sign in sign up
<<
Home , TRPM, TRPM1, TRPM2, TRPM3, TRPM4, TRPM5

in vivo 29: 763-770 (2015)

Association of TRPM Channel Polymorphisms with Systemic Sclerosis

SERDAR OZTUZCU1, AHMET M. ONAT2, YAVUZ PEHLIVAN2, FATMA ALIBAZ-ONER3, SALIM DONMEZ4, GOZDE Y. CETIN5, SERVET YOLBAS6, IBRAHIM BOZGEYIK1, NESLIHAN YILMAZ7, METIN OZGEN8, YONCA CAGATAY7, BUNYAMIN KISACIK2, SULEYMAN S. KOCA6, OMER NURI PAMUK9, MEHMET SAYARLIOGLU10, HANER DIRESKENELI3 and ABDULLAH T. DEMIRYUREK11

Departments of 1Medical Biology, 2Rheumatology, and 11Medical Pharmacology, Faculty of Medicine, University of Gaziantep, Gaziantep, Turkey; 3Department of Rheumatology, Faculty of Medicine, Marmara University, Istanbul, Turkey; 4Department of Rheumatology, Faculty of Medicine, Yüzüncü Yil University, Van, Turkey; 5Department of Rheumatology, Faculty of Medicine, Kahramanmaras Sütcü Imam University, Kahramanmaras, Turkey; 6Department of Rheumatology, Faculty of Medicine, Firat University, Elazig, Turkey; 7Department of Rheumatology, Faculty of Medicine, Istanbul Bilim University, Istanbul, Turkey; 8Department of Rheumatology, Faculty of Medicine, Inönü University, Malatya, Turkey; 9Department of Rheumatology, Faculty of Medicine, Trakya University, Edirne, Turkey; 10Department of Rheumatology, Faculty of Medicine, Ondokuz Mayıs University, Samsun, Turkey

Abstract. Background/Aim: Systemic sclerosis (SSc) is an polymorphisms studied were found. Conclusion: This is the inflammatory disease characterized by vascular first study to examine the involvement of TRPM channel abnormalities and fibrosis. The aim of the present study was gene variations on the risk of SSc incidence. Our results to investigate the possible role of transient suggest roles of TRPM3 and TRPM5 gene variants in the potential melastatin (TRPM) channel in the susceptibility to or clinical expression of SSc in the Turkish susceptibility and phenotype expression of SSc. Materials population. and Methods: A total of 339 patients with SSc and 302 healthy controls were studied. Genomic DNA was extracted Systemic sclerosis (SSc, scleroderma) is a rare complex, from leukocytes of the peripheral blood, and 25 single multi-system connective tissue disorder affecting the skin nucleotide polymorphisms in the TRPM channel genes were and internal organs characterised by widespread analyzed by the BioMark HD dynamic array system. microangiopathy, fibrosis, autoimmunity and autoantibody Results: There were marked increases in the CC genotype production (1, 2). Tissue fibrosis with an excessive (94.7% vs 81.8%, p<0.0001) and C allele frequencies accumulation of collagen-rich extracellular matrix disrupts (97.0% vs. 90.1%, p<0.0001) in the TRPM3 rs1328142, and the physiological tissue architecture and causes dysfunction TT genotype (19.0% vs. 7.8%, p=0.0002) in TRPM5 of the affected organs. Organ dysfunction can lead to organ rs34551253 (Ala456Thr) polymorphism in SSc patients failure and contributes profoundly to increased mortality due when compared to controls. TRPM3 gene rs1328142 due to SSc. Accumulating evidence indicates that the primary polymorphism was also markedly associated with disease target for both initiating and propagating SSc is blood phenotype. However, no associations with the other 23 vessels (3). The primary insult in targeted organs of SSc patients is directed at blood vessels, and this insult results in tissue ischemia, fibrosis, and ultimately major organ malfunction. Epigenetic modifications caused by Correspondence to: Assoc. Prof. Dr. Serdar Oztuzcu, Department environmental factors also play a role in the pathogenesis of of Medical Biology, Faculty of Medicine, University of Gaziantep, the disease (4, 5). Candidate gene and genome-wide Gaziantep 27310, Turkey. Tel: +90 5054579453, Fax: +90 association studies (GWAS) performed in SSc have identified 3423601617, e-mail: [email protected] several loci implicated in SSc susceptibility (5). Key Words: Cation channels, haplotype, polymorphism, scleroderma, Nevertheless, the complete genetic components of SSc have systemic sclerosis, transient receptor potential melastatin. not been yet completely understood.

0258-851X/2015 $2.00+.40 763 in vivo 29: 763-770 (2015)

