Mb Of DNA Research Articles

Variants in Candidate Genes for Phenotype Heterogeneity in Patients with the 22q11.2 Deletion Syndrome.

22q11.2 deletion syndrome (22q11.2DS) is a microdeletion syndrome with a broad and heterogeneous phenotype, even though most of the deletions present similar sizes, involving ∼3 Mb of DNA. In a relatively large population of a Brazilian 22q11.2DS cohort (60 patients), we investigated genetic variants that could act as genetic modifiers and contribute to the phenotypic heterogeneity, using a targeted NGS (Next Generation Sequencing) with a specific Ion AmpliSeq panel to sequence nine candidate genes (CRKL, MAPK1, HIRA, TANGO2, PI4KA, HDAC1, ZDHHC8, ZFPM2, and JAM3), mapped in and outside the 22q11.2 hemizygous deleted region. In silico prediction was performed, and the whole-genome sequencing annotation analysis package (WGSA) was used to predict the possible pathogenic effect of single nucleotide variants (SNVs). For the in silico prediction of the indels, we used the genomic variants filtered by a deep learning model in NGS (GARFIELD-NGS). We identified six variants, 4 SNVs and 2 indels, in MAPK1, JAM3, and ZFPM2 genes with possibly synergistic deleterious effects in the context of the 22q11.2 deletion. Our results provide the opportunity for the discovery of the co-occurrence of genetic variants with 22q11.2 deletions, which may influence the patients´ phenotype.

Copy number alteration features in pan-cancer homologous recombination deficiency prediction and biology

Homologous recombination deficiency (HRD) renders cancer cells vulnerable to unrepaired double-strand breaks and is an important therapeutic target as exemplified by the clinical efficacy of poly ADP-ribose polymerase (PARP) inhibitors as well as the platinum chemotherapy drugs applied to HRD patients. However, it remains a challenge to predict HRD status precisely and economically. Copy number alteration (CNA), as a pervasive trait of human cancers, can be extracted from a variety of data sources, including whole genome sequencing (WGS), SNP array, and panel sequencing, and thus can be easily applied clinically. Here we systematically evaluate the predictive performance of various CNA features and signatures in HRD prediction and build a gradient boosting machine model (HRDCNA) for pan-cancer HRD prediction based on these CNA features. CNA features BP10MB[1] (The number of breakpoints per 10MB of DNA is 1) and SS[ > 7 & <=8] (The log10-based size of segments is greater than 7 and less than or equal to 8) are identified as the most important features in HRD prediction. HRDCNA suggests the biallelic inactivation of BRCA1, BRCA2, PALB2, RAD51C, RAD51D, and BARD1 as the major genetic basis for human HRD, and may also be applied to effectively validate the pathogenicity of BRCA1/2 variants of uncertain significance (VUS). Together, this study provides a robust tool for cost-effective HRD prediction and also demonstrates the applicability of CNA features and signatures in cancer precision medicine.

Cas12a-Capture: A Novel, Low-Cost, and Scalable Method for Targeted Sequencing.

Targeted sequencing remains a valuable technique for clinical and research applications. However, many existing technologies suffer from pervasive guanine-cytosine (GC) sequence content bias, high input DNA requirements, and high cost for custom panels. We have developed Cas12a-Capture, a low-cost and highly scalable method for targeted sequencing. The method utilizes preprogrammed guide RNAs to direct CRISPR-Cas12a cleavage of double-stranded DNA in vitro and then takes advantage of the resulting four to five nucleotide overhangs for selective ligation with a custom sequencing adapter. Addition of a second sequencing adapter and enrichment for ligation products generates a targeted sequence library. We first performed a pilot experiment with 7176 guides targeting 3.5 Mb of DNA. Using these data, we modeled the sequence determinants of Cas12a-Capture efficiency, then designed an optimized set of 11,438 guides targeting 3.0 Mb. The optimized guide set achieves an average 64-fold enrichment of targeted regions with minimal GC bias. Cas12a-Capture variant calls had strong concordance with Illumina Platinum Genome calls, especially for single nucleotide variants, which could be improved by applying basic variant quality heuristics. We believe Cas12a-Capture has a wide variety of potential clinical and research applications and is amendable for selective enrichment for any double-stranded DNA template or genome.

