Age and sex are excellent predictors of bone complications in patients with type 2 diabetes with no history of osteoporotic fracture or treatment for osteoporosis.

OBJECTIVE: To identify patients with type 2 diabetes mellitus (T2DM) with no history of fracture or osteoporosis treatment who are at risk of bone complications through the assessment of bone quality and quantity. METHODS: Of the outpatients attending our clinic during 2021 to 2022, we retrospectively enrolled 137 (men/women: 85/52, median age: 65 years) consecutive patients aged ≥40 years who had T2DM but no history of fracture or osteoporosis treatment. The lumbar spine and femoral neck bone mineral density and the trabecular bone score were determined using dual-energy X-ray absorptiometry. Independent factors associated with bone disease were identified using logistic regression analysis, and odds ratios (ORs) were calculated. RESULTS: Age and female sex were significantly associated with high ORs for development of bone disease. The integrated risk of bone complications was nearly 40-fold higher in older (≥65 years) women than in younger (<65 years) men. This difference remained after adjustment for the duration of T2DM, body mass index, and HbA1c level. CONCLUSIONS: Older women have the highest risk of osteopenia and osteoporosis among patients with T2DM who have no history of fracture or osteoporosis treatment. These patients should undergo intensive monitoring for bone fragility from an early stage of their disease.

A two-dose viral-vectored Plasmodium vivax multistage vaccine confers durable protection and transmission-blockade in a pre-clinical study.

Among Plasmodium spp. responsible for human malaria, Plasmodium vivax ranks as the second most prevalent and has the widest geographical range; however, vaccine development has lagged behind that of Plasmodium falciparum, the deadliest Plasmodium species. Recently, we developed a multistage vaccine for P. falciparum based on a heterologous prime-boost immunization regimen utilizing the attenuated vaccinia virus strain LC16m8Δ (m8Δ)-prime and adeno-associated virus type 1 (AAV1)-boost, and demonstrated 100% protection and more than 95% transmission-blocking (TB) activity in the mouse model. In this study, we report the feasibility and versatility of this vaccine platform as a P. vivax multistage vaccine, which can provide 100% sterile protection against sporozoite challenge and >95% TB efficacy in the mouse model. Our vaccine comprises m8Δ and AAV1 viral vectors, both harboring the gene encoding two P. vivax circumsporozoite (PvCSP) protein alleles (VK210; PvCSP-Sal and VK247; -PNG) and P25 (Pvs25) expressed as a Pvs25-PvCSP fusion protein. For protective efficacy, the heterologous m8Δ-prime/AAV1-boost immunization regimen showed 100% (short-term; Day 28) and 60% (long-term; Day 242) protection against PvCSP VK210 transgenic Plasmodium berghei sporozoites. For TB efficacy, mouse sera immunized with the vaccine formulation showed >75% TB activity and >95% transmission reduction activity by a direct membrane feeding assay using P. vivax isolates in blood from an infected patient from the Brazilian Amazon region. These findings provide proof-of-concept that the m8Δ/AAV1 vaccine platform is sufficiently versatile for P. vivax vaccine development. Future studies are needed to evaluate the safety, immunogenicity, vaccine efficacy, and synergistic effects on protection and transmission blockade in a non-human primate model for Phase I trials.

Immunoprecipitation with an Anti-Epitope Tag Affinity Gel to Study Protein-Protein Interactions.

Protein-protein interactions (PPIs) play a pivotal role in biological phenomena, such as cellular organization, intracellular signal transduction, and transcriptional regulation. Therefore, understanding PPIs is an important starting point for further investigation of the function of the target protein. In this study, we propose a simple method to determine the binding of two target proteins by introducing mammalian expression vectors into HEK-293 cells using the polyethylenimine method, lysing the cells in homemade protein lysis buffer, and pulling down the target proteins on an epitope tag affinity gel. In addition, the PPI between the various epitope tag fused proteins can be confirmed by using affinity antibodies against each tag instead of the epitope tag affinity gel. This protocol could also be used to verify various PPIs, including nuclear extracts, from other cell lines. Therefore, it can be used as a basic method in a variety of PPI experiments. Proteins degrade by extended time course and repeated freeze-thaw cycles. Therefore, cell lysis, immunoprecipitation, and immunoblotting should be performed as seamlessly as possible.

