The N125S polymorphism in the cathepsin G gene (rs45567233) is associated with susceptibility to osteomyelitis in a Spanish population.

BACKGROUND: Osteomyelitis is a bone infection, most often caused by Staphylococcus aureus, in which neutrophils play a key role. Cathepsin G (CTSG) is a bactericidal serine protease stored in the neutrophil azurophilic granules. CTSG regulates inflammation, activating matrix metalloproteinases (MMPs), and coagulation. Lactoferrin (LF), a neutrophil glycoprotein, increases CTSG catalytic activity and induces inflammation. The aim of this study was to analyze a potential association between a CTSG gene polymorphism (Asn125Ser or N125S, rs45567233), that modifies CTSG activity, and could affect susceptibility to, or outcome of, bacterial osteomyelitis.
METHODS: CTSG N125S polymorphism was genotyped in 329 osteomyelitis patients and 415 controls), Blood coagulation parameters, serum CTSG activity, LF, MMP-1, MMP-13, and soluble receptor activator for nuclear factor κ B ligand (sRANKL) levels were assessed in carriers of the different CTSG genotypes.
RESULTS: CTSG N125S (AG) genotype was significantly more frequent among osteomyelitis patients than controls (15.5% vs. 9.4%, p = 0.014). CTSG N125S variant G allele (AG +GG) was also more frequent among osteomyelitis patients (8.1% vs. 4.7%, p = 0.01). Serum CTSG activity and LF levels were significantly higher in osteomyelitis patients carrying the G allele compared to those with the AA genotype, (p<0.04). Serum MMP-1 was lower in the G allele carriers (p = 0.01). There was no association between these genotypes and clinical characteristics of osteomyelitis, or coagulation parameters, MMP-13, and sRANKL serum levels.
CONCLUSIONS: Differences in the CTSG gene might enhance osteomyelitis susceptibility by increasing CTSG activity and LF levels.

Introduction

Cathepsin G (CTSG) is a 26-kDa serine protease stored in the azurophilic granules of the polymorphonuclear leukocytes. It is released as a consequence of neutrophil stimulation by platelet-activating factor and different cytokines, and CTSG then enhances platelet aggregation. CTSG has antimicrobial activity against several different species of bacteria. It also plays an important role on the breakdown of extracellular matrix (ECM) components by activating different extracellular matrix metalloproteinases (MMPs) [1-3]. CTSG also stimulates the production of receptor activator for nuclear factor κ B ligand (RANKL), critical in bone remodeling, by virtue of activating osteoclast precursors [4]. CTSG has been reported to play an important role in a variety of inflammatory diseases including rheumatoid arthritis, coronary artery diseases, and ischemic reperfusion injury, and also response to bone metastasis. It is also implicated in several dental and respiratory infectious and inflammatory diseases, including periodontitis, chronic obstructive pulmonary disease, acute respiratory distress syndrome, and cystic fibrosis [1, 2].

The CTSG gene is located in chromosome 14q11.2, spans 2.7 kb and consists of 5 exons and 4 introns. An A→G missense mutation in exon 4 of the CTSG gene (Asn125Ser or N125S, rs45567233) was reported [5]. This point mutation at position 2108 changes the codon AAC (125Asn) to AGC (125Ser). Single carriers of the variant G genotype are heterozygous (GA), double carriers are homozygous (GG), while those that do not carry the variant G allele are wild type and have the AA genotype. The N125S polymorphism, that might modify CTSG activity, associates with elevated plasma fibrinogen levels in addition to increased platelets activation, contributing to cardiovascular and cerebrovascular disease [6]. No associations of the CTSG N125S polymorphism with any infections have been reported [7].

Lactoferrin (LF), a glycoprotein also stored in neutrophil granules, and it increases CTSG catalytic activity at physiological concentrations. LF also enhances CTSG-induced platelets activation and inflammatory mediators, modulating the inflammatory response [8, 9]. Interestingly high LF parotid saliva levels have been observed in patients with periodontitis, an inflammatory condition that can progress to mandibular osteomyelitis [9].

Osteomyelitis is a bacterial bone infection characterized by progressive bone inflammatory destruction and necrosis sequestra and new bone formation (involucrum). Adult osteomyelitis is frequently a complication of compound fractures, and/or open bone surgery. This infection can also develop from contiguous spread of infection from open wounds such as chronic pressure ulcers. Hematogenous osteomyelitis occurs mostly in prepuberal children. In adults, hematogenous osteomyelitis usually affects the axial skeleton. Staphylococcus aureus is the microorganism most frequently isolated in both post-traumatic and hematogenous osteomyelitis [10]. In spite of its frequency, complicated therapy and frequent relapses, osteomyelitis pathogenesis has been scarcely studied. Neutrophils, one of the main host defenses against bacterial infections, play a key role in osteomyelitis pathogenesis. Our group has previously reported that there are changes in the life span of neutrophils of osteomyelitis patients [11]. CTSG N125S polymorphism, a genetic variation that might modify neutrophils CTSG activity could influence osteomyelitis pathogenesis by modifying LF production and subsequent cleavage, MMPs-mediated bone inflammation and remodeling, and activating coagulation.

The aim of this study was to analyze a potential association between bacterial osteomyelitis and the CTSG N125S polymorphism, and the effect of that genetic polymorphism on CTSG activity. We also assessed associations with potential downstream consequences of CTSG release on some aspects of coagulation, and levels of circulating LF, MMP -1 and -13 and the soluble receptor activator for nuclear factor κ B ligand (sRANKL).

Patients and methods

Patients

We enrolled 329 adult patients admitted to the Hospital Universitario Central de Asturias (HUCA) and three affiliated hospitals in the same Northern Spanish region with a diagnosis of bacterial osteomyelitis, between January 1998 and December 2018. These hospitals provide the health coverage to the region of Asturias (total population of 1 million people). Patients with acute and chronic osteomyelitis were included in the study and followed for one year. Clinical information regarding these patients has been published in detail [12]. A diagnosis of osteomyelitis was made by clinical and roentgenographic criteria [10]. Surgical and sinus tract pus samples were cultured in all the osteomyelitis patients to make an etiological diagnosis. The presence of bone sequestra and/or sinus tract in bone X ray, CT or MRI, a positive Ga67 uptake bone scan, or a positive culture of the surgical bone sequestra or sinus tract were diagnostic of osteomyelitis [10]. Patients were diagnosed with hematogenous osteomyelitis if their bone infection was acquired in absence of trauma or bone surgery, lower limb vascular insufficiency, or a contiguous focus of infection. Overall, 56 cases of hematogenous osteomyelitis, 27 (48.2%), with positive blood cultures, were included in the study Bacterial osteomyelitis in immunosuppressed patients undergoing chemotherapy or with bone tuberculosis were excluded. Osteomyelitis that was present for more than 3 months was considered as chronic and if it did not relapse within a year of follow-up was considered as “cured”. The DNA from all enrolled patients was stored at -20° C and has been used previously for several gene association studies [13-19]. In addition, 415 healthy HUCA Blood Bank donors, matched for sex and age with the osteomyelitis patients, were used as controls. Patients and controls were members of a homogeneous Caucasian population, and were residents of Asturias, a Northwestern Spanish region with a small foreign immigrant population (less than 5%). Each participant gave informed written consent for the study, which was approved by the Ethical Committee of Regional Clinical Research of the Principality of Asturias.

