Journal of the Neurological Sciences 351 (2015) 154–159
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Two functional promoter polymorphisms of neuregulin 1 gene are associated with progressive forms of multiple sclerosis Zahra Bahadori a, Mehrdad Behmanesh a,⁎, Mohammad Ali Sahraian b a b
Department of Genetics, Faculty of Biological Sciences, Tarbiat Modares University, Tehran, Iran Department of Neurology, Faculty of Medical Science, Tehran University of Medical Sciences, Tehran, Iran
a r t i c l e
i n f o
Article history: Received 12 May 2014 Received in revised form 6 February 2015 Accepted 4 March 2015 Available online 11 March 2015 Keywords: Multiple sclerosis NRG1 SNP Association study Remyelination PCR-RFLP
a b s t r a c t Multiple sclerosis (MS) is an inflammatory, autoimmune, demyelinating disease of the central nervous system (CNS). Spontaneous remyelination happens in most of MS lesions but it is incomplete and inadequate and eventually fails in majority of lesions of damaged areas. According to the fact that the accuracy of this phenomenon depends on distinct temporal expression profiles of molecules, especially growth factors, we decided to study the association of two functional polymorphisms of NRG1 as a myelin-related growth factor which can promote oligodendrocyte proliferation and differentiation in CNS and PNS. SNP rs6994992 and SNP rs7014762 were genotyped by PCR-RFLP and mismatch PCR-RFLP methods, respectively, in 165 subjects with MS and 200 healthy controls. Totally, our results showed no significant difference in the allelic and genotype frequencies of these two NRG1 polymorphisms between MS patients and control group in Iranian population; but statistically significant association was found for the progressive forms of MS (secondary progressive-MS and primary progressive-MS) for functional SNP of rs6994992 polymorphism. Also, considering rs7014762 polymorphism frequencies, significant difference was observed between primary progressive MS group in comparison to the control group. Furthermore, when the samples were stratified by gender, significant association was revealed between male MS subjects and rs7014762 polymorphism. These results indicate that progression of MS disease could be influenced by functional nucleotide variations in NRG1 gene, which might have an impact on remyelination capacity in different patients. © 2015 Elsevier B.V. All rights reserved.
1. Introduction Multiple sclerosis (MS) is a chronic inflammatory disease in the central nervous system (CNS) with autoimmune and neurodegenerative properties. The most common pathologic hallmarks of the disease include variable degrees of inflammation, demyelination, axonal damage, and scar formation (gliosis) [4]. In demyelinating diseases such as MS, remyelination process is followed by pathological loss of myelin in adult CNS. This regenerative process consists of two phases; at first oligodendrocyte progenitor cells (OPCs) are recruited to demyelinated regions, then they differentiate to myelinating oligodendrocytes, and finally they contact to denuded axons for producing myelin sheaths and improving function of viable axons [19,24]. Due to inaccuracies in differentiation of OPCs into mature myelin forming cells, the remyelination and regeneration process fails in most of the lesions of MS [7]. Indeed, understanding the etiology of this failure in remyelination is essential for finding appropriate therapeutic strategies in MS disease. Some ⁎ Corresponding author at: Department of Genetics, Faculty of Biological Sciences, Tarbiat Modares University, P.O. Box 14115-154, Tehran, Iran. Tel.: +98 21 82884451; fax: +98 21 8288 4717. E-mail address: [email protected] (M. Behmanesh).
http://dx.doi.org/10.1016/j.jns.2015.03.009 0022-510X/© 2015 Elsevier B.V. All rights reserved.
studies indicated that improvement in the ability of endogenous OP cells for efficient remyelination is obtained by meliorating the activity of growth factor signaling pathway [1]. NRG1-ErbB signaling pathway plays a critical role in migration, differentiation and survival of neural and oligodendrocyte precursors and in central nervous system development, also this signaling networks regulate myelination, neurotransmission, and synaptic plasticity [14]. Neuregulin 1 (NRG1) is a signaling protein that mediates cell–cell interactions and plays critical roles in the growth of the nervous system. This gene has 15 isoforms which are generated through alternative splicing and different promoter usage. Previous studies represented that NRG1 has pro-myelinating effect in PNS [11,26] and promotes myelination by oligodendrocyte in the CNS [30]. Taveggia et al. reported cortical hypomyelination in NRG1 type III heterozygous mice [28]. The amount of NRG1 type III expressed on myelinated axons determines myelin sheath thickness [15]. NRG1 type II can promote OPC proliferation in vivo and in vitro and is expressed in OPCs during their proliferative response to demyelination [3]. Based on genetic epidemiological studies, MS is a disease with complex genetic criteria. Since association study is an effective way to assign the degree of genetic contribution in complex diseases, this method was chosen for investigating effects of two important polymorphisms in
Z. Bahadori et al. / Journal of the Neurological Sciences 351 (2015) 154–159
NRG1 gene on MS. Rs6994992 (SNP8NRG243177) and rs7014762 are two functional promoter polymorphisms that are candidates for this study. Rs6994992 is located near to the promoter and 1.2 kb upstream of initiation transcription site of type IV NRG1. Indeed, bioinformatics analysis on rs6994992 has revealed that this polymorphism changes binding site of transcription factor implicated in NRG1 regulation. In one study Tan et al. showed that T allele conversion to C allele in rs6994992 results in 60% decrease in the promoter activity [27]. It is known as a functional polymorphism and the TT genotype has been associated with higher levels of NRG1 type IV transcription [8]. McIntosh et al. showed that homozygotes for rs6994992 T allele was associated with decreased white matter density in the right anterior limb of the internal capsule and reduced structural connectivity [13]. Reduced white matter integrity in the left anterior thalamic radiation was shown in T allele carriers [23]. Rs7014762 is a single nucleotide polymorphism that is located in core promoter region and has 87 base pair physical distance from rs6994992. Nicodemus et al. found significant association between this SNP and NRG1 type III expression in the hippocampus of schizophrenia cases [16]. So in this study, we assessed association between SNP rs6994992 and rs7014762 in NRG1 gene and MS disease for the first time in Iranian population.
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designed using PRIMER EXPRESS software (Applied Biosystems). PCR was conducted with 5 min of denaturation 94 °C, 35 cycles of 94 °C for 30 s, annealing at 61 °C for 30 s, 72 °C for 40 s and a final extension at 72 °C for 5 min. PCR products were digested by TaiI enzyme (Fermentas) at 65 °C and visualized by electrophoresis on 1% agarose gel. TaiI cannot cut the C allele, but cuts the T allele and produces two fragments with 446 bp and 138 bp in length. Mismatch PCR-RFLP technique was used for genotyping of SNP rs7014762. The region containing rs7014762 was amplified using a mismatch reverse primer to produce a recognition site for Bam HI enzyme (Fermentas) in the presence of T allele (Fig. 1). Sequences of primers were as follows: 5′-AGTAGGATTGGATGTTTGAACC-3′ as forward primer and 5′-GGGTCATCACACTCCCTGTGTGTCTTGGCAAGGGGGG-3′ as reverse primer. The mismatched nucleotide in reverse primer is underlined. In PCR reaction, following an initial 94 °C denaturation step (5 min), the samples were subjected to 35 cycles at 94 °C (30 s), 60 °C (30 s), 72 °C (30 s) and ending with a final extension at 72 °C (5 min). After digestion by Bam HI at 37 °C, products were subjected to electrophoresis on 12% polyacrylamide gel. The amplicon was 195 bp and Bam HI cannot cut the A allele. Genotyping of some samples from each genotype was confirmed by DNA sequencing with ABI automated DNA sequencer (Macrogen, Korea).
