Publications by authors named "Lars Feuk"

89 Publications

Transcriptome Analysis of Post-Mortem Brain Tissue Reveals Up-Regulation of the Complement Cascade in a Subgroup of Schizophrenia Patients.

Genes (Basel) 2021 08 13;12(8). Epub 2021 Aug 13.

Department of Immunology, Genetics and Pathology, Science for Life Laboratory, Uppsala University, 751 85 Uppsala, Sweden.

Schizophrenia is a genetically complex neuropsychiatric disorder with largely unresolved mechanisms of pathology. Identification of genes and pathways associated with schizophrenia is important for understanding the development, progression and treatment of schizophrenia. In this study, pathways associated with schizophrenia were explored at the level of gene expression. The study included post-mortem brain tissue samples from 68 schizophrenia patients and 44 age and sex-matched control subjects. Whole transcriptome poly-A selected paired-end RNA sequencing was performed on tissue from the prefrontal cortex and orbitofrontal cortex. RNA expression differences were detected between case and control individuals, focusing both on single genes and pathways. The results were validated with RT-qPCR. Significant differential expression between patient and controls groups was found for 71 genes. Gene ontology analysis of differentially expressed genes revealed an up-regulation of multiple genes in immune response among the patients (corrected -value = 0.004). Several genes in the category belong to the complement system, including , , , , , and . The increased complement expression is primarily driven by a subgroup of patients with increased expression of immune/inflammatory response genes, pointing to important differences in disease etiology within the patient group. Weighted gene co-expression network analysis highlighted networks associated with both synaptic transmission and activation of the immune response. Our results demonstrate the importance of immune-related pathways in schizophrenia and provide evidence for elevated expression of the complement cascade as an important pathway in schizophrenia pathology.
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http://dx.doi.org/10.3390/genes12081242DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8393670PMC
August 2021

Interpretable Machine Learning Reveals Dissimilarities Between Subtypes of Autism Spectrum Disorder.

Front Genet 2021 25;12:618277. Epub 2021 Feb 25.

Science for Life Laboratory, Department of Cell and Molecular Biology, Uppsala University, Uppsala, Sweden.

Autism spectrum disorder (ASD) is a heterogeneous neuropsychiatric disorder with a complex genetic background. Analysis of altered molecular processes in ASD patients requires linear and nonlinear methods that provide interpretable solutions. Interpretable machine learning provides legible models that allow explaining biological mechanisms and support analysis of clinical subgroups. In this work, we investigated several case-control studies of gene expression measurements of ASD individuals. We constructed a rule-based learning model from three independent datasets that we further visualized as a nonlinear gene-gene co-predictive network. To find dissimilarities between ASD subtypes, we scrutinized a topological structure of the network and estimated a centrality distance. Our analysis revealed that autism is the most severe subtype of ASD, while pervasive developmental disorder-not otherwise specified and Asperger syndrome are closely related and milder ASD subtypes. Furthermore, we analyzed the most important ASD-related features that were described in terms of gene co-predictors. Among others, we found a strong co-predictive mechanism between and , which may suggest a co-regulation between these genes. The present study demonstrates the potential of applying interpretable machine learning in bioinformatics analyses. Although the proposed methodology was designed for transcriptomics data, it can be applied to other omics disciplines.
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http://dx.doi.org/10.3389/fgene.2021.618277DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7946989PMC
February 2021

R.ROSETTA: an interpretable machine learning framework.

BMC Bioinformatics 2021 Mar 6;22(1):110. Epub 2021 Mar 6.

Department of Cell and Molecular Biology, Uppsala University, Uppsala, Sweden.

Background: Machine learning involves strategies and algorithms that may assist bioinformatics analyses in terms of data mining and knowledge discovery. In several applications, viz. in Life Sciences, it is often more important to understand how a prediction was obtained rather than knowing what prediction was made. To this end so-called interpretable machine learning has been recently advocated. In this study, we implemented an interpretable machine learning package based on the rough set theory. An important aim of our work was provision of statistical properties of the models and their components.

Results: We present the R.ROSETTA package, which is an R wrapper of ROSETTA framework. The original ROSETTA functions have been improved and adapted to the R programming environment. The package allows for building and analyzing non-linear interpretable machine learning models. R.ROSETTA gathers combinatorial statistics via rule-based modelling for accessible and transparent results, well-suited for adoption within the greater scientific community. The package also provides statistics and visualization tools that facilitate minimization of analysis bias and noise. The R.ROSETTA package is freely available at https://github.com/komorowskilab/R.ROSETTA . To illustrate the usage of the package, we applied it to a transcriptome dataset from an autism case-control study. Our tool provided hypotheses for potential co-predictive mechanisms among features that discerned phenotype classes. These co-predictors represented neurodevelopmental and autism-related genes.

Conclusions: R.ROSETTA provides new insights for interpretable machine learning analyses and knowledge-based systems. We demonstrated that our package facilitated detection of dependencies for autism-related genes. Although the sample application of R.ROSETTA illustrates transcriptome data analysis, the package can be used to analyze any data organized in decision tables.
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http://dx.doi.org/10.1186/s12859-021-04049-zDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7937228PMC
March 2021

Characterization of the nuclear and cytosolic transcriptomes in human brain tissue reveals new insights into the subcellular distribution of RNA transcripts.

Sci Rep 2021 02 18;11(1):4076. Epub 2021 Feb 18.

Department of Immunology, Genetics and Pathology, Uppsala University, BMC B11:4, Box 815, 751 08, Uppsala, Sweden.

