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The NOS3 (27-bp repeat, intron 4) polymorphism is associated with susceptibility to osteomyelitis

The NOS3 (27-bp repeat, intron 4) polymorphism is associated with susceptibility to osteomyelitis
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  Nitric Oxide 16 (2007) 44–$ - see front matter ©  2006 Elsevier Inc. All rights reserved.doi:10.1016/j.niox.2006.06.005 The NOS3  (27-bp repeat, intron 4) polymorphism is associated with susceptibility to osteomyelitis  Victor Asensi a, ¤ , A. Hugo Montes b , Eulalia Valle b , Marcos G. Ocaña b , Aurora Astudillo c , Victoria Alvarez d , Esteban López-Anglada e , Angeles Solís e , Eliecer Coto d , Alvaro Meana a , Pelayo Gonzalez d , Jose A. Carton a , Jose Paz e , Joshua Fierer f  , Antonio Celada g a Infectious Diseases Unit, Hospital Central de Asturias, Oviedo University Medical School, Celestino Villamil s/n, 33006 Oviedo, Spain b Biochemistry and Molecular Biology Department, Oviedo University Medical School, Celestino Villamil s/n, 33006 Oviedo, Spain c Department of Pathology, Hospital Central de Asturias, Oviedo University Medical School, Celestino Villamil s/n, 33006 Oviedo, Spain d Molecular Genetics Unit-Nephrology Research Institute, Hospital Central de Asturias, Oviedo University Medical School, Celestino Villamil s/n, 33006 Oviedo, Spain e Traumatology Department, Hospital Central de Asturias, Oviedo University Medical School, Celestino Villamil s/n, 33006 Oviedo, Spain f Infectious Diseases Section, VAMC, University of California, San Diego, USA g Institute for Research in Biomedicine, Barcelona Science Park, University of Barcelona, Spain Received 12 February 2006; revised 4 June 2006Available online 23 June 2006 Abstract Cytokines generate nitric oxide (NO) in osteoblasts and neutrophils through the induction of NO synthase isoforms, endothelial(NOS3) and inducible (NOS2), thereby producing bone loss. In osteomyelitis (OM), a chronic infection of the bone, homozygosity for the NOS3  (27-bp repeat, intron 4 polymorphism) 4 allele was signi W cantly more frequent among the 80 patients than in 300 healthy controls(  p D 0.044). No signi W cant di V  erences were found for other polymorphisms of the NOS   genes such as NOS3 , the promoter ( ¡ 786T/C), andthe missense change (E298D) in exon 7, and for NOS2 , the G/A substitution at position 37498 in exon 22, the (CCTTT) n , and (TAAA) n micro-satellites and the ¡ 954G/C in the promoter. Serum NO levels were signi W cantly higher only in the OM patients homozygous for the NOS3  (27-bp repeat, intron 4 polymorphism) 4 allele, compared to controls. In the presence of bacteria or bacterial products, the neutro-phils of these patients produced more NO. However, immunolabelling of osteoblasts for NOS3 in biopsy tissues did not correlate with thecarriage of a determined NOS   polymorphism but with the presence of bone in X ammation. This is the W rst report of an associationbetween a NOS3  polymorphism and the risk of developing OM. ©  2006 Elsevier Inc. All rights reserved. Keywords: Osteomyelitis; Nitric oxide synthase; Polymorphisms; Neutrophils; Osteoblasts Osteomyelitis (OM) is an infection characterized by pro-gressive in X ammatory destruction of the infected bone andnew apposition of bone at the site of infection. In adults,OM is usually a complication of open wounds involving thebone, caused by fractures, surgery or both, and occursbetween 0.4% and 7% of patients [1–4]. In spite of appropri- ate combined medical and surgical therapies, up to 30% of bone infections become chronic, causing major economiclosses, and morbidity and mortality [1,2]. Much attention has been dedicated to improving the surgical and medicaltreatment of this infection, but little progress has beenmade towards understanding its pathogenesis. The risk of OM is mainly in X uenced by local factors related to thenature and severity of the underlying bone injury, and by  These results were presented in part at the 43rd Intersciences Confer-ence on Antimicrobial Agents and Chemotherapy (ICAAC), Chicago,USA, 14–17 September 2003, Abstract B-625. * Corresponding author. Fax: +34 985107963. E-mail address: (V. Asensi).  