Publications by authors named "Bjorn Bakker"

41 Publications

Systemic complement levels in patients with age-related macular degeneration carrying rare or low frequency variants in the CFH gene.

Hum Mol Genet 2021 Sep 11. Epub 2021 Sep 11.

Department of Ophthalmology, Donders Institute for Brain, Cognition and Behavior, Radboud University Medical Center, 6525 GA Nijmegen, The Netherlands.

Age-related macular degeneration (AMD) is a major cause of vision loss among the elderly in the Western world. Genetic variants in the complement factor H (CFH) gene are associated with AMD, but the functional consequences of many of these variants are currently unknown. In this study we aimed to determine the effect of 64 rare and low frequency variants in the CFH gene on systemic levels of factor H (FH) and complement activation marker C3bBbP using plasma samples of 252 carriers and 159 non-carriers. Individuals carrying a heterozygous nonsense, frameshift or missense variant in CFH presented with significantly decreased FH levels, and significantly increased C3bBbP levels in plasma compared to non-carrier controls. FH and C3bBbP plasma levels were relatively stable over time in samples collected during follow-up visits. Decreased FH and increased C3bBbP concentrations were observed in carriers compared to non-carriers of CFH variants among different AMD stages, with the exception of C3bBbP levels in advanced AMD stages, which were equally high in carriers and non-carriers. In AMD families, FH levels were decreased in carriers compared to non-carriers, but C3bBbP levels did not differ. Rare variants in the CFH gene can lead to reduced FH levels or reduced FH function as measured by increased C3bBbP levels. The effects of individual variants in the CFH gene reported in this study will improve the interpretation of rare and low frequency variants observed in AMD patients in clinical practice.
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http://dx.doi.org/10.1093/hmg/ddab256DOI Listing
September 2021

Transient genomic instability drives tumorigenesis through accelerated clonal evolution.

Genes Dev 2021 Aug 15;35(15-16):1093-1108. Epub 2021 Jul 15.

Ludwig Cancer Research, Department of Cellular and Molecular Medicine, University of California at San Diego, La Jolla, California 92093, USA.

Abnormal numerical and structural chromosome content is frequently found in human cancer. To test the role of aneuploidy in tumor initiation and progression, we generated mice with random aneuploidies by transient induction of polo-like kinase 4 (Plk4), a master regulator of centrosome number. Short-term chromosome instability (CIN) from transient Plk4 induction resulted in formation of aggressive T-cell lymphomas in mice with heterozygous inactivation of one allele and accelerated tumor development in the absence of p53. Transient CIN increased the frequency of lymphoma-initiating cells with a specific karyotype profile, including trisomy of chromosomes 4, 5, 14, and 15 occurring early in tumorigenesis. Tumor development in mice with chronic CIN induced by an independent mechanism (through inactivation of the spindle assembly checkpoint) gradually trended toward a similar karyotypic profile, as determined by single-cell whole-genome DNA sequencing. Overall, we show how transient CIN generates cells with random aneuploidies from which ones that acquire a karyotype with specific chromosome gains are sufficient to drive cancer formation, and that distinct CIN mechanisms can lead to similar karyotypic cancer-causing outcomes.
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http://dx.doi.org/10.1101/gad.348319.121DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8336898PMC
August 2021

Common haplotypes at the CFH locus and low-frequency variants in CFHR2 and CFHR5 associate with systemic FHR concentrations and age-related macular degeneration.

Am J Hum Genet 2021 08 13;108(8):1367-1384. Epub 2021 Jul 13.

Department of Ophthalmology, Donders Institute for Brain, Cognition and Behaviour, Radboud University Medical Center, Nijmegen, 6525EX, the Netherlands; Department of Human Genetics, Donders Institute for Brain, Cognition and Behaviour, Radboud University Medical Centre, 6525GA, the Netherlands. Electronic address:

Age-related macular degeneration (AMD) is the principal cause of blindness in the elderly population. A strong effect on AMD risk has been reported for genetic variants at the CFH locus, encompassing complement factor H (CFH) and the complement-factor-H-related (CFHR) genes, but the underlying mechanisms are not fully understood. We aimed to dissect the role of factor H (FH) and FH-related (FHR) proteins in AMD in a cohort of 202 controls and 216 individuals with AMD. We detected elevated systemic levels of FHR-1 (p = 1.84 × 10), FHR-2 (p = 1.47 × 10), FHR-3 (p = 1.05 × 10) and FHR-4A (p = 1.22 × 10) in AMD, whereas FH concentrations remained unchanged. Common AMD genetic variants and haplotypes at the CFH locus strongly associated with FHR protein concentrations (e.g., FH p.Tyr402His and FHR-2 concentrations, p = 3.68 × 10), whereas the association with FH concentrations was limited. Furthermore, in an International AMD Genomics Consortium cohort of 17,596 controls and 15,894 individuals with AMD, we found that low-frequency and rare protein-altering CFHR2 and CFHR5 variants associated with AMD independently of all previously reported genome-wide association study (GWAS) signals (p = 5.03 × 10 and p = 2.81 × 10, respectively). Low-frequency variants in CFHR2 and CFHR5 led to reduced or absent FHR-2 and FHR-5 concentrations (e.g., p.Cys72Tyr in CFHR2 and FHR-2, p = 2.46 × 10). Finally, we showed localization of FHR-2 and FHR-5 in the choriocapillaris and in drusen. Our study identifies FHR proteins as key proteins in the AMD disease mechanism. Consequently, therapies that modulate FHR proteins might be effective for treating or preventing progression of AMD. Such therapies could target specific individuals with AMD on the basis of their genotypes at the CFH locus.
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http://dx.doi.org/10.1016/j.ajhg.2021.06.002DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8387287PMC
August 2021

Development of a Genotype Assay for Age-Related Macular Degeneration: The EYE-RISK Consortium.

Ophthalmology 2020 Jul 25. Epub 2020 Jul 25.

Department of Ophthalmology, Donders Institute for Brain, Cognition and Behaviour, Radboud University Medical Center, Nijmegen, The Netherlands. Electronic address:

Purpose: To develop a genotype assay to assess associations with common and rare age-related macular degeneration (AMD) risk variants, to calculate an overall genetic risk score (GRS), and to identify potential misdiagnoses with inherited macular dystrophies that mimic AMD.

