Association between brain imaging biomarkers and continuous glucose monitoring-derived glycemic control indices in Japanese patients with type 2 diabetes mellitus.

INTRODUCTION: Although type 2 diabetes mellitus (T2DM) is associated with alterations in brain structure, the relationship between glycemic control indices and brain imaging markers remains unclear. This study aimed to investigate the association between continuous glucose monitoring (CGM)-derived glycemic control indices and brain imaging biomarkers assessed by MRI. RESEARCH DESIGN AND METHODS: This cross-sectional study included 150 patients with T2DM. The severity of cerebral white matter lesions (WMLs) was assessed using MRI for deep and subcortical white matter and periventricular hyperintensities. The degree of medial temporal lobe atrophy (MTA) was assessed using voxel-based morphometry. Each participant wore a retrospective CGM for 14 consecutive days, and glycemic control indices, such as time in range (TIR) and glycemia risk index (GRI), were calculated. RESULTS: The proportion of patients with severe WMLs showed a decreasing trend with increasing TIR (P for trend=0.006). The proportion of patients with severe WMLs showed an increasing trend with worsening GRI (P for trend=0.011). In contrast, no significant association was observed between the degree of MTA and CGM-derived glycemic control indices, including TIR (P for trend=0.325) and GRI (P for trend=0.447). CONCLUSIONS: The findings of this study indicate that the severity of WMLs is associated with TIR and GRI, which are indices of the quality of glycemic control. TRIAL REGISTRATION NUMBER: UMIN000032143.

Association between continuous glucose monitoring-derived glycemic control indices and urinary biomarkers of diabetic kidney disease: Hyogo Diabetes Hypoglycemia Cognition Complications study.

AIMS: Glomerular damage and proximal tubular damage play an important role in the pathogenesis of diabetic kidney disease. This study aimed to investigate the relationship between the urinary markers of proximal tubular injury, including urinary liver-type fatty acid-binding protein-to-creatinine ratio (uL-FABP/Cr) and urinary N-acetyl-ß-D-glucosaminidase-to-creatinine ratio (uNAG/Cr), and glycemic control status. METHODS: This cross-sectional study included 245 and 39 patients with type 2 diabetes mellitus (T2DM) and non-T2DM (NDM), respectively. The participants of this study were fitted with retrospective CGM, and glycemic control indices, such as time in range (TIR) and glycemia risk index (GRI), were calculated. RESULTS: The results were presented as medians (interquartile ranges). The uL-FABP/Cr was significantly higher in the microalbuminuria than in the normo-albuminuria group [4.2 (2.7-7.1) and 2.2 (1.4-3.4) µg/gCr, respectively, P < 0.001], while the uNAG/Cr in the normo-albuminuria group [6.3 (4.5-10.1) U/gCr] was significantly higher than that in the NDM group [5.3 (3.8-6.3) U/gCr, P = 0.048] but significantly lower than that in the microalbuminuria group [9.2 (6.4-11.1) U/gCr, P = 0.004]. The multivariate logistic regression analysis indicated that CGM-derived TIR was significantly associated with the urinary albumin-to-creatinine ratio [uAlb/Cr, odds ratio (OR) 0.985, 95% confidence interval (CI) 0.971-0.998, P = 0.029] and uNAG/Cr (OR 0.973, 95% CI 0.957-0.989, P = 0.001) independent of renal function. GRI was similarly associated with uAlb/Cr and uNAG/Cr. CONCLUSION: The findings of this study indicated that uNAG/Cr was elevated before albuminuria development and was associated with CGM-derived TIR and GRI.

Association between low-carbohydrate diets and continuous glucose monitoring-derived time in ranges.

AIMS: Low-carbohydrate diets have become popular in the general community. The mutual relationship between the percentage of total energy intake from carbohydrates (CHO/E), glycemic control indices, and diabetes complications remains unclear. MATERIALS AND METHODS: This cross-sectional study included 177 patients with type 2 diabetes mellitus who regularly visited outpatient clinics. In this study, dietary questionnaires were used to assess the intake ratio of the three macronutrients, and the low-carbohydrate-diet score was calculated. We investigated the association between the low-carbohydrate-diet score, continuous glucose monitoring (CGM)-derived short-term glycemic control indices, and diabetes complications in patients with type 2 diabetes mellitus. RESULTS: The results are presented as medians (interquartile ranges) unless otherwise stated. Hemoglobin A1c was 7.1% (6.6-7.7%), CGM-derived time in range (TIR) was 75.3% (62.8-87.0%), body mass index (BMI) was 24.0 (22.1-26.3) kg/m2, and CHO/E was 49.8% (44.8-55.6%). BMI, triglycerides, and CGM-derived time above range decreased significantly with increasing low-carbohydrate-diet scores. However, no significant association was found between the low-carbohydrate-diet score and glycemic control indices, including TIR, mean amplitude of glycemic excursions, and vascular complications of type 2 diabetes mellitus. CONCLUSION: Moderate-carbohydrate diets positively impact weight control and lipid metabolism but may have a limited effect on short-term glycemic variability in Japanese patients with type 2 diabetes mellitus.

