Time-Course Metabolomic Analysis: Production of Betaine Structural Analogs by Fungal Fermentation of Seaweed.

Betaine structural analogs are compounds characterized by the presence of positive and negative charges in a single molecule and have been reported to have physiological properties, such as anti-inflammatory activities. In this study, we performed a metabolomic analysis of metabolite composition changes during the fermentation of Neopyropia yezoensis, an edible red alga, with Aspergillus oryzae for 72 h. The results indicated that three specific betaine structural analogs (betaine, stachydrine, and carnitine) exhibited significant changes in production by the end of the 72 h fermentation period. Time-course analysis suggested that betaine was generated from the precursor choline at 12-24 h during the late stage of fungal growth, while stachydrine was generated from the precursor-related compound glutamic acid at 48-72 h during the sporulation stage. However, the contribution of the precursor lysine to the increased production of carnitine during the 12-72 h period was unclear. This study provides useful information on the efficient production of betaine structural analogs by the fungal fermentation of seaweed as well as various other food materials.

Ozonated Olive Oil Intake Attenuates Hepatic Steatosis in Obese db/db Mice.

Chronic inflammation and insulin resistance lead to metabolic syndrome and there is an urgent need to establish effective treatments and prevention methods. Our previous study reported that obese model Zucker (fa/fa) rats fed with ozonated olive oil alleviated fatty liver and liver damage by suppressing inflammatory factors. However, differences among animal species related to the safety and efficacy of ozonated olive oil administration remain unclear. Therefore, this study investigated the effects of oral intake of ozonated olive oil on lipid metabolism in normal mice and mice in the obesity model. C57BL/6J and db/db mice were fed the following AIN-76 diets for four weeks: the mice were either fed a 0.5% olive oil diet (Control diet) or 0.5% ozonated olive oil diet (Oz-Olive diet) in addition to 6.5% corn oil. The results indicated that four weeks of Oz-Olive intake did not adversely affect growth parameters, hepatic lipids or serum parameters in normal C57BL/6J mice. Subsequent treatment of db/db mice with Oz-Olive for four weeks reduced the levels of hepatic triglycerides, serum alkaline phosphatase, and serum insulin. These effects of Oz-Olive administration might be due to suppression of fatty acid synthesis activity and expression of lipogenic genes, as well as suppression of inflammatory gene expression. In conclusion, this study confirmed the safety of Oz-Olive administration in normal mice and its ability to alleviate hepatic steatosis by inhibiting fatty acid synthesis and inflammation in obese mice.

Structural evidence for protein-protein interaction between the non-canonical methyl-CpG-binding domain of SETDB proteins and C11orf46.

SETDB1 and SETDB2 mediate trimethylation of histone H3 lysine 9 (H3K9), an epigenetic hallmark of repressive chromatin. They contain a non-canonical methyl-CpG-binding domain (MBD) and bifurcated SET domain, implying interplay between H3K9 trimethylation and DNA methylation in SETDB functions. Here, we report the crystal structure of human SETDB2 MBD bound to the cysteine-rich domain of a zinc-binding protein, C11orf46. SETDB2 MBD comprises the conserved MBD core and a unique N-terminal extension. Although the MBD core has the conserved basic concave surface for DNA binding, it utilizes it for recognition of the cysteine-rich domain of C11orf46. This interaction involves the conserved arginine finger motif and the unique N-terminal extension of SETDB2 MBD, with a contribution from intermolecular ß-sheet formation. Thus, the non-canonical MBD of SETDB1/2 seems to have lost methylated DNA-binding ability but gained a protein-protein interaction surface. Our findings provide insight into the molecular assembly of SETDB-associated repression complexes.

Identification of genes regulated by lipids from seaweed Susabinori (Pyropia yezoensis) involved in the improvement of hepatic steatosis: Insights from RNA-Seq analysis in obese db/db mice.