Molecular and Pathological Profiling of Corresponding Treatment-Naïve and Neoadjuvant Pazopanib-Treated High-Risk Soft Tissue Sarcoma Samples of the GISG-04/NOPASS Study.

Simple SummaryPatients with malignant soft tissue tumors, called soft tissue sarcoma (STS), show alterations in their deoxyribonucleic acid (DNA). Pathologists use these alterations for classification of STS and as drug-related biomarkers. Some drugs show better effectiveness in association with certain genetic alterations. In this study, we examined STS tumor tissue from a specific sarcoma study (GISG-04/NOPASS) with massively parallel DNA sequencing in order to find genetic biomarkers for pazopanib, a multi-target tyrosine kinase inhibitor approved for the treatment of advanced STS. While we could not clearly identify a specific genetic target, we were able to improve diagnostic accuracy and could detect mutations that are potentially useful for individualized therapy.In the framework of the German Interdisciplinary Sarcoma Group GISG-04/NOPASS trial, we evaluated soft tissue sarcoma samples taken before and after neoadjuvant pazopanib therapy using histopathology and next generation sequencing (NGS) to find potential predictive biomarkers. We also aimed to improve the genetically based sarcoma classification and to elucidate additional potentially druggable mutations. In total, 30 tumor samples from 18 patients consisting of 12 pre-therapeutic biopsies and 18 resection specimens following neoadjuvant pazopanib therapy were available for analyses. NGS was performed with the Oncomine Focus Assay (Ion Torrent) covering 0.03 Mb of DNA and enabled the detection of genetic variants in 52 cancer-relevant genes. Pathological analysis showed significant regression (≥50%) after pazopanib treatment in only one undifferentiated (pleomorphic) sarcoma. NGS analyses revealed a very high frequency of CDK4 amplification (88%; 7/8) in the group of dedifferentiated liposarcoma. In addition, two potentially druggable mutations, a MAP2K1 missense mutation (E203K) and a BRAF missense mutation (V600E), were traceable in two undifferentiated (pleomorphic) sarcoma patients (11%; 2/18). Our findings demonstrate that NGS testing is a powerful technology helping to improve diagnostic accuracy and offering some patients the chance for personalized medicine even in a “mutation unlikely” cohort like STS.

Abstract PS5-04: Therapeutic considerations in microsatellite instability high (MSI- H) breast cancers (BC) identified by comprehensive genomic profiling (CGP)

