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Abstract Carbohydrate-binding modules (CBMs) are non-catalytic proteins found appended to carbohydrate-active enzymes. Soil and marine bacteria secrete such enzymes to scavenge nutrition, and they often use CBMs to improve reaction rates and retention of released sugars. Here we present a structural and functional analysis of the recently established CBM family 92. All proteins analysed bind preferentially to β−1,6-glucans. This contrasts with the diversity of predicted substrates among the enzymes attached to CBM92 domains. We present crystal structures for two proteins, and confirm by mutagenesis that tryptophan residues permit ligand binding at three distinct functional binding sites on each protein. Multivalent CBM families are uncommon, so the establishment and structural characterisation of CBM92 enriches the classification database and will facilitate functional prediction in future projects. We propose that CBM92 proteins may cross-link polysaccharides in nature, and might have use in novel strategies for enzyme immobilisation.

Abstract Most Gram-negative bacteria synthesize osmo-regulated periplasmic glucans (OPG) in the periplasm or extracellular space. Pathogenicity of many pathogens is lost by knocking out opgG, an OPG-related gene indispensable for OPG synthesis. However, the biochemical functions of OpgG and OpgD, a paralog of OpgG, have not been elucidated. In this study, structural and functional analyses of OpgG and OpgD from Escherichia coli revealed that these proteins are β-1,2-glucanases with remarkably different activity from each other, establishing a new glycoside hydrolase family, GH186. Furthermore, a reaction mechanism with an unprecedentedly long proton transfer pathway among glycoside hydrolase families is proposed for OpgD. The conformation of the region that forms the reaction pathway differs noticeably between OpgG and OpgD, which explains the observed low activity of OpgG. The findings enhance our understanding of OPG biosynthesis and provide insights into functional diversity for this novel enzyme family.

Ossification of the posterior longitudinal ligament of the spine (OPLL) is an intractable disease leading to severe neurological deficits. Its etiology and pathogenesis are primarily unknown. The relationship between OPLL and comorbidities, especially type 2 diabetes (T2D) and high body mass index (BMI), has been the focus of attention; however, no trait has been proven to have a causal relationship. We conducted a meta-analysis of genome-wide association studies (GWASs) using 22,016 Japanese individuals and identified 14 significant loci, 8 of which were previously unreported. We then conducted a gene-based association analysis and a transcriptome-wide Mendelian randomization approach and identified three candidate genes for each. Partitioning heritability enrichment analyses observed significant enrichment of the polygenic signals in the active enhancers of the connective/bone cell group, especially H3K27ac in chondrogenic differentiation cells, as well as the immune/hematopoietic cell group. Single-cell RNA sequencing of Achilles tendon cells from a mouse Achilles tendon ossification model confirmed the expression of genes in GWAS and post-GWAS analyses in mesenchymal and immune cells. Genetic correlations with 96 complex traits showed positive correlations with T2D and BMI and a negative correlation with cerebral aneurysm. Mendelian randomization analysis demonstrated a significant causal effect of increased BMI and high bone mineral density on OPLL. We evaluated the clinical images in detail and classified OPLL into cervical, thoracic, and the other types. GWAS subanalyses identified subtype-specific signals. A polygenic risk score for BMI demonstrated that the effect of BMI was particularly strong in thoracic OPLL. Our study provides genetic insight into the etiology and pathogenesis of OPLL and is expected to serve as a basis for future treatment development.

Adolescent idiopathic scoliosis (AIS) is a common pediatric disease leading to spinal deformities. Here, the authors report GWAS followed by genome-wide meta-analysis in up to 79,211 Japanese individuals, identifying 20 genetic loci for AIS, 14 of which were previously unreported, and perform in vitro validation for rs1978060.

Cryptococcus is a mycosis founded in immunocompromised patients. Cryptococcus in the oral cavity is rare and skeletal infection is uncommon.We report the case of a 31-year-old man in whom cellulitis developed due to infection after tooth extraction complicated by acute myeloid leukemia (AML). Cellulitis of the left mandible did not improve after conservative therapy, including antimicrobial therapy, because of AML and chemotherapy, and gas was generated in the left cervical and supraclavicular regions. We considered the infection symptoms to be life-threatening, and surgery was performed for the infection of the head and neck under poor general conditions. As histopathological examination of the removed tissue revealed cryptococcus, antifungal agents were administered for cryptococcal infection. The surgical site healed after the operation.Surgical treatment, including debridement and drainage, should be avoided for patients with a poor general condition caused by AML and chemotherapy. However, the detection of Cryptococcus in the surgical site in such a condition is important. Keywords: Cryptococcus, Acute myeloid leukemia, Skeletal infection

