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\begin{document}

\title{Polymorphisms and haplotypes of IL2RA, IL-10, IFN-\selectlanguage{greek}γ, \selectlanguage{english}IRF5, and CCR2 are
associated with Epstein-Barr virus-associated hemophagocytic
lymphohistiocytosis in children\selectlanguage{english}}



\author[1]{Nannan Tang}%
\author[1]{Junbin Huang}%
\author[1]{Chun Chen}%
\author[2]{Xiaojun Wu}%
\author[3]{Hong Xu}%
\author[4]{Guohua Chen}%
\author[1]{Hongman Xue}%
\affil[1]{The Seventh Affiliated Hospital Sun Yat-sen University}%
\affil[2]{Sun Yat-sen Memorial Hospital , Sun Yat-sen University, Department of Pediatrics}%
\affil[3]{The 2nd affiliated hospital of Sun Yat-Sen university}%
\affil[4]{Huizhou First Hospital}%


\vspace{-1em}



  
  \date{May 26, 2024}


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\selectlanguage{english}
\begin{abstract}
Objective: Cytokine storms are central to the development of
Epstein-Barr virus-associated hemophagocytic lymphohistiocytosis
(EBV-HLH). Previous studies showed that single nucleotide polymorphisms
(SNPs) of cytokine genes may be associated with the development of
EBV-HLH in children. We investigated the associations between SNPs and
haplotypes of interleukin-2 receptor subunit alpha (IL2RA),
interleukin-10 (IL-10), interferon gamma (IFN-\selectlanguage{greek}γ\selectlanguage{english}), IFN regulatory factor
5 (IRF5), and C-C chemokine receptor 2 (CCR2) and susceptibility to
EBV-HLH in children. Methods: 66 children with EBV-HLH and 58 healthy
EBV-seropositive controls were enrolled in the study. SNPs of IL2RA
rs2104286, rs12722489, and rs11594656, IL-10 rs1800896, rs1800871, and
rs1800872, IFN-\selectlanguage{greek}γ \selectlanguage{english}rs2430561, IRF5 rs2004640, and CCR2 rs1799864 were
assayed and genotyped using the SNaPshot technique. Results: The
frequencies of the AA genotype and A allele of IL2RA rs2104286 and IL-10
rs1800896, and the CC genotype and C allele of IL-10 rs1800872 were
significantly higher in the EBV-HLH group compared with those in the
control group, respectively. The frequencies of genotypes and alleles of
IL2RA rs2104286, IL-10 rs1800871, IFN-\selectlanguage{greek}γ \selectlanguage{english}rs2430561, IRF5 rs2004640, and
CCR2 rs1799864 were similar in both groups. In addition, the IL2RA AGT
(rs2104286-rs12722489-rs11594656) and IL-10 ACC
(rs1800896-rs1800871-rs1800872) haplotypes were also significantly more
frequent in the EBV-HLH group. Conclusions: The SNPs of IL2RA rs2104286,
IL-10 rs1800896 and rs1800872 and the haplotypes of IL2RA AGT and IL-10
ACC are highly associated with susceptibility to EBV-HLH in children.%
\end{abstract}%



\sloppy


\textbf{Materials and methods}

\textbf{Clinical data}

Sixty-six children with EBV-HLH diagnosed and treated at the Seventh
Affiliated Hospital of Sun Yat-Sen University, Sun Yat-Sen Memorial
Hospital of Sun Yat-Sen University, and Huizhou First Hospital from 2008
to 2019 were included in the study (EBV-HLH group; 28 males and 38
females, age 1.5--17 years, median age 5 years). Fifty-eight healthy
children diagnosed as EBV-seropositive during the same period were
included as the control group (32 males and 26 females, age 1--16.7
years, median age 4.3 years).

The inclusion criteria for EBV-HLH subjects were: (i) a diagnosis of HLH
and exclusion of congenital HLH\textsuperscript{{[}6{]}}, and (ii)
diagnosis of EBV infection (positive for EBV antibodies or significantly
increased EBV-DNA in tissues or peripheral blood).

\textbf{Selection of SNPs}

SNPs retrieved from the HapMap database (http://www.hapmap.org/) were
imported into Haploview 4.2 software (Broad Institute, Cambridge, MA,
USA) and SNPs with a minor allele frequency (MAF) [?]0.01 were selected.
\emph{IL2RA} rs2104286, rs12722489, and rs11594656,\emph{IL-10}
rs1800896, rs1800871, and rs1800872, \selectlanguage{greek}\emph{IFN-γ}\selectlanguage{english}rs2430561, \emph{IRF5}
rs2004640, and \emph{CCR2} rs1799864 were finally identified for further
study.

\textbf{Genotyping}

DNA was extracted from 1--2 ml peripheral whole blood using a blood DNA
extraction kit (Tiangen, China) according to the manufacturer's
directions. Gene polymorphism typing was performed using a SNaPshot
Multiplex Kit (Thermo Fisher Scientific, Applied Biosystems, Foster
City, CA ) according to the manufacturer's directions.

