Mice lacking nucleotide sugar transporter SLC35A3 exhibit lethal chondrodysplasia with vertebral anomalies and impaired glycosaminoglycan biosynthesis.

SLC35A3 is considered an uridine diphosphate N-acetylglucosamine (UDP-GlcNAc) transporter in mammals and regulates the branching of N-glycans. A missense mutation in SLC35A3 causes complex vertebral malformation (CVM) in cattle. However, the biological functions of SLC35A3 have not been fully clarified. To address these issues, we have established Slc35a3-/-mice using CRISPR/Cas9 genome editing system. The generated mutant mice were perinatal lethal and exhibited chondrodysplasia recapitulating CVM-like vertebral anomalies. During embryogenesis, Slc35a3 mRNA was expressed in the presomitic mesoderm of wild-type mice, suggesting that SLC35A3 transports UDP-GlcNAc used for the sugar modification that is essential for somite formation. In the growth plate cartilage of Slc35a3-/-embryos, extracellular space was drastically reduced, and many flat proliferative chondrocytes were reshaped. Proliferation, apoptosis and differentiation were not affected in the chondrocytes of Slc35a3-/-mice, suggesting that the chondrodysplasia phenotypes were mainly caused by the abnormal extracellular matrix quality. Because these histological abnormalities were similar to those observed in several mutant mice accompanying the impaired glycosaminoglycan (GAG) biosynthesis, GAG levels were measured in the spine and limbs of Slc35a3-/-mice using disaccharide composition analysis. Compared with control mice, the amounts of heparan sulfate, keratan sulfate, and chondroitin sulfate/dermatan sulfate, were significantly decreased in Slc35a3-/-mice. These findings suggest that SLC35A3 regulates GAG biosynthesis and the chondrodysplasia phenotypes were partially caused by the decreased GAG synthesis. Hence, Slc35a3-/- mice would be a useful model for investigating the in vivo roles of SLC35A3 and the pathological mechanisms of SLC35A3-associated diseases.

CANT1 deficiency in a mouse model of Desbuquois dysplasia impairs glycosaminoglycan synthesis and chondrocyte differentiation in growth plate cartilage.

Desbuquois dysplasia (DD) type 1 is a rare skeletal dysplasia characterized by a short stature, round face, progressive scoliosis, and joint laxity. The causative gene has been identified as calcium-activated nucleotidase 1 (CANT1), which encodes a nucleotidase that preferentially hydrolyzes UDP to UMP and phosphate. In this study, we generated Cant1 KO mice using CRISPR/Cas9-mediated genome editing. All F0 mice possessing frameshift deletions at both Cant1 alleles exhibited a dwarf phenotype. Germline transmission of the edited allele was confirmed in an F0 heterozygous mouse, and KO mice were generated by crossing of the heterozygous breeding pairs. Cant1 KO mice exhibited skeletal defects, including short stature, thoracic kyphosis, and delta phalanx, all of which are observed in DD type 1 patients. The glycosaminoglycan (GAG) content and extracellular matrix space were reduced in the growth plate cartilage of mutants, and proliferating chondrocytes lost their typical flat shape and became round. Chondrocyte differentiation, especially terminal differentiation to hypertrophic chondrocytes, was impaired in Cant1 KO mice. These findings indicate that CANT1 is involved in the synthesis of GAG and regulation of chondrocyte differentiation in the cartilage and contribute to a better understanding of the pathogenesis of DD type 1.

Crim1C140S mutant mice reveal the importance of cysteine 140 in the internal region 1 of CRIM1 for its physiological functions.

Cysteine-rich transmembrane bone morphogenetic protein regulator 1 (CRIM1) is a type I transmembrane protein involved in the organogenesis of many tissues via its interactions with growth factors including BMP, TGF-ß, and VEGF. In this study, we used whole-exome sequencing and linkage analysis to identify a novel Crim1 mutant allele generated by ENU mutagenesis in mice. This allele is a missense mutation that causes a cysteine-to-serine substitution at position 140, and is referred to as Crim1C140S. In addition to the previously reported phenotypes in Crim1 mutants, Crim1C140S homozygous mice exhibited several novel phenotypes, including dwarfism, enlarged seminal vesicles, and rectal prolapse. In vitro analyses showed that Crim1C140S mutation affected the formation of CRIM1 complexes and decreased the amount of the overexpressed CRIM1 proteins in the cell culture supernatants. Cys140 is located in the internal region 1 (IR1) of the N-terminal extracellular region of CRIM1 and resides outside any identified functional domains. Inference of the domain architecture suggested that the Crim1C140S mutation disturbs an intramolecular disulfide bond in IR1, leading to the protein instability and the functional defects of CRIM1. Crim1C140S highlights the functional importance of the IR1, and Crim1C140S mice should serve as a valuable model for investigating the functions of CRIM1 that are unidentified as yet.

