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Additional file 1. Supplementary Figures S1 ��� S8

Genes in the major histocompatibility complex (MHC, also known as HLA) play a critical role in the immune response and variation within the extended 4-Mb region shows association with major risks of many diseases. Yet, deciphering the underlying causes of these associations is difficult because the MHC is the most polymorphic region of the genome with a complex linkage disequilibrium structure. Here, we reconstruct full MHC haplotypes from de novo assembled trios without relying on a reference genome and perform evolutionary analyses. We report 100 full MHC haplotypes and call a large set of structural variants in the regions for future use in imputation with GWAS data. We also present the first complete analysis of the recombination landscape in the entire region and show how balancing selection at classical genes have linked effects on the frequency of variants throughout the region.

Summary The truncated mutant form of the charged multivesicular body protein 2B (CHMP2B) is causative for frontotemporal dementia linked to chromosome 3 (FTD3). CHMP2B is a constituent of the endosomal sorting complex required for transport (ESCRT) and, when mutated, disrupts endosome-to-lysosome trafficking and substrate degradation. To understand the underlying molecular pathology, FTD3 patient induced pluripotent stem cells (iPSCs) were differentiated into forebrain-type cortical neurons. FTD3 neurons exhibited abnormal endosomes, as previously shown in patients. Moreover, mitochondria of FTD3 neurons displayed defective cristae formation, accompanied by deficiencies in mitochondrial respiration and increased levels of reactive oxygen. In addition, we provide evidence for perturbed iron homeostasis, presenting an in vitro patient-specific model to study the effects of iron accumulation in neurodegenerative diseases. All phenotypes observed in FTD3 neurons were rescued in CRISPR/Cas9-edited isogenic controls. These findings illustrate the relevance of our patient-specific in vitro models and open up possibilities for drug target development., Graphical Abstract, Highlights • FTD3 neurons show abnormalities in endosomes and mitochondria • Parkinson's and Alzheimer's disease core genes are altered in FTD3 neurons • Iron homeostasis is perturbed in FTD3 neurons • Impairments in FTD3 neurons are rescued in CRISPR/Cas9-edited isogenic controls, In this article, Freude, Zhang, and colleagues describe a patient iPSC-derived neuronal model for FTD3. This cellular model shows endosome abnormalities previously reported in patients. Furthermore, it provides insights into the role of impaired mitochondria function and imbalanced iron homeostasis in FTD3 pathology. All observed phenotypes were rescued in CRISPR/Cas9-edited isogenic controls.

Derivation and stable maintenance of porcine induced pluripotent stem cells (piPSCs) is challenging. We herein systematically analyzed two piPSC lines, derived by lentiviral transduction and cultured under either leukemia inhibitory factor (LIF) or fibroblast growth factor (FGF) conditions, to shed more light on the underlying biological mechanisms of porcine pluripotency. LIF-derived piPSCs were more successful than their FGF-derived counterparts in the generation of in vitro chimeras and in teratoma formation. When LIF piPSCs chimeras were transferred into surrogate sows and allowed to develop, only their prescence within the embryonic membranes could be detected. Whole transcriptome analysis of the piPSCs and porcine neonatal fibroblasts showed that they clustered together, but apart from the two pluripotent cell populations of early porcine embryos, indicating incomplete reprogramming. Indeed, bioinformatic analysis of the pluripotency-related gene network of the LIF- versus FGF-derived piPSCs revealed that ZFP42 (REX1) expression was absent in both piPSC-like cells, whereas it was expressed in the porcine inner cell mass at Day 7/8. A second striking difference was the expression of ATOH1 in piPSC-like cells, which was absent in the inner cell mass. Moreover, our gene expression analyses plus correlation analyses of known pluripotency genes identified unique relationships between pluripotency genes in the inner cell mass, which are to some extend, in the piPSC-like cells. This deficiency in downstream gene activation and divergent gene expression may be underlie the inability to derive germ line-transmitting piPSCs, and provides unique insight into which genes are necessary to achieve fully reprogrammed piPSCs. This article is protected by copyright. All rights reserved.