All the primers were designed using Primer Premier 6.0 (PREMIER Biosoft,
San Francisco, CA, USA) and synthesized by BGI Tech Solutions (Beijing
Liuhe, China). Primer sequences are listed in Table 1.\selectlanguage{english}
\begin{longtable}[]{@{}llll@{}}
\toprule
\textbf{Table 1. Primers used for polymerase chain reaction
amplification} & \textbf{Table 1. Primers used for polymerase chain
reaction amplification} & \textbf{Table 1. Primers used for polymerase
chain reaction amplification} & \textbf{Table 1. Primers used for
polymerase chain reaction amplification}\tabularnewline
SNP & & Primer sequence (5'--3') & Primer for SNP
(5'--3')\tabularnewline
rs2104286 & F & 5'-CAACGCAAAAACTCCACTCA-3' &
AAAACTCAGTAGATCTTACCACATA\tabularnewline
& R & 5'-CCTTTCCATGTCACCCTCAT-3' &\tabularnewline
rs12722489 & F & 5'-ACATCCACAGGGTCTTTGCT-3' &
TTATTTCTAGCTATTGGTGACTTATCCAAGG\tabularnewline
& R & 5'-GAGAATGGCGTGAAGGAGTC-3' &\tabularnewline
rs11594656 & F & 5'-TGGTCCATTTTCCCAGTCTC-3' &
GACATTCTAGAGAACTGGCACACTCCCCAAGGCGGTTCCTTGGTCTGTAGAG\tabularnewline
& R & 5'-TAAGGTGATCCACCCACCTC-3' &\tabularnewline
rs1800896 & F & 5'-TTCCCCAGGTAGAGCAACAC-3' &
ACACAAATCCAAGACAACACTACTAAGGCTTCTTTGGGA\tabularnewline
& R & 5'-ATGGAGGCTGGATAGGAGGT-3' &\tabularnewline
rs1800871 & F & 5'-TCAACTTCTTCCACCCCATC-3' &
GGCACTGGTGTACCCTTGTACAGGTGATGTAA\tabularnewline
& R & 5'-GTGCTCACCATGACCCCTAC-3' &\tabularnewline
rs1800872 & F & 5'-TGGAAACATGTGCCTGAGAA-3' &
CAAGCAGCCCTTCCATTTTACTTTCCAGAGACTGGCTTCCTACAG\tabularnewline
& R & 5'-GAGGGGGTGGGCTAAATATC-3' &\tabularnewline
rs2430561 & F & 5'-CGAGCTTTAAAAGATAGTTCCAAA-3' &
TAATATTCAGACATTCACAATTGATTTTATTCTTACAACACAAAATCAAATC\tabularnewline
& R & 5'-CTTCGTTGCTCACTGGGATT-3' &\tabularnewline
rs1799864 & F & 5'-CGGTGCTCCCTGTCATAAAT-3' &
ATGTCAGTCAAGCACTTCAGCTTTTTGCAGTTTATTAAGATGAGGA\tabularnewline
& R & 5'-GAGCCCACAATGGGAGAGTA-3' &\tabularnewline
rs2004640 & F & 5'-GCGGGATGAAGACTGGAGTA-3' &
GTCCCAGGCCGGCCGGGCGCACCCTGCTGTAGGCACCC\tabularnewline
& R & 5'-GAGGGAGAGCAGCAGAGC-3' &\tabularnewline
PCR: polymerase chain reaction; SNP: single nucleotide polymorphism; F:
forward primer; R: reverse primer. & PCR: polymerase chain reaction;
SNP: single nucleotide polymorphism; F: forward primer; R: reverse
primer. & PCR: polymerase chain reaction; SNP: single nucleotide
polymorphism; F: forward primer; R: reverse primer. & PCR: polymerase
chain reaction; SNP: single nucleotide polymorphism; F: forward primer;
R: reverse primer.\tabularnewline
\bottomrule
\end{longtable}

\textbf{Linkage disequilibrium analysis and haplotype construction}

The degree of linkage disequilibrium between the SNPs was analyzed using
SHEsis software\textsuperscript{{[}7{]}} and expressed as D' and
r\textsuperscript{2}, with larger values of D' and
r\textsuperscript{2}indicating a higher degree of linkage disequilibrium
between the two associated SNPs. Values of D' \textgreater{} 0.9 and
r\textsuperscript{2} \textgreater{} 0.33 are generally considered to
indicate highly interlocked genes. If D' [?] 0.8, the SNPs were considered
to be sites for the construction of haplotypes; haplotype frequencies
\textless{} 0.03 were not included in the statistical analysis.

\textbf{Statistical analysis}

Statistical analysis was performed using SPSS 26.0 (International
Business Machine Corp, Armonk, NY, USA). The frequencies of genotypes,
alleles, and haplotypes between the two groups were analyzed by
\selectlanguage{greek}χ\selectlanguage{english}\textsuperscript{2} or Fisher's exact methods. Odds ratios (ORs) with
95\% confidence intervals (CIs) were used to calculate the risk of
susceptibility. The Hardy--Weinberg equilibrium was determined to assess
the representativeness of the sample. P \textless{} 0.05 was considered
statistically significant.

