e.
presence of oligoclonal bands, OCBs) assessments
were available in 36 (72%) of the patients, with
31 (86.1%) being positive for OCBs. With regards
to the presence of OCBs in the CSF, only some
patients had been subjected to CSF analysis, as this
was not a prerequisite for the MS diagnosis accord-
ing to the revised 2010 McDonald criteria for AOMS
and the IPMSSG 2013 criteria for POMS (Table 1).
The time point of HLA sampling was the time point
of all disease characteristics and MRI performance.
None of our patients was on relapse.
HLA-DRB1 genotyping
HLA genotyping was performed at the
Immunogenetics Laboratory of the First Department
of Neurology at Aeginition Hospital. High molecular
weight DNA was extracted from peripheral blood
samples (8 mL peripheral blood in sodium citrate,
ACD VacutainerV
Rtube) using the DNA extraction,
Maxi Kit (QIAGEN, Germany) as per manufac-
turer’s guidelines. HLA class II (HLA-DRB1) fre-
quencies were determined by molecular techniques
for all the specificities included in the HLA nomen-
clature of 2012 (we present only the first two digits
of each allele, for low resolution respectively).
24
The
HLA-DRB1 genotyping was performed using a PCR-
SSO (polymerase chain reaction, sequence-specific
oligonucleotide) technique, based on a method that
depends on reverse hybridization (HLA-DRB1 Plus,
INNO-LiPA, low resolution, Innogenetics, Fujirebio,
Europe) according to the manufacturer’s protocol.
Quality control has been secured. First, our HCs
are already genotyped individuals from the large
histocompatibility study of Papassavas et al. in
2000.
22
Second, we have used external controls for
our laboratory; thus we have typed random MS sam-
ples in different HLA-genotyping laboratories in
Athens and found no discrepancies in our results.
National and Kapodistrian
University of Athens,
NKUA, Aeginition Hospital,
Athens, Greece
Artemios Artemiadis,
Immunogenetics Laboratory,
First Department of
Neurology, Medical School,
National and Kapodistrian
University of Athens,
NKUA, Aeginition Hospital,
Athens, Greece
Medical School, University
of Cyprus, Cyprus
Roser Pons,
First Department of
Pediatrics, National and
Kapodistrian University of
Athens, Medical School,
Aghia Sophia Children’s
Hospital, Athens, Greece
Sotiria Mastroyianni,
Department of Neurology,
Children’s Hospital of
Athens “P. and A.
Kyriakou”, Athens, Greece
George Vartzelis,
Second Department of
Pediatrics, National and
Kapodistrian University of
Athens, School of Medicine
P. & A. Kyriakou Children’s
Hospital, Athens, Greece
Virginia Theodorou,
Department of Pediatric
Neurology, “Aghia Sophia”
Children’s Hospital, Greece
Konstantinos Kilindireas,
Demyelinating Diseases
Unit, First Department of
Neurology, Medical School,
National and Kapodistrian
University of Athens,
NKUA, Aeginition Hospital,
Athens, Greece
Leonidas Stefanis,
First Department of
Neurology, Medical School,
National and Kapodistrian
University of Athens,
NKUA, Aeginition Hospital,
Athens, Greece
Marinos Dalakas,
Neuroimmunology Unit,
Department of
Pathophysiology, National
and Kapodistrian University
of Athens Medical School,
Athens, Greece
George Chrousos,
University Research Institute
of Maternal and Child Health
& Precision Medicine,
National and Kapodistrian
University of Athens, Aghia
Sophia Children’s Hospital,
Greece
Maria Anagnostouli ,
Demyelinating Diseases Unit
& Director of
Immunogenetics Laboratory,
First Department of
Neurology, Medical School,
National and Kapodistrian
Multiple Sclerosis Journal—Experimental, Translational and Clinical
2 www.sagepub.com/msjetc

Finally, our laboratory has participated in EFI
(European Federation of Immunogenetics) quality
control in 2001.
