Neprilysin Inhibition Promotes Skeletal Growth via the CNP/NPR-B Pathway.

C-type natriuretic peptide (CNP) plays a crucial role in enhancing endochondral bone growth and holds promise as a therapeutic agent for impaired skeletal growth. To overcome CNP's short half-life, we explored the potential of dampening its clearance system. Neprilysin (NEP) is an endopeptidase responsible for catalyzing the degradation of CNP. Thus, we investigated the effects of NEP inhibition on skeletal growth by administering sacubitril, a NEP inhibitor, to C57BL/6 mice. Remarkably, we observed a dose-dependent skeletal overgrowth phenotype in mice treated with sacubitril. Histological analysis of the growth plate revealed a thickening of the hypertrophic and proliferative zones, mirroring the changes induced by CNP administration. The promotion of skeletal growth observed in wild-type mice treated with sacubitril was nullified by the knockout of cartilage-specific natriuretic peptide receptor B (NPR-B). Notably, sacubitril promoted skeletal growth in mice only at 3 to 4 weeks of age, a period when endogenous CNP and NEP expression was higher in the lumbar vertebrae. Additionally, sacubitril facilitated endochondral bone growth in organ culture experiments using tibial explants from fetal mice. These findings suggest that NEP inhibition significantly promotes skeletal growth via the CNP/NPR-B pathway, warranting further investigations for potential applications in people with short stature.

Time-study research on maxillofacial prosthetic treatment.

Unreasonable medical fees can cause problems such as increased medical costs, greater medical disparities, decreased medical standards, and physician shortages. To prevent such problems, it is important to set appropriate medical fees, ensure their proper use, and improve the efficiency of medical care. The treatment of patients with maxillofacial defects is generally more expensive compared with general prosthodontic treatment because it involves more materials and requires more frequently follow-ups for longer period. However, the actual time required for maxillofacial prosthetic treatment is unclear. Therefore, in this study, we aimed to clarify the amount of time spent treating maxillofacial prosthetic patients. We analyzed clinical data from patients undergoing routine maxillofacial prosthetic treatment, irrespective of difficulty level, at 8 university hospitals and 2 dental clinics. We also collected data from maxillofacial prosthodontists on the treatment time required for various Japanese health insurance items, including the fabrication of maxillofacial prostheses. The results revealed that some aspects of maxillofacial prosthetic treatment may take longer to perform and are more costly to perform than previously thought, suggesting the need for some adjustments to the health insurance reimbursement system. Maintaining an appropriate balance between expenditures and fees will greatly benefit patients and physicians, ensuring positive health outcomes and a healthy society.

Prediction-based prompt levothyroxine replacement to prevent a hypothyroid state after immune-related adverse events involving the thyroid gland.

Immune checkpoint inhibitors (ICIs) are used for various malignancies, although they frequently cause immune-related adverse events involving the thyroid gland (thyroid irAEs). We conducted a retrospective cohort study to elucidate thyroid function outcomes. Fifty of 639 patients who received PD-1 blockade therapy met criteria and were divided into the following groups: thyrotoxicosis with subsequent hypothyroidism (Toxic-Hypo, n = 21); thyrotoxicosis without subsequent hypothyroidism (Toxic, n = 9); and hypothyroidism without prior thyrotoxicosis (Hypo, n = 20). The Toxic-Hypo group developed thyroid irAEs earlier than the Toxic group (26 vs. 91 days; p < 0.001), and had higher serum free T4 levels (3.210 vs. 1.880 ng/dL; p = 0.011). In addition, positive anti-thyroglobulin antibodies (TgAbs) at thyroid irAE onset were more common in the Toxic-Hypo group (93.3%) than in the Toxic group (0.0%; p = 0.005) and Hypo group (44.4%; p = 0.007). The Toxic-Hypo group developed severe hypothyroidism and required larger levothyroxine (LT4) doses than the Hypo group (75 vs. 25 µg/day; p = 0.007). We predicted that patients with positive TgAbs who developed severe thyrotoxicosis within 4 weeks after the first ICI administration would develop subsequent hypothyroidism. We treated 4 such patients with prompt LT4 replacement, characterized by LT4 initiation after thyrotoxicosis improvement and quick dose titration. Their euthyroid state was successfully maintained, in contrast with patients receiving conventional replacement. In conclusion, rapid-onset severe thyrotoxicosis in patients with TgAbs correlated with a high likelihood of subsequent hypothyroidism. Accordingly, prompt LT4 replacement is suggested to prevent a severely hypothyroid state.

