The rs4759314 SNP within Hotair lncRNA is associated with risk of multiple sclerosis

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The rs4759314 SNP within HOTAIR lncRNA is associated with risk of multiple sclerosis Mohammad Taheri , Rezvan Noroozi , Saba Sadeghpour , Mir Davood Omrnai , Soudeh Ghafouri-Fard PII: DOI: Reference:

S2211-0348(20)30062-6 https://doi.org/10.1016/j.msard.2020.101986 MSARD 101986

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Multiple Sclerosis and Related Disorders

Received date: Revised date: Accepted date:

16 December 2019 27 January 2020 5 February 2020

Please cite this article as: Mohammad Taheri , Rezvan Noroozi , Saba Sadeghpour , Mir Davood Omrnai , Soudeh Ghafouri-Fard , The rs4759314 SNP within HOTAIR lncRNA is associated with risk of multiple sclerosis, Multiple Sclerosis and Related Disorders (2020), doi: https://doi.org/10.1016/j.msard.2020.101986

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Highlights 

We genotyped three HOTAIR SNPs in Iranian MS patients and healthy subjects.



The rs4759314 SNP was associated with risk of MS in allelic model.



The C G A haplotype was less prevalent in cases compared with controls.



HOTAIR might be regarded as a risk locus for MS in Iranian population.

The rs4759314 SNP within HOTAIR lncRNA is associated with risk of multiple sclerosis. Mohammad Taheri1, Rezvan Noroozi2, Saba Sadeghpour3, Mir Davood Omrnai1*, Soudeh Ghafouri-Fard3* 1. Urogenital Stem Cell Research Center, Shahid Beheshti University of Medical Science, Tehran, Iran. 2. Malopolska Centre of Biotechnology of the Jagiellonian University, Kraków, Poland 3. Department of Medical Genetics, Shahid Beheshti University of Medical Science, Tehran, Iran Corresponding authors: Mir Davood Omrani, Soudeh Ghafouri-Fard Emails: [email protected], [email protected] Tel & Fax: 00982123872572Tehran, Iran.

Abstract Recent studies have demonstrated the role of long non-coding RNAs (lncRNAs) in the pathophysiology of autoimmune disorders such as multiple sclerosis (MS). Among these transcripts is HOX transcript antisense intergenic RNA (HOTAIR) whose contribution in MS has been verified both in animal models and in human studies. In the current study, we genotyped three single nucleotide polymorphisms (SNPs) with this lncRNA (rs12826786, rs1899663 and rs4759314) in 403 Iranian MS patients and 420 healthy subjects. After correction of P values for multiple comparisons, the rs4759314 SNP was associated with risk of MS in allelic model (OR (95% CI)= 1.34 (1.08-1.67), adjusted P value=0.02). The other SNPs were not associated with risk of MS in any inheritance model. The C G A haplotype (rs12826786, rs1899663 and rs4759314, respectively) was less prevalent in cases compared with controls (OR (95% CI)= 0.73 (0.59–0.90), adjusted P value=0.03). The T G A haplotype was more common among cases compared with controls (OR (95% CI)= 1.58 (1.20-2.08), adjusted P value=0.01). Taken together, HOTAIR might be regarded as a risk locus for MS in Iranian population.

Key words: HOTAIR, lncRNA, multiple sclerosis, rs12826786, rs1899663, rs4759314

Introduction Multiple sclerosis (MS) is an autoimmune disorder with several genetic and environmental risk factors. Dysregulation of numerous immune-related pathways and genes has been associated with development of this disorder (1). HOX transcript antisense intergenic RNA (HOTAIR) is a long non-coding RNA (lncRNA) transcribed from the HOXC gene cluster on chromosome 12 in an antisense orientation from the HOXC genes (2). This lncRNA participates in inhibition of expression of the HOXD locus through direct interaction with chromatin‐remodeling complexes (2). Recent studies have shown the role of this lncRNA in modulation of immune responses. Obaid et al. have shown induction of expression of HOTAIR in immune cells after treatment with lipopolysaccharide (LPS). Moreover, HOTAIR silencing has diminished NF-κB-induced expression of cytokines in immune cells. Based on the results of this study, HOTAIR has been regarded as a crucial regulator of NF-κB pathway and a modulator of immune responses (3). Another recent study has demonstrated higher levels of HOTAIR in peripheral blood mononuclear cells (PBMCs) from vitamin D‐deficient MS patients compared with healthy individuals (4). This lncRNA has putative functional interactions with vitamin D as a previous animal study has shown over-expression of Hotair in mouse keratinocyte/epidermis following vitamin D receptor knock-down (5). Based on the important role of vitamin D signaling in the pathogenesis of MS (6), HOTAIR might also contribute in the development of MS via this mechanism as well. Experiments in animal model of MS have also shown participation of Hotair in neuroinflammatory and neurodegenerative routes (4). Based on the results of mentioned studies, we hypothesized that HOTAIR might be regarded as a risk locus of development of MS. So, we designed a case-control study to genotype three single nucleotide polymorphisms (SNPs) namely rs12826786, rs1899663 and rs4759314 within this