Statistical analyses
Univariate comparisons were made using Mann–
Whitney U and chi-square tests (with Yates correc-
tion for 2 2 tables) for interval and categorical
variables, respectively. The Hardy–Weinberg pro-
portions (HWPs) for HLA-DRB1 haplotypes were
ascertained by using PyPoP software. Ewens–
Watterson (EW) homozygosity test for neutrality
was also performed. Calculation of the normalized
deviate of the homozygosity (i.e. Fnd) was done,
with positive and negative values implying direc-
tional and balancing selection, respectively. HLA-
DRB1 genotypic frequency in the subgroups of MS
patients was compared with that reported in a previ-
ous study of Hellenic HCs using chi-square tests.
22
Significance was set at 0.05. In HLA-DRB1 genotype
-related tests (except those for clinical parameters),
pvalue correction was made according to the
Benjamini–Yekutieli method (or B-Y) based on the
following formula: p(B-Y) ¼a/(R1/i), where i
denotes the number of comparisons and a¼0.05.
25
Statistical analyses were performed using SPSS 22
(SPSS Inc, Chicago, IL, USA).
Results
Non-genetic comparisons between POMS and
AOMS
POMS patients had a significantly younger age,
which primarily reflected the time of blood sam-
pling. Compared to AOMS, POMS patients also
presented with significantly lower EDSS (POMS
2.8 1.5, AOMS 3.4 1.7, p¼0.011) and clinically
Table 1. Clinical characteristics in POMS and AOMS.
POMS (N¼50)
mean SD or N(%)
AOMS (N¼144)
mean SD or N(%)p-value
Age (years) 26.7 10.4 42.3 11.9 <0.001*
Females 32/50 (64) 87/144 (60.4) 0.78
Age at onset (years) 15.3 2.8 33.5 10 <0.001*
Type of MS
Relapsing–remitting 39/50 (78) 113/144 (78.5)
Primary progressive 0/50 (0) 12/144 (8.3) 0.049*
Secondary progressive 11/50 (22) 19/144 (13.2)
Family history of MS 15/50 (30) 37/144 (25.7) 0.684
Duration of disease (months) 138.6 114.1 108.2 103 0.212
EDSS 2.8 1.5 3.4 1.7 0.011*
CIS onset 33/50 (66) 60/144 (41.7) 0.005*
CIS to CDMS (months) 26.2 35.9 27.3 27.3 0.276
First symptoms
Motor 24/48 (50) 53/135 (39.3)
Sensory 15/48 (31.3) 55/135 (40.7) 0.4
Optic neuritis 9/48 (18.8) 27/135 (20)
Number of relapses 4.9 4.6 4.1 3.2 0.794
Oligoclonal bands 31/36 (86.1) 79/103 (76.7) 0.338
Periventricular lesions 43/50 (86) 118/144 (81.9) 0.661
Subcortical lesions 27/50 (54) 79/144 (54.9) 1.0
Brainstem lesions 24/50 (48) 71/144 (49.3) 1.0
Cerebellar lesions 29/50 (58) 69/144 (47.9) 0.287
Cervical cord lesions 31/50 (62) 75/144 (52.1) 0.294
Thoracic cord lesions 18/50 (36) 56/144 (38.9) 0.847
Infratentorial lesions 32/50 (64) 86/144 (59.7) 0.715
Spinal cord lesions 32/50 (64) 90/144 (62.5) 0.985
MS ¼multiple sclerosis, POMS ¼pediatric-onset MS, AOMS ¼adult-onset MS, EDSS ¼Expanded Disability Status
Scale, CIS ¼clinically isolated syndrome, CDMS ¼clinically definite MS.
*p0.05.
University of Athens,
NKUA, Aeginition Hospital,
Athens, Greece
Gontika et al.
www.sagepub.com/msjetc 3

isolated syndrome onset (POMS 33/50 (66%),
AOMS 60/144 (41.7%), p¼0.005). We did not
find any POMS patients with PPMS (Table 1).
