Translational regulation and protein-coding capacity of the 5' untranslated region of human TREM2.

TREM2 is a transmembrane receptor expressed in microglia and macrophages. Elevated TREM2 levels in these cells are associated with age-related pathological conditions, including Alzheimer's disease. However, the regulatory mechanism underlying the protein expression of TREM2 remains unclear. In this study, we uncover the role of the 5' untranslated region (5'-UTR) of human TREM2 in translation. An upstream start codon (uAUG) in the 5'-UTR of TREM2 is specific to some primates, including humans. The expression of the conventional TREM2 protein, starting from the downstream AUG (dTREM2), is repressed by the 5'-UTR in a uAUG-mediated manner. We also detect a TREM2 protein isoform starting from uAUG (uTREM2) that is largely degraded by proteasomes. Finally, the 5'-UTR is essential for the downregulation of dTREM2 expression in response to amino acid starvation. Collectively, our study identifies a species-specific regulatory role of the 5'-UTR in TREM2 translation.

Alzheimer's Disease-Associated Alternative Splicing of CD33 Is Regulated by the HNRNPA Family Proteins.

Genetic variations of CD33 have been implicated as a susceptibility factor of Alzheimer's disease (AD). A polymorphism on exon 2 of CD33, rs12459419, affects the alternative splicing of this exon. The minor allele is associated with a reduced risk of AD and promotes the skipping of exon 2 to produce a shorter CD33 isoform lacking the extracellular ligand-binding domain, leading to decreased suppressive signaling on microglial activity. Therefore, factors that regulate the splicing of exon 2 may alter the disease-associated properties of CD33. Herein, we sought to identify the regulatory proteins of CD33 splicing. Using a panel of RNA-binding proteins and a human CD33 minigene, we found that exon 2 skipping of CD33 was promoted by HNRNPA1. Although the knockdown of HNRNPA1 alone did not reduce exon 2 skipping, simultaneous knockdown of HNRNPA1 together with that of HNRNPA2B1 and HNRNPA3 promoted exon 2 inclusion, suggesting functional redundancy among HNRNPA proteins. Similar redundant regulation by HNRNPA proteins was observed in endogenous CD33 of THP-1 and human microglia-like cells. Although mouse Cd33 showed a unique splicing pattern of exon 2, we confirmed that HNRNPA1 promoted the skipping of this exon. Collectively, our results revealed novel regulatory relationships between CD33 and HNRNPA proteins.

Inhibition of BACE1 and amyloid ß aggregation by polyketide from Streptomyces sp.

Alzheimer's disease (AD) causes cognitive impairment in the elderly and is a severe problem worldwide. One of the major reasons for the pathogenesis of AD is thought to be due to the accumulation of amyloid beta (Aß) peptides that result in neuronal cell death in the brain. In this study, bioassay-guided fractionation was performed to develop seed compounds for anti-AD drugs that can act as dual inhibitors of BACE1 and Aß aggregation from secondary metabolites produced by Streptomyces sp. To improve the solubility, the crude extracts were methylated with trimethylsilyl (TMS) diazomethane and then purified to yield polyketides 1-5, including the new compound 1. We synthesized the compounds 6 and 7 (original compounds 2 and 3, respectively), and their activities were evaluated. KS-619-1, the demethylated form of 4 and 5, was isolated and evaluated for its inhibitory activity. The IC50 values for BACE1 and Aß aggregation were found to be 0.48 and 1.1 µM, respectively, indicating that KS-619-1 could be a lead compound for the development of therapeutic agents for AD.

Inhibition of BACE1 and Amyloid-ß Aggregation by Meroterpenoids from the Mushroom Albatrellus yasudae.

To develop drugs to treat Alzheimer's disease (AD) on the basis of the amyloid cascade hypothesis, the amyloid-ß (Aß) aggregation inhibitory activities of 110 extracts from mushrooms were evaluated by thioflavin T (Th-T) assays. The MeOH extract of Albatrellus yasudae inhibited Aß aggregation, and the bioactivity-guided fractionation of the extract afforded four novel meroterpenoids, named scutigeric acid (1), albatrelactone methyl ester (2), albatrelactone (3), and 10',11'-dihydroxygrifolic acid (4), together with two known compounds, grifolin (5) and grifolic acid (6). The structures of 1-4 were elucidated using NMR, MS, UV, IR, and induced ECD spectral data. The structure of 1 was determined as a methyl ester (1a) by 2D NMR spectroscopy. Th-T assays showed that compounds 1-4 and 1a possessed inhibitory activities against Aß aggregation, with IC50 values of 6.6, 40.7, 51.4, 53.3, and 50.3 µM, respectively. Notably, 1 possessed an inhibitory activity against Aß aggregation comparable to that of myricetin as a positive control. Moreover, 1-6 exhibited inhibitory activities against BACE1, with IC50 values of 1.6, 10.9, 10.5, 34.4, 6.1, and 1.4 µM, respectively.