Summary: Human cancer tissues are heterogeneous in nature and become differentiated during expansion of cancer stem cells (CSCs). CSCs initiate tumorigenesis, and are involved in tumor recurrence and metastasis. Furthermore, data show that CSCs are highly resistant to anticancer drugs. Cetuximab, a specific anti-epidermal growth factor receptor (EGFR) monoclonal antibody, is used in cancer treatment. Although development of resistance to cetuximab is well recognized, the underlying mechanisms remain unclear. Lapatinib, a dual inhibitor of epidermal growth factor receptor (EGFR)/ErbB2, has antiproliferative effects and is used to treat patients with ErbB2-positive metastatic breast cancer. In this review, cetuximab and lapatinib-resistant oral squamous cell carcinoma (OSCC) cells proliferation and migration signal transduction passway is discussed by introducing our research. Keywords: Cancer stem cells, Cetuximab, Lapatinib, Epidermal growth factor receptor, Oral squamous cell carcinoma, Molecularly-targeted therapy, Sphere formation

Mechanical and resorbable scaffolds are in high demand for stem cell-based regenerative medicine, to treat refractory bone defects in craniofacial abnormalities and injuries. Multipotent progenitor cells, such as dedifferentiated fat (DFAT) cells, are prospective sources for regenerative therapies. Herein, we aimed to demonstrate that a composite gelatin sponge (α-TCP/GS) of alfa-tricalcium phosphate (α-TCP) mixed with gelatin scaffolds (GS), with/without DFATs, induced bone regeneration in a rat calvarial defect model in vivo. α-TCP/GS was prepared by mixing α-TCP and 2% GS using vacuum-heated methods. α-TCP/GS samples with/without DFATs were transplanted into the model. After 4 weeks of implantation, the samples were subjected to micro-computed tomography (μ-CT) and histological analysis. α-TCP/GS possessed adequate mechanical strength; α-TCP did not convert to hydroxyapatite upon contact with water, as determined by X-ray diffraction. Moreover, stable α-TCP/GS was formed by electrostatic interactions, and verified based on the infrared peak shifts. μ-CT analyses showed that bone formation was higher in the α-TCP/GS+ DFAT group than in the α-TCP/GS group. Therefore, the implantation of α-TCP/GS comprising DFAT cells enhanced bone regeneration and vascularization, demonstrating the potential for healing critical-sized bone defects.

Bone regeneration using mesenchymal stem cells has several limitations. We investigated adipose-derived dedifferentiated fat (DFAT) cells as an alternative, and evaluated their cell proliferation rate, osteoblast differentiation, and bone regeneration ability in combination with activated platelet-rich plasma (aPRP). Rat DFATs and aPRP were isolated using ceiling culture and centrifugation, respectively. The cell proliferation rate was measured, and the cells were cultured in an osteoblast differentiation medium under varying concentrations of aPRP for 21 days and stained with Alizarin red. Gene expression was evaluated using real time polymerase chain reaction. Critical defects were implanted with DFAT seeded gelatin sponges under aPRP, and four weeks later, the bone regeneration ability was evaluated using micro-computed tomography and hematoxylin-eosin staining. The cell proliferation rate was significantly increased by the addition of aPRP. Alizarin red staining was positive 21 days after the start of induction, with significantly higher Runt-related transcription factor 2 (Runx2) and osteocalcin (OCN) expression levels than those in the controls. A 9 mm critical defect was largely closed (60.6%) after four weeks of gelatin sponge implantation with DFAT and aPRP. Therefore, materials combining DFAT cells and aPRP may be an effective approach for bone regeneration. Further research is needed to explore the long-term effects of these materials.