Design: Case-control study.

Participants: Individuals (n = 4740) from 5 European cohorts.

Methods: We designed single-molecule molecular inversion probes for target selection and used next generation sequencing to sequence 87 single nucleotide polymorphisms (SNPs), coding and splice-site regions of 10 AMD-(related) genes (ARMS2, C3, C9, CD46, CFB, CFH, CFI, HTRA1, TIMP3, and SLC16A8), and 3 genes that cause inherited macular dystrophies (ABCA4, CTNNA1, and PRPH2). Genetic risk scores for common AMD risk variants were calculated based on effect size and genotype of 52 AMD-associated variants. Frequency of rare variants was compared between late AMD patients and control individuals with logistic regression analysis.

Main Outcome Measures: Genetic risk score, association of genetic variants with AMD, and genotype-phenotype correlations.

Results: We observed high concordance rates between our platform and other genotyping platforms for the 69 successfully genotyped SNPs (>96%) and for the rare variants (>99%). We observed a higher GRS for patients with late AMD compared with patients with early/intermediate AMD (P < 0.001) and individuals without AMD (P < 0.001). A higher proportion of pathogenic variants in the CFH (odds ratio [OR] = 2.88; P = 0.006), CFI (OR = 4.45; P = 0.005), and C3 (OR = 6.56; P = 0.0003) genes was observed in late AMD patients compared with control individuals. In 9 patients, we identified pathogenic variants in the PRPH2, ABCA4, and CTNNA1 genes, which allowed reclassification of these patients as having inherited macular dystrophy.

Conclusions: This study reports a genotype assay for common and rare AMD genetic variants, which can identify individuals at intermediate to high genetic risk of late AMD and enables differential diagnosis of AMD-mimicking dystrophies. Our study supports sequencing of CFH, CFI, and C3 genes because they harbor rare high-risk variants. Carriers of these variants could be amendable for new treatments for AMD that currently are under development.
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http://dx.doi.org/10.1016/j.ophtha.2020.07.037DOI Listing
July 2020

Effect of rare coding variants in the CFI gene on Factor I expression levels.

Hum Mol Genet 2020 08;29(14):2313-2324

Department of Ophthalmology, Donders Institute for Brain, Cognition and Behavior, Radboud University Medical Center, 6525 GA Nijmegen, The Netherlands.

Factor I (FI) is one of the main inhibitors of complement activity, and numerous rare coding variants have been reported in patients with age-related macular degeneration, atypical hemolytic uremic syndrome and C3 glomerulopathy. Since many of these variants are of unknown clinical significance, this study aimed to determine the effect of rare coding variants in the complement factor I (CFI) gene on FI expression. We measured FI levels in plasma samples of carriers of rare coding variants and in vitro in the supernatants of epithelial cells expressing recombinant FI. FI levels were measured in 177 plasma samples of 155 individuals, carrying 24 different rare coding variants in CFI. In carriers of the variants p.Gly119Arg, p.Leu131Arg, p.Gly188Ala and c.772G>A (r.685_773del), significantly reduced FI plasma levels were detected. Furthermore, recombinant FI expression levels were determined for 126 rare coding variants. Of these variants 68 (54%) resulted in significantly reduced FI expression in supernatant compared to wildtype (WT). The recombinant protein expression levels correlated significantly with the FI level in plasma of carriers of CFI variants. In this study, we performed the most comprehensive FI expression level analysis of rare coding variants in CFI to date. More than half of CFI variants lead to reduced FI expression, which might impair complement regulation in vivo. Our study will aid the interpretation of rare coding CFI variants identified in clinical practice, which is in particular important in light of patient inclusion in ongoing clinical trials for CFI gene supplementation in AMD.
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http://dx.doi.org/10.1093/hmg/ddaa114DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7424754PMC
August 2020

A living biobank of ovarian cancer ex vivo models reveals profound mitotic heterogeneity.

Nat Commun 2020 02 13;11(1):822. Epub 2020 Feb 13.

Division of Cancer Sciences, Faculty of Biology, Medicine and Health, University of Manchester, Manchester Cancer Research Centre, Wilmslow Road, Manchester, M20 4GJ, UK.

High-grade serous ovarian carcinoma is characterised by TP53 mutation and extensive chromosome instability (CIN). Because our understanding of CIN mechanisms is based largely on analysing established cell lines, we developed a workflow for generating ex vivo cultures from patient biopsies to provide models that support interrogation of CIN mechanisms in cells not extensively cultured in vitro. Here, we describe a "living biobank" of ovarian cancer models with extensive replicative capacity, derived from both ascites and solid biopsies. Fifteen models are characterised by p53 profiling, exome sequencing and transcriptomics, and karyotyped using single-cell whole-genome sequencing. Time-lapse microscopy reveals catastrophic and highly heterogeneous mitoses, suggesting that analysis of established cell lines probably underestimates mitotic dysfunction in advanced human cancers. Drug profiling reveals cisplatin sensitivities consistent with patient responses, demonstrating that this workflow has potential to generate personalized avatars with advantages over current pre-clinical models and the potential to guide clinical decision making.
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http://dx.doi.org/10.1038/s41467-020-14551-2DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7018727PMC
February 2020

Extensive Clonal Branching Shapes the Evolutionary History of High-Risk Pediatric Cancers.

Cancer Res 2020 04 10;80(7):1512-1523. Epub 2020 Feb 10.

Division of Clinical Genetics, Department of Laboratory Medicine, Lund University, Lund, Sweden.