Hepatic steatosis is an early stage in the progression of non-alcoholic fatty liver disease (NAFLD) and can lead to the development of non-alcoholic steatohepatitis (NASH), a major cause of liver-related morbidity and mortality. Identification of dietary components that can alleviate hepatic steatosis is crucial for developing effective therapeutic strategies for NAFLD. Recently, we demonstrated the impact of lipids extracted from the marine red alga Susabinori (Pyropia yezoensis) in a murine model of type 2-diabete (db/db). We found that Susabinori lipids (SNL), abundant in eicosapentaenoic acid (EPA)-containing polar lipids, protected against obesity-induced hepatic steatosis in db/db mice. To understand the specific genes or biological pathways underlying the effects of SNL, we conducted RNA-Seq analysis of the hepatic transcriptome. By performing comparative analysis of differentially expressed genes between normal mice and db/db mice consuming a control diet, as well as SNL-fed db/db mice, we identified the 15 SNL-dependent up-regulated genes that were down-regulated in db/db mice but up-regulated by SNL feeding. Gene ontology and pathway analysis on these 15 genes demonstrated a significant association with the metabolisms of arachidonic acid (AA) and linoleic acid (LA). Furthermore, we observed alterations in the expression levels of monoacylglycerol lipase (Magl) and fatty acid-binding protein 4 (Fabp4) in the SNL-fed db/db mice, both of which are implicated in AA and LA metabolism. Additionally, the livers of SNL-fed db/db mice exhibited reduced levels of AA and LA, but a high accumulation of EPA. In conclusion, the SNL diet might affect the metabolisms of AA and LA, which contribute to the improvement of hepatic steatosis. Our findings provide insights into the molecular mechanisms underlying the beneficial effects of SNL.

Replication dynamics identifies the folding principles of the inactive X chromosome.

Chromosome-wide late replication is an enigmatic hallmark of the inactive X chromosome (Xi). How it is established and what it represents remains obscure. By single-cell DNA replication sequencing, here we show that the entire Xi is reorganized to replicate rapidly and uniformly in late S-phase during X-chromosome inactivation (XCI), reflecting its relatively uniform structure revealed by 4C-seq. Despite this uniformity, only a subset of the Xi became earlier replicating in SmcHD1-mutant cells. In the mutant, these domains protruded out of the Xi core, contacted each other and became transcriptionally reactivated. 4C-seq suggested that they constituted the outermost layer of the Xi even before XCI and were rich in escape genes. We propose that this default positioning forms the basis for their inherent heterochromatin instability in cells lacking the Xi-binding protein SmcHD1 or exhibiting XCI escape. These observations underscore the importance of 3D genome organization for heterochromatin stability and gene regulation.

Identification, Chemical Synthesis, and Sweetness Evaluation of Rhamnose or Xylose Containing Steviol Glycosides of Stevia (Stevia rebaudiana) Leaves.

Steviol glycosides obtained from Stevia rebaudiana leaves are increasingly used in the food industry as natural low-calorie sweeteners. Among them, the sweetness of major glycosides composed of glucose residues (e.g., stevioside and rebaudioside A) has been widely studied. However, the properties of minor natural products containing rhamnose or xylose residues are poorly investigated. In this study, five unreported steviol glycosides containing rhamnose or xylose were extracted from our developing stevia leaves, and their sweetness was evaluated. The highly glycosylated steviol glycosides were identified, and their structures were examined by fragmentation analysis using mass spectrometry. Chemical synthesis of these glycosides confirmed their structures and allowed sensory evaluation of minor steviol glycosides. Our study revealed that a xylose-containing glycoside, rebaudioside FX1, exhibits a well-balanced sweetness, and thus, it is a promising candidate for natural sweeteners used in the food industry.

Dominant-negative variants in CBX1 cause a neurodevelopmental disorder.

PURPOSE: This study aimed to establish variants in CBX1, encoding heterochromatin protein 1ß (HP1ß), as a cause of a novel syndromic neurodevelopmental disorder. METHODS: Patients with CBX1 variants were identified, and clinician researchers were connected using GeneMatcher and physician referrals. Clinical histories were collected from each patient. To investigate the pathogenicity of identified variants, we performed in vitro cellular assays and neurobehavioral and cytological analyses of neuronal cells obtained from newly generated Cbx1 mutant mouse lines. RESULTS: In 3 unrelated individuals with developmental delay, hypotonia, and autistic features, we identified heterozygous de novo variants in CBX1. The identified variants were in the chromodomain, the functional domain of HP1ß, which mediates interactions with chromatin. Cbx1 chromodomain mutant mice displayed increased latency-to-peak response, suggesting the possibility of synaptic delay or myelination deficits. Cytological and chromatin immunoprecipitation experiments confirmed the reduction of mutant HP1ß binding to heterochromatin, whereas HP1ß interactome analysis demonstrated that the majority of HP1ß-interacting proteins remained unchanged between the wild-type and mutant HP1ß. CONCLUSION: These collective findings confirm the role of CBX1 in developmental disabilities through the disruption of HP1ß chromatin binding during neurocognitive development. Because HP1ß forms homodimers and heterodimers, mutant HP1ß likely sequesters wild-type HP1ß and other HP1 proteins, exerting dominant-negative effects.

