Akihiko Mabuchi

An intronic SNP in a RUNX1 binding site of SLC22A4 , encoding an organic cation transporter, is associated with rheumatoid arthritis

Rheumatoid arthritis is a common inflammatory disease with complex genetic components. We investigated the genetic contribution of the cytokine gene cluster in chromosome 5q31 to susceptibility to rheumatoid arthritis in the Japanese population by case-control linkage disequilibrium (LD) mapping using single nucleotide polymorphisms (SNPs). Here we report that there is significant association between rheumatoid arthritis and the organic cation transporter gene SLC22A4 (P = 0.000034). We show that expression of SLC22A4 is specific to hematological and immunological tissues and that SLC22A4 is also highly expressed in the inflammatory joints of mice with collagen-induced arthritis. A SNP affects the transcriptional efficiency of SLC22A4 in vitro, owing to an allelic difference in affinity to Runt-related transcription factor 1 (RUNX1), a transcriptional regulator in the hematopoietic system. A SNP in RUNX1 is also strongly associated with rheumatoid arthritis (P = 0.00035). Our data indicate that the regulation of SLC22A4 expression by RUNX1 is associated with susceptibility to rheumatoid arthritis, which may represent an example of an epistatic effect of two genes on this disorder.

An aspartic acid repeat polymorphism in asporin inhibits chondrogenesis and increases susceptibility to osteoarthritis.

Osteoarthritis is the most common form of human arthritis. We investigated the potential role of asporin, an extracellular matrix component expressed abundantly in the articular cartilage of individuals with osteoarthritis, in the pathogenesis of osteoarthritis. Here we report a significant association between a polymorphism in the aspartic acid (D) repeat of the gene encoding asporin (ASPN) and osteoarthritis. In two independent populations of individuals with knee osteoarthritis, the D14 allele of ASPN is over-represented relative to the common D13 allele, and its frequency increases with disease severity. The D14 allele is also over-represented in individuals with hip osteoarthritis. Asporin suppresses TGF-β–mediated expression of the genes aggrecan (AGC1) and type II collagen (COL2A1) and reduced proteoglycan accumulation in an in vitro model of chondrogenesis. The effect on TGF-β activity is allele-specific, with the D14 allele resulting in greater inhibition than other alleles. In vitro binding assays showed a direct interaction between asporin and TGF-β. Taken together, these findings provide another functional link between extracellular matrix proteins, TGF-β activity and disease, suggesting new therapeutic strategies for osteoarthritis.

A functional polymorphism in the 5′ UTR of GDF5 is associated with susceptibility to osteoarthritis

Osteoarthritis (MIM 165720), characterized by degeneration of articular cartilage, is the most common form of human arthritis and a major concern for aging societies worldwide. Epidemiological and genetic studies have shown that osteoarthritis is a polygenic disease. Here, we report that the gene encoding growth differentiation factor 5 (GDF5) is associated with osteoarthritis in Asian populations. A SNP in the 5′ UTR of GDF5 (+104T/C; rs143383) showed significant association (P = 1.8 × 10−13) with hip osteoarthritis in two independent Japanese populations. This association was replicated for knee osteoarthritis in Japanese (P = 0.0021) and Han Chinese (P = 0.00028) populations. This SNP, located in the GDF5 core promoter, exerts allelic differences on transcriptional activity in chondrogenic cells, with the susceptibility allele showing reduced activity. Our findings implicate GDF5 as a susceptibility gene for osteoarthritis and suggest that decreased GDF5 expression is involved in the pathogenesis of osteoarthritis.

Transcriptional regulation of endochondral ossification by HIF-2α during skeletal growth and osteoarthritis development

Chondrocyte hypertrophy followed by cartilage matrix degradation and vascular invasion, characterized by expression of type X collagen (COL10A1), matrix metalloproteinase-13 (MMP-13) and vascular endothelial growth factor (VEGF), respectively, are central steps of endochondral ossification during normal skeletal growth and osteoarthritis development. A COL10A1 promoter assay identified hypoxia-inducible factor-2α (HIF-2α, encoded by EPAS1) as the most potent transactivator of COL10A1. HIF-2α enhanced promoter activities of COL10A1, MMP13 and VEGFA through specific binding to the respective hypoxia-responsive elements. HIF-2α, independently of oxygen-dependent hydroxylation, was essential for endochondral ossification of cultured chondrocytes and embryonic skeletal growth in mice. HIF-2α expression was higher in osteoarthritic cartilages versus nondiseased cartilages of mice and humans. Epas1-heterozygous deficient mice showed resistance to osteoarthritis development, and a functional single nucleotide polymorphism (SNP) in the human EPAS1 gene was associated with knee osteoarthritis in a Japanese population. The EPAS1 promoter assay identified RELA, a nuclear factor-κB (NF-κB) family member, as a potent inducer of HIF-2α expression. Hence, HIF-2α is a central transactivator that targets several crucial genes for endochondral ossification and may represent a therapeutic target for osteoarthritis.