Abstract Background: Non-colorectal MSI-H tumors are increasingly identified by CGP. Rare types such as MSI-H BC remain poorly defined with an evidence gap on how to optimally sequence or combine with standard of care treatment. MSI can be measured by either IHC, PCR, or CGP and can be caused by both sporadic and germline variants within different tumor types. Prior studies in BC have shown evidence of dMMR by IHC cases MSS based on PCR. This could be due to intra-tumor heterogeneity, specific microsatellite loci evaluated, or penetrance of germline, somatic, or epigenetic alterations. Published data suggests carriers of germline pathogenic MMR variants have a BC risk equivalent to the normal population and currently germline testing is recommended only for BRCA. Currently in advanced BC, standard tumor biomarker testing includes IHC, PCR, and FISH; however, with increasing use of CGP we demonstrate additional actionable biomarkers as well as potential germline variants in MSI-H BC. Methods: DNA was extracted and hybrid capture CGP was performed on 29,160 BC cases. TMB was determined on 0.8-1.2 Mb of DNA and MSI status on 95-114 loci. Genomic LOH was also evaluated. Comparative analysis was done with 101 MSI-H BC, 841 MSS BC and 4,988 non-breast MSI-H cancers. Histological subtype was obtained from the pathology along with orthogonal testing for ER/PR/HER2 status. Somatic-germline-zygosity (SGZ) status was predicted using a published research use algorithm. Select case reports with clinical outcomes will be presented. Results: We identified 101 (0.35% of total) MSI-H BC cases: 29 ER+/HER2-, 5 HER2+, 29 TNBC, and 28 unknown. Amongst BC cases with known subtype, TNBC was enriched for MSI-H vs MSS (53.4 vs 35.8%, p=0.005). The median TMB in MSI-H BC (26.1 mut/Mb, IQR 17.4;42.8) was significantly lower than that of MSI-H colon (46.1mut/MB) and higher than that of MSI-H uterine tumors (22.6mut/Mb) in our comparison group (p&amp;lt;0.001 for both, Kruskal-Wallis test). Pathogenic variants in an MMR gene were found in 61.4% of MSI-H BC with MLH1 loss being the most common (13.6%) and much higher vs. the non-breast MSI-H cohort (2.4%, p&amp;lt;0.0001). Germline mutations in MMR genes in BC are rare yet 5/52 MMR short variants identified in 101 MSI-H BCs were predicted to be germline, 34 somatic, and 13 could not be determined. We identified 21 MSI-H BC patients with a total of 25 pathogenic BRCA1/2 alterations of which 4 were likely germline, 10 were homozygous, and were enriched in TNBC. These were mainly frameshift mutations, including BRCA2 T3033fs* in 5/18 (28%) cases; however, 7/25 were deletions, rearrangements, or nonsense mutations. Median gLOH was significantly higher in BRCA altered (19.7%) compared to BRCA wild-type MSI-H BC cases (9.6%) (p=0.007, Wilcox test). Additional potentially targetable biomarkers included 26 CDx eligible PIK3CA mutations, 11 ERBB2 activating point mutations in the TKD or ECD domain, 1 FGFR2 rearrangement, and 6 AKT1 E17K mutations. Four cases also had concurrent (CD274) PD-L1 amplifications. Conclusion: MSI-H BC is rare but CGP can identify additional therapeutic options for rational combination with targeted therapies such as PI3K, PARP, and HER2 inhibitors. BRCA alterations may be of germline or somatic origin and they may be targetable, as demonstrated by gLOH, rather than passenger mutations. Further characterization of these tumors and comparison to both MSS BC and non-breast MSI-H tumor types, combined with treatment outcomes, can provide insights on rationale combinations and/or sequencing of therapeutic agents. Citation Format: Kimberly McGregor, Natalie Danzinger, Jeffrey S. Ross, Kyle Gowen, Alexa B. Schrock, Garrett M. Frampton, Dean C. Pavlick, Jan W. Davis, Carl R. Gray, Jeffrey M. Venstrom. Therapeutic considerations in microsatellite instability high (MSI- H) breast cancers (BC) identified by comprehensive genomic profiling (CGP) [abstract]. In: Proceedings of the 2020 San Antonio Breast Cancer Virtual Symposium; 2020 Dec 8-11; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2021;81(4 Suppl):Abstract nr PS5-04.

Urothelial cancer harbours EGFR and HER2 amplifications and exon 20 insertions.

To review the genomic landscape of advanced urothelial carcinoma (UC) to assess the frequencies of EGFR and ERBB2 (HER2) alterations. Tumour specimens from 3753 patients with advanced UC were assayed with hybrid capture-based comprehensive genomic profiling of 180-395 genes. Tumour mutational burden (TMB) was assessed on 0.8 or 1.1Mb of DNA, and is reported as mutations per megabase. In 3753 cases of UC, EGFR alterations were detected in 4.1% (154) and were most commonly amplifications (64%; 99/154), while exon 20 insertions (EGFRexon20ins ) were the second most common alteration (18%; 27/154). Alterations in ERBB2 were observed in 15% (552/3753) of cases and, similarly, ERBB2 amplification was the most commonly observed alteration (278/552; 50%); ERBB2exon20ins occurred in 3.6% (20/552) of cases. EGFRexon20ins and ERBB2exon20ins occurred in younger patients (median age 62 vs 69years, P = 2.6E-2 and 60 vs 68years, P = 7.8E-4), and these cases had significantly lower TMB (median 3.6 vs 7.2, P = 2.7E-4 and 2.5 vs 10, P = 1.2E-7) and less frequent TP53 alterations (3.7% vs 83%, P = 4.3E-14 and 20% vs 68%, P = 9.8E-4) compared to cases with other EGFR or ERBB2 alterations. EGFR and ERBB2 alterations occur in 4% and 15% of UC, respectively. EGFRexon20ins and ERBB2exon20ins were present in 0.7% and 0.5% of UC overall and collectively define a small, but distinct, subset of UC with infrequent co-occurrence of other drivers and low TMB. Given recent promising clinical studies of inhibitors with activity against exon 20 insertions in non-small cell lung cancer, consideration should be given to developing a trial inclusive of patients with UC harbouring these alterations.

