Genetics, Cellular and Natural History of X-Linked Dystonia Parkinsonism
ABSTRACT: X-linked Dystonia-Parkinsonism (XDP) is a long-standing quandary in human disease genetics. XDP is predominantly observed on Panay island in the Philippines. This study is one of the first of its kind to interrogate an unsolved Mendelian disorder by integrating genome and transcriptome assembly methods using Illumina, 10X Genomics, Pacific Biosciences, and Agilent genome targeting technologies. These data provide strong evidence for a pathogenic link between a noncoding SVA retrotransposon and XDP. We demonstrate that this Mendelian disorder is associated with a sine-VNTR-Alu (SVA) retrotransposon that inserted into the TAF1 gene and is shared by all XDP probands, yet never observed in controls from worldwide populations. Transcriptome assembly in iPSC-derived neural stem cells (NSCs) and neurons revealed that this SVA caused aberrant splicing and significant intron retention, which was negatively correlated with TAF1 expression. Remarkably, CRISPR/Cas9... (for more see dbGaP study page.)
Project description:DYT1 dystonia is an autosomal-dominantly inherited movement disorder, which is usually caused by a GAG deletion in the TOR1A gene. Due to the reduced penetrance of ~30-40%, the determination of the mutation in a subject is of limited use with regard to actual manifestation of symptoms. In the present study, we used Affymetrix oligonucleotide microarrays to analyze global gene expression in blood samples of 15 manifesting and 15 non-manifesting mutation carriers in order to identify a susceptibility profile beyond the GAG deletion which is associated with the manifestation of symptoms in DYT1 dystonia.We identified a genetic signature which distinguished between asymptomatic mutation carriers and symptomatic DYT1 patients with 86.7% sensitivity and 100% specificity. This genetic signature could correctly predict the disease state in an independent test set with a sensitivity of 87.5% and a specificity of 85.7%.Conclusively, this genetic signature might provide a possibility to distinguish DYT1 patients from asymptomatic mutation carriers. Comparison of whole blood expression profiles of patients with DYT1 dystonia with non manifesting mutation carriers and non mutation carriers
Project description:Translational regulation can be studied on a global scale by integrating polysome fractionation of mRNAs with microarray hybridization. This approach is based on the fact that translationally quiescent mRNAs are sequestered within messenger ribonucleoprotein (mRNP) particles or associated with single ribosomes (monosomes), whereas actively translated mRNAs are associated with multiple ribosomes (polysomes). The mRNAs associated within these fractions are then used to interrogate microarrays, providing insight into how the translational state of individual mRNAs is modified by environmental cues. In this study, we coupled polysome fractionation with microarray detection in order to identify changes in the translation state of the A. fumigatus transcriptome under conditions that perturb ER homeostasis such as chemical stress (DTT, tunicamycin) or thermal stress (shift from 25 degrees celsius to 37 degrees celsius).