The lignin-degrading abilities of Gelatoporia subvermispora gat1 and pex1 mutants generated via CRISPR/Cas9.

White-rot fungi efficiently degrade wood lignin; however, the mechanisms involved remain largely unknown. Recently, a forward genetics approach to identify several genes in Pleurotus ostreatus (Agaricales) in which mutations cause defects in wood lignin degradation was used. For example, pex1 encodes a peroxisome biogenesis factor and gat1 encodes a putative Agaricomycetes-specific DNA-binding transcription factor. In this study, we examined the effects of single-gene mutations in pex1 or gat1 on wood lignin degradation in another white-rot fungus, Gelatoporia (Ceriporiopsis) subvermispora (Polyporales), to investigate conserved and derived degradation mechanisms in white-rot fungi. G. subvermispora pex1 and gat1 single-gene mutant strains were generated from a monokaryotic wild-type strain, FP-90031-Sp/1, using plasmid-based CRISPR/Cas9. As in P. ostreatus, Gsgat1 mutants were nearly unable to degrade lignin sourced from beech wood sawdust medium (BWS), while Gspex1 mutants exhibited a delay in lignin degradation. We also found that the transcripts of lignin-modifying enzyme-encoding genes, mnp4, mnp5, mnp6, mnp7, and mnp11, which predominantly accumulate in FP-90031-Sp/1 cultured with BWS, were greatly downregulated in Gsgat1 mutants. Taken together, the results suggest that Gat1 may be a conserved regulator of the ligninolytic system of white-rot fungi and that the contribution of peroxisomes to the ligninolytic system may differ among species.

CRISPR/Cas9 using a transient transformation system in Ceriporiopsis subvermispora.

Ceriporiopsis subvermispora is a white-rot fungus with great potential for industrial and biotechnological applications, such as the pretreatment of lignocellulose in biorefineries, as it decomposes the lignin in the plant cell wall without causing severe cellulose degradation. A genetic transformation system was recently developed; however, gene-targeting experiments to disrupt or modify the gene(s) of interest remain challenging, and this is a bottleneck for further molecular genetic studies and breeding of C. subvermispora. Herein, we report efficient clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated protein 9 (Cas9)-assisted gene mutagenesis in this fungus. Two plasmids expressing Cas9 together with a different pyrG-targeting single-guide RNA were separately introduced into the monokaryotic C. subvermispora strain FP-90031-Sp/1, which frequently generated strains that exhibited resistance to 5-fluoroorotic acid and uridine/uracil auxotrophy. Southern blot analyses and genomic polymerase chain reaction followed by DNA sequencing of some mutants revealed that they were pyrG mutants. We also observed that hygromycin resistance of the pyrG mutants was frequently lost after repeated subcultivations, indicating that a maker-free genome editing occurred successfully. It is also suggested that a gene mutation(s) can be introduced via a transient expression of Cas9 and a single-guide RNA; this feature, together with high-frequency gene targeting using the CRISPR/Cas9 system, would be helpful for studies on lignocellulose-degrading systems in C. subvermispora. KEY POINTS: • Efficient plasmid-based CRISPR/Cas9 was established in C. subvermispora. • The mutations can be introduced via a transient expression of Cas9 and sgRNA. • A maker-free CRISPR/Cas9 is established in this fungus.

Efficient genome editing with CRISPR/Cas9 in Pleurotus ostreatus.