\textbf{Results}

\textbf{Genotype and allele frequencies}

Genotype distributions were in Hardy--Weinberg equilibrium, indicating
that the subjects were representative of the general population. The
frequencies of the genotypes and alleles of \emph{IL2RA} , \emph{IL-10}
,\selectlanguage{greek}\emph{IFN-γ, IRF5}\selectlanguage{english} , and \emph{CCR2} between EBV-HLH and control groups
are shown in Table 2.

The \emph{IL2RA} rs2104286 AA genotype and A allele were significantly
more frequent in the EBV-HLH group than in the control group, suggesting
that they might be associated with a significantly increased risk of
EBV-HLH (OR = 2.894, 95\% CI = 1.374--6.094 and OR = 2.077, 95\% CI =
1.232--3.502, respectively). However, the frequencies of the\emph{IL2RA}
rs12722489 and rs11594656 genotypes and alleles were similar in the
EBV-HLH and control groups (P \textgreater{} 0.05).

The \emph{IL-10} rs1800896 AA genotype and A allele were more frequent
in the EBV-HLH group, suggesting that they might be associated with a
significantly increased risk of EBV-HLH (OR = 3.792, 95\% CI =
1.779--8.080 and OR = 2.194, 95\% CI = 1.279--3.763, respectively). The
frequencies of \emph{IL-10} rs1800872 AC and CC genotypes and C allele
were significantly higher in the EBV-HLH group, and the CC genotype and
C allele were also associated with a significantly increased risk of
EBV-HLH (OR = 6.222, 95\% CI = 1.330--29.109 and OR = 2.176, 95\% CI =
1.257--3.767,respectively). Meanwhile, the distribution of
\emph{IL-10}rs1800871 was similar in both groups (P \textgreater{}
0.05).