Regeneration process of myotendinous junction injury induced by collagenase injection between Achilles tendon and soleus muscle in mice.

Recently, it has become clear that peri-muscular tissues play a significant role in the deterioration of muscle function. Understanding the function and regeneration of muscle, as well as its surrounding tissues, is crucial to determining the causes of muscular illnesses. However, the regeneration process of the myotendinous junction (MTJ), the most closely related peri-muscular tissue, is still unknown. Therefore, we generated a mouse model of MTJ injury by collagenase injection and searched for the process of regeneration of the MTJ and its adjacent regions. The MTJ region was damaged by collagenase injection, which greatly increased the tendon cross sectional area. Collagenase injections increased the proportion of myofibers with a central nucleus, which is a characteristic of regenerating muscle. The collagenase injection group had myofibers with central nuclei and expressing MTJ markers. Additionally, we measured the length of MTJs using serial cross sections of the soleus muscle and discovered that MTJs at 2 weeks after collagenase injection were shorter compared to the control group, with a propensity to progressively recover their length over time. The results showed that MTJs undergo morphological regeneration even when severely damaged, and that this regeneration occurs in conjunction with muscle regeneration. We anticipate that these findings will be valuable in upcoming research on motor unit regeneration.

Selective activation of PPARα maintains thermogenic capacity of beige adipocytes.

Beige adipocytes are inducible thermogenic adipocytes used for anti-obesity treatment. Beige adipocytes rapidly lose their thermogenic capacity once external cues are removed. However, long-term administration of stimulants, such as PPARγ and ß-adrenergic receptor agonists, is unsuitable due to various side effects. Here, we reported that PPARα pharmacological activation was the preferred target for maintaining induced beige adipocytes. Pemafibrate used in clinical practice for dyslipidemia was developed as a selective PPARα modulator (SPPARMα). Pemafibrate administration regulated the thermogenic capacity of induced beige adipocytes, repressed body weight gain, and ameliorated impaired glucose tolerance in diet-induced obese mouse models. The transcriptome analysis revealed that the E-twenty-six transcription factor ELK1 acted as a cofactor of PPARα. ELK1 was mobilized to the Ucp1 transcription regulatory region with PPARα and modulated its expression by pemafibrate. These results suggest that selective activation of PPARα by pemafibrate is advantageous to maintain the function of beige adipocytes.

Development of immunohistochemistry for detecting fluvoxamine in rat tissues using newly prepared monoclonal antibody: its precise localization in small intestine, kidney, and liver of rats.

A monoclonal antibody (mAb) was produced against a fluvoxamine (FLV)-bovine serum albumin conjugate that was specific to both the conjugate and free form of FLV. The mAb enabled us to develop an immunohistochemistry (IHC) method for pharmacokinetic analysis of FLV at the cell and tissue levels. We demonstrated that IHC can be used to detect the localization of FLV in the small intestine, kidney, and liver 1 h after drug administration at the cell and tissue levels. Protease digestion is an important factor for obtaining appropriate IHC staining results for localization of drugs. In this study, precise FLV localization could be determined with only 1 h of protease digestion in the kidneys, but in the small intestine and liver, the staining results with two digestive conditions had to be merged. IHC provided new findings, such as (1) nerve cells are likely to uptake more FLV than other cells and tissues; (2) the ability of reabsorption and secretion in the kidney varies depending on the site, and the amount of FLV in the primary urine is regulated downstream of the proximal tubule S3 segment; and (3) some of the FLV is excreted in the bile.