Polycomb repressive complexes 1 and 2 are each essential for maintenance of X inactivation in extra-embryonic lineages.

In female mammals, one of the two X chromosomes becomes inactivated during development by X-chromosome inactivation (XCI). Although Polycomb repressive complex (PRC) 1 and PRC2 have both been implicated in gene silencing, their exact roles in XCI during in vivo development have remained elusive. To this end, we have studied mouse embryos lacking either PRC1 or PRC2. Here we demonstrate that the loss of either PRC has a substantial impact on maintenance of gene silencing on the inactive X chromosome (Xi) in extra-embryonic tissues, with overlapping yet different genes affected, indicating potentially independent roles of the two complexes. Importantly, a lack of PRC1 does not affect PRC2/H3K27me3 accumulation and a lack of PRC2 does not impact PRC1/H2AK119ub1 accumulation on the Xi. Thus PRC1 and PRC2 contribute independently to the maintenance of XCI in early post-implantation extra-embryonic lineages, revealing that both Polycomb complexes can be directly involved and differently deployed in XCI.

RNaseH2A downregulation drives inflammatory gene expression via genomic DNA fragmentation in senescent and cancer cells.

Cellular senescence caused by oncogenic stimuli is associated with the development of various age-related pathologies through the senescence-associated secretory phenotype (SASP). SASP is mediated by the activation of cytoplasmic nucleic acid sensors. However, the molecular mechanism underlying the accumulation of nucleotide ligands in senescent cells is unclear. In this study, we revealed that the expression of RNaseH2A, which removes ribonucleoside monophosphates (rNMPs) from the genome, is regulated by E2F transcription factors, and it decreases during cellular senescence. Residual rNMPs cause genomic DNA fragmentation and aberrant activation of cytoplasmic nucleic acid sensors, thereby provoking subsequent SASP factor gene expression in senescent cells. In addition, RNaseH2A expression was significantly decreased in aged mouse tissues and cells from individuals with Werner syndrome. Furthermore, RNaseH2A degradation using the auxin-inducible degron system induced the accumulation of nucleotide ligands and induction of certain tumourigenic SASP-like factors, promoting the metastatic properties of colorectal cancer cells. Our results indicate that RNaseH2A downregulation provokes SASP through nucleotide ligand accumulation, which likely contributes to the pathological features of senescent, progeroid, and cancer cells.

SmcHD1 underlies the formation of H3K9me3 blocks on the inactive X chromosome in mice.

Stable silencing of the inactive X chromosome (Xi) in female mammals is crucial for the development of embryos and their postnatal health. SmcHD1 is essential for stable silencing of the Xi, and its functional deficiency results in derepression of many X-inactivated genes. Although SmcHD1 has been suggested to play an important role in the formation of higher-order chromatin structure of the Xi, the underlying mechanism is largely unknown. Here, we explore the epigenetic state of the Xi in SmcHD1-deficient epiblast stem cells and mouse embryonic fibroblasts in comparison with their wild-type counterparts. The results suggest that SmcHD1 underlies the formation of H3K9me3-enriched blocks on the Xi, which, although the importance of H3K9me3 has been largely overlooked in mice, play a crucial role in the establishment of the stably silenced state. We propose that the H3K9me3 blocks formed on the Xi facilitate robust heterochromatin formation in combination with H3K27me3, and that the substantial loss of H3K9me3 caused by SmcHD1 deficiency leads to aberrant distribution of H3K27me3 on the Xi and derepression of X-inactivated genes.

Dried and Fermented Powders of Edible Algae (Neopyropia yezoensis) Attenuate Hepatic Steatosis in Obese Mice.

