De Novo Variants in TAOK1 Cause Neurodevelopmental Disorders.

De novo variants represent a significant cause of neurodevelopmental delay and intellectual disability. A genetic basis can be identified in only half of individuals who have neurodevelopmental disorders (NDDs); this indicates that additional causes need to be elucidated. We compared the frequency of de novo variants in patient-parent trios with (n = 2,030) versus without (n = 2,755) NDDs. We identified de novo variants in TAOK1 (thousand and one [TAO] amino acid kinase 1), which encodes the serine/threonine-protein kinase TAO1, in three individuals with NDDs but not in persons who did not have NDDs. Through further screening and the use of GeneMatcher, five additional individuals with NDDs were found to have de novo variants. All eight variants were absent from gnomAD (Genome Aggregation Database). The variant carriers shared a non-specific phenotype of developmental delay, and six individuals had additional muscular hypotonia. We established a fibroblast line of one mutation carrier, and we demonstrated that reduced mRNA levels of TAOK1 could be increased upon cycloheximide treatment. These results indicate nonsense-mediated mRNA decay. Further, there was neither detectable phosphorylated TAO1 kinase nor phosphorylated tau in these cells, and mitochondrial morphology was altered. Knockdown of the ortholog gene Tao1 (Tao, CG14217) in Drosophila resulted in delayed early development. The majority of the Tao1-knockdown flies did not survive beyond the third instar larval stage. When compared to control flies, Tao1 knockdown flies revealed changed morphology of the ventral nerve cord and the neuromuscular junctions as well as a decreased number of endings (boutons). Furthermore, mitochondria in mutant flies showed altered distribution and decreased size in axons of motor neurons. Thus, we provide compelling evidence that de novo variants in TAOK1 cause NDDs.

Novel pathogenic variants and multiple molecular diagnoses in neurodevelopmental disorders.

BACKGROUND: Rare denovo variants represent a significant cause of neurodevelopmental delay and intellectual disability (ID). METHODS: Exome sequencing was performed on 4351 patients with global developmental delay, seizures, microcephaly, macrocephaly, motor delay, delayed speech and language development, or ID according to Human Phenotype Ontology (HPO) terms. All patients had previously undergone whole exome sequencing as part of diagnostic genetic testing with a focus on variants in genes implicated in neurodevelopmental disorders up to January 2017. This resulted in a genetic diagnosis in 1336 of the patients. In this study, we specifically searched for variants in 14 recently implicated novel neurodevelopmental disorder (NDD) genes. RESULTS: We identified 65 rare, protein-changing variants in 11 of these 14 novel candidate genes. Fourteen variants in CDK13, CHD4, KCNQ3, KMT5B, TCF20, and ZBTB18 were scored pathogenic or likely pathogenic. Of note, two of these patients had a previously identified cause of their disease, and thus, multiple molecular diagnoses were made including pathogenic/likely pathogenic variants in FOXG1 and CDK13 or in TMEM237 and KMT5B. CONCLUSIONS: Looking for pathogenic variants in newly identified NDD genes enabled us to provide a molecular diagnosis to 14 patients and their close relatives and caregivers. This underlines the relevance of re-evaluation of existing exome data on a regular basis to improve the diagnostic yield and serve the needs of our patients.

MINA-1 and WAGO-4 are part of regulatory network coordinating germ cell death and RNAi in C. elegans.

