High-resolution breakpoint junction mapping of proximally extended D4Z4 deletions in FSHD1 reveals evidence for a founder effect.

Facioscapulohumeral muscular dystrophy (FSHD) is an inherited myopathy clinically characterized by weakness in the facial, shoulder girdle and upper a muscles. FSHD is caused by chromatin relaxation of the D4Z4 macrosatellite repeat, mostly by a repeat contraction, facilitating ectopic expression of DUX4 in skeletal muscle. Genetic diagnosis for FSHD is generally based on the sizing and haplotyping of the D4Z4 repeat on chromosome 4 by Southern blotting (SB), molecular combing or single-molecule optical mapping, which is usually straight forward but can be complicated by atypical rearrangements of the D4Z4 repeat. One of these rearrangements is a D4Z4 proximally extended deletion (DPED) allele, where not only the D4Z4 repeat is partially deleted, but also sequences immediately proximal to the repeat are lost, which can impede accurate diagnosis in all genetic methods. Previously, we identified several DPED alleles in FSHD and estimated the size of the proximal deletions by a complex pulsed-field gel electrophoresis and SB strategy. Here, using the next-generation sequencing, we have defined the breakpoint junctions of these DPED alleles at the base pair resolution in 12 FSHD families and 4 control individuals facilitating a PCR-based diagnosis of these DPED alleles. Our resultsshow that half of the DPED alleles are derivates of an ancient founder allele. For some DPED alleles, we found that genetic elements are deleted such as DUX4c, FRG2, DBE-T and myogenic enhancers necessitating re-evaluation of their role in FSHD pathogenesis.

New locus underlying auriculocondylar syndrome (ARCND): 430 kb duplication involving TWIST1 regulatory elements.

BACKGROUND: Auriculocondylar syndrome (ARCND) is a rare genetic disease that affects structures derived from the first and second pharyngeal arches, mainly resulting in micrognathia and auricular malformations. To date, pathogenic variants have been identified in three genes involved in the EDN1-DLX5/6 pathway (PLCB4, GNAI3 and EDN1) and some cases remain unsolved. Here we studied a large unsolved four-generation family. METHODS: We performed linkage analysis, resequencing and Capture-C to investigate the causative variant of this family. To test the pathogenicity of the CNV found, we modelled the disease in patient craniofacial progenitor cells, including induced pluripotent cell (iPSC)-derived neural crest and mesenchymal cells. RESULTS: This study highlights a fourth locus causative of ARCND, represented by a tandem duplication of 430 kb in a candidate region on chromosome 7 defined by linkage analysis. This duplication segregates with the disease in the family (LOD score=2.88) and includes HDAC9, which is located over 200 kb telomeric to the top candidate gene TWIST1. Notably, Capture-C analysis revealed multiple cis interactions between the TWIST1 promoter and possible regulatory elements within the duplicated region. Modelling of the disease revealed an increased expression of HDAC9 and its neighbouring gene, TWIST1, in neural crest cells. We also identified decreased migration of iPSC-derived neural crest cells together with dysregulation of osteogenic differentiation in iPSC-affected mesenchymal stem cells. CONCLUSION: Our findings support the hypothesis that the 430 kb duplication is causative of the ARCND phenotype in this family and that deregulation of TWIST1 expression during craniofacial development can contribute to the phenotype.

Breakpoint characterization of a rare alpha0 -thalassemia deletion using targeted locus amplification on genomic DNA.

