Project description:Chromosome segregation errors have been linked to DNA damage and genomic rearrangements. Accumulating evidence has shown that catastrophic genomic rearrangements, like chromothripsis, can result from lagging chromosomes undergoing aberrant DNA replication and DNA damage in micronuclei. Detailed characterization of genomic rearrangements resulting from DNA damage in micronuclei has been hampered because of difficulties in culturing daughter cells with DNA damage. Here, we employ a method by which a specific single chromosome is trapped in a micronucleus, followed by transfer to an acceptor cell. Next, stably propagating clonal cell lines with an extra chromosome were established and analyzed by copy number profiling and whole genome sequencing. While non-transformed, p53 proficient and telomerase-immortalized RPE1 cells showed a stable genome following addition of the transferred chromosome, we observed frequent de novo genomic rearrangements in cells derived from the HCT116 colorectal cancer cell line after chromosome transfer. The de novo rearrangements varied from simple deletions and duplications to complex rearrangements. Phase-informative SNPs revealed that the rearrangements specifically occurred on the transferred chromosome. We found that the complex rearrangements recapitulated all features of chromothripsis, including massive oscillation between two copy number states, localization to a single chromosome, random joining of chromosome fragments and non-homologous or micro-homologous repair. We describe an approach that enables the isolation of clonal cell lines with genomic rearrangements and chromothripsis on a specific chromosome in p53 proficient cells. The procedure enables further investigation of the exact mechanism leading to chromothripsis and the analysis of its consequences for cell survival (viability) and cancer development. We analyzed 38 cell clones, originating from HCT116 or RPE1 cells respectively, with Illumina beadchip arrays to test for unique de novo copy number variants and to determine the chromosome affacted by the CNAs.

Project description:Immunoglobulin A Nephropathy (IgAN) is a complex multifactorial disease whose genetic bases remain unknown. Distinct linkage and genome-wide association studies in both familial and sporadic IgAN suggest that there is a strong genetic component in IgAN. In this context, an intriguing role could be ascribed to copy number variants (CNVs) that have been recognized as an important source of genetic variation in humans. Here, we performed a whole-genome screening of CNVs in IgAN patients, their healthy relatives and healthy subjects (HS). A total of 217 individuals consisting of 51 IgAN cases and 166 healthy relatives were included in the initial screening. The high-throughput analysis of structural genetic variations, to find concordant aberrations across classes of samples, identified 178 IgAN-specific aberrations, specifically 114 loss and 64 gain. Several CNVs overlapped with regions evidenced by previous genome-wide genetic studies. Moreover, we found that IgAN patients characterized by deteriorated renal function carried low copy numbers of a CNV in chromosome 3 (chr3_loss:52031010-52260722). This CNV contained the TLR9 gene whose expression significantly correlated with the loss aberration in patients with progressive renal damage. Conversely, IgAN patients with normal renal function had no chr3_loss:52031010-52260722 and the TLR9 mRNA was expressed at the same level as in HS, still maintaining a strong correlation with the CNV. In conclusion, here we performed the first genome-wide CNV study in IgAN identifying some structural variants specific to IgAN patients and providing a collection of new candidate genes and loci that could help to dissect the complex genomic setting of the disease. Moreover, we identified a specific CNV, spanning the TLR9 gene, which could explain the disease severity in IgAN patients. To perform a genome-wide CNV study in IgAN identifying some structural variants specific to IgAN patients and providing a collection of new candidate genes and loci that could help to dissect the complex genomic setting of the disease.

Project description:Sakha M-bM-^@M-^S an area connecting South and Northeast Siberia M-bM-^@M-^S is significant for understanding the history of peopling of Northeast Eurasia and the Americas. Previous studies have shown a genetic contiguity between Siberia and East Asia and the key role of South Siberia in the colonization of Siberia. We report the results of a high-resolution phylogenetic analysis of 701 mtDNAs and 318 Y chromosomes from five native populations of Sakha (Yakuts, Evenks, Evens, Yukaghirs and Dolgans) and of the analysis of more than 500,000 autosomal SNPs of 758 individuals from 55 populations, including 40 previously unpublished samples from Siberia. Phylogenetically terminal clades of East Asian mtDNA haplogroups C and D and Y-chromosome haplogroups N1c, N1b and C3, constituting the core of the gene pool of the native populations from Sakha, connect Sakha and South Siberia. Analysis of autosomal SNP data confirms the genetic continuity between Sakha and South Siberia. Maternal lineages D5a2a2, C4a1c, C4a2, C5b1b and the Yakut-specific STR sub-clade of Y-chromosome haplogroup N1c can be linked to a migration of Yakut ancestors, while the paternal lineage C3c was most likely carried to Sakha by the expansion of the Tungusic people. MtDNA haplogroups Z1a1b and Z1a3, present in Yukaghirs, Evens and Dolgans, show traces of different and probably more ancient migration(s). Analysis of both haploid loci and autosomal SNP data revealed only minor genetic components shared between Sakha and the extreme Northeast Siberia. Although the major part of West Eurasian maternal and paternal lineages in Sakha could originate from recent admixture with East Europeans, mtDNA haplogroups H8, H20a and HV1a1a, as well as Y-chromosome haplogroup J, more probably reflect an ancient gene flow from West Eurasia through Central Asia and South Siberia. Our high-resolution phylogenetic dissection of mtDNA and Y-chromosome haplogroups as well as analysis of autosomal SNP data suggests that Sakha was colonized by repeated expansions from South Siberia with minor gene flow from the Lower Amur/Southern Okhotsk region and/or Kamchatka. The minor West Eurasian component in Sakha attests to both recent and ongoing admixture with East Europeans and an ancient gene flow from West Eurasia. 40 samples were analysed with the Illumina platform Human660W-Quad v1.0 and are described herein.