Transient receptor potential (TRP) form a All investigations were performed as part of a prospective routine superfamily of non-selective cation channels comprising of clinical protocol. Blood samples were collected for serology (for 28 members in mammals. TRP channels are sub-divided into antibodies) and DNA extraction. Measurement of main SSc-specific autoantibodies was performed by using standard methods. Patients six sub-families based on and with possible pulmonary arterial hypertension (PAH) were evaluated functional aspects: TRPA (ankyrin), TRPC (canonical), with right cardiac catheterization. Patients with ≥25 mm Hg mean TRPM (melastatin), TRPML (mucolipin), TRPP (polycystin) pulmonary arterial were regarded as PAH according to the and TRPV (vanilloid) (6, 7). The TRPM sub-family consists European Society of Cardiology guidelines (16). Electrocardiography of eight members (TRPM1-8). TRPM channels are and echocardiography examinations were used to evaluate the structurally and functionally diverse non-selective cation cardiovascular system involvement. We regarded patients with channels, that are involved in processes ranging from arrhythmia, pericarditis, pericardial effusion, heart failure, and/or PAH as having cardiac involvement. To evaluate the patients’ respiratory detection of cold, , osmolarity, redox state and pH to 2+ systems, we used lung X-rays for all patients. Patients with possible control of Mg homeostasis and cell proliferation or death lung involvement were also evaluated with respiratory function tests (7, 8). Three members of the TRPM sub-family (TRPM2, and high-resolution computed tomography. We regarded patients with TRPM6 and TRPM7) have enzymatic activity. TRPM2 is an ground glass opacification and honeycomb formation as having lung oxidant-sensitive, non-selective cation channel that is widely involvement. Esophagus involvement was evaluated through expressed in mammalian tissues, including the vascular esophagography. Disease activity was assessed according to Valentini endothelium (8). TRPM4 controls pressure-mediated smooth Disease Activity Index (17, 18), and the severity of the disease was defined by Medsger Disease Severity Scale (19). muscle cell and vasoconstriction of cerebral arteries, so it may participate in the autoregulation of cerebral DNA isolation and genotyping. Genomic DNA was extracted from blood flow (9, 10). TRPM6 and TRPM7 are close members peripheral blood leukocytes using a standard salting-out technique, of the TRP superfamily of ion channels having both ion and stored at –20˚C. The genotype was determined in all patients channel and protein kinase activities and have been shown to and controls by using the Fluidigm dynamic array system, as play important roles in cellular magnesium homeostasis. previously described (20). Polymorphisms were analyzed in TRPM8 has a major role in the cold sensation (11, 12). genomic DNA using a 96.96 dynamic array on the BioMark HD However, the role of TRPM channels in SSc is not known. system (Fluidigm, South San Francisco, CA, USA). The Digital Since TRPM channels are widely expressed in the PCR Analysis software (Fluidigm, South San Francisco, CA, USA) was used to process the data after the reaction. Chambers that endothelium and vascular smooth muscles and present in yielded signals were detected and counted. Criteria for choice of cutaneous cells (8, 11, 12), we hypothesize that TRPM SNPs used were: (i) relatively high minor allele frequencies in the channels gene variants play a role in the risk of SSc Caucasian populations; (ii) location within the promoter region, and development. The purpose of the present study was to exonic and intronic sites that could potentially impact on TRPM investigate a possible association between TRPM channel expression and function; and (iii) suitable for the Fluidigm dynamic gene polymorphisms and SSc in a Turkish population. array chip designing, i.e. with no high G/C levels. In the present study, 25 SNPs [TRPM1: rs28441327 in 1, rs11070811 in Materials and Methods intron 1, rs2241493 (Ser32Asn) in exon 5; TRPM2: rs1618355 in intron 18, rs1556314 (Asp543Glu) in exon 11; TRPM3: rs1328142 in intron 9; TRPM4: rs3760663 in 5’-untranslated region, Study population. A total of 339 cases, diagnosed as SSc at various rs71352737 (Trp525Ter) in exon 11; TRPM5: rs34364959 Rheumatology clinics in Turkey, and 302 controls were included in the (Gly900Ser) in exon 18, rs886277 (Asn235Ser) in exon 5, study. All patients met the criteria established by the American College rs4929982 (Arg578Gln) in exon 11, rs34551253 (Ala456Thr) in of Rheumatology classification criteria for SSc (13). In addition, exon 9, rs3986599 (Val254Ala) in exon 6; TRPM6: rs3750425 patients were classified as having limited or diffuse SSc according to (Val1388Ile) in exon 28, rs2274924 (Lys1579Glu) in exon 29; the definitions developed by LeRoy et al. (14). Limited cutaneous TRPM8: rs6431648 in intron 1, rs10803666 in intron 2, rs12472151 disease (lcSSc) was defined as definite skin thickening confined to the in intron 6, rs2215173 in intron 7, rs6740118 in intron 9, rs1016062 distal extremities, whereas cases of diffuse cutaneous disease (dcSSc) in intron 21, rs2362294 in intron 21, rs2362295 in intron 22, required also the involvement of skin proximal to the knees and elbow rs10490018 in intron 24, and rs2052029 in 3’-untranslated region] (14, 15). The control population consisted of healthy individuals were studied for TRPM gene polymorphisms. recruited at the same geographical regions as SSc patients and matched by age, sex and ethnicity to the SSc patient group. The study was Statistical analyses. Results are expressed as the mean±S.D. or approved by the local Ethics Committee, in compliance with the percentage otherwise indicated. The Chi-square test for Declaration of Helsinki. Both patients and controls were included in independence, Chi-square test with Yate’s correction or Fisher’s the study after written informed consent. Since the organ involvement exact tests was used for calculation of the significance of differences of SSc has been investigated, possible co-morbidities were excluded in genotype and allele frequencies. All probability values were from this study. Therefore, patients with the diagnosis of myocardial based on two-tailed tests. The Bonferroni correction for multiple infarction before the initiation of SSc, patients with hypertension, testing was used for all statistical comparisons and a p-value <0.002 hyperlipidemia, diabetes mellitus, renal dysfunction, additional chronic (0.05/25) was considered statistically significant. The effects of and other than autoimmune diseases were excluded. genetic polymorphisms on the risk of SSc were estimated with the