Prevalence of Possible Dementia in Patients with Maxillofacial Defects and Difficulty of Inserting Obturator in Maxillectomy Patients: Toward Better Provision of Supportive Care.

As society ages, it is important to understand the prevalence of dementia and the difficulties of inserting prostheses in patients with maxillofacial defects in order to clarify issues in supportive care. We screened 183 patients for dementia using the revised Hasegawa's dementia scale (HDS-R) at the Clinic for Maxillofacial prosthetics, Tokyo Medical and Dental University, and investigated age and sex differences in HDS-R score. We asked 47 of the 183 participants about the difficulty of inserting a maxillofacial obturator prosthesis and collected subjective comments, information about the prosthesis, and data from five assessments. Multiple regression analysis was used to reveal factors associated with insertion difficulty. Overall, 8.7% of the participants were judged to have possible dementia. Men were more likely than women to have possible dementia, and the risk increased with age. Of the 47 participants, 26 reported difficulty inserting their prosthesis, 12 of whom attributed it to their oral defect. Fourteen patients advised following doctor's instructions to practice insertion in order to become accustomed to it. A lower HDS-R score had a significant impact on insertion difficulty. Cognitive function and difficulty inserting maxillary obturator prostheses should be considered in the provision of continuous supportive care to patients with maxillary defects.

Nicotine Reduces Reactive Oxygen Species and Enhances Cell Proliferation via the α4 Nicotinic Acetylcholine Receptor Subunit in Human Induced Pluripotent Stem Cells.

The effects of smoking on fetal development and stem cell differentiation are not fully understood. Although nicotinic acetylcholine receptors (nAChRs) are expressed in many organs of the human body, their significance in human induced pluripotent stem cells (hiPSCs) remains unclear. After expression levels of nAChR subunits in hiPSCs were determined, the effects of the nAChR agonist, nicotine, on undifferentiated hiPSCs were evaluated using a Clariom S Array. We also determined the effect of nicotine alone and with a nAChR subunit antagonist on hiPSCs. nAChR α4, α7, and ß4 subunits were strongly expressed in hiPSCs. cDNA microarray, gene ontology, and enrichment analyses showed that exposing hiPSCs to nicotine altered expression of genes associated with immune responses, neurological system, carcinogenesis, cell differentiation, and cell proliferation. Particularly affected was metallothionein, which acts to decrease reactive oxygen species (ROS). The nicotine-induced reduction of ROS in hiPSCs was canceled by an α4 subunit or nonselective nAChR antagonist. HiPSC proliferation was increased by nicotine, and this effect, too, was canceled by an α4 antagonist. In conclusion, nicotine reduces ROS and enhances cell proliferation through the α4 nAChR subunit in hiPSCs. These findings provide new insight into the significance of nAChRs on human stem cells and fertilized human ova.

A 20-year clinical survey of radiotherapy prostheses at the Clinic for Maxillofacial Prosthetics of Tokyo Medical and Dental University Hospital.

PURPOSE: To identify secular trends in the use of radiotherapy prostheses in the Clinic for Maxillofacial Prosthetics at Tokyo Medical and Dental University Hospital and to ascertain patient characteristics and future needs. METHODS: The study included all patients who visited the clinic between January 1, 2000, and December 31, 2019. Data on numbers of cases, gender and age, numbers and types of radiotherapy prostheses, primary cancer site, and time required for prosthesis fabrication during the study period were collected from medical records and retrospectively analyzed. RESULTS: During the 20-year study period, 1,066 prostheses were fabricated for 1,135 cases. In most years, at least 60% of cases were male. The mean age increased from 60.6 to 67.1 years during the study period, and the number of cases older than 70 years increased over time. The most common cancer site was the tongue, and the most common type of radiotherapy prosthesis was a spacer. The mean time for spacer fabrication was 11 days. CONCLUSION: During a recent 20-year period, mean age and the proportion of elderly patients requiring savers increased. When treating elderly patients, prosthodontists responsible for fabricating radiotherapy prostheses must collaborate effectively with radiation oncologists.

Delayed-onset immune-related adverse events involving the thyroid gland by immune checkpoint inhibitors in combination with chemotherapy: a case report and retrospective cohort study.