Pleurotus ostreatus is one of the most commercially produced edible mushrooms worldwide. Improved cultivated strains with more useful traits have been obtained using classical breeding, which is laborious and time-consuming. Here, we attempted efficient gene mutagenesis using plasmid-based CRISPR/Cas9 as the first step for non-genetically modified (non-GM) P. ostreatus generation. Plasmids harboring expression cassettes of Cas9 and different single guide RNAs targeting fcy1 and pyrG were individually transferred into fungal protoplasts of the PC9 strain, which generated some strains exhibiting resistance to 5-fluorocytosine and 5-fluoroorotic acid, respectively. Genomic PCR followed by sequencing revealed small insertions/deletions or insertion of a fragment from the plasmid at the target site in some of the drug-resistant strains. The results demonstrated efficient CRISPR/Cas9-assisted genome editing in P. ostreatus, which could contribute to the molecular breeding of non-GM cultivated strains in the future. Furthermore, a mutation in fcy1 via homology-directed repair using this CRISPR/Cas9 system was also efficiently introduced, which could be applied not only for precise gene disruption, but also for insertions leading to heterologous gene expression in this fungus.

A promoter assay system using gene targeting in agaricomycetes Pleurotus ostreatus and Coprinopsis cinerea.

A novel promoter assay was developed for Agaricomycetes, using a gene-targeting approach, with or without the CRISPR/Cas9 technique. It enables precise evaluation of promoter activity at the original site of the chromosome without random and multiple integrations in conventional transformation experiments.

An Early Arising Role of the MicroRNA156/529-SPL Module in Reproductive Development Revealed by the Liverwort Marchantia polymorpha.

In angiosperms, the phase transition from vegetative to reproductive growth involves the de-repression of the squamosa promoter-binding-protein-like (SPL) class of transcription factors, which is negatively regulated by the specific microRNAs (miRNAs/miRs) miR156/529 [1]. Non-vascular land plants also undergo growth-phase transition to the reproductive state, but knowledge regarding the controlling mechanisms is limited. Here, we investigate the reproductive transition in the liverwort Marchantia polymorpha, focusing on the roles of miR529c [2-4] and MpSPL2. First, we established mir529c-null mutants using CRISPR/Cas9. Even in the absence of far-red light-supplemented long-day condition, which is usually needed to induce reproductive development [5, 6], the mutant thalli developed sexual reproductive organs (gametangia) and produced gametes. Transgenic plants expressing a miR529-resistant MpSPL2 transgene also showed a similar phenotype of reproductive transition in the absence of inductive far-red light signals. In these mutants and transgenic plants, the MpSPL2 mRNA abundance was elevated. Mpspl2ko mutant plants showed successful gamete development and fertilization, which suggests that MpSPL2 is involved in, but not essential for, sexual reproduction in M. polymorpha. Furthermore, analysis of Mpspl2ko mutant and its complemented lines suggests that MpSPL2 may have a role in promotion of reproductive transition. These findings support the notion that the transition to reproductive development in liverworts is controlled by a system similar to that in angiosperms, and the miR156/529-SPL module has common significance in the control of the vegetative-to-reproductive transition during development in many land plants, including liverworts.

[Adverse Events after the Introduction of Quadrivalent Influenza Vaccine in Comparison with AH1pdm Vaccine (2009) in Japan].

Inactivated quadrivalent influenza vaccine (IIV4) has been used as seasonal influenza vaccine since 2016 in Japan. This study examined the safety of IIV4 in comparison with the AH1pdm monovalent vaccine used for novel influenza in 2009. Questionnaire surveillance associated with adverse events (AEs) was conducted at Chiba University Hospital, Japan. After being vaccinated, all health care workers (HCWs) were given a daily AEs check sheet on which they recorded solicited events, the same surveillance program used after AH1pdm vaccination in 2009. The frequency of injection site AEs with IIV4 was significantly higher than with the monovalent vaccine, but there was no significant difference with systemic AEs. Injection site and systemic AEs were reported as 83.7% and 25.5%, respectively, with IIV4. The grades of AE, mild, moderate and severe, were 67.2%, 16.4% and 0.1% with IIV4, respectively, indicating that almost all of the AEs reported with IIV4 were mild or moderate. Systemic AEs with IIV4 and monovalent vaccine were reported to be 25.5% and 23.1%, respectively, with the difference not being significant. The grade of AEs with IIV4, mild, moderate and severe, was 19.1%, 5.6% and 0.9%, respectively. The ratio of HCWs reporting AEs peaked at around 80% on day 1, then decreasing to less than 5% by day 7. AEs with IIV4 were reported more frequently compared with the AH1pdm monovalent vaccine. However, in consideration of the grade and duration of AEs, IIV4 was a well-tolerated, safe vaccine.

Medaka and zebrafish contactin1 mutants as a model for understanding neural circuits for motor coordination.