Post-transcriptional control of mRNAs by RNA-binding proteins (RBPs) has a prominent role in the regulation of gene expression. RBPs interact with mRNAs to control their biogenesis, splicing, transport, localization, translation, and stability. Defects in such regulation can lead to a wide range of human diseases from neurological disorders to cancer. Many RBPs are conserved between Caenorhabditis elegans and humans, and several are known to regulate apoptosis in the adult C. elegans germ line. How these RBPs control apoptosis is, however, largely unknown. Here, we identify mina-1(C41G7.3) in a RNA interference-based screen as a novel regulator of apoptosis, which is exclusively expressed in the adult germ line. The absence of MINA-1 causes a dramatic increase in germ cell apoptosis, a reduction in brood size, and an impaired P granules organization and structure. In vivo crosslinking immunoprecipitation experiments revealed that MINA-1 binds a set of mRNAs coding for RBPs associated with germ cell development. Additionally, a system-wide analysis of a mina-1 deletion mutant compared with wild type, including quantitative proteome and transcriptome data, hints to a post-transcriptional regulatory RBP network driven by MINA-1 during germ cell development in C. elegans. In particular, we found that the germline-specific Argonaute WAGO-4 protein levels are increased in mina-1 mutant background. Phenotypic analysis of double mutant mina-1;wago-4 revealed that contemporary loss of MINA-1 and WAGO-4 strongly rescues the phenotypes observed in mina-1 mutant background. To strengthen this functional interaction, we found that upregulation of WAGO-4 in mina-1 mutant animals causes hypersensitivity to exogenous RNAi. Our comprehensive experimental approach allowed us to describe a phenocritical interaction between two RBPs controlling germ cell apoptosis and exogenous RNAi. These findings broaden our understanding of how RBPs can orchestrate different cellular events such as differentiation and death in C. elegans.

Development of an evidence-based algorithm that optimizes sensitivity and specificity in ES-based diagnostics of a clinically heterogeneous patient population.

PURPOSE: Next-generation sequencing (NGS) is rapidly replacing Sanger sequencing in genetic diagnostics. Sensitivity and specificity of NGS approaches are not well-defined, but can be estimated from applying NGS and Sanger sequencing in parallel. Utilizing this strategy, we aimed at optimizing exome sequencing (ES)-based diagnostics of a clinically diverse patient population. METHODS: Consecutive DNA samples from unrelated patients with suspected genetic disease were exome-sequenced; comparatively nonstringent criteria were applied in variant calling. One thousand forty-eight variants in genes compatible with the clinical diagnosis were followed up by Sanger sequencing. Based on a set of variant-specific features, predictors for true positives and true negatives were developed. RESULTS: Sanger sequencing confirmed 81.9% of ES-derived variants. Calls from the lower end of stringency accounted for the majority of the false positives, but also contained ~5% of the true positives. A predictor incorporating three variant-specific features classified 91.7% of variants with 100% specificity and 99.75% sensitivity. Confirmation status of the remaining variants (8.3%) was not predictable. CONCLUSIONS: Criteria for variant calling in ES-based diagnostics impact on specificity and sensitivity. Confirmatory sequencing for a proportion of variants, therefore, remains a necessity. Our study exemplifies how these variants can be defined on an empirical basis.

Clinical Exome Sequencing unravels new disease-causing mutations in the myeloproliferative neoplasms: A pilot study in patients from the state of Qatar.

Clinical Exome Sequencing (CES) has increasingly become a popular diagnostic tool in patients suffering from genetic disorders that are clinically and genetically complicated. Myeloproliferative Neoplasms (MPNs) is an example of a heterogeneous disorder. In Qatar, familial cases of MPNs are more frequently seen than described in the literature. In this study, we aimed to use CES to classify six Qatari subjects that were suspected of clinical diagnosis of MPNs, according to the WHO 2008 diagnostic criteria for hematologic malignancies, and identify variants that can potentially explain the phenotypic diversity of MPNs. We sequenced six Qatari subjects using CES, of whom, three probands were unrelated families and three members were from the same family, all probands come from consanguineous families, and had a positive family history of MPNs. CES identified 61 variants in 50 genes; of which, 13 were recurrently mutated in our patients. Ten novel variants were identified in ten known genes related to MPNs and seven variants were identified in seven novel candidate genes. The genotype of the six subjects was due to a combination of different variants in different genes. This study serves as a pilot study to investigate the complexity of the genotype of patients with MPNS in Qatar, and serves as a guide for further well-controlled genetic epidemiological studies for patients with MPNs. CES is a powerful tool to be used in the genetic clinics for differential and definitive diagnosis of patients with MPNs.

17q23.2q23.3 de novo duplication in association with speech and language disorder, learning difficulties, incoordination, motor skill impairment, and behavioral disturbances: a case report.