HLA genotypic comparisons between POMS/AOMS
and POMS/general population
Twelve distinct HLA-DRB1 alleles were identified
(total alleles: 388) in all MS patients. There were
23 homozygotes and 179 heterozygotes. The most
common genotypes were HLA-DRB1*15/DRB1*11
(6.2%) and HLA-DRB1*16/DRB1*11 (6.2%).
There were no deviations from the HWP (F(8) ¼
8.74, p¼0.364). The EW homozygosity test of neu-
trality was significantly negative (i.e. Fnd ¼1.509,
p¼0.0013), denoting a balanced selection. In sum-
mary, the most common HLA genotypes in POMS
were HLA-DRB1*11 (38%), and HLA-DRB1*15
(28%), followed by HLA-DRB1*03 (26%), HLA-
DRB1*04 (26%), and HLA-DRB1*16 (26%).
As shown in Table 2, the frequency of the HLA-
DRB1*03-positive MS patients was significantly
higher in the POMS than the AOMS population
(26%vs. 12.5%,p¼0.042), but not reaching statis-
tical significance after B-Y correction (i.e.
p0.017). The presence of the HLA-DRB1*03
genotype was also significantly higher in POMS
than in the general population (26%vs. 12.6%,
p¼0.004; Table 3), while HLA-DRB1*11 was sig-
nificantly lower in these patients (38%vs. 52%,
p¼0.033), suggesting a protective trend.
A total of 14 out of 50 POMS patients (28%) were
positive for HLA-DRB1*15 genotype, which is sig-
nificantly higher than the 16.7%genotype frequency
in the general population (p¼0.025), confirming
the well-established role of this allele in MS patho-
genesis. HLA- DRB1*15 genotype positivity was
31.9%(46/144) in AOMS similar to that of POMS
patients (p¼0.723).
Clinical comparisons among most frequent HLA
genotypes in POMS
POMS patients who were positive for the HLA-
DRB1*03 had significantly more relapses since
onset than HLA-DRB1*03-negative POMS patients
(6.9 4.9 vs. 4.2 4.4, p¼0.005), while they also
had more thoracic spinal cord lesions (61.5%
vs. 27%,p¼0.043) (Table 4). It is noted that
HLA-DRB1*03:01-positive patients had the same
disease duration with HLA-DRB1*03-negative
patients (161.6 110.9 vs. 130.5 115.6 respective-
ly, p¼0.432).
A sensitivity analysis was conducted for annual
relapse rate (ARR) by using a negative binomial
model with log(duration of disease in years) as an
offset variable after adjusting for the HLA-
DRB1*03, gender, and disease type predictors. The
HLA-DRB1*03 allele remained a strong significant
predictor of relapses (ARR for HLA-DRB1*03-pos-
itive patients 0.85 0.25 and for HLA-DRB1*
03-negative patients 0.43 0.08, p¼0.037). Also,
after adjusting for disease duration and type of MS
in a logistic regression model, HLA-DRB1*03-
Table 2. HLA genotypic comparisons between POMS and AOMS.
HLADRB1
*
allele
POMS (N¼50)
N(%)
AOMS (N¼144)
N(%) OR, 95%CI p-value
HLADRB1
*
01 4 (8) 23 (16) 0.46, 0.15–1.4 0.235
HLADRB1
*
03 13 (26) 18 (12.5) 2.46, 1.1–5.49 0.042
*
HLADRB1
*
04 13 (26) 28 (19.4) 1.46, 0.68–3.1 0.323
HLADRB1
*
07 3 (6) 14 (9.7) 0.59, 0.16–2.16 0.567
HLADRB1
*
10 3 (6) 5 (3.5) 1.77, 0.41–7.71 0.427
HLADRB1
*
11 19 (38) 53 (36.8) 1.05, 0.54–2.04 0.867
HLADRB1
*
12 2 (4) 5 (3.5) 1.16, 0.22–6.17 1.0
HLADRB1
*
13 8 (16) 25 (17.4) 0.91, 0.38–2.17 1.0
HLADRB1
*
14 4 (8) 12 (8.3) 0.96, 0.29–3.11 1.0
HLADRB1
*
15 14 (28) 46 (31.9) 0.83, 0.41–1.69 0.723
HLADRB1
*
16 13 (26) 42 (29.2) 0.85, 0.41–1.77 0.719
MS ¼multiple sclerosis, POMS ¼pediatric-onset MS, AOMS ¼adult-onset MS, OR ¼odds ratio, CI ¼confidence
interval.