INTRODUCTION: The high-sequence homology of the α-globin-gene cluster is responsible for microhomology-mediated recombination events during meiosis, resulting in a high density of deletion breakpoints within a 10 kb region. Commonly used deletion detection methods, such as multiplex ligation-dependent probe amplification (MLPA) and Southern blot, cannot exactly define the breakpoints. This typically requires long-range PCR, which is not always successful. Targeted locus amplification (TLA) is a targeted enrichment method that can be used to sequence up to 70 kb of neighboring DNA sequences without prior knowledge about the target site. METHODS: Genomic DNA (gDNA) TLA is a technique that folds isolated DNA, ensuring that adjacent loci are in a close spatial proximity. Subsequent digestion and religation form DNA circles that are amplified using fragment-specific inverse primers, creating a library that is suitable for Illumina sequencing. RESULTS: Here, we describe the characterization of a rare 16 771 bp deletion, utilizing gDNA TLA with a single inverse PCR primer set on one end of the breakpoint. Primers for breakpoint PCR were designed to confirm the deletion breakpoints and were consequently used to characterize the same deletion in 10 additional carriers sharing comparable hematologic data and similar MLPA results. CONCLUSIONS: The gDNA TLA technology was successfully used to identify deletion breakpoints within the alpha-globin cluster. The deletion was described only once in an earlier study as the --gb , but as it was not registered correctly in the available databases, it was not initially recognized as such.

Age-Associated Salivary MicroRNA Biomarkers for Oculopharyngeal Muscular Dystrophy.

Small non-coding microRNAs (miRNAs) are involved in the regulation of mRNA stability. Their features, including high stability and secretion to biofluids, make them attractive as potential biomarkers for diverse pathologies. This is the first study reporting miRNA as potential biomarkers for oculopharyngeal muscular dystrophy (OPMD), an adult-onset myopathy. We hypothesized that miRNA that is differentially expressed in affected muscles from OPMD patients is secreted to biofluids and those miRNAs could be used as biomarkers for OPMD. We first identified candidate miRNAs from OPMD-affected muscles and from muscles from an OPMD mouse model using RNA sequencing. We then compared the OPMD-deregulated miRNAs to the literature and, subsequently, we selected a few candidates for expression studies in serum and saliva biofluids using qRT-PCR. We identified 126 miRNAs OPMD-deregulated in human muscles, but 36 deregulated miRNAs in mice only (pFDR < 0.05). Only 15 OPMD-deregulated miRNAs overlapped between the in humans and mouse studies. The majority of the OPMD-deregulated miRNAs showed opposite deregulation direction compared with known muscular dystrophies miRNAs (myoMirs), which are associated. In contrast, similar dysregulation direction was found for 13 miRNAs that are common between OPMD and aging muscles. A significant age-association (p < 0.05) was found for 17 OPMD-deregulated miRNAs (13.4%), whereas in controls, only six miRNAs (1.4%) showed a significant age-association, suggesting that miRNA expression in OPMD is highly age-associated. miRNA expression in biofluids revealed that OPMD-associated deregulation in saliva was similar to that in muscles, but not in serum. The same as in muscle, miRNA expression levels in saliva were also found to be associated with age (p < 0.05). Moreover, the majority of OPMD-miRNAs were found to be associated with dysphagia as an initial symptom. We suggest that levels of specific miRNAs in saliva can mark muscle degeneration in general and dysphagia in OPMD.

Diagnostics of short tandem repeat expansion variants using massively parallel sequencing and componential tools.

Short tandem repeats (STRs) are scattered throughout the human genome. Some STRs, like trinucleotide repeat expansion (TRE) variants, cause hereditable disorders. Unambiguous molecular diagnostics of TRE disorders is hampered by current technical limitations imposed by traditional PCR and DNA sequencing methods. Here we report a novel pipeline for TRE variant diagnosis employing the massively parallel sequencing (MPS) combined with an opensource software package (FDSTools), which together are designed to distinguish true STR sequences from STR sequencing artifacts. We show that this approach can improve TRE diagnosis, such as Oculopharyngeal muscular dystrophy (OPMD). OPMD is caused by a trinucleotide expansion in the PABPN1 gene. A short GCN expansion, (GCN[10]), coding for a 10 alanine repeat is not pathogenic, but an alanine expansion is pathogenic. Applying this novel procedure in  a Dutch OPMD patient cohort, we found expansion variants from GCN[11] to GCN[16], with the GCN[16] as the most abundant variant. The repeat expansion length did not correlate with clinical features. However, symptom severity was found to correlate with age and with the initial affected muscles, suggesting that aging and muscle-specific factors can play a role in modulating OPMD.

Human Bone Marrow-Resident Natural Killer Cells Have a Unique Transcriptional Profile and Resemble Resident Memory CD8+ T Cells.

