Lectin-like oxidized low density lipoprotein receptor 1-deficient mice show resistance to instability-induced osteoarthritis.

OBJECTIVES: The lectin-like oxidized low density lipoprotein (ox-LDL) receptor 1 (LOX-1)/ox-LDL system, which contributes to the pathogenesis of atherosclerosis, may be involved in the development of osteoarthritis (OA). However, the mechanisms by which the LOX-1/ox-LDL system contributes to OA development in vivo are unclear. In this study, we investigated the direct involvement of LOX-1/ox-LDL in OA development by using LOX-1-knockout (LOX-1(-)/(-)) mice in a joint instability-induced model of OA. METHOD: OA development was evaluated with histological scoring at 4 and 8 weeks after surgery to induce knee destabilization in LOX-1(+)/(+) and LOX-1(-)/(-) mice. Immunohistological analysis was used to evaluate the expression of LOX-1, ox-LDL, Runt-related transcription factor 2 (Runx2), and type X collagen (COL X) in articular chondrocytes and osteophyte-forming cells. In addition, double immunofluorescence staining was performed to determine the relationships between LOX-1 and Runx2 or COL X expression. RESULTS: In the model of knee destabilization, symptoms were significantly suppressed in LOX-1(-)/(-) mice. LOX-1, ox-LDL, Runx2, and COL X were overexpressed in articular chondrocytes and osteophyte-forming cells in LOX-1(+)/(+) mice and were significantly downregulated in articular chondrocytes and osteophyte-forming cells in LOX-1(-)/(-) mice compared with those in LOX-1(+)/(+) mice. Double immunostaining indicated that LOX-1 localization coincided with Runx2 and COL X expression. CONCLUSIONS: These data indicate that the LOX-1/ox-LDL system plays a pivotal role in the pathogenesis of instability-induced OA through endochondral ossification. LOX-1-positive chondrocytes and osteophyte-forming cells may be possible targets to prevent disease progression in OA.

Expressions of local renin-angiotensin system components in chondrocytes.

In 2013, we reported that local renin-angiotensin system (local RAS) components express during the hypertrophic differentiation of chondrocytes and can modulate it, using ATDC5 cell line that involves differentiation from mesenchymal stem cells to calcified hypertrophic chondrocytes. However, the expressions of local RAS components in normal chondrocytes have not been revealed yet. The purpose of this study is to examine the expression of the local RAS components in chondrocytes in vivo and the conditions allowing the expression. We stained five major regions of 8-week-old C57BL/6 adult mice in which chondrocytes exist, including epiphyseal plates and hyaline cartilages, with antibodies to local RAS components. We also examined the expression of local RAS components in the cultured bovine's articular cartilage chondrocytes using quantitative reverse transcription polymerase chain reaction and western blot analysis. In result, hypertrophic chondrocytes of epiphyseal plates included in the tibia and the lamina terminals expressed local RAS components. However, hyaline chondrocytes, including the knee articular cartilages, the parenchyma of nasal septums and of the tracheal walls, did not express local RAS components. Cultured bovine's articular cartilage chondrocytes also did not express local RAS components. However, inducing hypertrophy by administering interleukin-1ß or tumor necrosis factor-α, the cultured articular chondrocytes also expressed angiotensin II type 1 receptor and angiotensin II type 2 receptor. In conclusion, local RAS components express particularly in chondrocytes which occur hypertrophy and do not in hyaline chondrocytes. The results are in accord with our previous in vitro study. We think this novel knowledge is important to investigate cartilage hypertrophy and diseases induced by hypertrophic changes like osteoarthritis.

Induction of hypertrophic chondrocyte-like phenotypes by oxidized LDL in cultured bovine articular chondrocytes through increase in oxidative stress.