BACKGROUND: Chromosomal rearrangements involving 17q23 have been described rarely. Deletions at 17q23.1q23.2 have been reported in individuals with developmental delay and growth retardation, whereas duplications at 17q23.1q23.2 appear to segregate with clubfoot. Dosage alterations in the TBX2 and TBX4 genes, located in 17q23.2, have been proposed to be responsible for the phenotypes observed in individuals with 17q23.1q23.2 deletions and duplications. In this report, we present the clinical phenotype of a child with a previously unreported de novo duplication at 17q23.2q23.3 located distal to the TBX2 and TBX4 region. CASE PRESENTATION: We report a 7.5-year-old boy with speech and language disorder, learning difficulties, incoordination, fine motor skill impairment, infrequent seizures with abnormal EEG, and behavior disturbances (mild self-inflicted injuries, hyperactivity-inattention, and stereotyped hand movements). Chromosomal microarray revealed a 2-Mb duplication of chromosome 17q23.2q23.3. Both parents did not have the duplication indicating that this duplication is de novo in the child. CONCLUSIONS: The duplicated region encompasses 16 genes. It is possible that increased dosage of one or more genes in this region is responsible for the observed phenotype. The TANC2 gene is one of the genes in the duplicated region.It encodes a member of the TANC (tetratricopeptide repeat, ankyrin repeat and coiled-coil containing) family which includes TANC1 and TANC2. These proteins are highly expressed in brain and play major roles in synapsis regulation. Hence, it is suggestive that TANC2 is the likely candidate gene responsible for the observed phenotype as an increased TANC2 dosage can potentially alter synapsis, resulting in neuronal dysfunction and the neurobehavioral phenotype observed in this child with 17q23.2q23.3 duplication.

Clinical exome sequencing: results from 2819 samples reflecting 1000 families.

We report our results of 1000 diagnostic WES cases based on 2819 sequenced samples from 54 countries with a wide phenotypic spectrum. Clinical information given by the requesting physicians was translated to HPO terms. WES processes were performed according to standardized settings. We identified the underlying pathogenic or likely pathogenic variants in 307 families (30.7%). In further 253 families (25.3%) a variant of unknown significance, possibly explaining the clinical symptoms of the index patient was identified. WES enabled timely diagnosing of genetic diseases, validation of causality of specific genetic disorders of PTPN23, KCTD3, SCN3A, PPOX, FRMPD4, and SCN1B, and setting dual diagnoses by detecting two causative variants in distinct genes in the same patient. We observed a better diagnostic yield in consanguineous families, in severe and in syndromic phenotypes. Our results suggest that WES has a better yield in patients that present with several symptoms, rather than an isolated abnormality. We also validate the clinical benefit of WES as an effective diagnostic tool, particularly in nonspecific or heterogeneous phenotypes. We recommend WES as a first-line diagnostic in all cases without a clear differential diagnosis, to facilitate personal medical care.

HIV-1 RNAs are Not Part of the Argonaute 2 Associated RNA Interference Pathway in Macrophages.

BACKGROUND: MiRNAs and other small noncoding RNAs (sncRNAs) are key players in post-transcriptional gene regulation. HIV-1 derived small noncoding RNAs (sncRNAs) have been described in HIV-1 infected cells, but their biological functions still remain to be elucidated. Here, we approached the question whether viral sncRNAs may play a role in the RNA interference (RNAi) pathway or whether viral mRNAs are targeted by cellular miRNAs in human monocyte derived macrophages (MDM). METHODS: The incorporation of viral sncRNAs and/or their target RNAs into RNA-induced silencing complex was investigated using photoactivatable ribonucleoside-induced cross-linking and immunoprecipitation (PAR-CLIP) as well as high-throughput sequencing of RNA isolated by cross-linking immunoprecipitation (HITS-CLIP), which capture Argonaute2-bound miRNAs and their target RNAs. HIV-1 infected monocyte-derived macrophages (MDM) were chosen as target cells, as they have previously been shown to express HIV-1 sncRNAs. In addition, we applied small RNA deep sequencing to study differential cellular miRNA expression in HIV-1 infected versus non-infected MDMs. RESULTS AND CONCLUSION: PAR-CLIP and HITS-CLIP data demonstrated the absence of HIV-1 RNAs in Ago2-RISC, although the presence of a multitude of HIV-1 sncRNAs in HIV-1 infected MDMs was confirmed by small RNA sequencing. Small RNA sequencing revealed that 1.4% of all sncRNAs were of HIV-1 origin. However, neither HIV-1 derived sncRNAs nor putative HIV-1 target sequences incorporated into Ago2-RISC were identified suggesting that HIV-1 sncRNAs are not involved in the canonical RNAi pathway nor is HIV-1 targeted by this pathway in HIV-1 infected macrophages.