Fisher’s exact chi-square tests were performed.
*p0.05.
Multiple Sclerosis Journal—Experimental, Translational and Clinical
4 www.sagepub.com/msjetc

positive patients were more likely to have thoracic
spinal cord lesions than HLA-DRB1*03-negative
patients (OR 5.66, 95%CI 1.17–27.35, p¼0.031).
No other significant association was found between
HLA and any other demographic or clinical
parameter.
Discussion
MHC represents a cluster of highly polymorphic
genes, which encode cell-surface proteins on
antigen-presenting cells of the peripheral (macro-
phages and dendritic cells) and central (astrocytes
and microglia) immune system, assisting antigen
presentation to T-lymphocytes and participating in
the initiation of the inflammatory cascade.
26
POMS
patients manifest increased activation of CD4þ
T memory cells and increased proportions of Th17
central memory cells compared to HCs and AOMS
patients, along with increased levels of pro-
inflammatory and fewer regulatory B cells.
27,28
Moreover, dozens of environmental factors and
exposures increase the risk for developing AOMS
or POMS through their interplay with the HLA
locus, including and, probably not limited to,
Epstein–Barr virus (EBV) remote infection, expo-
sure to ultraviolet radiation and vitamin D status,
obesity, puberty, and smoking.
18,19,29–31
We report the results of low-resolution HLA-DRB1
genotyping, as done in other Caucasian populations,
and its possible clinical and imaging impact on
POMS in a representative Hellenic sample of 50
POMS patients, comparing them to 144 AOMS
patients and 246 HCs. On a rough epidemiological
basis, no major or meaningful differences were
noticed regarding the clinical and imaging character-
istics of our POMS patients compared to other pedi-
atric populations.
1
More than 95%of POMS
patients follow a relapsing–remitting course at
onset, while PPMS is exceptionally rare, occurring
in less than 2%of children.
1,32
In our study, none of
the POMS patients presented with primary progres-
sive disease, which can be attributed to our sample
size compared to that of large, multicenter series.
32
There are no sufficient data regarding the exact time
interval for progression of our POMS to SPMS.
Nevertheless, 11 patients (22%) progressed to sec-
ondary progressive disease, a percentage in accor-
dance with other series.
32
Regarding HLA genotyping, we reconfirm HLA-
DRB1*15 allele as more frequent in POMS patients
than HCs, but do not observe any association
between the presence of HLA-DRB1*15 and earlier
age of MS onset.
5
In our research we had no infor-
mation regarding the genotypes of the patients’
parents. As a result, a possible parent of origin
effect which could correlate lower age of onset
with the maternally or paternally transmitted genes
could not be tested. A protective effect of
HLA-DRB1*11 allele was also noticed, as MS
Table 3. HLA genotypic comparisons between POMS and general population.