In addition, there were no significant differences in the frequencies
of\selectlanguage{greek}\emph{IFN-γ} \selectlanguage{english}rs2430561 and \emph{IRF5} rs2004640 genotypes and alleles
between the two groups (P \textgreater{} 0.05) (Table 2).\selectlanguage{english}
\begin{longtable}[]{@{}lllllll@{}}
\toprule
\textbf{Table 2. Genotype and allele frequencies in children with
Epstein-Barr virus-associated hemophagocytic lymphohistiocytosis and
controls} & \textbf{Table 2. Genotype and allele frequencies in children
with Epstein-Barr virus-associated hemophagocytic lymphohistiocytosis
and controls} & \textbf{Table 2. Genotype and allele frequencies in
children with Epstein-Barr virus-associated hemophagocytic
lymphohistiocytosis and controls} & \textbf{Table 2. Genotype and allele
frequencies in children with Epstein-Barr virus-associated
hemophagocytic lymphohistiocytosis and controls} & \textbf{Table 2.
Genotype and allele frequencies in children with Epstein-Barr
virus-associated hemophagocytic lymphohistiocytosis and controls} &
\textbf{Table 2. Genotype and allele frequencies in children with
Epstein-Barr virus-associated hemophagocytic lymphohistiocytosis and
controls} & \textbf{Table 2. Genotype and allele frequencies in children
with Epstein-Barr virus-associated hemophagocytic lymphohistiocytosis
and controls}\tabularnewline
Gene & SNPs & & Patients (freq) & Controls (freq) & \selectlanguage{greek}χ\selectlanguage{english}\textsuperscript{2}
& P value\tabularnewline
\emph{IL2RA} & rs2104286 & Genotype & & & &\tabularnewline
& & AA & 36(54.55\%) & 17(29.31\%) & 8.056 & 0.018*\tabularnewline
& & AG & 21(31.82\%) & 28(48.28\%) & &\tabularnewline
& & GG & 9(13.64\%) & 13(22.41\%) & &\tabularnewline
& & allele & & & &\tabularnewline
& & A & 93(70.45\%) & 62(53.45\%) & 7.619 & 0.006*\tabularnewline
& & G & 39(29.55\%) & 54(46.55\%) & &\tabularnewline
& rs12722489 & Genotype & & & &\tabularnewline
& & GG & 49(74.24\%) & 42(72.41\%) & 0.060 & 0.971\tabularnewline
& & AG & 14(21.21\%) & 13(22.41\%) & &\tabularnewline
& & AA & 3(4.55\%) & 3(5.18\%) & &\tabularnewline
& & allele & & & &\tabularnewline
& & G & 112(84.85\%) & 97(83.62\%) & 0.070 & 0.791\tabularnewline
& & A & 20(15.15\%) & 19(16.38\%) & &\tabularnewline
& rs11594656 & Genotype & & & &\tabularnewline
& & TT & 60(90.91\%) & 53(91.38\%) & 0.008 & 0.927\tabularnewline
& & AT & 6(9.09\%) & 5(8.62\%) & &\tabularnewline
& & allele & & & &\tabularnewline
& & T & 126(95.45\%) & 111(95.69\%) & 0.008 & 0.928\tabularnewline
& & A & 6(4.55\%) & 5(4.31\%) & &\tabularnewline
\emph{IL-10} & rs1800896 & Genotype & & & &\tabularnewline
& & AA & 39(59.09\%) & 16(27.59\%) & 12.732 & 0.002*\tabularnewline
& & AG & 21(31.82\%) & 35(60.34\%) & &\tabularnewline
& & GG & 6(9.09\%) & 7(12.07\%) & &\tabularnewline
& & allele & & & &\tabularnewline
& & A & 99(75.00\%) & 67(61.21\%) & 8.293 & 0.004*\tabularnewline
& & G & 33(25.00\%) & 49(38.79\%) & &\tabularnewline
& rs1800871 & Genotype & & & &\tabularnewline
& & TT & 18(27.27\%) & 20(34.48\%) & 1.169 & 0.557\tabularnewline
& & TC & 35(53.03\%) & 30(51.72\%) & &\tabularnewline
& & CC & 13(19.70\%) & 8(13.79\%) & &\tabularnewline
& & allele & & & &\tabularnewline
& & T & 71(53.79\%) & 70(60.34\%) & 1.082 & 0.298\tabularnewline
& & C & 61(46.21\%) & 46(39.66\%) & &\tabularnewline
& rs1800872 & Genotype & & & &\tabularnewline
& & AA & 24(36.36\%) & 32(55.17\%) & 8.472 & 0.014*\tabularnewline
& & AC & 30(45.45\%) & 24(41.38\%) & &\tabularnewline
& & CC & 12(18.18\%) & 2(3.45\%) & &\tabularnewline
& & allele & & & &\tabularnewline
& & A & 78(59.09\%) & 88(75.86\%) & 7.847 & 0.005*\tabularnewline
& & C & 54(40.91\%) & 28(24.14\%) & &\tabularnewline
\selectlanguage{greek}\emph{IFN-γ} & rs2430561 & Genotype & & & &\tabularnewline
& & TT & 50(75.76\%) & 36(62.07\%) & 4.665 & 0.097\tabularnewline
& & AT & 13(19.70\%) & 21(36.21\%) & &\tabularnewline
& & AA & 3(4.55\%) & 1(1.72\%) & &\tabularnewline
& & allele & & & &\tabularnewline
& & T & 113(85.61\%) & 93(80.17\%) & 1.296 & 0.255\tabularnewline
& & A & 19(14.39\%) & 23(19.83\%) & &\tabularnewline
\emph{IRF5} & rs2004640 & Genotype & & & &\tabularnewline
& & TT & 16(24.24\%) & 7(12.07\%) & 4.058 & 0.131\tabularnewline
& & TG & 37(56.06\%) & 33(56.90\%) & &\tabularnewline
& & GG & 13(19.70\%) & 18(31.03\%) & &\tabularnewline
& & allele & & & &\tabularnewline
& & T & 69(52.27\%) & 47(40.52\%) & 3.427 & 0.064\tabularnewline
& & G & 63(47.73\%) & 69(59.48\%) & &\tabularnewline
\emph{CCR2} & rs1799864 & Genotype & & & &\tabularnewline
& & GG & 39(59.09\%) & 38(65.52\%) & 2.390 & 0.303\tabularnewline
& & AG & 20(30.30\%) & 18(31.03\%) & &\tabularnewline
& & AA & 7(10.61\%) & 2(3.45\%) & &\tabularnewline
& & allele & & & &\tabularnewline
& & G & 98(74.24\%) & 94(81.03\%) & 1.629 & 0.202\tabularnewline
& & A & 34(25.76\%) & 22(18.97\%) & &\tabularnewline
SNP: single nucleotide polymorphism values calculated from two-sided \selectlanguage{greek}χ\selectlanguage{english}2
tests. & SNP: single nucleotide polymorphism values calculated from
two-sided \selectlanguage{greek}χ\selectlanguage{english}2 tests. & SNP: single nucleotide polymorphism values
calculated from two-sided \selectlanguage{greek}χ\selectlanguage{english}2 tests. & SNP: single nucleotide
polymorphism values calculated from two-sided \selectlanguage{greek}χ\selectlanguage{english}2 tests. & SNP: single
nucleotide polymorphism values calculated from two-sided \selectlanguage{greek}χ\selectlanguage{english}2 tests. &
SNP: single nucleotide polymorphism values calculated from two-sided \selectlanguage{greek}χ\selectlanguage{english}2
tests. & SNP: single nucleotide polymorphism values calculated from
two-sided \selectlanguage{greek}χ\selectlanguage{english}2 tests.\tabularnewline
\bottomrule
\end{longtable}

\textbf{Linkage disequilibrium analysis of identified SNPs}

The levels of linkage disequilibrium for \emph{IL2RA} rs2104286,
rs12722489, and rs11594656 and \emph{IL-10} rs1800896, rs1800871, and
rs1800872 were analyzed using SHEsis software. There was a strong
interlocking disequilibrium between \emph{IL2RA} rs2104286 and
rs12722489 (D' = 1, r\textsuperscript{2} = 0.311), and
between\emph{IL-10} rs1800871 and rs1800872 (D' = 1,
r\textsuperscript{2} = 0.651) (Fig. 1).\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image1/image1}