Disruption of the mouse Slc39a14 gene encoding zinc transporter ZIP14 is associated with decreased bone mass, likely caused by enhanced bone resorption.

Osteoclasts are bone-resorbing cells that play an essential role in maintaining bone homeostasis. Zinc (Zn) has been reported to inhibit osteoclast-mediated bone resorption, but the mechanism of this action has not been clarified. Zn homeostasis is tightly controlled by the coordinated actions of many Zn transporters. The Zn transporter ZIP14/Slc39a14 is involved in various physiological functions; hence, Zip14-knockout (KO) mice exhibit multiple phenotypes. In this study, we thoroughly investigated the bone phenotypes of Zip14-KO mice, demonstrating that the KO mice exhibited osteopenia in both trabecular and cortical bones. In Zip14-KO mice, bone resorption was increased, whereas the bone formation rate was unchanged. Zip14 mRNA was expressed in normal osteoclasts both in vivo and in vitro, but receptor activator of NF-κB ligand (RANKL)-induced osteoclastogenesis was not impaired in bone marrow-derived macrophages prepared from Zip14-KO mice. These results suggest that ZIP14 regulates bone homeostasis by inhibiting bore resorption and that in Zip14-KO mice, bone resorption is increased due to the elimination of this inhibitory regulation. Further studies are necessary to conclude whether the enhancement of bone resorption in Zip14-KO mice is due to a cell-autonomous or a non-cell-autonomous osteoclast defect.

An ENU-induced p.C225S missense mutation in the mouse Tgfb1 gene does not cause Camurati-Engelmann disease-like skeletal phenotypes.

Camurati-Engelmann disease (CED) is a rare sclerosing bone disorder in humans with autosomal dominant inheritance. Mutations in the gene (TGFB1) that encodes transforming growth factor-ß1 (TGF-ß1) are causative for CED. TGF-ß1 signaling is enhanced by the CED-causing mutations. In this study, we performed Tgfb1 mutation screening in an ENU-mutagenized mouse genomic DNA library. We identified a missense mutation in which cysteine was substituted by serine at position 225 (p.C225S), that corresponded to the CED-causing mutation (p.C225R). TGF-ß1 mutant protein carrying p.C225S was secreted normally into the extracellular space. Reporter gene assays showed that the p.C225S mutants enhanced TGF-ß signaling at the same level as p.C225R mutants. We generated p.C225S homozygous mice and confirmed that the mature TGF-ß1 levels in the culture supernatants of the calvarial cells from the homozygotes were significantly higher than those from wild-type mice. Although the skull and femur are sclerotic in CED, these phenotypes were not observed in p.C225S homozygous mice. These results suggest that human and mouse bone tissue react differently to TGF-ß1. These findings are useful to pharmacological studies using mouse models in developing drugs that will target TGF-ß signaling.

Overexpression of BCLXL in Osteoblasts Inhibits Osteoblast Apoptosis and Increases Bone Volume and Strength.

The Bcl2 family proteins, Bcl2 and BclXL, suppress apoptosis by preventing the release of caspase activators from mitochondria through the inhibition of Bax subfamily proteins. We reported that BCL2 overexpression in osteoblasts using the 2.3 kb Col1a1 promoter increased osteoblast proliferation, failed to reduce osteoblast apoptosis, inhibited osteoblast maturation, and reduced the number of osteocyte processes, leading to massive osteocyte death. We generated BCLXL (BCL2L1) transgenic mice using the same promoter to investigate BCLXL functions in bone development and maintenance. Bone mineral density in the trabecular bone of femurs was increased, whereas that in the cortical bone was similar to that in wild-type mice. Osteocyte process formation was unaffected and bone structures were similar to those in wild-type mice. A micro-CT analysis showed that trabecular bone volume in femurs and vertebrae and the cortical thickness of femurs were increased. A dynamic bone histomorphometric analysis revealed that the mineralizing surface was larger in trabecular bone, and the bone-formation rate was increased in cortical bone. Serum osteocalcin but not TRAP5b was increased, BrdU-positive osteoblastic cell numbers were increased, TUNEL-positive osteoblastic cell numbers were reduced, and osteoblast marker gene expression was enhanced in BCLXL transgenic mice. The three-point bending test indicated that femurs were stronger in BCLXL transgenic mice than in wild-type mice. The frequency of TUNEL-positive primary osteoblasts was lower in BCLXL transgenic mice than in wild-type mice during cultivation, and osteoblast differentiation was enhanced but depended on cell density, indicating that enhanced differentiation was mainly owing to reduced apoptosis. Increased trabecular and cortical bone volumes were maintained during aging in male and female mice. These results indicate that BCLXL overexpression in osteoblasts increased the trabecular and cortical bone volumes with normal structures and maintained them majorly by preventing osteoblast apoptosis, implicating BCLXL as a therapeutic target of osteoporosis. © 2016 American Society for Bone and Mineral Research.