Conventional Karyotype and Fluorescence in situ Hybridization (FISH) Technology

Karyotype analysis of cells has been in use for many years and has led to the causative genetic change in numerous clinical syndromes, including trisomy 21, Klinefelter, Turner, Prader-Willi and Angelman syndromes. The resolution of the test depends on the degree of condensation of the chromosomes in the karyotype, but even at high resolution (&gt; 800 bands per haploid set) the changes identified are in the order of 5 Mb of DNA. Fluorescence in situ hybridization (FISH) bridges the gap between the relatively low resolution of karyotype analysis and the very high resolution of DNA analysis. With FISH it is possible to identify smaller changes in individual cells. The size of the change identified correlates with the size of the probe, which vary from 120 kb to 600 kb in size. FISH is widely used to confirm deletions or duplications identified by newer methods, such as array analysis. This review contains 8 figures, 3 tables, and 25 references. Keywords: Cytogenetics, chromosome, karyotype, chromosomal resolution, tissue culture, fluorescence, hybridization, probe

Resequencing Study Confirms That Host Defense and Cell Senescence Gene Variants Contribute to the Risk of Idiopathic Pulmonary Fibrosis.

Rationale: Several common and rare genetic variants have been associated with idiopathic pulmonary fibrosis, a progressive fibrotic condition that is localized to the lung. Objectives: To develop an integrated understanding of the rare and common variants located in multiple loci that have been reported to contribute to the risk of disease. Methods: We performed deep targeted resequencing (3.69 Mb of DNA) in cases (n = 3,624) and control subjects (n = 4,442) across genes and regions previously associated with disease. We tested for associations between disease and 1) individual common variants via logistic regression and 2) groups of rare variants via sequence kernel association tests. Measurements and Main Results: Statistically significant common variant association signals occurred in all 10 of the regions chosen based on genome-wide association studies. The strongest risk variant is the MUC5B promoter variant rs35705950, with an odds ratio of 5.45 (95% confidence interval, 4.91-6.06) for one copy of the risk allele and 18.68 (95% confidence interval, 13.34-26.17) for two copies of the risk allele (P = 9.60 × 10-295). In addition to identifying for the first time that rare variation in FAM13A is associated with disease, we confirmed the role of rare variation in the TERT and RTEL1 gene regions in the risk of IPF, and found that the FAM13A and TERT regions have independent common and rare variant signals. Conclusions: A limited number of common and rare variants contribute to the risk of idiopathic pulmonary fibrosis in each of the resequencing regions, and these genetic variants focus on biological mechanisms of host defense and cell senescence.

Novel idiopathic pulmonary fibrosis susceptibility variants revealed by deepsequencing.

BackgroundSpecific common and rare single nucleotide variants (SNVs) increase the likelihood of developing sporadic idiopathic pulmonary fibrosis (IPF). We performed target-enriched sequencing on three loci previously identified by a genome-wide association study to gain a deeper understanding of the full spectrum of IPF genetic risk and performed a two-stage case–control association study.MethodsA total of 1.7 Mb of DNA from 181 IPF patients was deep sequenced (>100×) across 11p15.5, 14q21.3 and 17q21.31 loci. Comparisons were performed against 501 unrelated controls and replication studies were assessed in 3968 subjects.Results36 SNVs were associated with IPF susceptibility in the discovery stage (p<5.0×10−8). After meta-analysis, the strongest association corresponded to rs35705950 (p=9.27×10−57) located upstream from the mucin 5B gene (MUC5B). Additionally, a novel association was found for two co-inherited low-frequency SNVs (<5%) in MUC5AC, predicting a missense amino acid change in mucin 5AC (lowest p=2.27×10−22). Conditional and haplotype analyses in 11p15.5 supported the existence of an additional contribution of MUC5AC variants to IPF risk.ConclusionsThis study reinforces the significant IPF associations of these loci and implicates MUC5AC as another key player in IPF susceptibility.

