Genetic factors responsible for long bone fractures non-union.

INTRODUCTION: Approximately 10-15% of all fractures of long bones heal with delay, prolonged immobilization and repetitive operative interventions. Despite intense investigations, the pathomechanism of impaired healing of skeletal tissue remains unclear. An important role in the pathomechanism of mal-union of close fractures plays subclinically proceeding infections. AIM: The question arises whether colonization and proliferation of bacteria in the fracture gap could be related to the mutation of genes for factors regulating local antimicrobial response, such as pathogen recognizing receptors (PRR), cytokines and chemokines. METHODS: We carried out studies in patients with delayed long bone fractures estimating the frequency of mutation of genes crucial for pathogen recognition (TLR2, TLR4 and CD14), and elimination (CRP, IL-6, IL-1ra), as well as wound healing (TGF-ß). The molecular milieu regulating healing process (IGF-1, COLL1a, TGF-ß, BMP-2, and PDGF) was validated by Western blot analysis of the gap tissue. RESULTS: Microbiological investigations showed the presence of viable bacterial strains in 34 out of 108 gaps in patients with non-healing fractures (31.5%) and in 20 out of 122 patients with uneventful healing (16.4%) (P < 0.05). The occurrence of mutated TLR4 1/W but not 2/W gene was significantly higher (P < 0.05) in the non-healing infected than sterile group. In the non-healing infected group 1/W mutated gene frequency was also higher than in healing infected. In the TGF-ß codon 10 a significantly higher frequency of mutated homozygote T and heterozygote C/T in the non-healing infected versus non-healing sterile subgroup was observed (P < 0.05). Similar difference was observed in the non-healing infected versus healing infected subgroup (P < 0.05). The CRP (G1059C), IL1ra (genotype 2/2), IL-6 (G176C), CD14 (G-159T), TLR2 (G2259A) and TLR4/2 (Thr399Ile) polymorphisms did not play evident role in the delay of fracture healing. CONCLUSIONS: Individuals bearing the mutant TLR 4 gene 1/W (Asp299Gly) and TGF-ß gene codon 10 mutant T and T/C allele may predispose to impaired pathogen recognition and elimination, leading to prolonged pathogen existence in the fracture gaps and healing delays.

[Genetic factors predisposing to bone fracture non-union. A role of single point mutation Asp299Gly TLR4 on pathogen-evoked healing]. / Genetyczna predyspozycja do zaburzen zrostu kostnego--wplyw punktowej mutacji Asp299Gly TLR4 na bakteriozalezne zaburzenia gojenia.

UNLABELLED: We previously reported the presence of the bacterial genetic material (16S rRNA) and viable pathogens in fracture gaps specimens, which suggests an impaired pathogen recognition and/or elimination. The aim of study was to validate the hypothesis that patients with delayed bone fracture healing express the higher frequency of TLR4 mutations. Observations were performed in 295 patients treated due to closed fractures of the long bones of the lower extremity; in 151 with delayed bone union (group A), and in 144 with uneventful healing (group B). Control group consisted of 125 healthy blood donors from ethnically the same as investigations groups polish population. Fracture gaps and deep tissue biopsies served for microbiological studies, and DNA isolated from venous blood leukocyteswas used for analysis of mutations of TLR4 gene at Asp299Gly (1/W) and Thr399Ile (2/W). RESULTS: Microbiological studies revealed positive isolates in 31.5% fracture gaps in group A and 16.4% in group B (p < 0.05). The most frequent isolates were S. epidermidis, S. aureus and S. warneri, capitis, sciuri and lentus, in lower percentage micrococci and enterococci. Amplification of 16S rRNA was positive in 56.8 and 65.2% of fracture gaps in both groups respectively. The frequency of occurrence of 1/W was significantly higher (p <0.05) in subgroups of patients with non-healing infected vs. sterile fractures. In all subgroups with viable pathogens isolated from fracture gaps the frequency of 1/W allele was higher when compared with subgroups, where fracture gaps occurred sterile. DISCUSSION: Performed investigations supported our previously reported observations that gaps of closed bone fractures are not sterile and are positive for 16S rRNA. Genetic predisposal to infection and inflammatory response evoked by a single TLR4 mutation may be one of the factors affecting bone union. Observed coexistence of bacterial colonization with decreased inflammatory reaction observed in individuals bearing TLR4 mutations have to be mentioned as a possible, etiologic factor responsible for delayed healing