OBJECTIVE: It has been reported that the lectin-like oxidized low-density lipoprotein (Ox-LDL) receptor 1 (LOX-1) is expressed by chondrocytes in osteoarthritis (OA) cartilage and that Ox-LDL binding to LOX-1 increases intracellular oxidative stress in cultured bovine articular chondrocytes (BACs). It was recently demonstrated that reactive oxygen species (ROS) induce hypertrophic differentiation of chondrocytes in the growth plate. It has also been shown that activated chondrocytes in OA have hypertrophic chondrocyte-like phenotypes. The purpose of this study was to determine whether Ox-LDL induces hypertrophic chondrocyte-like phenotypes in BACs. DESIGN: Changes in type X collagen (COL10) and runt-related transcription factor 2 (Runx2) mRNA expression in BACs after Ox-LDL stimulation were investigated using real-time polymerase chain reaction (PCR). Western blotting and immunofluorescent cell staining were used to investigate changes in protein level. The antioxidant N-acetyl cysteine (NAC) was used to ascertain whether oxidative stress is involved in COL10 and Runx2 expression. We induced LOX-1 knockdown cells using small interfering RNA (siRNA) to examine the receptor specificity of Ox-LDL. RESULTS: COL10 expression was upregulated by Ox-LDL in a time- and dose-dependent manner. Immunofluorescent staining showed that Ox-LDL increased COL10 production in the extracellular matrix. Ox-LDL-induced upregulation of COL10 was suppressed by pretreatment with NAC and siRNA. Expression of Runx2 was upregulated by Ox-LDL and H(2)O(2), and these effects were suppressed by NAC pretreatment. CONCLUSION: Ox-LDL binding to LOX-1 induces a hypertrophic chondrocyte-like phenotype through oxidative stress, indicating that Ox-LDL plays a role in the degeneration of cartilage.

Functional restoration of rabbit spinal cord using collagen-filament scaffold.

We report the first success of functional restoration of transected rabbit spinal cord using collagen-filament nerve scaffold. We grafted 5 mm-long 6000 collagen filaments parallel to the axis of the spinal cord to bridge 3 mm defects of 21 adult rabbit spinal cords; 18 rabbits were used as controls. Of the 39 rabbits, 22 survived the experimental period. At 12 weeks postoperatively, regenerated axons crossed the proximal spinal cord-implant interfaces in four out of six rabbits. At 24 weeks postoperatively, regenerated axons crossed the proximal and distal spinal cord-implant interfaces in four out of six rabbits. At 24 weeks postoperatively, the Basso-Beattie-Bresnahan (BBB) locomotor rating scale scores of the rabbits in the collagen-filament grafted group were 4.7 +/- 2.3, while the score in the control group was 2.8 +/- 0.5. The BBB scale scores of the grafted group were significantly better than the control group. The results suggest that the collagen-filament nerve scaffold supports the axonal regeneration of the transected spinal cord and the restoration of function when grafted parallel to the axis of the spinal cord. The functional restoration appeared to be permanent, raising the possibility of therapeutic application in humans.

Oxidized LDL binding to LOX-1 enhances MCP-1 expression in cultured human articular chondrocytes.

OBJECTIVE: It has been suggested that oxidized low-density lipoprotein (ox-LDL) has some roles in progression of osteoarthritis. The purpose of this study is to investigate whether ox-LDL binding to lectin-like ox-LDL receptor 1 (LOX-1) enhances monocyte chemoattractant protein 1 (MCP-1) expression in cultured human articular chondrocytes (HACs). METHOD: The time course and dose response of MCP-1 mRNA expression and MCP-1 protein release into medium following ox-LDL stimulation were investigated using quantitative Real time PCR (delta-delta Ct method) and enzyme-linked immunosorbent assay (ELISA), respectively. To examine the receptor specificity of ox-LDL action, HACs were preincubated with anti-human LOX-1 monoclonal antibody (TS92). RESULTS: A time-course study revealed that MCP-1 mRNA expression increased 5.09+/-0.86 fold 12h after ox-LDL stimulation compared to time-0. ox-LDL stimulation increased MCP-1 protein level in conditioned medium in a time-dependent manner. Increased MCP-1 level was evident 6h after stimulation, reaching 830+/-91 pg/ml at 24h (33+/-8 pg/ml at time-0). Dose responses of MCP-1 expression were also evident in mRNA and protein levels. Pretreatment with TS92 markedly suppressed these stimulating effects of ox-LDL, although that with non-specific IgG did not. Native LDL did not affect MCP-1 expression. CONCLUSION: Our results suggest that ox-LDL enhances MCP-1 expression in HACs and supports the hypothesis that ox-LDL is involved in cartilage degeneration.