A genetic locus for sensory epilepsy precipitated by contact with hot water maps to chromosome 9p24.3-p23

Hot water epilepsy (HWE) is a rare form of sensory epilepsy where seizures are precipitated by a stimulus of contact with hot water. While earlier studies have suggested causal role of genes for HWE, specific underpinnings are beginning to be explored only recently. We carried out a whole genome-based linkage analysis in a family where most of its members affected by HWE and found evidence of a previously unknown locus at chromosome 9p24.3-p23. Parametric two-point analysis suggested linkage with the greatest LOD score of 3.42 for the marker D9S286 at 9p24.1 at recombination fraction (θ) = 0, 90% penetrance value and 1% phenocopy rate. The highest multipoint LODscore of 3.42 was obtained for same marker at 9p24. The critical genetic interval of about 10 Mb of DNA was defined by the markers D9S917 and D9S168 corresponding to the centromere-distal and centromere-proximal recombination boundaries, respectively. This observation along with our previous findings of hot water genetic loci at 10q21.3-q22.3 (OMIM: 613339) and 4q24-q28 (OMIM: 613340), indicates unanticipated genetic heterogeneity for the disorder in families from a relatively small geographic region in the southern parts of India.

Inversion variants in human and primate genomes.

For many years, inversions have been proposed to be a direct driving force in speciation since they suppress recombination when heterozygous. Inversions are the most common large-scale differences among humans and great apes. Nevertheless, they represent large events easily distinguishable by classical cytogenetics, whose resolution, however, is limited. Here, we performed a genome-wide comparison between human, great ape, and macaque genomes using the net alignments for the most recent releases of genome assemblies. We identified a total of 156 putative inversions, between 103 kb and 91 Mb, corresponding to 136 human loci. Combining literature, sequence, and experimental analyses, we analyzed 109 of these loci and found 67 regions inverted in one or multiple primates, including 28 newly identified inversions. These events overlap with 81 human genes at their breakpoints, and seven correspond to sites of recurrent rearrangements associated with human disease. This work doubles the number of validated primate inversions larger than 100 kb, beyond what was previously documented. We identified 74 sites of errors, where the sequence has been assembled in the wrong orientation, in the reference genomes analyzed. Our data serve two purposes: First, we generated a map of evolutionary inversions in these genomes representing a resource for interrogating differences among these species at a functional level; second, we provide a list of misassembled regions in these primate genomes, involving over 300 Mb of DNA and 1978 human genes. Accurately annotating these regions in the genome references has immediate applications for evolutionary and biomedical studies on primates.

Sequence analysis of European maize inbred line F2 provides new insights into molecular and chromosomal characteristics of presence/absence variants

BackgroundMaize is well known for its exceptional structural diversity, including copy number variants (CNVs) and presence/absence variants (PAVs), and there is growing evidence for the role of structural variation in maize adaptation. While PAVs have been described in this important crop species, they have been only scarcely characterized at the sequence level and the extent of presence/absence variation and relative chromosomal landscape of inbred-specific regions remain to be elucidated.ResultsDe novo genome sequencing of the French F2 maize inbred line revealed 10,044 novel genomic regions larger than 1 kb, making up 88 Mb of DNA, that are present in F2 but not in B73 (PAV). This set of maize PAV sequences allowed us to annotate PAV content and to analyze sequence breakpoints. Using PAV genotyping on a collection of 25 temperate lines, we also analyzed Linkage Disequilibrium in PAVs and flanking regions, and PAV frequencies within maize genetic groups.ConclusionsWe highlight the possible role of MMEJ-type double strand break repair in maize PAV formation and discover 395 new genes with transcriptional support. Pattern of linkage disequilibrium within PAVs strikingly differs from this of flanking regions and is in accordance with the intuition that PAVs may recombine less than other genomic regions. We show that most PAVs are ancient, while some are found only in European Flint material, thus pinpointing structural features that may be at the origin of adaptive traits involved in the success of this material. Characterization of such PAVs will provide useful material for further association genetic studies in European and temperate maize.