764 Oztuzcu et al: TRPM Polymorphisms in Systemic Sclerosis odds ratio (OR) and 95% confidence intervals (CI). For comparisons Table I. Clinical characteristics of the study population. of the differences between mean values of 2 groups, the unpaired Student’s t test was used. Statistical analysis was performed using Patients Controls p-Value GraphPad Instat version 3.05 (GraphPad Software Inc., San Diego, (n=339) (n=302) CA, USA). Haplotypes was analyzed with the SHEsis software (http://analysis.bio-x.cn/myAnalysis.php). Age (years)* 47.1±12.8 46.4±13.2 0.5386 Gender Results Male (n, %) 27 (8.0) 31 (10.3) Female (n, %) 312 (92.0) 271 (89.7) 0.3361 Raynaud's phenomenon (n, %) 308 (90.9) Clinical characteristics of the study population are presented in ANA (n, %) 296 (87.3) Table I. In the present study, 339 patients with SSc admitted to lcSSc (n, %) 196 (57.8) the Rheumatology clinics were investigated. The mean age and Systemic involvement (n, %) 192 (56.6) gender of the patients and control groups were similar. The Lung (n, %) 182 (53.7) majority of patients had limited and diffuse SSc, but other forms Digital ulceration (n, %) 153 (45.1) Anti-Scl 70 (n, %) 137 (40.4) such as systemic sclerosis sine SSc and overlap syndrome were Active Valentini score (n, %) 137 (40.4) also observed. Organ involvement in accordance to disease type dcSSc (n, %) 117 (34.5) and antibody positivity is documented in Table I. Mean disease ACA (n, %) 88 (26.0) duration was 8.8±6.5 years. Finger flexion deformity (n, %) 83 (24.5) Tables II and III show the distributions of genotype and allele GIS (n, %) 78 (23.0) Cardiac (n, %) 57 (16.8) frequencies for the TRPM channel gene polymorphisms in our PAH (n, %) 55 (16.2) study participants. Marked associations were detected in the Renal (n, %) 16 (4.7) rs1328142 (TRPM3) and rs34551253 (Ala456Thr) (TRPM5) Rodnan skin score* 14.3±7.3 polymoprhisms. In the rs1328142 polymorphism, there were Disease severity scale* 5.5±3.0 increases in the CC genotype (94.4%) and C allele frequencies *Data are mean±SD. ACA: Anticentromere antibody (≤15 was positive); (97.0%) in SSc patients when compared to the control group ANA: antinuclear antibody (≤1/160 was positive); anti-Scl-70: (CC, 81.8%, C, 90.1%, p<0.0001). The presence of C allele antitopoisomerase I antibodies (≤15 was positive); dcSSc: diffuse cutaneous decreased the risk of SSc (OR=0.276, 95% CI=0.165-0.464, systemic sclerosis; GIS: gastrointestinal system involvement; lcSSc: limited p<0.0001). A lower frequency of the CA genotype was observed cutaneous systemic sclerosis; PAH: pulmonary arterial hypertension. in SSc (5.3%) compared to controls (16.6%, p<0.0001). There was an increase in TT genotype (19.0% in patients vs. 7.8% in controls) and decrease in CT genotype frequencies (80.3% in Discussion control vs. 67.9% in patients) in SSc group (p=0.0002) in rs34551253 (Ala456Thr) polymorphism. In the presence of TT genotype, the risk of SSc increased 2.21-fold (OR=2.210, 95% In this case-control study, we showed that TRPM3 rs1328142 CI=1.146-4.263, p=0.0263). There were no marked associations and TRPM5 rs34551253 (Ala456Thr) polymorphisms were with the other 23 TRPM polymorhisms studied. significantly associated with SSc, and could be the risk factor We found that the TRPM3 gene rs1328142 polymorphism of developing SSc. Our results suggest that the CC genotype was also significantly associated with the limited cutaneous of the rs1328142 and the TT genotype of the rs34551253 systemic sclerosis, but not with diffuse cutaneous systemic (Ala456Thr) polymorphisms may increase susceptibility to sclerosis. This polymorphism was markedly associated with SSc. We observed that TRPM3 rs1328142 was markedly SSc-specific autoantibodies, active Valentini score, and associated with manifestations of SSc. A allele of this Raynaud's phenomenon bot not with pulmonary arterial polymorphism appeared to be protective allele. Two hypertension (Table IV). The TRPM3 gene rs1328142 haplotypes in TRPM5 gene polymoprhisms were found to be polymorphism A allele appeared to be a protective allele markedly associated with SSc. This is the first study to (Table IV). However, allele frequencies of the TRPM5 gene examine the genetic association of the TRPM gene rs34551253 polymorphism were not significantly associated polymorphisms with the risk of developing SSc. When with the measured parameters (Table V). patients with SSc were classified as having either lcSSc or Haplotypes based on the studied TRPM5 gene dcSSc and sub-analyses were performed, TRPM3 rs1328142 polymorphisms were constructed, and ten haplotypes with displayed marked associations with limited cutaneous high frequency were detected (Table VI). Only two of these systemic sclerosis in our study. haplotypes (CACCG, and CACTA) were markedly The functional roles of TRPM genes and channels in SSc associated with SSc. Although the CACCG haplotype was are unknown. TRPM2 channels may be involved in vessel less frequently seen, the CACTA haplotype was more injury or atherosclerosis development, which is associated frequently found in cases than controls (Table VI). with mitochondrial dysfunction, reactive oxygen species

765 in vivo 29: 763-770 (2015)

Table II. Genotype and allele frequencies and distribution of probable Table III. Genotype and allele frequencies and distribution of probable haplotypes of the TRPM1, TRPM2, TRPM3, TRPM4, and TRPM5 gene haplotypes of the TRPM6 and TRPM8 gene polymorphisms in patients polymorphisms in patients with systemic sclerosis (SSc) and controls. with systemic sclerosis (SSc) and controls.