Reactive astrocytes express Aggregatin (FAM222A) in the brains of Alzheimer's disease and Nasu-Hakola disease.

By combining genomic data and brain imaging data, a recent study has identified a novel gene named FAM222A that participates in the formation of amyloid-ß (Aß) plaques and brain atrophy in Alzheimer's disease (AD). FAM222A encodes a 47-kDa protein designated Aggregatin that accumulates in the center of amyloid plaques and physically interacts with Aß to facilitate Aß aggregation. Aggregatin is expressed predominantly in the central nervous system (CNS) and its levels are increased in brains of the patients with AD and in mouse models of AD. However, at present, the precise cell types that express Aggregatin in the human CNS remain unknown. By immunohistochemistry, we studied Aggregatin expression in the frontal lobe of the patients with AD, Nasu-Hakola disease (NHD), and the subjects who died of non-neurological causes (NNC). We identified the clusters of Aggregatin-positive reactive astrocytes distributed widely in the cerebral cortex of most cases examined. In contrast, small numbers of cortical neurons showed variable immunoreactivities for Aggregatin, whereas microglia and oligodendrocytes did not express Aggregatin. Importantly, amyloid plaques were not clearly labelled with anti-Aggregatin antibody. These results suggest that Aggregatin plays a primarily role in generation of reactive astrocytes in the human CNS.

CELF2 regulates the species-specific alternative splicing of TREM2.

Genetic variations of TREM2 have been implicated as a risk factor of Alzheimer's disease (AD). Recent studies suggest that the loss of TREM2 function compromises microglial responses to the accumulation of amyloid beta. Previously, we found that exon 3 of TREM2 is an alternative exon whose skipping leads to a reduction in full-length TREM2 protein by inducing nonsense-mediated mRNA decay. Here, we aimed to identify factors regulating TREM2 splicing. Using a panel of RNA-binding proteins, we found that exon 3 skipping of TREM2 was promoted by two paralogous proteins, CELF1 and CELF2, which were both linked previously with risk loci of AD. Although the overexpression of both CELF1 and CELF2 enhanced exon 3 skipping, only CELF2 reduced the expression of full-length TREM2 protein. Notably, the TREM2 ortholog in the green monkey, but not in the mouse, showed alternative splicing of exon 3 like human TREM2. Similarly, splicing regulation of exon 3 by CELF1/2 was found to be common to humans and monkeys. Using chimeric minigenes of human and mouse TREM2, we mapped a CELF-responsive sequence within intron 3 of human TREM2. Collectively, our results revealed a novel regulatory factor of TREM2 expression and highlighted a species-dependent difference of its regulation.

FACS-array-based cell purification yields a specific transcriptome of striatal medium spiny neurons in a murine Huntington disease model.

Huntington disease (HD) is a neurodegenerative disorder caused by expanded CAG repeats in the Huntingtin gene. Results from previous studies have suggested that transcriptional dysregulation is one of the key mechanisms underlying striatal medium spiny neuron (MSN) degeneration in HD. However, some of the critical genes involved in HD etiology or pathology could be masked in a common expression profiling assay because of contamination with non-MSN cells. To gain insight into the MSN-specific gene expression changes in presymptomatic R6/2 mice, a common HD mouse model, here we used a transgenic fluorescent protein marker of MSNs for purification via FACS before profiling gene expression with gene microarrays and compared the results of this "FACS-array" with those obtained with homogenized striatal samples (STR-array). We identified hundreds of differentially expressed genes (DEGs) and enhanced detection of MSN-specific DEGs by comparing the results of the FACS-array with those of the STR-array. The gene sets obtained included genes ubiquitously expressed in both MSNs and non-MSN cells of the brain and associated with transcriptional regulation and DNA damage responses. We proposed that the comparative gene expression approach using the FACS-array may be useful for uncovering the gene cascades affected in MSNs during HD pathogenesis.