Transcriptome analysis has mainly relied on analyzing RNA sequencing data from whole cells, overlooking the impact of subcellular RNA localization and its influence on our understanding of gene function, and interpretation of gene expression signatures in cells. Here, we separated cytosolic and nuclear RNA from human fetal and adult brain samples and performed a comprehensive analysis of cytosolic and nuclear transcriptomes. There are significant differences in RNA expression for protein-coding and lncRNA genes between cytosol and nucleus. We show that transcripts encoding the nuclear-encoded mitochondrial proteins are significantly enriched in the cytosol compared to the rest of protein-coding genes. Differential expression analysis between fetal and adult frontal cortex show that results obtained from the cytosolic RNA differ from results using nuclear RNA both at the level of transcript types and the number of differentially expressed genes. Our data provide a resource for the subcellular localization of thousands of RNA transcripts in the human brain and highlight differences in using the cytosolic or the nuclear transcriptomes for expression analysis.
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http://dx.doi.org/10.1038/s41598-021-83541-1DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7893067PMC
February 2021

DLG4-related synaptopathy: a new rare brain disorder.

Genet Med 2021 05 17;23(5):888-899. Epub 2021 Feb 17.

Institute of Human Genetics, Friedrich-Alexander-Universität Erlangen-Nürnberg (FAU), Erlangen, Germany.

Purpose: Postsynaptic density protein-95 (PSD-95), encoded by DLG4, regulates excitatory synaptic function in the brain. Here we present the clinical and genetic features of 53 patients (42 previously unpublished) with DLG4 variants.

Methods: The clinical and genetic information were collected through GeneMatcher collaboration. All the individuals were investigated by local clinicians and the gene variants were identified by clinical exome/genome sequencing.

Results: The clinical picture was predominated by early onset global developmental delay, intellectual disability, autism spectrum disorder, and attention deficit-hyperactivity disorder, all of which point to a brain disorder. Marfanoid habitus, which was previously suggested to be a characteristic feature of DLG4-related phenotypes, was found in only nine individuals and despite some overlapping features, a distinct facial dysmorphism could not be established. Of the 45 different DLG4 variants, 39 were predicted to lead to loss of protein function and the majority occurred de novo (four with unknown origin). The six missense variants identified were suggested to lead to structural or functional changes by protein modeling studies.

Conclusion: The present study shows that clinical manifestations associated with DLG4 overlap with those found in other neurodevelopmental disorders of synaptic dysfunction; thus, we designate this group of disorders as DLG4-related synaptopathy.
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http://dx.doi.org/10.1038/s41436-020-01075-9DOI Listing
May 2021

Hybrid sequencing resolves two germline ultra-complex chromosomal rearrangements consisting of 137 breakpoint junctions in a single carrier.

Hum Genet 2021 May 14;140(5):775-790. Epub 2020 Dec 14.

Department of Molecular Medicine and Surgery, Karolinska Institutet, Karolinska University Hospital Solna, 171 76, Stockholm, Sweden.

Chromoanagenesis is a genomic event responsible for the formation of complex structural chromosomal rearrangements (CCRs). Germline chromoanagenesis is rare and the majority of reported cases are associated with an affected phenotype. Here, we report a healthy female carrying two de novo CCRs involving chromosomes 4, 19, 21 and X and chromosomes 7 and 11, respectively, with a total of 137 breakpoint junctions (BPJs). We characterized the CCRs using a hybrid-sequencing approach, combining short-read sequencing, nanopore sequencing, and optical mapping. The results were validated using multiple cytogenetic methods, including fluorescence in situ hybridization, spectral karyotyping, and Sanger sequencing. We identified 137 BPJs, which to our knowledge is the highest number of reported breakpoint junctions in germline chromoanagenesis. We also performed a statistical assessment of the positioning of the breakpoints, revealing a significant enrichment of BPJ-affecting genes (96 intragenic BPJs, 26 genes, p < 0.0001), indicating that the CCRs formed during active transcription of these genes. In addition, we find that the DNA fragments are unevenly and non-randomly distributed across the derivative chromosomes indicating a multistep process of scattering and re-joining of DNA fragments. In summary, we report a new maximum number of BPJs (137) in germline chromoanagenesis. We also show that a hybrid sequencing approach is necessary for the correct characterization of complex CCRs. Through in-depth statistical assessment, it was found that the CCRs most likely was formed through an event resembling chromoplexy-a catastrophic event caused by erroneous transcription factor binding.
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http://dx.doi.org/10.1007/s00439-020-02242-3DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8052244PMC
May 2021

Evaluation of Single-Molecule Sequencing Technologies for Structural Variant Detection in Two Swedish Human Genomes.

Genes (Basel) 2020 11 30;11(12). Epub 2020 Nov 30.

Science for Life Laboratory, Department of Immunology, Genetics and Pathology, Uppsala University, 752 36 Uppsala, Sweden.

Long-read single molecule sequencing is increasingly used in human genomics research, as it allows to accurately detect large-scale DNA rearrangements such as structural variations (SVs) at high resolution. However, few studies have evaluated the performance of different single molecule sequencing platforms for SV detection in human samples. Here we performed Oxford Nanopore Technologies (ONT) whole-genome sequencing of two Swedish human samples (average 32× coverage) and compared the results to previously generated Pacific Biosciences (PacBio) data for the same individuals (average 66× coverage). Our analysis inferred an average of 17k and 23k SVs from the ONT and PacBio data, respectively, with a majority of them overlapping with an available multi-platform SV dataset. When comparing the SV calls in the two Swedish individuals, we find a higher concordance between ONT and PacBio SVs detected in the same individual as compared to SVs detected by the same technology in different individuals. Downsampling of PacBio reads, performed to obtain similar coverage levels for all datasets, resulted in 17k SVs per individual and improved overlap with the ONT SVs. Our results suggest that ONT and PacBio have a similar performance for SV detection in human whole genome sequencing data, and that both technologies are feasible for population-scale studies.
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http://dx.doi.org/10.3390/genes11121444DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7760597PMC
November 2020

Amplification-free long-read sequencing reveals unforeseen CRISPR-Cas9 off-target activity.

Genome Biol 2020 12 1;21(1):290. Epub 2020 Dec 1.

Science for Life Laboratory, Department of Immunology, Genetics and Pathology, Uppsala University, Uppsala, Sweden.