Endoplasmic reticulum stress-mediated apoptosis contributes to a skeletal dysplasia resembling platyspondylic lethal skeletal dysplasia, Torrance type, in a novel Col2a1 mutant mouse line.

In humans, mutations in the COL2A1 gene encoding the α1(II) chain of type II collagen, create many clinical phenotypes collectively termed type II collagenopathies. However, the mechanisms generating this diversity remain to be determined. Here we identified a novel Col2a1 mutant mouse line by screening a large-scale N-ethyl-N-nitrosourea mutant mouse library. This mutant possessed a p.Tyr1391Ser missense mutation in the C-propeptide coding region, and this mutation was located in positions corresponding to the human COL2A1 mutation responsible for platyspondylic lethal skeletal dysplasia, Torrance type (PLSD-T). As expected, p.Tyr1391Ser homozygotes exhibited lethal skeletal dysplasias resembling PLSD-T, including extremely short limbs and severe dysplasia of the spine and pelvis. The secretion of the mutant proteins into the extracellular space was disrupted, accompanied by an abnormally expanded endoplasmic reticulum (ER) and the up-regulation of ER stress-related genes in chondrocytes. Chondrocyte apoptosis was severely induced in the growth plate of the homozygotes. These findings strongly suggest that ER stress-mediated apoptosis caused by the accumulated mutant proteins in ER contributes to skeletal dysplasia in Co12a1 mutant mice and PLSD-T patients.

[Collagen abnormalities and endoplasmic reticulum stress in bone and cartilage].

There are many steps in the post-translational modification of collagen molecules. When abnormality occurs in some step, the unfolded collagen molecules are accumulated in the endoplasmic reticulum (ER) , leading to ER stress. ER stress also occurs downstream of the defective modification of collagen in bone and cartilage. ER stress-induced apoptosis or ER stress response without inducing apoptosis may be associated with the pathogenesis of genetic collagen disorders in bone and cartilage.

Zinc signal: a new player in osteobiology.

Disturbed zinc (Zn) homeostasis in mammals is mainly characterized by impaired bone generation accompanied with growth retardation. However, the underlying mechanisms that determine how Zn controls bone homeostasis remain to be defined. Zn homeostasis is tightly controlled by Zn transporter families. Recent studies have shown that Zn transporter-mediated Zn ion (Zn(2+)) behaves as a signaling factor, called Zn signal, that exerts a multiple function in cellular events, showing why Zn has a vital effect on mammalian bone growth and regeneration. This perspective put importance on the principal mechanisms of Zn participation in mammalian bone homeostasis, shifting our focus on the role of Zn from simply a nutrient to a signaling molecule that fine-tunes intracellular signaling events.

SP7 inhibits osteoblast differentiation at a late stage in mice.