Isolation of three new meroterpenoids and seven known compounds from Albatrellus yasudae and their Aß-aggregation inhibitory activity.

Alzheimer's disease is a serious neurologic disorder that cannot be cured completely. In this study, we targeted compounds that inhibit amyloid-beta (Aß) aggregation, based on the amyloid cascade hypothesis. Ten compounds (1-10) were isolated from CHCl3 extracts of the mushroom Albatrellus yasudae using Aß-aggregation inhibitory activity-guided separation. The structures of these compounds were elucidated from 1D and 2D NMR and MS spectral data. Compounds 1-3 were novel, whereas 4-10 were identified as the known compounds grifolin, grifolic acid, neogrifolin, confluentin, 2-hydroxyneogrifolin, daurichromenic acid, and a cerebroside derivative. Compounds 1-10 were tested for Aß-aggregation inhibitory activity. Compounds 1, 3, 5, 6, 8, and 9 have potential as Aß-aggregation inhibitory activity.

Long-read sequencing identifies GGC repeat expansions in NOTCH2NLC associated with neuronal intranuclear inclusion disease.

Neuronal intranuclear inclusion disease (NIID) is a progressive neurodegenerative disease that is characterized by eosinophilic hyaline intranuclear inclusions in neuronal and somatic cells. The wide range of clinical manifestations in NIID makes ante-mortem diagnosis difficult1-8, but skin biopsy enables its ante-mortem diagnosis9-12. The average onset age is 59.7 years among approximately 140 NIID cases consisting of mostly sporadic and several familial cases. By linkage mapping of a large NIID family with several affected members (Family 1), we identified a 58.1 Mb linked region at 1p22.1-q21.3 with a maximum logarithm of the odds score of 4.21. By long-read sequencing, we identified a GGC repeat expansion in the 5' region of NOTCH2NLC (Notch 2 N-terminal like C) in all affected family members. Furthermore, we found similar expansions in 8 unrelated families with NIID and 40 sporadic NIID cases. We observed abnormal anti-sense transcripts in fibroblasts specifically from patients but not unaffected individuals. This work shows that repeat expansion in human-specific NOTCH2NLC, a gene that evolved by segmental duplication, causes a human disease.

Meroterpenoids with BACE1 Inhibitory Activity from the Fruiting Body of Boletinus asiaticus.

BACE1 inhibitory activity-guided fractionation of an extract of the fruiting body of Boletinus asiaticus yielded five novel meroterpenoids (1-5) and one known compound (6; asiaticusin A). The structures of these compounds were determined by interpretation of NMR, MS, and IR spectral data. The five new compounds contain 4-hydroxybenzoic acid and geranylgeranoic acid units. Compounds 4-6 possessed BACE1 inhibitory activity (IC50 values: 14.7, 11.4, and 2.0 µM, respectively).

Microglia express GPNMB in the brains of Alzheimer's disease and Nasu-Hakola disease.

Glycoprotein non-metastatic melanoma protein B (GPNMB) is a type I transmembrane glycoprotein first identified in low-metastatic human melanoma cell lines as a regulator of tumor growth. GPNMB is widely expressed in various tissues, where it is involved in cell differentiation, migration, inflammation/anti-inflammation, tissue regeneration, and neuroprotection. GPNMB is identified in microglia of adult rat brains, neurons and astrocytes of GPNMB transgenic (Tg) mouse brains, and motor neurons of amyotrophic lateral sclerosis (ALS) patients. Nasu-Hakola disease (NHD) is a rare autosomal recessive disorder, characterized by progressive presenile dementia and formation of multifocal bone cysts, caused by genetic mutations of either TYROBP (DAP12) or TREM2. TREM2 and DAP12 constitute a receptor/adaptor signaling complex expressed exclusively on osteoclasts, dendritic cells, macrophages, and microglia. Pathologically, the brains of NHD patients exhibit leukoencephalopathy, astrogliosis, accumulation of axonal spheroids, and remarkable activation of microglia predominantly in the white matter of frontal and temporal lobes and the basal ganglia. At present, molecular mechanisms responsible for development of leukoencephaolpathy in NHD brains remain totally unknown. Recent evidence indicates that disease-associated microglia (DAM) that cluster around amyloid plaques express high levels of GPNMB in Alzheimer's disease (AD) brains. Because microglia act as a key regulator of leukoencephalopathy in NHD brains, it is proposed that GPNMB expressed on microglia might play a protective role in progression of leukoencephalopathy possibly via active phagocytosis of myelin debris. In the present study using immunohistochemistry, we have attempted to clarify the expression of GPNMB in NHD brains, compared with AD brains. We found that microglia accumulating in the white matter express an intense GPNMB immunoreactivity in both NHD and AD brains, suggesting that the accumulation of GPNMB-immunoreactive microglia is a general phenomenon in neurodegenerative brains.