Prevalence of radiographic knee osteoarthritis and its association with knee pain in the elderly of Japanese population-based cohorts: The ROAD study

OBJECTIVE We investigated the prevalence of radiographic knee osteoarthritis (OA) and knee pain in the Japanese elderly using a large-scale population of a nationwide cohort study, Research on Osteoarthritis Against Disability (ROAD), and examined their association. METHODS From the baseline survey of the ROAD study, 2,282 participants > or =60 years (817 men and 1,465 women) living in urban, mountainous and seacoast communities were analyzed. The radiographic severity at both knees was determined by the Kellgren/Lawrence (KL) grading system. KL> or =2 and KL> or =3 knee OA were examined separately to assess osteophytosis and joint space narrowing (JSN). RESULTS The prevalence of KL> or =2 OA (47.0% and 70.2% in men and women, respectively) was much higher than that of previous studies in Caucasians, while that of KL> or =3 OA was not much different in men. Age, BMI, female sex and rural residency were risk factors for radiographic knee OA, knee pain and their combination. The prevalence of knee pain was age-dependent in women, but not in men. Knee pain was more strongly associated with KL> or =3 OA than with KL=2, and the association was higher in men than in women. Female sex was a strong risk factor even in the subgroup without radiographic knee OA (KL=0/1). CONCLUSION The present cross-sectional study revealed a high prevalence of radiographic knee OA in the Japanese elderly. Knee pain was strongly associated with JSN especially in men, while women tended to have knee pain even without radiographic OA.

Meta-analysis of genome-wide association studies confirms a susceptibility locus for knee osteoarthritis on chromosome 7q22

Objectives Osteoarthritis (OA) is the most prevalent form of arthritis and accounts for substantial morbidity and disability, particularly in older people. It is characterised by changes in joint structure, including degeneration of the articular cartilage, and its aetiology is multifactorial with a strong postulated genetic component. Methods A meta-analysis was performed of four genome-wide association (GWA) studies of 2371 cases of knee OA and 35 909 controls in Caucasian populations. Replication of the top hits was attempted with data from 10 additional replication datasets. Results With a cumulative sample size of 6709 cases and 44 439 controls, one genome-wide significant locus was identified on chromosome 7q22 for knee OA (rs4730250, p=9.2×10−9), thereby confirming its role as a susceptibility locus for OA. Conclusion The associated signal is located within a large (500 kb) linkage disequilibrium block that contains six genes: PRKAR2B (protein kinase, cAMP-dependent, regulatory, type II, β), HPB1 (HMG-box transcription factor 1), COG5 (component of oligomeric golgi complex 5), GPR22 (G protein-coupled receptor 22), DUS4L (dihydrouridine synthase 4-like) and BCAP29 (B cell receptor-associated protein 29). Gene expression analyses of the (six) genes in primary cells derived from different joint tissues confirmed expression of all the genes in the joint environment.

C/EBPβ and RUNX2 cooperate to degrade cartilage with MMP-13 as the target and HIF-2α as the inducer in chondrocytes

To elucidate the molecular mechanism underlying the endochondral ossification process during the skeletal growth and osteoarthritis (OA) development, we examined the signal network around CCAAT/enhancer-binding protein-β (C/EBPβ, encoded by CEBPB), a potent regulator of this process. Computational predictions and a C/EBP motif-reporter assay identified RUNX2 as the most potent transcriptional partner of C/EBPβ in chondrocytes. C/EBPβ and RUNX2 were induced and co-localized in highly differentiated chondrocytes during the skeletal growth and OA development of mice and humans. The compound knockout of Cebpb and Runx2 in mice caused growth retardation and resistance to OA with decreases in cartilage degradation and matrix metalloproteinase-13 (Mmp-13) expression. C/EBPβ and RUNX2 cooperatively enhanced promoter activity of MMP13 through specific binding to a C/EBP-binding motif and an osteoblast-specific cis-acting element 2 motif as a protein complex. Human genetic studies failed to show the association of human CEBPB gene polymorphisms with knee OA, nor was there a genetic variation around the identified responsive region in the human MMP13 promoter. However, hypoxia-inducible factor-2α (HIF-2α), a functional and genetic regulator of knee OA through promoting endochondral ossification, was identified as a potent and functional inducer of C/EBPβ expression in chondrocytes by the CEBPB promoter assay. Hence, C/EBPβ and RUNX2, with MMP-13 as the target and HIF-2α as the inducer, control cartilage degradation. This molecular network in chondrocytes may represent a therapeutic target for OA.