A spontaneous medaka ro mutant shows abnormal wobbling and rolling swimming behaviors. By positional cloning, we mapped the ro locus to a region containing the gene encoding Contactin1b (Cntn1b), which is an immunoglobulin (Ig)-superfamily domain-containing membrane-anchored protein. The ro mutant had a deletion in the cntn1b gene that introduced a premature stop codon. Furthermore, cntn1b mutants generated by the CRISPR/Cas9 system and trans-heterozygotes of the CRISPR mutant allele and ro had abnormal swimming behavior, indicating that the cntn1b gene was responsible for the ro-mutant phenotype. We also established zebrafish cntn1a and cntn1b mutants by transcription activator-like effector nucleases (TALENs). Zebrafish cntn1b but not cntn1a mutants showed abnormal swimming behaviors similar to those in the ro mutant, suggesting that Cntn1b plays a conserved role in the formation or function of the neural circuits that control swimming in teleosts. Although Cntn1-deficient mice have abnormal cerebellar neural circuitry, there was no apparent histological abnormality in the cerebellum of medaka or zebrafish cntn1b mutants. The medaka cntn1b mutants had defective optokinetic response (OKR) adaptation and abnormal rheotaxis (body positioning relative to water flow). Medaka and zebrafish cntn1b mutants are effective models for studying the neural circuits involved in motor learning and motor coordination.

Sox5 functions as a fate switch in medaka pigment cell development.

Mechanisms generating diverse cell types from multipotent progenitors are crucial for normal development. Neural crest cells (NCCs) are multipotent stem cells that give rise to numerous cell-types, including pigment cells. Medaka has four types of NCC-derived pigment cells (xanthophores, leucophores, melanophores and iridophores), making medaka pigment cell development an excellent model for studying the mechanisms controlling specification of distinct cell types from a multipotent progenitor. Medaka many leucophores-3 (ml-3) mutant embryos exhibit a unique phenotype characterized by excessive formation of leucophores and absence of xanthophores. We show that ml-3 encodes sox5, which is expressed in premigratory NCCs and differentiating xanthophores. Cell transplantation studies reveal a cell-autonomous role of sox5 in the xanthophore lineage. pax7a is expressed in NCCs and required for both xanthophore and leucophore lineages; we demonstrate that Sox5 functions downstream of Pax7a. We propose a model in which multipotent NCCs first give rise to pax7a-positive partially fate-restricted intermediate progenitors for xanthophores and leucophores; some of these progenitors then express sox5, and as a result of Sox5 action develop into xanthophores. Our results provide the first demonstration that Sox5 can function as a molecular switch driving specification of a specific cell-fate (xanthophore) from a partially-restricted, but still multipotent, progenitor (the shared xanthophore-leucophore progenitor).

Comparison of tutored group with tutorless group in problem-based mixed learning sessions: a randomized cross-matched study.

BACKGROUND: Problem-based learning (PBL) involves discussions among students who resolve loosely-structured problems to facilitate learning. In the PBL curriculum, faculty tutors are employed as facilitators for small groups of students. Because of lack of time and staff shortage, the effectiveness of tutorless PBL has been discussed as an alternate option. METHODS: Sessions in which tutored and tutorless PBL groups are mixed were presented by 1st-year medical students, who experienced both tutored and tutorless groups alternately in the two sessions of a year. To examine the effectiveness of tutored and tutorless PBL, written examination scores (WES) and self-contentment scores (SCS) were statistically analysed. RESULTS: WES averages did not significantly differ between the tutored and tutorless groups; however, a significantly greater variation was observed in WES in the tutorless group. SCS averages tended to be higher in the tutored PBL than in tutorless PBL groups. CONCLUSIONS: Students in these tutorless PBL groups performed well in their written examinations, whereas those in the tutored PBL groups, achieved this and reported better self-contentment with their learning experience. Tutorless PBL sessions were considered to be comparable to tutored PBL sessions at least in the early stages.

Pitfalls in training simulated patients to respond appropriately to questions from medical students in family history-taking activities: the current situation surrounding the training of simulated patients for learning activities at Nippon Medical School.