Tandem-genotypes: robust detection of tandem repeat expansions from long DNA reads.

Tandemly repeated DNA is highly mutable and causes at least 31 diseases, but it is hard to detect pathogenic repeat expansions genome-wide. Here, we report robust detection of human repeat expansions from careful alignments of long but error-prone (PacBio and nanopore) reads to a reference genome. Our method is robust to systematic sequencing errors, inexact repeats with fuzzy boundaries, and low sequencing coverage. By comparing to healthy controls, we prioritize pathogenic expansions within the top 10 out of 700,000 tandem repeats in whole genome sequencing data. This may help to elucidate the many genetic diseases whose causes remain unknown.

Microglia express TMEM119 in the brains of Nasu-Hakola disease.

We previously identified an evolutionarily conserved protein named transmembrane protein 119 (TMEM119) as the most reliable maker for human microglia. Recent studies showed that under homeostatic conditions, microglia intensely express TMEM119, whereas the expression levels are greatly reduced in disease-associated microglia (DAM) activated at the site of neurodegeneration. Nasu-Hakola disease (NHD) is a rare autosomal recessive disorder, pathologically characterized by leukoencephalopathy, astrogliosis, axonal spheroids, and accumulation of microglia. However, it remains unknown whether microglia are homeostatic or activated in NHD brains. In the present study, we identified TMEM119 on microglia in NHD brains by immunohistochemistry. TMEM119 was expressed on microglia in NHD brains as well as in the brains of non-neurological controls (NC) and Alzheimer's disease (AD) patients, although TMEM119-immunolabeled areas exhibited great variability from case to case without significant differences among the study population. These results suggest that TMEM119 expression on microglia might play a key role in steady-state brain maintenance in NHD, AD and controls.

Microglia express gamma-interferon-inducible lysosomal thiol reductase in the brains of Alzheimer's disease and Nasu-Hakola disease.

Gamma-interferon-inducible lysosomal thiol reductase (GILT), expressed in antigen-presenting cells (APCs), facilitates the reduction of disulfide bonds of endocytosed proteins in the endocytic pathway and they are further processed for presentation of immunogenic peptides loaded on major histocompatibility complex (MHC) class II. Although the constitutive and IFNγ-inducible expression of GILT was observed in various APCs, such as dendritic cells, monocytes/macrophages, and B cells, GILT-expressing cell types remain unknown in the human central nervous system (CNS). Nasu-Hakola disease (NHD) is a rare autosomal recessive disorder characterized by sclerosing leukoencephalopathy and multifocal bone cysts, caused by a loss-of-function mutation of either TYROBP (DAP12) or TREM2, both of which are expressed on microglia. A rare heterozygous variant of the TREM2 gene encoding p.Arg47His causes a 3-fold increase in the risk for late-onset Alzheimer's disease (LOAD), suggesting that both NHD and AD are induced by dysfunction of the microglial TREM2 signaling pathway in the brains. We studied by immunohistochemistry GILT expression in NHD and AD brains. GILT was expressed on amoeboid microglia with the highest levels of expression in AD brains, compared with those in non-neurological control (NC) brains and in NHD brains. In AD brains, the clusters of amoeboid microglia surrounding amyloid-beta (Aꞵ) deposition strongly expressed GILT. Furthermore, a human microglial cell line expressed GILT in response to IFNγ. These results indicate that microglia, expressing constitutively high levels of GILT, act as a principal cell type of APCs in AD brains, in contrast to baseline levels of GILT expression in NHD brains.

Small nuclear RNA-mediated modulation of splicing reveals a therapeutic strategy for a TREM2 mutation and its post-transcriptional regulation.

