[Automation of Damage Detection and Damage Area Measurement of X-ray Protective Clothing Using Deep Learning].

PURPOSE: Damage to shielding sheets on X-ray protective clothing may be a cause of increased radiation exposure. To prevent increased radiation exposure, periodic quality control of shielding sheets is needed. For quality management, a record of the size of damage is required after checking for the existence of damage, and this requires a great deal of effort and time. Additionally, the detection model created from the images of the shielding sheets, limited by the number of samples, is predicted to have a low detection precision. The purpose of this study was to automate damage area detection and area measurement using artificial damage images and a damage detection model created using deep learning. METHOD: By synthesizing the X-ray protective clothing CT localizer image and the image simulating damage, we created an artificial damage image. We then found the detection precision of the damage detection model created by the artificial damage image and YOLOv5s, and error of the automatically measured damage area. RESULT: The accuracy rate of the damage detection model was 0.746, the precision was 0.645, the reproduction rate was 0.741, the F value was 0.690, and 48 mm2 or the detectable area of damage ranged from 2 mm2 to 113 mm2. The mean value of the damage area error was 7.58% for areas not including the hem and 43.39% for areas including the hem. In the areas not including the hem, with a detected damage area of 91%, the damage area error was 0%. Additionally, the process from damage area detection to damage area measurement was completed in 20 seconds. CONCLUSION: By using a damage detection model created with only artificial damage areas, it was possible to automate damage detection and damage area measurement, and this saved time for X-ray protective clothing management.

Impact of nonsynonymous mutations of factor X on the functions of factor X and anticoagulant activity of edoxaban.

Edoxaban is an oral direct factor Xa (FXa) inhibitor and its efficacy as an oral anticoagulant is less subject to drug-food and drug-drug interaction than existing vitamin K antagonists. Although this profile of edoxaban suggests it is well suited for clinical use, it is not clear whether genetic variations of factor X influence the activity of edoxaban. Our aim was to investigate a possible impact of single-nucleotide polymorphisms (SNPs) in the factor X gene on the functions of factor X and the activity of edoxaban. Two nonsynonymous SNPs within mature factor X, Ala152Thr and Gly192Arg, were selected as possible candidates that might affect the functions of FXa and the activity of edoxaban. We measured catalytic activities of wild type and mutant FXas in a chromogenic assay using S-2222 and coagulation times including prothrombin time (PT) and activated partial thrombin time (aPTT) of plasma-containing recombinant FXs in the presence and absence of edoxaban. Michaelis-Menten kinetic parameters of FXas, Km and Vmax values, PT and aPTT were not influenced by either mutation indicating these mutations do not affect the FXa catalytic and coagulation activities. The Ki values of edoxaban for the FXas and the concentrations of edoxaban required to double PT and aPTT were not different between wild type and mutated FXas indicating that both mutations have little impact on the activity of edoxaban. In conclusion, these data suggest that edoxaban has little interpatient variability stemming from SNPs in the factor X gene.

Effect of lime pretreatment of brown midrib sorghums.

The effect of lime pretreatment of brown midrib sorghums on enzymatic saccharification was investigated. Under most of the pretreatment conditions, the saccharification yields of bmrs were higher than those of the normal counterparts. This result suggests that bmr is useful to reduce pretreatment costs, because the amount of lime necessary for the pretreatment of biomass can reduced by using bmr mutants.

Alpha 2A adrenergic receptor polymorphism is associated with plasma von Willebrand factor levels in a general population.

High levels of plasma von Willebrand factor are a proposed risk factor for atherothrombotic disorders. Previously, ABO blood type and the von Willebrand factor -1793C/G polymorphism were shown to affect interindividual variations in plasma von Willebrand factor levels. We previously reported that polymorphisms of the alpha 2A adrenergic receptor, an epinephrine receptor, were associated with platelet function as assessed by platelet function analyzer-100. The measurement value of platelet function analyzer-100 has been reportedly associated with plasma von Willebrand factor levels. Also, it was demonstrated that epinephrine administration increases plasma von Willebrand factor levels in vivo. Thus, the present study investigated the association between plasma von Willebrand factor levels and genetic polymorphisms as follows: ABO blood type, von Willebrand factor -1051G/A (linked with -1793C/G), alpha 2A adrenergic receptor 1780A/G, and alpha 2A adrenergic receptor 2372A/G. Study subjects were genetically unrelated Japanese men (n = 277) recruited at their regular medical examinations. Genotyping of the polymorphisms was performed using the single nucleotide primer extension-based method. Plasma von Willebrand factor levels were measured as ristocetin-cofactor activities. The O blood type and alpha 2A adrenergic receptor 2372AA genotype were significantly associated with lower von Willebrand factor levels, though von Willebrand factor -1051G/A polymorphism did not affect them. In stratification analysis of the group according to blood type O and non-O, the significant association between the 2372AA genotype and lower plasma von Willebrand factor levels was observed in non-O subjects, but not O subjects. In conclusion, the alpha 2A adrenergic receptor 2372A/G polymorphism is associated with plasma von Willebrand factor levels in a general population.