Darwinian evolution of tumor cells remains underexplored in childhood cancer. We here reconstruct the evolutionary histories of 56 pediatric primary tumors, including 24 neuroblastomas, 24 Wilms tumors, and 8 rhabdomyosarcomas. Whole-genome copy-number and whole-exome mutational profiling of multiple regions per tumor were performed, followed by clonal deconvolution to reconstruct a phylogenetic tree for each tumor. Overall, 88% of the tumors exhibited genetic variation among primary tumor regions. This variability typically emerged through collateral phylogenetic branching, leading to spatial variability in the distribution of more than 50% (96/173) of detected diagnostically informative genetic aberrations. Single-cell sequencing of 547 individual cancer cells from eight solid pediatric tumors confirmed branching evolution to be a fundamental underlying principle of genetic variation in all cases. Strikingly, cell-to-cell genetic diversity was almost twice as high in aggressive compared with clinically favorable tumors (median Simpson index of diversity 0.45 vs. 0.88; = 0.029). Similarly, a comparison of multiregional sampling data from a total of 274 tumor regions showed that new phylogenetic branches emerge at a higher frequency per sample and carry a higher mutational load in high-risk than in low-risk tumors. Timelines based on spatial genetic variation showed that the mutations most influencing relapse risk occur at initiation of clonal expansion in neuroblastoma and rhabdomyosarcoma, whereas in Wilms tumor, they are late events. Thus, from an evolutionary standpoint, some high-risk childhood cancers are born bad, whereas others grow worse over time. SIGNIFICANCE: Different pediatric cancers with a high risk of relapse share a common generic pattern of extensively branching evolution of somatic mutations. GRAPHICAL ABSTRACT: http://cancerres.aacrjournals.org/content/canres/80/7/1512/F1.large.jpg.
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http://dx.doi.org/10.1158/0008-5472.CAN-19-3468DOI Listing
April 2020

E2F-Family Members Engage the PIDDosome to Limit Hepatocyte Ploidy in Liver Development and Regeneration.

Dev Cell 2020 02 23;52(3):335-349.e7. Epub 2020 Jan 23.

Division of Developmental Immunology, Biocenter, Medical University of Innsbruck, 6020 Innsbruck, Austria; Ludwig Boltzmann Institute for Rare and Undiagnosed Diseases, 1090 Vienna, Austria; CeMM Research Center for Molecular Medicine of the Austrian Academy of Sciences, 1090 Vienna, Austria. Electronic address:

E2F transcription factors control the cytokinesis machinery and thereby ploidy in hepatocytes. If or how these proteins limit proliferation of polyploid cells with extra centrosomes remains unknown. Here, we show that the PIDDosome, a signaling platform essential for caspase-2-activation, limits hepatocyte ploidy and is instructed by the E2F network to control p53 in the developing as well as regenerating liver. Casp2 and Pidd1 act as direct transcriptional targets of E2F1 and its antagonists, E2F7 and E2F8, that together co-regulate PIDDosome expression during juvenile liver growth and regeneration. Of note, whereas hepatocyte aneuploidy correlates with the basal ploidy state, the degree of aneuploidy itself is not limited by PIDDosome-dependent p53 activation. Finally, we provide evidence that the same signaling network is engaged to control ploidy in the human liver after resection. Our study defines the PIDDosome as a primary target to manipulate hepatocyte ploidy and proliferation rates in the regenerating liver.
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http://dx.doi.org/10.1016/j.devcel.2019.12.016DOI Listing
February 2020

BubR1 allelic effects drive phenotypic heterogeneity in mosaic-variegated aneuploidy progeria syndrome.

J Clin Invest 2020 01;130(1):171-188

Departments of Biochemistry and Molecular Biology.

Mosaic-variegated aneuploidy (MVA) syndrome is a rare childhood disorder characterized by biallelic BUBR1, CEP57, or TRIP13 aberrations; increased chromosome missegregation; and a broad spectrum of clinical features, including various cancers, congenital defects, and progeroid pathologies. To investigate the mechanisms underlying this disorder and its phenotypic heterogeneity, we mimicked the BUBR1L1012P mutation in mice (BubR1L1002P) and combined it with 2 other MVA variants, BUBR1X753 and BUBR1H, generating a truncated protein and low amounts of wild-type protein, respectively. Whereas BubR1X753/L1002P and BubR1H/X753 mice died prematurely, BubR1H/L1002P mice were viable and exhibited many MVA features, including cancer predisposition and various progeroid phenotypes, such as short lifespan, dwarfism, lipodystrophy, sarcopenia, and low cardiac stress tolerance. Strikingly, although these mice had a reduction in total BUBR1 and spectrum of MVA phenotypes similar to that of BubR1H/H mice, several progeroid pathologies were attenuated in severity, which in skeletal muscle coincided with reduced senescence-associated secretory phenotype complexity. Additionally, mice carrying monoallelic BubR1 mutations were prone to select MVA-related pathologies later in life, with predisposition to sarcopenia correlating with mTORC1 hyperactivity. Together, these data demonstrate that BUBR1 allelic effects beyond protein level and aneuploidy contribute to disease heterogeneity in both MVA patients and heterozygous carriers of MVA mutations.
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http://dx.doi.org/10.1172/JCI126863DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6934189PMC
January 2020

Premature mitotic entry induced by ATR inhibition potentiates olaparib inhibition-mediated genomic instability, inflammatory signaling, and cytotoxicity in BRCA2-deficient cancer cells.

Mol Oncol 2019 11 21;13(11):2422-2440. Epub 2019 Oct 21.

Department of Medical Oncology, University Medical Center Groningen, University of Groningen, The Netherlands.

Poly(ADP-ribose) polymerase (PARP) inhibitors are selectively cytotoxic in cancer cells with defects in homologous recombination (HR) (e.g., due to BRCA1/2 mutations). However, not all HR-deficient tumors efficiently respond to PARP inhibition and often acquire resistance. It is therefore important to uncover how PARP inhibitors induce cytotoxicity and develop combination strategies to potentiate PARP inhibitor efficacy in HR-deficient tumors. In this study, we found that forced mitotic entry upon ATR inhibition potentiates cytotoxic effects of PARP inhibition using olaparib in BRCA2-depleted and Brca2 knockout cancer cell line models. Single DNA fiber analysis showed that ATR inhibition does not exacerbate replication fork degradation. Instead, we find ATR inhibitors accelerate mitotic entry, resulting in the formation of chromatin bridges and lagging chromosomes. Furthermore, using genome-wide single-cell sequencing, we show that ATR inhibition enhances genomic instability of olaparib-treated BRCA2-depleted cells. Inhibition of CDK1 to delay mitotic entry mitigated mitotic aberrancies and genomic instability upon ATR inhibition, underscoring the role of ATR in coordinating proper cell cycle timing in situations of DNA damage. Additionally, we show that olaparib treatment leads to increased numbers of micronuclei, which is accompanied by a cGAS/STING-associated inflammatory response in BRCA2-deficient cells. ATR inhibition further increased the numbers of cGAS-positive micronuclei and the extent of cytokine production in olaparib-treated BRCA2-deficient cancer cells. Altogether, we show that ATR inhibition induces premature mitotic entry and mediates synergistic cytotoxicity with PARP inhibition in HR-deficient cancer cells, which involves enhanced genomic instability and inflammatory signaling.
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http://dx.doi.org/10.1002/1878-0261.12573DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6822251PMC
November 2019

Ongoing chromosomal instability and karyotype evolution in human colorectal cancer organoids.