Adeno-associated virus-based malaria booster vaccine following attenuated replication-competent vaccinia virus LC16m8Δ priming.

We previously demonstrated that boosting with adeno-associated virus (AAV) type 1 (AAV1) can induce highly effective and long-lasting protective immune responses against malaria parasites when combined with replication-deficient adenovirus priming in a rodent model. In the present study, we compared the efficacy of two different AAV serotypes, AAV1 and AAV5, as malaria booster vaccines following priming with the attenuated replication-competent vaccinia virus strain LC16m8Δ (m8Δ), which harbors the fusion gene encoding both the pre-erythrocytic stage protein, Plasmodium falciparum circumsporozoite (PfCSP) and the sexual stage protein (Pfs25) in a two-dose heterologous prime-boost immunization regimen. Both regimens, m8Δ/AAV1 and m8Δ/AAV5, induced robust anti-PfCSP and anti-Pfs25 antibodies. To evaluate the protective efficacy, the mice were challenged with sporozoites twice after immunization. At the first sporozoite challenge, m8Δ/AAV5 achieved 100% sterile protection whereas m8Δ/AAV1 achieved 70% protection. However, at the second challenge, 100% of the surviving mice from the first challenge were protected in the m8Δ/AAV1 group whereas only 55.6% of those in the m8Δ/AAV5 group were protected. Regarding the transmission-blocking efficacy, we found that both immunization regimens induced high levels of transmission-reducing activity (>99%) and transmission-blocking activity (>95%). Our data indicate that the AAV5-based multistage malaria vaccine is as effective as the AAV1-based vaccine when administered following an m8Δ-based vaccine. These results suggest that AAV5 could be a viable alternate vaccine vector as a malaria booster vaccine.

Sterile protection and transmission blockade by a multistage anti-malarial vaccine in the pre-clinical study.

The Malaria Vaccine Technology Roadmap 2013 (World Health Organization) aims to develop safe and effective vaccines by 2030 that will offer at least 75% protective efficacy against clinical malaria and reduce parasite transmission. Here, we demonstrate a highly effective multistage vaccine against both the pre-erythrocytic and sexual stages of Plasmodium falciparum that protects and reduces transmission in a murine model. The vaccine is based on a viral-vectored vaccine platform, comprising a highly-attenuated vaccinia virus strain, LC16m8Δ (m8Δ), a genetically stable variant of a licensed and highly effective Japanese smallpox vaccine LC16m8, and an adeno-associated virus (AAV), a viral vector for human gene therapy. The genes encoding P. falciparum circumsporozoite protein (PfCSP) and the ookinete protein P25 (Pfs25) are expressed as a Pfs25-PfCSP fusion protein, and the heterologous m8Δ-prime/AAV-boost immunization regimen in mice provided both 100% protection against PfCSP-transgenic P. berghei sporozoites and up to 100% transmission blocking efficacy, as determined by a direct membrane feeding assay using parasites from P. falciparum-positive, naturally-infected donors from endemic settings. Remarkably, the persistence of vaccine-induced immune responses were over 7 months and additionally provided complete protection against repeated parasite challenge in a murine model. We propose that application of the m8Δ/AAV malaria multistage vaccine platform has the potential to contribute to the landmark goals of the malaria vaccine technology roadmap, to achieve life-long sterile protection and high-level transmission blocking efficacy.

Localized Pancreatitis in an Elderly Patient Without Suspected Etiology.

Once pancreatic inflammation is triggered, it spreads throughout the pancreas. Here, we present a case of localized pancreatitis wherein the inflammation was confined to the pancreatic head. A 91-year-old woman was admitted with complaints of vomiting and epigastric pain. Blood tests showed elevated pancreatic enzyme levels, whereas imaging studies showed an enlarged pancreatic head and an area of increased density in the surrounding fatty tissue extending along the retroperitoneum below the subrenal pole. Atrophy of the pancreatic parenchyma in the pancreatic body and tail and dilatation of the main pancreatic duct were observed. The patient was diagnosed with acute pancreatitis, was kept nil by mouth, and was administered supplemental fluids. The symptoms resolved within two weeks. Age-related anatomical and histological changes in the pancreas may influence the development of pancreatitis, making it difficult to rule out the possibility of cancer. As age-related changes in the pancreas could lead to the development of pancreatitis, it is an important differential diagnosis of abdominal pain, even in older patients without suspected etiologies.