Background: One ongoing concern about CRISPR-Cas9 genome editing is that unspecific guide RNA (gRNA) binding may induce off-target mutations. However, accurate prediction of CRISPR-Cas9 off-target activity is challenging. Here, we present SMRT-OTS and Nano-OTS, two novel, amplification-free, long-read sequencing protocols for detection of gRNA-driven digestion of genomic DNA by Cas9 in vitro.

Results: The methods are assessed using the human cell line HEK293, re-sequenced at 18x coverage using highly accurate HiFi SMRT reads. SMRT-OTS and Nano-OTS are first applied to three different gRNAs targeting HEK293 genomic DNA, resulting in a set of 55 high-confidence gRNA cleavage sites identified by both methods. Twenty-five of these sites are not reported by off-target prediction software, either because they contain four or more single nucleotide mismatches or insertion/deletion mismatches, as compared with the human reference. Additional experiments reveal that 85% of Cas9 cleavage sites are also found by other in vitro-based methods and that on- and off-target sites are detectable in gene bodies where short-reads fail to uniquely align. Even though SMRT-OTS and Nano-OTS identify several sites with previously validated off-target editing activity in cells, our own CRISPR-Cas9 editing experiments in human fibroblasts do not give rise to detectable off-target mutations at the in vitro-predicted sites. However, indel and structural variation events are enriched at the on-target sites.

Conclusions: Amplification-free long-read sequencing reveals Cas9 cleavage sites in vitro that would have been difficult to predict using computational tools, including in dark genomic regions inaccessible by short-read sequencing.
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http://dx.doi.org/10.1186/s13059-020-02206-wDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7706270PMC
December 2020

Exploring autoantibody signatures in brain tissue from patients with severe mental illness.

Transl Psychiatry 2020 11 18;10(1):401. Epub 2020 Nov 18.

Science for Life Laboratory, Department of Immunology, Genetics and Pathology, Uppsala University, Uppsala, Sweden.

In recent years, studies have shown higher prevalence of autoantibodies in patients with schizophrenia compared to healthy individuals. This study applies an untargeted and a targeted affinity proteomics approach to explore and characterize the autoantibody repertoire in brain tissues from 73 subjects diagnosed with schizophrenia and 52 control subjects with no psychiatric or neurological disorders. Selected brain tissue lysates were first explored for IgG reactivity on planar microarrays composed of 11,520 protein fragments representing 10,820 unique proteins. Based on these results of ours and other previous studies of autoantibodies related to psychosis, we selected 226 fragments with an average length of 80 amino acids, representing 127 unique proteins. Tissue-based analysis of IgG reactivities using antigen suspension bead arrays was performed in a multiplex and parallel fashion for all 125 subjects. Among the detected autoantigens, higher IgG reactivity in subjects with schizophrenia, as compared to psychiatrically healthy subjects, was found against the glutamate ionotropic receptor NMDA type subunit 2D (anti-GluN2D). In a separate cohort with serum samples from 395 young adults with a wider spectrum of psychiatric disorders, higher levels of serum autoantibodies targeting GluN2D were found when compared to 102 control individuals. By further validating GluN2D and additional potential autoantigens, we will seek insights into how these are associated with severe mental illnesses.
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http://dx.doi.org/10.1038/s41398-020-01079-8DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7676257PMC
November 2020

Identification and rescue of a tRNA wobble inosine deficiency causing intellectual disability disorder.

RNA 2020 11 6;26(11):1654-1666. Epub 2020 Aug 6.

Department of Biology, Center for RNA Biology, University of Rochester, Rochester, New York 14627, USA.

The deamination of adenosine to inosine at the wobble position of tRNA is an essential post-transcriptional RNA modification required for wobble decoding in bacteria and eukaryotes. In humans, the wobble inosine modification is catalyzed by the heterodimeric ADAT2/3 complex. Here, we describe novel pathogenic ADAT3 variants impairing adenosine deaminase activity through a distinct mechanism that can be corrected through expression of the heterodimeric ADAT2 subunit. The variants were identified in a family in which all three siblings exhibit intellectual disability linked to biallelic variants in the locus. The biallelic ADAT3 variants result in a missense variant converting alanine to valine at a conserved residue or the introduction of a premature stop codon in the deaminase domain. Fibroblast cells derived from two ID-affected individuals exhibit a reduction in tRNA wobble inosine levels and severely diminished adenosine tRNA deaminase activity. Notably, the ADAT3 variants exhibit impaired interaction with the ADAT2 subunit and alterations in ADAT2-dependent nuclear localization. Based upon these findings, we find that tRNA adenosine deaminase activity and wobble inosine modification can be rescued in patient cells by overexpression of the ADAT2 catalytic subunit. These results uncover a key role for the inactive ADAT3 deaminase domain in proper assembly with ADAT2 and demonstrate that ADAT2/3 nuclear import is required for maintaining proper levels of the wobble inosine modification in tRNA.
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http://dx.doi.org/10.1261/rna.076380.120DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7566568PMC
November 2020

Whole genome sequencing of familial isolated oesophagus atresia uncover shared structural variants.

BMC Med Genomics 2020 06 26;13(1):85. Epub 2020 Jun 26.

Department of Immunology, Genetics and Pathology, Science for Life Laboratory, Uppsala, Sweden.

Background: Oesophageal atresia (OA) is a life-threatening developmental defect characterized by a lost continuity between the upper and lower oesophagus. The most common form is a distal connection between the trachea and the oesophagus, i.e. a tracheoesophageal fistula (TEF). The condition may be part of a syndrome or occurs as an isolated feature. The recurrence risk in affected families is increased compared to the population-based incidence suggesting contributing genetic factors.

Methods: To gain insight into gene variants and genes associated with isolated OA we conducted whole genome sequencing on samples from three families with recurrent cases affected by congenital and isolated TEF.