We proposed gel-fibers reeling system (Gel-FRS) with embedding biological cells for small diameter cellular vessels. To realize artificial 3D tissue, capillary blood vessels are important to be built in it as one of the typical small diameter cellular vessels. As previous works, a method to fabricate patient-specific vascular scaffolds was proposed using a biodegradable polymer, PLCL (poly(L-lactide-co-ε-caprolactone)). In this paper, we demonstrated a way for seeding smooth muscle cells and fibroblast cells outside the PLCL scaffold using the gel-fibers reeling system. At first, the mechanical stiffness of gel fibers was determined to achieve fiber reeling. The tensile stress of gel fibers was also evaluated without breaking down. The reeling speed of gel fiber was determined for seeding cells uniformly on a scaffold. The multiple hydrogel fibers embedding biological cells were reeled onto tubular PLCL scaffold, and then the alginate gel were melted by an alginate lyase. A uniform cell seeding onto upper and under surfaces of PLCL scaffold was achieved by rotating scaffold at 180 degrees every one hour. As a result, we achieved seeding cells onto scaffold and the cells viability was more than 90%. The proposed gel-fibers reeling system is important to be applicable in wide areas such as tissue engineering and bio simulators.

Despite the presence of β-1,2-glucan in nature, few β-1,2-glucan degrading enzymes have been reported to date. Recently, the Lin1839 protein from Listeria innocua was identified as a 1,2-β-oligoglucan phosphorylase. Since the adjacent lin1840 gene in the gene cluster encodes a putative glycoside hydrolase family 3 β-glucosidase, we hypothesized that Lin1840 is also involved in β-1,2-glucan dissimilation. Here we report the functional and structural analysis of Lin1840. A recombinant Lin1840 protein (Lin1840r) showed the highest hydrolytic activity toward sophorose (Glc-β-1,2-Glc) among β-1,2-glucooligosaccharides, suggesting that Lin1840 is a β-glucosidase involved in sophorose degradation. The enzyme also rapidly hydrolyzed laminaribiose (β-1,3), but not cellobiose (β-1,4) or gentiobiose (β-1,6) among β-linked gluco-disaccharides. We determined the crystal structures of Lin1840r in complexes with sophorose and laminaribiose as productive binding forms. In these structures, Arg572 forms many hydrogen bonds with sophorose and laminaribiose at subsite +1, which seems to be a key factor for substrate selectivity. The opposite side of subsite +1 from Arg572 is connected to a large empty space appearing to be subsite +2 for the binding of sophorotriose (Glc-β-1,2-Glc-β-1,2-Glc) in spite of the higher Km value for sophorotriose than that for sophorose. The conformations of sophorose and laminaribiose are almost the same on the Arg572 side but differ on the subsite +2 side that provides no interaction with a substrate. Therefore, Lin1840r is unable to distinguish between sophorose and laminaribiose as substrates. These results provide the first mechanistic insights into β-1,2-glucooligosaccharide recognition by β-glucosidase.

Axial spondylometaphyseal dysplasia (axial SMD) is an autosomal recessive disease characterized by dysplasia of axial skeleton and retinal dystrophy. We conducted whole exome sequencing and identified C21orf2 (chromosome 21 open reading frame 2) as a disease gene for axial SMD. C21orf2 mutations have been recently found to cause isolated retinal degeneration and Jeune syndrome. We found a total of five biallelic C21orf2 mutations in six families out of nine: three missense and two splicing mutations in patients with various ethnic backgrounds. The pathogenic effects of the splicing (splice-site and branch-point) mutations were confirmed on RNA level, which showed complex patterns of abnormal splicing. C21orf2 mutations presented with a wide range of skeletal phenotypes, including cupped and flared anterior ends of ribs, lacy ilia and metaphyseal dysplasia of proximal femora. Analysis of patients without C21orf2 mutation indicated genetic heterogeneity of axial SMD. Functional data in chondrocyte suggest C21orf2 is implicated in cartilage differentiation. C21orf2 protein was localized to the connecting cilium of the cone and rod photoreceptors, confirming its significance in retinal function. Our study indicates that axial SMD is a member of a unique group of ciliopathy affecting skeleton and retina.

Spinal extradural arachnoid cyst (SEDAC) is a cyst in the spinal canal that protrudes into the epidural space from a defect in the dura mater and leads to neurological disturbances. We previously showed that familial SEDAC is caused by FOXC2 mutation; however, the causal gene of sporadic SEDAC has not been identified. To identify the causal gene of sporadic SEDAC, we performed whole exome sequencing for 12 subjects with sporadic SEDAC and identified heterozygous HOXD4 loss-of-function mutations in three subjects. HOXD4 haplo-insufficiency causes SEDAC and a transcriptional network containing HOXD4 and FOXC2 is involved in the development of the dura mater and the etiology of SEDAC.