V. Asensi et al. / Nitric Oxide 16 (2007) 44–53 45 the micro-organisms inoculated in the bone. However,genetic factors of the host could also play crucial roles.Nitric oxide (NO), a free radical produced through themetabolism of arginine by the nitric oxide synthase (NOS),has crucial e V  ects on bone cell function. The endothelialisoform of NOS (eNOS or NOS3) is constitutivelyexpressed at low levels in bone, whereas inducible NOS(iNOS or NOS2) is expressed by bone cells in response toin X ammatory stimuli. Pro-in X ammatory cytokines such asIL-1-   and TNF-   cause activation of the NOS2-pathway,and NO derived from this pathway stimulates the bone lossinduced by cytokines and in X ammation [5,6]. Also, there is some evidence that NOS3 expression is regulated byin X ammatory stimuli through the Akt-kinase pathway [7].Knock-out mice for NOS3  have marked defects in osteo-blast maturation and activity [8,9].There are several NOS3  polymorphisms and at least threeof these have been linked to distinct NO levels in blood or todi V  erences in protein expression in response to several stim-uli: the 27-bp repeat in intron 4, the ( ¡ 786T/C) in the pro-moter region, and the missense (E298D) in exon 7. These NOS3  polymorphisms are associated with the risk of devel-oping a number of diseases, such as coronary artery disease[10–13]. Several polymorphisms of NOS2 , such as the highlypolymorphic (CCTTT) n  and (TAAA) n  micro-satellites, the ¡ 954G/C and the ¡ 1173 C/T at the promoter region, and aG/A substitution at position 37498 in exon 22 (iNOS 22), areassociated with rheumatoid arthritis, Parkinson disease, andpredisposition to or protection against malaria, tuberculosis,brucellosis and other infections [14–20].However, the association between NOS3  or NOS2  poly-morphisms and bone diseases, such as OM, has not beenreported. The research of our group focuses on the patho-genesis of OM. We previously described that a promoterpolymorphism of IL-1-   is a risk factor for this bone infec-tion [21]. Given the possible implications of NO in the path-ogenesis of this infection, here we analyzed severalpolymorphisms of NOS   genes and whether they constituterisk factors for developing OM. We also determined therole of these NOS   polymorphisms in the production of NOby circulating neutrophils invitro, and NOS expression byosteoblasts in bone biopsies. Patients and methods Patients Eighty adult patients (54 men and 26 women, with amean age of 52.3 § 18.3 years, range 16–89), with a diagno-sis of OM and admitted to the Hospital Central de Asturias and to three other a Y liated hospitals of the same region of Northern Spain between January 1998 and June 2004, werestudied. These hospitals give health coverage to the regionof Asturias (total population of 1 million). Patients withacute (24 cases) and chronic (56 cases) OM were included inthe study and followed for one year. OM was diagnosed byclinical, roentgenographic, computerized tomography (CT),magnetic resonance imaging (MRI) and isotopic boneimaging criteria. The demonstration of bone sequestra and/or sinus tract in bone X-ray, CT or MRI, a positive 67 Gauptake bone scan and a positive culture of the sequestra orsinus tract were considered diagnostic of OM [1,4]. OM patients with no history of trauma or bone surgery, nolower limb vascular insu Y ciency, and no contiguous focusof infection were considered as having hematogenous OM.Thirteen immunocompetent patients had hematogenousOM and 67 post-traumatic OM. Patients with infectedorthopaedic prostheses were excluded to avoid an