Prevalence of radiographic lumbar spondylosis and its association with low back pain in elderly subjects of population-based cohorts: the ROAD study

Objectives: Although lumbar spondylosis is a major cause of low back pain and disability in elderly people, few epidemiological studies have been performed. The prevalence of radiographic lumbar spondylosis was investigated in a large-scale population study and the association with low back pain was examined. Methods: From a nationwide cohort study (Research on Osteoarthritis Against Disability; ROAD), 2288 participants aged ⩾60 years (818 men and 1470 women) living in urban, mountainous and coastal communities were analysed. The radiographic severity at lumbar intervertebral levels from L1/2 to L5/S was determined by Kellgren/Lawrence (KL) grading. Results: In the overall population the prevalence of radiographic spondylosis with KL⩾2 and ⩾3 at the severest intervertebral level was 75.8% and 50.4%, respectively, and that of low back pain was 28.8%. Although KL⩾2 spondylosis was more prevalent in men, KL⩾3 spondylosis and low back pain were more prevalent in women. Age and body mass index were risk factors for both KL ⩾2 and KL⩾3 spondylosis. Although KL = 2 spondylosis was not significantly associated with low back pain compared with KL = 0 or 1, KL⩾3 spondylosis was related to the pain only in women. Conclusions: This cross-sectional study in a large population revealed a high prevalence of radiographic lumbar spondylosis in elderly subjects. Gender seems to be distinctly associated with KL⩾2 and KL⩾3 lumbar spondylosis, and disc space narrowing with or without osteophytosis in women may be a risk factor for low back pain.

Novel SBDS mutations caused by gene conversion in Japanese patients with Shwachman-Diamond syndrome.

Shwachman-Diamond syndrome (SDS; OMIM 260400) is an autosomal recessive disorder characterized by exocrine pancreatic insufficiency, bone marrow dysfunction and metaphyseal chondrodysplasia. SDS is caused by mutations in SBDS, an uncharacterized gene. A previous study in SDS patients largely of European ancestry found that most SBDS mutations occurred within a ~240-bp region of exon 2 and resulted from gene conversion due to recombination with a pseudogene, SBDSP. It is unknown, however, whether these findings are applicable to other ethnic groups. To address this question, we examined SBDS mutations in six Japanese families with SDS by direct sequencing. We identified compound heterozygous mutations in four families: two were recurrent (96–97insA, 258+2T>C), and three were novel [292–295delAAAG, (183–184TA>CT +201A>G), (141C>T+183–184TA>CT+201A>G)] mutations. Most of these mutations also appear to result from gene conversion, but the conversion events occurred at various sites between intron 1 and exon 3. Thus, gene conversion mutations in SBDS are common to different ethnic groups, but they are not confined to a limited region of the gene.

Nucleotide Pyrophosphatase Gene Polymorphism Associated With Ossification of the Posterior Longitudinal Ligament of the Spine

Ossification of the posterior longitudinal ligament (OPLL) of the spine is a disease that causes paralysis by compressing the spinal cord. Based on the fact that the nucleotide pyrophosphatase (Npps) gene is responsible for ectopic ossification in ttw, an OPLL model mouse, the possibility was explored whether the human NPPS gene is associated with susceptibility to and severity of OPLL. First, we screened for single‐nucleotide polymorphisms (SNPs) in the human NPPS locus using selected 25 OPLL patients with young onset (<35 years old) or severe ossification (>10 ossified vertebrae), and identified three novel SNPs in the locus. A case‐control association study between 180 OPLL patients and 265 non‐OPLL controls showed that one of these SNPs, IVS15‐14T → C substitution, was more frequently observed in OPLL patients (p = 0.022), especially in those with severe ossification (p < 0.0001) and young onset (p = 0.002), than in controls. A stratified study with the number of ossified vertebrae in OPLL patients revealed that IVS15‐14T → C substitution (p = 0.013) as well as young onset (p = 0.046) and female sex (p = 0.006) were associated with severe ossification. We conclude that the IVS15‐14T → C substitution in the human NPPS gene is associated not only with susceptibility to, but also with severity of OPLL.