Severe mandibuloacral dysplasia caused by novel compound heterozygous ZMPSTE24 mutations in two Japanese siblings.

Mandibuloacral dysplasia (MAD) is a rare autosomal recessive progeroid syndrome, characterized by mandibular hypoplasia, acroosteolysis affecting distal phalanges and clavicles, delayed closure of the cranial sutures, atrophic skin, and lipodystrophy. Recently, mutations in lamin A/C (LMNA) and zinc metalloprotease (ZMPSTE24), involved in post-translational processing of prelamin A to mature lamin A, have been identified in MAD kindreds. We now report novel compound heterozygous mutations in exon 1 (c.121C>T; p.Q41X) and exon 6 (c.743C>T; p.P248L) in ZMPSTE24 in two Japanese sisters, 7- and 3-year old, with severe MAD and characteristic facies and atrophic skin. The older sister had lipodystrophy affecting the chest and thighs but sparing abdomen. Their parents and a brother, who were healthy, had heterozygous mutations. The missense mutation, P248L, was not found in 100 normal subjects of Japanese origin. The mutant Q41X was inactive in a yeast halo assay; however, the mutant P248L retained near normal ZMPSTE24 activity. Immunoblots demonstrated accumulation of prelamin A in the patients' cell lysates from lymphoblasts. The lymphoblasts from the patients also revealed less intense staining for lamin A/C on immunofluorescence. We conclude that ZMPSTE24 deficiency results in accumulation of farnesylated prelamin A, which may be responsible for cellular toxicity and the MAD phenotype.

Bacterial components regulate the expression of Toll-like receptor 4 on human mast cells.

OBJECTIVES AND DESIGN: The aim of this study was to investigate whether the exposure of mast cells (MCs) to bacterial components affects the expression of Toll-like receptor (TLR) 4, and to elucidate the behavior of MCs during the early response to infection. MATERIALS: Two human MC lines, HMC-1 and LAD2, were employed. Messenger RNA expression was observed by RT and real-time PCR. TLR4 expression was determined by Western blotting. TNF-alpha secretion was analyzed with ELISA. The degranulation ratio was measured with betahexosaminidase assay. RESULTS: Although bacterial components increased TLR4 mRNA, only lipopolysaccharide (LPS) augmented the TLR4 protein expression. LAD2 pre-treated with LPS for 8 h resulted in 2-fold increased TNF-alpha secretion on LPS restimulation. CONCLUSION: These results suggest that the exposure of MCs to LPS may reinforce the innate immune system due to up-regulation of MC TLR4, followed by increased TNF-alpha release.

Possible involvement of the oxidized low-density lipoprotein/lectin-like oxidized low-density lipoprotein receptor-1 system in pathogenesis and progression of human osteoarthritis.