Immune checkpoint inhibitors (ICIs) frequently cause immune-related adverse events (irAEs) that often involve endocrine organs. Pembrolizumab and atezolizumab are currently administered in combination with chemotherapy for several malignancies. Although transient thyrotoxicosis within 6 weeks after the first ICI dose is the typical course of thyroid irAEs with ICI monotherapy, we encountered a unique course of a thyroid irAE in a patient who received combination therapy consisting of pembrolizumab plus pemetrexed and carboplatin. Delayed onset of thyrotoxicosis occurred at 22 weeks after the first dose of pembrolizumab. To understand more about this curious event, we conducted a retrospective cohort study of the following groups: pembrolizumab monotherapy (Pem-mono), pembrolizumab plus chemotherapy (Pem-combi), atezolizumab monotherapy (Atezo-mono), and atezolizumab plus chemotherapy (Atezo-combi). There were no differences in the incidence of overt thyroid irAEs: Pem-mono, 12 of 151 patients (7.9%) versus Pem-combi, 4 of 56 patients (7.1%) (p = 0.85) and Atezo-mono, 5 of 27 patients (18.5%) versus Atezo-combi, 5 of 57 patients (8.8%) (p = 0.20). Through detailed analyses of patients with thyrotoxicosis, we found some patients with delayed-onset thyroid irAE, defined as development at 16 weeks or more after the first ICI dose. Delayed-onset thyroid irAEs were only observed in the combination therapy groups: Pem-combi or Atezo-combi, 3 of 8 patients versus Pem-mono or Atezo-mono, 0 of 10 patients. Our observation that thyroid irAE development can be delayed with ICIs when used in combination with chemotherapy suggests longer monitoring of thyroid function is needed to avoid missing irAEs.

Two-piece detachable interlocking mold for low-dose-rate brachytherapy: A dental technique for radiotherapy prosthesis fabrication.

Brachytherapy is a type of radiotherapy used to eradicate malignancies in the head-and-neck region. Early-stage mucosal carcinomas, such as carcinomas of the hard palate and gingival mucosa, can be treated with low-dose-rate brachytherapy by using a radiotherapy prosthesis called a mold. Prosthodontists play a significant role in the design and fabrication of these molds, which deliver therapeutic doses to the primary lesion and protect adjacent tissues from radiation exposure. However, planning and observing ongoing treatment is difficult for radiation oncologists because the shield part cannot be removed from the carrier part or requires an attachment to do so. This article describes a technique for fabricating a 2-piece detachable interlocking mold without any special attachment for low-dose-rate brachytherapy.

Hypomorphic mutation of PEX3 with peroxisomal mosaicism reveals the oscillating nature of peroxisome biogenesis coupled with differential metabolic activities.

Impaired peroxisome assembly caused by mutations in PEX genes results in a human congenital metabolic disease called Zellweger spectrum disorder (ZSD), which impacts the development and physiological function of multiple organs. In this study, we revealed a long-standing problem of heterogeneous peroxisome distribution among cell population, so called "peroxisomal mosaicism", which appears in patients with mild form of ZSD. We mutated PEX3 gene in HEK293 cells and obtained a mutant clone with peroxisomal mosaicism. We found that peroxisomal mosaicism can be reproducibly arise from a single cell, even if the cell has many or no peroxisomes. Using time-lapse imaging and a long-term culture experiment, we revealed that peroxisome biogenesis oscillates over a span of days; this was also confirmed in the patient's fibroblasts. During the oscillation, the metabolic activity of peroxisomes was maintained in the cells with many peroxisomes while depleted in the cells without peroxisomes. Our results indicate that ZSD patients with peroxisomal mosaicism have a cell population whose number and metabolic activities of peroxisomes can be recovered. This finding opens the way to develop novel treatment strategy for ZSD patients with peroxisomal mosaicism, who currently have very limited treatment options.

High-throughput Screening in Combination With a Cohort Study for Iodothyronine Deiodinases.

Regulatory mechanisms of iodothyronine deiodinases (DIOs) require further elucidation, and conventional methods for evaluating DIOs are unsuitable for high-throughput screening (HTS). Here we explored factors of transcriptional regulation of 3 types of DIOs (DIO1, DIO2, and DIO3) from a chemical library using our designed HTS. We constructed HTS based on a promoter assay and performed a screen of 2480 bioactive compounds. For compounds that were clinically approved, we validated hit compounds through a retrospective cohort study in our department that evaluated changes in thyroid function in patients using the compounds as drug therapy. Furthermore, we verified the involvement of DIOs using mice treated with the compounds. Of the hit compounds, 6 and 7 compounds transcriptionally up- and downregulated DIO1, respectively; 34 transcriptionally upregulated DIO2; and 5 and 2 compounds transcriptionally up- and downregulated DIO3, respectively. The cohort study clarified the clinical effects of some hit compounds: ritodrine increased free triiodothyronine (fT3)/free thyroxine (fT4) ratio and decreased serum thyroid-stimulating hormone (TSH) levels, tadalafil increased serum fT3 levels, and tyrosine kinase inhibitors (TKIs) decreased serum fT3 and fT4 levels and increased serum TSH levels. Following in vivo experiments using treated mice, consistent results were observed in ritodrine, which upregulated DIO2 in the thyroid gland. In conclusion, we completed HTS for DIOs and obtained attractive hit compounds. Our cohort study revealed the clinical significance of ritodrine, sildenafil, and TKIs. We hope our unique method will contribute to analyzing various targets and lists of hit compounds will promote understanding of DIOs.