Gifted microbes for genome mining and natural product discovery

Actinomycetes are historically important sources for secondary metabolites (SMs) with applications in human medicine, animal health, and plant crop protection. It is now clear that actinomycetes and other microorganisms with large genomes have the capacity to produce many more SMs than was anticipated from standard fermentation studies. Indeed ~90% of SM gene clusters (SMGCs) predicted from genome sequencing are cryptic under conventional fermentation and analytical analyses. Previous studies have suggested that among the actinomycetes with large genomes, some have the coding capacity to produce many more SMs than others, and that strains with the largest genomes tend to be the most gifted. These contentions have been evaluated more quantitatively by antiSMASH 3.0 analyses of microbial genomes, and the results indicate that many actinomycetes with large genomes are gifted for SM production, encoding 20-50 SMGCs, and devoting 0.8-3.0Mb of coding capacity to SM production. Several Proteobacteria and Firmacutes with large genomes encode 20-30 SMGCs and devote 0.8-1.3Mb of DNA to SM production, whereas cultured bacteria and archaea with small genomes devote insignificant coding capacity to SM production. Fully sequenced genomes of uncultured bacteria and archaea have small genomes nearly devoid of SMGCs.

Centromere Structure and Function.

The centromere is the genetic locus that specifies the site of kinetochore assembly, where the chromosome will attach to the kinetochore microtubule. The pericentromere is the physical region responsible for the geometry of bi-oriented sister kinetochores in metaphase. In budding yeast the 125bp point centromere is sufficient to specify kinetochore assembly. The flanking region is enriched (3X) in cohesin and condensin relative to the remaining chromosome arms. The enrichment spans about 30-50kb around each centromere. We refer to the flanking chromatin as the pericentromere in yeast. In mammals, a 5-10Mb region dictates where the kinetochore is built. The kinetochore interacts with a very small fraction of DNA on the surface of the centromeric region. The remainder of the centromere lies between the sister kinetochores. This is typically called centromere chromatin. The chromatin sites that directly interface to microtubules cannot be identified due to the repeated sequence within the mammalian centromere. However in both yeast and mammals, the total amount of DNA between the sites of microtubule attachment in metaphase is highly conserved. In yeast the 16 chromosomes are clustered into a 250nm diameter region, and 800kb (16×50kb) or~1Mb of DNA lies between sister kinetochores. In mammals, 5-10Mb lies between sister kinetochores. In both organisms the sister kinetochores are separated by about 1μm. Thus, centromeres of different organisms differ in how they specify kinetochore assembly, but there may be important centromere chromatin functions that are conserved throughout phylogeny. Recently, centromeric chromatin has been reconstituted in vitro using alpha satellite DNA revealing unexpected features of centromeric DNA organization, replication, and response to stress. We will focus on the conserved features of centromere in this review.

Integrative analysis of DNA copy number, DNA methylation and gene expression in multiple myeloma reveals alterations related to relapse.

Multiple myeloma (MM) remains incurable despite the introduction of novel agents, and a relapsing course is observed in most patients. Although the development of genomic technologies has greatly improved our understanding of MM pathogenesis, the mechanisms underlying relapse have been less thoroughly investigated. In this study, an integrative analysis of DNA copy number, DNA methylation and gene expression was conducted in matched diagnosis and relapse samples from MM patients. Overall, the acquisition of abnormalities at relapse was much more frequent than the loss of lesions present at diagnosis, and DNA losses were significantly more frequent in relapse than in diagnosis samples. Interestingly, copy number abnormalities involving more than 100 Mb of DNA at relapse significantly affect the gene expression of these samples, provoking a particular deregulation of the IL-8 pathway. On the other hand, no significant modifications of gene expression were observed in those samples with less than 100 Mb affected by chromosomal changes. Although several statistical approaches were used to identify genes whose abnormal expression at relapse was regulated by methylation, only two genes that were significantly deregulated in relapse samples (SORL1 and GLT1D1) showed a negative correlation between methylation and expression. Further analysis revealed that DNA methylation was involved in regulating SORL1 expression in MM. Finally, relevant changes in gene expression observed in relapse samples, such us downregulation of CD27 and P2RY8, were most likely not preceded by alterations in the corresponding DNA. Taken together, these results suggest that the genomic heterogeneity described at diagnosis remains at relapse.