HLADRB1
*
alleles
POMS (N¼50)
N(%)
General population
(N¼246)
N(%) OR, 95%CI p-value
HLADRB1
*
01 4 (8) 36 (14.7) 0.51, 0.17–1.50 0.128
HLADRB1
*
03 13 (26) 31 (12.6) 2.44, 1.17–5.10 0.004*
¥
HLADRB1
*
04 13 (26) 45 (18.3) 1.57, 0.77–3.19 0.11
HLADRB1
*
07 3 (6) 33 (13.4) 0.41, 0.12–1.40 0.092
HLADRB1
*
10 3 (6) 6 (2.4) 2.55, 0.62–10.57 0.115
HLADRB1
*
11 19 (38) 128 (52.0) 0.09, 0.04–0.18 0.033
*
HLADRB1
*
12 2 (4) 5 (2) 2.01, 0.38–10.66 0.307
HLADRB1
*
13 8 (16) 44 (17.8) 0.87, 0.38–1.99 0.441
HLADRB1
*
14 4 (8) 22 (8.9) 1.94, 0.81–4.65 0.5
HLADRB1
*
15 14 (28) 41 (16.7) 1.94, 1.01–3.93 0.025
*
HLADRB1
*
16 13 (26) 65 (26.5) 0.98, 0.49–1.96 1.0
MS ¼multiple sclerosis, POMS ¼pediatric-onset MS, AOMS ¼adult-onset MS, OR ¼odds ratio, CI ¼confidence
interval.
Fisher’s exact chi-square tests were performed.
*p0.05.
¥
p0.017 according to the B-Y correction method.
Gontika et al.
www.sagepub.com/msjetc 5

patients show decreased frequencies of this allele,
irrespectively of age at disease onset, a finding
also confirmed in older studies.
5,33
We have previously shown that the HLA-DRB1*03
allele is associated with disease risk.
5
This allele
has been associated with MS susceptibility,
34
and
with the presence of IgM OCBs. In older studies,
the HLA-DRB1*03 allele did not affect clinical
disease severity, cognition, or cerebral atrophy,
although in one study it was associated with better
MS prognosis.
3,15
Interestingly, the HLA-DRB1*03
allele has been strongly associated with NMO,
a mainly humoral, anti-AQP4 disease entity.
35
The
higher frequency of HLA-DRB1*03 in POMS than
AOMS patients (p¼0.042*) and especially HCs
(p¼0.004, p0.017 in B-Y correction method)
was clearly demonstrated. This correlation, and at
such a statistically significant degree, could indicate
a probably enhanced role of humoral immunity in
POMS, which, if confirmed, could shift our thera-
peutic strategies to B-cell-directed therapies in the
future. To our knowledge, such a finding concerning
the HLA-DRB1*03 allele risk in POMS has not been
reported in other populations.
The clinical significance of HLA-DRB1*03 predom-
inance is strengthened further by the comparative
analysis of patients’ clinical characteristics on the
basis of the most frequently observed HLA geno-
types. HLA-DRB1*03-positive POMS patients pre-
sent an increased number of clinical relapses and
an enhanced lesion burden at the thoracic level,
compared to HLA-DRB1*03-negative patients. It is
noted that HLA-DRB1*03:01-positive patients had
the same disease duration and the above effects
remained significant in the sensitivity analysis that
has been accomplished. On the basis of this finding,
Table 4. Demographic and clinical comparisons between HLA-DRB1
*
03-positive and HLA-DRB1
*
03-neg-
ative POMS patients.
