Transcriptional Alterations in X-Linked Dystonia–Parkinsonism Caused by the SVA Retrotransposon

X-linked dystonia–parkinsonism (XDP) is a severe neurodegenerative disorder that manifests as adult-onset dystonia combined with parkinsonism. A SINE-VNTR-Alu (SVA) retrotransposon inserted in an intron of the TAF1 gene reduces its expression and alters splicing in XDP patient-derived cells. As a consequence, increased levels of the TAF1 intron retention transcript TAF1-32i can be found in XDP cells as compared to healthy controls. Here, we investigate the sequence of the deep intronic region included in this transcript and show that it is also present in cells from healthy individuals, albeit in lower amounts than in XDP cells, and that it undergoes degradation by nonsense-mediated mRNA decay. Furthermore, we investigate epigenetic marks (e.g., DNA methylation and histone modifications) present in this intronic region and the spanning sequence. Finally, we show that the SVA evinces regulatory potential, as demonstrated by its ability to repress the TAF1 promoter in vitro. Our results enable a better understanding of the disease mechanisms underlying XDP and transcriptional alterations caused by SVA retrotransposons

The importance of genetic testing for dystonia patients and translational research

Genetic testing through a variety of methods is a fundamental but underutilized approach for establishing the precise genetic diagnosis in patients with heritable forms of dystonia. Our knowledge of numerous dystonia-related genes, variants that they may contain, associated clinical presentations, and molecular disease mechanism may have significant translational potential for patients with genetically confirmed dystonia or their family members. Importantly, genetic testing permits the assembly of patient cohorts pertinent for dystonia-related research and developing therapeutics. Here we review the genetic testing approaches relevant to dystonia patients, and summarize and illustrate the multifold benefits of establishing an accurate molecular diagnosis for patients imminently or for translational research in the long run

Hidden Mutations in CdLS - Limitations of Sanger Sequencing in Molecular Diagnostics

International audienceCornelia de Lange syndrome (CdLS) is a well characterized developmental disorder. The genetic cause of CdLS is a mutation in one of five associated genes (NIPBL, SMC1A, SMC3, RAD21 and HDAC8) accounting for about 70 % of cases. To improve our current molecular diagnostic and to analyze some of CdLS candidate genes we developed and established a gene panel approach. Because recent data indicate a high frequency of mosaic NIPBL mutations that were not detected by conventional sequencing approaches of blood DNA, we started to collected buccal mucosa samples of our patients that were negative for mutations in the known CdLS genes. Here we report the identification of three mosaic NIPBL mutations by our high-coverage gene panel sequencing approach that were undetected by classical Sanger sequencing analysis of buccal mucosa DNA. All mutations were confirmed by the use of highly sensitive SNaPshot fragment analysis using DNA from buccal mucosa, urine and fibroblast samples. In blood samples we could not detect the respective mutation. Finally, in fibroblast samples from all three patients, Sanger sequencing could identify all the mutations. Thus, our study highlights the need for highly sensitive technologies in molecular diagnostic of CdLS to improve genetic diagnosis and counseling of patients and their families. This article is protected by copyright. All rights reserved

Regulation of the cohesin-loading factor NIPBL: Role of the lncRNA NIPBL-AS1 and identification of a distal enhancer element

International audienceCohesin is crucial for genome stability, cell division, transcription and chromatin organization. Its functions critically depend on NIPBL, the cohesin-loader protein that is found to be mutated in > 60% of the cases of Cornelia de Lange syndrome (CdLS). Other mutations are described in the cohesin subunits SMC1A, RAD21, SMC3 and the HDAC8 protein. In 25-30% of CdLS cases no mutation in the known CdLS genes is detected. Until now, functional elements in the noncoding genome were not characterized in the molecular etiology of CdLS and therefore are excluded from mutation screening, although the impact of such mutations has now been recognized for a wide range of diseases. We have identified different elements of the noncoding genome involved in regulation of the NIPBL gene. NIPBLAS1 is a long non-coding RNA transcribed upstream and antisense to NIPBL. By knockdown and transcription blocking experiments, we could show that not the NIPBL-AS1 gene product, but its actual transcription is important to regulate NIPBL expression levels. This reveals a possibility to boost the transcriptional activity of the NIPBL gene by interfering with the NIPBL-AS1 lncRNA. Further, we have identified a novel distal enhancer regulating both NIPBL and NIPBLAS1. Deletion of the enhancer using CRISPR genome editing in HEK293T cells reduces expression of NIPBL, NIPBL-AS1 as well as genes found to be dysregulated in CdLS