Blood coagulation parameters

All the osteomyelitis patients enrolled in the study had 20 ml of blood drawn at hospital admission. Ten ml were used for hemogram, standard biochemistry, and coagulation assays. Routine coagulation parameters included fibrinogen levels, prothrombin time (PT), and international normalized ratio (INR), and activated partial thromboplastin time (aPTT).

Measurement of the N125S (rs45567233) genetic polymorphism of CTSG gen

Ten ml of blood from each patient and controls were collected in potassium-EDTA glass tubes. Genomic DNA was extracted from peripheral leukocytes with a salting-out method and stored at -20° C until use. The N125S (rs45567233) polymorphism of the CTSG was analyzed in patients and controls by PCR. The PCR primers used for amplifying the region of the gene containing the N125S polymorphism (rs45567233) were: Forward: and Reverse: 5 [6--7]. These primers amplified a fragment of 263 bp. The amplification protocol consists on an initial denaturation at 94° C for 5 minutes, followed by 35 cycles of denaturation at 94° C for 1 minute, annealing at 61° C for 1 minute and elongation at 72° C for 1 minute, and a final extension at 72° C for 7 minutes. PCR products were incubated overnight at 37° C with the restriction enzyme SduI (Thermo Fisher Scientific Inc, Waltham, MA, USA) followed by capillary electrophoresis on ABI PRISM® 3130xl Genetic Analyzer (Applied Biosystem, Foster City, CA, USA). Results were assessed using the Peak Scanner™ Software v1.0 (Applied Biosystem, Foster City, CA, USA). The CTSG N125S polymorphism was also genotyped individually for each osteomyelitis patient and control. PCR products were electrophoresed on a 2% low-melting agarose gel, and the fragments were then excised from the gel, purified with spin columns (DNA gel extraction Kit; Millipore, Billerica, MA,USA), and the fragments were directly sequenced on an ABIPrism 310 Genetic Analyser (Applied Biosystems, Foster City, CA, USA).(Fig 1).

Fig 1

Detection of the N125S (rs 45567233) polymorphism in the CTSG gene.

(A): polyacrylamide gel analysis of genomic DNA from 5 patients with acute osteomyelitis (OM) processed as in methods. AA denotes a wild-type sample with two distinct bands of 143 and 89 bp (lanes 1 and 2); AG denotes a heterozygous sample showing three distinct bands of 143, 89 and 31 bp (lanes 3 and 4); GG denotes a double variant sample showing two distinct bands of 143 and 31 bp (lane 5). MW, molecular weight markers; (B) Peak scan analysis of 3 patients with the genotypes AA, AG and GG; (C) Sequencing of the restriction fragment length polymorphism. Samples with no alteration: wild type (upper); with a heterozygous A-to-G replacement (middle) and with a double A-to-G replacement (lower).

CTSG, LF, MMPs and sRANKL assessment

Serum from patients with acute osteomyelitis was used for CTSG, LF, MMPs and sRANKL assessment. All the patients used in CTSG, LF, MMPs and sRANKL assessment assays had been consecutively admitted to the HUCA between 2016–18.

Serum CTSG activity

Cathepsin G activity was determined in serum of 21 patients with acute osteomyelitis and 21 blood donors were used as controls by a commercially available colorimetric assay (Cathepsin G Activity Assay Kit, PK-CA577-K146, PromoCell GmbH, Heidelberg, Germany). CTSG activity levels were expressed as μU/ml.

Serum Lactoferrin (LF)

LF was determined in serum of 27 patients with acute osteomyelitis by an ELISA assay (ab200015 human lactoferrin simpleStep ELISA Kit Abcam, Cambridge, MA, USA). LF levels were expressed as ng/ml.

Serum MMPs and sRANKL

MMP-1 and -13 and sRANKL were determined in 21 patients with acute osteomyelitis by ELISA and by commercial available colorimetric assays (Raybiotech, Norcross, GA, USA for MMP-1 and 13, Immunodiagnostics, Bensheim, Gerrmany for sRANKL). Serum MMPs levels are expressed in pg/ml.and sRANKL levels in pmol/L.

Statistics

Sample size calculation

Power calculation using one control for each osteomyelitis patients indicated a need for 746 individuals, 373 osteomyelitis patients and 373 controls to have a power of 80% at a confidence interval of 90% to detect a prevalence of 17% of the CTSG N125S polymorphism carriage among osteomyelitis patients considering that this polymorphism has a known prevalence of 10% in the Caucasian European population.

Results are expressed as median and inter-quartile range (IQR) or proportions as appropriate. As the distribution of some continuous variables was non-Gaussian, natural logarithmic transformation was done for analysis. The reported values are the result of back-transformation into the original units. The Pearson X2 test was used to compare allele and genotype frequencies between the groups. Yates’ correction and Mantel–Haenszel test were also used when indicated. Odds ratios (OR´s) and their 95% confidence intervals (CI) were also calculated. Serum CTSG, LF, MMP-1, -3, sRANKL were compared for the genotypes with the Student t test or the Mann-Whitney test when appropriate. Serum CTSG and LF, were compared by using the Pearson correlation coefficient. All the reported p values are two-sided. A p-value < 0.05 was considered as significant. The statistical analysis was performed with the computer program statistical package SPSS (IBM SPSS Statistics 25.0 package, IBM New York, NY, USA) and with the GraphPad Prism software (GraphPad Software, version 7.0, San Diego, CA, USA).