HLA-DRB1
*
03
positive
HLA-DRB1
*
03
negative OR, 95%CI p-value
a
Age (years) 27.9 11.4 26.3 10.1 - 0.723
Females 10/13 (76.9%) 22/37 (59.5%) 2.27, 0.53–9.67 0.328
Age of onset (years) 14.4 3.4 15.6 2.6 – 0.337
Relapsing MS 8/13 (61.5%) 26/37 (70.3%) 0.68, 0.18–2.54 0.731
Family history of MS 4/13 (30.8%) 11/37 (29.7%) 1.05, 0.27–4.15 1.0
Duration of disease (months) 161.6 110.9 130.5 115.6 – 0.432
EDSS 3.2 1.5 2.6 1.5 – 0.180
CIS onset 4/13 (30.8%) 13/37 (35.1%) 0.82, 0.21–3.19 1.0
CIS to CDMS (months) 30.6 44.5 24.6 33.0 – 0.619
First symptoms
Motor 6/12 (50%) 18/36 (50%)
Sensory 3/12 (25%) 12/36 (33.3%) – 0.766
Optic neuritis 3/12 (25%) 6/36 (16.7%)
Number of relapses 6.9 4.9 4.2 4.4 – 0.005
*
Oligoclonal bands 5/6 (83.3%) 26/30 (86.7%) 0.77, 0.7–8.41 1.0
Periventricular lesions 11/13 (84.6%) 32/37 (86.5%) 0.86, 0.15–5.08 1.0
Subcortical lesions 8/13 (61.5%) 19/37 (51.4%) 1.52, 0.42–5.51 0.747
Brainstem lesions 4/13 (30.8%) 20/37 (54.1%) 0.38, 0.10–1.45 0.202
Cerebellar lesions 7/13 (53.8%) 22/37 (59.5%) 0.80, 0.22–2.84 0.754
Cervical Cord lesions 10/13 (76.9%) 21/37 (56.8%) 2.54, 0.60–10.77 0.320
Thoracic cord lesions 8/13 (61.5%) 10/37 (27%) 4.32, 1.14–16.37 0.043
*
Infratentorial lesions 7/13 (53.8%) 25/37 (67.6%) 0.56, 0.154–2.03 0.504
Spinal cord lesions 10/13 (76.9%) 22/37 (59.5%) 2.27, 0.53–9.67 0.328
MS ¼multiple sclerosis, POMS ¼pediatric-onset MS, EDSS ¼Expanded Disability Status Scale, CIS ¼clinically
isolated syndrome, CDMS ¼clinically definite MS, OR ¼odds ratio, CI ¼confidence interval.
a
Mann–Whitney U tests for numerical and Fisher’s chi-square exact test for categorical characteristics.
*p0.05.
Multiple Sclerosis Journal—Experimental, Translational and Clinical
6 www.sagepub.com/msjetc

we suggest that the HLA-DRB1*03 allele not only
predisposes to POMS, but is also correlated with
increased disease activity. This finding needs further
verification in larger samples, even in different
ethnic groups, for safer conclusions. There is paucity
of HLA genotypic/clinical correlation studies in
POMS, with only a few exceptions. Specifically,
there are some isolated correlations of HLA-
DRB1*15 with a low vitamin D-dependent relapse
rate,
18,19
and possibly a later age at menarche.
19
Limitations in our study are the low-resolution tech-
nique performed for HLA genotyping and the rela-
tively small sample size. Nevertheless, POMS
accounts for approximately 3–5%of all MS cases
and, thus, all studies in POMS have inherent diffi-
culties. In this regard, our sample of 50 Hellenic
POMS patients represents a proportionally satisfac-
tory number. We would like to underline that all the
participants of our POMS, AOMS, and HC groups
come from reference centers from all over Greece.
In the Hellenic population no allelic association of
HLA-DRB1 has been found concerning the age and
gender, and our POMS and HC groups studied here
have the same age range (approximately 20–55 years
old). Thus, the low-resolution HLA genotyping rep-
resents a satisfactory process, as a first step concern-
ing the POMS sample in the Hellenic population and
the HLA-DRB1*03 allele cannot be altered by high-
resolution typing, but only to be enriched with more
digits, corresponding each high-resolution level.
In conclusion, our study reports in a relatively large
country-wide pediatric MS sample the HLA-DRB1
frequencies, as well as the correlations of specific
HLA-DRB1 alleles with clinical and MRI character-
istics in Hellenic POMS.
Conflict of interests
The author(s) declared no potential conflicts of interest
with respect to the research, authorship, and/or publicatio-
nof this article.
Funding
The author(s) received no financial support for the
research, authorship, and/or publication of this article.