Characterization of Somatically-Eliminated Genes During Development of the Sea Lamprey (Petromyzon marinus).

The sea lamprey (Petromyzon marinus) is a basal vertebrate that undergoes developmentally programmed genome rearrangements (PGRs) during early development. These events facilitate the elimination of ∼20% of the genome from the somatic cell lineage, resulting in distinct somatic and germline genomes. Thus far only a handful of germline-specific genes have been definitively identified within the estimated 500 Mb of DNA that is deleted during PGR, although a few thousand germline-specific genes are thought to exist. To improve our understanding of the evolutionary/developmental logic of PGR, we generated computational predictions to identify candidate germline-specific genes within a new transcriptomic dataset derived from adult germline and the early embryonic stages during which PGR occurs. Follow-up validation studies identified 44 germline-specific genes and further characterized patterns of transcription and DNA loss during early embryogenesis. Expression analyses reveal that many of these genes are differentially expressed during early embryogenesis and presumably function in the early development of the germline. Ontology analyses indicate that many of these germline-specific genes play known roles in germline development, pluripotency, and oncogenesis (when misexpressed). These studies provide support for the theory that PGR serves to segregate molecular functions related to germline development/pluripotency in order to prevent their potential misexpression in somatic cells. This larger set of eliminated genes also allows us to extend the evolutionary/developmental breadth of this theory, as some deleted genes (or their gnathostome homologs) appear to be associated with the early development of somatic lineages, perhaps through the evolution of novel functions within gnathostome lineages.

Nucleosome eviction in mitosis assists condensin loading and chromosome condensation.

Condensins associate with DNA and shape mitotic chromosomes. Condensins are enriched nearby highly expressed genes during mitosis, but how this binding is achieved and what features associated with transcription attract condensins remain unclear. Here, we report that condensin accumulates at or in the immediate vicinity of nucleosome-depleted regions during fission yeast mitosis. Two transcriptional coactivators, the Gcn5 histone acetyltransferase and the RSC chromatin-remodelling complex, bind to promoters adjoining condensin-binding sites and locally evict nucleosomes to facilitate condensin binding and allow efficient mitotic chromosome condensation. The function of Gcn5 is closely linked to condensin positioning, since neither the localization of topoisomerase II nor that of the cohesin loader Mis4 is altered in gcn5 mutant cells. We propose that nucleosomes act as a barrier for the initial binding of condensin and that nucleosome-depleted regions formed at highly expressed genes by transcriptional coactivators constitute access points into chromosomes where condensin binds free genomic DNA.

Karyotype features of trematode Himasthla elongata.

BackgroundTrematodes have a complex life cycle with animal host changes and alternation of parthenogenetic and hermaphrodite generations. The parthenogenetic generation of the worm (rediae) from the first intermediate host Littorina littorea was used for chromosome spreads production. Karyotype description of parasitic flatworm Himasthla elongata Mehlis, 1831 (Digenea: Himasthlidae) based on fluorochrome banding and 18S rDNA mapping.ResultsChromosome spreads were obtained from cercariae embryos and redial tissue suspensions with high pressure squash method.74.4 % of the analysed spreads contained 12 chromosome pairs (2n = 24). Chromosome classification was performed according to the morphometry and nomenclature published. H. elongata spread chromosomes had a rather bead-like structure. Ideograms of DAPI-banded chromosomes contained 130 individual bands. According to flow cytometry data, the H. elongata genome contains 1.25 pg of DNA, so one band contains, on average, 9.4 Mb of DNA. Image bank captures of individual high-resolution DAPI-banded chromosomes were provided. Differential DAPI- and CMA3-staining revealed the chromatin areas that differed in AT- or GC-content. Both dyes stained chromosomes all along but with varying intensities in different areas. FISH revealed that vast majority (95.0 %) of interphase nuclei contained one signal for 18S rDNA. This corresponded to the number of nucleoli per cell detected by observations in vivo. The rDNA signal was observed on one or two homologs of chromosome 10 in 72.2 % of analysed chromosome spreads, therefore chromosome 10 possessed the main rDNA cluster and minor ones on chromosomes 3 and 6, that corresponds with AgNOR results.ConclusionsHimasthla elongata chromosomes variations presented as image bank. Differential chromosome staining with fluorochromes and FISH used for 18S rDNA mapping let us to conclude: (1) Himasthla elongata karyotype is 2n = 24; (2) chromosome number deviates from the previously studied echinostomatids (2n = 14–22); (3). Chromosome 10 possesses the main rDNA cluster with the minor ones existing on chromosomes 3 and 6.Electronic supplementary materialThe online version of this article (doi:10.1186/s13039-016-0246-8) contains supplementary material, which is available to authorized users.