Elucidating Hexanucleotide Repeat Number and Methylation within the X-Linked Dystonia-Parkinsonism (XDP)-Related SVA Retrotransposon in <i>TAF1</i> with Nanopore Sequencing

Background: X-linked dystonia-parkinsonism (XDP) is an adult-onset neurodegenerative disorder characterized by progressive dystonia and parkinsonism. It is caused by a SINE-VNTR-Alu (SVA) retrotransposon insertion in the TAF1 gene with a polymorphic (CCCTCT)n domain that acts as a genetic modifier of disease onset and expressivity. Methods: Herein, we used Nanopore sequencing to investigate SVA genetic variability and methylation. We used blood-derived DNA from 96 XDP patients for amplicon-based deep Nanopore sequencing and validated it with fragment analysis which was performed using fluorescence-based PCR. To detect methylation from blood- and brain-derived DNA, we used a Cas9-targeted approach. Results: High concordance was observed for hexanucleotide repeat numbers detected with Nanopore sequencing and fragment analysis. Within the SVA locus, there was no difference in genetic variability other than variations of the repeat motif between patients. We detected high CpG methylation frequency (MF) of the SVA and flanking regions (mean MF = 0.94, SD = ±0.12). Our preliminary results suggest only subtle differences between the XDP patient and the control in predicted enhancer sites directly flanking the SVA locus. Conclusions: Nanopore sequencing can reliably detect SVA hexanucleotide repeat numbers, methylation and, lastly, variation in the repeat motif

<i>NIPBL-AS1</i> does not influence <i>NIPBL</i> transcription.

<p>A) Overview of the genomic position of <i>NIPBL</i> and <i>NIPBL-AS1</i> genes. Strand-specific read coverage of RNA-sequencing data (positive in green; negative in red) from HEK293T cells shows the transcription of <i>NIPBL-AS1</i> antisense to <i>NIPBL</i> [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1007137#pgen.1007137.ref001" target="_blank">1</a>]. CTCF binding sites in HEK293 cells (ENCODE hg18) are shown. Primers used in the transcript analysis are indicated as green bars. (B-C) Transcript levels of (B) <i>NIPBL-AS1</i> and (C) <i>NIPBL</i> after antisense oligonucleotide knockdown (ASO2, ASO3) of <i>NIPBL-AS1</i> in HEK293T cells. ASO C was used as control. Transcript levels were normalized against the control sample (ASO C) and the housekeeping <i>SNAPIN</i> using the ΔΔCt method (mean n = 3, error bars +/- s.d., p-values determined with t-Test).</p

Interactions of <i>NIPBL</i> and <i>NIPBL-AS1</i> with a potential distal enhancer.

<p>A) Long-range chromosomal interactions of the <i>NIPBL</i> and <i>NIPBL-AS1</i> promoter detected by chromosome conformation capture (3C-seq) in HEK293T cells using an ApoI digest. The positions of the different viewpoints used are marked in yellow. Three different viewpoints at the promoter (VP4, blue track) and the candidate enhancers regions R1 (VP5, green track) and R2 (R2—VP6, red track) were used. B) CTCF ChIP sequencing track from HEK293 cells (ENCODE). The orientations of the CTCF motifs as determined with JASPAR are shown below the track (red triangle–forward orientation, green triangle–reverse orientation). The CTCF sites involved in the promoter-enhancer interaction are indicated with yellow triangles above the track. C) DNAse clusters as well as histone modification profiles—H2A.z, H3K4me1, H3K4me2 and H3K4me3—of six different cell lines (G312878, K562, HeLa-S3, HEMEC, HSMM and HUVEC, available from ENCODE) are displayed as density graph. Black represents areas with the highest enrichment of the signals.</p

Implications for CdLS.