Edible algae Neopyropia yezoensis is used as "Nori", its dried sheet product, in Japanese cuisine. Its lipid components reportedly improve hepatic steatosis in obese db/db mice. In this study, we prepared "Nori powder (NP)" and "fermented Nori powder (FNP)" to utilize the functional lipids contained in "Nori" and examined their nutraceutical effects in vivo. Male db/db mice were fed a basal AIN-76 diet, a 10% NP-supplemented diet, or a 10% FNP-supplemented diet for 4 weeks. We detected eicosapentaenoic acid (EPA) present in both NP and FNP in the serum and liver of db/db mice in a dose-dependent manner. The NP diet reduced hepatic triglyceride accumulation (by 58%) in db/db mice by modulating gene expression, which resulted in the inhibition of lipogenic enzyme activity. Additionally, NP intake significantly suppressed the expression of inflammatory genes in the liver and hepatic injury marker levels in the sera (by 26%) of db/db mice. The FNP diet also led to a marked reduction in hepatic triglyceride accumulation (by 50%) and hepatic injury (by 28%) in db/db mice, and the mechanism of these alleviative actions was similar to that of the NP diet. Although the EPA content of FNP was one-third that of NP, metabolomic analysis revealed that bioactive betaine analogs, such as stachydrine, betaine, and carnitine, were detected only in FNP. In conclusion, we suggest that (1) mechanical processing of "Nori" makes its lipid components readily absorbable by the body to exert their lipid-lowering effects, and (2) fermentation of "Nori" produces anti-inflammatory molecules and lipid-lowering molecules, which together with the lipid components, can exert hepatic steatosis-alleviating effects.

Comparison of Lipoprotein Cholesterol Levels in Golden Syrian Hamster Administrated trans-Octadecenoic Acid Positional Isomers.

We previously conducted a study using HepG2 cells to compare the effect on the secreted apolipoprotein B-100 and apolipoprotein A-1 ratio (B-100/A-1) corresponding to the ratio of low-density to high-density lipoprotein cholesterol (LDL/HDL) among 13 types of trans-octadecenoic acid (t-18:1) positional isomers. The results revealed that trans-5-18:1 (t5) significantly increased B-100/A-1. In this study, 1% of t5 in the diet, corresponding to 2.08 energy%, was administrated golden Syrian hamsters for 4 weeks to reveal the effects on lipid profiles, including LDL/HDL, by comparing cis-9-octadecenoic acid (OA, oleic acid), trans-9-octadecenoic acid (EA), trans-11-octadecenoic acid (VA), and trans-9,trans-12- octadecadienoic acid (TT). LDL/HDL was not significantly different among the groups. However, the cholesterol concentration of medium very low-density lipoprotein (VLDL) was significantly lower in the TT diet than in the OA and t5 diets. The cholesterol concentration of small VLDL was significantly lower in the TT diet than in the OA, t5, and EA diets. The cholesterol concentration of large LDL was significantly lower in the TT diet than in the t5 and EA diets. However, no significant difference was detected between the TT and OA diets. In contrast, the cholesterol concentration of very small HDL was significantly higher in the TT diet than in the t5 diet. These results would support that lipid metabolism is affected by the structure of TFA in animals. However, t5-18:1 did not significantly change any lipid profile compared to OA existing in nature, and the previous result from the cell experiment showing that t5 increased B-100/A-1 (LDL/HDL) was not confirmed in this animal experiment.

Ozonated Olive Oil Alleviates Hepatic Steatosis in Obese Zucker (fa/fa) Rats.

Excessive lipid accumulation in organs and adipocytes results in chronic inflammation. This causes irreversible organ dysfunction and the development of metabolic syndrome, atherosclerosis, and cancer. Ozonated olive oil shows anti-inflammatory effects when applied directly to the skin; however, there are no reports on its effects on lipid metabolism through its oral administration in rats. Hence, this study investigates the effects of oral ingestion of ozonated olive oil on the pathologies of obese model rats. Obese model Zucker (fa/fa) rats were fed one of the following AIN-76 diets for four weeks: control diet: 6.5% corn oil + 0.5% olive oil, low ozonated oil diet: 6.5% corn oil + 0.45% olive oil + 0.05% ozonated olive oil, high ozonated oil diet: 6.5% corn oil + 0.5% ozonated olive oil. Control diet fed-Zucker lean rats were used as the reference. Growth parameters, hepatic lipids, hepatic enzyme activities, and serum parameters were determined. As the results, there was a dose-dependent improvement of hepatomegaly, fatty liver and elevated levels of hepatic injury markers in Zucker (fa/fa) rat upon ozonated olive oil consumption. Activities of hepatic enzymes related to lipid synthesis and lipid degradation were not affected by ozonated olive oil intake. On the other hand, there was a dose-dependent elimination of hepatic lipid secretion deficiency and suppression of inflammatory factors upon ozonated olive oil consumption. In conclusion, ozonated olive oil intake by Zucker (fa/fa) rats alleviates hepatic steatosis through the inhibition of triglyceride accumulation in the liver and suppression of inflammatory factors.