Production of transgenic animals via somatic cell nuclear transfer (SCNT) has been widely used worldwide. However, the application of SCNT is impeded by overall high costs and low efficiency. Here, we reported a modification of the existing technology in order to overcome some of the disadvantages associated with SCNT. Firstly, a marker gene, enhanced green fluorescent gene (EGFP), was transfected into pig fetal fibroblast cells, and was subsequently screened by fluorescent expression to ensure donor cells expressing EGFP. Porcine embryos expressing EGFP were then produced by a method called handmade cloning (HMC), a simplified method for micromanipulation. To demonstrate the concept, we collected a total of 378 fresh swine oocytes, from which 266 with the nucleus removed, obtained a total of 127 viable recombinant oocytes after fusion with EGFP-expressing cells. In vitro incubation of the 127 recombinant oocytes for approximately 144 hours resulted in successful generation of 65 viable embryos, with an average success rate of 52.1±8.3%. Compared with the traditional SCNT, the method of HMC is not only easy to operate, but also increases the rate of recombinant embryo significantly. Furthermore, the modified method no longer relies on expensive instrument like micromanipulator, facilitating the industrialization of transgenic animal production.

Gene ontology (GO) and Kyoto encyclopedia of genes and genomes (KEGG) analysis by the web based software ChIP-Enrich of FUS and EWS ChIP-seq peaks [51]. (DOCX 19 kb)

qPCR validation of enrichment peaks identified by FUS and EWS ChIP-seq. The enrichment of DNA was quantified as percentage of the amount in the input sample and the p-values for enrichment of FUS and EWS were calculated. IFG28 was used as a negative control for enrichment. A control ChIP experiment was also performed with inclusion of pre-immune antiserum added to the AG beads used for chromatin purification (AG) instead of FUS and EWS antibodies. Data represents three independent experiments and standard deviation shown by error bars. A. C19orf48 and ACPT; B. RCC1 and SNHG3; C. HNRNPK. (PDF 23 kb)

Analysis of ChIP-seq FUS and EWS enrichment peaks in the RCC1 and SNHG3 gene complex, including SNORA73A and SNORA73B genes. A. Graphic distribution of ChIP-seq reads aligned to the RCC1 and SNHG3 genes from the input, FUS, EWS and Ac-H3K9 ChIP-seq samples. The number of reads is shown on the scale to the left of each figure. The transcripts from the genes in the UCSC hg19 genomic database are shown in the bottom. The arrows beneath peaks illustrate location of the several qPCR amplicons used. The arrows above the transcripts illustrate location of RT-qPCR amplicons. Numbers above arrows denotes target exon numbers. B-C. FUS and EWS effect for RCC1, SNHG3 and snoRNA expression. The expression levels were determined by qPCR from HEK-293 cells transfected with siRNA for FUS and EWS or control siRNA. Three independent experiments were performed and error bars indicate standard deviation. In B., simultaneous depletion of FUS and EWS was performed, whereas in C. FUS and EWS were individually depleted. (PDF 552 kb)

Consequences of siRNA mediated depletion of FUS and EWS for gene expression. HEK-293 cells were double-transfected with specific siRNAs for FUS, EWS and FUS plus EWS, and as well as with a control siRNA (siControl). A. siRNA-depleted cells were used for relative mRNA quantification of FUS and EWS by qPCR. FUS and EWS expression was normalized to reference gene TBP. Experiments were performed in triplicates. Data presented as mean + SEM. Similar qPCR expression analyses showed a 1.5 to 2 fold increase in TAF15 mRNA by EWS and FUS depletion (not shown). B. Protein quantification by western blot of FUS and EWS from siRNA and control transfected cells. 4 F4 antibody recognizing HNRNP C1 + C2 was used as a loading control. (PDF 276 kb)

Comparison of the distribution of FindPeaks called ChIP-seq peaks across a model gene using data from this study or the study by Schwarts et al. [30]. For A. and B. legend details see Additional file 16. (DOCX 693 kb)

Genes co-identified in the hereby presented ChIP-seq data and RNA-CLIP from Hoell et al. [26]. (DOCX 37 kb)

Comparative analysis and alignment to the human genome (UCSC hg19) of raw FUS ChIP-seq reads from this study and recalculated from the few data files presented in the study by Schwartz et al. [30]. (DOCX 16 kb)