age biasbecause these devices are generally placed in older patientswith osteoarthritis. Cases of tuberculosis and bacterial OMin immunosuppressed patients under chemotherapy wereexcluded. OM was considered chronic when present formore than 3 months, and cured when it did not relapse dur-ing a year of follow-up. Surgical and sinus tract pus sam-ples were cultured for the OM patients. Overall, 57 patientshad Gram-positive OM, 47 by Staphylococus aureus , and 23had Gram-negative OM, 10 by Pseudomonas aeruginosa . Inaddition, 300 healthy Blood Bank donors, matched for sexand age with the OM patients, were used as controls.Patients and controls were members of a homogeneouspopulation, all Caucasians and residents of the same region(Asturias, Northern Spain), and were in Hardy–Weinbergequilibrium. Each participant gave informed consent forthe study, which was approved by the Ethics Committee of the Hospital Central de Asturias . Serum nitrate and nitrite levels NO is a short-lived free radical gas that rapidly reactsrapidly with oxygen to generate the stable metabolitesnitrate and nitrite. Thus NO was assessed indirectly bymeasuring the accumulation of nitrates and nitrites. Fivemillilitres of whole blood was obtained from each patientand control; serum was removed and stored at ¡ 70°C upto 6 months until use. Serum nitrate and nitrite (NO x )determinations were performed using the Griess reaction,as previously described [22]. Results were expressed as  Mof NO x  /sample. For some experiments the increment inNO x (  NO x ) was calculated as follows:  NO x D NO x  inautologous serum after a 12-h incubation of neutrophilsminus NO x  levels in autologous serum before incubation. DNA genotyping analysis Another 10ml of blood from each patient and controlwas simultaneously collected in a tube containing potas-sium-EDTA. Genomic DNA was extracted from peripheralleukocytes following a salting-out method [23]. NOS3 (27-bp repeat, intron 4) genotyping  For analysis of the NOS3  (27-bp repeat, intron 4) poly-morphism, we used the primers described in Table 1. Thepolymerase chain reaction (PCR) was performed in a W nal  46 V. Asensi et al. / Nitric Oxide 16 (2007) 44–53 volume of 15  l, containing 100ng of genomic DNA, andconsisted of an initial denaturation at 95°C for 2min, fol-lowed by 32 cycles of 30s at 95°C, 1min at 63°C, and 1minat 72°C and a W nal extension of 5min at 72°C. PCR prod-ucts of 195bp (5 repeat alleles) and 168bp (4 repeat alleles)were separated by electrophoresis on a 3% agarose gel andvisualized after ethidium bromide staining, as previouslydescribed [10,13]. The results were con W rmed by sequencingrepresentative samples for genotypes 44 and 55. PCR prod-ucts were electrophoresed on a 2% low-melting agarose geland the fragments were excised from the gel, puri W ed withspin columns (DNA gel extraction Kit, Millipore, Billerica,MA, USA) and sequenced on an ABI Prism 310 GeneticAnalyser (Applied Biosystems, Foster City, CA, USA). NOS3 ( ¡ 786T/C) genotyping  The T to C change at nucleotide ¡ 786 introduces a sitefor the restriction enzyme Msp I (New England Biolabs,Beverly, MA, USA). A sequence of 180bp was PCR-ampli- W ed, as described for the NOS3  (27-bp repeat, intron 4)polymorphism, with primers shown in Table 1 and anannealing temperature of 62°C was applied. After digestionwith Msp I, fragments of 40bp (constant) and 140 ( ¡ 786T)or 90+50bp ( ¡ 786C) were visualized. NOS3 (E298D) genotyping  The coding sequence variant was a G ! T substitutionin exon 7 (at position 894) in codon 298, which alters theamino acids at this residue from Glu to Asp. Genotyping of this polymorphism was performed by PCR ampli W cation of exon 7 with the X anking intronic primers described in Table1, followed by Cfo I (New England Biolabs, Beverly, MA,USA) restriction endonuclease digestion for 16h at 37°Cand resolution by electrophoresis on a 