Loss-of-function mutations in TREM2 cause Nasu-Hakola disease (NHD), a rare genetic disease characterized by early-onset dementia with leukoencephalopathy and bone cysts. An NHD-associated mutation, c.482 + 2 T > C, disrupts the splice donor site of intron 3 and causes aberrant skipping of exon 3, resulting in the loss of full-length TREM2 protein. Here, we examined the efficacy of artificial U1 and U7 small nuclear RNAs (snRNAs) designed to enhance exon 3 inclusion. Using mutant TREM2 minigenes, we found that some modified U1, but not U7, snRNAs enhanced exon 3 inclusion and restored TREM2 protein expression. Unexpectedly, we found that exon 3 of wild-type TREM2 is an alternative exon, whose skipping leads to reduced expression of the full-length protein. Indeed, TREM2 protein levels were modulated by modified snRNAs that either promoted or repressed exon 3 inclusion. The splice donor site flanking exon 3 was predicted to be weak, which may explain both the alternative splicing of exon 3 under normal conditions and complete exon skipping when the c.482 + 2 T > C mutation was present. Collectively, our snRNA-based approaches provide a potential therapeutic strategy for NHD-associated mis-splicing and novel insights into the post-transcriptional regulation of TREM2.

Alzheimer's disease pathology in Nasu-Hakola disease brains.

Nasu-Hakola disease (NHD) is a rare autosomal recessive disorder, characterized by progressive presenile dementia and formation of multifocal bone cysts, caused by genetic mutations of either triggering receptor expressed on myeloid cells 2 (TREM2) or TYRO protein tyrosine kinase binding protein (TYROBP), alternatively named DNAX-activation protein 12 (DAP12), both of which are expressed on microglia in the brain and form the receptor-adaptor complex that chiefly recognizes anionic lipids. TREM2 transmits the signals involved in microglial survival, proliferation, chemotaxis, and phagocytosis. A recent study indicated that a loss of TREM2 function causes greater amounts of amyloid-ß (Aß) deposition in the hippocampus of a mouse model of Alzheimer's disease (AD) owing to a dysfunctional response of microglia to amyloid plaques, suggesting that TREM2 facilitates Aß clearance by microglia. TREM2/DAP12-mediated microglial response limits diffusion and toxicity of amyloid plaques by forming a protective barrier. However, the levels of Aß deposition in postmortem brains of NHD, where the biological function of the TREM2/DAP12 signaling pathway is completely lost, remain to be investigated. By immunohistochemistry, we studied the expression of Aß and phosphorylated tau (p-tau) in the frontal cortex and the hippocampus of five NHD cases. Although we identified several small Aß-immunoreactive spheroids, amyloid plaques were almost undetectable in NHD brains. We found a small number of p-tau-immunoreactive neurofibrillary tangle (NFT)-bearing neurons in NHD brains. Because AD pathology is less evident in NHD than the full-brown AD, it does not play an active role in the development of NHD.

Microglia express ABI3 in the brains of Alzheimer's disease and Nasu-Hakola disease.

Nasu-Hakola disease (NHD) is a rare autosomal recessive leukoencephalopathy caused by a loss-of-function mutation of either TYROBP (DAP12) or TREM2 expressed in microglia. A rare variant of the TREM2 gene encoding p.Arg47His causes a 3-fold increase in the risk for late-onset Alzheimer's disease (LOAD). A recent study demonstrated that a rare coding variant p.Ser209Phe in the ABI family member 3 (ABI3) gene, a regulator of actin cytoskeleton organization, confers risk of developing of LOAD, although the pattern of ABI3 expression in AD and NHD brains with relevance to microglial pathology remains to be characterized. We investigated the cell type-specific expression of ABI3 in the brains derived from four non-neurological controls (NC), ten AD and five NHD cases by immunohistochemistry. We identified an intense ABI3 immunoreactivity chiefly on a subset of microglia with ramified or amoeboid morphology located in the grey matter and the white matter of the frontal cortex and the hippocampus of NC, AD, and NHD cases. The immunolabeled area of ABI3-positive microglia was not significantly different among NC, AD, and NHD cases due to great variability from case to case. The clusters of ABI3-immunoreactive microglia were found exclusively in AD brains and they were associated with amyloid plaques. Although these observations do not actively support the view that ABI3-immunoreactive microglia play a central role in the development of leukoencephalopathy in NHD brains and the neurodegeneration in AD brains, the intense expression of ABI3 on microglia might regulate their migration under conditions of health and disease in the central nervous system (CNS).

Gastric and intestinal proteases resistance of chicken acidic chitinase nominates chitin-containing organisms for alternative whole edible diets for poultry.

