Identification of PSMB5 as a genetic modifier of fragile X-associated tremor/ataxia syndrome.

Fragile X­associated tremor/ataxia syndrome (FXTAS) is a debilitating late-onset neurodegenerative disease in premutation carriers of the expanded CGG repeat in FMR1 that presents with a spectrum of neurological manifestations, such as gait ataxia, intention tremor, and parkinsonism [P. J. Hagerman, R. J. Hagerman, Ann. N. Y. Acad. Sci. 1338, 58­70 (2015); S. Jacquemont et al., JAMA 291, 460­469 (2004)]. Here, we performed whole-genome sequencing (WGS) on male premutation carriers (CGG55­200) and prioritized candidate variants to screen for candidate genetic modifiers using a Drosophila model of FXTAS. We found 18 genes that genetically modulate CGG-associated neurotoxicity in Drosophila, such as Prosbeta5 (PSMB5), pAbp (PABPC1L), e(y)1 (TAF9), and CG14231 (OSGEPL1). Among them, knockdown of Prosbeta5 (PSMB5) suppressed CGG-associated neurodegeneration in the fly as well as in N2A cells. Interestingly, an expression quantitative trait locus variant in PSMB5, PSMB5rs11543947-A, was found to be associated with decreased expression of PSMB5 and delayed onset of FXTAS in human FMR1 premutation carriers. Finally, we demonstrate evidence that PSMB5 knockdown results in suppression of CGG neurotoxicity via both the RAN translation and RNA-mediated toxicity mechanisms, thereby presenting a therapeutic strategy for FXTAS.

Intercepting IRE1 kinase-FMRP signaling prevents atherosclerosis progression.

Fragile X Mental Retardation protein (FMRP), widely known for its role in hereditary intellectual disability, is an RNA-binding protein (RBP) that controls translation of select mRNAs. We discovered that endoplasmic reticulum (ER) stress induces phosphorylation of FMRP on a site that is known to enhance translation inhibition of FMRP-bound mRNAs. We show ER stress-induced activation of Inositol requiring enzyme-1 (IRE1), an ER-resident stress-sensing kinase/endoribonuclease, leads to FMRP phosphorylation and to suppression of macrophage cholesterol efflux and apoptotic cell clearance (efferocytosis). Conversely, FMRP deficiency and pharmacological inhibition of IRE1 kinase activity enhances cholesterol efflux and efferocytosis, reducing atherosclerosis in mice. Our results provide mechanistic insights into how ER stress-induced IRE1 kinase activity contributes to macrophage cholesterol homeostasis and suggests IRE1 inhibition as a promising new way to counteract atherosclerosis.

CXCR6 is required for antitumor efficacy of intratumoral CD8+ T cell.

BACKGROUND: Increasing infiltration of CD8+ T cells within tumor tissue predicts a better prognosis and is essential for response to checkpoint blocking therapy. Furthermore, current clinical protocols use unfractioned T cell populations as the starting point for transduction of chimeric antigen receptors (CARs)-modified T cells, but the optimal T cell subtype of CAR-modified T cells remains unclear. Thus, accurately identifying a group of cytotoxic T lymphocytes with high antitumor efficacy is imperative. Inspired by the theory of yin and yang, we explored a subset of CD8+ T cell in cancer with the same phenotypic characteristics as highly activated inflammatory T cells in autoimmune diseases. METHODS: Combination of single-cell RNA sequencing, general transcriptome sequencing data and multiparametric cytometric techniques allowed us to map CXCR6 expression on specific cell type and tissue. We applied Cxcr6-/- mice, immune checkpoint therapies and bone marrow chimeras to identify the function of CXCR6+CD8+ T cells. Transgenic Cxcr6-/- OT-I mice were employed to explore the functional role of CXCR6 in antigen-specific antitumor response. RESULTS: We identified that CXCR6 was exclusively expressed on intratumoral CD8+ T cell. CXCR6+CD8+ T cells were more immunocompetent, and chimeras with specific deficiency on CD8+ T cells showed weaker antitumor activity. In addition, Cxcr6-/- mice could not respond to anti-PD-1 treatment effectively. High tumor expression of CXCR6 was not mainly caused by ligand-receptor chemotaxis of CXCL16/CXCR6 but induced by tumor tissue self. Induced CXCR6+CD8+ T cells possessed tumor antigen specificity and could enhance the effect of anti-PD-1 blockade to retard tumor progression. CONCLUSIONS: This study may contribute to the rational design of combined immunotherapy. Alternatively, CXCR6 may be used as a biomarker for effective CD8+ T cell state before adoptive cell therapy, providing a basis for tumor immunotherapy.