A Nonsense Variant in COL6A1 in Landseer Dogs with Muscular Dystrophy.

A novel canine muscular dystrophy in Landseer dogs was observed. We had access to five affected dogs from two litters. The clinical signs started at a few weeks of age, and the severe progressive muscle weakness led to euthanasia between 5 and 15 months of age. The pedigrees of the affected dogs suggested a monogenic autosomal-recessive inheritance of the trait. Linkage and homozygosity mapping indicated two potential genome segments for the causative variant on chromosomes 10 and 31 harboring a total of 4.8 Mb of DNA or 0.2% of the canine genome. Using the Illumina sequencing technology, we obtained a whole-genome sequence from one affected Landseer. Variants were called with respect to the dog reference genome and compared with the genetic variants of 170 control dogs from other breeds. The affected Landseer dog was homozygous for a single, private nonsynonymous variant in the critical intervals, a nonsense variant in the COL6A1 gene (Chr31:39,303,964G>T; COL6A1:c.289G>T; p.E97*). Genotypes at this variant showed perfect concordance with the muscular dystrophy phenotype in all five cases and more than 1000 control dogs. Variants in the human COL6A1 gene cause Bethlem myopathy or Ullrich congenital muscular dystrophy. We therefore conclude that the identified canine COL6A1 variant is most likely causative for the observed muscular dystrophy in Landseer dogs. On the basis of the nature of the genetic variant in Landseer dogs and their severe clinical phenotype these dogs represent a model for human Ullrich congenital muscular dystrophy.

MG-134 Validation of a customizable next generation sequencing panel for ear and eye diseases

<h3>Background</h3> Hearing and/or vision loss can result from both genetic and non-genetic etiologies. Up to 70% of prelingual hearing loss has a genetic basis, as does up to 60% of congenital blindness among infants. Next generation sequencing (NGS) technology is ideal for diagnostic testing for such disorders due to extreme locus heterogeneity and phenotypic overlap. <h3>Objectives</h3> The aim is to design and validate a comprehensive, customizable NGS panel for detection of pathogenic variants in genes involved in hearing and/or vision loss. <h3>Design/method</h3> 380 genes were selected for inclusion based on literature review and comparison with commercially available assays, including 245 genes for vision loss and 83 genes for hearing loss. Agilent SureSelect biotinylated RNA baits were designed to capture 1.5 Mb of DNA using SureSelect XT target enrichment followed by Illumina HiSeq 2500 sequencing. DNA from six HapMap cell lines and five Coriell repository samples with known pathogenic variants were used to assess intra-run and inter-run variability. Additionally, DNA from a family with a known hearing loss aetiology was included. <h3>Results</h3> An average yield of 6 Gb of sequence per sample was obtained, where 94% of bases were covered at a depth &gt;20X. Genotype calls from HapMap samples were highly concordant across three runs. Eight known pathogenic variants were confirmed in <i>GJB2, USH1C, MARVELD2, TRIOBP, and OAT.</i> <h3>Conclusions</h3> This NGS panel targets an extensive list of genes implicated in diseases of the ear and the eye. Customizable testing allows ordering of an ear-specific or eye-specific gene panel, or both.