\end{center}
\end{figure}

\textbf{Differences in the distributions of SNP haplotypes}

Haplotypes were constructed using SHEsis software in the
order\emph{IL2RA} rs2104286-rs12722489-rs11594656 and \emph{IL
10}rs1800896-rs1800871-rs1800872, respectively, and the distributions of
the \emph{IL2RA} and \emph{IL-10} haplotypes were compared between the
EBV-HLH and control groups. As shown in Table 3, there were no
significant differences in \emph{IL2RA} AGA, GAA, GAT, and GGA between
the two groups (P \textgreater{} 0.05); however, the frequencies of AGT
and GGT were significantly higher (P = 0.006) and lower (P = 0.001),
respectively, in the EBV-HLH group. This suggested that \emph{IL2RA} AGT
was associated with an increased risk of EBV-HLH (OR = 2.102, 95\% CI =
1.229--3.593), while GGT significantly reduced the susceptibility to
EBV-HLH (OR = 0.364, 95\% CI = 0.192--0.689). There was no significant
difference in the distribution of \emph{IL-10} ATA, GCA, or GCC
haplotypes between the two groups (P \textgreater{} 0.05). However, the
frequency of \emph{IL-10} ACC was significantly higher in the EBV-HLH
group (P = 0.003, OR = 2.962, 95\% CI = 1.429--6.141), suggesting that
this haplotype may be associated with an increased risk of HLH in
children with EBV, while the significantly lower frequencies of ACA and
GTA (P = 0.020, OR = 0.253, 95\% CI = 0.073--0.876; P \textless{} 0.05,
OR = 0.218, 95\% CI = 0.093--0.513, respectively) indicated
that\emph{IL-10} ACA and GTA haplotypes may be protective factors for
EBV-HLH.\selectlanguage{english}
\begin{longtable}[]{@{}lllllll@{}}
\toprule
\textbf{Table 3. Haplotype frequencies in patients with Epstein-Barr
virus-associated hemophagocytic lymphohistiocytosis and controls} &
\textbf{Table 3. Haplotype frequencies in patients with Epstein-Barr
virus-associated hemophagocytic lymphohistiocytosis and controls} &
\textbf{Table 3. Haplotype frequencies in patients with Epstein-Barr
virus-associated hemophagocytic lymphohistiocytosis and controls} &
\textbf{Table 3. Haplotype frequencies in patients with Epstein-Barr
virus-associated hemophagocytic lymphohistiocytosis and controls} &
\textbf{Table 3. Haplotype frequencies in patients with Epstein-Barr
virus-associated hemophagocytic lymphohistiocytosis and controls} &
\textbf{Table 3. Haplotype frequencies in patients with Epstein-Barr
virus-associated hemophagocytic lymphohistiocytosis and controls} &
\textbf{Table 3. Haplotype frequencies in patients with Epstein-Barr
virus-associated hemophagocytic lymphohistiocytosis and
controls}\tabularnewline
Haplotype & Patients (freq) & Controls (freq) & \selectlanguage{greek}χ\selectlanguage{english}\textsuperscript{2} & P
& OR & 95\%CI\tabularnewline
\emph{IL2RA} rs2104286-rs12722489-rs11594656 & & & & & &\tabularnewline
A G A & 3.43(2.6\%) & 2.15(1.9\%) & NA & NA & NA & NA\tabularnewline
A G T & 89.57(67.9\%) & 59.85(51.6\%) & 7.470 &
0.006\textsuperscript{\textbf{*}} & 2.102 &
1.229\textasciitilde{}3.593\tabularnewline
G A A & 1.63(1.2\%) & 2.83(2.4\%) & NA & NA & NA & NA\tabularnewline
G A T & 18.37(13.9\%) & 16.17(13.9\%) & 0.000 & 0.998 & 1.001 &
0.486\textasciitilde{}2.063\tabularnewline
G G A & 0.94(0.7\%) & 0.02(0\%) & NA & NA & NA & NA\tabularnewline
G G T & 18.06(13.7\%) & 34.98(30.2\%) & 10.030 &
0.002\textsuperscript{\textbf{*}} & 0.364 &
0.192\textasciitilde{}0.689\tabularnewline
\emph{IL-10} rs1800896-rs1800871-rs1800872 & & & & & &\tabularnewline
A C A & 3.41(2.60\%) & 11.01(9.50\%) & 5.374 &
0.020\textsuperscript{\textbf{*}} & 0.253 &
0.073\textasciitilde{}0.876\tabularnewline
A C C & 32.21(24.40\%) & 11.40(9.80\%) & 9.05 &
0.003\textsuperscript{\textbf{*}} & 2.962 &
1.429\textasciitilde{}6.141\tabularnewline
A T A & 63.38(48.00\%) & 44.60(38.40\%) & 2.3 & 0.129 & 1.479 &
0.891\textasciitilde{}2.454\tabularnewline
G C A & 3.59(2.70\%) & 6.99(6.00\%) & 1.657 & 0.198 & 0.436 &
0.119\textasciitilde{}1.595\tabularnewline
G C C & 21.79(16.50\%) & 16.60(14.30\%) & 0.228 & 0.633 & 1.184 &
0.592\textasciitilde{}2.369\tabularnewline
G T A & 7.62(5.80\%) & 25.40(21.90\%) & 13.912 &
0.000\textsuperscript{\textbf{*}} & 0.218 &
0.093\textasciitilde{}0.513\tabularnewline
OR: odds ratio; CI: confidence interval; NA: not applicable. *P < 0.05.
& OR: odds ratio; CI: confidence interval; NA: not applicable. *P <
0.05. & OR: odds ratio; CI: confidence interval; NA: not applicable. *P
< 0.05. & OR: odds ratio; CI: confidence interval; NA: not applicable.
*P < 0.05. & OR: odds ratio; CI: confidence interval; NA: not
applicable. *P < 0.05. & OR: odds ratio; CI: confidence interval; NA:
not applicable. *P < 0.05. & OR: odds ratio; CI: confidence interval;
NA: not applicable. *P < 0.05.\tabularnewline
\bottomrule
\end{longtable}

