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Background Addressing the laborious nature of traditional biological experiments by using an efficient computational approach to analyze RNA-binding proteins (RBPs) binding sites has always been a challenging task. RBPs play a vital role in post-transcriptional control. Identification of RBPs binding sites is a key step for the anatomy of the essential mechanism of gene regulation by controlling splicing, stability, localization and translation. Traditional methods for detecting RBPs binding sites are time-consuming and computationally-intensive. Recently, the computational method has been incorporated in researches of RBPs. Nevertheless, lots of them not only rely on the sequence data of RNA but also need additional data, for example the secondary structural data of RNA, to improve the performance of prediction, which needs the pre-work to prepare the learnable representation of structural data. Results To reduce the dependency of those pre-work, in this paper, we introduce DeepPN, a deep parallel neural network that is constructed with a convolutional neural network (CNN) and graph convolutional network (GCN) for detecting RBPs binding sites. It includes a two-layer CNN and GCN in parallel to extract the hidden features, followed by a fully connected layer to make the prediction. DeepPN discriminates the RBP binding sites on learnable representation of RNA sequences, which only uses the sequence data without using other data, for example the secondary or tertiary structure data of RNA. DeepPN is evaluated on 24 datasets of RBPs binding sites with other state-of-the-art methods. The results show that the performance of DeepPN is comparable to the published methods. Conclusion The experimental results show that DeepPN can effectively capture potential hidden features in RBPs and use these features for effective prediction of binding sites.

Rapid, cost-effective identification of genetic variants in small candidate genomic regions remains a challenge, particularly for less well equipped or lower throughput laboratories. The application of Oxford Nanopore Technologies’ MinION sequencer has the potential to fulfil this requirement. We demonstrate a proof of concept for a multiplexing assay that pools PCR amplicons for MinION sequencing to enable sequencing of multiple templates from multiple individuals, which could be applied to gene-targeted diagnostics. A combined strategy of barcoding and sample pooling was developed for simultaneous multiplex MinION sequencing of 100 PCR amplicons. The amplicons are family-specific, spanning a total of 30 loci in DNA isolated from 82 human neurodevelopmental cases and family members. The target regions were chosen for further interrogation because a potentially disease-causative variant had been identified in affected individuals following Illumina exome sequencing. The pooled MinION sequences were deconvoluted by aligning to custom references using the minimap2 aligner software. Our multiplexing approach produced an interpretable and expected sequence from 29 of the 30 targeted genetic loci. The sequence variant which was not correctly resolved in the MinION sequence was adjacent to a five nucleotide homopolymer. It is already known that homopolymers present a resolution problem with the MinION approach. Interestingly despite equimolar quantities of PCR amplicon pooled for sequencing, significant variation in the depth of coverage (127×–19,626×; mean = 8321×, std err = 452.99) was observed. We observed independent relationships between depth of coverage and target length, and depth of coverage and GC content. These relationships demonstrate biases of the MinION sequencer for longer templates and those with lower GC content. We demonstrate an efficient approach for variant discovery or confirmation from short DNA templates using the MinION sequencing device. With less than 130 × depth of coverage required for accurate genotyping, the methodology described here allows for rapid highly multiplexed targeted sequencing of large numbers of samples in a minimally equipped laboratory with a potential cost as much 200 × less than that from Sanger sequencing.