Chitin, a polymer of N-acetyl-D-glucosamine (GlcNAc), functions as a major structural component in crustaceans, insects and fungi and is the second most abundant polysaccharide in the nature. Although these chitin-containing organisms have been suggested as novel animal feed resources, chitin has long been considered as indigestible fibers in the animal body. Recently, we reported that acidic chitinase (Chia) is a protease-resistant major glycosidase in mouse gastrointestinal tract (GIT) and that it digests chitin in the mouse stomach. However, the physiological role of Chia in other animals including poultry remains unknown. Here, we report that Chia can function as a digestive enzyme that breaks down chitin-containing organisms in chicken GIT. Chia mRNA is predominantly expressed in the glandular stomach tissue in normal chicken. We also show that chicken Chia has a robust chitinolytic activity at pH 2.0 and is highly resistant to proteolysis by pepsin and trypsin/chymotrypsin under conditions mimicking GIT. Chia degraded shells of mealworm larvae in the presence of digestive proteases and produced (GlcNAc)2. Thus, functional similarity of chicken Chia with the mouse enzyme suggests that chitin-containing organisms can be used for alternative poultry diets not only as whole edible resources but also as enhancers of their nutritional value.

Expression of GPR17, a regulator of oligodendrocyte differentiation and maturation, in Nasu-Hakola disease brains.

The G protein-coupled receptor 17 (GPR17), a Gi-coupled GPCR, acts as an intrinsic timer of oligodendrocyte differentiation and myelination. The expression of GPR17 is upregulated during differentiation of oligodendrocyte precursor cells (OPCs) into premyelinating oligodendrocytes (preoligodendrocytes), whereas it is markedly downregulated during terminal maturation of myelinating oligodendrocytes. Nasu-Hakola disease (NHD) is a rare autosomal recessive disorder caused by a loss-of-function mutation of either TYROBP (DAP12) or TREM2. Pathologically, the brains of NHD patients exhibit extensive demyelination designated leukoencephalopathy, astrogliosis, accumulation of axonal spheroids, and activation of microglia predominantly in the white matter of frontal and temporal lobes. Although GPR17 is a key regulator of oligodendrogenesis, a pathological role of GPR17 in NHD brains with relevance to development of leukoencephalopathy remains unknown. We studied the expression of GPR17 in five NHD brains and eight control brains by immunohistochemistry. We identified GPR17-immunoreactive preoligodendrocytes with a multipolar ramified morphology distributed in the white matter and the grey matter of all cases examined. However, we did not find statistically significant differences in the number of GPR17-expressing cells between NHD and control brains both in the white matter and the grey matter due to great variability from case to case. These observations do not support the view that GPR17-positive preoligodendrocytes play a central role in the development of leukoencephalopathy in NHD brains.

Targeted sequencing approach to identify genetic mutations in Nasu-Hakola disease.

Nasu-Hakola disease (NHD) is a rare autosomal recessive disorder characterized by sclerosing leukoencephalopathy and multifocal bone cysts, caused by a loss-of-function mutation of either TYROBP (DAP12) or TREM2. TREM2 and DAP12 constitute a receptor/adaptor signaling complex expressed exclusively on osteoclasts, dendritic cells, macrophages, and microglia. Premortem molecular diagnosis of NHD requires genetic analysis of both TYROBP and TREM2, in which 20 distinct NHD-causing mutations have been reported. Due to genetic heterogeneity, it is often difficult to identify the exact mutation responsible for NHD. Recently, the revolution of the next-generation sequencing (NGS) technology has greatly advanced the field of genome research. A targeted sequencing approach allows us to investigate a selected set of disease-causing genes and mutations in a number of samples within several days. By targeted sequencing using the TruSight One Sequencing Panel, we resequenced genetic mutations of seven NHD cases with known molecular diagnosis and two control subjects. We identified homozygous variants of TYROBP or TREM2 in all NHD cases, composed of a frameshift mutation of c.141delG in exon 3 of TYROBP in four cases, a missense mutation of c.2T>C in exon 1 of TYROBP in two cases, or a splicing mutation of c.482+2T>C in intron 3 of TREM2 in one case. The results of targeted resequencing corresponded to those of Sanger sequencing. In contrast, causative variants were not detected in control subjects. These results indicate that targeted sequencing is a useful approach to precisely identify genetic mutations responsible for NHD in a comprehensive manner.