lncRNA in a population of Iranian MS patients and healthy subjects. These SNPs were selected based on the previous evidences regarding their functionality (7) and participation in the pathogenesis of human disorders (8, 9). Material and Methods Study population The study was performed on genomic DNA isolated from 403 unrelated patients with sporadic relapsing-remitting MS (RRMS) and 420 age and sex matched controls. Heathy controls were biologically unrelated to the MS patients. Cases were recruited from Hamadan University of Medical Sciences affiliated hospitals during 2017-2018. The revised McDonald criteria (10) was used for validation of MS diagnosis. Written consent forms were signed by all study participants. The study protocol was approved by ethical committee of Shahid Beheshti University of Medical Sciences. Genotyping Three HOTAIR SNPs were selected. Table 1 shows the features of these SNPs. Table

1.

General

information

of

selected

SNPs

as

obtained

from

(https://www.ncbi.nlm.nih.gov/snp/). SNP

Position

Minor Allele

rs12826786 rs1899663 rs4759314

Chr12:53961717 Chr12:53967210 Chr12:53968051

T T G

Minor Allele Frequency 0.36 0.25 0.10

Minor Allele Count 1791 1271 476

dbSNP

These SNPs were genotyped using tetra-primer amplification refractory mutation system PCR method. First, genomic DNA was isolated from peripheral blood samples using conventional salting out strategy. Then, reactions were prepared using Taq 2x red master mix (Ampliqon, Denmark) and inner and outer primers. Thermal cycling reactions were conducted in FlexCycler system (Analytik Jena, Germany). Negative control containing no DNA template was used in each run. Samples verified by Sanger sequencing were used as positive controls. The detailed information of primers and PCT conditions are shown in Table 2. Table 2. The detailed information of primers used in tetra-ARMS PCR. SNP

Primer sequence

Tm

Annealing

PCR product size (bp)

temperature rs12826786

Forward inner primer (C allele): 5′-

73 °C

64 °C

203 bp (C allele)

CGCAGGACGGGCTTCGTGAATTAAAC Reverse inner primer (T allele): 5′-

73 °C

145 bp (T allele)

73 °C

293 bp (two outer primers)

CGGCAGAGGGAAGGAGCTTAGGATAATGA Forward outer primer: 5′ATCTGTCCAGTCGCTCGTACCTGAG Reverse outer primer: 5′-

73 °C

TGTGTTTGGTCTTGTCGAGGTCCAGTTT rs1899663

Forward inner primer (T allele):

68 °C

62 °C

226 bp (T allele)

CCATTATTCCAGTTGAGGAGGGTGAAT Reverse inner primer (G allele):

68 °C

284 bp (G allele)

68 °C

457 bp (two outer primers)

CCAAAAGCCTCTAATTGTTGTCGCC Forward outer primer: TGAAAGCCACGATCATTTAACATAACCA Reverse outer primer:

68 °C

TATCTACGGAGGACTTACCTTATTCCTG rs4759314

Forward inner primer (A allele):

65 °C

61 °C

181 bp (A allele)

GCATGGAAGAGATATAAACAGGCGAA Reverse inner primer (G allele):

65 °C

121 bp (G allele)

TTATCACGTTTTATTAACTTGCATCCTCC Forward outer primer:

65 °C

247 bp (two outer primers)

AAACCATATCCTGACAGAAGCCAAATAC Reverse outer primer:

65 °C

CCAAGGTAGGGAAGTCTCTATTTCTCTG

Statistical methods Association between mentioned SNPs and MS risk was appraised using the SNP Analyzer 2.0 (11). The detected genotype frequencies were compared with the expected frequencies using Chi-square test. Linkage disequilibrium (LD) between rs12826786, rs1899663 and rs4759314 SNPs was appraised through computing D' and r factors. Associations were judged in four inheritance models namely co-dominant, dominant, recessive and allelic models. P values were corrected for multiple comparisons using Bonferroni test. P-values less than 0.05 were regarded as significant. Results General data of study population Table 3 shows the demographic and clinical data of study participants. Table 3. General data of study population (All patients were under treatment with Interferon beta). Variables

Patient

Control

Male/Female [no. (%)]

103 (26%)/300 (74%)