Comparative distribution of p-values for ChIP-seq peaks called by FindPeaks and MACS of data from this study and the study by Schwarts et al. [30]. A. Distribution of p-values for ChIP-seq peaks called by FindPeaks (F). Most of the ChIP-seq peaks from the hereby presented FUS ChIP-seq data (unFUS_JB) have a low p-value. Most of the ChIP-seq peaks from Schwartz’s et al. (unFUS_ref) have a high P-value. B. Distribution of p-values for ChIP-seq peaks called by MACS. A lower number of ChIP-seq peaks were identified compared to with usage of FindPeaks and the p-value distribution is more comparable between the two datasets. (DOCX 106 kb)

Annotated genes overlapping the significant enrichment peaks determined from FUS, EWS and Ac-H3K9 ChIP-seq analysis. Chromosome (CH): the peak start and end in base pairs; Length: the peak length in base pairs; Tags: the number of sequences included in the peak; p-value: p-value on log2 scale; fold-change (fc): relative to input sample; FDR: false discovery rate in percentage; ENS ID: gene ID in the ensemble database; gene location: location of the enrichment in the gene (upstream, downstream, intron, exon); and T: number of known transcript variants produced from the given gene. (PDF 1497 kb)

Cross-comparison of the genes identified to be associated with FUS, EWS or Ac-H3K9 by ChIP-seq analysis. The displayed overlap categories are FUS and EWS, FUS and Ac-H3K9, EWS and Ac-H3K9, FUS and EWS and Ac-H3K9, FUS and EWS. Chromosome (CH): the peak start and end in base pairs; Length: the peak length in base pairs; Tags: the number of sequences included in the peak; p-value: p-value on log2 scale; fold-change (fc): relative to input sample; FDR: false discovery rate in percent; ENS ID: gene ID in the ensemble database; gene location: location of the enrichment in the gene (upstream, downstream, intron, exon); and T: number of known transcript variants produced from the given gene. (DOCX 325 kb)

Analysis of ChIP-seq FUS and EWS enrichment peaks in the ACPT and C19orf48 gene complex. A. Graphic distribution of ChIP-seq reads aligned to the ACPT and C19orf48 genes from the input, FUS, EWS and Ac-H3K9 ChIP-seq samples. The number of reads is shown on the scale to the left of each figure. The transcripts from the genes in the UCSC hg19 genomic database are shown in the bottom. The arrows beneath peaks illustrate location of the several qPCR amplicons used. The arrows above the transcripts illustrate location of RT-qPCR amplicons. Numbers above arrows denotes target exon numbers. B-C. FUS and EWS effect in ACPT and C19orf48 expression. The expression levels of C19orf48 transcripts were determined by qPCR from HEK-293 cells transfected with siRNA for FUS and EWS or control siRNA. Three independent experiments were performed and error bars indicate standard deviation. In B. simultaneous depletion of FUS and EWS was performed, whereas in C. FUS and EWS were individually depleted. (PDF 483 kb)

FUS and EWS regulate RNA processing in the 3’-end of HNRNPK . A. Graphic distribution of ChIP-seq reads aligned to HNRNPK. The number of reads is shown on the scale to the left of each figure. The transcripts from the genes in the UCSC hg19 genomic database are shown in the bottom. The arrows beneath peaks illustrate location of qPCR amplicons. The arrows above the transcripts illustrate location of RT-qPCR amplicons. Numbers above arrows denote target exon numbers. B-C. The expression levels were determined by qPCR from HEK-293 cells transfected with siRNA for FUS and EWS or control siRNA. In B, simultaneous depletion of FUS and EWS was performed, whereas in C FUS and EWS were individually depleted. All values were normalized to reference gene TBP. All experiments were performed in triplicates. *P

List of primer sequences for PCR. (DOCX 20 kb)

In this paper, we mine full mtDNA sequences from an exome capture data set of 2000 Danes, showing that it is possible to get high-quality full-genome sequences of the mitochondrion from this resource. The sample includes 1000 individuals with type 2 diabetes and 1000 controls. We characterise the variation found in the mtDNA sequence in Danes and relate the variation to diabetes risk as well as to several blood phenotypes of the controls but find no significant associations. We report 2025 polymorphisms, of which 393 have not been reported previously. These 393 mutations are both very rare and estimated to be caused by very recent mutations but individuals with type 2 diabetes do not possess more of these variants. Population genetics analysis using Bayesian skyline plot shows a recent history of rapid population growth in the Danish population in accordance with the fact that >40% of variable sites are observed as singletons.