Results

Clinical characteristics of osteomyelits patients

Osteomyelitis patients were mainly men over sixty, with infected bones after fractures (38.9%). Pressure ulcers (23.4%), prosthetic joint infection (20.7%) and hematogenous spread (17.2%) were the risk factors for about half the cases of osteomyelitis. The most commonly involved bones were tibia (24.1%) and femur (21.8%). The most common pathogen was S. aureus and most of the bone infections were chronic (Table 1). The controls were blood donors mentioned in methods; 295 (71.1%) were males and 120 (28.9%) were females with a median age of 59.1 years (41–60). Although the controls were on average 2.9 years younger than the patients, there was not a significant difference between the ages of the patients and controls (p = 0.14).

Table 1

Clinical characteristics of the 329 osteomyelitis (OM) patients enrolled in the study.

Clinical Characteristics	Patients(n = 329)
Median age (years, range)	62.0 (49–74)
Male gender (n, %)	243 (73.9)
Chronic OM (n, %)	215 (65.3)
Post-traumatic source of infection (n, %)	128 (38.9)
Hematogenous source of infection (n, %)	56 (17.2)
Paraplegia/pressure ulcers infection (n, %)	77 (23.4)
Orthopedic prosthesis infection (n, %)	68 (20.7)
Staphylococcus aureus OM (n, %)	139 (67.5) *
Gram negative OM (n, %)	48 (23.8) *
Other microorganisms (n, %)	19 (23.4) *

* Positive cultures were available only in 206 osteomyelitis patients

Frequency of the CTSG N125S (rs 45567233) polymorphism in osteomyelitis

Tables 2 and 3 show the genotypic and allelic frequencies of the CTSG N125S (rs 45567233) polymorphism in osteomyelitis patients and controls. The CTSG N125S AG heterozygous genotype was significantly more frequent among osteomyelitis patients compared to controls (15.5% vs. 9.4%; χ2 = 6.5, OR = 1.78, 95% CI = 1.11–2.84, p = 0.011 by the Mantel-Haenszel test, χ2 = 5.95, p = 0.014 by the Yates correction). The CTSG N125S variant G allele was also more frequent among osteomyelitis patients (8.1% vs 4.7%, χ2 = 7.12, OR = 1.78, 95% CI = 1.14–2.78, p = 0.0076 by the Mantel-Haenszel test, χ2 = 6.56, P = 0.01 by the Yates correction). Only one carrier of the double variant GG genotype was found among osteomyelitis patients (0.3%) and none among the controls. The CTSG N125S polymorphism was in Hardy–Weinberg equilibrium among patients with osteomyelitis and controls. No association of the CTSG N125S polymorphism with type of bone infection (acute vs. chronic) (p = 0.7), source of infection (hematogenous vs. non hematogenous, p = 0.9), (pressure ulcers vs. non pressure ulcers, p = 0.5) or microorganism isolated (S. aureus vs. Gram negative bacteria p = 0.9) was found.

Table 2

Frequency of CTSG N125S (rs 45567233) genotypes in osteomyelitis (OM) patients and blood donor (controls).

CTSG N125S Genotypes	OM patients(n, %)	Controls(n, %)
AA	277 (84.2)	376 (90.6)
AG	51 (15.5) *	39 (9.4)
GG	1 (0.3)	0 (0)

* p = 0.011 by the Mantel-Haenszel test; p = 0.015 by the Yates’s correction while comparing OM patients vs. controls.

Table 3

Frequency of CTSG N125S (rs 45567233) alleles in osteomyelitis (OM) patients and blood donor controls.

CTSG Alleles	OM patients(n, %)	Controls(n, %)
A	605 (91.9)	791 (95.3)
G	53 (8.1) *	39 (4.7)

* p = 0.008 by the Mantel-Haenszel test, p = 0.01 by the Yates correction comparing OM patients vs. controls.

Serum CTSG activity and CTSG N125S (rs 45567233) polymorphism

Serum CTSG activity was significantly higher in carriers of the G allele of the N125S polymorphism compared to those with the AA genotype (p = 0.016) and to uninfected controls (p = 0.005) (Fig 2). No significant differences in serum CTSG activity between osteomyelitis patients and controls were observed (p = 0.1).

Fig 2

Serum cathepsin G (CTSG) activity in carriers of the different genotypes of the CTSG N125S (rs 45567233) polymorphism.

Cathepsin G (CTSG) activity was determined in serum of osteomyelitis patients and controls by a colorimetric assay. Data are shown as the median and IQR of 21 patients with acute osteomyelitis, 10 carriers of the AA, 10 of the AG and 1 of the GG genotypes of the CTSG N125S (rs 45567233) polymorphism and 10 blood donors were assessed.

Serum LF and CTSG N125S (rs 45567233) polymorphism

Serum LF was significantly higher in carriers of the G allele of CTNG compared to those with the AA genotype (p = 0.006) (Fig 3). On the other hand, LF levels and CTSG activity were correlated but the association was just below the limit of significance (r = 0.501, p = 0.057 by Pearson correlation coefficient).

Fig 3

Serum Lactoferrrin (LF) levels in patients with acute osteomyelitis according to their alleles of CTSG (rs 45567233).

Lactoferrin (LF) was measured in serum of osteomyelitis patients by an ELISA assay. Results from individual AA subjects are shown as inverted triangles and AG + GG subjects as squares. The long horizontal bars are the median values and shorter bars show IQR.

Serum MMP-1, -13 and sRANKL levels and CTSG N125S (rs 45567233) polymorphism

Serum MMP-1 levels were significantly lower in osteomyelitis patients with acute infection who were carriers of the G allele of the N125S polymorphism compared to those with the AA genotype (p = 0.024) (Fig 4). We found no differences in MMP-13 or sRANKL serum levels between osteomyelitis patients who were carriers of the different alleles of CTSG 125 (124.3 [8.6–240.0] and 89 [55.05–304.0] pg/ml, p = 0.9 for MMP-13, and 0.165 [0.04–0.250] and 0.08 [0.04–0.210] pmol/L, p = 0.7 for sRANKL for osteomyelitis patients carriers of the AA and AG + GG genotypes of the CTSG).

Fig 4

Serum MMP-1 levels in carriers of the different genotypes of the CTSG (rs 45567233).

Serum MMP-1 levels from 27 patients with acute osteomyelitis were determined after the end of treatment by ELISA and the group was divided according to whether or not they carried the G allele. Individual patients’ results are shown and the median and IQR. There were 10 carriers of the AA, 10 of the AG and 1 of the GG genotypes of the CTSG N125S (rs 45567233) polymorphism.