Gene Genotypes/ Controls n* Patients n* p-Value Gene Genotypes/ Controls n* Patients n* p-Value SNP Alleles SNP Alleles

TRPM1 CC/CT/TT 167/112/22 301 200/106/24 330 0.3886 TRPM6 GG/GA/AA 216/77/6 299 223/102/9 334 0.3206 rs28441327 C/T 446/156 506/154 0.3182 rs3750425 G/A 509/89 548/120 0.1620 TRPM1 GG/GA/AA 162/123/17 302 200/112/20 332 0.1887 (Val1388Ile) rs11070811 G/A 447/157 512/152 0.2227 TRPM6 GG/GC/CC 110/158/0 268 160/152/0 312 0.0173 TRPM1 AA/AG/GG 160/113/28 301 173/136/22 331 0.3805 rs2274924 G/C 378/158 472/152 0.0578 rs2241493 A/G 433/169 482/180 0.7736 (Lys1579Glu) (Ser32Asn) TRPM8 CC/CT/TT 180/103/18 301 189/128/20 337 0.6058 TRPM2 TT/TG/GG 168/111/23 302 193/123/14 330 0.1919 rs6431648 C/T 463/139 506/168 0.4837 rs1618355 T/G 447/157 509/151 0.2214 TRPM8 GG/GC/CC 224/70/8 302 239/92/8 339 0.5111 TRPM2 CC/CA/AA 213/74/11 298 229/64/23 316 0.0814 rs10803666 G/C 518/86 570/108 0.4442 rs1556314 C/A 500/96 522/110 0.5948 TRPM8 CC/CT/TT 104/183/0 287 148/167/4 319 0.0046 (Asp543Glu) rs12472151 C/T 391/183 463/175 0.1024 TRPM3 CC/CA/AA 247/50/5 302 319/18/1 338 <0.0001 TRPM8 CC/CT/TT 212/80/9 301 240/86/10 336 0.9602 rs1328142 C/A 544/60 656/20 <0.0001 rs2215173 C/T 504/98 566/106 0.8658 TRPM4 CC/CT/TT 148/118/33 299 177/120/28 325 0.3802 TRPM8 GG/GA/AA 215/76/9 300 240/87/10 337 0.9903 rs3760663 C/T 414/184 474/176 0.1689 rs6740118 G/A 506/94 567/107 0.9800 TRPM4 GG/GA/AA 295/6/0 301 329/8/0 337 0.7930 TRPM8 CC/CT/TT 213/80/8 301 243/86/9 338 0.9478 rs71352737 G/A 596/6 666/8 0.7941 rs1016062 C/T 506/96 572/104 0.8422 (Trp525Ter) TRPM8 CC/CT/TT 68/128/59 255 120/122/64 306 0.0062 TRPM5 CC/CT/TT 280/21/1 302 315/19/3 337 0.5365 rs2362294 C/T 264/246 362/250 0.0155 rs34364959 C/T 581/23 649/25 1.0000 TRPM8 AA/AG/GG 173/110/18 301 190/121/27 338 0.6124 (Gly900Ser) rs2362295 A/G 456/146 501/175 0.5431 TRPM5 AA/AG/GG 123/144/34 301 138/159/41 338 0.9437 TRPM8 GG/GA/AA 149/134/18 301 151/149/36 336 0.0863 rs886277 A/G 390/212 435/241 0.9174 rs10490018 G/A 312/170 451/221 0.4352 (Asn235Ser) TRPM8 GG/GC/CC 164/124/13 301 188/138/13 339 0.9377 TRPM5 CC/CT/TT 121/134/42 297 139/113/66 318 0.0217 rs2052029 G/C 452/150 514/164 0.8126 rs4929982 C/T 376/218 391/245 0.5485 (Arg578Gln) *Numbers do not always add up to total numbers because of missing TRPM5 CC/CT/TT 35/237/23 295 42/218/61 321 0.0002 values on the BioMark dynamic array system. SNP, Single nucleotide rs34551253 C/T 307/283 302/340 0.0902 polymorphism. (Ala456Thr) TRPM5 GG/GA/AA 205/73/22 300 193/109/26 328 0.0372 rs3986599 G/A 483/117 495/161 0.0373 (Val254Ala) The progressive vascular injury includes persistent endothelial *Numbers do not always add-up to total numbers because of missing cell activation/damage and apoptosis, intimal thickening, de- values on the BioMark dynamic array system. SNP, Single nucleotide lamination, vessel narrowing and obliteration. These changes polymorphism. lead to vascular tone dysfunction and reduced capillary blood flow, with consequent tissue ischemia and severe clinical manifestations, such as digital ulceration, pulmonary arterial production and (21). It is also known that hypertension, and scleroderma renal crisis (28). Collectively, decreases TRPM6 channel activity (22). these findings suggest that TRP channels are critical for Since reactive oxygen species are implicated as potential aspects of vascular function and may involve the vasculopathy mediators in the initial vascular injury in SSc, and are also a seen in SSc. potential cause of constitutive fibroblastic activation (23, 24), It is generally accepted that first-line therapy for TRPM channels may contribute to pathogenesis of SSc. Raynaud’s phenomenon is dihydropyridine-type TRPM7-dependent Mg2+ influx is required for vascular channel blockers, such as nifedipine and nicardipine, in order smooth muscle cell proliferation in response to Ang II (25). to inhibit peripheral vasospasm (29). It is known that calcium TRPM4 requires high levels of intracellular Ca2+ for activation channel blockers do not inhibit TRP channels (30). In fact, (26). TRPM4 channels are important for pressure-induced high concentrations of nifedipine can activate TRPM3 smooth muscle cell de-polarization and vasoconstriction (9). channel (31). Our study showed that TRPM3 rs1328142 SSc vasculopathy is characterized by a variety of changes that polymorphism was significantly associated with Raynaud's mainly affect the microcirculation and small arterioles (27). phenomenon.