Results: We identified a combination of single nucleotide variants (SNVs), splice site variants (SSV) and structural variants (SV) annotated to altogether 100 coding genes in the six affected individuals.

Conclusion: This study highlights rare SVs among candidate gene variants in our individuals with OA and provides a gene framework for further investigations of genetic factors behind this malformation.
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http://dx.doi.org/10.1186/s12920-020-00737-6DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7318369PMC
June 2020

Transcriptome analysis of fibroblasts from schizophrenia patients reveals differential expression of schizophrenia-related genes.

Sci Rep 2020 01 20;10(1):630. Epub 2020 Jan 20.

Department of Immunology, Genetics and Pathology, Science for Life Laboratory Uppsala, Uppsala University, Uppsala, Sweden.

Schizophrenia is a complex neurodevelopmental disorder with high rate of morbidity and mortality. While the heritability rate is high, the precise etiology is still unknown. Although schizophrenia is a central nervous system disorder, studies using peripheral tissues have also been established to search for patient specific biomarkers and to increase understanding of schizophrenia etiology. Among all peripheral tissues, fibroblasts stand out as they are easy to obtain and culture. Furthermore, they keep genetic stability for long period and exhibit molecular similarities to cells from nervous system. Using a unique set of fibroblast samples from a genetically isolated population in northern Sweden, we performed whole transcriptome sequencing to compare differentially expressed genes in seven controls and nine patients. We found differential fibroblast expression between cases and controls for 48 genes, including eight genes previously implicated in schizophrenia or schizophrenia related pathways; HGF, PRRT2, EGR1, EGR3, C11orf87, TLR3, PLEKHH2 and PIK3CD. Weighted gene correlation network analysis identified three differentially co-expressed networks of genes significantly-associated with schizophrenia. All three modules were significantly suppressed in patients compared to control, with one module highly enriched in genes involved in synaptic plasticity, behavior and synaptic transmission. In conclusion, our results support the use of fibroblasts for identification of differentially expressed genes in schizophrenia and highlight dysregulation of synaptic networks as an important mechanism in schizophrenia.
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http://dx.doi.org/10.1038/s41598-020-57467-zDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6971273PMC
January 2020

Linkage and exome analysis implicate multiple genes in non-syndromic intellectual disability in a large Swedish family.

BMC Med Genomics 2019 11 6;12(1):156. Epub 2019 Nov 6.

Department of Immunology, Genetics and Pathology, Science for Life Laboratory Uppsala, Uppsala University, Box 815, SE-751 08, Uppsala, Sweden.

Background: Non-syndromic intellectual disability is genetically heterogeneous with dominant, recessive and complex forms of inheritance. We have performed detailed genetic studies in a large multi-generational Swedish family, including several members diagnosed with non-syndromic intellectual disability. Linkage analysis was performed on 22 family members, nine affected with mild to moderate intellectual disability and 13 unaffected family members.

Methods: Family members were analyzed with Affymetrix Genome-Wide Human SNP Array 6.0 and the genetic data was used to detect copy number variation and to perform genome wide linkage analysis with the SNP High Throughput Linkage analysis system and the Merlin software. For the exome sequencing, the samples were prepared using the Sure Select Human All Exon Kit (Agilent Technologies, Santa Clara, CA, USA) and sequenced using the Ion Proton™ System. Validation of identified variants was performed with Sanger sequencing.

Results: The linkage analysis results indicate that intellectual disability in this family is genetically heterogeneous, with suggestive linkage found on chromosomes 1q31-q41, 4q32-q35, 6p25 and 14q24-q31 (LOD scores of 2.4, simulated p-value of 0.000003 and a simulated genome-wide p-value of 0.06). Exome sequencing was then performed in 14 family members and 7 unrelated individuals from the same region. The analysis of coding variation revealed a pathogenic and candidate variants in different branches of the family. In three patients we find a known homozygous pathogenic mutation in the Homo sapiens solute carrier family 17 member 5 (SLC17A5), causing Salla disease. We also identify a deletion overlapping KDM3B and a duplication overlapping MAP3K4 and AGPAT4, both overlapping variants previously reported in developmental disorders.

Conclusions: DNA samples from the large family analyzed in this study were initially collected based on a hypothesis that affected members shared a major genetic risk factor. Our results show that a complex phenotype such as mild intellectual disability in large families from genetically isolated populations may show considerable genetic heterogeneity.
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http://dx.doi.org/10.1186/s12920-019-0606-4DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6833288PMC
November 2019

Novel Y-Chromosome Long Non-Coding RNAs Expressed in Human Male CNS During Early Development.

Front Genet 2019 24;10:891. Epub 2019 Sep 24.

Department of Organismal Biology, EBC, Uppsala University, Uppsala, Sweden.

Global microarray gene expression analyses previously demonstrated differences in female and male embryos during neurodevelopment. In particular, before sexual maturation of the gonads, the differences seem to concentrate on the expression of genes encoded on the X- and Y-chromosomes. To investigate genome-wide differences in expression during this early developmental window, we combined high-resolution RNA sequencing with qPCR to analyze brain samples from human embryos during the first trimester of development. Our analysis was tailored for maximum sensitivity to discover Y-chromosome gene expression, but at the same time, it was underpowered to detect X-inactivation escapees. Using this approach, we found that 5 out of 13 expressed gametolog pairs showed unbalanced gene dosage, and as a consequence, a male-biased expression. In addition, we found six novel non-annotated long non-coding RNAs on the Y-chromosome with conserved expression patterns in newborn chimpanzee. The tissue specific and time-restricted expression of these long non-coding RNAs strongly suggests important functions during central nervous system development in human males.
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http://dx.doi.org/10.3389/fgene.2019.00891DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6769107PMC
September 2019

Copy number determination of the gene for the human pancreatic polypeptide receptor NPY4R using read depth analysis and droplet digital PCR.

BMC Biotechnol 2019 06 4;19(1):31. Epub 2019 Jun 4.