\textbf{Discussion}

EBV-HLH is a typical cytokine storm syndrome, and cytokines play a
crucial role in the pathogenesis of EBV-HLH. Wada \emph{et al}
\textsuperscript{{[}8{]}}. observed that EBV-infected patients had
different serum cytokine levels and different severities of clinical
manifestations. The synthesis, secretion, and biological effects of
cytokines are regulated by their genes, and many studies have
demonstrated important roles of cytokine gene polymorphisms in
regulating cytokine levels and influencing the development of
diseases\textsuperscript{{[}9-11{]}}. SNPs are the most common form of
gene polymorphisms, and we therefore hypothesized that cytokine SNPs
might play an important role in the pathogenesis of and susceptibility
to EBV-HLH.

Soluble IL-2 receptor (sIL-2R, also known as sCD25) is a marker of
T-lymphocyte activation and is elevated in conditions such as infection,
trauma, malignant hematological disorders, and autoimmune diseases.
sIL-2R is currently used as an indicator of the severity of several
diseases, and its level was shown to be directly proportional to the
degree of inflammation\textsuperscript{{[}12{]}}. Excessive activation
of CD8+ T cells and significantly increased serum levels of sIL-2R have
made sIL-2R an important diagnostic and disease marker for HLH in
patients with EBV. The HLH-2004 diagnostic guidelines even added serum
sIL-2R level [?]2400 U/ml as a new diagnostic criterion for
HLH\textsuperscript{{[}13{]}}. Furthermore, SNPs of the \emph{IL2RA}gene
encoding sIL-2R are associated with the development of autoimmune
diseases, such as diabetes, multiple sclerosis, and
baldness\textsuperscript{{[}14-16{]}}.

In the current study, we investigated the three most common
\emph{IL2RA}loci, rs2104286, rs12722489, and rs11594656, and found that
the rs2104286 AA genotype and A allele frequencies were significantly
higher in children with EBV-HLH than in controls, suggesting that these
might be risk factors for the development of EBV-HLH in children.
rs2104286 is located in the first intron of the \emph{IL2RA} gene, which
is not a coding or regulatory region; however, this SNP has been
associated with several autoimmune diseases, including multiple
sclerosis and rheumatoid arthritis\textsuperscript{{[}17{]}}. Carriers
of the rs2104286 A allele have elevated sIL-2R levels, diminished IL-2R
signaling, increased granulocyte-macrophage colony-stimulating factor
production by memory CD4+ T cells, increased frequency of CD25+ na\selectlanguage{ngerman}ïve T
cells, and decreased CD25 expression on the surface of memory
CD127+CD25+ and Treg cells\textsuperscript{{[}15, 18{]}}. Moreover, the
rs2104286 polymorphism can affect the activity of enhancer elements in
the first intron of the \emph{IL2RA} gene and the binding affinity of
the transcription factor TRAF4, as well as mRNA processing and
half-life, to influence \emph{IL2RA}
expression\textsuperscript{{[}17{]}}. Dendrou\emph{et al.}
\textsuperscript{{[}19{]}} found that the rs2104286 AA genotype
upregulated CD25 (IL-2R\selectlanguage{greek}α\selectlanguage{english}) expression levels on CD4+ na\selectlanguage{ngerman}ïve T cells and
CD14+CD16+ monocytes, thereby enhancing T-cell activation, and this
genotypic effect persisted after T cell activation, leading to a high
proportion of CD69+CD4+ naïve T cells that upregulated CD25 in donors
carrying the susceptible rs2104286 allele. We thus deduced that the
rs2104286 polymorphism might affect the pathogenesis of EBV-HLH by
upregulating CD25 expression on CD4+ naïve T cells, thus increasing
sIL-2R levels.