Project description:DYT1 dystonia is an autosomal-dominantly inherited movement disorder, which is usually caused by a GAG deletion in the TOR1A gene. Due to the reduced penetrance of ~30-40%, the determination of the mutation in a subject is of limited use with regard to actual manifestation of symptoms. In the present study, we used Affymetrix oligonucleotide microarrays to analyze global gene expression in blood samples of 15 manifesting and 15 non-manifesting mutation carriers in order to identify a susceptibility profile beyond the GAG deletion which is associated with the manifestation of symptoms in DYT1 dystonia.We identified a genetic signature which distinguished between asymptomatic mutation carriers and symptomatic DYT1 patients with 86.7% sensitivity and 100% specificity. This genetic signature could correctly predict the disease state in an independent test set with a sensitivity of 87.5% and a specificity of 85.7%.Conclusively, this genetic signature might provide a possibility to distinguish DYT1 patients from asymptomatic mutation carriers. Overall design: Comparison of whole blood expression profiles of patients with DYT1 dystonia with non manifesting mutation carriers and non mutation carriers
Project description:Diamond-Blackfan anemia (DBA) is a rare bone marrow failure disorder that affects 7 out of 1,000,000 live births and has been associated with mutations in components of the ribosome. In order to characterize the genetic landscape of this heterogeneous disorder, we recruited a cohort of 472 individuals with a clinical diagnosis of DBA and performed whole exome sequencing (WES). We identified rare and predicted damaging mutations in likely causal genes for 78% of individuals. The majority of mutations were singletons, absent from population databases, predicted to cause loss of function, and in one of 19 previously reported ribosomal protein (RP) encoding genes. Using exon coverage estimates, we identified and validated 31 deletions in RP genes. We also observed an enrichment for extended splice site mutations and validated their diverse effects using RNA sequencing in individual-derived cell lines. Leveraging the size of our cohort, we observed robust genotype-phenotype associations with congenital abnormalities and treatment outcomes. We further identified rare mutations in 7 previously unreported RP genes that may cause DBA, as well as several distinct disorders that appear to phenocopy DBA, including 9 individuals with biallelic CECR1 mutations that result in deficiency of ADA2. However, no new genes were identified at exome-wide significance, suggesting that there are no unidentified genes containing mutations readily identified by WES that explain > 5% of DBA cases. Overall, this report should not only inform clinical practice for DBA individuals, but also the design and analysis of rare variant studies for heterogeneous Mendelian disorders. Overall design: 9 individuals with DBA with putative splice mutations and 5 control individuals were processed for RNA-seq.
Project description:Gene transcription is an essential step of gene function and transcriptome variation is of agronomical, ecological and evolutionary importance. To explore global expression patterns and dissect the underlying genetic mechanisms are important scientific inquires which are still largely unknown, especially between a segregating population and the parents. In our study, we used RNA-Seq to profile the shoot apex transcriptome variation (including protein coding genes and non-coding genes) in maize IBM RIL population, to map eQTLs underlying the transcriptome variations and to utilize eQTLs to clone genes involved in maize shoot apices development. We revealed that: Much of the variation (the population mean, the coefficient of variation) of gene expression levels in RILs is reflective of differences present among the parents; These transcriptome variations could be explained by 30,774 eQTLs with 96 trans-eQTL hotspots; In many cases, the genes commonly regulated by a trans-eQTL hotspot are enriched for a specific function or act in the same genetic pathway; Structural variation within and near genes contributs to cis-regulatory variation. All of these results indicate Mendelian factors play as major contributors to the transcriptome variation. Meanwhile, non-Mendelian regulations were also observed as paramutation-like expression pattern for 145 genes, of which 88% genes were predicted to be potential targets of miRNAs or ta-siRNAs, and as unexpected presence/absence expression patterns for 210 genes. These genes with unexpected presence/absence expression patterns in the RILs likely include examples of functional genes as well as transposed gene fragments that may contribute to regulatory variation of their ancestral syntenic genes.
