Clustering Of Variants Research Articles

The effects of clusterin gene variant on coronary artery disease risk factors

Background and Aims: Coronary artery disease (CAD) which is a complex cardiovascular disease has high mortality and morbidity worldwide, despite all measures. Identifying the genetic background and environmental contributors of the disease is important for public health. In previous studies, clusterin (CLU) gene variants were found associated with CAD and disease-related risk factors. In this study, we aimed to identify the effects of the CLU variant on CAD risk factors.

Astrocyte-derived clusterin suppresses amyloid formation in vivo

BackgroundAccumulation of amyloid-β (Aβ) peptide in the brain is a pathological hallmark of Alzheimer’s disease (AD). The clusterin (CLU) gene confers a risk for AD and CLU is highly upregulated in AD patients, with the common non-coding, protective CLU variants associated with increased expression. Although there is strong evidence implicating CLU in amyloid metabolism, the exact mechanism underlying the CLU involvement in AD is not fully understood or whether physiologic alterations of CLU levels in the brain would be protective.ResultsWe used a gene delivery approach to overexpress CLU in astrocytes, the major source of CLU expression in the brain. We found that CLU overexpression resulted in a significant reduction of total and fibrillar amyloid in both cortex and hippocampus in the APP/PS1 mouse model of AD amyloidosis. CLU overexpression also ameliorated amyloid-associated neurotoxicity and gliosis. To complement these overexpression studies, we also analyzed the effects of haploinsufficiency of Clu using heterozygous (Clu+/−) mice and control littermates in the APP/PS1 model. CLU reduction led to a substantial increase in the amyloid plaque load in both cortex and hippocampus in APP/PS1; Clu+/− mice compared to wild-type (APP/PS1; Clu+/+) littermate controls, with a concomitant increase in neuritic dystrophy and gliosis.ConclusionsThus, both physiologic ~ 30% overexpression or ~ 50% reduction in CLU have substantial impacts on amyloid load and associated pathologies. Our results demonstrate that CLU plays a major role in Aβ accumulation in the brain and suggest that efforts aimed at CLU upregulation via pharmacological or gene delivery approaches offer a promising therapeutic strategy to regulate amyloid pathology.

Association of clusterin (CLU) variants and exfoliation syndrome: An analysis in two Caucasian studies and a meta-analysis

Exfoliation syndrome (XFS) is an important risk factor for glaucoma (XFG) worldwide. LOXL1 variants are highly associated with XFS in most populations; however, the high frequency of risk alleles in normal individuals and the reversal of risk alleles in different ethnic populations suggest that other factors contribute to XFS pathogenesis. Clusterin (CLU) is an extracellular matrix chaperone that prevents protein aggregation and is highly expressed in ocular tissues affected by XFS. Studies examining common CLU variants for association with XFS have been inconsistent. The purpose of this study was to evaluate CLU variants for association with XFS in two independent datasets from the United States (222 cases and 344 controls) and Israel (92 cases and 102 controls). Seven tag SNPs that captured >95% of alleles at r2 greater than 0.8 across the CLU genomic region were genotyped using TaqMan assays. Genotypes for an additional SNP, rs2279590, were imputed using phased haplotypes of HapMap reference CEU samples. Of the 8 CLU SNPs selected for the study, none were significantly associated with XFS in either case–control group (age and sex adjusted P > 0.14 and 0.36, respectively, in the US and Israeli datasets), or when they were meta-analyzed together (age and sex adjusted P > 0.13). Haplotype analysis using all 8 SNPs or only the promoter region SNPs also did not show significant associations of CLU with XFS in the combined US and Israeli dataset (P > 0.28). Meta-analysis of the data from this study and previous studies in Caucasian populations (1184 cases and 978 controls) resulted in statistically significant association of rs2279590 with XFS (summary OR = 1.18, 95% CI: 1.03–1.33, P = 0.01). Significant association between rs2279590 and XFS was also found in Indian populations (summary OR = 0.76, 95% CI: 0.61–0.96; P = 0.02); however, significant heterogeneity between the Caucasian and Indian populations possibly due to reversal of the risk allele precluded an overall meta-analysis for rs2279590 (Q = 0.001, I2 = 91%). No significant association was identified for rs3087554 in either Caucasian populations (summary OR = 0.90, 95% CI: 0.77–1.05, P = 0.17) or Indian populations (summary OR = 0.89, 95% CI: 0.72–1.10, P = 0.28), or in both populations combined (1705 cases and 3713 controls; summary OR = 0.90, 95% CI: 0.79–1.01, P = 0.08). Significant heterogeneity precluded the addition of the Japanese data to the meta-analysis for rs3087554 (Q = 0.006, I2 = 87%). Our results suggest that common CLU variants may contribute to modest XFS risk but even larger datasets are required to confirm these findings.