2. Material & method 2.1. Collection of MS patients and control samples
2.4. Statistical analysis
Case samples were collected from patients with MS diseases referred to Sina Hospital, and they were diagnosed by physician specialty based on diagnostic criteria by [32,33] and according to Brain magnetic resonance imaging. Control subjects were acquired from volunteers with no neurological disease history (Table 1). Totally peripheral blood samples were collected from 165 subjects with MS and 200 healthy controls and genotyped for two rs6994992 and rs7014762 polymorphisms. The control groups are coincident with case groups in sexuality, age and race. Informed consent from each case was acquired before blood sampling and the Ethics Committee of Tarbiat Modares University approved the study.
The calculated genotype frequencies of studied SNPs were compared with expected genotype frequencies according to the Hardy–Weinberg law. The association of SNP rs6994992 and SNP rs7014762 with MS was analyzed with Chi-square test and (version 16) SPSS software to understand difference of allele and genotype frequencies between the study groups. A P-value of ≤0.05 was considered as significant level.
2.2. Genomic DNA extraction DNA extraction from whole blood was done using DNG Plus™ kit (Cinnagen, Iran). According to producer instructions, blood cells are lysed by kit lysis buffer followed by DNA precipitation using isopropanol. Then extracted DNA was washed by 75% ethanol and dissolved in distilled water. Quality and concentration of extracted DNAs were examined by electrophoresis in 1% agarose gel or spectrophotometer, respectively. 2.3. Genotyping SNP rs6994992 was genotyped by PCR-RFLP method. For each sample approximately 100 ng of extracted DNA was used to amplify 584 bp amplicon that containing rs6994992 SNP using recombinant Taq polymerase (Cinnagen, Iran). Amplification was carried out using specific primers: 5′-ACTCTATTGAAACACAACCAGC-3′ as forward primer and 5′-AGTTTGGAGGGACAGGGTCA-3′ as reverse primer. All primers were
3. Results In this study, we used PCR-RFLP and mismatch PCR-RFLP methods for genotyping SNP rs6994992 and rs7014762, respectively. By these methods, we distinguished different genotypes of individuals for rs7014762 and rs6994992 in 12% acrylamide gel (Fig. 1D) and 1% agarose gel (Fig. 2), respectively. Sequencing results were matched with genotyped results based on used methods (Fig. 3). Two mentioned SNPs in NRG1 gene were genotyped in 165 MS patients and 200 healthy controls. Determined genotype frequencies were in Hardy–Weinberg equilibrium in the control and patient groups for both SNP. The allelic frequency of rs6994992 (C, T) was 50.3%, 49.7% in patient group vs. 46.6% and 53.4% in healthy group, respectively; and genotype frequency (CC, CT, TT) was 25.4%, 49.8%, 24.8% in MS group vs. 20.47%, 52.38%, 27.15% in healthy group, respectively. But for rs7014762 (A, T), in MS group allelic frequency was 25.8%, 74.2%, while in control group was 27.1%, 72.9%, respectively, and genotype frequency (AA, AT, TT) was 7.1%, 37.4%, 55.5% in MS group vs. 6.7%, 40.8%, 52.5%, in control group, respectively. According to statistical analysis and χ2 test result, totally there was no significant difference in allelic and genotype frequency between case and control groups (Table 2).
Table 1 Demographic features of MS patient and control individuals. Male
MS Control
Female
Frequency of MS types
Number
Age range
Mean onset age
Number
Age range
Mean onset age
Relapsing remitting (RR)
Secondary progressive (SP)
Primary progressive (PP)
40 45
23–50 21–52
29.7 –
125 155
17–53 16–60
29.7 –
69.7% –
25.3% –
5% –
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Z. Bahadori et al. / Journal of the Neurological Sciences 351 (2015) 154–159
Fig. 1. Schematic diagram of mismatch PCR-RFLP technique for rs7014762 genotyping and results of restriction digestion of PCR product by BamHI enzyme on 12% polyacryleamide gel. (A) There is no restriction site at the T/A allelic location of rs7014762. (B) The T nucleotide near the SNP is changed to C nucleotide in PCR reaction by using a reverse primer that has a mismatched nucleotide. (C) BamHI restriction Enzyme can cut PCR product only in T allelic form. (D) If there is T allele form, BamHI restriction enzyme can cut PCR product and 158 bp and 37 bp fragments are produced, but 195 bp fragment represents A allelic form genotyping of each sample were determined on top of the gel.
By classification for clinical MS groups, significant difference in the genotype frequency was observed between SPMS patients and controls for SNP rs6994992; genotype frequency (CC, CT, TT) was 34.28%, 34.28%, 31.42% in patients vs. 20.47%, 52.38%, 27.15%, in control group, respectively (P = 0.038, OR = 2.06, Table 2) and also, a significant difference in the allelic distributions between PPMS patients and controls was revealed; frequency of alleles (C, T) in PPMS group were 31.25%, 68.75% vs. control group, 46.6%, 53.4%, respectively (P = 0.029, OR = 0.5); genotype frequencies (CC, CT, TT) were 0%, 62.5%, 37.5% vs. control group, 20.47%, 52.38%, 27.15%, respectively (P = 0.0001, OR = 0.04, Table 2); there was a significant shift in frequency of C allele between PPMS group vs. other type of MS. Significant association was found for rs7014762 with MS only in PPMS group; allelic frequency (A, T) in PPMS group was 12.5%, 87.5% vs. control group, 27.1%, 72.9% (P = 0.014, OR = 2.5) and genotype frequencies (AA, AT, TT) were 0%, 25%, 75% in patients vs., 6.7%, 40.8%, 52.5%, in control group respectively (P = 0.001, OR = 2.7, Table 2). Gender stratification showed significant difference in the genotype and allelic frequencies between male patients and male controls for SNP rs7014762; allele frequencies (T, A) in male MS group were
82.8%, 17.2% vs., 70%, 30%, in male control group respectively (P = 0.045, OR = 2.09); genotype frequencies (TT, AT, AA) were 65.5%, 34.5%, 0% in male patients vs. male control group, 46.7%, 46.7%, 6.7%, respectively (P = 0.01, OR = 2.1, Table 3). 4. Discussion In this study, we could demonstrate that nucleotide variations in NRG1 gene may have an influence in the progression of MS disease. We also could reveal significant association of functional rs6994992 polymorphism with progressive MS groups (secondary and primary progressive MS), which means that CC genotype frequency increases significantly in secondary progressive (SP) MS group (P = 0.038, OR = 2.06) and heterozygote genotype (CT) frequency decreases in comparison to the healthy group. Allelic and genotype frequencies have different distributions in primary progressive (PP) MS group in comparison to other MS groups, and T allele frequency significantly increases (P = 0.029, OR = 0.5) in comparison to healthy group, CT and TT genotype frequency also increase (P = 0.0001, OR = 0.04) and we could not detect any CC genotype in PPMS.