Drug delivery, minimally-invasive surgery, and a hospital-in-the-body are highly desirable for meeting the rapidly growing needs of nanorobot. This paper reports a Z-shaped gold/platinum (Au/Pt) hybrid nanorobot which realizes the self-rotational movement without an external force field. The Z-shaped Au/Pt hybrid nanorobot was fabricated by focused ion beam (FIB) and plasma sputtering. The purity of the nanorobot was tested by energy dispersive X-ray analysis (EDS). The weight percentage of Pt and Au at the tip were 94.28% and 5.72%, respectively. The weight percentage of Pt and Au at the bottom were 17.39% and 82.75%, respectively. The size of the nanorobot was 2.58 × 10−16 m2 and the mass of the nanorobot was 8.768 × 10−8 kg. The driving force of the nanorobot was 9.76 × 10−14 N at the 6.9% concentration of hydrogen peroxide solution. The rotation speed was 13 rpm, 14 rpm, and 19 rpm at 5.6%, 6.2%, and 7.8% concentrations, respectively.

In this study, we introduce novel method of flow cytometry for cell detection based on impedance measurements. The state of the art method for impedance flow cytometry detection utilizes an embedded electrode in the microfluidic to perform measurement of electrical impedance of the presence of cells at the sensing area. Nonetheless, this method requires an expensive and complicated electrode fabrication process. Furthermore, reuse of the fabricated electrode also requires an intensive and tedious cleaning process. Due to that, we present a microfluidic device with integrated microneedles. The two microneedles are placed at the half height of the microchannel for cell detection and electrical measurement. A commercially-available Tungsten needle was utilized for the microneedles. The microneedles are easily removed from the disposable PDMS (Polydimethylsiloxane) microchannel and can be reused with a simple cleaning process, such as washing by ultrasonic cleaning. Although this device was low cost, it preserves the core functionality of the sensor, which is capable of detecting passing cells at the sensing area. Therefore, this device is suitable for low-cost medical and food safety screening and testing process in developing countries.

The 3D assembly of micro-/nano-building blocks with multi-nanomanipulator coordinated manipulation is one of the central elements of nanomanipulation. A novel rail-guided nanomanipulation system was proposed for the assembly of a cellular vascular-like hydrogel microchannel. The system was equipped with three nanomanipulators and was restricted on the rail in order to realize the arbitrary change of the end-effectors during the assembly. It was set up with hybrid motors to achieve both a large operating space and a 30 nm positional resolution. The 2D components such as the assembly units were fabricated through the encapsulation of cells in the hydrogel. The coordinated manipulation strategies among the multi-nanomanipulators were designed with vision feedback and were demonstrated through the bottom-up assembly of the vascular-like microtube. As a result, the multi-layered microchannel was assembled through the cooperation of the nanomanipulation system.

The 3D assembly of cellular structures is important for the fabrication of biological substitutes in tissue engineering. In particular, a micro-channel with a 200 μm diameter is of interest because of its promising ability to construct the vascular network for oxygen and nutrition delivery in thick biological substitutes in the future. In this paper, a novel rail-guided micro-robot-team system is proposed for the micro-assembly of a cellular structure. The cellular two-dimensional (2D) component was fabricated by ultraviolet (UV) illumination of a cross-linkable hydrogel. The modular rail-guided micro-robotic system was set up with multi-micromanipulators as the modules and controlled with hybrid motors to achieve an operation resolution of 30 nm. To realize the bottom-up fabrication of the cellular micro-channel, different micro-assembly strategies with multi-manipulators were developed. The micro-assembly success rate and the efficiency of the different strategies were evaluated based on the assembly of micro-donuts. Through the novel, designed, concentric movement of the multi-manipulators along the rail, arbitrary change of the approaching angle and the coordination posture was achieved to improve the micro-assembly's flexibility. The operation range for every micromanipulator in different coordinated manipulation modes was analysed to avoid the breakdown of the assembled 3D structure. The image processing for the target location and end-effector identification was conducted to improve assembly efficiency in the micro-robot-team system. Finally, the assembly of the cellular vascular-like micro-channel was achieved with coordinated manipulation in the rail-guided micro-robot-team system.