<p>A) Transcript levels of the genes <i>BBX</i>, <i>GLCCI1</i> and <i>ZNF695</i> that were described as dysregulated genes in CdLS [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1007137#pgen.1007137.ref020" target="_blank">20</a>] and previously confirmed as NIPBL-dependent genes with NIPBL binding sites at the promoter [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1007137#pgen.1007137.ref008" target="_blank">8</a>] were analysed in the different enhancer deletion clones D1 and D2 (mean n = 5 for D1 and n = 4 for D2, error bars +/- s.d., p-values determined with t-Test, the transcript levels of the individual clones are shown in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1007137#pgen.1007137.s007" target="_blank">S7 Fig</a>). B) Average transcript levels of <i>NIPBL</i> and <i>NIPBL-AS1</i> in lymphoblastoid cell lines (LCLs) derived from CdLS patients and controls. The details of the four LCL controls and three CdLS LCLs as well as the individual transcript levels are shown in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1007137#pgen.1007137.s008" target="_blank">S8 Fig</a> and in [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1007137#pgen.1007137.ref008" target="_blank">8</a>,<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1007137#pgen.1007137.ref020" target="_blank">20</a>]. Two primer pairs for <i>NIPBL</i> and one for <i>NIPBL-AS1</i> were used. Transcript levels were normalized against the housekeeping gene <i>NADH</i> (mean n = 4 for control LCLs and n = 3 for CdLS LCLs, error bars +/- s.d., p-values determined with t-Test).</p

Expanding the clinical spectrum of the "HDAC8-phenotype" - Implications for molecular diagnostics, counselling and risk prediction

International audienceCornelia de Lange syndrome (CdLS) is a clinically heterogeneous disorder characterized by typical facial dysmorphism, cognitive impairment and multiple congenital anomalies. Approximately 75% of patients carry a variant in one of the five cohesin-related genes NIPBL, SMC1A, SMC3, RAD21 and HDAC8. Herein we report on the clinical and molecular characterization of eleven patients carrying ten distinct variants in HDAC8. Given the high number of variants identified so far, we advise sequencing of HDAC8 as an indispensable part of the routine molecular diagnostic for patients with CdLS or CdLS-overlapping features. The phenotype of our patients is very broad whereas males tend to be more severely affected than females, who instead often present with less canonical CdLS features. The extensive clinical variability observed in the heterozygous females might be at least partially associated with a completely skewed X-inactivation, observed in seven out of eight female patients. Our cohort also includes two affected siblings whose unaffected mother was found to be mosaic for the causative mutation inherited to both affected children. This further supports the urgent need for an integration of highly sensitive sequencing technology to allow an appropriate molecular diagnostic, genetic counselling and risk predictio

Transcriptional Alterations in X-Linked Dystonia–Parkinsonism Caused by the SVA Retrotransposon

X-linked dystonia–parkinsonism (XDP) is a severe neurodegenerative disorder that manifests as adult-onset dystonia combined with parkinsonism. A SINE-VNTR-Alu (SVA) retrotransposon inserted in an intron of the TAF1 gene reduces its expression and alters splicing in XDP patient-derived cells. As a consequence, increased levels of the TAF1 intron retention transcript TAF1-32i can be found in XDP cells as compared to healthy controls. Here, we investigate the sequence of the deep intronic region included in this transcript and show that it is also present in cells from healthy individuals, albeit in lower amounts than in XDP cells, and that it undergoes degradation by nonsense-mediated mRNA decay. Furthermore, we investigate epigenetic marks (e.g., DNA methylation and histone modifications) present in this intronic region and the spanning sequence. Finally, we show that the SVA evinces regulatory potential, as demonstrated by its ability to repress the TAF1 promoter in vitro. Our results enable a better understanding of the disease mechanisms underlying XDP and transcriptional alterations caused by SVA retrotransposons