3% agarose gel. The160-bp PCR product was cleaved into 140-bp and 20-bpfragments in the presence of G at position 894. NOS2 (exon 22) genotyping  For NOS2  (NOS2A), we studied a G/A substitution atposition 37498 in exon 22 (NOS2 22), which does not leadto an amino acid change [16]. This polymorphism was gen-otyped by PCR ampli W cation of exon 22 with the X ankingintronic primers described in Table 2 (annealing at 60°C),followed by Nco I (New England Biolabs, Beverly, MA,USA) restriction endonuclease digestion for 16h at 37°Cand resolution by electrophoresis on a 3% agarose gel. The140-bp PCR product was cleaved in two fragments of 120and 20bp, which de W ned the presence of the G allele, andone fragment of 140bp in the presence of the A allele. NOS2 (CCTTT) n  and (TAAA) n  genotyping  For these polymorphisms the PCR ampli W cation wasdone with the primers described in Table 2. The forwardprimers were 5   labelled with the X uorescent dye 6-FAMfor (CCTTT) n  and with PET for (TAAA) n . The ampli W ca-tion was at 95°C for 5min, 35 cycles of 95°C for 30s/ 62°C ae Oligonucleotide primer sequences, PCR conditions and restriction enzymes used for genotyping and sequencing the nitric oxide synthase ( NOS3 ) poly-morphisms studiedThe underlined bases in the primers di V  er from the srcinal sequences and served to introduce a restriction site or to disrupt a natural restriction sitewithin the primer sequence.GenePolymorphismPrimersPCR length (bp)Annealing temperature (°C)Restriction enzyme NOS3 27-bp, intron 4Forward: 5  -CTATGGTAGTGCCTTGGCTGGAGG-3  19563Not applicableReverse: 5  -ACCGCCCAGGGAACTCCGCT-3  NOS3  ¡ 786T/CForward: 5  -TGGAGAGTGCTGGTGACCCCA-3  18062 Msp IReverse: 5  -GCCTCCACCCCCACCCTGTC-3  NOS3 E298DForward: 5  -CTGCTGCAGGCCCCAGATGC-3  16062 Cfo IReverse: 5  -CACCCCCTTGCAGGCCCT-3  Table 2Oligonucleotide primer sequences, PCR conditions and restriction enzymes used for genotyping and sequencing the nitric oxide synthase (NOS2) poly-morphisms studiedThe underlined bases in the primers di V  er from the srcinal sequences and served to introduce a restriction site or to disrupt a natural restriction sitewithin the primer sequence.GenePolymorphismPrimersPCR length (bp)Annealing temperature (°C)Restriction enzyme NOS2 (CCTTT) n Forward: 5  -ACCCCTGGAAGCCTACAACTGCAT-3  19662Not applicableReverse: 5  -GCCACTGCACCCTAGCCTGTCTCA-3  NOS2 (TAAA) n Forward: 5  -TGCCACTCCGCTCCAG-3  22062Not applicableReverse: 5  -GGCCTCTGAGATGTTGGTCTT-3  NOS2  ¡ 954G/CForward: 5  -CATATGTATGGGAATACTGTATTTCAGGC-3  57062 Bsa IReverse: 5  -TCTGAACTAGTCACTTGAGG-3  NOS2 Exon 22Forward: 5  -CTCCCGGGATCACACGCCCAT-3  14060 Nco IReverse: 5  -GCTGAATCTGAGTTGATGAACAGATG-3   V. Asensi et al. / Nitric Oxide 16 (2007) 44–53 47 for 30s/ 72°C for 30s, followed by 72°C for 10min [24].Samples were analyzed using AbiPrism 3100 Genetic Ana-lyzer (Applied Biosystem, Foster City, CA, USA) and sizedusing GeneScan ®  Analysis Software ver. 3.7 (Applied Bio-system, Foster City, CA, USA). NOS2 ¡ 954G/C genotyping  For this SNP we used the primers described in Table 2(annealing temperature at 62°C). The PCR product wasincubated with the restriction enzyme Bsa I (New EnglandBiolabs, Beverly, MA, USA) overnight at 50°C [25]. Thefragments were separated on a 2.5% agarose gel and visual-ized with ethidium bromide and UV-light. Neutrophil isolation and incubation with bacteria or bacterial  products For each assay, 10ml of peripheral blood from one ormore OM patients and one or two healthy donors wassimultaneously collected in vacutainer plastic tubes con-taining potassium-EDTA (Vacuette, Greiner Bio-One,Kremsmuenster, Austria). Neutrophils were separated aspreviously described [26], adjusted to 0.5 £ 10 7 cells/ml inHam’s medium (Biochrom KG, Berlin, Germany) and kepton ice until use. Cells collected from the gradient interfacewere >95% neutrophils by Coulter identi W cation and >95%viable