Nat Genet 2019 05 29;51(5):824-834. Epub 2019 Apr 29.

Oncode Institute, Hubrecht Institute-KNAW, University Medical Centre Utrecht, Utrecht, the Netherlands.

Chromosome segregation errors cause aneuploidy and genomic heterogeneity, which are hallmarks of cancer in humans. A persistent high frequency of these errors (chromosomal instability (CIN)) is predicted to profoundly impact tumor evolution and therapy response. It is unknown, however, how prevalent CIN is in human tumors. Using three-dimensional live-cell imaging of patient-derived tumor organoids (tumor PDOs), we show that CIN is widespread in colorectal carcinomas regardless of background genetic alterations, including microsatellite instability. Cell-fate tracking showed that, although mitotic errors are frequently followed by cell death, some tumor PDOs are largely insensitive to mitotic errors. Single-cell karyotype sequencing confirmed heterogeneity of copy number alterations in tumor PDOs and showed that monoclonal lines evolved novel karyotypes over time in vitro. We conclude that ongoing CIN is common in colorectal cancer organoids, and propose that CIN levels and the tolerance for mitotic errors shape aneuploidy landscapes and karyotype heterogeneity.
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http://dx.doi.org/10.1038/s41588-019-0399-6DOI Listing
May 2019

Association of rs10490924 in ARMS2/HTRA1 with age-related macular degeneration in the Pakistani population.

Ann Hum Genet 2019 07 20;83(4):285-290. Epub 2019 Mar 20.

Transalational Genomics Laboratory, Department of Biosciences, COMSATS University Islamabad, Pakistan.

Age-related macular degeneration (AMD) is a disease of the elderly in which central vision is lost because of degenerative changes of the macula. The current study investigated the association of single-nucleotide polymorphisms (SNPs) with AMD in the Pakistani population. Four SNPs were analyzed in this study: rs1061170 in the CFH, rs429608 near CFB, rs2230199 in the C3, and rs10490924 in ARMS2/HTRA1. This case-control association study was conducted on 300 AMD patients (125 wet AMD and 175 dry AMD) and 200 unaffected age- and gender-matched control individuals. The association of the SNP genotypes and allele frequency distributions were compared between patients and healthy controls, keeping age, gender, and smoking status as covariates. A significant genotype and variant allele association was found of rs10490924 in ARMS2/HTRA1 with wet AMD, while the SNPs in CFH, CFB, and C3 were not associated with AMD in the current Pakistani cohort. The lack of association of CFH, CFB, and C3 may be attributed to limited sample size. This study demonstrates that genetic causative factors of AMD differ among populations and supports the need for genetic association studies among cohorts from various populations to increase our global understanding of the disease pathogenesis.
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http://dx.doi.org/10.1111/ahg.12311DOI Listing
July 2019

DNA Replication Vulnerabilities Render Ovarian Cancer Cells Sensitive to Poly(ADP-Ribose) Glycohydrolase Inhibitors.

Cancer Cell 2019 03;35(3):519-533.e8

Division of Cancer Sciences, Faculty of Biology, Medicine and Health, University of Manchester, Manchester Cancer Research Centre, 555 Wilmslow Road, Manchester M20 4GJ, UK. Electronic address:

Inhibitors of poly(ADP-ribose) polymerase (PARP) have demonstrated efficacy in women with BRCA-mutant ovarian cancer. However, only 15%-20% of ovarian cancers harbor BRCA mutations, therefore additional therapies are required. Here, we show that a subset of ovarian cancer cell lines and ex vivo models derived from patient biopsies are sensitive to a poly(ADP-ribose) glycohydrolase (PARG) inhibitor. Sensitivity is due to underlying DNA replication vulnerabilities that cause persistent fork stalling and replication catastrophe. PARG inhibition is synthetic lethal with inhibition of DNA replication factors, allowing additional models to be sensitized by CHK1 inhibitors. Because PARG and PARP inhibitor sensitivity are mutually exclusive, our observations demonstrate that PARG inhibitors have therapeutic potential to complement PARP inhibitor strategies in the treatment of ovarian cancer.
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http://dx.doi.org/10.1016/j.ccell.2019.02.004DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6428690PMC
March 2019

Quantification of Aneuploidy in Mammalian Systems.

Methods Mol Biol 2019 ;1896:159-190

European Research Institute for the Biology of Ageing (ERIBA), University of Groningen, University Medical Center Groningen, Groningen, The Netherlands.

High-throughput next generation sequencing karyotyping has emerged as a powerful tool for the detection of genomic heterogeneity in normal tissues and cancers. Here we describe a single-cell whole genome sequencing (scWGS) platform to assess whole-chromosome aneuploidy, structural aneuploidies involving only chromosome fragments and more local small copy number alterations in individual cells. We provide a detailed protocol for the isolation, library preparation, low coverage sequencing and data analysis of single cells. Since our approach does not involve a whole-genome preamplification step, our method allows for acquisition of reliable high-resolution single-cell copy number profiles. Moreover, the protocol allows multiplexing of 384 single-cell libraries in one sequencing run, thereby significantly reducing sequencing costs and can be completed in 3-4 days starting from single cell isolation to analysis of sequencing data.
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http://dx.doi.org/10.1007/978-1-4939-8931-7_15DOI Listing
August 2019

The p38α Stress Kinase Suppresses Aneuploidy Tolerance by Inhibiting Hif-1α.