Coagulation parameters and CTSG N125S (rs 45567233) polymorphism

There were no differences in blood fibrinogen levels, PT, or PTT between the osteomyelitis patients who carried the CTSG and those that did not (median [IQR] 621[433-772] and 622 [492.3–783.8] mg/dl, for the AG and AA genotypes, respectively [p>0.6]), (PT: 11.7 [110–12.6] and 11.6 [10.7–12.5] seconds, p = 0.1; INR: 1.1 [1.0–1.2] and 1.1 [1.0–1.1], p = 0.3; aPTT: 31.4 [28.8–33.8] and 32.0[28.4–33.8] seconds, p = 0.4, for the AG and AA genotypes, respectively).

Discussion

We describe for the first time an association in adults between the CTSG N125S polymorphism, previously associated with cardiovascular and neurovascular diseases, and osteomyelitis, a bacterial infection of the bone. This CTSG polymorphism is associated with increased serum CTSG activity and LF levels in our study. Carriers of the G allele were over-represented in patients with osteomyelitis compared to a control population drawn from the same area of Spain where the patients were hospitalized, suggesting that this allele increases the risk of developing osteomyelitis. The carriage of this variant G allele is relatively frequent (9.4%) in the healthy Spanish Caucasian controls in this study, but was even higher (15.5%) in our patients with osteomyelitis. We have previously found that variants in the genes of IL-1α, MMP1, TLR4 receptor, endothelial nitric oxide synthase (NOS3), Bax and tPA also contribute to the risk of developing osteomyelitis with prevalences of between 3.8% and 65.3% [14-19]. The CTSG N125S polymorphism frequency in our control population was very similar to that reported by others (10% in healthy Caucasian Americans and 11.7% in healthy French and British Caucasians) [6, 20]. This encourages us to consider that our finding was not due to a peculiarity of the population we studied.

Previous reports did not find increased in vitro transcriptional activities due to the CTSG N125S polymorphism in human monocyte-like U937 cells, a cell-line that expresses 25% more CTSG activity than human neutrophils [6, 21, 22]. We report here for the first time that the CTSG N125S polymorphism is associated with increased plasma CTSG activity in carriers of the variant G allele. This increased activity was due to the effect of the G allele of the CTSG N125S polymorphism and not to the bone infection as it also was true of the uninfected controls.

We also observed that serum LF levels were significant higher in carriers of the variant G allele of the CTSG N125S polymorphism. These individuals also had increased serum CTSG serum activity. Considering that LF increases the catalytic activity of CTSG at physiological concentration and broadens the substrate specificity of CTSG [8], our LF levels finding could partially explain the increased activity of serum CTSG. However, there was not a statistically significant correlation between serum CTSG activity and LF levels, which could mean that the N→S change in CSTG also changed its enzymatic activity.

We also explored the mechanisms whereby the CTSG N125S polymorphism might increase susceptibility to osteomyelitis. CTSG is released after neutrophil stimulation by platelet-activating factor, tumor necrosis factor-alpha, and interleukin-8 which, via the CTSG platelet receptor protease -activated receptor 4, leads to calcium mobilization, platelet secretion and aggregation and a systemic release of the platelet thrombogenic products producing intravascular thrombosis [3, 6]. The CTSG N125S G allele has been associated with elevated plasma fibrinogen levels in myocardial infarct patients [6, 23]. Prothrombotic polymorphisms such as the tPA Alu (I/D) have been associated with osteomyelitis [19]. However, fibrinogen levels and other blood coagulation parameters were similar in carriers of the different genotypes of the CTSG N125S polymorphism among osteomyelitis patients in our study.

CTSG plays a role in the inflammatory response and it has been associated with different inflammatory diseases, especially some related to the bone such as rheumatoid arthritis, periodontitis, and bone metastasis. CTSG degrades collagen and proteoglycan and digests ECM components at inflammatory sites to which neutrophils are recruited by activating MMP-1, -2, -3 and RANKL [4, 24–26]. CTSG increases MMP expression in normal human fibroblasts through fibronectin fragmentation, and induces the conversion of pro-MMP-1 to active MMP-1 [24]. CTSG is also capable of activating pro-MMP-9, that cleaves and releases active transforming growth factor-β (TGFβ), MMP-13 and RANKL at the tumor-bone interface of mammary tumor-induced osteolytic lesions [26]. These MMPs and RANKL interfere in the bone remodelling by enhancing osteoclasts activation and bone resorption, potentially aggravating the bone damage already induced by infection in osteomyelitis. We have reported increased plasma levels of MMP-1 and MMP-13 in osteomyelitis patients in a previous work [18]. However, although MMP-1 plasma levels were increased in osteomyelitis AA carriers, MMP-1 plasma levels were not increased but were significantly decreased in the osteomyelitis patients with the CTSG N125S allele in this study. There were no differences in plasma levels of MMP-13 and RANKL among osteomyelitis patients with the different genotypes of the CTSG. Therefore, mechanisms other than increased MMP-1, -13 and sRANKL might explain the increased susceptibility to osteomyelitis in carriers of the CTSG N125S polymorphism.

The CTSG N125S polymorphism, increased LF serum and probably bone levels as well As an inducer of inflammation, LF obtained from human parotid saliva increased the production of IL-6, monocyte chemoattractant protein-1 (MCP-1) and the activation of NF-κB in human epithelial HSC-2 cells [9]. Perhaps the association of the CTSG N125S polymorphism with osteomyelitis might be mediated by LF. However Komine et al found that the activity was in fragments of LF not in the whole protein and the response was measured with an epithelial cell line that might not be pertinent to bones.

However, there are still other plausible explanations of the association of the CTSG polymorphism with osteomyelitis. Neutrophil CTSG is important during the early inflammation stage of wound healing. CTSG may be involved in processing different soluble mediators in the wound milieu that are responsible for neutrophil chemotaxis. CTSG KO mice have abnormalities in wound healing manifested by a decrease in wound tensile strength in spite of having an increased number of neutrophils and more myeloperoxidase activity in the wound [27]. In spite of the increased serum CTSG activity associated with the CTSG polymorphism we could speculate that there was abnormal wound healing that could play some role in the enhanced susceptibility to osteomyelitis. Many of the patients contracted bone infection as a complication of unhealed open wounds after orthopedic surgery or from contiguous spread to bone from chronic pressure ulcers in patients with spinal cord lesions. However, no association between osteomyelitis due chronic pressure ulcers and the CTSG N125S polymorphism was found in our study. Finally, a linkage disequilibrium of the CTSG gene with another gene located on chromosome 14q11-2 cannot be ruled-out.