by trypan blue exclusion. Staphylococcus aureus ATCC 29213 and Escherichia coli   ATCC 15922 from theAmerican Type Culture Collection (Manassas, VA) weregrown on tripticase soy broth (TSB, Difco Laboratories,Augsburg, Germany) at 37°C overnight and adjusted byspectrophotometry to 0.5 £ 10 8 cells/ml in Ham’s medium.Neutrophils (0.5 £ 10 7 ) were incubated at 37°C with 200  lof autologous serum for 12h at 37°C with/out the additionof 0.5 £ 10 8 CFU/ml of S. aureus , E. coli  , lyophilized LPSfrom E. coli   serotype 0111:B4 (10  g/ml, Sigma, St. Louis,MO, USA) or lipotheicoic acid from S. aureus  (10  g/ml,Sigma). Immunohistochemistry Bone biopsies obtained at the time of curettage seques-trum from OM patients were examined. Six bone surgicalbiopsies from six patients with aseptic loosening of ortho-paedic prostheses and from twelve patients with osteopo-rotic femoral fractures were used as controls. In addition,10ml of peripheral blood from these patients with bone dis-eases di V  erent from OM was simultaneously collected.Samples were stored at ¡ 70°C until use. Bone biopsieswere thawed and W xed in 10% formol-saline for 24h beforepara Y n embedding, and then sectioned (3–5  m) in amicrotome. Samples then underwent antigenic recovery byrinsing in 10mM citrate bu V  er, pH 6.0 and boiling for2min. After rinsing the samples in TBS, the presence of NOS3 or NOS2 was assessed using immunohistochemistrywith a primary antibody raised against an epitope mappingat the carboxyterminus of human NOS3 or NOS2, as previ-ously described [27]. Sections were dried under a fan atroom temperature for 30min, rinsed in Tris-bu V  ered saline(TBS; pH 7.4, Sigma) for 5min, and incubated for 2h witha 1/100 primaries anti-human NOS3 and anti-humanNOS2 antibodies (Lab Vision Corporation, CA, 0.2  g/mL,in TBS plus 0.1% Tween-20 and 10% heat-inactivated sheepserum) in a dark chamber at 4°C overnight. Sections werewashed with TBS (3 £ 5min), incubated with the secondaryantibody (ChemMate Dako Envision Dual, HHP rabbit/mouse), and revealed using the peroxidase/DAB DakoEnvision detection kit. Sections were examined using aZeiss confocal microscope and images were stored in RGBTIFF format. Statistics The Pearson  2  and the Fisher’s exact tests were used tocompare allelic and genotypic frequencies between thegroups. Odds ratios (ORs) and their 95% con W dence inter-vals (CI) were also calculated. All the p values reported aretwo-sided. A  p -value <0.05 was considered signi W cant.Average NO x  and  NO x  values between genotypes werecompared with the Student’s t  test. The statistical analysiswas performed with two statistical packages: SPSS Soft-ware version 13.0 (SPSS, Chicago, IL, USA) and BMDPStatistical Software Ltd. (Cork, Ireland). Results To determine the polymorphisms in NOS2  and NOS3 ,genomic DNA from 80 OM patients and 300 healthy con-trols was ampli W ed by PCR. The frequency of NOS3  (27-bprepeat, intron 4) 4 allele of NOS3  in homozygous or hetero-zygous individuals was signi W cantly higher in OM patientscompared to controls (  p D 0.044 and  p D 0.039, respectively)(Table 3 and Fig.1). However, patients with this NOS3 polymorphism did not have any special clinical presenta-tion. Thus, there were no di V  erences between these patientsand the ones carrying other genotypes of this polymor-phism regarding the type of OM, rate of cure, age of onsetof the bone infection, or micro-organisms isolated from thebone. Genotypic and allelic frequencies of the ( ¡ 786T/C),and (E298D) polymorphisms of NOS3  or of the (TAAA) n ,(CCTTT) n  and exon 22 of NOS2  did not di V  er between OMpatients and controls (Tables 3 and 4 and Fig.2). The geno- typing of the NOS2 ¡ 954G/C polymorphism in 300 normalhealthy Caucasians and in 80 OM patients did not yield anypolymorphic variant at all (data not shown). Our W ndingsregarding the NOS2   ¡ 954G/C polymorphism