RUNX2 and SP7 are essential transcription factors for osteoblast differentiation at an early stage. Although RUNX2 inhibits osteoblast differentiation at a late stage, the function of SP7 at the late stage of osteoblast differentiation is not fully elucidated. Thus, we pursued the function of SP7 in osteoblast differentiation. RUNX2 induced Sp7 expression in Runx2(-/-) calvarial cells. Adenoviral transfer of sh-Sp7 into primary osteoblasts reduced the expression of Alpl, Col1a1, and Bglap2 and mineralization, whereas that of Sp7 reduced Bglap2 expression and mineralization at a late stage of osteoblast differentiation. Sp7 transgenic mice under the control of 2.3 kb Col1a1 promoter showed osteopenia and woven-bone like structure in the cortical bone, which was thin and less mineralized, in a dose-dependent manner. Further, the number of processes in the osteoblasts and osteocytes was reduced. Although the osteoblast density was increased, the bone formation was reduced. The frequency of BrdU incorporation was increased in the osteoblastic cells, while the expression of Col1a1, Spp1, Ibsp, and Bglap2 was reduced. Further, the osteopenia in Sp7 or Runx2 transgenic mice was worsened in Sp7/Runx2 double transgenic mice and the expression of Col1a1 and Bglap2 was reduced. The expression of Sp7 and Runx2 was not increased in Runx2 and Sp7 transgenic mice, respectively. The expression of endogenous Sp7 was increased in Sp7 transgenic mice and Sp7-transduced cells; the introduction of Sp7 activated and sh-Sp7 inhibited Sp7 promoter; and ChIP assay showed the binding of endogenous SP7 in the proximal region of Sp7 promoter. These findings suggest that SP7 and RUNX2 inhibit osteoblast differentiation at a late stage in a manner independent of RUNX2 and SP7, respectively, and SP7 positively regulates its own promoter.

Overexpression of Bcl2 in osteoblasts inhibits osteoblast differentiation and induces osteocyte apoptosis.

Bcl2 subfamily proteins, including Bcl2 and Bcl-X(L), inhibit apoptosis. As osteoblast apoptosis is in part responsible for osteoporosis in sex steroid deficiency, glucocorticoid excess, and aging, bone loss might be inhibited by the upregulation of Bcl2; however, the effects of Bcl2 overexpression on osteoblast differentiation and bone development and maintenance have not been fully investigated. To investigate these issues, we established two lines of osteoblast-specific BCL2 transgenic mice. In BCL2 transgenic mice, bone volume was increased at 6 weeks of age but not at 10 weeks of age compared with wild-type mice. The numbers of osteoblasts and osteocytes increased, but osteoid thickness and the bone formation rate were reduced in BCL2 transgenic mice with high expression at 10 weeks of age. The number of BrdU-positive cells was increased but that of TUNEL-positive cells was unaltered at 2 and 6 weeks of age. Osteoblast differentiation was inhibited, as shown by reduced Col1a1 and osteocalcin expression. Osteoblast differentiation of calvarial cells from BCL2 transgenic mice also fell in vitro. Overexpression of BCL2 in primary osteoblasts had no effect on osteoclastogenesis in co-culture with bone marrow cells. Unexpectedly, overexpression of BCL2 in osteoblasts eventually caused osteocyte apoptosis. Osteocytes, which had a reduced number of processes, gradually died with apoptotic structural alterations and the expression of apoptosis-related molecules, and dead osteocytes accumulated in cortical bone. These findings indicate that overexpression of BCL2 in osteoblasts inhibits osteoblast differentiation, reduces osteocyte processes, and causes osteocyte apoptosis.

ENU-induced missense mutation in the C-propeptide coding region of Col2a1 creates a mouse model of platyspondylic lethal skeletal dysplasia, Torrance type.

The COL2A1 gene encodes the α1(II) chain of the homotrimeric type II collagen, the most abundant protein in cartilage. In humans, COL2A1 mutations create many clinical phenotypes collectively termed type II collagenopathies; however, the genetic basis of the phenotypic diversity is not well elucidated. Therefore, animal models corresponding to multiple type II collagenopathies are required. In this study we identified a novel Col2a1 missense mutation--c.44406A>C (p.D1469A)--produced by large-scale N-ethyl-N-nitrosourea (ENU) mutagenesis in a mouse line. This mutation was located in the C-propeptide coding region of Col2a1 and in the positions corresponding to a human COL2A1 mutation responsible for platyspondylic lethal skeletal dysplasia, Torrance type (PLSD-T). The phenotype was inherited as a semidominant trait. The heterozygotes were mildly but significantly smaller than wild-type mice. The homozygotes exhibited lethal skeletal dysplasias, including extremely short limbs, severe spondylar dysplasia, severe pelvic hypoplasia, and brachydactyly. As expected, these skeletal defects in the homozygotes were similar to those in PLSD-T patients. The secretion of the mutant proteins into the extracellular space was disrupted, accompanied by abnormally expanded rough endoplasmic reticulum (ER) and upregulation of ER stress-related genes, such as Grp94 and Chop, in chondrocytes. These findings suggested that the accumulation of mutant type II collagen in the ER and subsequent induction of ER stress are involved, at least in part in the PLSD-T-like phenotypes of the mutants. This mutant should serve as a good model for studying PLSD-T pathogenesis and the mechanisms that create the great diversity of type II collagenopathies.