Thyroid hormone economy in mice overexpressing iodothyronine deiodinases.

In peripheral tissues, triiodothyronine (T3) production and consequent thyroid hormone actions are mainly regulated by iodothyronine deiodinases (DIOs) classified into 3 types: D1, D2, and D3. We aimed to investigate the effects of peripheral DIOs on thyroid hormone economy independent of the hypothalamus-pituitary-thyroid axis. We cloned coding sequences of human DIOs with FLAG-tag and HiBiT-tag sequences into a pcDNA3.1 vector. To obtain full-length proteins, we modified these vectors by cloning the selenocysteine insertion sequence of each DIO (SECIS vectors). Western blot analyses and HiBiT lytic assay using HEK293T cells revealed that SECIS vectors expressed full-length proteins with substantial activity. Subsequently, in vivo transfections of pLIVE-based SECIS vectors into male C57BL/6J mice were performed by hydrodynamic gene delivery to generate mice overexpressing DIOs predominantly in the liver (D1, D2, and D3 mice). After 7 days from transfections, mice were analyzed to clarify phenotypes. To summarize, serum thyroid hormone levels did not change in D1 mice but D2 mice had higher serum free T3 levels. D3 mice developed hypothyroidism with higher serum reverse T3 (rT3) levels. Transfections with levothyroxine administration suggested that thyroid hormone action was upregulated in D2 mice. Our DIO-overexpressing mice provided insights on the physiological properties of upregulated DIOs: D2 augments local thyroid hormone action and recruits T3 into the circulation: D3 decreases circulating T3 and T4 levels with elevated rT3, leading to consumptive hypothyroidism. As D3 mice are expected to be a novel hypothyroidism model, they can contribute to progress in the field of thyroid hormone economy and action.

Human language evolution: a view from theoretical linguistics on how syntax and the lexicon first came into being.

Human language is a multi-componential function comprising several sub-functions each of which may have evolved in other species independently of language. Among them, two sub-functions, or modules, have been claimed to be truly unique to the humans, namely hierarchical syntax (known as "Merge" in linguistics) and the "lexicon." This kind of species-specificity stands as a hindrance to our natural understanding of human language evolution. Here we challenge this issue and advance our hypotheses on how human syntax and lexicon may have evolved from pre-existing cognitive capacities in our ancestors and other species including but not limited to nonhuman primates. Specifically, we argue that Merge evolved from motor action planning, and that the human lexicon with the distinction between lexical and functional categories evolved from its predecessors found in animal cognition through a process we call "disintegration." We build our arguments on recent developments in generative grammar but crucially depart from some of its core ideas by borrowing insights from other relevant disciplines. Most importantly, we maintain that every sub-function of human language keeps evolutionary continuity with other species' cognitive capacities and reject a saltational emergence of language in favor of its gradual evolution. By doing so, we aim to offer a firm theoretical background on which a promising scenario of language evolution can be constructed.

Clinical features and thyroid dysfunction in adverse events involving the pituitary gland during PD-1 blockade therapy.