Cell Rep 2018 10;25(3):749-760.e6

Division of Cancer Sciences, Faculty of Biology, Medicine and Health, University of Manchester, Manchester Cancer Research Centre, Wilmslow Road, Manchester M20 4QL, UK. Electronic address:

Deviating from the normal karyotype dramatically changes gene dosage, in turn decreasing the robustness of biological networks. Consequently, aneuploidy is poorly tolerated by normal somatic cells and acts as a barrier to transformation. Paradoxically, however, karyotype heterogeneity drives tumor evolution and the emergence of therapeutic drug resistance. To better understand how cancer cells tolerate aneuploidy, we focused on the p38 stress response kinase. We show here that p38-deficient cells upregulate glycolysis and avoid post-mitotic apoptosis, leading to the emergence of aneuploid subclones. We also show that p38 deficiency upregulates the hypoxia-inducible transcription factor Hif-1α and that inhibiting Hif-1α restores apoptosis in p38-deficent cells. Because hypoxia and aneuploidy are both barriers to tumor progression, the ability of Hif-1α to promote cell survival following chromosome missegregation raises the possibility that aneuploidy tolerance coevolves with adaptation to hypoxia.
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http://dx.doi.org/10.1016/j.celrep.2018.09.060DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6205844PMC
October 2018

Genetic screening for macular dystrophies in patients clinically diagnosed with dry age-related macular degeneration.

Clin Genet 2018 12 15;94(6):569-574. Epub 2018 Oct 15.

Department of Ophthalmology, Donders Institute for Brain, Cognition and Behaviour, Radboud University Medical Center, Nijmegen, the Netherlands.

It can be clinically challenging to distinguish dry age-related macular degeneration (AMD) from AMD-mimicking dystrophies, and sometimes misdiagnosis occurs. With upcoming therapies for dry AMD it is important to exclude patients with a different retinal disease from clinical trials. In this study we evaluated the occurrence of AMD-mimicking dystrophies in an AMD cohort. Whole-exome sequencing (WES) was performed in 218 patients with intermediate AMD or geographic atrophy secondary to AMD and 133 control individuals. WES data was analyzed for rare variants in 19 genes associated with autosomal dominant and recessive macular dystrophies mimicking AMD. In three (1.4%) of 218 cases we identified a pathogenic heterozygous variant (PRPH2 c.424C > T; p.R142W) causal for autosomal dominant central areolar choroidal dystrophy (CACD). Phenotypically, these patients all presented with geographic atrophy. In 12 (5.5%) of 218 cases we identified a heterozygous variant of unknown clinical significance, but predicted to be highly deleterious, in genes previously associated with autosomal dominant macular dystrophies. The distinction between AMD and AMD-mimicking dystrophies, such as CACD, can be challenging based on fundus examination alone. Genetic screening for genes associated with macular dystrophies, especially PRPH2, can be beneficial to help identify AMD-mimicking dystrophies.
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http://dx.doi.org/10.1111/cge.13447DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6282796PMC
December 2018

Role of the Complement System in Chronic Central Serous Chorioretinopathy: A Genome-Wide Association Study.

JAMA Ophthalmol 2018 10;136(10):1128-1136

Department of Ophthalmology, Donders Institute of Brain, Cognition and Behaviour, Radboud university medical centre, Nijmegen, the Netherlands.

Importance: To date, several targeted genetic studies on chronic central serous chorioretinopathy (cCSC) have been performed; however, unbiased genome-wide studies into the genetics of cCSC have not been reported. To discover new genetic loci associated with cCSC and to better understand the causative mechanism of this disease, we performed a genome-wide association study (GWAS) on patients with cCSC.

Objective: To discover new genetic loci and pathways associated with cCSC and to predict the association of genetic variants with gene expression in patients with cCSC.

Design, Setting, And Participants: This case-control GWAS was completed in the general community, 3 referral university medical centers, and outpatient care on Europeans individuals with cCSC and population-based control participants. Genotype data was collected from May 2013 to August 2017, and data analysis occurred from August 2017 to November 2017.

Main Outcomes And Measures: Associations of single-nucleotide polymorphisms, haplotypes, genetic pathways, and predicted gene expression with cCSC.

Results: A total of 521 patients with cCSC (median age, 51 years; interquartile range [IQR], 44-59 years; 420 [80.6%] male) and 3577 European population-based control participants (median age, 52 years; IQR, 37-71 years; 1630 [45.6%] male) were included. One locus on chromosome 1 at the complement factor H (CFH) gene reached genome-wide significance and was associated with an increased risk of cCSC (rs1329428; odds ratio [OR], 1.57 [95% CI, 1.38-1.80]; P = 3.12 × 10-11). The CFH haplotypes H1 and H3 were protective for cCSC (H1: OR, 0.64 [95% CI, 0.53-0.77]; P = 2.18 × 10-6; H3: OR, 0.54 [95% CI, 0.42-0.70]; P = 2.49 × 10-6), whereas haplotypes H2, H4, H5, and the aggregate of rare CFH haplotypes conferred increased risk (H2: OR, 1.57 [95% CI, 1.30-1.89]; P = 2.18 × 10-6; H4: OR, 1.43 [95% CI, 1.13-1.80]; P = 2.49 × 10-3; H5: OR, 1.80 [95% CI, 1.36-2.39]; P = 4.61 × 10-5; rare haplotypes: OR, 1.99 [95% CI, 1.43-2.77]; P = 4.59 × 10-5). Pathway analyses showed involvement of the complement cascade and alternative open reading frame (ARF) pathway in cCSC. Using PrediXcan, we identified changes in predicted expression of complement genes CFH, complement factor H related 1 (CFHR1), complement factor related 4 (CFHR4), and membrane cofactor protein (MCP/CD46). Additionally, the potassium sodium-activated channel subfamily T member 2 (KCNT2) and tumor necrosis factor receptor superfamily member 10a (TNFRSF10A) genes were differentially expressed in patients with cCSC.