Maintenance therapy involving a tapering dose of danazol or mid/low doses of oral contraceptive after gonadotropin-releasing hormone agonist treatment for endometriosis-associated pelvic pain.

Pelvic pain associated with endometriosis was treated by the long-term administration of a tapering dose of danazol or mid/low doses of oral contraceptives after the end of therapy involving a GnRH agonist (GnRH-a). Results demonstrated that each of these three therapies is a practical and efficient treatment regimen to maintain the relief of pelvic pain achieved by GnRH-a therapy, at least for a period of 12 months.

Platelet responsiveness to in vitro aspirin is independent of COX-1 and COX-2 protein levels and polymorphisms.

Aspirin's inhibitory effect on platelet function has been shown to be highly heterogeneous. However, due to the considerable individual variation in pharmacokinetics after aspirin intake, it has been difficult to investigate the mechanism of aspirin resistance empirically. Our objective was to examine whether platelet responsiveness to in vitro aspirin treatment could be affected by cyclooxygenase (COX)-1/2 protein levels in platelets or single-nucleotide polymorphisms (SNPs), which could possibly change specific activity of enzymes and/or aspirin susceptibility. Collagen/epinephrine closure time (CEPI-CT) of PFA-100 in blood from 178 healthy males was assessed with/without aspirin. Platelet COX-1 protein levels and the sequences of COX-1 gene exons were examined in three groups categorized by CEPI-CT: PR (Poor responders to aspirin), 10 people showing the shortest CEPI-CT under aspirin; GR-High or GR-Low (good responders to aspirin with high or low platelet basal reactivity), 10 people showing CEPI-CT over 300 s under aspirin and having the shortest or longest basal CEPI-CT, respectively. We analyzed the three groups, representing phenotypic extremes, aiming to increase statistical power to investigate the possible relevance of COXs to platelet response to aspirin. Western blot analysis revealed that COX-1 was abundantly expressed in platelets at comparable levels among the three groups, whereas COX-2 was undetectable. The frequencies of nonsynonymous COX-1/2 SNPs were unlikely to explain the difference in aspirin responsiveness considering the observed genotype frequencies and wide individual variation in platelet response. These results suggest that heterogeneity in platelet responsiveness to in vitro aspirin is independent of COX-1/2 protein levels and SNPs.

Association of killer cell immunoglobulin-like receptor genotypes with susceptibility to endometriosis.

PROBLEM: Endometriosis is an immune-related chronic inflammatory disease with a polygenic predisposition. The aim of this study was to investigate whether polymorphisms in killer cell immunoglobulin-like receptors (KIRs) is responsible, in part, for genetic susceptibility to endometriosis. METHOD OF STUDY: The KIRs genotype was determined in 186 patients with endometriosis and 165 control women using polymerase chain reaction with sequence-specific primers. RESULTS: The frequency of KIR3DS1 was significantly decreased in patients compared with controls (32% versus 44%, P=0.028). KIR data were analyzed using a model comprised of three large groups, in which a gradient of activation/inhibitory potential derived from the combination of KIR and human leukocyte antigen (HLA) ligand genes was taken into account. The frequency of inhibitory KIRs/HLA-class I combination genotypes was significantly higher in patients than in controls (chi2=6.010, 2 df, P=0.0496). CONCLUSION: Our results suggest that polymorphism in KIRs may be associated with susceptibility for endometriosis.

Increased basal platelet activity, plasma adiponectin levels, and diabetes mellitus are associated with poor platelet responsiveness to in vitro effect of aspirin.