Protein phosphatase 1 acts as a RIF1 effector to suppress DSB resection prior to Shieldin action.

DNA double-strand breaks (DSBs) are repaired mainly by non-homologous end joining (NHEJ) or homologous recombination (HR). RIF1 negatively regulates resection through the effector Shieldin, which associates with a short 3' single-stranded DNA (ssDNA) overhang by the MRN (MRE11-RAD50-NBS1) complex, to prevent further resection and HR repair. In this study, we show that RIF1, but not Shieldin, inhibits the accumulation of CtIP at DSB sites immediately after damage, suggesting that RIF1 has another effector besides Shieldin. We find that protein phosphatase 1 (PP1), a known RIF1 effector in replication, localizes at damage sites dependent on RIF1, where it suppresses downstream CtIP accumulation and limits the resection by the MRN complex. PP1 therefore acts as a RIF1 effector distinct from Shieldin. Furthermore, PP1 deficiency in the context of Shieldin depletion elevates HR immediately after irradiation. We conclude that PP1 inhibits resection before the action of Shieldin to prevent precocious HR in the early phase of the damage response.

The Cholesterol Metabolite Cholest-5-en-3-One Alleviates Hyperglycemia and Hyperinsulinemia in Obese (db/db) Mice.

Dietary sterols are catabolized into various substances in the intestinal tract. Dietary 3-oxo derivatives of cholesterol and plant sterols (e.g., cholest-4-en-3-one and campest-5-en-3-one) have been shown to have anti-obesity effects. In this study, we tested whether feeding cholest-5-en-3-one (5-cholestenone), a cholesterol metabolite, to db/db mice protects them from obesity-associated metabolic disorders. In db/db mice, dietary 5-cholestenone significantly alleviated hepatomegaly and elevated serum triglyceride levels; however, the effect was not sufficient to improve hepatic steatosis and obesity. On the other hand, hyperglycemia and severe hyperinsulinemia in control db/db mice were markedly attenuated in 5-cholestenone-fed db/db mice. The production of inflammatory cytokines, such as monocyte chemoattractant protein-1, interleukin-6, and tumor necrosis factor-alpha (TNFα), was decreased, suggesting that the suppressive actions of 5-cholestenone were attributable to the alleviation of chronic inflammation in db/db mice. Additionally, 5-cholestenone showed an inhibitory effect on TNFα-induced nuclear factor kappa B (NFκB) activation in the NFκB luciferase gene reporter assay. These results suggest that obesity-induced abnormal glucose metabolism could be alleviated in 5-cholestenone-fed db/db mice by reducing the production of inflammatory cytokines through suppression of the NFκB signaling pathway.

Mapping replication timing domains genome wide in single mammalian cells with single-cell DNA replication sequencing.

Replication timing (RT) domains are stable units of chromosome structure that are regulated in the context of development and disease. Conventional genome-wide RT mapping methods require many S-phase cells for either the effective enrichment of replicating DNA through bromodeoxyuridine (BrdU) immunoprecipitation or the determination of copy-number differences during S-phase, which precludes their application to non-abundant cell types and single cells. Here, we provide a simple, cost-effective, and robust protocol for single-cell DNA replication sequencing (scRepli-seq). The scRepli-seq methodology relies on whole-genome amplification (WGA) of genomic DNA (gDNA) from single S-phase cells and next-generation sequencing (NGS)-based determination of copy-number differences that arise between replicated and unreplicated DNA. Haplotype-resolved scRepli-seq, which distinguishes pairs of homologous chromosomes within a single cell, is feasible by using single-nucleotide polymorphism (SNP)/indel information. We also provide computational pipelines for quality control, normalization, and binarization of the scRepli-seq data. The experimental portion of this protocol (before sequencing) takes 3 d.

Anti-stress effects of rosemary (Rosmarinus officinalis L.) leaf extract on intestinal goblet cells and immobility of forced-swimming test in BALB/c mice.

We investigated the anti-stress effect of rosemary (Rosmarinus officinalis L.) leaf extract (RLE) on restraint-stressed mice and found that RLE alleviated decreases in the number of intestinal goblet cells and amount of hepatic triglycerides. It also decreased the immobility time in the forced-swimming test and activation of microglia in the brain, suggesting that RLE has beneficial effects on stress-induced dysfunctions.

Rare variant of the epigenetic regulator SMCHD1 in a patient with pituitary hormone deficiency.