Conclusions And Relevance: In this GWAS on cCSC, we identified a locus on chromosome 1 at the CFH gene that was significantly associated with cCSC, and we report protective and risk-conferring haplotypes in this gene. Pathway analyses were enriched for complement genes, and gene expression analysis suggests a role for CFH, CFHR1, CFHR4, CD46, KCNT2, and TNFRSF10A in the disease. Taken together, these results underscore the potential importance of the complement pathway in the causative mechanisms of cCSC.
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http://dx.doi.org/10.1001/jamaophthalmol.2018.3190DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6233836PMC
October 2018

FoxM1 repression during human aging leads to mitotic decline and aneuploidy-driven full senescence.

Nat Commun 2018 07 19;9(1):2834. Epub 2018 Jul 19.

Aging and Aneuploidy Laboratory, IBMC, Instituto de Biologia Molecular e Celular, Universidade do Porto, 4200-135, Porto, Portugal.

Aneuploidy, an abnormal chromosome number, has been linked to aging and age-associated diseases, but the underlying molecular mechanisms remain unknown. Here we show, through direct live-cell imaging of young, middle-aged, and old-aged primary human dermal fibroblasts, that aneuploidy increases with aging due to general dysfunction of the mitotic machinery. Increased chromosome mis-segregation in elderly mitotic cells correlates with an early senescence-associated secretory phenotype (SASP) and repression of Forkhead box M1 (FoxM1), the transcription factor that drives G2/M gene expression. FoxM1 induction in elderly and Hutchison-Gilford progeria syndrome fibroblasts prevents aneuploidy and, importantly, ameliorates cellular aging phenotypes. Moreover, we show that senescent fibroblasts isolated from elderly donors' cultures are often aneuploid, and that aneuploidy is a key trigger into full senescence phenotypes. Based on this feedback loop between cellular aging and aneuploidy, we propose modulation of mitotic efficiency through FoxM1 as a potential strategy against aging and progeria syndromes.
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http://dx.doi.org/10.1038/s41467-018-05258-6DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6053425PMC
July 2018

Non-random Mis-segregation of Human Chromosomes.

Cell Rep 2018 06;23(11):3366-3380

Barts Cancer Institute, Queen Mary University of London, London EC1M 6BQ, UK. Electronic address:

A common assumption is that human chromosomes carry equal chances of mis-segregation during compromised cell division. Human chromosomes vary in multiple parameters that might generate bias, but technological limitations have precluded a comprehensive analysis of chromosome-specific aneuploidy. Here, by imaging specific centromeres coupled with high-throughput single-cell analysis as well as single-cell sequencing, we show that aneuploidy occurs non-randomly following common treatments to elevate chromosome mis-segregation. Temporary spindle disruption leads to elevated mis-segregation and aneuploidy of a subset of chromosomes, particularly affecting chromosomes 1 and 2. Unexpectedly, we find that a period of mitotic delay weakens centromeric cohesion and promotes chromosome mis-segregation and that chromosomes 1 and 2 are particularly prone to suffer cohesion fatigue. Our findings demonstrate that inherent properties of individual chromosomes can bias chromosome mis-segregation and aneuploidy rates, with implications for studies on aneuploidy in human disease.
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http://dx.doi.org/10.1016/j.celrep.2018.05.047DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6019738PMC
June 2018

Functional analyses of rare genetic variants in complement component C9 identified in patients with age-related macular degeneration.

Hum Mol Genet 2018 08;27(15):2678-2688

Division of Medical Protein Chemistry, Department of Translational Medicine, Lund University, Malmö Sweden.

Age-related macular degeneration (AMD) is a progressive disease of the central retina and the leading cause of irreversible vision loss in the western world. The involvement of abnormal complement activation in AMD has been suggested by association of variants in genes encoding complement proteins with disease development. A low-frequency variant (p.P167S) in the complement component C9 (C9) gene was recently shown to be highly associated with AMD; however, its functional outcome remains largely unexplored. In this study, we reveal five novel rare genetic variants (p.M45L, p.F62S, p.G126R, p.T170I and p.A529T) in C9 in AMD patients, and evaluate their functional effects in vitro together with the previously identified (p.R118W and p.P167S) C9 variants. Our results demonstrate that the concentration of C9 is significantly elevated in patients' sera carrying the p.M45L, p.F62S, p.P167S and p.A529T variants compared with non-carrier controls. However, no difference can be observed in soluble terminal complement complex levels between the carrier and non-carrier groups. Comparing the polymerization of the C9 variants we reveal that the p.P167S mutant spontaneously aggregates, while the other mutant proteins (except for C9 p.A529T) fail to polymerize in the presence of zinc. Altered polymerization of the p.F62S and p.P167S proteins associated with decreased lysis of sheep erythrocytes and adult retinal pigment epithelial-19 cells by carriers' sera. Our data suggest that the analyzed C9 variants affect only the secretion and polymerization of C9, without influencing its classical lytic activity. Future studies need to be performed to understand the implications of the altered polymerization of C9 in AMD pathology.
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http://dx.doi.org/10.1093/hmg/ddy178DOI Listing
August 2018

Whole-Exome Sequencing in Age-Related Macular Degeneration Identifies Rare Variants in COL8A1, a Component of Bruch's Membrane.

Ophthalmology 2018 09 26;125(9):1433-1443. Epub 2018 Apr 26.

Department of Ophthalmology, Donders Institute for Brain, Cognition and Behaviour, Radboud University Medical Center, Nijmegen, The Netherlands; Department of Human Genetics, Donders Institute for Brain, Cognition and Behaviour, Radboud University Medical Center, Nijmegen, The Netherlands. Electronic address:

Purpose: Genome-wide association studies and targeted sequencing studies of candidate genes have identified common and rare variants that are associated with age-related macular degeneration (AMD). Whole-exome sequencing (WES) studies allow a more comprehensive analysis of rare coding variants across all genes of the genome and will contribute to a better understanding of the underlying disease mechanisms. To date, the number of WES studies in AMD case-control cohorts remains scarce and sample sizes are limited. To scrutinize the role of rare protein-altering variants in AMD cause, we performed the largest WES study in AMD to date in a large European cohort consisting of 1125 AMD patients and 1361 control participants.

Design: Genome-wide case-control association study of WES data.

Participants: One thousand one hundred twenty-five AMD patients and 1361 control participants.