Development and Regeneration of Muscle, Tendon, and Myotendinous Junctions in Striated Skeletal Muscle.

Owing to a rapid increase in aging population in recent years, the deterioration of motor function in older adults has become an important social problem, and several studies have aimed to investigate the mechanisms underlying muscle function decline. Furthermore, structural maintenance of the muscle-tendon-bone complexes in the muscle attachment sites is important for motor function, particularly for joints; however, the development and regeneration of these complexes have not been studied thoroughly and require further elucidation. Recent studies have provided insights into the roles of mesenchymal progenitors in the development and regeneration of muscles and myotendinous junctions. In particular, studies on muscles and myotendinous junctions have-through the use of the recently developed scRNA-seq-reported the presence of syncytia, thereby suggesting that fibroblasts may be transformed into myoblasts in a BMP-dependent manner. In addition, the high mobility group box 1-a DNA-binding protein found in nuclei-is reportedly involved in muscle regeneration. Furthermore, studies have identified several factors required for the formation of locomotor apparatuses, e.g., tenomodulin (Tnmd) and mohawk (Mkx), which are essential for tendon maturation.

Immunohistochemical Localization of Alogliptin, a DPP-4 Inhibitor, in Tissues of Normal and Type 2 Diabetes Model Rat.

We investigated the pharmacokinetics of alogliptin (AG) at the cell and tissue level in healthy Wistar rats and a type 2 diabetic Goto-Kakizaki (GK) rat model. Immunohistochemistry of the renal tissue in these rats, post 1 hr of AG administration, showed that the signal was observed in the glomeruli, proximal tubule S3 segments, distal tubules, collecting ducts, and only in the brush border of the epithelial cells of the proximal tubule S1, S2 segments. After 6 hr of AG administration, the staining intensity of the regions other than the S3 segments was considerably reduced in Wistar rats, with no change observed in GK rats. At 24 hr, the staining intensity was considerably reduced, even in GK rats; however, the staining of the S3 segment remained unaltered in both. Hepatocytes in zone III of the hepatic lobule were more intensely stained than those in zone I in Wistar rats at 1 hr. However, almost no staining was observed in the hepatocytes of GK rats at 1 hr. Complete loss of signal was observed in the hepatocytes of the Wistar rats after 6 hr. This study revealed that the pharmacokinetics of AG in GK rats are different from those in Wistar rats.

Immunohistochemical localization and pharmacokinetics of the anti-MRSA drug teicoplanin in rat kidney using a newly developed specific antibody.

We prepared a polyclonal antibody against a teicoplanin (TEIC)-bovine serum albumin conjugate that was specific to both conjugated and free forms of TEIC. We demonstrated that this antibody could be used to detect the time-dependent localization of TEIC in rat kidneys. Immunohistochemistry revealed immunoreactivity specifically in the microvilli and apical cytoplasm of epithelial cells in proximal tubule segments S1 and S2, 1 h after intravenous TEIC injection, with higher staining intensity in the S2 segments. The epithelial cells of S3 segments showed moderate immunostaining with a few cells exhibiting nuclear staining. Furthermore, we found that the distal tubules and collecting ducts contained both TEIC-positive and -negative cells. TEIC immunoreactivity decreased rapidly over time; only weak staining remained in the S3 segments, distal tubules, and collecting ducts 24 h after administration. No staining was detected 7 days after injection. These results were significantly different from those of our previous study obtained using vancomycin, which showed moderate staining in the proximal tubule segments S1 and S2, distal tubules, and the collecting ducts 8 days after administration. The lower TEIC accumulation in tissues may account for a lower risk of adverse events compared to that using vancomycin.