766 Oztuzcu et al: TRPM Polymorphisms in Systemic Sclerosis

Table IV. Allele frequencies of TRPM3 gene rs1328142 polymorphism in systemic sclerosis cases and controls.

C allele A alelle p-Value OR (95% CI) n (%) n (%)

Control 544 (90.1) 60 (9.9) Raynaud's phenomenon 594 (96.7) 20 (3.3) <0.0001 0.305 (0.182-0.513) Positive antinuclear antibody (ANA) 574 (97.0) 18 (3.0) <0.0001 0.284 (0.166-0.488) Cases with limited cutaneous systemic sclerosis (lcSSc) 385 (98.2) 7 (1.8) <0.0001 0.165 (0.075-0.365) Cases with lung involvement 351 (96.4) 13 (3.6) 0.0002 0.336 (0.182-0.621) Cases with digital ulceration 300 (98.0) 6 (2.0) <0.0001 0.181 (0.077-0.425) Positive anti-Scl-70 (anti-topoisomerase I antibodies; ATA) 264 (96.4) 10 (3.6) 0.0011 0.343 (0.173-0.682) Active Valentini score 265 (96.7) 9 (3.3) 0.0004 0.308 (0.151-0.630) Cases with diffuse cutaneous systemic sclerosis (dcSSc) 224 (95.7) 10 (4.3) 0.0077 0.405 (0.204-0.805) Positive anticentromere antibody (ACA) 172 (97.7) 4 (2.3) 0.0005 0.211 (0.076-0.589) Cases with finger flexion deformity 162 (97.6) 4 (2.4) 0.0012 0.224 (0.080-0.626) Cases with gastrointestinal system involvement 152 (97.4) 4 (2.6) 0.0018 0.239 (0.085-0.667) Cases with cardiac involvement 112 (98.2) 2 (1.8) 0.0018 0.162 (0.039-0.672) Cases with PAH 106 (96.4) 4 (3.6) 0.0299 0.342 (0.122-0.962)

OR, Odds ratio; CI, Confidence interval.

Table V. Allele frequencies of TRPM5 gene rs34551253 (Ala456Thr) polymorphism in systemic sclerosis cases and controls.

G allele A alelle p-Value OR (95% CI) n (%) n (%)

Control 307 (52.0) 283 (48.0) Raynaud's phenomenon 268 (45.9) 316 (54.1) 0.0407 1.279 (1.017-1.609) Positive antinuclear antibody (ANA) 268 (47.5) 296 (52.5) 0.1402 1.198 (0.951-1.510) Cases with limited cutaneous systemic sclerosis (lcSSc) 182 (48.4) 194 (51.6) 0.3011 1.156 (0.893-1.498) Cases with lung involvement 157 (45.9) 185 (54.1) 0.0827 1.278 (0.979-1.670) Cases with digital ulceration 147 (50.7) 143 (49.3) 0.7616 1.055 (0.797-1.398) Positive anti-Scl-70 (anti-topoisomerase I antibodies; ATA) 120 (45.1) 146 (54.9) 0.0718 1.320 (0.987-1.765) Active Valentini score 120 (46.2) 140 (53.8) 0.1322 1.266 (0.945-1.696) Cases with diffuse cutaneous systemic sclerosis (dcSSc) 101 (45.5) 121 (54.5) 0.1136 1.300 (0.954-1.771) Positive anticentromere antibody (ACA) 81 (48.8) 85 (51.2) 0.5159 1.138 (0.807-1.607) Cases with finger flexion deformity 80 (50.6) 78 (49.4) 0.8233 1.058 (0.744-1.503) Cases with gastrointestinal system involvement 62 (40.8) 90 (59.2) 0.0172 1.575 (1.097-2.261) Cases with cardiac involvement 54 (49.1) 56 (50.9) 0.6433 1.125 (0.749-1.691) Cases with PAH 48 (45.3) 58 (54.7) 0.2402 1.311 (0.865-1.986)

OR, Odds ratio; CI, confidence interval.

PAH is the main pulmonary vascular complication of SSc, SSc-PAH (35). We found no evidence regarding association of and leads to mortality (32). There exists evidence of TRPM variants with SSc-related PAH. underlying endothelial injury and dysfunction in PAH Primary myocardial involvement is likely to result from development. Many mediators that modulate arterial tone and the general vasospastic mechanism that is thought to play a re-modelling are involved in the genesis of PAH, and all genes key role in this disease. Vasospasm of the small coronary encoding these mediators are potential target-candidate genes. arteries or arterioles would initially impair perfusion and While potential common molecular pathways for pulmonary function, with reversible involvement. This would be vascular disease have been regularly discovered (33), genes followed by structural coronary arteriolar lesion leading to creating susceptibility to the disease are only just emerging irreversible abnormalities. TRPM3 rs1328142 polymorphism (34). Recently, the first evidence for the was found to be significantly associated with cardiac KCNA5 rs10744676 variant was found to be associated with involvement in the present study.