Table 2 Allele and genotype frequencies of two NRG1 polymorphisms in MS patients compared to controls. SNP
Cohort
Maj.allele%
Min.allele %
P-Value
OR (CI 95%)
Genotypes Maj./Maj.%
Genotypes Maj./Min.%
Genotypes Min./Min.%
P-Value
OR (CI 95%)
rs6994992 Maj. allele: C Min. allele: T
Total MS RR MS SP MS PP MS Male MS Female MS Control Male control Female control Total MS RR MS SP MS PP MS Male MS Female MS Control Male control Female control
50.3 52.3 51.4 31.25 52.4 49.6 46.6 44.6 47.3 74.2 75.6 70.7 87.5 82.8 71.65 72.9 70 73
49.7 47.7 48.6 68.75 47.6 50.4 53.4 55.4 52.7 25.8 24.4 29.3 12.5 17.2 28.35 27.1 30 27
0.77 0.57 0.67 0.029 0.396 0.77
1.13 (0.65–1.96) 1.22 (0.7–2.13) 1.17 (0.67–2.04) 0.5 (0.28–0.9) 1.32 (0.76–2.31) 1.13 (0.65–1.96)
24.8 21.8 31.42 37.5 14.65 28.2 27.15 29.7 26 7.1 6.09 10.34 – – 9.3 6.7 6.7 7
1.33 (0.68–2.5) 1.4 (0.72–2.73) 2.06 (1.08–3.91) 0.04 (0.005–0.3) 1.05 (0.52–2.12) 1.39 (0.72–2.67)
1.1 (0.59–2.1) 1.17 (0.62–2.21) 0.9 (0.49–1.68) 2.5 (1.2–5.1) 2.09 (1.06–4.1) 0.95 (0.51–1.77)
49.8 51.8 34.28 62.5 65.85 44.5 52.38 51.3 53.3 37.4 36.58 37.93 25 34.5 38.5 40.8 46.7 40
0.498 0.4 0.038 0.0001 1.00 0.4
0.88 0.74 0.875 0.014 0.045 0.89
25.4 26.36 34.28 – 19.5 27.3 20.47 19 20.7 55.5 57.3 51.72 75 65.5 52.5 52.5 46.7 53
0.78 0.57 0.89 0.001 0.01 1.00
1.13 (0.65–1.96) 1.2 (0.69–2.1) 0.91 (0.53–1.58) 2.7 (1.5–4.9) 2.1 (1.2–3.75) 0.98 (0.56–1.7)
rs7014762 Maj. allele: T Min. allele: A
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Table 3 Allele and genotype frequencies of two NRG1 polymorphisms in MS sub type patients according to gender. SNP
Cohort
Maj.allele%
Min.allele%
P-Value
OR (CI 95%)
Genotypes Maj./Maj.%
Genotypes Maj./Min.%
Genotypes Min./Min.%
P-Value
OR (CI 95%)
rs6994992 Maj. allele: C Min. allele: T
Total MS Male MS Female MS RR male RR female SP male SP female PP male PP female Control Male control Female control Total MS Male MS Female MS RR male RR female SP male SP female PP female Control Male control Female control
50.3 52.4 49.6 58 50.60 45 56.5 50 12.5 46.6 44.6 47.3 74.2 82.8 71.65 86.80 72.30 64.3 71.50 83.3 72.9 70 73
49.7 47.6 50.4 42 49.40 55 43.5 50 87.5 53.4 55.4 52.7 25.8 17.2 28.35 13.20 27.70 35.7 28.50 16.7 27.1 30 27
0.77 0.396 0.77 0.089 0.67 1 0.2 0.57 0.00
1.13 (0.65–1.96) 1.32 (0.76–2.31) 1.13 (0.65–1.96) 1.68 (0.96–2.9) 1.17 (0.67–2) 1 (0.5–1.7) 1.46 (0.84–2.5) 1.2 (0.7–2.1) 0.16 (0.083–0.33)
24.8 14.65 28.2 8.00 25.88 30.00 30.43 – 75.00 27.15 29.7 26 7.1 – 9.3 0.00 7.94 0.00 14.29 – 6.7 6.7 7
1.33 (0.68–2.5) 1.05 (0.52–2.12) 1.39 (0.72–2.67) 1.35 (0.68–2.6) 1.39 (0.7–2.67) 1.06 (0.53–2.15) 2.8 (1.5–5.3) 0.043 (0.006–0.33) 0.038 (0.005–0.289)
1.1 (0.59–2.1) 2.09 (1.06–4.1) 0.95 (0.51–1.77) 2.86 (1.4–5.9) 0.95 (0.51–1.7) 0.76 (0.42–1.37) 0.91 (0.49–1.7) 1.8 (0.91–3.57)
49.8 65.85 44.5 68.00 47.06 50.00 26.09 100 25.00 52.38 51.3 53.3 37.4 34.5 38.5 26.32 39.68 71.43 28.57 33.33 40.8 46.7 40
0.498 1.00 0.4 0.49 0.4 1 0.001 0.00 0.00
0.88 0.045 0.89 0.005 1 0.45 0.87 0.124
25.4 19.5 27.3 24.00 27.06 20.00 43.48 – – 20.47 19 20.7 55.5 65.5 52.5 73.68 52.38 28.57 57.14 66.67 52.5 46.7 53
0.78 0.01 1.00 0.00 1 0.013 0.67 0.06
1.13 (0.65–1.96) 2.1 (1.2–3.75) 0.98 (0.56–1.7) 3.2 (1.8–5.8) 0.96 (0.55–1.67) 0.46 (0.25–0.83) 1.17 (0.67–2) 1.8 (1–3.2)
rs7014762 Maj. allele: T Min. allele: A
Also we have found a significant association of PPMS with SNP rs7014762 located in NRG1 promoter. Frequency of T allele (P = 0.014, OR = 2.5) and TT genotype (P = 0.001, OR = 2.7) increased significantly, but there is no significant association with other types of MS group (Table 4). Interestingly, a significant association was found in male MS subjects for T allele (P = 0.045, OR = 2.09) and TT genotype (P = 0.01, OR = 2.1) when the samples were stratified by gender. The results of several independent studies indicated that SNP rs6994992 (SNP8NRG243177) is a functional polymorphism in NRG1 regulatory domain that affects NRG1 function. This polymorphism is located on 1.2 kb upstream of the transcription start of type IV NRG1 [8, 27]. Recent meta-analysis confirmed the association of this haplotype with schizophrenia and the risk haplotype denoted HAPice [9]. On the basis of bioinformatics prediction by MATINSPECTOR software (Genomatix, Munich), SNP rs6994992 is located on the putative binding sites for serum response factor (SRF) and myelin transcription factor 1 (Myt-1); but in the presence of T allele, both of these transcription factor binding sites are abolished and a high mobility group box protein-1 (HMGBP-1) binding site is formed [8]. Myt-1 transcription factor is a zinc-finger DNA binding protein that regulates oligodendrocyte regeneration and remyelination. Myt-1 is present inside the nucleus of immature cells during oligodendrocyte lineage development, but it reduced after final differentiation and accumulation of myelin proteins in mature oligodendrocyte [29]. Also, SRF transcription factor plays a substantial role in neural precursor cells (NPCs) and regulates astrocyte and oligodendrocyte specificity and
migration in brain. SRF loss in neurons of mice forebrain increased oligodendrocyte precursors but inhibited oligodendrocyte final differentiation and reduced myelin expression [10,25]. Since SNP rs6994992 is located within mentioned transcription binding sites, interpretation of nucleotide variation is difficult. In one in vitro study Tan et al. found that activity of the 1.5-kb human NRG1 type IV promoter-luciferase fusion gene was 65% higher in T allele constructs (pGL4.SNPT) compared with that of the C allele construct (pGL4.SNPC) in transfected HEK293 Cells [27]. In addition Law et al. reported that there is a substantial difference in NRG1 type IV transcription level between carriers of T allele and C allele in schizophrenia patient brains. Acquisition of two HMGBP1 binding motifs (Homozigocity for TT) but not for one motif (heterozigocity for T) associates with significantly increased type IV NRG1 expression, showing that this effect is recessive [8]. Another study revealed that NRG1-mediated cell migration tendency reduces in schizophrenia patients carrying T allele compared with those carrying C allele, in addition since α7 acetylcholine receptor is regulated by NRG1, carrying T allele showed increased expression of the receptor [22]. In addition SNP rs6994992 was associated with endophenotypes related to schizophrenia. Homozygotes for rs6994992 T allele were associated with deficits in cognitive function, impairment and decreased activation of frontal and temporal lobe regions in individuals at high risk for developing schizophrenia [5,6], which suggests an inefficacy in their neural processing. Besides, normal individuals carrying (T/T) showed reduced white matter in the anterior limb of the internal
Fig. 2. The results of restriction digestion of PCR product by TaiI enzyme on 1% agarose gel for genotyping of SNP rs6994992. The PCR product has a restriction site for TaiI enzyme in T allelic form but not in C allelic form, so digesting PCR product containing T allele produce 137 bp and 446 bp fragments whereas 583 bp fragment represents C allelic form. Genotyping of each sample was determined on top of the gel.