OBJECTIVE: Using human cartilage samples and cultured chondrocytes, to assess the possible involvement of oxidized low-density lipoprotein (ox-LDL) and lectin-like ox-LDL receptor-1 (LOX-1) in pathogenesis and progression of osteoarthritis (OA). METHODS: Thirty-two cartilage samples were obtained from 16 patients with knee OA, and 12 Control samples from six with femoral neck fracture. LOX-1 mRNA expressions in 12 OA and six Control samples were analyzed by reverse transcription-polymerase chain reaction (RT-PCR). Immunohistochemistry for ox-LDL and LOX-1 was performed in all samples. The histological OA grade was assessed with the modified Mankin score. The relative percentage of the ox-LDL and LOX-1 immunopositive chondrocytes was calculated in all samples. The effects of ox-LDL on cell viability in cultured human chondrocytes were investigated by the 3-(4,5-dimethylthiazolyl-2)-2,5-diphenyltetrazolium bromide (MTT) assay and on proteoglycan synthesis by monitoring [35S] sulfate incorporation. RESULTS: There was a statistically significant difference between mean LOX-1/GAPDH (LOX-1/human glyceraldehyde-3-phosphate dehydrogenase) ratio of OA samples and that of Control samples (40.6%+/-10.3 and 11.9%+/-2.8, respectively, P<0.0001). The mean percentage of ox-LDL-positive cells was 23.0+/-15.7% in OA and 4.3+/-3.7% in Control cells (P=0.0002). The mean percentage of LOX-1-positive cells was 51.7+/-29.5% in OA and 10.0+/-8.1% in Control cells (P<0.0001). Both the ox-LDL immunoreactivity and the LOX-1 immunoreactivity were significantly correlated with the modified Mankin scores (R2=0.67 and 0.48, respectively; P<0.0001 for each). ox-LDL significantly reduced the human chondrocyte viability and proteoglycan synthesis, and pretreatment with anti-human LOX-1 monoclonal antibody reversed these effects. CONCLUSION: The ox-LDL/LOX-1 system may be involved in human OA.

ZD6126 inhibits orthotopic growth and peritoneal carcinomatosis in a mouse model of human gastric cancer.

The purpose of this study was to examine the effects of ZD6126, a novel vascular-targeting agent, on tumour growth and angiogenesis in an orthotopic model of gastric cancer. TMK-1 human gastric adenocarcinoma cells were injected into the gastric wall of nude mice. After the tumours were established (day 14), therapy was initiated. Mice (n=11-12/group) received (a). vehicle, (b). ZD6126 at 100 mg x kg day(-1) i.p. one time per week or (c) ZD6126 at 100 mg x kg day(-1) i.p. five times per week. Tumour mass, volume and the presence or absence of peritoneal carcinomatosis were determined at sacrifice on day 38. Tumours from each group were stained for markers of blood vessels, proliferation and apoptosis. To further define the time frame of the vascular-targeting effects of chronic therapy with ZD6126, TMK-1 cells were again injected into the gastric wall of mice in a second experiment. On day 14, a single i.p. injection of ZD6126 100 mg x kg(-1) mouse(-1) or vehicle was delivered. Groups of three mice each were killed and the tumours harvested at days 1, 3 and 5 post-ZD6126 injection. Tumours were processed and stained for endothelial and tumour cell apoptosis and proliferation. No overt toxicity was observed with ZD6126 therapy. ZD6126 led to a marked inhibition of tumour growth (82% decrease vs control (P<0.001)). ZD6126 also led to a significant decrease in the incidence of peritoneal carcinomatosis (10 out of 12 controls, vs one out of 12 ZD6126) (P<0.01). Histological analysis of tumours revealed large regions of central necrosis in the treated group, as well as a dramatic increase in tumour cell apoptosis (7.4-fold increase (P<0.001)), consistent with the vascular-targeting activity of ZD6126. Mice treated with ZD6126 demonstrated a 59% decrease in PCNA-positive cells (P< 0.02), indicating reduced tumour cell proliferation. In addition, tumours treated with ZD6126 exhibited a 40% decrease in microvessel density (P<0.05). Results from mice treated with a single injection of ZD6126 demonstrated the acute effects this agent has on the tumour vasculature. The ratio of endothelial cell apoptosis to endothelial cell proliferation was increased within 24 h of a single injection. In conclusion, ZD6126 significantly inhibited tumour growth and metastasis in an orthotopic model of human gastric adenocarcinoma, without detectable problematic adverse effects. These data suggest that ZD6126 may be worthy of investigation in the treatment of primary gastric adenocarcinoma.