[Development of a Sensitive Enzyme-linked Immunosorbent Assay for Daptomycin].

Daptomycin (DAP) has a completely different mechanism of action compared with conventional drugs for methicillin-resistant Staphylococcus aureus (MRSA) and is widely used as the first-line drug for treatment of dermal soft tissue infection and sepsis caused by MRSA infection in clinical practice. However, DAP has serious side effects, including renal dysfunction and rhabdomyolysis, and thus therapeutic drug monitoring of DAP is recommended. The purpose of this study was to develop an enzyme-linked immunosorbent assay (ELISA) for DAP that is simpler and more sensitive compared with existing assay methods and can be used in pharmacokinetic studies. Anti-DAP antibody was obtained by immunizing mice with an antigen conjugated with mercaptosuccinyl bovine serum albumin using N-(4-maleimidobutyryloxy) succinimide as a heterobifunctional coupling agent. Enzyme labeling of DAP with horseradish peroxidase was performed using pyromellitic dianhydride. The generated antibody and enzyme conjugate were used to develop a highly sensitive and specific ELISA for DAP in human serum. This ELISA shows a linear range of detection from 0.3 to 72.9 ng/mL, and a limit of quantification of approximately 0.3 ng/mL. The developed ELISA should be a valuable tool for pharmacokinetic studies and therapeutic drug monitoring of DAP.

Effects of resistance training using elastic bands on muscle strength with or without a leucine supplement for 48 weeks in elderly patients with type 2 diabetes.

Type 2 diabetes is associated with sarcopenia. Resistance training and appropriate nutritional therapy are reported to be effective for muscle strength and mass. This study aimed to evaluate the effect of resistance training using elastic bands at home combined with a leucine-rich amino acid supplement on muscle strength, physical function, and muscle mass in elderly type 2 diabetes. We conducted a 48-week prospective single-center randomized controlled trial in 60 patients who were randomly allocated to one of three groups: control (C), resistance exercise (R), and resistance exercise plus supplement (RL). R and RL groups performed daily bodyweight resistance training with elastic bands exercises at home, and the RL group also took 6 g of a leucine-rich amino acid supplement daily. Knee extension strength (muscle strength), grip strength, usual gait speed (physical function), muscle mass, and cognitive function were assessed at 0 and 48 weeks. Although the change in knee extension strength from baseline was significantly increased by 6.4 Nm (95% CI 1.0, 11.7) in the RL group (p = 0.036), no significant difference was observed among the three groups (p = 0.090). Physical function, muscle mass, and cognitive function also had no changes during the study period among the three groups. No additive effect of a leucine-rich amino acid supplement on muscle strength or mass was observed. Although a post hoc analysis comparing with or without resistance training (C group vs. R + RL group) found that knee extension strength was significantly increased (p = 0.028), and cognitive decline was less (p = 0.046) than in the C group.

Immunohistochemical Pharmacokinetics of the Anti-diabetes Drug Alogliptin in Rat Kidney and Liver.

Alogliptin is one of a new class of therapeutic agents for type 2 diabetes called dipeptidyl peptidase-4 inhibitors. Here, we used immunohistochemistry to investigate the pharmacokinetics of alogliptin at the cell and tissue levels in the rat kidney and liver. One hour after alogliptin administration, the most noticeable immunoreactivity in the kidney was a moderate-to-strong staining in proximal tubule S3 segment epithelial cells. On the other hand, immunostaining was found only in the microvilli of S1 and S2 segment cells. Immunoreactivity was also observed in the glomerulus and distal tubules. Positive cells and almost negative cells coexisted in the collecting ducts. Twenty-four hours after administration, moderate immunostaining remained in the S3 segment but staining in other regions had almost disappeared. In the liver 1 hr after administration, hepatocyte staining differed in the hepatic lobule, with zone III being stronger than zone I. Immunostaining had almost disappeared 24 hr after administration. These findings suggest that alogliptin reabsorption at the kidney and uptake at the hepatocyte vary from region to region and that one or more types of transporter are involved in these processes. In addition, long-term alogliptin use may cause the drug to accumulate in S3 segment, leading to adverse events.