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Fig. 3. Sequencing result of PCR product that is consistent with genotyping results of PCR-RFLP and mismatch PCR-RFLP technique. (A) Sequencing result of heterozygote sample of SNP rs6994992. (B) Sequencing result of heterozygote sample of SNP rs7014762.
capsule [13]. Also, in normal subjects, rs6994992 (T) allele was found to predict lower fractional anisotropy (FA) and decreased white matter in the left anterior thalamic radiation [23]. All of these studies confirm the high importance of this SNP rs6994992 polymorphism. SNP rs7014752 is 87 bp from rs6994992 and is located in the core promoter region; it is also in moderate linkage disequilibrium with rs6994992 (D′ = 0.96, r2 = 0.21). Bioinformatics analysis was performed for finding transcription factor binding sites in the location of this SNP by Transfac software and revealed that the location has a binding site for NF-κB in the presence of A allele but not T allele [31]. Nicodemus et al. indicated that NRG1 type III expression significantly reduce in the presence of T allele (Homozygote and Heterozygote) in comparison to AA homozygote group in schizophrenia patient hippocampus. Of course rs7014762 effect on NRG1 type III expression was significant only in patients in this study [16]. Abnormalities in normal appearing white matter (NAWM) have been detected in almost all MS phenotypes. FA in RR MS patients was lower than in benign MS patients. Most, but not all of studies have shown differences in NAWM diffusion matrices between healthy subjects and RR MS patients. SP MS patients exhibit more certain white matter diffusion abnormalities in comparison with
RR and benign MS patients [20]. Progressive NAWM changes in PP MS patients have been detected by serial diffusion MRI images which proved to be related to both lesion volume and development of clinical disability [21]. At the point of molecular view, demyelination without remyelination might be an important mechanism in MS disease progression [11,18]. The capacity for repair and extent of remyelination in MS lesions is dramatically variable. The reason of incomplete and failed remyelination in MS lesions is unknown yet [17]. The result that two functional polymorphisms in NRG1 gene have different frequencies in different MS types may be interpreted by the fact that primary progressive MS differs from other types of MS (relapse–remitting and secondary progressive MS) in pathologic properties, clinical course, response to treatment, and differential diagnosis. The lesions in PPMS patients are more diffused and less inflammatory in comparison to RRMS and SPMS, and less likely to remyelinate than those occurring in relapsing–remitting MS and secondary progressive MS [2,12]. Hence investigations about molecular mechanisms of different remyelination capacity in different types of MS is essential and could open new avenues for finding appropriate treatment according to repair capacity of different patients.
Table 4 Allele and genotype frequencies of two NRG1 polymorphisms in MS subtype patients according to age. SNP
Cohort
Maj.allele%
Min.allele%
P-Value
rs6994992 Maj. allele: C Min. allele: T
Total MS MS onset age N 25 MS onset age ≤ 25 RR onset age N 25 RR onset age ≤ 25 SP onset age N 25 SP onset age ≤ 25 PP onset age N 25 PP onset age ≤ 25 Control Total MS MS onset age N 25 MS onset age ≤ 25 RR onset age N 25 RR onset age ≤ 25 SP onset age N 25 SP onset age ≤ 25 PP onset age N 25 PP onset age ≤ 25 Control
50.3 54.8 44.28 58 44.80 58.30 42.85 31.25 – 46.6 74.2 79.4 68.52 79.50 71.10 78.10 54.55 87.5 – 72.9
49.7 45.2 55.72 42 55.20 41.70 57.15 68.75 – 53.4 25.8 20.6 31.48 20.50 28.90 21.90 45.45 12.5 – 27.1
0.77 0.32 0.77 0.157 0.88 0.157 0.67 0.029
1.13 (0.65–1.96) 0.72 (0.4–1.26) 1.12 (0.64–1.97) 1.55 (0.89–2.7) 0.9 (0.5–1.6) 1.55 (0.89–2.7) 0.85 (0.48–1.48) 0.5 (0.28–0.9)
0.88 0.4 0.64 0.3 0.87 0.5 0.012 0.014
1.1 (0.59–2.1) 1.39 (0.7–2.6) 0.823 (0.44–1.5) 1.4 (0.7–2.7) 0.9 (0.48–1.68) 1.31 (0.68–2.5) 0.45 (0.25–0.817) 2.5 (1.2–5.1)
rs7014762 Maj. allele: T Min. allele: A
OR (CI 95%)
Genotypes Maj./Maj.%
Genotypes Maj./Min.%
Genotypes Min./Min.%
P-Value
OR (CI 95%)
25.4 28.72 20 30.65 20.83 38.89 28.57 – – 20.47 55.5 61.76 48.15 63.64 50.00 56.25 36.36 75.00 – 52.5
49.8 52.13 48.57 54.84 47.92 38.89 28.57 62.50 – 52.38 37.4 35.29 40.74 31.82 42.11 43.75 36.36 25.00 – 40.8
24.8 19.15 31.43 14.52 31.25 22.22 42.86 37.50 – 27.15 7.1 2.94 11.11 4.55 7.89 – 27.27 – – 6.7
0.498 0.188 1 0.1 1 0.005 0.188 0.00
1.33 (0.68–2.5) 1.63 (0.85–3.14) 1 (0.5–2) 1.8 (0.9–3.4) 1 (0.5–2.1) 2.55 (1.33–4.8) 1.6 (0.85–3.1) 0.04 (0.005–0.3)
0.78 0.2 0.57 0.116 0.77 0.67 0.02 0.001
1.13 (0.65–1.96) 1.47 (0.84–2.59) 0.83 (0.48–1.45) 1.6 (0.9–2.8) 0.9 (0.5–1.57) 1.15 (0.6–2) 0.5 (0.29–0.89) 2.7 (1.5–4.9)
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Conflict of interest The authors declare that they have no conflict of interest. Acknowledgments The authors gratefully acknowledge the contribution of the patients, healthy controls for their blood donations and also the institutions for their help in this study. The Iran National Science Foundation and the Department of Research Affairs of Tarbiat Modares University provide the funding of this work. References [1] Armstrong RC. Growth factor regulation of remyelination: behind the growing interest in endogenous cell repair of the CNS. Future Neurol 2007;2(6):689–97. [2] Bramow S, Frischer JM, Lassmann H, Koch-Henriksen N, Lucchinetti CF, Sørensen PS, et al. Demyelination versus remyelination in progressive multiple sclerosis. Brain 2010;133(10):2983–98. [3] Cannella B, Hoban CJ, Gao YL, Garcia-Arenas R, Lawson D, Marchionni M, et al. The neuregulin, glial growth factor 2, diminishes autoimmune demyelination and enhances remyelination in a chronic relapsing model for multiple sclerosis. Proc Natl Acad Sci 1998;95:10100–5. [4] Compston A, Coles A. Multiple sclerosis. Lancet 2008;359(9648):1221–31. [5] Hall J, Whalley HC, Job DE, Baig BJ, McIntosh AM, Evans KL, et al. A neuregulin 1 variant associated with abnormal cortical function and psychotic symptoms. Nat Neurosci 2006;9:1477–8. [6] Keri S, Kiss I, Kelemen O. Effects of a neuregulin 1 variant on conversion to schizophrenia and schizophreniform disorder in people at high risk for psychosis. Mol Psychiatry 2009;14:118–9. [7] Kotter MR, Stadelmann C, Hrtung HP. Enhancing remyelination in disease — can we wrap it up? Brain 2011;1:1–19. [8] Law AJ, Lipska BK, Weickert CS, Hyde TM, Straub RE, Hashimoto R, et al. Neuregulin 1 transcripts are differentially expressed in schizophrenia and regulated by 5′ SNPs associated with the disease. Proc Natl Acad Sci U S A 2006;103(17):6747–52. [9] Li D, Collier DA, He L. Meta-analysis shows strong positive association of the neuregulin 1 (NRG1) gene with schizophrenia. Hum Mol Genet 2006;15:1995–2002. [10] Lu PP, Ramanan N. A critical cell-intrinsic role for serum response factor in glial specification in the CNS. J Neurosci 2012;32(23):8012–23. [11] Lucchinetti C, Brück W, Parisi J, Scheithauer B, Rodriguez M, Lassmann H, et al. A quantitative analysis of oligodendrocytes in multiple sclerosis lesions. A study of 113 cases. Brain 1999;122:2279–95. [12] Lucchinetti C, Brück W, Parisi J, Scheithauer B, Rodriguez M, Lassmann H, et al. Heterogeneity of multiple sclerosis lesions: implications for the pathogenesis of demyelination. Ann Neurol 2000;47:707–17. [13] McIntosh AM, Moorhead TW, Job D, Lymer GK, Muñoz Maniega S, McKirdy J, et al. The effects of a neuregulin 1 variant on white matter density and integrity. Mol Psychiatry 2008;13:1054–9. [14] Mei L, Nave KA. Neuregulin-ERBB signaling in the nervous system and neuropsychiatric diseases. Neuron 2014;83(1):27–49. [15] Michailov GV, Sereda MW, Brinkmann BG, Fischer TM, Haug B, Birchmeier C, et al. Axonal neuregulin-1 regulates myelin sheath thickness. Science 2004;304:700–3.