Optimization of specific multiplex DNA primers to detect variable CLU genomic lesions in patients with Alzheimer’s disease

Recently, polymorphisms in the clusterin (CLU) or Apolipoprotein J (ApoJ) gene were reported to be involved in lipid metabolism, atherogenesis, and being associated with the risk of developing Alzheimer’s disease (AD). The influences from genetic variation has not been examined among Koreans. To screen genes with abnormal exon expression profiles, we developed PCR primer pairs for CLU gene. The new primer set can target specific regions of previously sequenced CLU gene. Primers were designed to target eight exon amplicons with flanking regions to optimize PCR amplification. One-hundred samples from clinically diagnosed Korean AD patients were selected for testing. We identified four single nucleotide polymorphisms (SNPs) in CLU through direct sequencing of the PCR products. These included three SNPs (rs7982, rs2279590 and rs3216167) that were previously reported variants in the SNP database (dbSNP), which could be found in NCBI. Interestingly, one new (NEW1) or extremely low-frequency mutation was found in more than one individual among the late-onset AD subjects. Our study suggests that CLU variants may also be an AD susceptibility factor among Koreans. Moreover, the findings of the study can be able to develop for simultaneous identify all of the genotypes of CLU mutation sites by DNA microarray PCR-based in the future.

Radicicol induces intracellular accumulation of glycan-deficient clusterin variant

Proteostasis regulation using naturally occurring small molecules has been considered as a promising strategy for manipulating cancer sensitivity and therapy. Here, we identify a small molecule Hsp90 inhibitor radicicol that induces intracellular accumulation of cytotoxic clusterin variant. In the mechanistic basis, this variant proved to be a product disposed from the stressed ER. During this process, inhibitory effect of radicicol on protein degradation results in cytosolic accumulation of glycan-deficient clusterin variant that signals cell death. These results provide a therapeutic insight into the targeted proteostasis perturbation of clusterin as an anti-cancer strategy.

Intracellular clusterin interacts with brain isoforms of the bridging integrator 1 and with the microtubule-associated protein Tau in Alzheimer's disease.

Sporadic or late-onset Alzheimer's disease (AD) is expected to affect 50% of individuals reaching 85 years of age. The most significant genetic risk factor for late-onset AD is the e4 allele of APOE gene encoding apolipoprotein E, a lipid carrier shown to modulate brain amyloid burden. Recent genome-wide association studies have uncovered additional single nucleotide polymorphisms (SNPs) linked to AD susceptibility, including those in the CLU and BIN1 genes encoding for clusterin (CLU) and the bridging integrator 1 (BIN1) proteins, respectively. Because CLU has been implicated in brain amyloid-β (Aβ) clearance in mouse models of amyloid deposition, we sought to investigate whether an AD-linked SNP in the CLU gene altered Aβ42 biomarker levels in the cerebrospinal fluid (CSF). Instead, we found that the CLU rs11136000 SNP modified CSF levels of the microtubule-associated protein Tau in AD patients. We also found that an intracellular form of CLU (iCLU) was upregulated in the brain of Tau overexpressing Tg4510 mice, but not in Tg2576 amyloid mouse model. By overexpressing iCLU and Tau in cell culture systems we discovered that iCLU was a Tau-interacting protein and that iCLU associated with brain-specific isoforms of BIN1, also recently identified as a Tau-binding protein. Through expression analysis of CLU and BIN1 variants, we found that CLU and BIN1 interacted via their coiled-coil motifs. In co-immunoprecipitation studies using human brain tissue, we showed that iCLU and the major BIN1 isoform expressed in neurons were associated with modified Tau species found in AD. Finally, we showed that expression of certain coding CLU variants linked to AD risk led to increased levels of iCLU. Together, our findings suggest that iCLU and BIN1 interaction might impact Tau function in neurons and uncover potential new mechanisms underlying the etiology of Tau pathology in AD.

Clusterin variants are not associated with southern Chinese patients with Alzheimer's disease

Recent genome-wide association studies identified clusterin (CLU) to be associated with sporadic Alzheimer's disease. To help clarify the relevance of CLU as genetic determinant of AD, we analyzed its association in southern Chinese Han population. This study comprised 499 sporadic Alzheimer's disease patients and 592 unrelated age- and sex-matched healthy controls. Four single-nucleotide polymorphisms (rs2279590, rs9331888, rs11136000, and rs1532278) within CLU were selected for genotyping. No positive association was found between the CLU variants and AD. Our study suggests that CLU variants may not be an AD susceptibility factor in southern Chinese Han population.