INTRODUCTION: Aspirin is one of the most effective antiplatelet agents and is now commonly used to prevent vascular events. In some patients, however, recurrent vascular events have been demonstrated despite aspirin therapy. Our objective was to characterize individuals showing poor response to in vitro effect of aspirin, using PFA-100. METHODS: One hundred sixty-eight healthy male subjects were analyzed. We assessed platelet function tests, including PFA-100, whole blood aggregation, and optical platelet aggregation. Also measured were hemostatic and other parameters including von Willebrand factor (VWF:Ag), VWF ristocetin cofactor activity (VWF:RCo), soluble vascular adhesion molecule-1 (sVCAM-1), high sensitive C-reactive protein (hs-CRP), and adiponectin. Poor responders were defined as having a collagen/epinephrine-induced closure time (CEPI-CT) under 250 s with PFA-100 when incubated with 10 microM aspirin, whereas good responders were defined as having a CEPI-CT of more than 250 s. RESULTS AND CONCLUSIONS: PFA-100 tests revealed that 40 subjects (24%) were poor responders (PR) and 128 (76%) were good responders (GR). Poor responsiveness was significantly associated with (1) higher basal platelet activities in PFA-100, as well as in whole blood aggregation and aggregometer;(2) increased level of adiponectin (8.8+/-4.1 micro g/mL [PR] vs 7.3+/-2.9 micro g/mL [GR], p=0.010);and (3) the presence of diabetes mellitus (17.5% [PR] vs 4.7% [GR], p=0.009). Importantly, whereas 24% of the subjects showed insufficient inhibition in PFA-100 when incubated with 10 microM aspirin, almost all subjects showed maximum inhibition with 30 microM aspirin. These observations suggest that higher doses of aspirin might overcome aspirin resistance.

Differential effects of progestogens, by type and regimen, on estrogen-metabolizing enzymes in human breast cancer cells.

OBJECTIVES: To investigate the in vitro effects of five progestogens commonly used in hormone replacement therapy (HRT) on estrogen-metabolizing enzymes in human breast cancer cells. METHODS: The human hormone-dependent breast cancer cell lines T47D, MCF-7, and MCF-7aro were cultured with estradiol (E(2)) and progestogens. The mRNA levels of estrogen-metabolizing enzymes were determined by RT-PCR or Northern blot, and enzyme activities by radiolabeled substrates. Cell proliferation was measured by bromodeoxyuridine incorporation. In vitro models for continuous combined regimen (CCR) and sequential combined regimen (SCR) were established to mimic the in vivo conditions of HRT. RESULTS: Medroxyprogesterone acetate (MPA) plus E(2) (10(-8)M) stimulated the mRNA levels and activities of estrogen-activating enzymes aromatase (at 10(-8)M MPA), 17beta-hydroxysteroid dehydrogenase type 1 (17betaHSD1) (at 10(-6)M), and sulfatase (at 10(-8) to 10(-6)M) compared to E(2) only. Progesterone also stimulated enzyme activity, but to a lower magnitude. Levonorgestrel, norethindrone, and dienogest showed no enzyme stimulation. The estrogen-inactivating enzymes 17beta-hydroxysteroid dehydrogenase type 2 and sulfotransferase were not affected by any of the progestogens tested. However, all the progestogens (at 10(-8) to 10(-6)M) inhibited E(2)-stimulated cell proliferation. While increased aromatase and 17betaHSD1 activities were observed in the CCR model, no significant enzyme stimulation was observed in the SCR model. CONCLUSIONS: The present study suggested that progestogens exert different actions on estrogen-metabolizing enzymes in breast cancer cells dependent on the specific progestogen and regimen used. Further studies are needed to elucidate whether MPA, a progestogen currently used in HRT, leads to a higher risk of breast cancer development than other progestogens.

Synergistic effect of interleukin-6 promoter (IL6 -634C/G) and intercellular adhesion molecule-1 (ICAM-1 469K/E) gene polymorphisms on the risk of endometriosis in Japanese women.

PROBLEM: Endometriosis is an immune-related, chronic inflammatory disease with a polygenic predisposition. The aim of this study was to investigate whether the interleukin-6 (IL-6) gene promoter region polymorphism (-634C/G) and the intercellular adhesion molecule-1 (ICAM-1) gene 469K/E polymorphism are responsible in part for the genetic susceptibility to endometriosis. METHODS OF STUDY: The IL-6 -634C/G and ICAM-1 469K/E genotypes were determined in 202 patients with endometriosis and 236 control women by polymerase chain reaction-restriction fragment length polymorphism. RESULTS: There were no differences in the IL-6 -634C/G or the ICAM-1 469K/E genotypes and allele frequencies between control women and endometriosis patients collectively, or between control women and each clinical subgroup of endometriosis patients. Interestingly, the frequency of ICAM-1 EE homozygotes who concomitantly carried the IL-6 -634G allele was significantly higher in patients with endometriosis (chi(2) = 6.458, P = 0.0396, d.f. = 2). CONCLUSION: Our results suggest that the IL-6 -634C/G and ICAM-1 469K/E polymorphisms synergistically affect the susceptibility for endometriosis in the Japanese population.