Simultaneous Screening of the FRAXA and FRAXE Loci for Rapid Detection of FMR1 CGG and/or AFF2 CCG Repeat Expansions by Triplet-Primed PCR.

Moderate to hyper-expansion of trinucleotide repeats at the FRAXA and FRAXE fragile sites, with or without concurrent hypermethylation, has been associated with intellectual disability and other conditions. Unlike molecular diagnosis of FMR1 CGG repeat expansions in FRAXA, current detection of AFF2 CCG repeat expansions in FRAXE relies on low-throughput and otherwise inefficient techniques combining Southern blot analysis and PCR. A novel triplet-primed PCR assay was developed for simultaneous screening for trinucleotide repeat expansions at the FRAXA and FRAXE fragile sites, and was validated using archived clinical samples of known FMR1 and AFF2 genotypes. Population samples and FRAXE-affected samples were sequenced for the evaluation of variations in the AFF2 CCG repeat structure. The duplex assay accurately identified expansions at the FMR1 and AFF2 trinucleotide repeat loci. On Sanger sequencing of the AFF2 CCG repeat, the single-nucleotide polymorphism variant rs868914124(C) that effectively adds two CCG repeats at the 5'-end, was enriched in the Malay population and with short repeats (<11 CCGs), and was present in all six expanded AFF2 alleles of this study. All expanded AFF2 alleles contained multiple non-CCG interruptions toward the 5'-end of the repeat. A sensitive, robust, and rapid assay has been developed for the simultaneous detection of expansion mutations at the FMR1 and AFF2 trinucleotide repeat loci, simplifying screening for FRAXA- and FRAXE-associated disorders.

Metabolic pathways modulate the neuronal toxicity associated with fragile X-associated tremor/ataxia syndrome.

Fragile X-associated tremor/ataxia syndrome (FXTAS) is an adult-onset neurodegenerative disorder that affects premutation carriers (55-200 CGG repeats) of the fragile X mental retardation 1 (FMR1) gene. Much remains unknown regarding the metabolic alterations associated with FXTAS, especially in the brain, and the most affected region, the cerebellum. Investigating the metabolic changes in FXTAS will aid in the identification of biomarkers as well as in understanding the pathogenesis of disease. To identify the metabolic alterations associated with FXTAS, we took advantage of our FXTAS mouse model that expresses 90 CGG repeats in cerebellar Purkinje neurons and exhibits the key phenotypic features of FXTAS. We performed untargeted global metabolic profiling of age-matched control and FXTAS mice cerebella at 16-20 weeks and 55 weeks. Out of 506 metabolites measured in cerebellum, we identified 186 metabolites that demonstrate significant perturbations due to the (CGG)90 repeat (P<0.05) and found that these differences increase dramatically with age. To identify key metabolic changes in FXTAS pathogenesis, we performed a genetic screen using a Drosophila model of FXTAS. Out of 28 genes that we tested in the fly, 8 genes showed significant enhanced neuronal toxicity associated with CGG repeats, such as Schlank (ceramide synthase), Sk2 (sphingosine kinase) and Ras (IMP dehydrogenase). By combining metabolic profiling with a Drosophila genetic screen to identify genetic modifiers of FXTAS, we demonstrate an effective method for functional validation of high-throughput metabolic data and show that sphingolipid and purine metabolism are significantly perturbed in FXTAS pathogenesis.

MBD5 haploinsufficiency is associated with sleep disturbance and disrupts circadian pathways common to Smith-Magenis and fragile X syndromes.

Individuals with autism spectrum disorders (ASD) who have an identifiable single-gene neurodevelopmental disorder (NDD), such as fragile X syndrome (FXS, FMR1), Smith-Magenis syndrome (SMS, RAI1), or 2q23.1 deletion syndrome (del 2q23.1, MBD5) share phenotypic features, including a high prevalence of sleep disturbance. We describe the circadian deficits in del 2q23.1 through caregiver surveys in which we identify several frequent sleep anomalies, including night/early awakenings, coughing/snoring loudly, and difficulty falling asleep. We couple these findings with studies on the molecular analysis of the circadian deficits associated with haploinsufficiency of MBD5 in which circadian gene mRNA levels of NR1D2, PER1, PER2, and PER3 were altered in del 2q23.1 lymphoblastoid cell lines (LCLs), signifying that haploinsufficiency of MBD5 can result in dysregulation of circadian rhythm gene expression. These findings were further supported by expression microarrays of MBD5 siRNA knockdown cells that showed significantly altered expression of additional circadian rhythm signaling pathway genes. Based on the common sleep phenotypes observed in del 2q23.1, SMS, and FXS patients, we explored the possibility that MBD5, RAI1, and FMR1 function in overlapping circadian rhythm pathways. Bioinformatic analysis identified conserved putative E boxes in MBD5 and RAI1, and expression levels of NR1D2 and CRY2 were significantly reduced in patient LCLs. Circadian and mTOR signaling pathways, both associated with sleep disturbance, were altered in both MBD5 and RAI1 knockdown microarray data, overlapping with findings associated with FMR1. These data support phenotypic and molecular overlaps across these syndromes that may be exploited to provide therapeutic intervention for multiple disorders.

