No evidence for a role of the proximal IL-6 G/C -174 single nucleotide polymorphism in Italian patients with systemic sclerosis.

Dear Editor,

In a recent issue of the JCMM, Sfrent-Cornateanu et al.[1] described an association between the G/A single nucleotide polymorphism (SNP) in the position -597 of the IL-6 gene, with disease activity and disability, but not with the disease per se, in 20 systemic sclerosis (SSc) patients.The idea of a disease-modifying, -rather than a disease-susceptibility, point mutation is intriguing and in line with others findings in the context of SSc [2-5]. Nonetheless, the inability to depict such an association could be attributable to the low number of subjects and indeed the possibility of a type II error in such a small group of patients is more than remote. Based on previous findings that indicate that the G/C substitution at position -174 -in complete linkage disequilibrium with the G/A-597 SNP-, is associated with an increased IL-6 production in vitro and in vivo[6], the authors anticipated a decreased risk for SSc in CC -174 (AA -597) individuals compared to GG -174 (GG -597) subjects and claimed further studies in larger patient groups to verify this hypothesis and their findings as well.

The present retrospective case-control study was conducted on 196 consecutive patients with a diagnosis of SSc (ACR criteria [7]), referring to our outpatient clinic. One-hundred-ninety-six healthy sex-, ageand ethnically-matched subjects were included as controls. The number of patients and the case-to control ratio were chosen so as to obtain a power of 0.75 with an alpha level equal to 0.05 with a putative difference of 9% in the prevalence of the G allele at position -174; power calculation was carried out with the PS Program [8] and by using the frequencies of the G/C -174 SNP previously published in the Italian population as reference [9]. Clinical procedures for the study were those described by Sfrent-Cornateanu [1]: patients were categorized as having the limited cutaneous (lcSSc) or the diffuse cutaneous (dcSSc) subset of the disease according to LeRoy et al.[10] and were asked to complete the Disability Index of the Health Assessment Questionnaire (HAQ-DI) [11]; disease activity was assessed according to the European Scleroderma Study Group (ESsSG) [12] and by an experienced physician (AS) on a 100 mm visual analogue scale (VAS). The G/C -174 SNP was determined with sequence-specific primers by a commercial kit (CTS PCR-SSP TRAY, from the Institute of Immunology, Department of Transplantation Immunology, University of Heidelberg, Heidelberg, Germany) as previously described [4, 5, 9]. All the participating subjects gave written consent for the research. Statistics were carried out with the SPSS package, ver 15.0 (SPSS Chicago, IL).

The majority of patients were females (n = 178, 90.8%), with the lcSSc subset of the disease (n = 142, 72.4%), with a mean age and disease duration of 59.6 ± 12.4 and 11.2 ± 6.8 years, respectively. The distribution of the G/C-174 genotypes in patients and controls was consistent with Hardy-Weinberg's equilibrium; the genotypes or the alleles of the G/C -174 SNP were equally distributed between cases and controls (Table). HAQDI, VAS or ESsSG activity scores, as well as the most recent forced vital capacity% of predicted values, diffusing capacity for carbon monoxide% of predicted values, pulmonary systolic pressure on echocardiogram and total skin scores [13] were equally distributed among the G/C 174 genotypes or alleles (Table).

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Interleukin-6 G/C -174 single nucleotide polymorphism in 196 Italian systemic sclerosis patients and 196 healthy ethnically-matched controls

Variable	Genotype				Allele
GG		CG			CC				G					C
Controls, n (%)	89 (45.4%)	87 (44.4%)	20 (10.2%)	265 (67.6%)	127 (32.4%)
SSc, n (%)	84 (42.9%)	94 (48%)	18 (9.2%)	262 (66.8%)	130 (33.2%)
lcSSc	63 (44.4%)	68 (47.9%)	11 (7.7%)	194 (68.3%)	90 (31.7%)
dcSSc	21 (38.9%)	26 (48.1%)	7 (13%)	68 (63%)	40 (37%)
HAQ-DI, score	0.86 ± 0.77	0.67 ± 0.6	1.2 ± 0.58	0.79 ± 0.71	0.8 ± 0.63
ESsSG, score	1.52 ± 1.25	1.71 + 1.21	1.33 ± 1.7	1.58 ± 1.27	1.61 ± 1.18
VAS, mm	38.3 ± 30.1	42.5 ± 28.7	42 ± 22.4	39.8 ± 29.3	42.4 ± 26.8
FVC,% pred	82.7 ± 23.7	81.2 ± 20.8	77.8 ± 24.9	82.2 ± 22.5	80.2 ± 21.8
DLco,% pred	56.2 ± 19.6	57.2 ± 19.6	57.2 ± 18.6	56.7 ± 19.5	57.4 ± 19.2
PAP, mmHg	32.2 ± 18	29 ± 14.2	38.9 ± 17.7	30.1 ± 16.7	31.6 ± 15.6
mRSS	5.3 ± 5	5.7 ± 5.6	6.7 ± 4.7	5.5 ± 5.2	6 ± 5.3

Variables expressed as mean ± standard deviation, except where otherwise indicated. Statistical analysis performed by the chi-square test, Student's t-test (between alleles) or ANOVA (among genotypes); P-value not significant for all the comparisons. SSc, systemic sclerosis, -lcSSc, limited- and -dcSSc, diffuse- [10]. HAQ-DI, Health Assessment Questionnaire Disability Index [11]; ESsSG, European Scleroderma Study Group activity score [12]; VAS, visual analog scale (1–100 mm); FVC, forced vital capacity; DLco, diffusing capacity for carbon monoxide; PAP, pulmonary systolic pressure; mRSS, modified Rodnan Skin Score [13].]

The G/C -174 (G/A -597) SNP seems thus to play no role either in SSc-susceptibility or in SSc activity or disability, as suggested by Sfrent-Cornateanu et al.[1]. The latter results are not unexpected, as functional e.g. HAQ-DI) and activity scores (e.g. ESsSG and VAS) are “dynamic” measures that reflect the patient's short term condition (e.g. within 1–4 weeks) and may change over time and in response to therapy [14, 15], being hence inadequate for comparison with a “static” genetic variable. Of course, at the moment we cannot definitively rule out the possibility that the G/C -174 (G/A -597) SNP might be associated with other aspects of the disease when a more thorough analysis is carried out in a prospective fashion on selected outcomes variables or in selected subgroup of patients, even if this possibility seems quite remote.