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Two functional promoter polymorphisms of neuregulin 1 gene are associated with progressive forms of multiple sclerosis Zahra Bahadori a, Mehrdad Behmanesh a,⁎, Mohammad Ali Sahraian b a b
Department of Genetics, Faculty of Biological Sciences, Tarbiat Modares University, Tehran, Iran Department of Neurology, Faculty of Medical Science, Tehran University of Medical Sciences, Tehran, Iran
a r t i c l e
i n f o
Article history: Received 12 May 2014 Received in revised form 6 February 2015 Accepted 4 March 2015 Available online 11 March 2015 Keywords: Multiple sclerosis NRG1 SNP Association study Remyelination PCR-RFLP
a b s t r a c t Multiple sclerosis (MS) is an inflammatory, autoimmune, demyelinating disease of the central nervous system (CNS). Spontaneous remyelination happens in most of MS lesions but it is incomplete and inadequate and eventually fails in majority of lesions of damaged areas. According to the fact that the accuracy of this phenomenon depends on distinct temporal expression profiles of molecules, especially growth factors, we decided to study the association of two functional polymorphisms of NRG1 as a myelin-related growth factor which can promote oligodendrocyte proliferation and differentiation in CNS and PNS. SNP rs6994992 and SNP rs7014762 were genotyped by PCR-RFLP and mismatch PCR-RFLP methods, respectively, in 165 subjects with MS and 200 healthy controls. Totally, our results showed no significant difference in the allelic and genotype frequencies of these two NRG1 polymorphisms between MS patients and control group in Iranian population; but statistically significant association was found for the progressive forms of MS (secondary progressive-MS and primary progressive-MS) for functional SNP of rs6994992 polymorphism. Also, considering rs7014762 polymorphism frequencies, significant difference was observed between primary progressive MS group in comparison to the control group. Furthermore, when the samples were stratified by gender, significant association was revealed between male MS subjects and rs7014762 polymorphism. These results indicate that progression of MS disease could be influenced by functional nucleotide variations in NRG1 gene, which might have an impact on remyelination capacity in different patients. © 2015 Elsevier B.V. All rights reserved.
1. Introduction Multiple sclerosis (MS) is a chronic inflammatory disease in the central nervous system (CNS) with autoimmune and neurodegenerative properties. The most common pathologic hallmarks of the disease include variable degrees of inflammation, demyelination, axonal damage, and scar formation (gliosis) [4]. In demyelinating diseases such as MS, remyelination process is followed by pathological loss of myelin in adult CNS. This regenerative process consists of two phases; at first oligodendrocyte progenitor cells (OPCs) are recruited to demyelinated regions, then they differentiate to myelinating oligodendrocytes, and finally they contact to denuded axons for producing myelin sheaths and improving function of viable axons [19,24]. Due to inaccuracies in differentiation of OPCs into mature myelin forming cells, the remyelination and regeneration process fails in most of the lesions of MS [7]. Indeed, understanding the etiology of this failure in remyelination is essential for finding appropriate therapeutic strategies in MS disease. Some ⁎ Corresponding author at: Department of Genetics, Faculty of Biological Sciences, Tarbiat Modares University, P.O. Box 14115-154, Tehran, Iran. Tel.: +98 21 82884451; fax: +98 21 8288 4717. E-mail address: [email protected] (M. Behmanesh).
http://dx.doi.org/10.1016/j.jns.2015.03.009 0022-510X/© 2015 Elsevier B.V. All rights reserved.
studies indicated that improvement in the ability of endogenous OP cells for efficient remyelination is obtained by meliorating the activity of growth factor signaling pathway [1]. NRG1-ErbB signaling pathway plays a critical role in migration, differentiation and survival of neural and oligodendrocyte precursors and in central nervous system development, also this signaling networks regulate myelination, neurotransmission, and synaptic plasticity [14]. Neuregulin 1 (NRG1) is a signaling protein that mediates cell–cell interactions and plays critical roles in the growth of the nervous system. This gene has 15 isoforms which are generated through alternative splicing and different promoter usage. Previous studies represented that NRG1 has pro-myelinating effect in PNS [11,26] and promotes myelination by oligodendrocyte in the CNS [30]. Taveggia et al. reported cortical hypomyelination in NRG1 type III heterozygous mice [28]. The amount of NRG1 type III expressed on myelinated axons determines myelin sheath thickness [15]. NRG1 type II can promote OPC proliferation in vivo and in vitro and is expressed in OPCs during their proliferative response to demyelination [3]. Based on genetic epidemiological studies, MS is a disease with complex genetic criteria. Since association study is an effective way to assign the degree of genetic contribution in complex diseases, this method was chosen for investigating effects of two important polymorphisms in
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NRG1 gene on MS. Rs6994992 (SNP8NRG243177) and rs7014762 are two functional promoter polymorphisms that are candidates for this study. Rs6994992 is located near to the promoter and 1.2 kb upstream of initiation transcription site of type IV NRG1. Indeed, bioinformatics analysis on rs6994992 has revealed that this polymorphism changes binding site of transcription factor implicated in NRG1 regulation. In one study Tan et al. showed that T allele conversion to C allele in rs6994992 results in 60% decrease in the promoter activity [27]. It is known as a functional polymorphism and the TT genotype has been associated with higher levels of NRG1 type IV transcription [8]. McIntosh et al. showed that homozygotes for rs6994992 T allele was associated with decreased white matter density in the right anterior limb of the internal capsule and reduced structural connectivity [13]. Reduced white matter integrity in the left anterior thalamic radiation was shown in T allele carriers [23]. Rs7014762 is a single nucleotide polymorphism that is located in core promoter region and has 87 base pair physical distance from rs6994992. Nicodemus et al. found significant association between this SNP and NRG1 type III expression in the hippocampus of schizophrenia cases [16]. So in this study, we assessed association between SNP rs6994992 and rs7014762 in NRG1 gene and MS disease for the first time in Iranian population.