In summary, we describe here a potential association between the CTSG N125S polymorphism and osteomyelitis. The effect might be due to bone inflammation enhanced by increased LF local levels. More research in populations with other ethnic background is needed to confirm this observation and to discover the mechanism underneath the increased susceptibility to bone infection due to this genetic variant.

23 Aug 2019

PONE-D-19-18899

The N125S polymorphism in the G gene is associated with susceptibility toginger-module-highlighter-mistake-type-3" id="gwmw-15664826668904541061438"> .

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7. We note that you have included the phrase “data not shown” in your manuscript. Unfortunately, this does not meet our data sharing requirements. PLOS does not permit references to inaccessible data. We require that authors provide all relevant data within the paper, Supporting Information files, or in an acceptable, public repository. Please add a citation to support this phrase or upload the data that corresponds with these findings to a stable repository (such as Figshare or Dryad) and provide and URLs, DOIs, or accession numbers that may be used to access these data. Or, if the data are not a core part of the research being presented in your study, we ask that you remove the phrase that refers to these data.

Additional Editor Comments:

1- The authors should use a language-editing service to refine the use of English in their manuscript and submit an "Editing Certificate" with the revised version of the manuscript.

2- Abbreviations must be spelled out on first mention particularly in the abstract.

3- Authors are advised to revise all the manuscript to ensure that all gene names are written in italic font.

4- Authors should pay attention to the use of "Gender" terminology and substitute by the "sex" terminology as it appears from the results they mean the normal biological differences between males and females.

5- Thanks to the authors for providing the PCR primer sequences. Are these primers -designed or derived from other published work? If the former, please provide the name of the program you applied in its formal citation in the text, or provide the citations you follow if they were derived from other publications.

6- The PCR results need more elaboration and clarification (recommended for acceptance). The authors wrote that "PCR products were incubated overnight at 37º C with the restriction enzymeginger-module-highlighter-mistake-type-3" id="gwmw-15664829754791295809559">. Results were determined by using the Peak ScannerTM Software v10". The authors should send a photo related to the original scanner and an edited one for publication (in which all the details of band size for each genotype are written clearly) to facilitate replication of the work by future readers.

7- The quality control measurements the authors followed either in their PCR or other laboratory works, including ELISA, etc. ginger-module-highlighter-mistake-type-1" id="gwmw-15664831811790536589857">should be written in details.

[Note: HTML markup is below. Please do not edit.]

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #1: Partly

Reviewer #2: Yes

**********

2. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: Yes

Reviewer #2: Yes

**********

3. Have the authors made all data underlying the findings in their manuscript fully available?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or ginger-module-highlighter-mistake-type-2" id="gwmw-15664829756224531275852">deposited a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—ginger-module-highlighter-mistake-type-1" id="gwmw-15664829756322768885661">eg. ginger-module-highlighter-mistake-type-1" id="gwmw-15664829756364873410917">privacy or use of data from a third party—those must be specified.

Reviewer #1: Yes

Reviewer #2: No

**********

4. Is the manuscript presented in an intelligible fashion and written in standard English?

PLOS ONE does not copyedit accepted manuscripts, so the language in ginger-module-highlighter-mistake-type-3" id="gwmw-15664829757670599906198">articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #1: Yes

Reviewer #2: Yes

**********

5. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: The study concept is interesting and can be of clinical significance.

My main concern is that the authors recruited an accepted number of patients (329) and controls (415) for and when they made the correlation with the evaluated parameters, they included very small groups of individuals (10 to 27 per group). The validity of these results is an issue and the bias in the selection of these individuals could be another problem.

Other points include

The title should determine the polymorphism ID and the study ethnic group.

-ginger-module-highlighter-mistake-type-1" id="gwmw-15664829758386099843190">value is sometimes mentioned as "equals" and other occasions as "less than". It should be consistent.

Page 4 last line. The authors mentioned that no similar association studies has been done so far. The reference dated 2006.

Page 5, last paragraph is a mixture of the hypothesis and information extracted from reference no.9. Please rewrite.

There are linguistic style and punctuation errors. The manuscript needs linguistic revision. For example a sentence is repeated page 7, parenthesis ginger-module-highlighter-mistake-type-3" id="gwmw-15664829758768011961931">in page 19, the tense in discussion (mixture of present and past tenses), first line in the second paragraph p23, .....

The authors provided some data analysis based on the gender. It would be better to remove that as the female group is a minority.

Table 2 is confusing. Please separate into two tables (one for and the other for frequency)

The use of Yates correction is not preferred by someginger-module-highlighter-mistake-type-3" id="gwmw-15664829759029781080455"> . I am not sure if it is the best to use here.

Reviewer #2: The authors presented an interesting study evaluating the association between CTSG polymorphisms and the occurrence of and its clinical characteristics. The study is well-conducted, but I suggest the following edits:

* Abstract: The authors did not mention the objective of their study in the abstract.

* Introduction: the authors should present the different alleles, in their study.

* Methods: "We enrolled 329 adult patients with a diagnosis of bacterial between January 1998 and December 2018" Was this a part of a hospital database system or a one study effort that lasted 20 years?

- Also, was there a systematic sample size calculation?

- " ginger-module-highlighter-mistake-type-1" id="gwmw-15664829759509741182248">was diagnosed using clinical and roentgenographic findings" Please clarify?

- The molecular techniques used in the current study are well-described.

* Results: The age and gender of control subjects and their comparisons to patients should be mentioned in the results' first paragraph. Because they are supposed to be matched, no significant difference should be noted.

- "No association of the CTSG N125S polymorphism with or type of bone infection (acute vs. ), source of infection or microorganism isolated was found" the authors should mention at least p for such associations!

* General: The manuscript needs a professional editing service because there are several grammatical errors across the entire manuscript.

- Data availability statement should be added to the manuscript after the conclusion explaining where the underlying data could be found.

**********

6. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.

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Reviewer #1: No

Reviewer #2: No

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While revising your submission, please upload your figure files to the Preflight Analysis and Conversion Engine (PACE) digital diagnostic tool, https://pacev2.apexcovantage.com/. PACE helps ensure that figures meet PLOS requirements. To use PACE, you must first register as a user. Registration is free. Then, login and navigate to the UPLOAD tab, where you will find detailed instructions on how to use the tool. If you encounter any issues or have any questions when using PACE, please email us at figures@plos.org. Please note that Supporting Information files do not need this step.