Follistatin-like 1 (Fstl1) is a bone morphogenetic protein (BMP) 4 signaling antagonist in controlling mouse lung development.

Lung morphogenesis is a well orchestrated, tightly regulated process through several molecular pathways, including TGF-ß/bone morphogenetic protein (BMP) signaling. Alteration of these signaling pathways leads to lung malformation. We investigated the role of Follistatin-like 1 (Fstl1), a secreted follistatin-module-containing glycoprotein, in lung development. Deletion of Fstl1 in mice led to postnatal lethality as a result of respiratory failure. Analysis of the mutant phenotype showed that Fstl1 is essential for tracheal cartilage formation and alveolar maturation. Deletion of the Fstl1 gene resulted in malformed tracheal rings manifested as discontinued rings and reduced ring number. Fstl1-deficient mice displayed septal hypercellularity and end-expiratory atelectasis, which were associated with impaired differentiation of distal alveolar epithelial cells and insufficient production of mature surfactant proteins. Mechanistically, Fstl1 interacted directly with BMP4, negatively regulated BMP4/Smad1/5/8 signaling, and inhibited BMP4-induced surfactant gene expression. Reducing BMP signaling activity by Noggin rescued pulmonary atelectasis of Fstl1-deficient mice. Therefore, we provide in vivo and in vitro evidence to demonstrate that Fstl1 modulates lung development and alveolar maturation, in part, through BMP4 signaling.

A founder mutation of CANT1 common in Korean and Japanese Desbuquois dysplasia.

Desbuquois dysplasia (DBQD) is a severe skeletal dysplasia of autosomal recessive inheritance. DBQD is classified into types 1 and 2 based on presence or absence of hand anomalies. In a previous study, we found a CANT1 (for calcium-activated nucleotidase 1) mutation, c.676G>A in five DBQD families. They were all East Asians (Japanese or Korean). The high prevalence of the same mutation among Japanese and Korean suggested that it is a common founder mutation in the two populations. To examine a possible common founder, we examined the region around CANT1 in chromosomes with c.676G>A mutation by genotyping polymorphic markers in the region for the families. We examined their haplotypes using the family data. We identified in all families a common haplotype containing the CANT1 mutation that ranged up to 550 kb. The two unrelated carriers of the mutation in general populations in Korea and Japan could also have the haplotype. We estimated the age of the founder mutation as ∼ 1420 years (95% CI=880-1940 years). The c.676G>A mutation of CANT1 commonly seen in Japanese and Korean DBQD should be derived from a common founder.

SMOC1 is essential for ocular and limb development in humans and mice.

Microphthalmia with limb anomalies (MLA) is a rare autosomal-recessive disorder, presenting with anophthalmia or microphthalmia and hand and/or foot malformation. We mapped the MLA locus to 14q24 and successfully identified three homozygous (one nonsense and two splice site) mutations in the SPARC (secreted protein acidic and rich in cysteine)-related modular calcium binding 1 (SMOC1) in three families. Smoc1 is expressed in the developing optic stalk, ventral optic cup, and limbs of mouse embryos. Smoc1 null mice recapitulated MLA phenotypes, including aplasia or hypoplasia of optic nerves, hypoplastic fibula and bowed tibia, and syndactyly in limbs. A thinned and irregular ganglion cell layer and atrophy of the anteroventral part of the retina were also observed. Soft tissue syndactyly, resulting from inhibited apoptosis, was related to disturbed expression of genes involved in BMP signaling in the interdigital mesenchyme. Our findings indicate that SMOC1/Smoc1 is essential for ocular and limb development in both humans and mice.

CANT1 mutation is also responsible for Desbuquois dysplasia, type 2 and Kim variant.