Department of Neuroscience, SciLifeLab, Uppsala University, Uppsala, Sweden.

Background: Copy number variation (CNV) plays an important role in human genetic diversity and has been associated with multiple complex disorders. Here we investigate a CNV on chromosome 10q11.22 that spans NPY4R, the gene for the appetite-regulating pancreatic polypeptide receptor Y4. This genomic region has been challenging to map due to multiple repeated elements and its precise organization has not yet been resolved. Previous studies using microarrays were interpreted to show that the most common copy number was 2 per genome.

Results: We have investigated 18 individuals from the 1000 Genomes project using the well-established method of read depth analysis and the new droplet digital PCR (ddPCR) method. We find that the most common copy number for NPY4R is 4. The estimated number of copies ranged from three to seven based on read depth analyses with Control-FREEC and CNVnator, and from four to seven based on ddPCR. We suggest that the difference between our results and those published previously can be explained by methodological differences such as reference gene choice, data normalization and method reliability. Three high-quality archaic human genomes (two Neanderthal and one Denisova) display four copies of the NPY4R gene indicating that a duplication occurred prior to the human-Neanderthal/Denisova split.

Conclusions: We conclude that ddPCR is a sensitive and reliable method for CNV determination, that it can be used for read depth calibration in CNV studies based on already available whole-genome sequencing data, and that further investigation of NPY4R copy number variation and its consequences are necessary due to the role of Y4 receptor in food intake regulation.
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http://dx.doi.org/10.1186/s12896-019-0523-9DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6549351PMC
June 2019

Analyzing DNA methylation patterns in subjects diagnosed with schizophrenia using machine learning methods.

J Psychiatr Res 2019 07 2;114:41-47. Epub 2019 Apr 2.

Department of Immunology, Genetics and Pathology, Science for Life Laboratory, Uppsala University, Uppsala, Sweden. Electronic address:

Schizophrenia is a common mental disorder with high heritability. It is genetically complex and to date more than a hundred risk loci have been identified. Association of environmental factors and schizophrenia has also been reported, while epigenetic analyses have yielded ambiguous and sometimes conflicting results. Here, we analyzed fresh frozen post-mortem brain tissue from a cohort of 73 subjects diagnosed with schizophrenia and 52 control samples, using the Illumina Infinium HumanMethylation450 Bead Chip, to investigate genome-wide DNA methylation patterns in the two groups. Analysis of differential methylation was performed with the Bioconductor Minfi package and modern machine-learning and visualization techniques, which were shown previously to be successful in detecting and highlighting differentially methylated patterns in case-control studies. In this dataset, however, these methods did not uncover any significant signals discerning the patient group and healthy controls, suggesting that if there are methylation changes associated with schizophrenia, they are heterogeneous and complex with small effect.
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http://dx.doi.org/10.1016/j.jpsychires.2019.04.001DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7416578PMC
July 2019

Expression profiling and in situ screening of circular RNAs in human tissues.

Sci Rep 2018 11 16;8(1):16953. Epub 2018 Nov 16.

Department of Immunology, Genetics and Pathology, Science for Life Laboratory, Uppsala University, Uppsala, Sweden.

Circular RNAs (circRNAs) were recently discovered as a class of widely expressed noncoding RNA and have been implicated in regulation of gene expression. However, the function of the majority of circRNAs remains unknown. Studies of circRNAs have been hampered by a lack of essential approaches for detection, quantification and visualization. We therefore developed a target-enrichment sequencing method suitable for screening of circRNAs and their linear counterparts in large number of samples. We also applied padlock probes and in situ sequencing to visualize and determine circRNA localization in human brain tissue at subcellular levels. We measured circRNA abundance across different human samples and tissues. Our results highlight the potential of this RNA class to act as a specific diagnostic marker in blood and serum, by detection of circRNAs from genes exclusively expressed in the brain. The powerful and scalable tools we present will enable studies of circRNA function and facilitate screening of circRNA as diagnostic biomarkers.
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http://dx.doi.org/10.1038/s41598-018-35001-6DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6240052PMC
November 2018

De Novo Assembly of Two Swedish Genomes Reveals Missing Segments from the Human GRCh38 Reference and Improves Variant Calling of Population-Scale Sequencing Data.

Genes (Basel) 2018 Oct 9;9(10). Epub 2018 Oct 9.

Science for Life Laboratory, Department of Immunology, Genetics and Pathology, Uppsala University, 752 36 Uppsala, Sweden.

The current human reference sequence (GRCh38) is a foundation for large-scale sequencing projects. However, recent studies have suggested that GRCh38 may be incomplete and give a suboptimal representation of specific population groups. Here, we performed a de novo assembly of two Swedish genomes that revealed over 10 Mb of sequences absent from the human GRCh38 reference in each individual. Around 6 Mb of these novel sequences (NS) are shared with a Chinese personal genome. The NS are highly repetitive, have an elevated GC-content, and are primarily located in centromeric or telomeric regions. Up to 1 Mb of NS can be assigned to chromosome Y, and large segments are also missing from GRCh38 at chromosomes 14, 17, and 21. Inclusion of NS into the GRCh38 reference radically improves the alignment and variant calling from short-read whole-genome sequencing data at several genomic loci. A re-analysis of a Swedish population-scale sequencing project yields > 75,000 putative novel single nucleotide variants (SNVs) and removes > 10,000 false positive SNV calls per individual, some of which are located in protein coding regions. Our results highlight that the GRCh38 reference is not yet complete and demonstrate that personal genome assemblies from local populations can improve the analysis of short-read whole-genome sequencing data.
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http://dx.doi.org/10.3390/genes9100486DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6210158PMC
October 2018

Detailed analysis of HTT repeat elements in human blood using targeted amplification-free long-read sequencing.

Hum Mutat 2018 09 12;39(9):1262-1272. Epub 2018 Jul 12.

Science for Life Laboratory, Department of Immunology, Genetics and Pathology, Uppsala University, Uppsala, Sweden.