IL-10 is produced by Th2 cells and is an important anti-inflammatory
factor. Cellular secretion of IL-10 is significantly increased during
microbial infections and autoimmune diseases. It is involved in the
development of HLH by modulating immunity and suppressing the
inflammatory response. Significantly elevated serum IL-10 levels are a
characteristic cytokine pattern of HLH\textsuperscript{{[}20{]}}.
Sánchez \emph{et al.} \textsuperscript{{[}21{]}} reported that certain
SNPs in the promoter region of the \emph{IL-10} gene might influence
IL-10 production and response intensity, and were associated with
disease development. \emph{IL-10} SNPs were shown to be associated with
various diseases, such as sepsis, asthma, multiple sclerosis, ankylosing
spondylitis, gastric cancer, and coronary heart
disease\textsuperscript{{[}4, 21, 22{]}}. In the current study, we
selected rs1800896, rs1800871, and rs1800872 for testing and showed that
the frequencies of the AA genotype and A allele of rs1800896 and the CC
genotype and C allele of rs1800872 were significant higher in children
with EBV-HLH than in controls, suggesting that these might be
predisposing risk factors for the development of EBV-HLH in children.
Wang \emph{et al} .\textsuperscript{{[}9{]}} studied the relationship
between the rs1800872 and children with EBV-HLH, and similarly showed
that the rs1800872 CC genotype and C allele frequencies were higher in
children with EBV-HLH than in patients with infectious mononucleosis
(IM) and in healthy controls. rs1800896 is located at a putative
ETS-like transcription factor-binding site and rs1800872 at a putative
signal transducer and activator of transcription 3-binding site and
regions of negative regulatory function\textsuperscript{{[}23, 24{]}}.
Polymorphisms at these sites may thus influence transcription factor
binding and consequent \emph{IL-10} transcription and IL-10 serum
levels, thus affecting disease susceptibility\textsuperscript{{[}25,
26{]}}. IL-10 levels are significantly elevated in children with
EBV-HLH. Numerous studies have shown that the C allele of rs1800872 is
associated with high levels of \textsuperscript{{[}27, 28{]}}, while the
relationships between the G and A alleles of rs1800896 and IL-10 levels
remain controversial. Rees \emph{et al.} \textsuperscript{{[}24,
29{]}}showed that the rs1800896 A allele enhanced transcription of
the\emph{IL-10} gene and resulted in high levels of IL-10. In contrast,
Engelhardt \emph{et al.} \textsuperscript{{[}30-32{]}} showed that the
rs1800896 G allele was associated with higher IL-10 production, while
the rs1800896 A allele resulted in lower IL-10 production. The
frequencies of \emph{IL-10} polymorphisms differ among
populations\textsuperscript{{[}33{]}}, and IL-10 plays different roles
in different diseases, although its regulatory mechanisms are not fully
understood. Numerous genetic correlation studies and a few functional
studies have shown that the regulation of IL-10 is complex and
multifactorial. Further studies are therefore needed to determine how
rs1800896 and rs1800872 regulate the production of IL-10 and affect the
susceptibility to and clinical course of EBV-HLH in children. In
addition, the rs1800896 polymorphism can also influence EBV infection
status. Consistent with the results of the present study,
Helminen\emph{et al.} \textsuperscript{{[}34{]}} also showed that
individuals carrying the rs1800896 A allele were more susceptible to
severe EBV infection. The rs1800896 and rs1800872 polymorphisms may thus
influence EBV-HLH susceptibility by synergistically regulating IL-10
expression and EBV infection status.

Haplotype analysis provides more accurate genetic information than
analysis of individual SNPs. Comparisons between children with EBV-HLH
and controls based on estimated haplotype frequencies thus provide a
better understanding of the role of cytokine SNPs in EBV-HLH
susceptibility. The present results showed that the frequency of
the\emph{IL2RA} AGT (rs2104286-rs12722489-rs11594656) haplotype was
significantly increased while that of the GGT haplotype was
significantly decreased in children with EBV-HLH. The frequency of
the\emph{IL-10} ACC (rs1800896-rs1800871-rs1800872) haplotype was also
significantly increased, whereas the frequencies of the ACA and GTA
haplotypes were significantly decreased. In addition, some of these
haplotypes showed strong linkage disequilibrium, suggesting
that\emph{IL2RA} and \emph{IL-10} haplotype association and linkage
disequilibrium may play important roles in the susceptibility to
EBV-HLH. The results of this study suggest that the \emph{IL2RA} AGT
haplotype may increase the risk of EBV-HLH; however, no other studies
have simultaneously tested the \emph{IL2RA} rs2104286, rs12722489, and
rs11594656 loci, and studies of the AGT haplotype in other diseases are
lacking, and further studies are therefore needed to validate the roles
of the AGT and GGT haplotypes. The common
\emph{IL-10}rs1800896-rs1800871-rs1800872 haplotypes are GCC, ACC, and
ATA. The ACC haplotype is associated with moderate IL-10
production\textsuperscript{{[}35{]}}, but its role in different diseases
is controversial. Marangon \emph{et al.}
\textsuperscript{{[}36{]}}showed that this haplotype was a protective
factor in diffuse large B-cell lymphoma, while Gao \emph{et al.}
\textsuperscript{{[}28{]}}showed that it was associated with an
increased risk of IgA nephropathy.