BACKGROUND: Desbuquois dysplasia (DD) is a recessively inherited condition characterised by short stature, generalised skeletal dysplasia and advanced bone maturation. DD is both clinically and radiographically heterogeneous, and two subtypes have been distinguished based on the presence (type 1) or absence (type 2) of an accessory metacarpal bone. In addition, an apparently distinct variant without additional metacarpal bone but with short metacarpals and long phalanges (Kim variant) has been described recently. Mutations in the gene that encodes for CANT1 (calcium-activated nucleotidase 1) have been identified in a subset of patients with DD type 1. METHODS: A series of 11 subjects with DD from eight families (one type 1, two type 2, five Kim variant) were examined for CANT1 mutations by direct sequencing of all coding exons and their flanking introns. RESULTS: Eight distinct mutations were identified in seven families (one type 1, one type 2 and all 5 Kim variant): three were nonsense and five were missense. All missense mutations occurred at highly conserved amino acids in the nucleotidase conserved regions of CANT1. Measurement of nucleotidase activity in vitro showed that the missense mutations were all associated with loss-of-function. CONCLUSION: The clinical-radiographic spectrum produced by CANT1 mutations must be extended to include DD type 2 and Kim variant. While presence or absence of an additional metacarpal ossification centre has been used to distinguish subtypes of DD, this sign is not a distinctive criterion to predict the molecular basis in DD.

Chondroitin sulfate N-acetylgalactosaminyltransferase-1 is required for normal cartilage development.

CS (chondroitin sulfate) is a glycosaminoglycan species that is widely distributed in the extracellular matrix. To understand the physiological roles of enzymes involved in CS synthesis, we produced CSGalNAcT1 (CS N-acetylgalactosaminyltransferase 1)-null mice. CS production was reduced by approximately half in CSGalNAcT1-null mice, and the amount of short-chain CS was also reduced. Moreover, the cartilage of the null mice was significantly smaller than that of wild-type mice. Additionally, type-II collagen fibres in developing cartilage were abnormally aggregated and disarranged in the homozygous mutant mice. These results suggest that CSGalNAcT1 is required for normal CS production in developing cartilage.

Loss-of-function mutations of CHST14 in a new type of Ehlers-Danlos syndrome.

Ehlers-Danlos syndrome (EDS) is a heterogeneous connective tissue disorder involving skin and joint laxity and tissue fragility. A new type of EDS, similar to kyphoscoliosis type but without lysyl hydroxylase deficiency, has been investigated. We have identified a homozygous CHST14 (carbohydrate sulfotransferase 14) mutation in the two familial cases and compound heterozygous mutations in four sporadic cases. CHST14 encodes dermatan 4-O-sulfotransferase 1 (D4ST1), which transfers active sulfate from 3'-phosphoadenosine 5'-phosphosulfate to position 4 of the N-acetyl-D-galactosamine (GalNAc) residues of dermatan sulfate (DS). Transfection experiments of mutants and enzyme assays using fibroblast lysates of patients showed the loss of D4ST1 activity. CHST14 mutations altered the glycosaminoglycan (GAG) components in patients' fibroblasts. Interestingly, DS of decorin proteoglycan, a key regulator of collagen fibril assembly, was completely lost and replaced by chondroitin sulfate (CS) in the patients' fibroblasts, leading to decreased flexibility of GAG chains. The loss of the decorin DS proteoglycan due to CHST14 mutations may preclude proper collagen bundle formation or maintenance of collagen bundles while the sizes and shapes of collagen fibrils are unchanged as observed in the patients' dermal tissues. These findings indicate the important role of decorin DS in the extracellular matrix and a novel pathomechanism in EDS.

A regulatory variant in CCR6 is associated with rheumatoid arthritis susceptibility.

Rheumatoid arthritis is a common autoimmune disease with a complex genetic etiology. Here, through a genome-wide association study of rheumatoid arthritis, we identified a polymorphism in CCR6, the gene encoding chemokine (C-C motif) receptor 6 (a surface marker for Th17 cells) at 6q27, that was associated with rheumatoid arthritis susceptibility and was validated in two independent replication cohorts from Japan (rs3093024, a total of 7,069 individuals with rheumatoid arthritis (cases) and 20,727 controls, overall odds ratio = 1.19, P = 7.7 x 10(-19)). We identified a triallelic dinucleotide polymorphism of CCR6 (CCR6DNP) in strong linkage disequilibrium with rs3093024 that showed effects on gene transcription. The CCR6DNP genotype was correlated with the expression level of CCR6 and was associated with the presence of interleukin-17 (IL-17) in the sera of subjects with rheumatoid arthritis. Moreover, CCR6DNP was associated with susceptibility to Graves' and Crohn's diseases. These results suggest that CCR6 is critically involved in IL-17-driven autoimmunity in human diseases.

New sequence variants in HLA class II/III region associated with susceptibility to knee osteoarthritis identified by genome-wide association study.

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.