The phenotypic heterogeneity may be the result of variable penetrance and expressivity, epigenetic influences or epistasis caused by gene-gene interactions. Because of genetic and phenotypic heterogeneity, there has been understandable reluctance to routinely sequence CVID patients because of the low yield (25). Given the rapid progress in the understanding of these conditions in recent years, we believe there is now a strong case for routine diagnostic genetic testing of patients with a CVID phenotype (Table 1). Establishing the diagnosis Confirming the clinical diagnosis of a CVID-like disorder Identifying novel presentations of other CVID-like disorders eg as LOCID Identifying atypical presentations of other PIDs with hypogammaglobulinemia eg XLP Distinguishing genetic from acquired disorders eg drug-induced hypogammaglobulinemiaIdentifying digenic disordersTHA-Variability of IgG levels over time: some of these patients may have CVID-like disordersDifferences in diagnostic criteria for CVID: the presence of a CVID-like disorder will obviate the need to apply CVID diagnostic criteria.Identifying CVID-like disorders in patients who have already developed malignancyIdentifying CVID-like disorders in patients on SCIG/IVIG or immunosuppression Treatment Offering early SCIG/IVIG treatment for individuals carrying causative mutationsIdentifying specific treatment options eg abatacept for CTLA-4/LRBA deficiency Identifying patients who may benefit from gene based therapy in the future Prognosis Asymptomatic patients with monogenic defects have a high probability of symptomatic disease, leading to long-term SCIG/IVIG treatmentMay distinguish patients with THI, who may not recover till adulthood where some have impaired vaccine responses Pre-symptomatic testing Where presymptomatic diagnosis (at any age) is not possible with protein based tests eg patients with CVID-like disorders who are asymptomatic with normal immunoglobulins Diagnosis in infancy where conventional diagnostic tests are unreliable eg because of transplacentally acquired IgG levels Screening Cascade screening of at-risk relatives with or without symptoms after genetic counselingIdentifying mutations from tissue samples from deceased relativesIdentifying mutations from Guthrie cards from deceased relatives PID prevention Prenatal diagnosis with chorionic villus sampling (CVS) Pre-implantation genetic diagnosis (PGD) Research Characterizing the role of molecules in cellular function Assisting with the classification of primary immunodeficiency disorders Identification of new genetic defects with trio analysisInvestigating animal models of CVID-like disordersIdentifying epistasis caused by digenic (or oligogenic) disorders Most of the clinical scenarios are described in the text.

Background A large proportion of pregnancy losses occur during the pre-implantation period, when the developing embryo is elongating rapidly and signalling its presence to the maternal system. The molecular mechanisms that prevent luteolysis and support embryo survival within the maternal environment are not well understood. To gain a more complete picture of these molecular events, genome-wide transcriptional profiles of reproductive day 17 endometrial tissue were determined in pregnant and cyclic Holstein-Friesian dairy cattle. Results Microarray analyses revealed 1,839 and 1,189 differentially expressed transcripts between pregnant and cyclic animals (with ≥ 1.5 fold change in expression; P-value < 0.05, MTC Benjamini-Hochberg) in caruncular and intercaruncular endometrium respectively. Gene ontology and biological pathway analysis of differentially expressed genes revealed enrichment for genes involved in interferon signalling and modulation of the immune response in pregnant animals. Conclusion The maternal immune system actively surveys the uterine environment during early pregnancy. The embryo modulates this response inducing the expression of endometrial molecules that suppress the immune response and promote maternal tolerance to the embryo. During this period of local immune suppression, genes of the innate immune response (in particular, antimicrobial genes) may function to protect the uterus against infection.

[...]these variants do not segregate with disease in family studies (11). [...]the epistatic role of these variants was directly shown in a family carrying mutations of both TNFSF13B/TACI (C104R, c.310TC) variant and a nonsense mutation of TCF3 (T168fsX191) (12). Epistasis in laboratory animals can be explored by inducing mutation by techniques such as gene editing and selective breeding. Conflict of interest The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

The existence of epistasis in humans was first predicted by Bateson in 1909. Epistasis describes the non-linear, synergistic interaction of two or more genetic loci, which can substantially modify disease severity or result in entirely new phenotypes. The concept has remained controversial in human genetics because of the lack of well-characterized examples. In humans, it is only possible to demonstrate epistasis if two or more genes are mutated. In most cases of epistasis, the mutated gene products are likely to be constituents of the same physiological pathway leading to severe disruption of a cellular function such as antibody production. We have recently described a digenic family, who carry mutations of /TACI as well as genes. Both genes lie in tandem along the immunoglobulin isotype switching and secretion pathway. We have shown they interact in an epistatic way causing severe immunodeficiency and autoimmunity in the digenic proband. With the advent of next generation sequencing, it is likely other families with digenic inheritance will be identified. Since digenic inheritance does not always cause epistasis, we propose an epistasis index which may help quantify the effects of the two mutations. We also discuss the clinical implications of digenic inheritance and epistasis in humans with primary immunodeficiency disorders.