Our goal was to train simulated patients (SPs) to respond appropriately to questions about family history from medical students in simulated medical interviews. To this end, we carried out a survey of 91 SPs and 76 4th-year medical students to investigate their notions of what constitutes a family. All of the SPs and students surveyed deemed parents and children living together to be members of a family. In a situation where one spouse's parents live together with the basic family unit, 93% of the SPs considered them to be members of the family, whereas only 79% of the students did. Married children living apart from their parents were considered members of the family by 18% of the SPs and 39% of the students. These results indicate clear differences between the SPs and students in their notions of the family. To verify the level of understanding of the definitions of family and blood relatives in particular scenarios used in simulated medical interviews, we administered a written test to 14 SPs who were training to assist in the nationwide common achievement test in medicine, the Objective Structured Clinical Examination (OSCE). The overall score of the SPs was 93.5%; the incorrect answers were "a sibling is not a blood relative" and "a spouse is a blood relative." We analyzed the performance of these 14 SPs in medical interviews carried out after training for the OSCE, in which they were asked questions that required them to reveal their understanding of blood relatives, cohabiting relatives, and the family. All of the SPs responded appropriately to the students' questions about family history. After the OSCE, we asked the SPs to assess themselves on how well they had given their family histories and to evaluate the usefulness of the SP training they had received. Their mean self-assessment score on providing a family history was 3.6 (scale: 1-4); on the usefulness of training, it was 3.4 (scale: 1-4). In conclusion, training SPs to respond appropriately to questions about family history in medical interviews is very important. Medical students have to learn how to take family histories accurately, so SP trainers should pay attention to training SPs in giving appropriate responses to students' questions, bearing in mind the differences between family history taking and everyday conversations about the family.

Polycystic kidney disease in the medaka (Oryzias latipes) pc mutant caused by a mutation in the Gli-Similar3 (glis3) gene.

Polycystic kidney disease (PKD) is a common hereditary disease in humans. Recent studies have shown an increasing number of ciliary genes that are involved in the pathogenesis of PKD. In this study, the Gli-similar3 (glis3) gene was identified as the causal gene of the medaka pc mutant, a model of PKD. In the pc mutant, a transposon was found to be inserted into the fourth intron of the pc/glis3 gene, causing aberrant splicing of the pc/glis3 mRNA and thus a putatively truncated protein with a defective zinc finger domain. pc/glis3 mRNA is expressed in the epithelial cells of the renal tubules and ducts of the pronephros and mesonephros, and also in the pancreas. Antisense oligonucleotide-mediated knockdown of pc/glis3 resulted in cyst formation in the pronephric tubules of medaka fry. Although three other glis family members, glis1a, glis1b and glis2, were found in the medaka genome, none were expressed in the embryonic or larval kidney. In the pc mutant, the urine flow rate in the pronephros was significantly reduced, which was considered to be a direct cause of renal cyst formation. The cilia on the surface of the renal tubular epithelium were significantly shorter in the pc mutant than in wild-type, suggesting that shortened cilia resulted in a decrease in driving force and, in turn, a reduction in urine flow rate. Most importantly, EGFP-tagged pc/glis3 protein localized in primary cilia as well as in the nucleus when expressed in mouse renal epithelial cells, indicating a strong connection between pc/glis3 and ciliary function. Unlike human patients with GLIS3 mutations, the medaka pc mutant shows none of the symptoms of a pancreatic phenotype, such as impaired insulin expression and/or diabetes, suggesting that the pc mutant may be suitable for use as a kidney-specific model for human GLIS3 patients.

Kidney regeneration through nephron neogenesis in medaka.

Although renal regeneration is limited to repair of the proximal tubule in mammals, some bony fish are capable of renal regeneration through nephron neogenesis in the event of renal injury. We previously reported that nephron development in the medaka mesonephros is characterized by four histologically distinct stages, generally referred to as condensed mesenchyme, nephrogenic body, relatively small nephron, and the mature nephron. Developing nephrons are positive for wt1 expression during the first three of these stages. In the present study, we examined the regenerative response to renal injury, artificially induced by the administration of sublethal amounts of gentamicin in adult medaka. Similar to previous reports in other animals, the renal tubular epithelium and the glomerulus of the medaka kidney exhibited severe damage after exposure to this agent. However, kidneys showed substantial recovery after gentamicin administration, and a significant number of developing nephrons appeared 14 days after gentamicin administration (P < 0.01). Similarly, the expression of wt1 in developing nephrons also indicated the early stages of nephrogenesis. These findings show that medaka has the ability to regenerate kidney through nephron neogenesis during adulthood and that wt1 is a suitable marker for detecting nephrogenesis.

Population data of six STR loci using hexaplex PCR amplification and typing system, "Midi-6" in four regional populations in Japan.