767 in vivo 29: 763-770 (2015)

Table VI. Distribution of haplotype frequencies of TRPM5 gene and thermosensor in the somatosensory system (41). TRPM3 polymorphisms in SSc patients and controls. is activated by heat, and its activation provokes pain. TRPM3-deficient mice also failed to develop heat hyper- Haplotypes* Controls Patients p-Value OR (95% CI) n (%) n (%) algesia following an inflammatory challenge (41). These results indicate that TRPM3 is likely to play a role in the C A C C A 41 (7.0) 42 (6.8) 0.8755 0.965 (0.617-1.508) pain perception in SSc. C A C C G 118 (20.3) 84 (13.5) 0.0014 0.607 (0.446-0.826) In conclusion, the present study provided first evidence for 0.0001 C A C T A 40 (6.8) 84 (13.6) 2.161 (1.452-3.215) a genetic association of TRPM gene polymorphisms and C A C T G 91 (15.6) 79 (12.7) 0.1506 0.787 (0.567-1.092) C A T C G 26 (4.5) 35 (5.6) 0.3760 1.264 (0.752-2.127) susceptibility of SSc. Our findings showed that TRPM3 and C A T T G 11 (1.8) 27 (4.3) 0.0121 2.454 (1.191-5.055) TRPM5 genes might be risk factors for SSc, and suggest that C G C C G 16 (2.8) 30 (4.8) 0.0644 1.777 (0.959-3.294) genetic polymorphisms in TRPM gene modify individual C G C T G 39 (6.7) 39 (6.2) 0.7252 0.921 (0.580-1.460) susceptibility to SSc in the Turkish population. The newly- C G T C G 82 (14.0) 73 (11.8) 0.2323 0.813 (0.579-1.142) identified genetic susceptibility loci can lead to identification C G T T G 72 (12.3) 78 (12.6) 0.8803 1.027 (0.727-1.449) of novel therapeutic targets and guide drug development. Our *The haplotype analysis was performed in the order of rs34364959, findings also imply that polymorphisms or haplotypes of the rs886277, rs4929982, rs34551253, and rs3986599. Haplotypes with TRPM gene might be used as a biomarker for the early frequencies <3% were excluded from analysis. OR, Odds ratio; CI, predictor of risk of developing SSc. However, further confidence interval. validation of these findings in different ethnic groups and independent cohorts is necessary. Conflicts of Ιnterest Positive autoantibody titers are a main feature of SSc, especially anticentromere autoantibodies (ACA) and The Authors declare no conflict of interest. antitopoisomerase autoantibodies (ATA) (1). We observed that ACA, ATA and anti-nuclear antibody (ANA) were References associated with TRPM3 rs1328142 polymorphism. To date, a number of genes have been implicated in an 1 Katsumoto TR, Whitfield ML and Connolly MK: The pathogenesis of systemic sclerosis. Annu Rev Pathol 6: 509-537, increased susceptibility to SSc, confirming the genetic 2011. component of this complex disease (4, 36). However, the 2 Bhattacharyya S, Wei J and Varga J: Understanding fibrosis in genetic basis for SSc is defined by multiple genes that have systemic sclerosis: shifting paradigms, emerging opportunities. only a modest effect on disease susceptibility (4, 5). GWAS Nat Rev Rheumatol 8: 42-54, 2012. studies have been performed on SSc patients, and strong 3 Matucci-Cerinic M, Kahaleh B and Wigley FM: Review: associations within the major histocompatibility have evidence that systemic sclerosis is a vascular disease. Arthritis been identified. These studies may indicate that adaptive Rheum 65: 1953-1962, 2013. 4 Martin J and Fonseca C: The genetics of scleroderma. Curr antigen-driven immunity contributes to SSc pathogenesis (36, Rheumatol Rep 13: 13-20, 2011. 37). We found that TRPM3 (located on 9q-21.11- 5 Martin JE, Bossini-Castillo L and Martin J: Unraveling the 12) and TRPM5 (11p15.5) genes may modify individual genetic component of systemic sclerosis. Hum Genet 131: 1023- susceptibility to SSc. Therefore, our results with TRPM gene 1037, 2012. polymorphisms add new loci of susceptibility to SSc. 6 Montell C: The TRP superfamily of cation channels. Sci STKE In addition to the TRPM3 gene rs1328142 polymorphism, 2005: re3, 2005. TRPM5 rs34551253 and haplotypes were markedly 7 Nilius B: TRP channels in disease. Biochim Biophys Acta 1772: associated with SSc in our study. These polymorphisms and 805-812, 2007. 8 Zholos A: Pharmacology of transient receptor potential haplotypes may be used as a biomarker for the early melastatin channels in the vasculature. Br J Pharmacol 159: prediction of risk of SSc development. 1559-1571, 2010. TRPM5 is highly expressed in the taste buds of the tongue 9 Earley S, Waldron BJ and Brayden JE: Critical role for transient where it has a central role in taste transduction (38). potential channel TRPM4 in myogenic constriction of impairment and a unilateral loss of taste with fasciculations of cerebral arteries. Circ Res 95: 922-929, 2004. the tongue have been reported in SSc patients with cranial 10 Gonzales AL, Amberg GC and Earley S: Ca2+ release from the nerve involvement (39). Thus, TRPM5 may have a role in SSc. sarcoplasmic reticulum is required for sustained TRPM4 activity in cerebral artery smooth muscle cells. Am J Physiol Cell Pain is found in a large percentage of SSc patients and Physiol 299: C279-C288, 2010. does not appear adequately treated (40). TRPM3 expressed 11 McKemy DD, Neuhausser WM and Julius D: Identification of a in a large subset of small-diameter sensory from cold receptor reveals a general role for TRP channels in dorsal root and trigeminal ganglia, and functions as a chemo- thermosensation. Nature 416: 52-58, 2002.