We also analyzed \selectlanguage{greek}\emph{IFN-γ} \selectlanguage{english}rs2430561, \emph{IRF5} rs2004640,
and\emph{CCR2} rs2004640, but found no significant associations between
these SNPs and susceptibility to EBV-HLH. IFN-\selectlanguage{greek}γ \selectlanguage{english}is an important cytokine
produced by a variety of immune cells in response to inflammatory
stimuli. It regulates the body's immune response, and is significantly
elevated in children with EBV-HLH. \selectlanguage{greek}\emph{IFN-γ} \selectlanguage{english}rs2430561 has been shown
to affect susceptibility to diseases such as systemic lupus
erythematosus, sepsis, ankylosing spondylitis, and breast
cancer\textsuperscript{{[}37-40{]}}, but no previous association between
this SNP and HLH has been reported. IRF5 is a member of the IRF
transcription factor family that plays important roles in both the
interferon pathway and the Toll-like receptor signaling pathway, and is
closely associated with many autoimmune diseases\textsuperscript{{[}41,
42{]}}. \emph{IRF5} rs2004640 has been associated with susceptibility to
diseases such as systemic juvenile idiopathic arthritis(sJIA), systemic
lupus erythematosus(SLE), and systemic
sclerosis(SS)\textsuperscript{{[}43, 44{]}}. Yanagimachi \emph{et al.}
\textsuperscript{{[}42{]}} investigated the relationship between
rs2004640 and susceptibility to secondary HLH, such as macrophage
activation syndrome and EBV-HLH, and found that the GT/TT genotype at
this locus increased overall susceptibility to secondary HLH, but not to
EBV-HLH, consistent with the results of the present study. The chemokine
receptor CCR2 plays a key role in several diseases, and the
\emph{CCR2}rs1799864 gene polymorphism has also been associated with
susceptibility to diseases such as ischemic stroke and multiple
sclerosis\textsuperscript{{[}45, 46{]}}. Ou \emph{et al}
.\textsuperscript{{[}47{]}} found no significant difference in the
rs1799864 polymorphism between children with and without HLH, suggesting
that this locus may be independent of HLH susceptibility, consistent
with the current study. However, the paucity of relevant studies and the
limited numbers of cases mean that further studies are needed to confirm
the relationships between the above genetic polymorphisms and EBV-HLH.

This study had some limitations. First, the relatively small sample size
may have limited the statistical power, and more multicenter studies
with larger sample sizes are therefore needed to validate our findings.
Second, because of technical limitations in the quantitative analysis of
cytokines before 2014, cytokine profiles were not available for more
than half of the patients. Unfortunately, we were therefore unable to
analyze the relationships among SNPs, cytokine secretion, and HLH.

\par\null

\textbf{Conclusions}

This study analyzed the associations between polymorphisms and
haplotypes of \selectlanguage{greek}\emph{sIL-2R, IL-10, IFN-γ, IRF5}\selectlanguage{english} , and \emph{CCR2} and
susceptibility to EBV-HLH in children. The \emph{IL2RA} rs2104286 AA
genotype and A allele, \emph{IL-10} rs1800896 AA genotype and A allele,
and \emph{IL-10} rs1800872 CC genotype and C allele, as well as
the\emph{IL2RA} AGT and \emph{IL-10} ACC haplotypes were identified as
possible susceptibility risk factors for HLH in children with EBV.

\textbf{Data Availability Statement}

The datasets for this article are not publicly available due to concerns
regarding participant/patient

anonymity. Requests to access the datasets should be directed to the
corresponding author.

\textbf{Ethics Statement}

The studies involving human participants were reviewed and approved by
the Ethics Committee of the Seventh Affiliated Hospital of Sun Yat-Sen
University, Sun Yat-Sen Memorial Hospital of Sun Yat-Sen University and
Huizhou First Hospital. Written informed consent to participate in this
study was provided by the participants' legal guardian/next of kin.

\textbf{Author Contributions}

HX and GC designed the experiments and drafted the manuscript. NT and JH
accomplished works of the data collection, DNA extraction, and
genotyping. CC and XW helped to enroll all of the patients. HX guided to
conduct the statistic works. All authors contributed to the article and
approved the submitted version.

\textbf{Funding}

This study was supported by Sanming Project of Medicine in Shenzhen
(SZSM202011004),

Science,Technology and Innovation Commission of Shenzhen Municipality(
JCYJ20190809160609727).

\textbf{Conflict of Interest}

The authors declare that the research was conducted in the absence of
any commercial or financial relationships that could be construed as a
potential conflict of interest.

\textbf{Acknowledgments}

The authors thank all the patients and their families. The authors also
thank the support and advise

from Doctor Junchao Dong, Department Immunology, Zhongshan School of
Medicine, Sun Yat-sen

University, Guangzhou, Guangdong, China.

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