120 (29%)/300(71%)

Age (mean ± SD, Y)

35 ± 9.47

35.66 ± 9.90

Age range (Y)

15-62

15-63

EDSS score (mean ± SD)

4.8 ± 2.95

-

Age at onset (mean ± SD, Y)

28.2 ± 3.25

-

Duration (mean ± SD, years)

8.76±0.89

-

Genotyping Frequencies of the three mentioned SNPs were in accordance with HWE in both cases and controls (Table 4). Table 4. Frequencies of genotypes in cases and controls and their accordance with HWE supposition. SNPs Groups Cases Controls

rs12826786 CC CT TT 130 200 73 158 211 52

Pvalue 0.80 0.15

rs1899663 GG GT TT 173 182 48 153 213 55

Pvalue 0.99 0.15

rs4759314 AA AG GG 201 161 41 243 151 27

Pvalue 0.30 0.59

After correction of P values for multiple comparisons, the rs4759314 SNP was associated with risk of MS in allelic model (OR (95% CI)= 1.34 (1.08-1.67), adjusted P value=0.02). The other SNPs were not associated with risk of MS in any inheritance model (Table 5). Table 5. Association between HOTAIR genotypes and risk of MS in four inheritance models.

rs12826786

SNP

Model Allele

T vs. C

Co-dominant

TT vs. CC TC vs. CC TC+TT vs. CC TT vs. TC+CC T vs. G

Dominant

rs189 9663

Recessive Allele

Case N (%) 346 (43) 460 (57) 73 (18.1) 200 (49.6) 273 (67.7) 130 (32.3) 73 (18.1) 330 (81.9) 278 (34) 528 (66)

Control N (%) 315 (37) 527 (63) 52 (12.3) 211 (50.1) 263 (62.5) 158 (37.5) 52 (12.3) 369 (87.7) 323 (38) 519 (62)

OR (95% CI)

P-value

1.26 (1.03-1.53)

0.02

Adjusted P-value 0.07

1.69 (1.11-2.63) 1.15 (0.85-1.56) 1.26 (0.95-1.68)

0.05

0.14

0.11

0.34

1.57 (1.07-2.31)

0.02

0.06

0.85 (0.69-1.03)

0.10

0.31

Co-dominant

TT vs. GG GT vs. GG

48 (11.9) 182 (45.2)

55 (13.1) 213 (50.6)

0.77 (0.50-1.20) 0.76 (0.56-1.01)

0.15

0.46

Dominant

0.16

0.9 (0.60-1.36)

0.62

1011

1.34 (1.08-1.67)

0.008

0.02

GG vs. AA

268 (63.7) 153 (36.3) 55 (13.1) 366 (86.9) 205 (24) 637 (76) 27 (6.4)

0.05

Co-dominant

230 (57.1) 173 (42.9) 48 (11.9) 355 (88.1) 243 (30) 563 (70) 41 (10.2)

0.76 (0.57-1.00)

Allele

GT+TT vs. GG TT vs. GG+GT G vs. A

1.85 (1.09-3.13)

0.03

0.10

Dominant

AG vs. AA AG+GG vs. AA

161 (40) 202 (50.1) 201 (49.9) 41 (10.2) 362 (89.8)

151 (35.9) 178 (42.3) 243 (57.7) 27 (6.4) 394 (93.6)

1.28 (0.96-1.72) 1.37 (1.04-1.81)

0.02

0.07

1.65 (1.00-2.74)

0.05

0.15

rs4759314

Recessive

Recessive

GG vs. AA+AG

The C G A haplotype (rs12826786, rs1899663 and rs4759314, respectively) was less prevalent in cases compared with controls (OR (95% CI)= 0.73 (0.59–0.90), adjusted P value=0.03). The T G A haplotype was more common among cases compared with controls (OR (95% CI)= 1.58 (1.20-2.08), adjusted P value=0.01). Table 6 shows the frequencies of estimated haplotypes between cases and controls. Table 6. Frequencies of estimated haplotypes between cases and controls. rs12826786 rs1899663 rs4759314 Total

Frequency Frequency

frequency

in cases

in controls

OR (95% CI)

P-value

Adjusted P-value

C

G

A

0.31

0.28

0.35

0.73 (0.59–0.90)

0.003

0.03

T

G

A

0.15

0.19

0.12

1.58 (1.20-2.08)

0.001

0.01

T

T

A

0.15

0.13

0.17

0.87 (0.69-1.11)

0.26

1.00

C

G

G

0.11

0.14

0.09

1.33 (1.02-1.74)

0.04

0.30

C

T

A

0.11

0.10

0.12

0.68 (0.46-1.00)

0.05

0.38

C

T

G

0.06

0.06

0.06

1.00 (0.67-1.45)

0.96

1.00

T

G

G

0.05

0.05

0.05

1.56 (0.86-2.81)

0.14

1.00

T

T

G

0.04

0.05

0.03

1.55 (0.80-3.00)

0.19

1.00

The mentioned SNPs were not in LD in the assessed population (Table 7).