In this paper, we present the fabrication and assembly of microstructures inside a microfluidic device based on a photocrosslinkable resin and optical tweezers. We also report a method of solution replacement inside the microfluidic channel in order to improve the manipulation performance and apply the assembled microstructures for single cell cultivation. By the illumination of patterned ultraviolet (UV) through a microscope, microstructures of arbitrary shape were fabricated by the photocrosslinkable resin inside a microfluidic channel. Based on the microfluidic channel with both glass and polydimethylsiloxane (PDMS) surfaces, immovable and movable microstructures were fabricated and manipulated. The microstructures were fabricated at the desired places and manipulated by the optical tweezers. A rotational microstructure including a microgear and a rotation axis was assembled and rotated in demonstrating this technique. The improved laser manipulation of microstructures was achieved based on the on-chip solution replacement method. The manipulation speed of the microstructures increased when the viscosity of the solvent decreased. The movement efficiency of the fabricated microstructures inside the lower viscosity solvent was evaluated and compared with those microstructures inside the former high viscosity solvent. A novel cell cage was fabricated and the cultivation of a single yeast cell ( w303 ) was demonstrated in the cell cage, inside the microfluidic device.

We characterized recombinant Lin1839 protein (Lin1839r) belonging to glycoside hydrolase family 94 from Listeria innocua. Lin1839r catalyzed the synthesis of a series of 1,2-β-oligoglucans (Sopn: n denotes degree of polymerization) using sophorose (Sop2) as the acceptor and α-D-glucose 1-phosphate (Glc1P) as the donor. Lin1839r recognized glucose as a very weak acceptor substrate to form polymeric 1,2-β-glucan. The degree of polymerization of the 1,2-β-glucan gradually decreased with long-term incubation to generate a series of Sopns. Kinetic analysis of the phosphorolytic reaction towards sophorotriose revealed that Lin1839r followed a sequential Bi Bi mechanism. The kinetic parameters of the phosphorolysis of sophorotetraose and sophoropentaose were similar to those of sophorotriose, although the enzyme did not exhibit significant phosphorolytic activity on Sop2. These results indicate that the Lin1839 protein is a novel inverting phosphorylase that catalyzes reversible phosphorolysis of 1,2-β-glucan with a degree of polymerization of ≥3. We propose 1,2-β-oligoglucan: phosphate α-glucosyltransferase as the systematic name and 1,2-β-oligoglucan phosphorylase as the short name for this Lin1839 protein.

This paper presents a method of particle manipulation by dielectrophoresis (DEP) and immobilization using photo-crosslinkable resin inside microfluidic devices. High speed particle manipulation, including patterning and concentration control by DEP was demonstrated. Immovable and movable microstructures embedding particles were fabricated on-chip. Several microelectrodes were fabricated using Indium Tin Oxides (ITO) and Cr/Au. The two kinds of DEP responses of yeast cells ( W303 ) and other particles were experimentally confirmed. Based on negative DEP phenomenon, cell traps generated by microelectrodes were demonstrated. Position control, transportation and patterning of cells were performed with cell traps. The on-chip fabrication of arbitrary shapes of microstructures based on Poly(ethylene glycol) diacrylate (PEGDA) was reported. With cell patterning by DEP and immobilization using on-chip fabrication, microstructures embedding line patterned cells were fabricated inside microfluidic channels. A novel microfluidic device was designed to separate patterning and fabrication areas and movable microstructures embedding concentration controllable particles were fabricated inside this device.

Spinal extradural arachnoid cyst (SEDAC) is a cyst in the spinal canal that protrudes into the epidural space from a defect in the dura mater. Most cases are sporadic; however, three familial SEDAC cases have been reported, suggesting genetic etiological factors. All familial cases are associated with lymphedema-distichiasis syndrome (LDS), whose causal gene is FOXC2. However, FOXC2 mutation analysis has been performed in only 1 family, and no mutation analysis has been performed on sporadic (non-familial) SEDACs. We recruited 17 SEDAC subjects consisting of 2 familial and 7 sporadic cases and examined FOXC2 mutations by Sanger sequencing and structural abnormalities by TaqMan copy number assay. We identified 2 novel FOXC2 mutations in 2 familial cases. Incomplete LDS penetrance was noted in both families. Four subjects presented with SEDACs only. Thus, SEDAC caused by the heterozygous FOXC2 loss-of-function mutation should be considered a feature of LDS, although it often manifests as the sole symptom. Seven sporadic SEDAC subjects had no FOXC2 mutations, no symptoms of LDS, and showed differing clinical characteristics from those who had FOXC2 mutations, suggesting that other gene(s) besides FOXC2 are likely to be involved in SEDAC.