Human lymphoid tissues harbor, in addition to CD56bright and CD56dim natural killer (NK) cells, a third NK cell population: CD69+CXCR6+ lymphoid tissue (lt)NK cells. The function and development of ltNK cells remain poorly understood. In this study, we performed RNA sequencing on the three NK cell populations derived from bone marrow (BM) and blood. In ltNK cells, 1,353 genes were differentially expressed compared to circulating NK cells. Several molecules involved in migration were downregulated in ltNK cells: S1PR1, SELPLG and CD62L. By flow cytometry we confirmed that the expression profile of adhesion molecules (CD49e-, CD29low, CD81high, CD62L-, CD11c-) and transcription factors (Eomeshigh, Tbetlow) of ltNK cells differed from their circulating counterparts. LtNK cells were characterized by enhanced expression of inhibitory receptors TIGIT and CD96 and low expression of DNAM1 and cytolytic molecules (GZMB, GZMH, GNLY). Their proliferative capacity was reduced compared to the circulating NK cells. By performing gene set enrichment analysis, we identified DUSP6 and EGR2 as potential regulators of the ltNK cell transcriptome. Remarkably, comparison of the ltNK cell transcriptome to the published human spleen-resident memory CD8+ T (Trm) cell transcriptome revealed an overlapping gene signature. Moreover, the phenotypic profile of ltNK cells resembled that of CD8+ Trm cells in BM. Together, we provide transcriptional and phenotypic data that clearly distinguish ltNK cells from both the CD56bright and CD56dim NK cells and substantiate the view that ltNK cells are tissue-resident cells, which are functionally restrained in killing and have low proliferative activity.

Comprehensive multi-center assessment of small RNA-seq methods for quantitative miRNA profiling.

RNA-seq is increasingly used for quantitative profiling of small RNAs (for example, microRNAs, piRNAs and snoRNAs) in diverse sample types, including isolated cells, tissues and cell-free biofluids. The accuracy and reproducibility of the currently used small RNA-seq library preparation methods have not been systematically tested. Here we report results obtained by a consortium of nine labs that independently sequenced reference, 'ground truth' samples of synthetic small RNAs and human plasma-derived RNA. We assessed three commercially available library preparation methods that use adapters of defined sequence and six methods using adapters with degenerate bases. Both protocol- and sequence-specific biases were identified, including biases that reduced the ability of small RNA-seq to accurately measure adenosine-to-inosine editing in microRNAs. We found that these biases were mitigated by library preparation methods that incorporate adapters with degenerate bases. MicroRNA relative quantification between samples using small RNA-seq was accurate and reproducible across laboratories and methods.

Immune stimuli shape the small non-coding transcriptome of extracellular vesicles released by dendritic cells.

The release and uptake of nano-sized extracellular vesicles (EV) is a highly conserved means of intercellular communication. The molecular composition of EV, and thereby their signaling function to target cells, is regulated by cellular activation and differentiation stimuli. EV are regarded as snapshots of cells and are, therefore, in the limelight as biomarkers for disease. Although research on EV-associated RNA has predominantly focused on microRNAs, the transcriptome of EV consists of multiple classes of small non-coding RNAs with potential gene-regulatory functions. It is not known whether environmental cues imposed on cells induce specific changes in a broad range of EV-associated RNA classes. Here, we investigated whether immune-activating or -suppressing stimuli imposed on primary dendritic cells affected the release of various small non-coding RNAs via EV. The small RNA transcriptomes of highly pure EV populations free from ribonucleoprotein particles were analyzed by RNA sequencing and RT-qPCR. Immune stimulus-specific changes were found in the miRNA, snoRNA, and Y-RNA content of EV from dendritic cells, whereas tRNA and snRNA levels were much less affected. Only part of the changes in EV-RNA content reflected changes in cellular RNA, which urges caution in interpreting EV as snapshots of cells. By comprehensive analysis of RNA obtained from highly purified EV, we demonstrate that multiple RNA classes contribute to genetic messages conveyed via EV. The identification of multiple RNA classes that display cell stimulation-dependent association with EV is the prelude to unraveling the function and biomarker potential of these EV-RNAs.