Induction of neuropilin-1 and vascular endothelial growth factor by epidermal growth factor in human gastric cancer cells.

The epidermal growth factor receptor (EGF-R) pathway plays a pivotal role in the progression of human gastric cancer. The angiogenic factor vascular endothelial growth factor (VEGF) has been shown to be induced by EGF in various cancer cell lines. Neuropilin-1 (NRP-1) acts as a coreceptor for VEGF-165 and increases its affinity for VEGF receptor 2 (VEGFR-2) in endothelial cells. Furthermore, NRP-1 has been found to be expressed by tumour cells and has been shown to enhance tumour angiogenesis and growth in preclinical models. We examined the expression of NRP-1 mRNA and EGF-R protein in seven human gastric cancer cell lines. NRP-1 expression was expressed in five of seven cell lines, and EGF-R expression closely mirrored NRP-1 expression. Moreover, in EGF-R-positive NCI-N87 and ST-2 cells, EGF induced both NRP-1 and VEGF mRNA expression. C225, a monoclonal antibody to EGF-R, blocked EGF-induced NRP-1 and VEGF expression in NCI-N87 cells in a dose-dependent manner. The treatment of NCI-N87 cells with EGF resulted in increases in phosphorylation of Erk1/2, Akt, and P38. Blockade of the Erk, phosphatidylinositol-3 kinase/Akt, or P38 pathways in this cell line prevented EGF induction of NRP-1 and VEGF. These results suggest that regulation of NRP-1 expression in human gastric cancer is intimately associated with the EGF/EGF-R system. Activation of EGF-R might contribute to gastric cancer angiogenesis by a mechanism that involves upregulation of VEGF and NRP-1 expression via multiple signalling pathways.

Functional analysis of the effect of the posterior stabilising cam in two total knee replacements. A comparison of the Insall/Burstein and Bisurface prostheses.

The Bisurface knee prosthesis (BP) has a posterior stabilising cam (ball-and-socket joint) in the mid-posterior region of the femorotibial joint in an attempt to improve the range of movement. Based on an in vitro weight-bearing study contact areas of the Insall/Burstein 2 (IB2) and the BP knee were compared using pressure-sensitive films. The stability afforded by the cam was evaluated by means of dislocation distances in the vertical and horizontal planes. Significant adverse anterior translation in mid-flexion was not observed with the BP knee since the cam was effective above 60 degrees of flexion. At flexion of 60 degrees or more, the total contact areas were larger, as the cam represented a weight-bearing surface. The dislocation distances for the BP knee compared favourably with those for the IB2 knee. We conclude that the cam of the BP knee allows good movement, stability and wear.

Effects of combination anti-vascular endothelial growth factor receptor and anti-epidermal growth factor receptor therapies on the growth of gastric cancer in a nude mouse model.

We hypothesised that the combination of anti-angiogenic and anti-epidermal growth factor (EFG)-receptor (R) therapies would more effectively inhibit gastric cancer growth than single-agent therapy. TMK-1 gastric cancer cells were injected into the gastric wall of nude mice to generate tumours. After 4 days, mice were randomly assigned to the following groups: control, DC101 ([vascular endothelial growth factor (VEGF)-receptor (R)-2 antibody], C225 (EGF-R antibody), or a combination of DC101 and C225. The combination therapy significantly inhibited gastric tumour growth compared with the control group, whereas the decrease in tumour growth in mice treated with DC101 or C225 alone did not reach statistical significance. All mice administered DC101 demonstrated decreased tumour vascularity and increased endothelial cell apoptosis. C225 alone did not affect angiogenesis, but inhibited tumour cell proliferation. The combination therapy led to a further decrease in tumour cell proliferation. The combination of anti-VEGF-R and anti-EGF-R therapies was effective in inhibiting gastric cancer growth. These findings support the hypothesis that inhibiting multiple biological pathways that mediate tumour growth may be an effective therapeutic strategy.