Project description:Chromosome segregation errors have been linked to DNA damage and genomic rearrangements. Accumulating evidence has shown that catastrophic genomic rearrangements, like chromothripsis, can result from lagging chromosomes undergoing aberrant DNA replication and DNA damage in micronuclei. Detailed characterization of genomic rearrangements resulting from DNA damage in micronuclei has been hampered because of difficulties in culturing daughter cells with DNA damage. Here, we employ a method by which a specific single chromosome is trapped in a micronucleus, followed by transfer to an acceptor cell. Next, stably propagating clonal cell lines with an extra chromosome were established and analyzed by copy number profiling and whole genome sequencing. While non-transformed, p53 proficient and telomerase-immortalized RPE1 cells showed a stable genome following addition of the transferred chromosome, we observed frequent de novo genomic rearrangements in cells derived from the HCT116 colorectal cancer cell line after chromosome transfer. The de novo rearrangements varied from simple deletions and duplications to complex rearrangements. Phase-informative SNPs revealed that the rearrangements specifically occurred on the transferred chromosome. We found that the complex rearrangements recapitulated all features of chromothripsis, including massive oscillation between two copy number states, localization to a single chromosome, random joining of chromosome fragments and non-homologous or micro-homologous repair. We describe an approach that enables the isolation of clonal cell lines with genomic rearrangements and chromothripsis on a specific chromosome in p53 proficient cells. The procedure enables further investigation of the exact mechanism leading to chromothripsis and the analysis of its consequences for cell survival (viability) and cancer development. We analyzed 38 cell clones, originating from HCT116 or RPE1 cells respectively, with Illumina beadchip arrays to test for unique de novo copy number variants and to determine the chromosome affacted by the CNAs.

Project description:A genome-wide eQTL analysis was performed in whole blood samples collected from 76 Japanese subjects. RNA microarray analysis was performed for 3 independent samples that were genotyped in a genome-wide scan. The correlations between the genotypes of 534,404 autosomal single nucleotide polymorphisms (SNPs) and the expression levels of 30,465 probes were examined for each sample. The SNP-probe pairs with combined correlation coefficients of all 3 samples corresponding to P < 3.10 × 10-12 (i.e., Bonferroni-corrected P < 0.05) were considered significant. SNP-probe pairs with a high likelihood of cross-hybridization and SNP-in-probe effects were excluded to exclude false positive results. We identified 102 cis-acting and 5 trans-acting eQTL regions. The cis-eQTL regions were widely distributed both upstream and downstream of the gene, as well as within the gene. RNA microarray data obtained from 3 independent samples originally recruited for other studies investigating the gene expression levels in psychiatric disorders were used in the present study. For the purpose of the present analyses, genomic DNA was collected from 24 subjects (13 men and 11 women, mean age [SD] = 39.9 [7.6] years) in sample 1, 24 subjects in sample 2 (12 men and 12 women, 34.1 [11.5] years), and 28 subjects (14 men and 14 women, 41.4 [11.8] years) in sample 3. Some of the subjects had depressive symptoms, but all were physically healthy and without clinically significant systemic disease (e.g., malignant disease, diabetes mellitus, hypertension, renal failure, or endocrine disorders). Subjects were recruited from the outpatient clinic of the National Center of Neurology and Psychiatry Hospital, Tokyo, Japan, through advertisements in free local information magazines or through our website announcement. All the subjects were biologically unrelated Japanese individuals who resided in the same geographical area (western Tokyo). The study protocol was approved by the ethics committee at the National Center of Neurology and Psychiatry, Japan. Written informed consent was obtained from every subject after the study was explained to them. Venous blood was collected between 1100 and 1200 h in PAXgene tubes (Qiagen, Valencia) from each subject and was incubated at room temperature for 24 h for RNA stabilization. RNA was extracted from whole blood according to the manufacturer’s guidelines by using the PAXgene Blood RNA System Kit (PreAnalytix GmbH, Hombrechtikon, Switzerland). The RNA was quantified by optical density readings at A260nm by using the NanoDrop ND-1000 (Thermo Scientific, Rockford). Gene expression analysis was performed using Agilent Human Genome 4 × 44 K arrays (Agilent Technologies, Santa Clara). Raw signal data for each of the 3 independent samples were analyzed separately by the GeneSpring GX software (Agilent Technologies). Data were filtered according to the expression level for quality control to eliminate genes that were below the 20th percentile threshold. The expression value of each gene was normalized to the median expression value of all genes in each chip. A total of 30,465 probes were included in the analysis. Genomic DNA was obtained from venous blood samples. Genotyping was performed by Riken Genesis (Yokohama, Japan) using the Illumina HumanOmni1-Quad BeadChip (Illumina, Inc., San Diego). A total of 713,495 autosomal SNPs were assessed for quality using the PLINK v1.07 software. All SNPs with a call rate below 95%, a deviation from Hardy-Weinberg equilibrium at an error level of P < 0.001, or a minor allele frequency of less than 10% were excluded. The remaining 534,404 SNPs were used for further analysis.

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