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designed using PRIMER EXPRESS software (Applied Biosystems). PCR was conducted with 5 min of denaturation 94 °C, 35 cycles of 94 °C for 30 s, annealing at 61 °C for 30 s, 72 °C for 40 s and a final extension at 72 °C for 5 min. PCR products were digested by TaiI enzyme (Fermentas) at 65 °C and visualized by electrophoresis on 1% agarose gel. TaiI cannot cut the C allele, but cuts the T allele and produces two fragments with 446 bp and 138 bp in length. Mismatch PCR-RFLP technique was used for genotyping of SNP rs7014762. The region containing rs7014762 was amplified using a mismatch reverse primer to produce a recognition site for Bam HI enzyme (Fermentas) in the presence of T allele (Fig. 1). Sequences of primers were as follows: 5′-AGTAGGATTGGATGTTTGAACC-3′ as forward primer and 5′-GGGTCATCACACTCCCTGTGTGTCTTGGCAAGGGGGG-3′ as reverse primer. The mismatched nucleotide in reverse primer is underlined. In PCR reaction, following an initial 94 °C denaturation step (5 min), the samples were subjected to 35 cycles at 94 °C (30 s), 60 °C (30 s), 72 °C (30 s) and ending with a final extension at 72 °C (5 min). After digestion by Bam HI at 37 °C, products were subjected to electrophoresis on 12% polyacrylamide gel. The amplicon was 195 bp and Bam HI cannot cut the A allele. Genotyping of some samples from each genotype was confirmed by DNA sequencing with ABI automated DNA sequencer (Macrogen, Korea).
2. Material & method 2.1. Collection of MS patients and control samples
2.4. Statistical analysis
Case samples were collected from patients with MS diseases referred to Sina Hospital, and they were diagnosed by physician specialty based on diagnostic criteria by [32,33] and according to Brain magnetic resonance imaging. Control subjects were acquired from volunteers with no neurological disease history (Table 1). Totally peripheral blood samples were collected from 165 subjects with MS and 200 healthy controls and genotyped for two rs6994992 and rs7014762 polymorphisms. The control groups are coincident with case groups in sexuality, age and race. Informed consent from each case was acquired before blood sampling and the Ethics Committee of Tarbiat Modares University approved the study.
The calculated genotype frequencies of studied SNPs were compared with expected genotype frequencies according to the Hardy–Weinberg law. The association of SNP rs6994992 and SNP rs7014762 with MS was analyzed with Chi-square test and (version 16) SPSS software to understand difference of allele and genotype frequencies between the study groups. A P-value of ≤0.05 was considered as significant level.
2.2. Genomic DNA extraction DNA extraction from whole blood was done using DNG Plus™ kit (Cinnagen, Iran). According to producer instructions, blood cells are lysed by kit lysis buffer followed by DNA precipitation using isopropanol. Then extracted DNA was washed by 75% ethanol and dissolved in distilled water. Quality and concentration of extracted DNAs were examined by electrophoresis in 1% agarose gel or spectrophotometer, respectively. 2.3. Genotyping SNP rs6994992 was genotyped by PCR-RFLP method. For each sample approximately 100 ng of extracted DNA was used to amplify 584 bp amplicon that containing rs6994992 SNP using recombinant Taq polymerase (Cinnagen, Iran). Amplification was carried out using specific primers: 5′-ACTCTATTGAAACACAACCAGC-3′ as forward primer and 5′-AGTTTGGAGGGACAGGGTCA-3′ as reverse primer. All primers were
3. Results In this study, we used PCR-RFLP and mismatch PCR-RFLP methods for genotyping SNP rs6994992 and rs7014762, respectively. By these methods, we distinguished different genotypes of individuals for rs7014762 and rs6994992 in 12% acrylamide gel (Fig. 1D) and 1% agarose gel (Fig. 2), respectively. Sequencing results were matched with genotyped results based on used methods (Fig. 3). Two mentioned SNPs in NRG1 gene were genotyped in 165 MS patients and 200 healthy controls. Determined genotype frequencies were in Hardy–Weinberg equilibrium in the control and patient groups for both SNP. The allelic frequency of rs6994992 (C, T) was 50.3%, 49.7% in patient group vs. 46.6% and 53.4% in healthy group, respectively; and genotype frequency (CC, CT, TT) was 25.4%, 49.8%, 24.8% in MS group vs. 20.47%, 52.38%, 27.15% in healthy group, respectively. But for rs7014762 (A, T), in MS group allelic frequency was 25.8%, 74.2%, while in control group was 27.1%, 72.9%, respectively, and genotype frequency (AA, AT, TT) was 7.1%, 37.4%, 55.5% in MS group vs. 6.7%, 40.8%, 52.5%, in control group, respectively. According to statistical analysis and χ2 test result, totally there was no significant difference in allelic and genotype frequency between case and control groups (Table 2).
Table 1 Demographic features of MS patient and control individuals. Male
MS Control
Female
Frequency of MS types
Number
Age range
Mean onset age
Number
Age range
Mean onset age
Relapsing remitting (RR)
Secondary progressive (SP)
Primary progressive (PP)
40 45
23–50 21–52
29.7 –
125 155
17–53 16–60
29.7 –
69.7% –
25.3% –
5% –
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Fig. 1. Schematic diagram of mismatch PCR-RFLP technique for rs7014762 genotyping and results of restriction digestion of PCR product by BamHI enzyme on 12% polyacryleamide gel. (A) There is no restriction site at the T/A allelic location of rs7014762. (B) The T nucleotide near the SNP is changed to C nucleotide in PCR reaction by using a reverse primer that has a mismatched nucleotide. (C) BamHI restriction Enzyme can cut PCR product only in T allelic form. (D) If there is T allele form, BamHI restriction enzyme can cut PCR product and 158 bp and 37 bp fragments are produced, but 195 bp fragment represents A allelic form genotyping of each sample were determined on top of the gel.
By classification for clinical MS groups, significant difference in the genotype frequency was observed between SPMS patients and controls for SNP rs6994992; genotype frequency (CC, CT, TT) was 34.28%, 34.28%, 31.42% in patients vs. 20.47%, 52.38%, 27.15%, in control group, respectively (P = 0.038, OR = 2.06, Table 2) and also, a significant difference in the allelic distributions between PPMS patients and controls was revealed; frequency of alleles (C, T) in PPMS group were 31.25%, 68.75% vs. control group, 46.6%, 53.4%, respectively (P = 0.029, OR = 0.5); genotype frequencies (CC, CT, TT) were 0%, 62.5%, 37.5% vs. control group, 20.47%, 52.38%, 27.15%, respectively (P = 0.0001, OR = 0.04, Table 2); there was a significant shift in frequency of C allele between PPMS group vs. other type of MS. Significant association was found for rs7014762 with MS only in PPMS group; allelic frequency (A, T) in PPMS group was 12.5%, 87.5% vs. control group, 27.1%, 72.9% (P = 0.014, OR = 2.5) and genotype frequencies (AA, AT, TT) were 0%, 25%, 75% in patients vs., 6.7%, 40.8%, 52.5%, in control group respectively (P = 0.001, OR = 2.7, Table 2). Gender stratification showed significant difference in the genotype and allelic frequencies between male patients and male controls for SNP rs7014762; allele frequencies (T, A) in male MS group were
82.8%, 17.2% vs., 70%, 30%, in male control group respectively (P = 0.045, OR = 2.09); genotype frequencies (TT, AT, AA) were 65.5%, 34.5%, 0% in male patients vs. male control group, 46.7%, 46.7%, 6.7%, respectively (P = 0.01, OR = 2.1, Table 3). 4. Discussion In this study, we could demonstrate that nucleotide variations in NRG1 gene may have an influence in the progression of MS disease. We also could reveal significant association of functional rs6994992 polymorphism with progressive MS groups (secondary and primary progressive MS), which means that CC genotype frequency increases significantly in secondary progressive (SP) MS group (P = 0.038, OR = 2.06) and heterozygote genotype (CT) frequency decreases in comparison to the healthy group. Allelic and genotype frequencies have different distributions in primary progressive (PP) MS group in comparison to other MS groups, and T allele frequency significantly increases (P = 0.029, OR = 0.5) in comparison to healthy group, CT and TT genotype frequency also increase (P = 0.0001, OR = 0.04) and we could not detect any CC genotype in PPMS.