Identification of ADRA2A polymorphisms related to shear-mediated platelet function.

alpha2A adrenergic receptor (ADRA2A) on platelets interacts with epinephrine, which has a key role in regulating platelet functions. There is familial clustering of inter-individual variations in the epinephrine-induced platelet aggregation, the molecular basis of which, however, has not been fully understood. In this study, we screened the sequence variations in the transcriptional region of ADRA2A gene and analyzed the relationship between the two common polymorphisms and platelet function using epinephrine/collagen cartridge in the platelet function analyzer-100 system, in a healthy Japanese male population (n=211). Among the identified 16 sequence variations including five novel variations, 1780GG genotype was associated with longer closure time which represents low platelet function under high shear-stress conditions (p=0.0478). We also observed enhanced effect of the combination of 1780GG and 2372AA genotypes on longer closure time (p=0.0319). These findings suggest that 1780A/G and 2372A/G polymorphisms are associated with platelet function in interactions with collagen/epinephrine.

Association of two polymorphisms in the peroxisome proliferator-activated receptor-gamma gene with adenomyosis, endometriosis, and leiomyomata in Japanese women.

OBJECTIVE: The peroxisome proliferator-activated receptor-gamma (PPAR-gamma) is a nuclear hormone receptor that plays an important role in many diseases. This study investigated whether two polymorphisms (Pro12Ala in exon B and C161T in exon 6) of the PPAR-gamma2 gene are related to adenomyosis, endometriosis, or leiomyomata. METHODS: A total of 390 patients with adenomyosis, endometriosis, and/or leiomyomata were classified into four groups: 103 patients with adenomyosis (21 adenomyosis only and 82 adenomyosis with endometriosis and/or leiomyomata), 95 patients with endometriosis only, 100 patients with leiomyomata only, and 92 patients with endometriosis and leiomyomata. RESULTS: There was no association between distribution of genotype or allele frequencies for the PPAR-gamma Pro12Ala polymorphism and the presence of adenomyosis, endometriosis, and/or leiomyomata. However, compared with results for controls, the PPAR-gamma 161CC genotype and 161C allele frequencies were significantly increased in patients with adenomyosis (genotype: chi2 = 8.185, corrected P value [Pc] = .0169; allele: chi2 = 8.337, Pc = .0155) and in patients with endometriosis (genotype: chi2 = 6.748, Pc = .0375; allele: chi2 = 6.413, Pc = .0453). CONCLUSION: The results suggest that the PPAR-gamma 161CC genotype could be a genetic risk factor for adenomyosis and endometriosis, whereas the Pro12Ala polymorphism was not associated with these estrogen-dependent benign uterine diseases in a Japanese population.

Usefulness and limits of CA-125 in diagnosis of endometriosis without associated ovarian endometriomas.

BACKGROUND: The aim of this study was to evaluate the diagnostic significance of CA-125 for endometriosis without ovarian endometriomas. METHODS: Preoperative serum CA-125 levels were measured in 775 consecutive women diagnosed by laparoscopy or laparotomy with endometriosis, adenomyosis, leiomyomas, or normal pelvis. RESULTS: Receiver operating characteristic curve analysis revealed that the area under the curve for endometriosis without endometriomas was 0.788, significantly smaller than that for endometriosis with endometriomas (0.935, P < 0.05). In diagnosis of endometriosis without endometriomas, both the maximal accuracy of 78.8% and the maximal diagnostic value of 61.2% were obtained at the cutoff value of 20 U/mL. Negative predictive value was 78.0% at the cutoff value of 20 U/mL, whereas positive predictive value was 92.9% at the cutoff value of 30 U/mL. This range is clearly superior to the empirical single cutoff of 35 U/mL. CONCLUSIONS: In the diagnosis of endometriosis without endometriomas, combined use of two cutoff values for CA-125, 20 and 30 U/mL, provides improved diagnostic performance. However, the accuracy of using only CA-125 testing for diagnosis is still limited. Serum CA-125 testing can be done during initial screenings of women with possible endometriosis.