Roles of superoxide dismutase in rat mast cell granules.

BACKGROUND: It has been suggested that in the granules of rat mast cells there is some kind of superoxide dismutase (SOD), but details of this SOD in mast cells remain unclear. In the present study, we studied the mode of existence of SOD in mast cells and its releasing mechanism from the granules. In addition, we discussed the physiological role of SOD in allergic events. METHODS: Purified rat mast cells were disrupted with a sonic disrupter and granules (sample I) were separated from supernatant (sample II) by centrifugation. The granules were treated with 1 mM Ca(2+), and the supernatant (sample III) was separated from the pellet (sample IV). Sample III was applied to a heparin column and the eluate was used as sample V. SOD activity was measured in these samples. RESULTS: SOD existed in mast cell granules as a heparin-binding and an inactive form. However, when granules were released and exposed to high Ca(2+) concentration, SOD was discharged from heparin and shifted to the active form. The expression of macrophage inflammatory protein-1 alpha mRNA was enhanced when hydrogen peroxide (H(2)O(2)) or sample III with the xanthine-xanthine oxidase system were added to the culture media. CONCLUSIONS: These findings suggest that in stimulated rat mast cells, the released SOD may transform the generated superoxide anion into H(2)O(2), and the generated H(2)O(2) may enhance the expression of chemokine mRNA in the mast cells.

Inhibitory effect of egualen sodium: a new stable derivative of azulene on histamine release from mast cell-like cells in the stomach.

We studied the inhibitory effect of egualen sodium (ES) (sodium 3-ethyl-7-isopropyl-1-azulenesulfonate 1/3 hydrate, KT1-32), a new derivative and more stable compound than azulene, on histamine release from the mucosal histaminocytes and elucidated the mechanism for this action. ES prevented the histamine release from isolated mast cell-like cells of the guinea pig stomach induced by A23187 in a dose-dependent fashion. ES dose-dependently inhibited the histamine release from lung pieces of sensitized guinea pigs induced by an antigen-antibody reaction. ES also inhibited histamine release from rat peritoneal mast cells induced by compound 48/80 or antigen-antibody reaction. ES exhibited the membrane stabilizing activity on DPPC liposomes. These findings suggest that ES may prevent histamine release from histaminocytes induced by various stimuli and the stabilizing action of the cell membrane may be responsible for the inhibition of histamine release.

Perception of lateral misarticulation and its physical correlates.

In order to clarify the relationship between perceptual diagnosis of lateral misarticulation (LM) by sophisticated listeners and the physical correlates of LM, three experiments using sustained speech /integral/ were conducted. Experiment 1 was designed to compare the spectral envelopes of normal speech (NS) /integral/ with those of LM /integral/. Experiment 2 was designed to collect the auditory impressions of sophisticated listeners listening to LM and NS /integral/ with specific spectral envelope bands replaced by LM. These two experiments showed that: (1) the spectral envelopes of LM are flat or decrease along the frequency axis in the frequency band above approximately 4 kHz, and there is a substantial peak at around 3.2 kHz in LM, which varies peculiarly with time; (2) the replacement of the spectral envelope between 2.5 and 4.5 kHz of NS with that of LM resulted in a remarkable increase in auditory impressions of LM. The facts suggest that the spectral envelope characteristic of LM has a peculiar variation at around 3.2 kHz. Additionally, experiment 3 estimated the spectrum of sustained speech /integral/ using vocal tract area functions. The results suggest that typical peaks of LM are related to the length and position of the vocal tract constriction region.

In vivo three-dimensional knee kinematics using a biplanar image-matching technique.