30 Sep 2019

ANSWERS TO EDITOR AND REVIEWERS

ANSWERS TO THE EDITOR

Dear Editor,

Below are the answers to all the questions raised by the Editor and Reviewers. Dr. Joshua Fierer, Professor of Medicine and Pathology of the University of California San Diego -UCSD did a complete scientific and English language editing of the original manuscript. This is the reason we decided to include him among the authors of the revised version of the paper. We have introduced a new Fig.1 including gel and peak scan analysis images and RFLP sequences analysis of the N125S (rs 45567233) polymorphism in the CTSG gene from the DNA of 5 osteomyelitis patients. In addition in this revised version of the manuscript we have deleted the S.aureus killing by neutrophils and ROS assays. No differences among genotypes regarding the N125S CTSG polymorphism were observed in these deleted assays and therefore the scientific value of the work has not decreased. Now the manuscript is shorter, and much more readable.

When submitting your revision, we need you to address these additional requirements.

1. Please ensure that your manuscript meets PLOS ONE's style requirements, including those for file naming. The PLOS ONE style templates can be found at

http://www.journals.plos.org/plosone/s/file?id=wjVg/PLOSOne_formatting_sample_main_body.pdf andhttp://www.journals.plos.org/plosone/s/file?id=ba62/PLOSOne_formatting_sample_title_authors_affiliations.pdf

Done

2. Please provide additional details regarding participant consent. In the ethics statement in the Methods and online submission information, please ensure that you have specified whether consent was written or verbal/oral. If consent was verbal/oral, please specify: a) whether the ethics committee approved the verbal/oral consent procedure, b) why written consent could not be obtained, and c) how verbal/oral consent was recorded. If your study included minors, please state whether you obtained consent from parents or guardians in these cases. If the need for consent was waived by the ethics committee, please include this information.

Done A paragraph was added in page 8 of the revised manuscript.” Each participant gave informed written consent for the study, which was approved by the Ethical Committee of Regional Clinical Research of the Principality of Asturias.”

3. Please note that all PLOS journals ask authors to adhere to our policies for sharing of data and materials:https://journals.plos.org/plosone/s/data-availability. According to PLOS ONE’s Data Availability policy, we require that the minimal dataset underlying results reported in the submission must be made immediately and freely available at the time of publication. As such, please remove any instances of 'unpublished data' or 'data not shown' in your manuscript and replace these with either the relevant data (in the form of additional figures, tables or descriptive text, as appropriate), a citation to where the data can be found, or remove altogether any statements supported by data not presented in the manuscript."

Done. All the not shown data were added to the text of the revised version of the manuscript .All the data of coagulation were added to page 16 of the revised manuscript.

4. We noticed you have some minor occurrence(s) of overlapping text with the following previous publication(s), which needs to be addressed:

https://doi.org/10.1016/j.molimm.2007.10.013

https://doi.org/10.1093/infdis/jit158

https://doi.org/10.1097/GIM.0b013e318039b23d

https://doi.org/10.1016/j.molimm.2007.10.013

Done. All the overlapping text with previous publications, mostly from our group, has been written again.

In your revision ensure you cite all your sources (including your own works), and quote or rephrase any duplicated text outside the Methods section. Further consideration is dependent on these concerns being addressed.

Done

5. Thank you for including your ethics statement:

"Each participant gave informed consent for the study, which was approved by the Ethics Committee of the HUCA.".

i) Please amend your current ethics statement to include the full name of the ethics committee/institutional review board(s) that approved your specific study.

Done. A paragraph was added in page 8 of the revised manuscript.” Each participant gave informed written consent for the study, which was approved by the Ethical Committee of Regional Clinical Research of the Principality of Asturias.”

ii) Once you have amended this/these statement(s) in the Methods section of the manuscript, please add the same text to the “Ethics Statement” field of the submission form (via “Edit Submission”).

Done

For additional information about PLOS ONE ethical requirements for human subjects research, please refer tohttp://journals.plos.org/plosone/s/submission-guidelines#loc-human-subjects-research.

6. Thank you for stating in your Funding Statement:"Partially funded by a Fondo de Investigaciones Sanitarias (FIS) grant PI16/01999 given to VA.T he funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript".

Done.A paragraph was added to page 22 ot the revised manuscript: “This study was partially funded by a Fondo de Investigaciones Sanitarias (FIS) grant PI16/01999 given to VA. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript . There was no additional external funding received for this study.”

Please provide an amended statement that declares *all* the funding or sources of support (whether external or internal to your organization) received during this study, as detailed online in our guide for authors at http://journals.plos.org/plosone/s/submit-now. Please also include the statement “There was no additional external funding received for this study.” in your updated Funding Statement.

Done. A paragraph was added to page 22 ot the revised manuscript: “This study was partially funded by a Fondo de Investigaciones Sanitarias (FIS) grant PI16/01999 given to VA. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript . There was no additional external funding received for this study.”

Please include your amended Funding Statement within your cover letter. We will change the online submission form on your behalf.

Done

7. We note that you have included the phrase “data not shown” in your manuscript. Unfortunately, this does not meet our data sharing requirements. PLOS does not permit references to inaccessible data. We require that authors provide all relevant data within the paper, Supporting Information files, or in an acceptable, public repository. Please add a citation to support this phrase or upload the data that corresponds with these findings to a stable repository (such as Figshare or Dryad) and provide and URLs, DOIs, or accession numbers that may be used to access these data. Or, if the data are not a core part of the research being presented in your study, we ask that you remove the phrase that refers to these data.

Done .All the not shown data were added to the text of the revised version of the manuscript .All the data of coagulation were added to pages 16 of the revised manuscript..

Additional Editor Comments:

1- The authors should use a language-editing service to refine the use of English in their manuscript and submit an "Editing Certificate" with the revised version of the manuscript.

Done. Joshua Fierer, Professor of Medicine and Pathology of the University of California San Diego (UCSD) did a complete scientific and English language editing of the original manuscript. This is the reason we decided to include him among the authors. A separate statement from Prof. Fierer confirming his contribution to the work is attached to the cover letter to the Editor.

2- Abbreviations must be spelled out on first mention particularly in the abstract.

Done

3- Authors are advised to revise all the manuscript to ensure that all gene names are written in italic font.

Done.

4- Authors should pay attention to the use of "Gender" terminology and substitute by the "sex" terminology as it appears from the results they mean the normal biological differences between males and females.

Done although all mentions to sex differences among CTSG N125S SNP genotypes have deleted from the revised version of the manuscript as Reviewer # 1 recommends.

5- Thanks to the authors for providing the PCR primer sequences. Are these primers -designed or derived from other published work?