Carbon nanotubes (CNT) have been developed in recent decades for nanodevices such as nanoradios, nanogenerators, carbon nanotube field effect transistors (CNTFETs) and so on, indicating that the application of CNTs for nanoscale electronics may play a key role in the development of nanotechnology. Nanorobotics manipulation systems are a promising method for nanodevice construction and assembly. For the purpose of constructing three-dimensional CNTFETs, a nanorobotics manipulation system with 16 DOFs was developed for nanomanipulation of nanometer-scale objects inside the specimen chamber of a scanning electron microscope (SEM). Nanorobotics manipulators are assembled into four units with four DOFs (X-Y-Z-θ) individually. The rotational one is actuated by a picomotor. That means a manipulator has four DOFs including three linear motions in the X, Y, Z directions and a 360-degree rotational one (X-Y-Z-θ stage, θ is along the direction rotating with X or Y axis). Manipulators are actuated by picomotors with better than 30 nm linear resolution and

This work presents a novel method to assess the condition of Caenorhabditis elegans (C. elegans) through a resistance measurement of its undulatory locomotion speed inside a micro channel. As the worm moves over the electrode inside the micro channel, the length of the electrode changes, consequently behaving like a strain gauge. In this paper, the electrotaxis was applied for controlling the direction of motion of C. elegans as an external stimulus, resulting in the worm moving towards the cathode of the circuit. To confirm the proposed measurement method, a microfluidic device was developed that employs a sinusoidal channel and a thin polydimethylsiloxane (PDMS) layer with an electrode. The PDMS layer maintains a porous structure to enable the flexibility of the electrode. In this study, 6 measurements were performed to obtain the speed of an early adult stage C. elegans, where the measured average speed was 0.35 (±0.05) mm/s. The results of this work demonstrate the application of our method to measure the speed of C. elegans undulatory locomotion. This novel approach can be applied to make such measurements without an imaging system, and more importantly, allows directly to detect the locomotion of C. elegans using an electrical signal (i.e., the change in resistance).

To perform specific analysis for the single cell, individual cells have to be captured and separated from each other before further treatments and analysis can be carried out. This paper presents the design, simulation, fabrication, and testing of a microfluidic device for trapping a single cell/particle based on a hydrodynamic technique. A T-channel trapping chip has been proposed to provide single-cell trapping and consequently could be a platform for cell treatments and manipulations. A finite element T-channel trapping model was developed using Abaqus FEA™ software to observe it’s trapping ability by optimizing the channel’s geometry and RhMain/RhTrap ratio. A proof of concept demonstration for cell trapping in the T-channel model was presented in the simulation analysis and experimental work using HUVEC cell aggregate. The T-channel was found to be able to trap a single cell via the hydrodynamic trapping concept using an appropriate channel geometry and RhMain/RhTrap ratio. The proposed T-channel single-cell trapping has potential application for single cell characterization and single 3D cell aggregates treatments and analysis.

This paper reports tungsten-platinum hybrid nanowire growth via field emission, based on nanorobotic manipulation within a field emission scanning electron microscope (FESEM). A multiwalled carbon nanotube (MWCNT) was used as the emitter, and a tungsten probe was used as the anode at the counterposition, by way of nanomanipulation. By independently employing trimethylcyclopentadienyl platinum (CpPtMe3) and tungsten hexacarbonyl (W(CO)6) as precursors, the platinum nanowire grew on the tip of the MWCNT emitter. Tungsten nanowires then grew on the tip of the platinum nanowire. The hybrid nanowire length wascontrolled by nanomanipulation. Their purity was evaluated using energy-dispersive X-ray spectroscopy (EDS). Thus, it is possible to fabricate various metallic hybrid nanowires by changing the precursor materials. Hybrid nanowires have various applications in nanoelectronics, nanosensor devices, and nanomechanical systems.

Osteoarthritis (OA) is a common disease that has a definite genetic component. Only a few OA susceptibility genes that have definite functional evidence and replication of association have been reported, however. Through a genome-wide association study and a replication using a total of approximately 4,800 Japanese subjects, we identified two single nucleotide polymorphisms (SNPs) (rs7775228 and rs10947262) associated with susceptibility to knee OA. The two SNPs were in a region containing HLA class II/III genes and their association reached genome-wide significance (combined P = 2.43x10(-8) for rs7775228 and 6.73x10(-8) for rs10947262). Our results suggest that immunologic mechanism is implicated in the etiology of OA.