ORCID iDs
Artemios Artemiadis https://orcid.org/0000-0001-
9435-9644
Maria Anagnostouli https://orcid.org/0000-0001-8934-
670X
References
1. Chitnis T. Pediatric central nervous system demyelin-
ating diseases. Continuum 2019; 25: 793–814.
2. Waubant E, Lucas R, Mowry E, et al. Environmental
and genetic risk factors for MS: an integrated review.
Ann Clin Transl Neurol 2019; 6: 1905–1922.
3. Katsavos S and Anagnostouli M. Biomarkers in mul-
tiple sclerosis: an up-to-date overview. Mult Scler Int
2013; 2013: 340508.
4. Moutsianas L, Jostins L, Beecham AH, et al. Class II
HLA interactions modulate genetic risk for multiple
sclerosis. Nat Genet 2015; 47: 1107–1113.
5. Anagnostouli MC, Manouseli A, Artemidiadis AK,
et al. HLA-DRB1* allele frequencies in pediatric,
adolescent and adult-onset multiple sclerosis
patients, in a Hellenic sample. Evidence for new and
established associations. J Mult Scler 2014; 1:
2376–0389.
6. Banwell B, Bar-Or A, Arnold DL, et al. Clinical, envi-
ronmental, and genetic determinants of multiple scle-
rosis in children with acute demyelination: a
prospective national cohort study. Lancet Neurol
2011; 10: 436–445.
7. Disanto G, Magalhaes S, Handel AE, et al. HLA-
DRB1 confers increased risk of pediatric-onset MS
in children with acquired demyelination. Neurology
2011; 76: 781–786.
8. Gontika MP and Anagnostouli MC. Anti-myelin
oligodendrocyte glycoprotein and human leukocyte
antigens as markers in pediatric and adolescent multi-
ple sclerosis: on diagnosis, clinical phenotypes, and
therapeutic responses. Mult Scler Int 2018; 2018:
8487471.
9. Wu J, Qiu W, Castley A, et al. Modifying effects of
HLA-DRB1 allele interactions on age at onset of mul-
tiple sclerosis in Western Australia. Mult Scler J 2009;
16: 15–20.
10. Hensiek AE, Sawcer SJ, Feakes R, et al. HLA-DR 15
is associated with female sex and younger age at diag-
nosis in multiple sclerosis. J Neurol Neurosurg
Psychiatry 2002; 72: 184–187.
11. Irizar H, Mu~
noz-Culla M, Zuriarrain O, et al.
HLA-DRB1*15:01 and multiple sclerosis: a female
association? Mult Scler 2012; 18: 569–577.
12. Barcellos LF, Oksenberg JR, Green AJ, et al. Genetic
basis for clinical expression in multiple sclerosis.
Brain 2002; 125: 150–158.
13. Briggs FBS, Yu JC, Davis MF, et al. Multiple sclero-
sis risk factors contribute to onset heterogeneity.
Mult Scler Relat Disord 2019; 28: 11–16.
14. Van Der Walt A, Stankovich J, Bahlo M, et al.
Heterogeneity at the HLA-DRB1 allelic variation
locus does not influence multiple sclerosis disease
severity, brain atrophy or cognition. Mult Scler J
2011; 17: 344–352.
15. Zivadinov R, Uxa L, Zacchi T, et al. HLA genotypes
and disease severity assessed by magnetic resonance
imaging findings in patients with multiple sclerosis.
J Neurol 2003; 250: 1099–1106.
Gontika et al.
www.sagepub.com/msjetc 7

16. Hoffmann S, Cepok S, Grummel V, et al.
HLA-DRB1*0401 and HLA-DRB1*0408 are strongly
associated with the development of antibodies against
interferon-beta therapy in multiple sclerosis. Am J
Hum Genet 2008; 83: 219–227.
17. Graves JS, Barcellos LF, Shao X, et al. Genetic pre-
dictors of relapse rate in pediatric MS. Mult Scler
2016; 22: 1528–1535.