The primers were derived from previous works (reference 5. Ludecke B, Poler W, Olek K, Bartholome K. Sequence variant of the human cathepsin G gene. Hum Genet 1993; 91.83-84.; reference 6. Hermann SM, Funke-Kaiser H, Schmidt-Petersen K, Nicaud V, Gautier-Bertrand M, et al. Characterization of polymorphic structure of cathepsin G. Role in cardiovascular and cerebrovascular diseases. Arterioscler Thromb Vasc Biol 2001, 21: 1538-1543.

If the former, please provide the name of the program you applied in its formal citation in the text, or provide the citations you follow if they were derived from other publications.

6- The PCR results need more elaboration and clarification (recommended for acceptance). The authors wrote that "PCR products were incubated overnight at 37º C with the restriction enzyme.

Done. The PCR products overnight incubation at 37ºC is correct. A complete paragraph on PCR assay was added to pages 8 and 9 of the revised version of the manuscript “Measurement of the N125S (rs45567233) genetic polymorphism of CTSG gen .Ten ml of blood from each patient and controls were collected in potassium-EDTA glass tubes. Genomic DNA was extracted from peripheral leukocytes with a salting-out method and stored at -70º C until use. The N125S (rs45567233) polymorphism of the CTSG was analyzed in patients and controls by PCR. The PCR primers used for amplifying the region of the gene containing the N125S polymorphism (rs45567233) were: Forward: 5’-6-FAM-GCTGAGCGGGAACGCCTACA-3’ and Reverse: 5´-GCTGAGCGGGAACGCCTACA-3’. These primers amplified a fragment of 263 bp. The amplification protocol consists on an initial denaturation at 94º C for 5 minutes, followed by 35 cycles of denaturation at 94º C for 1 minute, annealing at 61º C for 1 minute and elongation at 72º C for 1 minute, and a final extension at 72º C for 7 minutes. PCR products were incubated overnight at 37º C with the restriction enzyme SduI (Thermo Fisher Scientific Inc, Waltham, MA, USA). Results were determined by using the Peak ScannerTM Software v1.0 (Applied Biosystem, Foster City, CA, USA) following capillary electrophoresis on ABI PRISM® 3130xl Genetic Analyzer (Applied Biosystem, Foster City, CA, USA). The CTSG N125S polymorphism was also genotyped individually for each osteomyelitis patient and control. PCR products were electrophoresed on a 2% low-melting agarose gel, and the fragments were then excised from the gel, purified with spin columns (DNA gel extraction Kit; Millipore, Billerica, MA,USA), and the fragments were directly sequenced on an ABIPrism 310 Genetic Analyser (Applied Biosystems, FosterCity, CA, USA).(Fig.1)

Results were determined by using the Peak ScannerTM Software v1.0". The authors should send a photo related to the original scanner and an edited one for publication (in which all the details of band size for each genotype are written clearly) to facilitate replication of the work by future readers.

Done. A new Figure 1 includes a photo of the gel (A), and of the original peak scan (B) in which the band size of genotypes GG, AG and AA are seen. This new Fig.1 also includes the sequencing information of each genotype (C).

7- The quality control measurements the authors followed either in their PCR or other laboratory works, including ELISA, etc. should be written in details.

Done

ANSWERS TO THE REVIEWERS

Reviewer #1:

The study concept is interesting and can be of clinical significance.

My main concern is that the authors recruited an accepted number of patients (329) and controls (415) for and when they made the correlation with the evaluated parameters, they included very small groups of individuals (10 to 27 per group). The validity of these results is an issue and the bias in the selection of these individuals could be another problem.

We started recruiting osteomyelitis (OM) patients for several genetic studies since January 1998. So far we have recruited 329 adult patients with OM and collected their DNA. A large population of OM patients and controls is needed for genetic studies. However for CTSG, LF, MMPs and sRANKL assessment assays much smaller sample sizes are needed. Therefore we recruited 27 OM patients with acute bone infection consecutively admitted to the HUCA between 2016-18. In addition, 415 healthy HUCA Blood Bank donors, matched for sex and age with the OM patients, were used as controls, of them 21 were used for CTSG, LF, MMPs and sRANKL assessment.

Other points include

The title should determine the polymorphism ID and the study ethnic group.

Done. The title has been changed to “The N125S polymorphism in the cathepsin G gene (rs45567233 )is associated with susceptibility to osteomyelitis in a Spanish population.”

-value is sometimes mentioned as "equals" and other occasions as "less than". It should be consistent.

Done. “Equals” was used for individual p values throughout the manuscript. However when several p values are aggregated “less than”was used.

Page 4 last line. The authors mentioned that no similar association studies has been done so far. The reference dated 2006.

Done. The sentence was modified to “No associations of the CTSG N125S polymorphism with infections have been reported �7�”

Page 5, last paragraph is a mixture of the hypothesis and information extracted from reference no.9. Please rewrite.

Done. The sentence mentioning the association of high lactoferrin parotid saliva levels with periodontitis was moved from the paragraph backwards in the Introduction section.

There are linguistic style and punctuation errors. The manuscript needs linguistic revision. For example a sentence is repeated page 7,

Done. English language was edited throughout the revised manuscript by Prof.Joshua Fierer from UCSD, a native English speaker. The repeated sentence was deleted.

parenthesis in page 19,

Done

the tense in discussion (mixture of present and past tenses), first line in the second paragraph p23, .....

Done

The authors provided some data analysis based on the gender. It would be better to remove that as the female group is a minority.

Done. All mentions to sex differences among CTSG N125S SNP genotypes have have deleted from the revised version of the manuscript and reference 23 from the original version of the paper as Reviewer # 1 recommends.

Table 2 is confusing. Please separate into two tables (one for and the other for frequency.

Done. Two new tables, Table 2 with the genotype frequencies and Table 3 with the allelic frequencies were introduced in the revised version of the manuscript.

The use of Yates correction is not preferred by some . I am not sure if it is the best to use here.

Done. Although the �2 obtained by the Yates correction is more exigent than the the �2 obtained by the Mantel -Haenszel test we have included both statistical tests in the revised version of the manuscript. as Reviewer # 1 suggests.

Reviewer #2: The authors presented an interesting study evaluating the association between CTSG polymorphisms and the occurrence of and its clinical characteristics. The study is well-conducted, but I suggest the following edits:

* Abstract: The authors did not mention the objective of their study in the abstract.

Done. A new paragraph was added to the abstract: “The aim of this study was to analyze a potential association between a CTSG gene polymorphism (Asn125Ser or N125S, rs45567233), that modifies CTSG activity, and could affect susceptibility to, or outcome of, bacterial osteomyelitis.“

* Introduction: the authors should present the different alleles, in their study.