are in agree-ment with previous studies on Caucasian population wherethe C allele was absent, with all individuals carrying thewild-type G allele, suggesting that this NOS2  polymor-phism is ethnic speci W c for African population [20,24,25].To determine the biological consequences of the (27-bprepeat, intron 4) 44 genotype on the functional activity of NOS3, we measured the amount of circulating NO in these  48 V. Asensi et al. / Nitric Oxide 16 (2007) 44–53 subjects. Serum nitrate and nitrite levels (NO x ) were signi W -cantly higher in patients with this genotype (  p <0.05)(Fig.3).After incubating the neutrophils for 12h in autologousserum, the increase in NO production was similar in OMpatients who were homozygous carriers of the 4 allele (27-bp repeat, intron 4) compared to the rest of the patientswithout this genotype (Fig.4). However, invitro incuba-tion with bacteria ( S. aureus , E. coli  ) or with bacterialproducts (LPS, lipotheicoic acid) for 12h increased theproduction of NO in these patients, signi W cantly for E.coli  (Fig.4). To determine the biological consequences of the(27-bp repeat, intron 4) 4 allele mutation on the func-tional activity of NOS3 in osteoblasts, we used immuno-histochemistry to assess NOS3 expression in biopsiesfrom OM patients, and from individuals with aseptic loos-ening of orthopaedic prostheses and osteoporotic femoralfractures; the last two were used as controls (Fig.5).Immunolabelling for NOS3 in osteoblasts did not corre-late with the NOS3  or NOS2  polymorphisms studied butwith the underlying bone disease. Thus, 90% of the bonebiopsies from OM patients had positive NOS3 immuno-staining while those from patients with diseases with amuch lower in X ammatory component, such as asepticloosening of orthopedic prostheses (33.3%,  p D 0.035) andosteoporotic femoral fractures (25%,  p D 0.0035), showeda signi W cantly lower staining (Table 5). No positive immu-nolabeling for NOS2 in osteoblasts was observed in biop-sies from patients with OM, aseptic loosening of orthopaedic prostheses or osteoporotic femoral fractures.There was, however, positive NOS2 immunolabeling intissue macrophages and endothelial cells in the bone fromOM patients (data not shown). ae Polymorphisms of NOS3  in patients with osteomyelitis (OM) and controlsAllelic and genotypic frequencies between OM patients and controls were compared by Pearson’s  2  and the Fisher’s exact tests. a 95% con W dence intervals.GeneGenotype frequenciesOMControlPearson  2 Odds ratio P Allele frequenciesOMControlPearson  2 Odds ratio  pNOS3  (27-bp repeat)Number of patients (%)80 (100.0)300 (100.0)445 (6.3)6 (2.0)4.053.27 (0.84–12.5) a 0.044433 (0.2)84 (0.14)4.251.6 (1.0–2.55) a 0.0394523 (28.8)72 (24.0)5552 (64.9)222 (74.0)5127 (0.8)516 (0.86) NOS 3  ( ¡ 786T/C)Number of patients (%)80 (100.0)300 (100.0)CC15 (18.8)43 (14.3)0.951.38 (0.69–2.75) a 0.33C70 (0.43)223 (0.37)2.311.31 (0.91–1.9) a 0.13CT40 (50.0)137 (46.7)TT25 (31.2)120 (40.0)T90 (0.57)375 (0.62) NOS 3  (E298D)Number of patients (%)80 (100.0)300 (100.0)TT9 (11.3)33 (11.0)0.0041.03 (0.43–2.36) a 0.95T54 (0.33)223 (0.37)0.680.86 (0.58–1.2) a 0.68CT36 (45.0)157 (52.3)CC35 (43.7)110 (36.3)C106 (0.67)377 (0.62)Fig.1. Detection of the NOS3  (27-bp repeat, intron 4) polymorphism.Genomic DNA of osteomyelitis (OM) patients was ampli W ed withoutenzymatic digestion. The agarose gel shows the RFLP of the NOS3  (27-bprepeat, intron 4) gene from OM patients with the following genotypes: 44homozygous (lane B), 45 heterozygous (lane C), and 55 wild-type (lane D).Lane A denotes molecular weight markers. Upon sequencing, the samplein lane B was homozygous 44, and the sample in lane D was wild-type 55for the NOS3  (27-bp repeat, intron 4) polymorphism (data not shown). 168 pb195 pb 3 S O N   )selella5 / 4()4nortni,taeper-pb72( DCBA sepytoneG 555444
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