miR-CLIP capture of a miRNA targetome uncovers a lincRNA H19-miR-106a interaction.

Identifying the interaction partners of noncoding RNAs is essential for elucidating their functions. We have developed an approach, termed microRNA crosslinking and immunoprecipitation (miR-CLIP), using pre-miRNAs modified with psoralen and biotin to capture their targets in cells. Photo-crosslinking and Argonaute 2 immunopurification followed by streptavidin affinity purification of probe-linked RNAs provided selectivity in the capture of targets, which were identified by deep sequencing. miR-CLIP with pre-miR-106a, a miR-17-5p family member, identified hundreds of putative targets in HeLa cells, many carrying conserved sequences complementary to the miRNA seed but also many that were not predicted computationally. miR-106a overexpression experiments confirmed that miR-CLIP captured functional targets, including H19, a long noncoding RNA that is expressed during skeletal muscle cell differentiation. We showed that miR-17-5p family members bind H19 in HeLa cells and myoblasts. During myoblast differentiation, levels of H19, miR-17-5p family members and mRNA targets changed in a manner suggesting that H19 acts as a 'sponge' for these miRNAs.

3'-UTR poly(T/U) tract deletions and altered expression of EWSR1 are a hallmark of mismatch repair-deficient cancers.

The genome-wide accumulation of DNA replication errors known as microsatellite instability (MSI) is the hallmark lesion of DNA mismatch repair (MMR)-deficient cancers. Although testing for MSI is widely used to guide clinical management, the contribution of MSI at distinct genic loci to the phenotype remains largely unexplored. Here, we report that a mononucleotide (T/U)16 tract located in the 3' untranslated region (3'-UTR) of the Ewing sarcoma breakpoint region 1 (EWSR1) gene is a novel MSI target locus that shows perfect sensitivity and specificity in detecting mismatch repair-deficient cancers in two independent populations. We further found a striking relocalization of the EWSR1 protein from nucleus to cytoplasm in MMR-deficient cancers and that the nonprotein-coding MSI target locus itself has a modulatory effect on EWSR1 gene expression through alternative 3' end processing of the EWSR1 gene. Our results point to a MSI target gene-specific effect in MMR-deficient cancers.

Modeling the binding specificity of the RNA-binding protein GLD-1 suggests a function of coding region-located sites in translational repression.

To understand the function of the hundreds of RNA-binding proteins (RBPs) that are encoded in animal genomes it is important to identify their target RNAs. Although it is generally accepted that the binding specificity of an RBP is well described in terms of the nucleotide sequence of its binding sites, other factors such as the structural accessibility of binding sites or their clustering, to enable binding of RBP multimers, are also believed to play a role. Here we focus on GLD-1, a translational regulator of Caenorhabditis elegans, whose binding specificity and targets have been studied with a variety of methods such as CLIP (cross-linking and immunoprecipitation), RIP-Chip (microarray measurement of RNAs associated with an immunoprecipitated protein), profiling of polysome-associated mRNAs and biophysical determination of binding affinities of GLD-1 for short nucleotide sequences. We show that a simple biophysical model explains the binding of GLD-1 to mRNA targets to a large extent, and that taking into account the accessibility of putative target sites significantly improves the prediction of GLD-1 binding, particularly due to a more accurate prediction of binding in transcript coding regions. Relating GLD-1 binding to translational repression and stabilization of its target transcripts we find that binding sites along the entire transcripts contribute to functional responses, and that CDS-located sites contribute most to translational repression. Finally, biophysical measurements of GLD-1 affinity for a small number of oligonucleotides appear to allow an accurate reconstruction of the sequence specificity of the protein. This approach can be applied to uncover the specificity and function of other RBPs.