Methods: A single variant association test of WES data was performed to detect variants that are associated individually with AMD. The cumulative effect of multiple rare variants with 1 gene was analyzed using a gene-based CMC burden test. Immunohistochemistry was performed to determine the localization of the Col8a1 protein in mouse eyes.

Main Outcome Measures: Genetic variants associated with AMD.

Results: We detected significantly more rare protein-altering variants in the COL8A1 gene in patients (22/2250 alleles [1.0%]) than in control participants (11/2722 alleles [0.4%]; P = 7.07×10). The association of rare variants in the COL8A1 gene is independent of the common intergenic variant (rs140647181) near the COL8A1 gene previously associated with AMD. We demonstrated that the Col8a1 protein localizes at Bruch's membrane.

Conclusions: This study supported a role for protein-altering variants in the COL8A1 gene in AMD pathogenesis. We demonstrated the presence of Col8a1 in Bruch's membrane, further supporting the role of COL8A1 variants in AMD pathogenesis. Protein-altering variants in COL8A1 may alter the integrity of Bruch's membrane, contributing to the accumulation of drusen and the development of AMD.
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http://dx.doi.org/10.1016/j.ophtha.2018.03.040DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6104593PMC
September 2018

Single-cell sequencing to quantify genomic integrity in cancer.

Int J Biochem Cell Biol 2018 01 23;94:146-150. Epub 2017 Sep 23.

European Research Institute for the Biology of Ageing (ERIBA), University of Groningen, University Medical Center Groningen, 9713 AV Groningen, The Netherlands. Electronic address:

The use of single-cell DNA sequencing (sc-seq) techniques for the diagnosis, prognosis and treatment of cancer is a rapidly developing field. Sc-seq research is gaining momentum by decreased sequencing costs and continuous improvements in techniques. In this review, we provide an overview of recent advancements in the field of sc-seq in cancer and we discuss how sc-seq can contribute to improved care for cancer patients. Sc-seq has made it possible to study the genomes of individual cancer cells from primary tumors, metastases and circulating tumor cells, revealing inter- and intra-tumor heterogeneity, which cannot be detected using other methods. We review studies on individual human cancer cells in relation to prognosis and treatment response. Finally, future perspectives of sc-seq in cancer diagnosis and treatment are discussed with a focus on the use of circulating tumor cells to monitor therapy response and the development of personalized treatments based on knowledge about the genomic heterogeneity.
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http://dx.doi.org/10.1016/j.biocel.2017.09.016DOI Listing
January 2018

p53 Prohibits Propagation of Chromosome Segregation Errors that Produce Structural Aneuploidies.

Cell Rep 2017 06;19(12):2423-2431

Department of Cell Biology and Cancer Genomics Center, Netherlands Cancer Institute, Plesmanlaan 121, 1066 CX Amsterdam, the Netherlands. Electronic address:

The presence of an abnormal karyotype has been shown to be profoundly detrimental at the cellular and organismal levels but is an overt hallmark of cancer. Aneuploidy can lead to p53 activation and thereby prevents proliferation, but the exact trigger for p53 activation has remained controversial. Here, we have used a system to induce aneuploidy in untransformed human cells to explore how cells deal with different segregation errors. We show that p53 is activated only in a subset of the cells with altered chromosome content. Importantly, we find that at least a subset of whole-chromosome aneuploidies can be propagated in p53-proficient cells, indicating that aneuploidy does not always lead to activation of p53. Finally, we demonstrate that propagation of structural aneuploidies (gain or loss of part of a chromosome) induced by segregation errors is limited to p53-deficient cells.
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http://dx.doi.org/10.1016/j.celrep.2017.05.055DOI Listing
June 2017

Deletion of the spindle assembly checkpoint gene is tolerated in mouse models of acute T-cell lymphoma and hepatocellular carcinoma.

Elife 2017 03 20;6. Epub 2017 Mar 20.

Department of Systems Biology, Harvard Medical School, Boston, United States.

Chromosome instability (CIN) is deleterious to normal cells because of the burden of aneuploidy. However, most human solid tumors have an abnormal karyotype implying that gain and loss of chromosomes by cancer cells confers a selective advantage. CIN can be induced in the mouse by inactivating the spindle assembly checkpoint. This is lethal in the germline but we show here that adult T cells and hepatocytes can survive conditional inactivation of the Mad2l1 SAC gene and resulting CIN. This causes rapid onset of acute lymphoblastic leukemia (T-ALL) and progressive development of hepatocellular carcinoma (HCC), both lethal diseases. The resulting DNA copy number variation and patterns of chromosome loss and gain are tumor-type specific, suggesting differential selective pressures on the two tumor cell types.
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http://dx.doi.org/10.7554/eLife.20873DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5400506PMC
March 2017

Centrosome Amplification Is Sufficient to Promote Spontaneous Tumorigenesis in Mammals.

Dev Cell 2017 02 26;40(3):313-322.e5. Epub 2017 Jan 26.

Department of Molecular Biology and Genetics, Johns Hopkins University School of Medicine, Baltimore, MD 21205, USA. Electronic address:

Centrosome amplification is a common feature of human tumors, but whether this is a cause or a consequence of cancer remains unclear. Here, we test the consequence of centrosome amplification by creating mice in which centrosome number can be chronically increased in the absence of additional genetic defects. We show that increasing centrosome number elevated tumor initiation in a mouse model of intestinal neoplasia. Most importantly, we demonstrate that supernumerary centrosomes are sufficient to drive aneuploidy and the development of spontaneous tumors in multiple tissues. Tumors arising from centrosome amplification exhibit frequent mitotic errors and possess complex karyotypes, recapitulating a common feature of human cancer. Together, our data support a direct causal relationship among centrosome amplification, genomic instability, and tumor development.
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http://dx.doi.org/10.1016/j.devcel.2016.12.022DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5296221PMC
February 2017

The Functional Effect of Rare Variants in Complement Genes on C3b Degradation in Patients With Age-Related Macular Degeneration.

JAMA Ophthalmol 2017 Jan;135(1):39-46

Department of Ophthalmology, Donders Institute for Brain, Cognition, and Behaviour, Radboud University Medical Center, Nijmegen, the Netherlands4Department of Human Genetics, Donders Institute for Brain, Cognition, and Behaviour, Radboud University Medical Center, Nijmegen, the Netherlands.