Brain Transcriptomic Analysis of Hereditary Cerebral Hemorrhage With Amyloidosis-Dutch Type.

Hereditary cerebral hemorrhage with amyloidosis-Dutch type (HCHWA-D) is an early onset hereditary form of cerebral amyloid angiopathy (CAA) caused by a point mutation resulting in an amino acid change (NP_000475.1:p.Glu693Gln) in the amyloid precursor protein (APP). Post-mortem frontal and occipital cortical brain tissue from nine patients and nine age-related controls was used for RNA sequencing to identify biological pathways affected in HCHWA-D. Although previous studies indicated that pathology is more severe in the occipital lobe in HCHWA-D compared to the frontal lobe, the current study showed similar changes in gene expression in frontal and occipital cortex and the two brain regions were pooled for further analysis. Significantly altered pathways were analyzed using gene set enrichment analysis (GSEA) on 2036 significantly differentially expressed genes. Main pathways over-represented by down-regulated genes were related to cellular aerobic respiration (including ATP synthesis and carbon metabolism) indicating a mitochondrial dysfunction. Principal up-regulated pathways were extracellular matrix (ECM)-receptor interaction and ECM proteoglycans in relation with an increase in the transforming growth factor beta (TGFß) signaling pathway. Comparison with the publicly available dataset from pre-symptomatic APP-E693Q transgenic mice identified overlap for the ECM-receptor interaction pathway, indicating that ECM modification is an early disease specific pathomechanism.

Inter- and intra-species variation in genome-wide gene expression of Drosophila in response to parasitoid wasp attack.

BACKGROUND: Parasitoid resistance in Drosophila varies considerably, among and within species. An immune response, lamellocyte-mediated encapsulation, evolved in a subclade of Drosophila and was subsequently lost in at least one species within this subclade. While the mechanisms of resistance are fairly well documented in D. melanogaster, much less is known for closely related species. Here, we studied the inter- and intra-species variation in gene expression after parasitoid attack in Drosophila. We used RNA-seq after parasitization of four closely related Drosophila species of the melanogaster subgroup and replicated lines of D. melanogaster experimentally selected for increased resistance to gain insights into short- and long-term evolutionary changes. RESULTS: We found a core set of genes that are consistently up-regulated after parasitoid attack in the species and lines tested, regardless of their level of resistance. Another set of genes showed no up-regulation or expression in D. sechellia, the species unable to raise an immune response against parasitoids. This set consists largely of genes that are lineage-restricted to the melanogaster subgroup. Artificially selected lines did not show significant differences in gene expression with respect to non-selected lines in their responses to parasitoid attack, but several genes showed differential exon usage. CONCLUSIONS: We showed substantial similarities, but also notable differences, in the transcriptional responses to parasitoid attack among four closely related Drosophila species. In contrast, within D. melanogaster, the responses were remarkably similar. We confirmed that in the short-term, selection does not act on a pre-activation of the immune response. Instead it may target alternative mechanisms such as differential exon usage. In the long-term, we found support for the hypothesis that the ability to immunologically resist parasitoid attack is contingent on new genes that are restricted to the melanogaster subgroup.

Detecting PKD1 variants in polycystic kidney disease patients by single-molecule long-read sequencing.

A genetic diagnosis of autosomal-dominant polycystic kidney disease (ADPKD) is challenging due to allelic heterogeneity, high GC content, and homology of the PKD1 gene with six pseudogenes. Short-read next-generation sequencing approaches, such as whole-genome sequencing and whole-exome sequencing, often fail at reliably characterizing complex regions such as PKD1. However, long-read single-molecule sequencing has been shown to be an alternative strategy that could overcome PKD1 complexities and discriminate between homologous regions of PKD1 and its pseudogenes. In this study, we present the increased power of resolution for complex regions using long-read sequencing to characterize a cohort of 19 patients with ADPKD. Our approach provided high sensitivity in identifying PKD1 pathogenic variants, diagnosing 94.7% of the patients. We show that reliable screening of ADPKD patients in a single test without interference of PKD1 homologous sequences, commonly introduced by residual amplification of PKD1 pseudogenes, by direct long-read sequencing is now possible. This strategy can be implemented in diagnostics and is highly suitable to sequence and resolve complex genomic regions that are of clinical relevance.