Amplification of DNA is required as a mandatory step during library preparation in most targeted sequencing protocols. This can be a critical limitation when targeting regions that are highly repetitive or with extreme guanine-cytosine (GC) content, including repeat expansions associated with human disease. Here, we used an amplification-free protocol for targeted enrichment utilizing the CRISPR/Cas9 system (No-Amp Targeted sequencing) in combination with single molecule, real-time (SMRT) sequencing for studying repeat elements in the huntingtin (HTT) gene, where an expanded CAG repeat is causative for Huntington disease. We also developed a robust data analysis pipeline for repeat element analysis that is independent of alignment of reads to a reference genome. The method was applied to 11 diagnostic blood samples, and for all 22 alleles the resulting CAG repeat count agreed with previous results based on fragment analysis. The amplification-free protocol also allowed for studying somatic variability of repeat elements in our samples, without the interference of PCR stutter. In summary, with No-Amp Targeted sequencing in combination with our analysis pipeline, we could accurately study repeat elements that are difficult to investigate using PCR-based methods.
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http://dx.doi.org/10.1002/humu.23580DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6175010PMC
September 2018

Copy number of pancreatic polypeptide receptor gene NPY4R correlates with body mass index and waist circumference.

PLoS One 2018 5;13(4):e0194668. Epub 2018 Apr 5.

Dept. of Neuroscience, Uppsala University, Uppsala, Sweden.

Multiple genetic studies have linked copy number variation (CNV) in different genes to body mass index (BMI) and obesity. A CNV on chromosome 10q11.22 has been associated with body weight. This CNV region spans NPY4R, the gene encoding the pancreatic polypeptide receptor Y4, which has been described as a satiety-stimulating receptor. We have investigated CNV of the NPY4R gene and analysed its relationship to BMI, waist circumference and self-reported dietary intake from 558 individuals (216 men and 342 women) representing a wide BMI range. The copy number for NPY4R ranged from 2 to 8 copies (average 4.6±0.8). Rather than the expected negative correlation, we observed a positive correlation between NPY4R copy number and BMI as well as waist circumference (r = 0.267, p = 2.65×10-7 and r = 0.256, p = 8×10-7, respectively). Each additional copy of NPY4R correlated with 2.6 kg/m2 increase in BMI and 5.67 cm increase in waist circumference (p = 3.3×10-7 and p = 1×10-6, respectively) for women. For men, there was no statistically significant correlation between CNV and BMI. Our results suggest that NPY4R genetic variation influences body weight in women, but the exact role of this receptor appears to be more complex than previously proposed.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0194668PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5886410PMC
July 2018

A 3-way hybrid approach to generate a new high-quality chimpanzee reference genome (Pan_tro_3.0).

Gigascience 2017 11;6(11):1-6

Institut de Biologia Evolutiva, (CSIC-Universitat Pompeu Fabra), PRBB, Doctor Aiguader 88, Barcelona, Catalonia 08003, Spain.

The chimpanzee is arguably the most important species for the study of human origins. A key resource for these studies is a high-quality reference genome assembly; however, as with most mammalian genomes, the current iteration of the chimpanzee reference genome assembly is highly fragmented. In the current iteration of the chimpanzee reference genome assembly (Pan_tro_2.1.4), the sequence is scattered across more then 183 000 contigs, incorporating more than 159 000 gaps, with a genome-wide contig N50 of 51 Kbp. In this work, we produce an extensive and diverse array of sequencing datasets to rapidly assemble a new chimpanzee reference that surpasses previous iterations in bases represented and organized in large scaffolds. To this end, we show substantial improvements over the current release of the chimpanzee genome (Pan_tro_2.1.4) by several metrics, such as increased contiguity by >750% and 300% on contigs and scaffolds, respectively, and closure of 77% of gaps in the Pan_tro_2.1.4 assembly gaps spanning >850 Kbp of the novel coding sequence based on RNASeq data. We further report more than 2700 genes that had putatively erroneous frame-shift predictions to human in Pan_tro_2.1.4 and show a substantial increase in the annotation of repetitive elements. We apply a simple 3-way hybrid approach to considerably improve the reference genome assembly for the chimpanzee, providing a valuable resource for the study of human origins. Furthermore, we produce extensive sequencing datasets that are all derived from the same cell line, generating a broad non-human benchmark dataset.
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http://dx.doi.org/10.1093/gigascience/gix098DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5714192PMC
November 2017

Exome sequencing reveals NAA15 and PUF60 as candidate genes associated with intellectual disability.

Am J Med Genet B Neuropsychiatr Genet 2018 Jan 9;177(1):10-20. Epub 2017 Oct 9.

Department of Immunology, Genetics and Pathology, Science for Life Laboratory Uppsala, Uppsala University, Uppsala, Sweden.

Intellectual Disability (ID) is a clinically heterogeneous condition that affects 2-3% of population worldwide. In recent years, exome sequencing has been a successful strategy for studies of genetic causes of ID, providing a growing list of both candidate and validated ID genes. In this study, exome sequencing was performed on 28 ID patients in 27 patient-parent trios with the aim to identify de novo variants (DNVs) in known and novel ID associated genes. We report the identification of 25 DNVs out of which five were classified as pathogenic or likely pathogenic. Among these, a two base pair deletion was identified in the PUF60 gene, which is one of three genes in the critical region of the 8q24.3 microdeletion syndrome (Verheij syndrome). Our result adds to the growing evidence that PUF60 is responsible for the majority of the symptoms reported for carriers of a microdeletion across this region. We also report variants in several genes previously not associated with ID, including a de novo missense variant in NAA15. We highlight NAA15 as a novel candidate ID gene based on the vital role of NAA15 in the generation and differentiation of neurons in neonatal brain, the fact that the gene is highly intolerant to loss of function and coding variation, and previously reported DNVs in neurodevelopmental disorders.
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http://dx.doi.org/10.1002/ajmg.b.32574DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5765476PMC
January 2018

SweGen: a whole-genome data resource of genetic variability in a cross-section of the Swedish population.