Importance: In age-related macular degeneration (AMD), rare variants in the complement system have been described, but their functional consequences remain largely unexplored.

Objectives: To identify new rare variants in complement genes and determine the functional effect of identified variants on complement levels and complement regulation in serum samples from carriers and noncarriers.

Design, Setting, And Participants: This study evaluated affected (n = 114) and unaffected (n = 60) members of 22 families with AMD and a case-control cohort consisting of 1831 unrelated patients with AMD and 1367 control individuals from the European Genetic Database from March 29, 2006, to April 26, 2013, in Nijmegen, the Netherlands, and Cologne, Germany. Exome sequencing data of families were filtered for rare variants in the complement factor H (CFH), complement factor I (CFI), complement C9 (C9), and complement C3 (C3) genes. The case-control cohort was genotyped with allele-specific assays. Serum samples were obtained from carriers of identified variants (n = 177) and age-matched noncarriers (n = 157). Serum concentrations of factor H (FH), factor I (FI), C9, and C3 were measured, and C3b degradation ability was determined.

Main Outcomes And Measures: Association of rare variants in the CFH, CFI, C9, and C3 genes with AMD, serum levels of corresponding proteins, and C3b degradation ability of CFH and CFI variant carriers.

Results: The 1831 unrelated patients with AMD had a mean (SD) age of 75.0 (9.4) years, and 60.5% were female. The 1367 unrelated control participants had a mean (SD) age of 70.4 (7.0), and 58.7% were female. All individuals were of European descent. Rare variants in CFH, CFI, C9, and C3 contributed to an increased risk of developing AMD (odds ratio, 2.04; 95% CI, 1.47-2.82; P < .001). CFI carriers had decreased median FI serum levels (18.2 μg/mL in Gly119Arg carriers and 16.2 μg/mL in Leu131Arg carriers vs 27.2 and 30.4 μg/mL in noncarrier cases and controls, respectively; both P < .001). Elevated C9 levels were observed in Pro167Ser carriers (10.7 µg/mL vs 6.6 and 6.1 µg/mL in noncarrier cases and controls, respectively; P < .001). The median FH serum levels were 299.4 µg/mL for CFH Arg175Gln and 266.3 µg/mL for CFH Ser193Leu carriers vs 302.4 and 283.0 µg/mL for noncarrier cases and controls, respectively. The median C3 serum levels were 943.2 µg/mL for C3 Arg161Trp and 946.7 µg/mL for C3 Lys155Gln carriers vs 874.0 and 946.7 µg/mL for noncarrier cases and controls, respectively. The FH and FI levels correlated with C3b degradation in noncarriers (R2 = 0.35 and R2 = 0.31, respectively; both P < .001).

Conclusions And Relevance: Reduced serum levels were associated with C3b degradation in carriers of CFI but not CFH variants, suggesting that CFH variants affect functional activity of FH rather than serum levels. Carriers of CFH (Arg175Gln and Ser193Leu) and CFI (Gly119Arg and Leu131Arg) variants have an impaired ability to regulate complement activation and may benefit more from complement-inhibiting therapy than patients with AMD in general.
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http://dx.doi.org/10.1001/jamaophthalmol.2016.4604DOI Listing
January 2017

Aneuploidy in stem cells.

World J Stem Cells 2016 Jun;8(6):216-22

Jorge Garcia-Martinez, Bjorn Bakker, Klaske M Schukken, Judith E Simon, Floris Foijer, European Research Institute for the Biology of Ageing, University of Groningen, University Medical Center Groningen, NL-9713 AV Groningen, The Netherlands.

Stem cells hold enormous promise for regenerative medicine as well as for engineering of model systems to study diseases and develop new drugs. The discovery of protocols that allow for generating induced pluripotent stem cells (IPSCs) from somatic cells has brought this promise steps closer to reality. However, as somatic cells might have accumulated various chromosomal abnormalities, including aneuploidies throughout their lives, the resulting IPSCs might no longer carry the perfect blueprint for the tissue to be generated, or worse, become at risk of adopting a malignant fate. In this review, we discuss the contribution of aneuploidy to healthy tissues and how aneuploidy can lead to disease. Furthermore, we review the differences between how somatic cells and stem cells respond to aneuploidy.
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http://dx.doi.org/10.4252/wjsc.v8.i6.216DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4919689PMC
June 2016

Single-cell whole genome sequencing reveals no evidence for common aneuploidy in normal and Alzheimer's disease neurons.

Genome Biol 2016 05 31;17(1):116. Epub 2016 May 31.

European Research Institute for the Biology of Ageing (ERIBA), University of Groningen, University Medical Center Groningen, 9713 AV, Groningen, The Netherlands.

Background: Alzheimer's disease (AD) is a neurodegenerative disease of the brain and the most common form of dementia in the elderly. Aneuploidy, a state in which cells have an abnormal number of chromosomes, has been proposed to play a role in neurodegeneration in AD patients. Several studies using fluorescence in situ hybridization have shown that the brains of AD patients contain an increased number of aneuploid cells. However, because the reported rate of aneuploidy in neurons ranges widely, a more sensitive method is needed to establish a possible role of aneuploidy in AD pathology.

Results: In the current study, we used a novel single-cell whole genome sequencing (scWGS) approach to assess aneuploidy in isolated neurons from the frontal cortex of normal control individuals (n = 6) and patients with AD (n = 10). The sensitivity and specificity of our method was shown by the presence of three copies of chromosome 21 in all analyzed neuronal nuclei of a Down's syndrome sample (n = 36). Very low levels of aneuploidy were found in the brains from control individuals (n = 589) and AD patients (n = 893). In contrast to other studies, we observe no selective gain of chromosomes 17 or 21 in neurons of AD patients.

Conclusion: scWGS showed no evidence for common aneuploidy in normal and AD neurons. Therefore, our results do not support an important role for aneuploidy in neuronal cells in the pathogenesis of AD. This will need to be confirmed by future studies in larger cohorts.
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http://dx.doi.org/10.1186/s13059-016-0976-2DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4888403PMC
May 2016
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