Flexible and Scalable Full-Length CYP2D6 Long Amplicon PacBio Sequencing.

Cytochrome P450 2D6 (CYP2D6) is among the most important genes involved in drug metabolism. Specific variants are associated with changes in the enzyme's amount and activity. Multiple technologies exist to determine these variants, like the AmpliChip CYP450 test, Taqman qPCR, or Second-Generation Sequencing, however, sequence homology between cytochrome P450 genes and pseudogene CYP2D7 impairs reliable CYP2D6 genotyping, and variant phasing cannot accurately be determined using these assays. To circumvent this, we sequenced CYP2D6 using the Pacific Biosciences RSII and obtained high-quality, full-length, phased CYP2D6 sequences, enabling accurate variant calling and haplotyping of the entire gene-locus including exonic, intronic, and upstream and downstream regions. Unphased diplotypes (Roche AmpliChip CYP450 test) were confirmed for 24 of the 25 samples, including gene duplications. Cases with gene deletions required additional specific assays to resolve. In total, 61 unique variants were detected, including variants that had not previously been associated with specific haplotypes. To further aid genomic analysis using standard reference sequences, we have established an LOVD-powered CYP2D6 gene-variant database, and added all reference haplotypes and data reported here. We conclude that our CYP2D6 genotyping approach produces reliable CYP2D6 diplotypes and reveals information about additional variants, including phasing and copy-number variation.

Full-Length Mitochondrial-DNA Sequencing on the PacBio RSII.

Conventional mitochondrial-DNA (MT DNA) sequencing approaches use Sanger sequencing of 20-40 partially overlapping PCR fragments per individual, which is a time- and resource-consuming process. We have developed a high-throughput, accurate, fast, and cost-effective human MT DNA sequencing approach. In this setup we first generate long-range PCR products for two partially overlapping 7.7 and 9.2 kb MT DNA-specific amplicons, add sample-specific barcodes, and sequence these on the PacBio RSII system to obtain full-length MT DNA sequences for genotyping/haplotyping purposes.

Whole-transcriptome analysis of flow-sorted cervical cancer samples reveals that B cell expressed TCL1A is correlated with improved survival.

Cervical cancer is typically well infiltrated by immune cells. Because of the intricate relationship between cancer cells and immune cells, we aimed to identify both cancer cell and immune cell expressed biomarkers. Using a novel approach, we isolated RNA from flow-sorted viable EpCAM+ tumor epithelial cells and CD45+ tumor-infiltrating immune cells obtained from squamous cell cervical cancer samples (n = 24). Total RNA was sequenced and differential gene expression analysis of the CD45+ immune cell fractions identified TCL1A as a novel marker for predicting improved survival (p = 0.007). This finding was validated using qRT-PCR (p = 0.005) and partially validated using immunohistochemistry (p = 0.083). Importantly, TCL1A was found to be expressed in a subpopulation of B cells (CD3-/CD19+/CD10+/CD34-) using multicolor immunofluorescence. A high TCL1A/CD20 (B cell) ratio, determined in total tumor samples from a separate patient cohort using qRT-PCR (n = 52), was also correlated with improved survival (p = 0.027). This is the first study demonstrating the prognostic value of separating tumor epithelial cells from tumor-infiltrating immune cells and determining their RNA expression profile for identifying putative cancer biomarkers. Our results suggest that intratumoral TCL1A+ B cells are important for controlling cervical cancer development.

KeyGenes, a Tool to Probe Tissue Differentiation Using a Human Fetal Transcriptional Atlas.

Differentiated derivatives of human pluripotent stem cells in culture are generally phenotypically immature compared to their adult counterparts. Their identity is often difficult to determine with certainty because little is known about their human fetal equivalents in vivo. Cellular identity and signaling pathways directing differentiation are usually determined by extrapolating information from either human adult tissue or model organisms, assuming conservation with humans. To resolve this, we generated a collection of human fetal transcriptional profiles at different developmental stages. Moreover, we developed an algorithm, KeyGenes, which uses this dataset to quantify the extent to which next-generation sequencing or microarray data resemble specific cell or tissue types in the human fetus. Using KeyGenes combined with the human fetal atlas, we identified multiple cell and tissue samples unambiguously on a limited set of features. We thus provide a flexible and expandable platform to monitor and evaluate the efficiency of differentiation in vitro.