Metabolism and disposition of oseltamivir (OS) in rats, determined by immunohistochemistry with monospecific antibody for OS or its active metabolite oseltamivir carboxylate (OC): A possibility of transporters dividing the drugs' excretion into the bile and kidney.

Among any drugs, no comparative pharmacological study on how prodrug and its active metabolite behave in animal bodies is available. Immunohistochemistry (IHCs) using newly prepared two monoclonal antibodies, AOS-96 and AOC-160, monospecific for oseltamivir (OS) and its metabolite oseltamivir carboxylate (OC) were developed, simultaneously detecting the uptake or excretion of OS and OC in the intestine, liver, and kidney of rats to which OS was orally administered. In the intestine, IHC for OS revealed OS highly distributed to the absorptive epithelia with heavily stained cytoplasmic small granules (CSGs). IHC for OC showed that OC also distributed highly in the epithelia, but without CSGs, suggesting that OS was partly converted to OC in the cells. In the liver, OS distributed in the hepatocytes and on their bile capillaries, as well as on the lumina from the bile capillaries to the interlobular bile ducts. OC distributed in the whole cell of the hepatocytes, but without CSGs nor on any lumina through the interlobular bile ducts. In the kidney, a few levels of OS distributed in the cytoplasm of almost all the renal tubule cells, but they contained numerous CSGs. In contrast, OC distributed highly in the proximal tubules, but very slightly in the lower renal tubules of the nephrons. Thus, it was concluded that the two drugs behave in completely different ways in rat bodies. This paper also discusses a possibility of the correlation of OS or OC levels in tissue cells with their known transporters.

[Localization and Accumulation Studies of Daptomycin in Rats Kidney Using Immunohistochemistry].

Daptomycin (DAP) has a completely different mechanism of action compared to conventional methicillin-resistant Staphylococcus aureus (MRSA) drugs and is widely used clinically as the first-line drug for the treatment of skin soft tissue infection and sepsis caused by MRSA infection. However, the most serious side effects of DAP include renal dysfunction and rhabdomyolysis. Knowledge of the time sequence of localization of DAP in cells and tissues of animals may help in developing a better understanding of the actual overall pharmacokinetics of DAP. We prepared DAP-specific antibodies by immunizing mice with DAP-GMBS-BSA conjugate. The Anti-DAP antibody was specific for DAP, which enabled us to develop an immunocytochemical method for detecting the uptake of DAP in the rat kidneys. One hour after a single intravenous (i.v.) injection of DAP at 12 mg/kg, immunohistochemical observation showed a strong ring-like positive reaction in the cytoplasm immediately below the microvilli of proximal tubule epithelial cells. The distal tubules and collecting ducts contained DAP-positive and negative cells in the cross section of one tubule. Twenty-four hours after DAP administration, several strong positive reactions of different sizes were observed in the cytoplasm of epithelial cells at the proximal tubule. No staining was detected after 7 days. This study will be a useful tool for analyzing the pharmacokinetics of DAP.

A Functional Parathyroid Cyst from the Hemorrhagic Degeneration of a Parathyroid Adenoma.

A 77-year-old man with a history of hypertension, prostate hyperplasia, and urolithiasis was admitted for acute kidney injury caused by hypercalcemia. Neck ultrasonography showed a large cyst adjacent to the right lower thyroid lobe. Although a 99mtechnetium sestamibi scan was negative, an extremely high intracystic intact parathyroid hormone level suggested that the cyst had a parathyroid origin and that a functional parathyroid cyst was present. Immunohistochemical staining for the calcium-sensing receptor (CaSR) after right lower parathyroidectomy revealed CaSR-positive cells lining the cyst, indicating that the functional parathyroid cyst had originated from the hemorrhagic degeneration of a parathyroid adenoma.