Insights into snoRNA biogenesis and processing from PAR-CLIP of snoRNA core proteins and small RNA sequencing.

BACKGROUND: In recent years, a variety of small RNAs derived from other RNAs with well-known functions such as tRNAs and snoRNAs, have been identified. The functional relevance of these RNAs is largely unknown. To gain insight into the complexity of snoRNA processing and the functional relevance of snoRNA-derived small RNAs, we sequence long and short RNAs, small RNAs that co-precipitate with the Argonaute 2 protein and RNA fragments obtained in photoreactive nucleotide-enhanced crosslinking and immunoprecipitation (PAR-CLIP) of core snoRNA-associated proteins. RESULTS: Analysis of these data sets reveals that many loci in the human genome reproducibly give rise to C/D box-like snoRNAs, whose expression and evolutionary conservation are typically less pronounced relative to the snoRNAs that are currently cataloged. We further find that virtually all C/D box snoRNAs are specifically processed inside the regions of terminal complementarity, retaining in the mature form only 4-5 nucleotides upstream of the C box and 2-5 nucleotides downstream of the D box. Sequencing of the total and Argonaute 2-associated populations of small RNAs reveals that despite their cellular abundance, C/D box-derived small RNAs are not efficiently incorporated into the Ago2 protein. CONCLUSIONS: We conclude that the human genome encodes a large number of snoRNAs that are processed along the canonical pathway and expressed at relatively low levels. Generation of snoRNA-derived processing products with alternative, particularly miRNA-like, functions appears to be uncommon.

A quantitative analysis of CLIP methods for identifying binding sites of RNA-binding proteins.

Cross-linking and immunoprecipitation (CLIP) is increasingly used to map transcriptome-wide binding sites of RNA-binding proteins. We developed a method for CLIP data analysis, and applied it to compare CLIP with photoactivatable ribonucleoside-enhanced CLIP (PAR-CLIP) and to uncover how differences in cross-linking and ribonuclease digestion affect the identified sites. We found only small differences in accuracies of these methods in identifying binding sites of HuR, which binds low-complexity sequences, and Argonaute 2, which has a complex binding specificity. We found that cross-link-induced mutations led to single-nucleotide resolution for both PAR-CLIP and CLIP. Our results confirm the expectation from original CLIP publications that RNA-binding proteins do not protect their binding sites sufficiently under the denaturing conditions used during the CLIP procedure, and we show that extensive digestion with sequence-specific RNases strongly biases the recovered binding sites. This bias can be substantially reduced by milder nuclease digestion conditions.

Deciphering the role of RNA-binding proteins in the post-transcriptional control of gene expression.

Eukaryotic cells express a large variety of ribonucleic acid-(RNA)-binding proteins (RBPs) with diverse affinity and specificity towards target RNAs that play a crucial role in almost every aspect of RNA metabolism. In addition, specific domains in RBPs impart catalytic activity or mediate protein-protein interactions, making RBPs versatile regulators of gene expression. In this review, we elaborate on recent experimental and computational approaches that have increased our understanding of RNA-protein interactions and their role in cellular function. We review aspects of gene expression that are modulated post-transcriptionally by RBPs, namely the stability of polymerase II-derived mRNA transcripts and their rate of translation into proteins. We further highlight the extensive regulatory networks of RBPs that implement a combinatorial control of gene expression. Taking cues from the recent development in the field, we argue that understanding spatio-temporal RNA-protein association on a transcriptome level will provide invaluable and unexpected insights into the regulatory codes that define growth, differentiation and disease.

The snoRNA MBII-52 (SNORD 115) is processed into smaller RNAs and regulates alternative splicing.