The genetic differences of the allele frequency distributions for six STR loci (D20S480, D6S2439, D6S1056, D9S1118, D4S2639, and D17S1290) among regions in Japan were examined using our recently designed hexaplex amplification and typing system, "Midi-6" newly named, to construct a database in the Japanese population. Genotypes at six loci were analyzed in 198, 200, 175, and 196 individuals from the area of Akita, Nagoya, Oita, and Okinawa, respectively, in Japan. The allele frequency distributions were significantly different (p<0.05) at from one to five loci among the four populations when compared pairwise. Significant differences were also observed at two or three loci between Oita- or Okinawa-Japanese and the "pooled" population (n=769), respectively. However, since F(ST) (theta) values were extremely low (<0.05), ranging from 0.0020 to 0.0118 for six loci, genetic differentiation within the pooled Japanese population was negligible. Therefore, it suggested that the data of the allele frequencies at six loci in the pooled population would be employed as the base of calculation for statistical probabilities.

The Tomita collection of medaka pigmentation mutants as a resource for understanding neural crest cell development.

All body pigment cells in vertebrates are derived from the neural crest. In fish the neural crest can generate up to six different types of pigment cells, as well as various non-pigmented derivatives. In mouse and zebrafish, extensive collections of pigmentation mutants have enabled dissection of many aspects of pigment cell development, including fate specification, survival, proliferation and differentiation. A collection of spontaneous mutations collected from wild medaka (Oryzias latipes) populations and maintained at Nagoya University includes more than 40 pigmentation mutations. The descriptions of their adult phenotypes have been previously published by Tomita and colleagues (summarised in Medaka (Killifish) Biology and Strains, 1975), but the embryonic phenotypes have not been systematically described. Here we examine these embryonic phenotypes, paying particular attention to the likely defect in pigment cell development in each, and comparing the spectrum of defects to those in the zebrafish and mouse collections. Many phenotypes parallel those of identified zebrafish mutants, although pigment cell death phenotypes are largely absent, presumably due to the different selective pressures under which the mutants were isolated. We have identified mutant phenotypes that may represent the Mitf/Kit pathway of melanophore specification and survival. We use in situ hybridisation with available markers to confirm a key prediction of this hypothesis. We also highlight a set of novel phenotypes not seen in the zebrafish collection. These mutants will be a valuable resource for pigment cell and neural crest studies and will strongly complement the mutant collections in other vertebrates.

Immunofluorescence analysis of neutrophil nonmuscle myosin heavy chain-A in MYH9 disorders: association of subcellular localization with MYH9 mutations.

The autosomal dominant macrothrombocytopenia with leukocyte inclusions, May-Hegglin anomaly, Sebastian syndrome, and Fechtner syndrome, are rare human disorders characterized by a triad of giant platelets, thrombocytopenia, and characteristic Döhle body-like cytoplasmic inclusions in granulocytes. Epstein syndrome is another autosomal dominant macrothrombocytopenia associated with Alport syndrome but without leukocyte inclusions. These disorders are caused by mutations in the same gene, the MYH9, which encodes the nonmuscle myosin heavy chain-A (NMMHCA). The term, MYH9 disorders, has been proposed, but the clinicopathologic basis of MYH9 mutations has been poorly investigated. In this study, a total of 24 cases with MYH9 disorders and suspected cases were subjected to immunofluorescence analysis by a polyclonal antibody against human platelet NMMHCA. Abnormal subcellular localization of NMMHCA was observed in every neutrophil from individuals with MYH9 mutations. Comparison with May-Grünwald-Giemsa staining revealed that the NMMHCA always coexisted with the neutrophil inclusion bodies, suggesting that NMMHCA is associated with such bodies. In three cases, neutrophil inclusions were not detected on conventional May-Grünwald-Giemsa-stained blood smears but immunofluorescence analysis revealed the abnormal NMMHCA localization. In contrast, cases with Epstein syndrome and the isolated macrothrombocytopenia with normal NMMHCA localization had no MYH9 mutations. An antibody that recognizes the C-terminal 12 mer peptides showed similar immunoreactivity from the patients heterozygous for truncated mutations that abolished the C-terminal epitope, suggesting that normal NMMHCA dimerizes with abnormal NMMHCA to form inclusion bodies. We further propose that the localization pattern can be classified into three groups according to the number, size, and shape of the fluorescence-labeled NMMHCA granule. Immunofluorescence analysis of neutrophil NMMHCA is useful as a screening test for the clear hematopathologic classification of MYH9 disorders.