768 Oztuzcu et al: TRPM Polymorphisms in Systemic Sclerosis

12 Peier AM, Moqrich A, Hergarden AC, Reeve AJ, Andersson DA, 25 He Y, Yao G, Savoia C and Touyz RM: Transient receptor Story GM, Earley TJ, Dragoni I, McIntyre P, Bevan S and potential melastatin 7 ion channels regulate magnesium Patapoutian A: A TRP channel that senses cold stimuli and homeostasis in vascular smooth muscle cells: role of angiotensin . Cell 108: 705-715, 2002. II. Circ Res 96: 207-215, 2005. 13 Preliminary criteria for the classification of systemic sclerosis 26 Launay P, Fleig A, Perraud AL, Scharenberg AM, Penner R and (scleroderma). Subcommittee for scleroderma criteria of the Kinet JP: TRPM4 is a Ca2+-activated nonselective cation channel American Rheumatism Association Diagnostic and Therapeutic mediating cell membrane depolarization. Cell 109: 397-407, Criteria Committee. Arthritis Rheum 23: 581-590, 1980. 2002. 14 LeRoy EC, Black C, Fleischmajer R, Jablonska S, Krieg T, 27 Rodnan GP, Myerowitz RL and Justh GO: Morphologic changes Medsger TA Jr, Rowell N and Wollheim F: Scleroderma in the digital arteries of patients with progressive systemic (systemic sclerosis): classification, subsets and pathogenesis. J sclerosis (scleroderma) and Raynaud phenomenon. Medicine Rheumatol 15: 202-205, 1988. (Baltimore) 59: 393-408, 1980. 15 LeRoy EC and Medsger TA Jr: Criteria for the classification of 28 Manetti M, Guiducci S, Ibba-Manneschi L and Matucci-Cerinic early systemic sclerosis. J Rheumatol 28: 1573-1576, 2001. M: Mechanisms in the loss of capillaries in systemic sclerosis: 16 Galiè N, Hoeper MM, Humbert M, Torbicki A, Vachiery JL, angiogenesis versus vasculogenesis. J Cell Mol Med 14: 1241- Barbera JA, Beghetti M, Corris P, Gaine S, Gibbs JS, Gomez- 1254, 2010. Sanchez MA, Jondeau G, Klepetko W, Opitz C, Peacock A, Rubin 29 Walker KM and Pope J: Expert agreement on EULAR/EUSTAR L, Zellweger M, Simonneau G and ESC Committee for Practice recommendations for the management of systemic sclerosis. J Guidelines (CPG): Guidelines for the diagnosis and treatment of Rheumatol 38: 1326-1328, 2011. pulmonary hypertension: The Task Force for the Diagnosis and 30 Yang XR, Lin MJ, McIntosh LS and Sham JS: Functional Treatment of Pulmonary Hypertension of the European Society of expression of transient receptor potential melastatin- and Cardiology (ESC) and the European Respiratory Society (ERS), vanilloid-related channels in pulmonary arterial and aortic endorsed by the International Society of Heart and Lung smooth muscle. Am J Physiol Lung Cell Mol Physiol 290: Transplantation (ISHLT). Eur Heart J 30: 2493-2537, 2009. L1267-L1276, 2006. 17 Valentini G, Della Rossa A, Bombardieri S, Bencivelli W, 31 Wagner TF, Loch S, Lambert S, Straub I, Mannebach S, Mathar Silman AJ, D'Angelo S, Cerinic MM, Belch JF, Black CM, I, Düfer M, Lis A, Flockerzi V, Philipp SE and Oberwinkler J: Bruhlmann P, Czirják L, De Luca A, Drosos AA, Ferri C, Transient receptor potential M3 channels are ionotropic steroid Gabrielli A, Giacomelli R, Hayem G, Inanc M, McHugh NJ, receptors in pancreatic beta cells. Nat Cell Biol 10: 1421-1430, Nielsen H, Rosada M, Scorza R, Stork J, Sysa A, van den 2008. Hoogen FH and Vlachoyiannopoulos PJ: European multicentre 32 Tyndall AJ, Bannert B, Vonk M, Airò P, Cozzi F, Carreira PE, study to define disease activity criteria for systemic sclerosis. II. Bancel DF, Allanore Y, Müller-Ladner U, Distler O, Iannone F, Identification of disease activity variables and development of Pellerito R, Pileckyte M, Miniati I, Ananieva L, Gurman AB, preliminary activity indexes. Ann Rheum Dis 60: 592-598, 2001. Damjanov N, Mueller A, Valentini G, Riemekasten G, Tikly M, 18 Valentini G, Silman AJ and Veale D: Assessment of disease Hummers L, Henriques MJ, Caramaschi P, Scheja A, Rozman activity. Clin Exp Rheumatol 21: S39-S41, 2003. B, Ton E, Kumánovics G, Coleiro B, Feierl E, Szucs G, Von 19 Medsger TA Jr, Bombardieri S, Czirjak L, Scorza R, Della Rossa Mühlen CA, Riccieri V, Novak S, Chizzolini C, Kotulska A, A and Bencivelli W: Assessment of disease severity and Denton C, Coelho PC, Kötter I, Simsek I, de la Pena Lefebvre prognosis. Clin Exp Rheumatol 21: S42-S46, 2003. PG, Hachulla E, Seibold JR, Rednic S, Stork J, Morovic-Vergles 20 Okumus S, Demiryürek S, Gürler B, Coskun E, Bozgeyik I, J and Walker UA: Causes and risk factors for death in systemic Oztuzcu S, Kaydu E, Celik O, Erbagcı I and Demiryürek AT. sclerosis: a study from the EULAR Scleroderma Trials and Association TRPM channel gene polymorphism with primary Research (EUSTAR) database. Ann Rheum Dis 69: 1809-1815, open-angle glaucoma. Mol Vis 19: 1852-1858, 2013. 2010. 21 Takahashi N, Kozai D, Kobayashi R, Ebert M and Mori Y: Roles 33 Humbert M, Sitbon O and Simonneau G: Treatment of of TRPM2 in oxidative stress. Cell Calcium 50: 279-287, 2011. pulmonary arterial hypertension. N Engl J Med 351: 1425-1436, 22 Cao G, Lee KP, van der Wijst J, de Graaf M, van der Kemp A, 2004. Bindels RJ and Hoenderop JG. Methionine sulfoxide reductase 34 Shah SJ: Genetics of systemic sclerosis-associated pulmonary B1 (MsrB1) recovers TRPM6 channel activity during oxidative arterial hypertension: recent progress and current concepts. Curr stress. J Biol Chem 285: 26081-26087, 2010. Rheumatol Rep 11: 89-96, 2009. 23 Sambo P, Baroni SS, Luchetti M, Paroncini P, Dusi S, Orlandini 35 Wipff J, Dieudé P, Guedj M, Ruiz B, Riemekasten G, Cracowski G and Gabrielli A: Oxidative stress in scleroderma: maintenance JL, Matucci-Cerinic M, Melchers I, Humbert M, Hachulla E, of scleroderma fibroblast phenotype by the constitutive up- Airo P, Diot E, Hunzelmann N, Caramaschi P, Sibilia J, Valentini regulation of reactive oxygen species generation through the G, Tiev K, Girerd B, Mouthon L, Riccieri V, Carpentier PH, NADPH oxidase complex pathway. Arthritis Rheum 44: 2653- Distler J, Amoura Z, Tarner I, Degano B, Avouac J, Meyer O, 2664, 2001. Kahan A, Boileau C and Allanore Y: Association of a KCNA5 24 Servettaz A, Guilpain P, Goulvestre C, Chéreau C, Hercend C, gene polymorphism with systemic sclerosis-associated Nicco C, Guillevin L, Weill B, Mouthon L and Batteux F: pulmonary arterial hypertension in the European Caucasian Radical oxygen species production induced by advanced population. Arthritis Rheum 62: 3093-3100, 2010. oxidation protein products predicts clinical evolution and 36 Radstake TR, Gorlova O, Rueda B, Martin JE, Alizadeh BZ, response to treatment in systemic sclerosis. Ann Rheum Dis 66: Palomino-Morales R, Coenen MJ, Vonk MC, Voskuyl AE, 1202-1209, 2007. Schuerwegh AJ, Broen JC, van Riel PL, van't Slot R, Italiaander