Platelet glycoprotein Ib alpha polymorphisms affect the interaction with von Willebrand factor under flow conditions.

Interaction of platelet glycoprotein (GP) Ibalpha with von Willebrand factor (VWF) is essential for thrombus formation, particularly under high shear conditions. Previous case-control studies indicated that two GPIb alpha polymorphisms, (145)Thr/Met and/or variable number (1-4) tandem repeats of 13 amino-acid sequences, are associated with arterial thrombosis. The (145)Met-allele and the 3R- or 4R-allele is associated with increased risk. However, there is little clear experimental data to support this association. To elucidate the functional effects of these polymorphisms, we prepared recombinant GPIb alpha fragments and tested them in vitro. The dissociation constants of ristocetin-induced (125)I-labelled VWF binding to two forms of soluble recombinant GPIb alpha [(1)His-(302)Ala, either (145)Thr (145T) or (145)Met (145M)] were not different. Four types of Chinese hamster ovary cells expressing full-length GPIb alpha beta/IX, 145T with one repeat (T1R), 145M with one repeat (M1R), 145T with four repeats (T4R), and 145M with four repeats (M4R), were prepared, and cell interactions with immobilized-VWF were examined under various shear conditions. The cell rolling velocity of M4R under a shear condition of 114/s was significantly slower than that of T1R. Intermediate values were obtained with M1R and T4R. The results suggest that M4R interacts more strongly with VWF under flow conditions.

Genetic analysis of hereditary factor X deficiency in a French patient of Sri Lankan ancestry: in vitro expression study identified Gly366Ser substitution as the molecular basis of the dysfunctional factor X.

We investigated a new family with cross-reactive material-positive factor X (FX) deficiency. The proband was an 11-year-old French girl from Sri Lanka with a tendency towards severe bleeding. The FX antigen level was 67%, although the activity with extrinsic pathway was 1 U/dl. The complete nucleotide sequences of all exons and exon/intron junctions of the patient's genomic DNA revealed a homozygous G <-- A substitution in exon 8, which would result in replacement of Gly366 with Ser. The proband is the first reported case of homozygote for the FX Gly366Ser mutation. Heterozygosity for Gly366Ser substitution was previously reported in a Japanese patient (FX Nagoya 2). We studied the functional consequences by expressing mutant FX Gly366Ser protein in HEK293 cells. FX Gly366Ser was secreted into the culture media at levels similar to wild-type FX; however, mutant FX activities were only 0.04, 1.05, and 0.75% of wild-type FX upon activation by the extrinsic system, the intrinsic system, and Russell's viper venom, respectively. Moreover, the activity of FX Gly366Ser was undetectable when analyzed with chromogenic-activated FX and thrombin generation assays. These data suggest that the Gly366Ser substitution would cause a major defect in function of the FX molecule.

Expression of allograft inflammatory factor-1 in human eutopic endometrium and endometriosis: possible association with progression of endometriosis.

Allograft inflammatory factor-1 (AIF-1) is a cytokine originally identified in rat cardiac allografts with chronic rejection. AIF-1 is expressed in various human immune-related tissues and is thought to play a role in inflammatory responses and the immune activation and function of macrophages. Expression has also been shown in human placentas and bovine embryos, suggesting that AIF-1 may be involved in reproductive function. Immune factors are thought to be involved in the pathogenesis of endometriosis. High concentrations of activated macrophages and various cytokines have been found in the peritoneal fluid of patients with endometriosis. In the current work we examined the expression of AIF-1 in human eutopic endometrium and endometriosis, and measured AIF-1 in peritoneal fluid samples from women with and without endometriosis. RT-PCR, Western blot analysis, and immunohistochemistry showed that AIF-1 mRNA and protein were expressed both in eutopic endometrium and in endometriotic tissue. In eutopic endometrium, expression was greater in the late secretory and menstrual phases than in other phases of the menstrual cycle (P < 0.01). AIF-1 protein was present in greater amounts in peritoneal fluid from patients with endometriosis than in women without it (P < 0.01), and its concentration correlated with the Revised American Society for Reproductive Medicine score (rs = 0.693; P < 0.0001). Peritoneal macrophages from endometriosis patients secreted more AIF-1 than those from unaffected women (P < 0.05). AIF-1 release from macrophages was stimulated by IL-1beta (P < 0.01) and interferon-gamma (P < 0.05). These results demonstrate for the first time that AIF-1 is expressed in eutopic endometrium and endometriotic tissue, suggesting that AIF-1 is one cytokine in the local network involved in the onset of menstruation. AIF-1 derived from peritoneal macrophages may also possibly play a significant role in the pathophysiology and progression of endometriosis.