Isolated hypogonadotropic hypogonadism (IHH), combined pituitary hormone deficiency (CPHD), and septo-optic dysplasia (SOD) constitute a disease spectrum whose etiology remains largely unknown. This study aimed to clarify whether mutations in SMCHD1, an epigenetic regulator gene, might underlie this disease spectrum. SMCHD1 is a causative gene for Bosma arhinia microphthalmia syndrome characterized by arhinia, microphthalmia and IHH. We performed mutation screening of SMCHD1 in patients with etiology-unknown IHH (n = 31) or CPHD (n = 43, 19 of whom also satisfied the SOD diagnostic criteria). Rare variants were subjected to in silico analyses and classified according to the American College of Medical Genetics and Genomics guidelines. Consequently, a rare likely pathogenic variant, p.Asp398Asn, was identified in one patient. The patient with p.Asp398Asn exhibited CPHD, optic nerve hypoplasia, and a thin retinal nerve fiber layer, and therefore satisfied the criteria of SOD. This patient showed a relatively low DNA methylation level of the 52 SMCHD1-target CpG sites at the D4Z4 locus. Exome sequencing for the patient excluded additional variants in other IHH/CPHD-causative genes. In vitro assays suggested functional impairment of the p.Asp398Asn variant. These results provide the first indication that SMCHD1 mutations represent a rare genetic cause of the HH-related disease spectrum.

Eicosapentaenoic acid-containing polar lipids from seaweed Susabinori (Pyropia yezoensis) alleviate hepatic steatosis in obese db/db mice.

Nonalcoholic fatty liver disease (NAFLD) is emerging as the most common liver disease in industrialized countries. Because hepatic steatosis is an early pathogenesis of NAFLD, the discovery of food components that could ameliorate hepatic steatosis is of interest. Susabinori (Pyropia yezoensis) is recognized as one of the most delicious edible brown algae, and we prepared lipid component of susabinori (SNL), which is rich in eicosapentaenoic acid (EPA)-containing polar lipids. In this study, we tested whether feeding SNL to db/db mice protects them from developing obesity-induced hepatic steatosis. After four weeks of feeding, hepatomegaly, hepatic steatosis, and hepatic injury were markedly alleviated in SNL-fed db/db mice. These effects were partly attributable to the suppression of activities and mRNA expressions of lipogenic enzymes and enhanced levels of adiponectin due to the SNL diet. Additionally, mRNA expression of monocyte chemoattractant protein-1, an inflammatory chemokine, was markedly suppressed, and the mRNA levels of PPARδ, the anti-inflammatory transcription factor, were strongly enhanced in the livers of db/db mice by the SNL diet. We speculate that the development and progression of obesity-induced hepatic steatosis was prevented by the suppression of chronic inflammation due to the combination of bioactivities of EPA, phospholipids, and glycolipids in the SNL diet.

Homozygous nonsense variant in LRIF1 associated with facioscapulohumeral muscular dystrophy.

OBJECTIVE: Facioscapulohumeral muscular dystrophy (FSHD) is a heterogenetic disorder predominantly characterized by progressive facial and scapular muscle weakness. Patients with FSHD either have a contraction of the D4Z4 repeat on chromosome 4q35 or mutations in D4Z4 chromatin modifiers SMCHD1 and DNMT3B, both causing D4Z4 chromatin relaxation and inappropriate expression of the D4Z4-encoded DUX4 gene in skeletal muscle. In this study, we tested the hypothesis whether LRIF1, a known SMCHD1 protein interactor, is a disease gene for idiopathic FSHD2. METHODS: Clinical examination of a patient with idiopathic FSHD2 was combined with pathologic muscle biopsy examination and with genetic, epigenetic, and molecular studies. RESULTS: A homozygous LRIF1 mutation was identified in a patient with a clinical phenotype consistent with FSHD. This mutation resulted in the absence of the long isoform of LRIF1 protein, D4Z4 chromatin relaxation, and DUX4 and DUX4 target gene expression in myonuclei, all molecular and epigenetic hallmarks of FSHD. In concordance, LRIF1 was shown to bind to the D4Z4 repeat, and knockdown of the LRIF1 long isoform in muscle cells results in DUX4 and DUX4 target gene expression. CONCLUSION: LRIF1 is a bona fide disease gene for FSHD2. This study further reinforces the unifying genetic mechanism, which postulates that FSHD is caused by D4Z4 chromatin relaxation, resulting in inappropriate DUX4 expression in skeletal muscle.