Combined effects of Alzheimer risk variants in the CLU and ApoE genes on ventricular expansion patterns in the elderly.

The C allele at the rs11136000 locus in the clusterin (CLU) gene is the third strongest known genetic risk factor for late-onset Alzheimer's disease (LOAD). A recent genome-wide association study of LOAD found the strongest evidence of association with CLU at rs1532278, in high linkage disequilibrium with rs11136000. Brain structure and function are related to the CLU risk alleles, not just in LOAD patients but also in healthy young adults. We tracked the volume of the lateral ventricles across baseline, 1-year, and 2-year follow-up scans in a large sample of elderly human participants (N = 736 at baseline), from the Alzheimer's Disease Neuroimaging Initiative, to determine whether these CLU risk variants predicted longitudinal ventricular expansion. The rs11136000 major C allele-previously linked with reduced CLU expression and with increased risk for dementia-predicted faster expansion, independently of dementia status or ApoE genotype. Further analyses revealed that the CLU and ApoE risk variants had combined effects on both volumetric expansion and lateral ventricle surface morphology. The rs1532278 locus strongly resembles a regulatory element. Its association with ventricular expansion was slightly stronger than that of rs11136000 in our analyses, suggesting that it may be closer to a functional variant. Clusterin affects inflammation, immune responses, and amyloid clearance, which in turn may result in neurodegeneration. Pharmaceutical agents such as valproate, which counteract the effects of genetically determined reduced clusterin expression, may help to achieve neuroprotection and contribute to the prevention of dementia, especially in carriers of these CLU risk variants.

Clusterin Is a Gene-Specific Target of microRNA-21 in Head and Neck Squamous Cell Carcinoma

MicroRNA-21 (miRNA-21) has proto-oncogenic properties, although no miRNA-21-specific targets have been found in head and neck squamous cell carcinoma (HNSCC). Further study of miRNA-21 and its specific targets is essential to understanding HNSCC biology. miRNA expression profiles of 10 HNSCCs and 10 normal mucosa samples were investigated using a custom miRNA microarray. Thirteen HNSCCs and five normal mucosa primary tissue specimens underwent mRNA expression microarray analysis. To identify miRNA-21 downstream targets, oral keratinocyte cells were subjected to microarray analysis after miRNA-21 transient transfection. miRNA and mRNA expression were validated by reverse transcription quantitative polymerase chain reaction (RT-qPCR) in a separate cohort of 16 HNSCCs and 15 normal mucosal samples. Microarray and bioinformatics analyses were integrated to identify potential gene targets. In vitro assays looked at the function and interaction of miRNA-21 and its specific gene targets. miRNA-21 was upregulated in HNSCCs and stimulated cell growth. Integrated analyses identified Clusterin (CLU) as a potential miRNA-21 gene target. CLU was downregulated after forced expression of miRNA-21 in normal and HNSCC cell lines. The activity of a luciferase construct containing the 3'-untranslated region (UTR) of CLU was repressed by the ectopic expression of miRNA-21. CLU was also downregulated in primary HNSCCs and correlated with miRNA-21 overexpression. CLU variant 1 (CLU-1) was the predominant splice variant in HNSCCs and showed growth suppression function that was reversed by miRNA-21 overexpression. CLU is a specific, functional target of oncogenic miRNA-21 in HNSCCs. CLU-1 isoform is the predominant growth-suppressive variant targeted by miRNA-21.

Cotransin induces accumulation of a cytotoxic clusterin variant that cotranslationally rerouted to the cytosol

Although clusterin (CLU) was originally identified as a secreted glycoprotein that plays cytoprotective role, several intracellular CLU variants have been recently identified in the diverse pathological conditions. The mechanistic basis of these variants is now believed to be alternative splicing and retrotranslocation. Here, we uncovered, an unglycosylated and signal sequence-unprocessed, CLU variant in the cytosol. This variant proved to be a product that cotranslationally rerouted to the cytosol during translocation. Cytosolic CLU was prone to aggregation at peri-nuclear region of cells and induced cell death. Signal sequence is shown to be an important determinant for cytosolic CLU generation and aggregation. These results provide not only a new mechanistic insight into the cytosolic CLU generation but also an idea for therapeutic mislocalization of CLU as a strategy for cancer treatment.