Table 2 Allele and genotype frequencies of two NRG1 polymorphisms in MS patients compared to controls. SNP
Cohort
Maj.allele%
Min.allele %
P-Value
OR (CI 95%)
Genotypes Maj./Maj.%
Genotypes Maj./Min.%
Genotypes Min./Min.%
P-Value
OR (CI 95%)
rs6994992 Maj. allele: C Min. allele: T
Total MS RR MS SP MS PP MS Male MS Female MS Control Male control Female control Total MS RR MS SP MS PP MS Male MS Female MS Control Male control Female control
50.3 52.3 51.4 31.25 52.4 49.6 46.6 44.6 47.3 74.2 75.6 70.7 87.5 82.8 71.65 72.9 70 73
49.7 47.7 48.6 68.75 47.6 50.4 53.4 55.4 52.7 25.8 24.4 29.3 12.5 17.2 28.35 27.1 30 27
0.77 0.57 0.67 0.029 0.396 0.77
1.13 (0.65–1.96) 1.22 (0.7–2.13) 1.17 (0.67–2.04) 0.5 (0.28–0.9) 1.32 (0.76–2.31) 1.13 (0.65–1.96)
24.8 21.8 31.42 37.5 14.65 28.2 27.15 29.7 26 7.1 6.09 10.34 – – 9.3 6.7 6.7 7
1.33 (0.68–2.5) 1.4 (0.72–2.73) 2.06 (1.08–3.91) 0.04 (0.005–0.3) 1.05 (0.52–2.12) 1.39 (0.72–2.67)
1.1 (0.59–2.1) 1.17 (0.62–2.21) 0.9 (0.49–1.68) 2.5 (1.2–5.1) 2.09 (1.06–4.1) 0.95 (0.51–1.77)
49.8 51.8 34.28 62.5 65.85 44.5 52.38 51.3 53.3 37.4 36.58 37.93 25 34.5 38.5 40.8 46.7 40
0.498 0.4 0.038 0.0001 1.00 0.4
0.88 0.74 0.875 0.014 0.045 0.89
25.4 26.36 34.28 – 19.5 27.3 20.47 19 20.7 55.5 57.3 51.72 75 65.5 52.5 52.5 46.7 53
0.78 0.57 0.89 0.001 0.01 1.00
1.13 (0.65–1.96) 1.2 (0.69–2.1) 0.91 (0.53–1.58) 2.7 (1.5–4.9) 2.1 (1.2–3.75) 0.98 (0.56–1.7)
rs7014762 Maj. allele: T Min. allele: A
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Table 3 Allele and genotype frequencies of two NRG1 polymorphisms in MS sub type patients according to gender. SNP
Cohort
Maj.allele%
Min.allele%
P-Value
OR (CI 95%)
Genotypes Maj./Maj.%
Genotypes Maj./Min.%
Genotypes Min./Min.%
P-Value
OR (CI 95%)
rs6994992 Maj. allele: C Min. allele: T
Total MS Male MS Female MS RR male RR female SP male SP female PP male PP female Control Male control Female control Total MS Male MS Female MS RR male RR female SP male SP female PP female Control Male control Female control
50.3 52.4 49.6 58 50.60 45 56.5 50 12.5 46.6 44.6 47.3 74.2 82.8 71.65 86.80 72.30 64.3 71.50 83.3 72.9 70 73
49.7 47.6 50.4 42 49.40 55 43.5 50 87.5 53.4 55.4 52.7 25.8 17.2 28.35 13.20 27.70 35.7 28.50 16.7 27.1 30 27
0.77 0.396 0.77 0.089 0.67 1 0.2 0.57 0.00
1.13 (0.65–1.96) 1.32 (0.76–2.31) 1.13 (0.65–1.96) 1.68 (0.96–2.9) 1.17 (0.67–2) 1 (0.5–1.7) 1.46 (0.84–2.5) 1.2 (0.7–2.1) 0.16 (0.083–0.33)
24.8 14.65 28.2 8.00 25.88 30.00 30.43 – 75.00 27.15 29.7 26 7.1 – 9.3 0.00 7.94 0.00 14.29 – 6.7 6.7 7
1.33 (0.68–2.5) 1.05 (0.52–2.12) 1.39 (0.72–2.67) 1.35 (0.68–2.6) 1.39 (0.7–2.67) 1.06 (0.53–2.15) 2.8 (1.5–5.3) 0.043 (0.006–0.33) 0.038 (0.005–0.289)
1.1 (0.59–2.1) 2.09 (1.06–4.1) 0.95 (0.51–1.77) 2.86 (1.4–5.9) 0.95 (0.51–1.7) 0.76 (0.42–1.37) 0.91 (0.49–1.7) 1.8 (0.91–3.57)
49.8 65.85 44.5 68.00 47.06 50.00 26.09 100 25.00 52.38 51.3 53.3 37.4 34.5 38.5 26.32 39.68 71.43 28.57 33.33 40.8 46.7 40
0.498 1.00 0.4 0.49 0.4 1 0.001 0.00 0.00
0.88 0.045 0.89 0.005 1 0.45 0.87 0.124
25.4 19.5 27.3 24.00 27.06 20.00 43.48 – – 20.47 19 20.7 55.5 65.5 52.5 73.68 52.38 28.57 57.14 66.67 52.5 46.7 53
0.78 0.01 1.00 0.00 1 0.013 0.67 0.06
1.13 (0.65–1.96) 2.1 (1.2–3.75) 0.98 (0.56–1.7) 3.2 (1.8–5.8) 0.96 (0.55–1.67) 0.46 (0.25–0.83) 1.17 (0.67–2) 1.8 (1–3.2)
rs7014762 Maj. allele: T Min. allele: A
Also we have found a significant association of PPMS with SNP rs7014762 located in NRG1 promoter. Frequency of T allele (P = 0.014, OR = 2.5) and TT genotype (P = 0.001, OR = 2.7) increased significantly, but there is no significant association with other types of MS group (Table 4). Interestingly, a significant association was found in male MS subjects for T allele (P = 0.045, OR = 2.09) and TT genotype (P = 0.01, OR = 2.1) when the samples were stratified by gender. The results of several independent studies indicated that SNP rs6994992 (SNP8NRG243177) is a functional polymorphism in NRG1 regulatory domain that affects NRG1 function. This polymorphism is located on 1.2 kb upstream of the transcription start of type IV NRG1 [8, 27]. Recent meta-analysis confirmed the association of this haplotype with schizophrenia and the risk haplotype denoted HAPice [9]. On the basis of bioinformatics prediction by MATINSPECTOR software (Genomatix, Munich), SNP rs6994992 is located on the putative binding sites for serum response factor (SRF) and myelin transcription factor 1 (Myt-1); but in the presence of T allele, both of these transcription factor binding sites are abolished and a high mobility group box protein-1 (HMGBP-1) binding site is formed [8]. Myt-1 transcription factor is a zinc-finger DNA binding protein that regulates oligodendrocyte regeneration and remyelination. Myt-1 is present inside the nucleus of immature cells during oligodendrocyte lineage development, but it reduced after final differentiation and accumulation of myelin proteins in mature oligodendrocyte [29]. Also, SRF transcription factor plays a substantial role in neural precursor cells (NPCs) and regulates astrocyte and oligodendrocyte specificity and
migration in brain. SRF loss in neurons of mice forebrain increased oligodendrocyte precursors but inhibited oligodendrocyte final differentiation and reduced myelin expression [10,25]. Since SNP rs6994992 is located within mentioned transcription binding sites, interpretation of nucleotide variation is difficult. In one in vitro study Tan et al. found that activity of the 1.5-kb human NRG1 type IV promoter-luciferase fusion gene was 65% higher in T allele constructs (pGL4.SNPT) compared with that of the C allele construct (pGL4.SNPC) in transfected HEK293 Cells [27]. In addition Law et al. reported that there is a substantial difference in NRG1 type IV transcription level between carriers of T allele and C allele in schizophrenia patient brains. Acquisition of two HMGBP1 binding motifs (Homozigocity for TT) but not for one motif (heterozigocity for T) associates with significantly increased type IV NRG1 expression, showing that this effect is recessive [8]. Another study revealed that NRG1-mediated cell migration tendency reduces in schizophrenia patients carrying T allele compared with those carrying C allele, in addition since α7 acetylcholine receptor is regulated by NRG1, carrying T allele showed increased expression of the receptor [22]. In addition SNP rs6994992 was associated with endophenotypes related to schizophrenia. Homozygotes for rs6994992 T allele were associated with deficits in cognitive function, impairment and decreased activation of frontal and temporal lobe regions in individuals at high risk for developing schizophrenia [5,6], which suggests an inefficacy in their neural processing. Besides, normal individuals carrying (T/T) showed reduced white matter in the anterior limb of the internal
Fig. 2. The results of restriction digestion of PCR product by TaiI enzyme on 1% agarose gel for genotyping of SNP rs6994992. The PCR product has a restriction site for TaiI enzyme in T allelic form but not in C allelic form, so digesting PCR product containing T allele produce 137 bp and 446 bp fragments whereas 583 bp fragment represents C allelic form. Genotyping of each sample was determined on top of the gel.