Table 7. Evaluation of linkage disequilibrium between HOTAIR SNPs.

D’ statistics

r statistic

rs1899663 rs4759314

rs1899663

rs4759314

0.12

0.04

0.004

0.11

-

1.4E-4

rs12826786 0.21 rs1899663

-

Discussion In the present case-control study, we compared genotype frequencies of three HOTAIR SNPs between MS patients and healthy subjects. Notably, we detected higher frequency of G allele of rs4759314 in MS patients compared with healthy controls. A recent study has shown association between the G allele of rs4759314 and risk of pancreatic cancer (12). Their functional studies have verified the role of this minor allele in the up-regulation of HOTAIR levels (12). This SNP has also been associated with higher risk of urothelial cell carcinoma and poor overall survival in a subset of patients (13). This intronic promoter SNP affects expression of HOTAIR gene and a neighboring gene, HOXC11 (14). Consequently, the observed association between rs4759314 and MS risk in the current study might be explained by the role of this SNP in over-expression of HOTAIR. Consistent with this speculation, Pahlevan Kakhki et al. have shown higher levels of HOTAIR in PBMCs from vitamin D‐deficient MS patients compared with healthy subjects (4). They also reported induction of HOTAIR in experimental autoimmune encephalomyelitis (4) which further supports contribution of this lncRNA in MS pathogenesis. Li et al. have reported the effect of G allele of this SNP in induction of transcription activity of HOTAIR promoter. Besides, they have shown that HOTAIR suppressed expression of miR‐545 (15), a microRNA which has been shown to be decreased in peripheral blood of all MS subtypes

compared with controls (16). Consequently, HOTAIR might be involved in the pathophysiology of MS through down-regulation of miR-454. Li et al. also demonstrated contribution of HOTAIR/miR‐545/EGFR/MAPK signaling cascade in the pathophysiology of congenital heart disease (15). Based on the fundamental role of MAPK signaling pathway in the development of MS (17), the same cascade might be involved during the course of MS. Although we detected association between the rs4759314 SNP and risk of MS in Iranian population, this SNP was not found in the recent MS GWAS by the International MS genetics consortium (18). This might imply a population-specific role for this SNP in the pathogenesis of MS. Alternatively; lack of association between this SNP and MS in the mentioned study might be due to the process of significant SNP selection in this study. It is worth mentioning the optimum selection of SNPs which are to be claimed as significantly associated with a certain trait is a problematic process in the interpretation of GWAS results. Notably, there have been inconsistencies across methods and data sets in this regard. This problem has been more highlighted in interpretation of results in diseases with complex mode of inheritance (19). We also reported that the C G A haplotype (rs12826786, rs1899663 and rs4759314, respectively) was less prevalent in MS patients compared with controls (OR (95% CI)= 0.73 (0.59–0.90), adjusted P value=0.03). Besides, the T G A haplotype was more common among cases compared with controls (OR (95% CI)= 1.58 (1.20-2.08), adjusted P value=0.01). The results of haplotype analysis was not consistent with our data regarding over-representation of G allele of rs4759314 in MS patients, so we suggest the presence of another functional variant within the haplotypes which has not been genotyped in the current study.

Finally, we did not detect LD between the mentioned SNPs. This result is in contrast with the results obtained from 1000 genomes project which showed strong LD between rs12826786 and rs1899663. This finding can be explained by the presence of population-based factors that determine LD. Previous studies also highlighted the difference in LD patterns among populations that differ in gene history (20). Our study is the first study to assess association between HOTAIR SNPs and risk of MS in a relative large cohort of Iranian patients. This study has the strength of finding risk loci for MS in this population. Simultaneous assessment of three putative functional SNPs helps in identification of risk haplotypes. However, lack of assessment of other functional SNPs and analysis of association with response to immunomodulatory drugs are is the main limitations of our study. Taken together, our study demonstrated association between the minor allele of the rs4759314 and risk of MS. Based on these results, HOTAIR might be regarded as a risk locus for MS in Iranian population. Acknowledgement The current study was supported by a grant from Shahid Beheshti University of Medical Sciences.

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Authors contribution SSP and MT performed the experiment. RN analyzed the data. SGF and MDO wrote the manuscript and revised it. MT designed the study. All the authors fully aware of submission and contributed equally.