Chromosomal microarray analysis (CMA) detects a large X chromosome deletion including FMR1, FMR2, and IDS in a female patient with mental retardation.

Chromosomal microarray analysis (CMA) by array-based comparative genomic hybridization (CGH) is a new clinical test for the detection of well-characterized genomic disorders caused by chromosomal deletions and duplications that result in gene copy number variation (CNV). This powerful assay detects an abnormality in approximately 7-9% of patients with various clinical phenotypes, including mental retardation. We report here on the results found in a 6-year-old girl with mildly dysmorphic facies, obesity, and marked developmental delay. CMA was requested and showed a heterozygous loss in copy number with clones derived from the genomic region cytogenetically defined as Xq27.3-Xq28. This loss was not cytogenetically visible but was seen on FISH analysis with clones from the region. Further studies confirmed a loss of one copy each of the FMR1, FMR2, and IDS genes (which are mutated in Fragile X syndrome, FRAXE syndrome, and Hunter syndrome, respectively). Skewed X-inactivation has been previously reported in girls with deletions in this region and can lead to a combined Fragile X/Hunter syndrome phenotype in affected females. X-inactivation and iduronate 2-sulfatase (IDS) enzyme activity were therefore examined. X-inactivation was found to be random in the child's peripheral leukocytes, and IDS enzyme activity was approximately half of the normal value. This case demonstrates the utility of CMA both for detecting a submicroscopic chromosomal deletion and for suggesting further testing that could possibly lead to therapeutic options for patients with developmental delay.

Cytogenetic analysis of obsessive-compulsive disorder (OCD): identification of a FRAXE fragile site.

Obsessive-compulsive disorder (OCD) is a chronic psychiatric disease characterized by recurrent obsessions, compulsions, or both. The prevalence rate of OCD is 2.1% in the general population. Here we report cytogenetic analysis of 26 patients affected with OCD. In one male patient (OCD-K33), we identified a fragile X chromosome by cytogenetic analysis with 21% of cells demonstrating a fragile site at Xq27-q28. Polymerase chain reaction (PCR) and Southern blot analysis demonstrated that the molecular basis of the OCD-K33 fragile X chromosome was expansion of the CCG repeat at FRAXE. The number of the expanded repeats was estimated to be more than 300 copies, qualifying it as a full FRAXE mutation. Further analysis of the family members of OCD-K33 revealed another member with a full FRAXE mutation (630-1,200 copies of the CCG repeat), who had the clinical phenotype of speech impairment, and two other members with normal phenotypes and no FRAXE expansion. The two FRAXE expansions lead to complete methylation at the CCG repeat. The co-segregation of the full FRAXE mutation with apparent neurologic disorders in the same family provides further support to the notion that FRAXE is a genetic neurologic condition. Our findings expand the spectrum of clinical phenotypes associated with FRAXE mutations.

Impaired conditioned fear and enhanced long-term potentiation in Fmr2 knock-out mice.

FRAXE mental retardation results from expansion and methylation of a CCG trinucleotide repeat located in exon 1 of the X-linked FMR2 gene, which results in transcriptional silencing. The product of FMR2 is a member of a family of proteins rich in serine and proline, members of which have been associated with transcriptional activation. We have developed a murine Fmr2 gene knock-out model by replacing a fragment containing parts of exon 1 and intron 1 with the Escherichia coli lacZ gene, placing lacZ under control of the Fmr2 promoter. Expression of lacZ in the knock-out animals indicates that Fmr2 is expressed in several tissues, including brain, bone, cartilage, hair follicles, lung, tongue, tendons, salivary glands, and major blood vessels. In the CNS, Fmr2 expression begins at the time that cells in the neuroepithelium differentiate into neuroblasts. Mice lacking Fmr2 showed a delay-dependent conditioned fear impairment. Long-term potentiation (LTP) was found to be enhanced in hippocampal slices of Fmr2 knock-out compared with wild-type littermates. To our knowledge, this mouse knock-out is the first example of an animal model of human mental retardation with impaired learning and memory performance and increased LTP. Thus, although a number of studies have suggested that diminished LTP is associated with memory impairment, our data suggest that increased LTP may be a mechanism that leads to impaired cognitive processing as well.