Project description:Chromatin immunoprecipitation followed by next-generation DNA sequencing (ChIP-seq) is a widely used technique for identifying transcription factor (TF) binding events throughout an entire genome. However, ChIP-seq is limited by the availability of suitable ChIP-seq grade antibodies, and the vast majority of commercially available antibodies fail to generate usable datasets. To ameliorate these technical obstacles, we present a robust methodological approach for performing ChIP-seq through epitope tagging of endogenous TFs. We used Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/Cas9-based genome editing technology to develop CRISPR Epitope Tagging ChIP-seq (CETCh-seq) of DNA-binding proteins. We assessed the feasibility of CETCh-seq by tagging several TFs spanning a wide range of endogenous expression levels in the hepatocellular carcinoma cell line HepG2. Our data exhibit strong correlations between both replicate types as well as with standard ChIP-seq approaches that use TF antibodies. Notably, we also observed minimal changes to the cellular transcriptome and to the expression of the tagged TF. To examine the robustness of our technique, we further performed CETCh-seq in the breast adenocarcinoma cell line MCF7 as well as mouse embryonic stem cells and observed similarly high correlations. Collectively, these data highlight the applicability of CETCh-seq to accurately define the genome-wide binding profiles of DNA-binding proteins, allowing for a straightforward methodology to potentially assay the complete repertoire of TFs, including the large fraction for which ChIPquality antibodies are not available. CRISPR/Cas9 mediated epitope tagging of transcription factors in human cell types
Project description:As CRISPR/Cas9 mediated screens with pooled sgRNA libraries in somatic cells become increasingly established, an unmet need for rapid and accurate companion informatics tools has emerged. We have developed a lightweight standalone software to easily manipulate raw large next generation sequencing (NGS) datasets derived from such screens into informative relational context with graphical support. We demonstrate the capabilities of the software to interrogate meaningful results from an in vitro viability screen using Tumor Necrosis Factor-alpha (TNFa). The results not only identified stereotypical players in extrinsic apoptotic signaling but two as yet uncharacterized members of the apoptotic cascade, Smg7 and Ces2a. We further characterized cell lines containing mutations in these genes against a panel of cell death stimuli. In summary, this software enables bench scientists without access to informatic cores to rapidly access and interpret results from large scale experiments resulting from CRISPR/Cas9 library screens. Overall design: Comparison of mouse fibroblast control cells with cells treated by TNFalpha
Project description:Originating from Northeast Asia, the Pacific oyster Crassostrea gigas has been introduced into a large number of countries for aquaculture purpose. Following introduction, the Pacific oyster has turned into an invasive species in an increasing number of coastal areas, notably in Northern Europe. To explore adaptation on reproductive traits of population considered as invasive, we set up a common garden experiment based on the comparison of progenies from two populations of Pacific oyster sampled in France and Denmark. A female-biased sex-ratio and a higher condition index were observed in the Danish progeny, possibly reflecting an evolutionary reproductive strategy to increase the potential success of natural recruitment in recently settled population. Using multifarious statistical approaches and accounting for sex differences we identified several genes differentially expressed between the Danish and French progenies, and with an intermediate expression level in hybrids (additive behavior). Candidate transcripts included mRNA coding for sperm quality and insulin metabolism known to be implicated in coordinated control of reproduction. Our results suggest adaptation of invasive populations during expansion acting on reproductive traits, and in particular on a female-biased sex-ratio, fertility and gamete quality. A common garden experiment was performed in order to compare progenies from two populations of Pacific oyster sampled in France and Denmark and their hybrids. Progenies were reared under standard hatchery and nursery conditions until gonadal maturation. The employed arrays were Agilent 60-mer 4x44K custom microarrays, containing 31,918 C. gigas ESTs, designed by Dheilly et al. (2011).
Project description:According to Mendel's laws, each parent makes an equal genetic contribution to an offspring in sexually reproducing organisms. The bipolar mitotic spindle controls the equal segregation of paternal and maternal chromosomes during the first cell division. By overexpression of a single protein, GPR-1, in the maternal strain we changed the structure of the mitotic spindle from bipolar to two monopolar spindles to segregate maternal and paternal chromosomes into different cell lineages, resulting in non-mendelian segregation for entire genomes. To follow maternal and paternal segregation of the chromosomes we used red and green histone markers respectively. By mating gpr-1-overexpressing hermaphrodites with wild-type males, mendelian F1 worms that express both markers simultaneously in all tissues and non-mendelian F1 worms that express red and green markers in different tissues will be produced representing embryos with bipolar and embryos with two monopolar spindles. Thus, we show that the rules of genetic inheritance can be changed, which may inspire the formation of a new field of synthetic zoology. Transcriptional profiling was done to investigate the differences in gene expression between mendelian and non-mendelian offspring. Approximately 60 adult worms were used per sample. Four conditions were collected: hermaphrodites of the paternal strain, hermaphrodites of the maternal strain, co-segregating (mendelian) F1 after crossing of parental strains, and (non-mendelian) F1 that segregates the paternal genotype to body wall muscle, intestine + germline and the maternal genotype to the nervous system after crossing of parental strains.