769 in vivo 29: 763-770 (2015)

A, Ophoff RA, Riemekasten G, Hunzelmann N, Simeon CP, 38 Liman ER: TRPM5 and taste transduction. Handb Exp Ortego-Centeno N, González-Gay MA, González-Escribano MF; Pharmacol 179: 287-298, 2007. Spanish Scleroderma Group, Airo P, van Laar J, Herrick A, 39 Teasdall RD, Frayha RA and Shulman LE: Cranial nerve Worthington J, Hesselstrand R, Smith V, de Keyser F, Houssiau involvement in systemic sclerosis (scleroderma): a report of 10 F, Chee MM, Madhok R, Shiels P, Westhovens R, Kreuter A, cases. Medicine (Baltimore) 59: 149-159, 1980. Kiener H, de Baere E, Witte T, Padykov L, Klareskog L, Beretta 40 Rehberger P, Müller H, Günther C and Schmitt J: Treatment L, Scorza R, Lie BA, Hoffmann-Vold AM, Carreira P, Varga J, satisfaction and health status in patients with systemic sclerosis. Hinchcliff M, Gregersen PK, Lee AT, Ying J, Han Y, Weng SF, J Dtsch Dermatol Ges 10: 905-912, 2012. Amos CI, Wigley FM, Hummers L, Nelson JL, Agarwal SK, 41 Vriens J, Owsianik G, Hofmann T, Philipp SE, Stab J, Chen X, Assassi S, Gourh P, Tan FK, Koeleman BP, Arnett FC, Martin J Benoit M, Xue F, Janssens A, Kerselaers S, Oberwinkler J, and Mayes MD: Genome-wide association study of systemic Vennekens R, Gudermann T, Nilius B and Voets T: TRPM3 is a sclerosis identifies CD247 as a new susceptibility locus. Nat channel involved in the detection of noxious heat. Genet 42: 426-429, 2010. 70: 482-494, 2011. 37 Allanore Y, Saad M, Dieudé P, Avouac J, Distler JH, Amouyel P, Matucci-Cerinic M, Riemekasten G, Airo P, Melchers I, Hachulla E, Cusi D, Wichmann HE, Wipff J, Lambert JC, Hunzelmann N, Tiev K, Caramaschi P, Diot E, Kowal-Bielecka O, Valentini G, Mouthon L, Czirják L, Damjanov N, Salvi E, Conti C, Müller M, Müller-Ladner U, Riccieri V, Ruiz B, Cracowski JL, Letenneur L, Dupuy AM, Meyer O, Kahan A, Munnich A, Boileau C and Martinez M: Genome-wide scan Received August 31, 2015 identifies TNIP1, PSORS1C1, and RHOB as novel risk loci for Revised October 15, 2015 systemic sclerosis. PLoS Genet 7: e1002091, 2011. Accepted October 16, 2015

770