Identification of novel mutations in ADAMTS13 in an adult patient with congenital thrombotic thrombocytopenic purpura.

Congenital thrombotic thrombocytopenic purpura/hemolytic uremic syndrome (TTP/HUS) is associated with an inherited von Willebrand factor-cleaving protease (ADAMTS13 [a disintegrin and metalloprotease with thrombospondin type I domains 13]) deficiency. In this study, we identified novel mutations in the ADAMTS13 gene in a patient with TTP. The patient was a 51-year-old Japanese male who exhibited TTP symptoms at frequent intervals. The ADAMTS13 activity during acute episodes was less than 3% that of normal. The enzyme activities of the patient's father and mother were both 46%, and both parents were asymptomatic. Genetic analysis revealed that the patient was a compound heterozygote for 2 mutations. One mutation was a missense mutation in the metalloprotease domain (A250V, exon 7), and the other was a guanine to adenine substitution at the 5' end of intron 3 (intron 3 G-->A). In vitro expression studies revealed that the A250V mutation markedly reduced ADAMTS13 activity and the intron 3 G-->A mutation caused abnormal mRNA synthesis.

Aromatase cytochrome P450 and estrogen and progesterone receptors in uterine sarcomas: correlation with clinical parameters.

We examined the immunohistochemical expression of aromatase cytochrome P450 (P450arom), estrogen receptor (ER), progesterone receptor (PR), and Ki-67 in postoperative uterine sarcomas (n = 31) and the corresponding eutopic endometria (n = 20) to evaluate the relationships between the endocrine character of uterine sarcomas and the clinical features. In sarcoma tissues, P450arom was detected in 55% of cases, ER in 42%, PR in 42%, and Ki-67 in 90%. In eutopic endometria, P450arom was detected in 60% of cases, ER in 60%, and PR in 35%. There were correlations in the steroid-related proteins between the tumors and endometria (P = 0.001-0.026). The positivity of endometrial P450arom (P = 0.04) and ER (P = 0.006) was higher in surviving patients than dead patients regardless of the menstrual state. The results demonstrate correlation between the expression of P450arom, ER, and PR in tumors and eutopic endometria. Intense expression of the steroid-related proteins was associated with better survival.

Detection of von Willebrand factor-cleaving protease (ADAMTS-13) in human platelets.

The hemostatic activity of von Willebrand factor (vWF) is strongly dependent on its multimeric structure, with the highest activity in 'unusually large' multimers secreted from endothelial cells. The multimeric structure is regulated by a plasma protease, vWF-cleaving protease (vWF-CP, or ADAMTS-13). ADAMTS-13 mRNA is variably expressed in liver and other tissues. Because 15-25% of circulating vWF is stored in platelets, the presence and function of ADAMTS-13 in platelets are important issues. Here we report ADAMTS-13 expression in human platelets. Western blot analysis and flow cytometric analysis on permeabilized platelets revealed the presence of ADAMTS-13 protein. Real-time PCR demonstrated that ADAMTS-13 mRNA is present in platelets of six healthy volunteers, with little quantitative difference. The presence of ADAMTS-13 in platelets may imply the functional role of this enzyme in the local regulation of platelet function at the site of vascular injury or thrombus formation, and provide a useful tool for the analysis of structure and function of this enzyme.

A highly sensitive detection method for somatic mutations in p53 gene.

A simple and highly sensitive detection method for somatic mutations, which is applicable to diagnostic samples is developed. This method detects somatic mutations that are lower than several percent in abundance. The unique feature with this method is that single-nucleotide primer extension is carried out in the absence of dye terminators that carry respective bases in the wild type gene. Data of mutation sites and bases in the target genes are obtained from databases. Primers of different nucleotide length are designed so that electrophoreses of the single-nucleotide primer extension products allow identification of respective mutations. Based on somatic mutation data in IARC TP53 mutation database, this method was applied to p53 somatic mutations. Results showed that extension reactions in 5 separate tubes allowed detection of mutations in human p53 gene for the most frequent 12 mutations in a nucleotide sequence of 14,049-14,522, which cover hot spot regions in exons 7 and 8. Confirming these results, reported mutations of p53 in human culture cells, MiaPaCa2, TE-6, RPMI8226, DLD1, and PC-3 are detected at 5% relative abundance in the sample DNA.