A biplanar image-matching technique was developed and applied to a study of normal knee kinematics in vivo under weightbearing conditions. Three-dimensional knee models of six volunteers were constructed using computed tomography. Projection images of the models were fitted onto anteroposterior and lateral radiographs of the knees at hyperextension and every 15 degrees from 0 degrees to 120 degrees flexion. Knee motion was reconstructed on the computer. The femur showed a medial pivoting motion relative to the tibia during knee flexion, and the average range of external rotation associated with flexion was 29.1 degrees . The center of the medial femoral condyle translated 3.8 mm anteriorly, whereas the center of the lateral femoral condyle translated 17.8 mm posteriorly. This rotational motion, with a medially offset center, could be interpreted as a screw home motion of the knee around the tibial knee axis and a posterior femoral rollback in the sagittal plane. However, the motion of the contact point differed from that of the center of the femoral condyle when the knee flexion angle was less than 30 degrees. Within this range, medial and lateral contact points translated posteriorly, and a posterior femoral rollback occurred. This biplanar image-matching technique is useful for investigating knee kinematics in vivo.

Relationship between frontal knee alignment and reference axes in the distal femur.

The two transepicondylar axes (the clinical and surgical epicondylar axes), the posterior condylar axis, and the anteroposterior axis were constructed using computed tomography scans in 111 (66 patients) knees with symptomatic arthritis. The relationships between angles made by these reference axes and two angles indicating frontal knee alignment (the tibiofemoral valgus angle and the femoral valgus angle) were investigated. In Y of the knees, the surgical epicondylar axis could not be constructed because the sulcus of the medial epicondyle was not recognizable. The condylar twist angle was almost constant and averaged 6 degrees when the femoral valgus angle was 9 degrees or less, but increased gradually when the angle was greater than 9 degrees. The difference between the condylar twist angle and the posterior condylar angle was constantly 3 degrees. The anteroposterior axis was almost at right angles to the clinical epicondylar axis, and the relationship between these axes was constant, independent of the femoral valgus angle. With 3 degrees to 6 degrees external rotation relative to the posterior condylar axis, the femoral component could be set parallel to the transepicondylar axis in common varus or neutral knees. In cases with a larger femoral valgus angle, the anteroposterior axis would be a more reliable reference axis. Preoperative computed tomography scans are recommended for patients with knees with severe valgus deformity or severe hypertrophic osteoarthritis.

Mutational analysis of MECP2 in Japanese patients with atypical Rett syndrome.

Rett syndrome (RTT) is one of the most common neurodevelopmental disorders in females. Recently, this disease was found to be linked with mutations in the methyl-CpG-binding protein 2 gene (MECP2) and various mutations have been reported. To explore the spectrum of phenotypes resulting from MECP2 mutations, we searched for mutations in the MECP2 of 20 Japanese patients who had more than five of the criteria necessary for RTT diagnosis proposed in 1988 (The Rett Syndrome Diagnostic Criteria Work Group, Ann Neurol 23 (1988) 425) and compared the phenotype between patients with and without mutation by giving a score to each diagnostic criterion. We found four missense mutations (T158M, R133C, Y120D, and R306C), two nonsense mutations (R168X and R270X), one frameshift (726delAAAG) mutation, and one polymorphism (A201V) in ten patients (50%). This included two novel mutations (726delAAAG and Y120D). All mutations were found in the highly conserved methyl-binding and transcription repression domains. Comparison of the mean total diagnostic criterion score of the groups with and without mutation did not reveal any statistically significantly difference (P=0.28). The only difference between the groups, which was of borderline significance (P=0.051), was the sum of the scores for diagnostic criteria 2 (apparently normal psychomotor development through the first 6 months) and 5 (loss of acquired purposeful hand skills between the ages of 6 and 30 months). From these results, it is suggested that the clinical phenotype of RTT is variable and it is important to investigate the MECP2 genotype for patients having more than five criteria and not only in those who exhibit all RTT diagnostic criteria. The diagnosis of RTT is clinically difficult before 3 years of age, especially in atypical cases, but molecular analysis of the MECP2 will assist diagnosis in some patients.