Unglycosylated clusterin variant accumulates in the endoplasmic reticulum and induces cytotoxicity

Clusterin is a stress-responsive and highly glycosylated secretory protein that plays cytoprotective role in most body fluids. In addition to extracellular clusterin, several intracellular clusterin variants that are rather cytotoxic have been recently uncovered under diverse pathological conditions. Although these variants revealed heterogeneity in their glycan modification, its significance in many diseases remains to be validated. Here, we found that clusterin is differentially metabolized by two well-characterized ER stress inducers. Thapsigargin induced retrotranslocation and rapid degradation of clusterin from the endoplasmic reticulum, whereas tunicamycin failed to degrade but rather retained clusterin in the endoplasmic reticulum. Important sorting determinant for these processes proved to be N-glycan moieties that are required for the prevention of terminal misfolding and aggregation of clusterin in the endoplasmic reticulum. This study provides a mechanistic insight into the generation of noble cytotoxic variant of intracellular clusterin and an idea about molecular pathogenesis of diseases associated with chronic endoplasmic reticulum stress, such as neurodegeneration.

Two More Gene Variants Linked to Alzheimer's Risk

Back to table of contents Previous article Next article Clinical & Research NewsFull AccessTwo More Gene Variants Linked to Alzheimer's RiskJoan Arehart-TreichelJoan Arehart-TreichelSearch for more papers by this authorPublished Online:16 Oct 2009https://doi.org/10.1176/pn.44.20.0021bIn the largest genome-wide association hunt to date for genes that contribute to increased Alzheimer's risk, scientists have identified two gene variants that seem to heighten risk for the disease. Their study included DNA samples made available by dozens of labs in Europe and the United States.One of the genes, called CLU (for clusterin), is located on chromosome 8. The other gene, called PICALM (for phosphatidylinositol-binding clathin assembly protein), is located on chromosome 11.Four other gene variants have already been firmly linked with Alzheimer's. A variant of the gene making amyloid precursor protein and variants of the genes making presenilin 1 and 2 appear to be capable of causing the rare, early-onset form of Alzheimer's. The e4 variant of the APOE gene is a notable risk factor for the more common late-onset type.The study was led by Julie Williams, Ph.D., and colleagues at Cardiff University in Wales. DNA samples were taken from 11,789 individuals—3,941 with Alzheimer's and 7,848 without. The well-established Alzheimer's risk gene variant APOE e4 was found to be present significantly more often in the samples from Alzheimer's subjects than from controls. But two other gene variants were as well—for the CLU gene and the PICALM gene.Williams and her team then attempted to replicate their findings, this time using DNA samples from 4,363 individuals—2,023 with Alzheimer's and 2,340 without. Again they found that not just APOE e4, but also the CLU and PICALM gene variants were present significantly more often in DNA from Alzheimer's subjects than that from controls.These findings, the researchers concluded, provide “compelling evidence” that both the CLU gene variant and the PICALM gene variant are risk factors for Alzheimer's. They published their findings online September 6 in Nature Genetics.The PICALM gene is known to produce its protein product in all types of mammalian tissues, but especially at nerve synapses, and this protein product in turn is known to ferry neurotransmitters between neurons. So perhaps the PICALM gene variant that Williams and her colleagues have linked with Alzheimer's interferes with normal nerve synapse activities, thereby increasing the risk for Alzheimer's.The CLU gene makes a major brain apolipoprotein, as does the APOE gene, called clusterin. The CLU gene makes more clusterin in areas of the brain that are injured or inflamed than in areas that are not. And clusterin has been shown to cooperate with the apolipoprotein made by the APOE gene in suppressing the deposit of amyloid plaques in the brain. So the CLU gene variant that Williams and her team have linked with Alzheimer's may malfunction in carrying out such activities and thereby increase Alzheimer's risk.“Identifying gene variants like CLU and PICALM advances our understanding of the many genetic factors that may contribute to overall risk for this devastating neurological disorder and how these genes affect the development of Alzheimer's,” Marcelle Morrison-Bogorad, Ph.D., said in a press release issued in conjunction with the publication of the study's results. “This knowledge may then lead to novel disease pathways that can be targeted to develop new treatments.” Morrison-Bogorad is director of the National Institute on Aging's Division of Neuroscience. The National Institute on Aging's Laboratory of Neurogenetics is one of the labs that contributed DNA samples to the study.An abstract of “Genome-wide Association Study Identifies Variants at CLU and PICALM Associated With Alzheimer's Disease” is posted at<www.nature.com/ng/journal/vaop/ncurrent/abs/ng.440.html>.▪ ISSUES NewArchived