OBJECTIVE: Programmed cell death-1 (PD-1) blockade therapy, an immune checkpoint treatment, can induce hypophysitis or hypopituitarism as an immune-related adverse event (pituitary irAE). We aimed to clarify the clinical features of pituitary irAEs during PD-1 blockade therapy. DESIGN, PATIENTS AND MEASUREMENTS: This retrospective study investigated consecutive patients treated with nivolumab, an anti-PD-1 antibody, at Kyoto University Hospital between 1 September 2014 and 31 August 2019. We examined patients' baseline characteristics and analysed the clinical data of those who developed pituitary irAEs. RESULTS: Of the 374 recruited patients, 7 (1.9%) developed pituitary irAEs, and each presented with isolated secondary adrenal insufficiency. In 4 patients, changes in ACTH were delayed relative to those in cortisol: when serum cortisol levels fell below the reference range, plasma ACTH levels were still normal. Pituitary irAEs were accompanied by elevated serum-free T3 (fT3) levels, which resolved with glucocorticoid replacement. Serum TSH levels were not suppressed despite elevated serum fT3 levels and 1 patient even presented with high fT3 level above the reference range (fT3, 7.1 pmol/L; free T4 (fT4), 13.9 pmol/L; and TSH, 5.1 mIU/L). CONCLUSIONS: Isolated secondary adrenal insufficiency was a common pituitary irAE during PD-1 blockade therapy. This condition was accompanied by thyroid dysfunction, including elevation of fT3 without TSH suppression.

Ceritinib-associated hyperglycemia in the Japanese Adverse Drug Event Report Database.

Genetic rearrangements of anaplastic lymphoma kinase contribute to the pathogenesis of non-small-cell lung cancer; the anaplastic lymphoma kinase inhibitor, ceritinib, is widely used, as it is effective even in patients with non-small-cell lung cancer resistant to other anaplastic lymphoma kinase inhibitors. Although a case of possible ceritinib-induced hyperglycemia was reported, the association of ceritinib with hyperglycemia remains to be investigated. Disproportionality analysis was carried out using the Japanese Adverse Drug Event Report database, which contains all pharmacovigilance data based on spontaneous reports of adverse events between April 2004 and November 2018 to the Pharmaceuticals and Medical Devices Agency. The reporting odds ratio of ceritinib for hyperglycemia was 2.25 (95% confidence interval [CI] 1.24-4.08], whereas those of crizotinib and alectinib were 0.07 (95% CI 0.01-0.40) and 0.94 (95% CI 0.30-2.94), respectively. Among reported events without antidiabetes agent use, the reporting odds ratio of ceritinib was still 2.54 (95% CI 1.27-5.12). Thus, the possibility of hyperglycemia should be carefully monitored in patients receiving ceritinib.

Prosthodontic Treatment of a Patient with Gnathodiaphyseal Dysplasia: 30-Year Follow-up.

This case history report describes the long-term prosthodontic treatment of a patient with gnathodiaphyseal dysplasia (GDD). The patient was initially diagnosed with osteomyelitis in the maxilla in 1986, followed by osteonecrosis spread throughout the mandible. GDD was genetically diagnosed in 2006. Despite the severe alveolar bone resorption, prosthodontic treatment improved the patient's satisfaction and ability to perform essential functions. Regular prosthesis adjustments and periodic follow-up should continue to avoid future complications.

Difference in cesium accumulation among rice cultivars grown in the paddy field in Fukushima Prefecture in 2011 and 2012.

After the accident of the Fukushima 1 Nuclear Power Plant in March 2011, radioactive cesium was released and paddy fields in a wide area including Fukushima Prefecture were contaminated. To estimate the levels of radioactive Cs accumulation in rice produced in Fukushima, it is crucial to obtain the actual data of Cs accumulation levels in rice plants grown in the actual paddy field in Fukushima City. We herein conducted a two-year survey in 2011 and 2012 of radioactive and non-radioactive Cs accumulation in rice using a number of rice cultivars grown in the paddy field in Fukushima City. Our study demonstrated a substantial variation in Cs accumulation levels among the cultivars of rice.

CCAR1/CoCoA pair-mediated recruitment of the Mediator defines a novel pathway for GATA1 function.

The MED1 subunit of the Mediator transcriptional coregulator complex coactivates GATA1 and induces erythropoiesis. Here, we show the dual mechanism of GATA1- and MED1-mediated transcription. MED1 expression levels in K562 erythroleukemia cells paralleled the levels of GATA1-targeted gene transcription and erythroid differentiation. An N-terminal fragment of MED1, MED1(1-602), which is incapable of interacting with GATA1, enhanced GATA1-targeted gene transcription and erythroid differentiation, and introduction of MED1(1-602) into Med1(-/-) mouse embryonic fibroblasts (MEFs) partially rescued GATA1-mediated transcription. The C-terminal zinc-finger domain of GATA1 interacts with the MED1(1-602)-interacting coactivator CCAR1, CoCoA and MED1(681-715). CCAR1 and CoCoA synergistically enhanced GATA1-mediated transcription from the γ-globin promoter in MEFs. Recombinant GATA1, CCAR1, CoCoA and MED1(1-602) formed a complex in vitro, and GATA1, CCAR1, CoCoA and MED1 were recruited to the γ-globin promoter in K562 cells during erythroid differentiation. Therefore, in addition to the direct interaction between GATA1 and MED1, CoCoA and CCAR1 appear to relay the GATA1 signal to MED1, and multiple modes of the GATA1-MED1 axis may help to fine-tune GATA1 function during GATA1-mediated homeostasis events.