Common variable immunodeficiency disorders (CVID) are an enigmatic group of often heritable conditions, which may manifest for the first time in early childhood or as late as the eighth decade of life. In the last 5 years, next generation sequencing (NGS) has revolutionised identification of genetic disorders. However, despite the best efforts of researchers around the globe, CVID conditions have been slow to yield their molecular secrets. We have previously described the many clinical advantages of identifying the genetic basis of primary immunodeficiency disorders (PIDs). In a minority of CVID patients, monogenic defects have now been identified. If a causative mutation is identified, these conditions are reclassified as CVID-like disorders. Here we discuss recent advances in the genetics of CVID and discuss how NGS can be optimally deployed to identify the causal mutations responsible for the protean clinical manifestations of these conditions. Diagnostic criteria such as the Ameratunga et al. criteria will continue to play an important role in patient management as well as case selection and sequencing strategy design until the genetic conundrum of CVID is solved.

In the last decade there has been a rapid increase in the rate of discovery of new genetic defects in primary immunodeficiency disorders (PIDs), largely due to the advent of Next Generation Sequencing (NGS) (2,3). Identification of a gain for function mutation of PIK3CD for example, may lead to specific treatments such as idelalisib in addition to subcutaneous or intravenous immunoglobulin (SCIG/IVIG) replacement. With the rapid increase in the discovery of new genetic defects, there has been a move to name these conditions inborn errors of immunity (IEI) (2). [...]only 25% patients from non-consanguineous populations have an underlying causative mutation, mostly late-onset autosomal dominant disorders (27).

Protein arginine N-methyltransferase 7 (PRMT7) encodes an arginine methyltransferase central to a number of fundamental biological processes, mutations in which result in an autosomal recessive developmental disorder characterized by short stature, brachydactyly, intellectual developmental disability and seizures (SBIDDS). To date, fewer than 15 patients with biallelic mutations in PRMT7 have been documented. Here we report brothers from a consanguineous Iraqi family presenting with a developmental disorder characterized by global developmental delay, shortened stature, facial dysmorphisms, brachydactyly, and kidney dysfunction. In both affected brothers, whole genome sequencing (WGS) identified a novel homozygous substitution in PRMT7 (ENST00000339507.5), c.1097 G > A (p.Cys366Tyr), considered to account for the majority of the phenotypic presentation. Rare compound heterozygous mutations in the dysplasia-associated perlecan-encoding HSPG2 gene (ENST00000374695.3) were also found (c.10721-2dupA, p.Ser71Asn and c.212 G > A), potentially accounting for the kidney dysfunction. In addition to expanding the known mutational spectrum of variably expressive PRMT7 mutations alongside potential digenic inheritance with HSPG2, this report underlines the diagnostic utility of a WGS-guided analysis in the detection of rare genetic disorders.

Background Segmental axonopathy is a recessively inherited neurodegenerative disorder that has affected Merino sheep since the early 1930s. Despite its long-standing recognition, the genetic basis of the condition remained unknown. This study aimed to identify the genetic cause of segmental axonopathy and confirm its pathological features to improve diagnostic accuracy and inform breeding strategies. Results Whole genome sequencing and genotyping of affected and unaffected Merino sheep identified a novel homozygous frameshift variant in the ALS2 gene that segregated with disease. RNA sequencing of cerebellar peduncle tissue confirmed the nonsense consequence on the ALS2 transcript. Histological analysis highlighted the hallmarks of the disease as large, foamy eosinophilic axonal swellings predominantly in the trigeminal ganglia, with additional degenerative changes in both the brain and spinal cord. These findings support the value of targeted sampling of sensory roots of the trigeminal nerve, spinal cord tracts, and dorsal nerve rootlets to enhance diagnostic accuracy. The same ALS2 variant was found across multiple unrelated flocks in both Australia and New Zealand, indicating a broader presence within the fine-wool Merino sheep population. Conclusions This study identifies a novel ALS2 frameshift variant associated with segmental axonopathy in Merino sheep and provides both genetic and histological evidence supporting its role in disease pathology. The development of a DNA diagnostic test will enable more informed breeding decisions, reduce the prevalence of this condition, and improve animal welfare and productivity in the Merino industry. Moreover, the findings offer a potential large-animal model for exploring early-onset forms of human motor neuron diseases, including amyotrophic lateral sclerosis, in which ALS2 variants are implicated.