Done. A new paragraph was added to the Introduction section:” Single carriers of the variant G genotype are heterozygous (GA), double carriers are homozygous (GG) while those that do not carry the variant G allele are wild type and have the AA genotype”

* Methods: "We enrolled 329 adult patients with a diagnosis of bacterial between January 1998 and December 2018" Was this a part of a hospital database system or a one study effort that lasted 20 years?

We started recruiting osteomyelitis (OM) patients for genetic studies since January 1998. So far we have recruited 329 adult patients with OM and collected their DNA.A large population of OM patients and controls is needed for genetic studies. However for CTSG, LF, MMPs and sRANKL assessment much smaller sample sizes are needed. For this purpose we recruited 27 with acute bone infection admitted to the HUCA between 2016-18. In addition, 415 healthy HUCA Blood Bank donors, matched for sex and age with the OM patients, were used as controls, 21 of them were used for CTSG, LF, MMPs and sRANKL assessment.

.

- Also, was there a systematic sample size calculation?

A paragraph regarding sample size calculation was added to the revised version of the manuscript:” Sample size calculation : Power calculation using one control for each osteomyelitis patients indicated a need for 746 individuals, 373 osteomyelitis patients and 373 controls to have a power of 80% at a confidence interval of 90% to detect a prevalence of 17% of the CTSG N125S polymorphism carriage among osteomyelitis patients considering that this polymorphism has a known prevalence of 10% in the Caucasian European population “

- "was diagnosed using clinical and roentgenographic findings" Please clarify?

Done. A paragraph was added to clarify this point. We have followed the osteomyelitis diagnostic criteria of Lew and Waldvogel. (Lancet 2004; 364: 369-79, reference 10): “A diagnosis of osteomyelitis was made by clinical and roentgenographic criteria. Surgical and sinus tract pus samples were cultured in all the osteomyelitis patients to make an etiologic diagnosis. The presence of bone sequestra and/or sinus tract in bone X ray, CT or MRI, a positive Ga67 uptake bone scan, or a positive culture of the surgical bone sequestra or sinus tract were diagnostic of osteomyelitis �10].”

- The molecular techniques used in the current study are well-described.

* Results: The age and gender of control subjects and their comparisons to patients should be mentioned in the results' first paragraph. Because they are supposed to be matched, no significant difference should be noted.

Done. A new paragraph was added to the results section: “The control group was made of 295 male (71.1%) and 120 female donors, with a median age of 59.1 years (41–60). Although the controls were 2.9 years younger than the patients there was not a significant difference between the groups (p=0.14) ”

- No association of the CTSG N125S polymorphism with or type of bone infection (acute vs. ), source of infection or microorganism isolated was found" the authors should mention at least p for such associations!

Done. A paragraph explaining this point was added to the revised version of the manuscript.: “No association of the CTSG N125S polymorphism with type of bone infection (acute vs. chronic) (p=0.7), source of infection (hematogenous vs.non hematogenous, p=0.9), (pressure ulcers vs. non pressure ulcers, p=0.5) or microorganism isolated (S. aureus vs. Gram negative bacteria p=0.9) was found “

”

* General: The manuscript needs a professional editing service because there are several grammatical errors across the entire manuscript.

Done. English language was edited throughout the revised manuscript by Prof.Joshua Fierer from UCSD, a native English speaker.

- Data availability statement should be added to the manuscript after the conclusion explaining where the underlying data could be found.

All the data are already shown in the revised version of the manuscript.

Submitted filename: ANSWERS TO EDITOR AND REVIEWERS 6-9-19.doc

Click here for additional data file.

8 Oct 2019

The N125S polymorphism in the ginger-module-highlighter-mistake-type-1" id="gwmw-15703449758395636918275">cathepsin G gene (rs45567233) is associated with susceptibility to ginger-module-highlighter-mistake-type-1" id="gwmw-15703449758393413330348">osteomyelitis in a Spanish population.

PONE-D-19-18899R1

Dear Dr. Asensi,

We are pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it complies with all outstanding technical requirements.

Within one week, you will receive an e-mail containing information on the amendments required prior to publication. When all required modifications have been addressed, you will receive a formal acceptance letter and your manuscript will proceed to our production department and be scheduled for publication.

Shortly after the formal acceptance letter is sent, an invoice for payment will follow. To ensure an efficient production and billing process, please log ginger-module-highlighter-mistake-type-1" id="gwmw-15703449930245554470188">into Editorial Manager at https://www.editorialmanager.com/pone/, click the "Update My Information" link at the top of the page, and update your user information. If you have any billing related questions, please contact our Author Billing department directly at authorbilling@plos.org.

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With kind regards,

Manal S. ginger-module-highlighter-mistake-type-1" id="gwmw-15703450150231143822811">Fawzy, Ph.D., M.D.

Academic Editor

PLOS ONE

Additional Editor Comments (optional):

The authors have adequately addressed the concerns raised by the editor and the reviewers. Thank you

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. If the authors have adequately addressed your comments raised in a previous round of review and you feel that this manuscript is now acceptable for publication, you may indicate that here to bypass the “Comments to the Author” section, enter your conflict of interest statement in the “Confidential to Editor” section, and submit your "Accept" recommendation.

Reviewer #2: All comments have been addressed

**********

2. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #2: Yes

**********

3. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #2: Yes

**********

4. Have the authors made all data underlying the findings in their manuscript fully available?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited ginger-module-highlighter-mistake-type-3" id="gwmw-15703450284479521618669">to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—eginger-module-highlighter-mistake-type-3" id="gwmw-15703450313334784379329">.g. ginger-module-highlighter-mistake-type-1" id="gwmw-15703450319090844961165">participant privacy or use of data from a third party—those must be specified.

Reviewer #2: Yes

**********

5. Is the manuscript presented in an intelligible fashion and written in standard English?

PLOS ONE does not copyedit accepted manuscripts, so the language in ginger-module-highlighter-mistake-type-3" id="gwmw-15703450342040199955324">submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #2: Yes

**********

6. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. ginger-module-highlighter-mistake-type-3" id="gwmw-15703450378082981490053">(Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #2: All my comments were adequately addressed. I have no further comments on this manuscript and I recommend it for publication.

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Reviewer #2: No

15 Oct 2019

PONE-D-19-18899R1

The N125S polymorphism in the cathepsin G gene (rs45567233) is associated with susceptibility to osteomyelitis in a Spanish population.

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