Production and degradation of N-acylhomoserine lactone quorum sensing signal molecules in bacteria isolated from activated sludge.

N-Acylhomoserine lactones (AHLs) function as quorum-sensing signaling molecules in many Gram-negative bacteria. We isolated a total of 672 bacterial strains from activated sludge obtained from seven sewage treatment plants in Tochigi Prefecture, Japan, and screened for AHL-producing and degrading strains. Isolates (n = 107) stimulated AHL-mediated purple pigment production in AHL reporter strains Chromobacterium violaceum CV026 and VIR07. Based on their 16S rRNA gene sequences, most of these AHL-producing isolates were assigned to the genus Aeromonas, and they were divided into six groups. Isolates (n = 46) degraded N-decanoyl-L-homoserine lactone (C10-HSL) within 24 h. Based on their 16S rRNA gene sequences, the most dominant AHL-degrading isolates were assigned to the genus Acinetobacter and divided into six groups. Strains Ooi24, Omo91, and Uzu81, which showed higher C10-HSL-degrading activity, showed putative AHL-acylase activity.

Stop codon mutations in the flavin-containing monooxygenase 3 (FMO3) gene responsible for trimethylaminuria in a Japanese population.

The reduced capacity of flavin-containing monooxygenase 3 (FMO3) to N-oxidize trimethylamine (TMA) is believed to cause a metabolic disorder. The aim of this study was to investigate the inter-individual variations of FMO3. Genomic DNA of case subjects that showed only 10-20% of FMO3 metabolic capacity among self-reported trimethylaminuria Japanese volunteers was sequenced. Functional analysis of recombinant FMO3 proteins was also performed. One homozygote for a novel single nucleotide substitution causing a stop codon at Arg500 was observed. The biological parents of this Proband A were heterozygous and showed >90% TMA N-oxygenation metabolic capacity. Another Proband B had the Arg500Stop and Cys197Stop codons. The TMA N-oxygenation metabolic capacities of the father and brother of this Proband B were apparently observed by possessing Arg205Cys mutant that coded for decreased TMA N-oxygenase. Recombinant Arg500Stop FMO3 cDNA expressed in Escherichia coli membranes and a series of highly purified truncation mutants at different positions of the C-terminus of FMO3 showed no detectable functional activity toward typical FMO3 substrates. The results suggest that individuals homozygous for either of the nonsense mutations, Arg500Stop and/or Cys197Stop alleles, in the FMO3 gene can possess abnormal TMA N-oxygenation.

Three novel single nucleotide polymorphisms of the FMO3 gene in a Japanese population.

We sequenced all exons and exon-intron junctions of the flavin-containing monooxygenase 3 (FMO3) gene from 2 Japanese individuals and their family members, who were case subjects that showed low FMO3 metabolic capacity among a population of self-reported trimethylaminuria Japanese volunteers. We found two novel single nucleotide polymorphisms (SNPs) (21,254 C>A and 24,006 A>G) causing amino acid substitutions, Thr(201)Lys in exon 5 and Met(260)Val in exon 6, respectively. The Thr(201)Lys and Met(260)Val also presented together with known SNPs (Glu(158)Lys-Glu(308)Gly and Val(257)Met, respectively) in the same alleles of the FMO3 gene to form novel haplotypes. A SNP (30,398 C>T) in the FMO3 gene causing a stop codon at Arg(500) in exon 9 was also discovered. These sequences are as follows: 1) SNP, 060116Shimizu001; GENE NAME, FMO3; ACCESSION NUMBER, AL021026; LENGTH, 25 base; 5'-GTGATATTGCCAC/AAGAACTCAGCCG-3'. 2) SNP, 060116Shimizu002; GENE NAME, FMO3; ACCESSION NUMBER, AL021026; LENGTH, 25 base; 5'-TAC(G/A)TGAAGCAGA/GTGAATGCAAGAT-3'. 3) SNP, 060116Shimizu003; GENE NAME, FMO3; ACCESSION NUMBER, AL021026; LENGTH, 25 base; 5'-CCCATGCAGACAC/TGAGTGGTCGGGA-3'.