MicroRNA classifier and nomogram for metastasis prediction in colon cancer.

BACKGROUND: Colon cancer prognosis and treatment are currently based on a classification system still showing large heterogeneity in clinical outcome, especially in TNM stages II and III. Prognostic biomarkers for metastasis risk are warranted as development of distant recurrent disease mainly accounts for the high lethality rates of colon cancer. miRNAs have been proposed as potential biomarkers for cancer. Furthermore, a verified standard for normalization of the amount of input material in PCR-based relative quantification of miRNA expression is lacking. METHODS: A selection of frozen tumor specimens from two independent patient cohorts with TNM stage II-III microsatellite stable primary adenocarcinomas was used for laser capture microdissection. Next-generation sequencing was performed on small RNAs isolated from colorectal tumors from the Dutch cohort (N = 50). Differential expression analysis, comparing in metastasized and nonmetastasized tumors, identified prognostic miRNAs. Validation was performed on colon tumors from the German cohort (N = 43) using quantitative PCR (qPCR). RESULTS: miR25-3p and miR339-5p were identified and validated as independent prognostic markers and used to construct a multivariate nomogram for metastasis risk prediction. The nomogram showed good probability prediction in validation. In addition, we recommend combination of miR16-5p and miR26a-5p as standard for normalization in qPCR of colon cancer tissue-derived miRNA expression. CONCLUSIONS: In this international study, we identified and validated a miRNA classifier in primary cancers, and propose a nomogram capable of predicting metastasis risk in microsatellite stable TNM stage II-III colon cancer. IMPACT: In conjunction with TNM staging, by means of a nomogram, this miRNA classifier may allow for personalized treatment decisions based on individual tumor characteristics.

Efficient and sensitive identification and quantification of airborne pollen using next-generation DNA sequencing.

Pollen monitoring is an important and widely used tool in allergy research and creation of awareness in pollen-allergic patients. Current pollen monitoring methods are microscope-based, labour intensive and cannot identify pollen to the genus level in some relevant allergenic plant groups. Therefore, a more efficient, cost-effective and sensitive method is needed. Here, we present a method for identification and quantification of airborne pollen using DNA sequencing. Pollen is collected from ambient air using standard techniques. DNA is extracted from the collected pollen, and a fragment of the chloroplast gene trnL is amplified using PCR. The PCR product is subsequently sequenced on a next-generation sequencing platform (Ion Torrent). Amplicon molecules are sequenced individually, allowing identification of different sequences from a mixed sample. We show that this method provides an accurate qualitative and quantitative view of the species composition of samples of airborne pollen grains. We also show that it correctly identifies the individual grass genera present in a mixed sample of grass pollen, which cannot be achieved using microscopic pollen identification. We conclude that our method is more efficient and sensitive than current pollen monitoring techniques and therefore has the potential to increase the throughput of pollen monitoring.

Novel variants in GNAI3 associated with auriculocondylar syndrome strengthen a common dominant negative effect.

Auriculocondylar syndrome is a rare craniofacial disorder comprising core features of micrognathia, condyle dysplasia and question mark ear. Causative variants have been identified in PLCB4, GNAI3 and EDN1, which are predicted to function within the EDN1-EDNRA pathway during early pharyngeal arch patterning. To date, two GNAI3 variants in three families have been reported. Here we report three novel GNAI3 variants, one segregating with affected members in a family previously linked to 1p21.1-q23.3 and two de novo variants in simplex cases. Two variants occur in known functional motifs, the G1 and G4 boxes, and the third variant is one amino acid outside of the G1 box. Structural modeling shows that all five altered GNAI3 residues identified to date cluster in a region involved in GDP/GTP binding. We hypothesize that all GNAI3 variants lead to dominant negative effects.