Effects of anagliptin on plasma glucagon levels and gastric emptying in patients with type 2 diabetes: An exploratory randomized controlled trial versus metformin.

AIMS: Glucagon has an important role in glucose homeostasis. Recently, a new plasma glucagon assay based on liquid chromatography-high resolution mass spectrometry was developed. We evaluated the influence of a dipeptidyl peptidase-4 inhibitor (anagliptin) on plasma glucagon levels in Japanese patients with type 2 diabetes by using this new assay. METHODS: Twenty-four patients with type 2 diabetes were enrolled in a prospective, single-center, randomized, open-label study and were randomly allocated to 4 weeks of treatment with metformin (1000 mg/day) or anagliptin (200 mg/day). A liquid test meal labeled with sodium [13C] acetate was ingested before and after the treatment period. Samples of blood and expired air were collected over 3 h. Plasma levels of glucose, glucagon, C-peptide, glucagon-like peptide-1 (GLP-1), and glucose-dependent insulinotropic polypeptide (GIP) were measured, and gastric emptying was also evaluated. RESULTS: Twenty-two patients completed the study (metformin group: n = 10; anagliptin group: n = 12). Glycemic control showed similar improvement in both groups. In the anagliptin group, there was a slight decrease of the incremental area under the plasma concentration versus time curve for glucagon after the test meal (P = 0.048). In addition, the plasma level of active GLP-1 and GIP was increased, and plasma C-peptide was also increased versus baseline. Neither anagliptin nor metformin delayed gastric emptying. CONCLUSIONS: In patients with type 2 diabetes maintained endogenous insulin secretion, anagliptin increased the plasma level of active GLP-1 and GIP in association with a slight stimulation of insulin secretion and slight inhibition of glucagon secretion, but did not delay gastric emptying. Clinical Trial Registry: University hospital Medical Information Network UMIN000028293.

ARMC5 Alterations in Primary Macronodular Adrenal Hyperplasia (PMAH) and the Clinical State of Variant Carriers.

CONTEXT: Primary macronodular adrenal hyperplasia (PMAH) is a rare type of Cushing or subclinical Cushing syndrome and is associated with bilateral multinodular formation. ARMC5 is one of the responsible genes for PMAH. OBJECTIVES: This study was performed to identify the genotype-phenotype correlation of ARMC5 in a cohort of Japanese patients. PATIENTS AND METHODS: Fourteen patients with clinically diagnosed PMAH and family members of selected patients were studied for ARMC5 gene alteration and clinical phenotype. The associated nonadrenal tumor tissues were also studied. RESULTS: Of fourteen patients with PMAH, 10 had pathogenic or likely pathogenic variants of ARMC5. We found two variants. Five unrelated patients had identical variants (p.R619*). In two patients, the variant was found in offspring with the asymptomatic or presymptomatic state. Six of ten patients who tested positive for the ARMC5 pathogenic or likely pathogenic variant carried nonadrenal tumors; however, no loss of heterozygosity (LOH) or second hit of the ARMC5 gene was evident. The ARMC5 variant-positive group showed a significantly higher basal cortisol level. Furthermore, age-dependent cortisol hypersecretion was seen in the ARMC5 variant-positive group. CONCLUSIONS: ARMC5 pathogenic variants are common (71%) in Japanese patients with PMAH. p.R619* might be a hot spot in Japanese patients with PMAH. Asymptomatic or presymptomatic pathogenic variant carriers were found among the family members of the patients. Although 50% of ARMC5 variant carriers had nonadrenal neoplastic lesions, no LOH or second hit of ARMC5 in the tumor tissues was evident. The ARMC5 variant-positive mutant group showed a higher basal cortisol level than the negative group.