18. Graves JS, Barcellos LF, Krupp L, et al. Vitamin D
genes influence MS relapses in children. Mult Scler.
Epub ahead of print 13 May 2019. DOI: 10.1177/
1352458519845842.
19. Bove R, Chua AS, Xia Z, et al. Complex relation
of HLA-DRB1*1501, age at menarche, and age at
multiple sclerosis onset. Neurol Genet 2016; 2: e88.
20. Krupp LB, Tardieu M, Amato MP, et al. International
Pediatric Multiple Sclerosis Study Group criteria for
pediatric multiple sclerosis and immune-mediated
central nervous system demyelinating disorders: revi-
sions to the 2007 definitions. Mult Scler 2013; 19:
1261–1267.
21. Kurtzke JF. Rating neurologic impairment in multiple
sclerosis: an Expanded Disability Status Scale
(EDSS). Neurology 1983; 33: 1444–1452.
22. Papassavas EC, Spyropoulou-Vlachou M, Papassavas
AC, et al. MHC class I and class II phenotype, gene,
and haplotype frequencies in Greeks using molecular
typing data. Hum Immunol 2000; 61: 615–623.
23. Polman CH, Reingold SC, Banwell B, et al.
Diagnostic criteria for multiple sclerosis: 2010 revi-
sions to the McDonald criteria. Ann Neurol 2011; 69:
292–302.
24. Marsh SG. Nomenclature for factors of the HLA
system, update June 2012. Tissue Antigens 2012; 80:
289–293.
25. Narum SR. Beyond Bonferroni: less conservative
analyses for conservation genetics. Conserv Genet
2006; 7: 783–787.
26. Chitnis T and Weiner HL. CNS inflammation and-
neurodegeneration. J Clin Invest 2017; 127:
3577–3587.
27. Vargas-Lowy D, Kivis€
akk P, Gandhi R, et al.
Increased Th17 response to myelin peptides in pedi-
atric MS. Clin Immunol 2013; 146: 176–184.
28. Schwarz A, Balint B, Korporal-Kuhnke M, et al. B-cell
populations discriminate between pediatric- and adult-
onset multiple sclerosis. Neurol Neuroimmunol
Neuroinflamm 2017; 4: e309.
29. Nourbakhsh B, Rutatangwa A, Waltz M, et al.
Heterogeneity in association of remote herpes virus
infections and pediatric MS. Ann Clin Transl Neurol
2018; 5: 1222–1228.
30. Hedstr€
om AK, Bomfim IL, Barcellos L, et al.
Interaction between adolescent obesity and HLA risk
genes in the etiology of multiple sclerosis. Neurology
2014; 82: 865–872.
31. Hedstr€
om AK, Sundqvist E, B€
a€
arnhielm M, et al.
Smoking and two human leukocyte antigen genes
interact to increase the risk for multiple sclerosis.
Brain 2011; 134: 653–664.
32. Renoux C, Vukusic S, Mikaeloff Y, et al. Natural his-
tory of multiple sclerosis with childhood onset. N Engl
J Med 2007; 356: 2603–2613.
33. Ramagopalan SV, Morris AP, Dyment DA, et al. The
inheritance of resistance alleles in multiple sclerosis.
PLoS Genet 2007; 3: 1607–1613.
34. Patsopoulos NA, Barcellos LF, Hintzen RQ, et al.
Fine-mapping the genetic association of the major his-
tocompatibility complex in multiple sclerosis: HLA
and non-HLA effects. PLoS Genet 2013; 9: e1003926.
35. Brum DG, Barreira AA, dos Santos AC, et al. HLA-
DRB association in neuromyelitis optica is different
from that observed in multiple sclerosis. Mult Scler
2010; 16: 21–29.
Multiple Sclerosis Journal—Experimental, Translational and Clinical
8 www.sagepub.com/msjetc