Eur J Hum Genet 2017 11 23;25(11):1253-1260. Epub 2017 Aug 23.

Science for Life Laboratory, Department of Immunology, Genetics and Pathology, Uppsala University, Uppsala, Sweden.

Here we describe the SweGen data set, a comprehensive map of genetic variation in the Swedish population. These data represent a basic resource for clinical genetics laboratories as well as for sequencing-based association studies by providing information on genetic variant frequencies in a cohort that is well matched to national patient cohorts. To select samples for this study, we first examined the genetic structure of the Swedish population using high-density SNP-array data from a nation-wide cohort of over 10 000 Swedish-born individuals included in the Swedish Twin Registry. A total of 1000 individuals, reflecting a cross-section of the population and capturing the main genetic structure, were selected for whole-genome sequencing. Analysis pipelines were developed for automated alignment, variant calling and quality control of the sequencing data. This resulted in a genome-wide collection of aggregated variant frequencies in the Swedish population that we have made available to the scientific community through the website https://swefreq.nbis.se. A total of 29.2 million single-nucleotide variants and 3.8 million indels were detected in the 1000 samples, with 9.9 million of these variants not present in current databases. Each sample contributed with an average of 7199 individual-specific variants. In addition, an average of 8645 larger structural variants (SVs) were detected per individual, and we demonstrate that the population frequencies of these SVs can be used for efficient filtering analyses. Finally, our results show that the genetic diversity within Sweden is substantial compared with the diversity among continental European populations, underscoring the relevance of establishing a local reference data set.
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http://dx.doi.org/10.1038/ejhg.2017.130DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5765326PMC
November 2017

Reduced cell surface levels of GPI-linked markers in a new case with PIGG loss of function.

Hum Mutat 2017 10 12;38(10):1394-1401. Epub 2017 Jun 12.

Department of Immunology, Genetics and Pathology, Science for Life Laboratory, Uppsala University, Uppsala, Sweden.

Glycosylphosphatidylinositol (GPI) is a glycolipid that tethers more than 150 different proteins to the cell surface. Aberrations in biosynthesis of GPI anchors cause congenital disorders of glycosylation with clinical features including intellectual disability (ID), seizures, and facial dysmorphism. Here, we present two siblings with ID, cerebellar hypoplasia, cerebellar ataxia, early-onset seizures, and minor facial dysmorphology. Using exome sequencing, we identified a homozygous nonsense variant (NM_001127178.1:c.1640G>A, p.Trp547*) in the gene Phosphatidylinositol Glycan Anchor Biosynthesis, Class G (PIGG) in both the patients. Variants in several other GPI anchor synthesis genes lead to a reduced expression of GPI-anchored proteins (GPI-APs) that can be measured by flow cytometry. No significant differences in GPI-APs could be detected in patient granulocytes, consistent with recent findings. However, fibroblasts showed a reduced global level of GPI anchors and of specific GPI-linked markers. These findings suggest that fibroblasts might be more sensitive to pathogenic variants in GPI synthesis pathway and are well suited to screen for GPI-anchor deficiencies. Based on genetic and functional evidence, we confirm that pathogenic variants in PIGG cause an ID syndrome, and we find that loss of function of PIGG is associated with GPI deficiency.
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http://dx.doi.org/10.1002/humu.23268DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6180480PMC
October 2017

Mutations in HECW2 are associated with intellectual disability and epilepsy.

J Med Genet 2016 10 22;53(10):697-704. Epub 2016 Jun 22.

Department of Immunology, Genetics and Pathology, Science for Life Laboratory Uppsala, Uppsala University, Uppsala, Sweden.

Background: De novo mutations are a frequent cause of disorders related to brain development. We report the results of screening patients diagnosed with both epilepsy and intellectual disability (ID) using exome sequencing to identify known and new causative de novo mutations relevant to these conditions.

Methods: Exome sequencing was performed on 39 patient-parent trios to identify de novo mutations. Clinical significance of de novo mutations in genes was determined using the American College of Medical Genetics and Genomics standard guidelines for interpretation of coding variants. Variants in genes of unknown clinical significance were further analysed in the context of previous trio sequencing efforts in neurodevelopmental disorders.

Results: In 39 patient-parent trios we identified 29 de novo mutations in coding sequence. Analysis of de novo and inherited variants yielded a molecular diagnosis in 11 families (28.2%). In combination with previously published exome sequencing results in neurodevelopmental disorders, our analysis implicates HECW2 as a novel candidate gene in ID and epilepsy.

Conclusions: Our results support the use of exome sequencing as a diagnostic approach for ID and epilepsy, and confirm previous results regarding the importance of de novo mutations in this patient group. The results also highlight the utility of network analysis and comparison to previous large-scale studies as strategies to prioritise candidate genes for further studies. This study adds knowledge to the increasingly growing list of causative and candidate genes in ID and epilepsy and highlights HECW2 as a new candidate gene for neurodevelopmental disorders.
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http://dx.doi.org/10.1136/jmedgenet-2016-103814DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5099177PMC
October 2016

A Role for the Chromatin-Remodeling Factor BAZ1A in Neurodevelopment.

Hum Mutat 2016 09 8;37(9):964-75. Epub 2016 Jul 8.

Department of Immunology, Genetics and Pathology, Science for Life Laboratory, Uppsala University, Sweden.