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Fig. 3. Sequencing result of PCR product that is consistent with genotyping results of PCR-RFLP and mismatch PCR-RFLP technique. (A) Sequencing result of heterozygote sample of SNP rs6994992. (B) Sequencing result of heterozygote sample of SNP rs7014762.
capsule [13]. Also, in normal subjects, rs6994992 (T) allele was found to predict lower fractional anisotropy (FA) and decreased white matter in the left anterior thalamic radiation [23]. All of these studies confirm the high importance of this SNP rs6994992 polymorphism. SNP rs7014752 is 87 bp from rs6994992 and is located in the core promoter region; it is also in moderate linkage disequilibrium with rs6994992 (D′ = 0.96, r2 = 0.21). Bioinformatics analysis was performed for finding transcription factor binding sites in the location of this SNP by Transfac software and revealed that the location has a binding site for NF-κB in the presence of A allele but not T allele [31]. Nicodemus et al. indicated that NRG1 type III expression significantly reduce in the presence of T allele (Homozygote and Heterozygote) in comparison to AA homozygote group in schizophrenia patient hippocampus. Of course rs7014762 effect on NRG1 type III expression was significant only in patients in this study [16]. Abnormalities in normal appearing white matter (NAWM) have been detected in almost all MS phenotypes. FA in RR MS patients was lower than in benign MS patients. Most, but not all of studies have shown differences in NAWM diffusion matrices between healthy subjects and RR MS patients. SP MS patients exhibit more certain white matter diffusion abnormalities in comparison with
RR and benign MS patients [20]. Progressive NAWM changes in PP MS patients have been detected by serial diffusion MRI images which proved to be related to both lesion volume and development of clinical disability [21]. At the point of molecular view, demyelination without remyelination might be an important mechanism in MS disease progression [11,18]. The capacity for repair and extent of remyelination in MS lesions is dramatically variable. The reason of incomplete and failed remyelination in MS lesions is unknown yet [17]. The result that two functional polymorphisms in NRG1 gene have different frequencies in different MS types may be interpreted by the fact that primary progressive MS differs from other types of MS (relapse–remitting and secondary progressive MS) in pathologic properties, clinical course, response to treatment, and differential diagnosis. The lesions in PPMS patients are more diffused and less inflammatory in comparison to RRMS and SPMS, and less likely to remyelinate than those occurring in relapsing–remitting MS and secondary progressive MS [2,12]. Hence investigations about molecular mechanisms of different remyelination capacity in different types of MS is essential and could open new avenues for finding appropriate treatment according to repair capacity of different patients.
Table 4 Allele and genotype frequencies of two NRG1 polymorphisms in MS subtype patients according to age. SNP
Cohort
Maj.allele%
Min.allele%
P-Value
rs6994992 Maj. allele: C Min. allele: T
Total MS MS onset age N 25 MS onset age ≤ 25 RR onset age N 25 RR onset age ≤ 25 SP onset age N 25 SP onset age ≤ 25 PP onset age N 25 PP onset age ≤ 25 Control Total MS MS onset age N 25 MS onset age ≤ 25 RR onset age N 25 RR onset age ≤ 25 SP onset age N 25 SP onset age ≤ 25 PP onset age N 25 PP onset age ≤ 25 Control
50.3 54.8 44.28 58 44.80 58.30 42.85 31.25 – 46.6 74.2 79.4 68.52 79.50 71.10 78.10 54.55 87.5 – 72.9
49.7 45.2 55.72 42 55.20 41.70 57.15 68.75 – 53.4 25.8 20.6 31.48 20.50 28.90 21.90 45.45 12.5 – 27.1
0.77 0.32 0.77 0.157 0.88 0.157 0.67 0.029
1.13 (0.65–1.96) 0.72 (0.4–1.26) 1.12 (0.64–1.97) 1.55 (0.89–2.7) 0.9 (0.5–1.6) 1.55 (0.89–2.7) 0.85 (0.48–1.48) 0.5 (0.28–0.9)
0.88 0.4 0.64 0.3 0.87 0.5 0.012 0.014
1.1 (0.59–2.1) 1.39 (0.7–2.6) 0.823 (0.44–1.5) 1.4 (0.7–2.7) 0.9 (0.48–1.68) 1.31 (0.68–2.5) 0.45 (0.25–0.817) 2.5 (1.2–5.1)
rs7014762 Maj. allele: T Min. allele: A
OR (CI 95%)
Genotypes Maj./Maj.%
Genotypes Maj./Min.%
Genotypes Min./Min.%
P-Value
OR (CI 95%)
25.4 28.72 20 30.65 20.83 38.89 28.57 – – 20.47 55.5 61.76 48.15 63.64 50.00 56.25 36.36 75.00 – 52.5
49.8 52.13 48.57 54.84 47.92 38.89 28.57 62.50 – 52.38 37.4 35.29 40.74 31.82 42.11 43.75 36.36 25.00 – 40.8
24.8 19.15 31.43 14.52 31.25 22.22 42.86 37.50 – 27.15 7.1 2.94 11.11 4.55 7.89 – 27.27 – – 6.7
0.498 0.188 1 0.1 1 0.005 0.188 0.00
1.33 (0.68–2.5) 1.63 (0.85–3.14) 1 (0.5–2) 1.8 (0.9–3.4) 1 (0.5–2.1) 2.55 (1.33–4.8) 1.6 (0.85–3.1) 0.04 (0.005–0.3)
0.78 0.2 0.57 0.116 0.77 0.67 0.02 0.001
1.13 (0.65–1.96) 1.47 (0.84–2.59) 0.83 (0.48–1.45) 1.6 (0.9–2.8) 0.9 (0.5–1.57) 1.15 (0.6–2) 0.5 (0.29–0.89) 2.7 (1.5–4.9)
Z. Bahadori et al. / Journal of the Neurological Sciences 351 (2015) 154–159
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