Chromatin-remodeling factors are required for a wide range of cellular and biological processes including development and cognition, mainly by regulating gene expression. As these functions would predict, deregulation of chromatin-remodeling factors causes various disease syndromes, including neurodevelopmental disorders. Recent reports have linked mutations in several genes coding for chromatin-remodeling factors to intellectual disability (ID). Here, we used exome sequencing and identified a nonsynonymous de novo mutation in BAZ1A (NM_182648.2:c.4043T > G, p.Phe1348Cys), encoding the ATP-utilizing chromatin assembly and remodeling factor 1 (ACF1), in a patient with unexplained ID. ACF1 has been previously reported to bind to the promoter of the vitamin D receptor (VDR)-regulated genes and suppress their expression. Our results show that the patient displays decreased binding of ACF1 to the promoter of the VDR-regulated gene CYP24A1. Using RNA sequencing, we find that the mutation affects the expression of genes involved in several pathways including vitamin D metabolism, Wnt signaling and synaptic formation. RNA sequencing of BAZ1A knockdown cells and Baz1a knockout mice revealed that BAZ1A carry out distinctive functions in different tissues. We also demonstrate that BAZ1A depletion influence the expression of genes important for nervous system development and function. Our data point to an important role for BAZ1A in neurodevelopment, and highlight a possible link for BAZ1A to ID.
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http://dx.doi.org/10.1002/humu.23034DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6681169PMC
September 2016

One CNV Discordance in NRXN1 Observed Upon Genome-wide Screening in 38 Pairs of Adult Healthy Monozygotic Twins.

Twin Res Hum Genet 2016 Apr 22;19(2):97-103. Epub 2016 Feb 22.

Department of Medical Epidemiology and Biostatistics,Karolinska Institutet,Stockholm,Sweden.

Monozygotic (MZ) twins stem from the same single fertilized egg and therefore share all their inherited genetic variation. This is one of the unequivocal facts on which genetic epidemiology and twin studies are based. To what extent this also implies that MZ twins share genotypes in adult tissues is not precisely established, but a common pragmatic assumption is that MZ twins are 100% genetically identical also in adult tissues. During the past decade, this view has been challenged by several reports, with observations of differences in post-zygotic copy number variations (CNVs) between members of the same MZ pair. In this study, we performed a systematic search for differences of CNVs within 38 adult MZ pairs who had been misclassified as dizygotic (DZ) twins by questionnaire-based assessment. Initial scoring by PennCNV suggested a total of 967 CNV discordances. The within-pair correlation in number of CNVs detected was strongly dependent on confidence score filtering and reached a plateau of r = 0.8 when restricting to CNVs detected with confidence score larger than 50. The top-ranked discordances were subsequently selected for validation by quantitative polymerase chain reaction (qPCR), from which one single ~120kb deletion in NRXN1 on chromosome 2 (bp 51017111-51136802) was validated. Despite involving an exon, no sign of cognitive/mental consequences was apparent in the affected twin pair, potentially reflecting limited or lack of expression of the transcripts containing this exon in nerve/brain.
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http://dx.doi.org/10.1017/thg.2016.5DOI Listing
April 2016

Spatial sexual dimorphism of X and Y homolog gene expression in the human central nervous system during early male development.

Biol Sex Differ 2016 12;7. Epub 2016 Jan 12.

Department of Organismal Biology, EBC, Uppsala University, Uppsala, Sweden.

Background: Renewed attention has been directed to the functions of the Y chromosome in the central nervous system during early human male development, due to the recent proposed involvement in neurodevelopmental diseases. PCDH11Y and NLGN4Y are of special interest because they belong to gene families involved in cell fate determination and formation of dendrites and axon.

Methods: We used RNA sequencing, immunocytochemistry and a padlock probing and rolling circle amplification strategy, to distinguish the expression of X and Y homologs in situ in the human brain for the first time. To minimize influence of androgens on the sex differences in the brain, we focused our investigation to human embryos at 8-11 weeks post-gestation.

Results: We found that the X- and Y-encoded genes are expressed in specific and heterogeneous cellular sub-populations of both glial and neuronal origins. More importantly, we found differential distribution patterns of X and Y homologs in the male developing central nervous system.

Conclusions: This study has visualized the spatial distribution of PCDH11X/Y and NLGN4X/Y in human developing nervous tissue. The observed spatial distribution patterns suggest the existence of an additional layer of complexity in the development of the male CNS.
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http://dx.doi.org/10.1186/s13293-015-0056-4DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4710049PMC
January 2016

Transcriptome Profiling Reveals Degree of Variability in Induced Pluripotent Stem Cell Lines: Impact for Human Disease Modeling.

Cell Reprogram 2015 Oct 8;17(5):327-37. Epub 2015 Sep 8.

Department of Immunology, Genetics and Pathology, Science for Life Laboratory, Uppsala University , Uppsala, Sweden .

Induced pluripotent stem cell (iPSC) technology has become an important tool for disease modeling. Insufficient data on the variability among iPSC lines derived from a single somatic parental cell line have in practice led to generation and analysis of several, usually three, iPSC sister lines from each parental cell line. We established iPSC lines from a human fibroblast line (HDF-K1) and used transcriptome sequencing to investigate the variation among three sister lines (iPSC-K1A, B, and C). For comparison, we analyzed the transcriptome of an iPSC line (iPSC-K5B) derived from a different fibroblast line (HDF-K5), a human embryonic stem cell (ESC) line (ESC-HS181), as well as the two parental fibroblast lines. All iPSC lines fulfilled stringent criteria for pluripotency. In an unbiased cluster analysis, all stem cell lines (four iPSCs and one ESC) clustered together as opposed to the parental fibroblasts. The transcriptome profiles of the three iPSC sister lines were indistinguishable from each other, and functional pathway analysis did not reveal any significant hits. In contrast, the expression profiles of the ESC line and the iPSC-K5B line were distinct from that of the sister lines iPSC-K1A, B, and C. Differentiation to embryoid bodies and subsequent analysis of germ layer markers in the five stem cell clones confirmed that the distribution of their expression profiles was retained. Taken together, our observations stress the importance of using iPSCs of different parental origin rather than several sister iPSC lines to distinguish disease-associated mechanisms from genetic background effects in disease modeling.
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http://dx.doi.org/10.1089/cell.2015.0009DOI Listing
October 2015
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