The loss of HBII-52 and related C/D box small nucleolar RNA (snoRNA) expression units have been implicated as a cause for the Prader-Willi syndrome (PWS). We recently found that the C/D box snoRNA HBII-52 changes the alternative splicing of the serotonin receptor 2C pre-mRNA, which is different from the traditional C/D box snoRNA function in non-mRNA methylation. Using bioinformatic predictions and experimental verification, we identified five pre-mRNAs (DPM2, TAF1, RALGPS1, PBRM1 and CRHR1) containing alternative exons that are regulated by MBII-52, the mouse homolog of HBII-52. Analysis of a single member of the MBII-52 cluster of snoRNAs by RNase protection and northern blot analysis shows that the MBII-52 expressing unit generates shorter RNAs that originate from the full-length MBII-52 snoRNA through additional processing steps. These novel RNAs associate with hnRNPs and not with proteins associated with canonical C/D box snoRNAs. Our data indicate that not a traditional C/D box snoRNA MBII-52, but a processed version lacking the snoRNA stem is the predominant MBII-52 RNA missing in PWS. This processed snoRNA functions in alternative splice-site selection. Its substitution could be a therapeutic principle for PWS.

Rapid generation of splicing reporters with pSpliceExpress.

Almost all human protein-coding transcripts undergo pre-mRNA splicing and a majority of them is alternatively spliced. The most common technique used to analyze the regulation of an alternative exon is through reporter minigene constructs. However, their construction is time-consuming and is often complicated by the limited availability of appropriate restriction sites. Here, we report a fast and simple recombination-based method to generate splicing reporter genes, using a new vector, pSpliceExpress. The system allows generation of minigenes within one week. Minigenes generated with pSpliceExpress show the same regulation as displayed by conventionally cloned reporter constructs and provide an alternate avenue to study splice site selection in vivo.

Common variations in the IL4R gene affect splicing and influence natural expression of the soluble isoform.

We previously found the soluble interleukin 4 receptor (sIL4R) to be differently expressed in allergic asthma patients compared to healthy individuals. Here we present data demonstrating the involvement of the sequence variations, c.912-1003A > G, c.912-833T > C, c. 912-630A > G, and c.912-577A > G, in the expressional regulation of IL4R splice variants. By using an IL4R minigene construct, genomic DNA and mRNA from asthma patients and nonasthmatic individuals, we analyzed the function of four highly-linked SNPs, flanking the alternatively-spliced exon in the IL4R gene. Results from the minigene assay showed that the form containing the minor alleles significantly decreased the expression of the soluble IL4R (exon 8+) variant, a decrease that could only be seen in the major construct after increasing amounts of either the splicing factor SRp20, or YT521-B. Analysis of mRNA expression in our human material confirmed the results, demonstrating lower expression of the sIL4R in patients and controls carrying the minor alleles. Together these results show sequence variations as a possible way of altering alternative splicing selection of IL4R in vivo.

The snoRNA HBII-52 regulates alternative splicing of the serotonin receptor 2C.

The Prader-Willi syndrome is a congenital disease that is caused by the loss of paternal gene expression from a maternally imprinted region on chromosome 15. This region contains a small nucleolar RNA (snoRNA), HBII-52, that exhibits sequence complementarity to the alternatively spliced exon Vb of the serotonin receptor 5-HT(2C)R. We found that HBII-52 regulates alternative splicing of 5-HT(2C)R by binding to a silencing element in exon Vb. Prader-Willi syndrome patients do not express HBII-52. They have different 5-HT(2C)R messenger RNA (mRNA) isoforms than healthy individuals. Our results show that a snoRNA regulates the processing of an mRNA expressed from a gene located on a different chromosome, and the results indicate that a defect in pre-mRNA processing contributes to the Prader-Willi syndrome.

Identification of karyopherin beta as an immunogenic antigen of the malaria parasite using immune mice and human sera.

A differential immunoscreening of the lambdagt11 Plasmodium falciparum genomic expression library was carried out using anti-P. yoelii sera (convalescent-phase mouse sera) and immune sera collected from healthy adults, to identify novel cross-reactive and possibly protective antigens of the parasite. One clone, with an insert size of 1132 bp that reacted strongly with both the sera was selected. The insert was found to be a part of the P. falciparum karyopherin beta (PfKbeta) homologue. RT-PCR and Northern blot analysis confirmed the expression of PfKbeta in the blood stages of the parasite. The approximately 110 kDa protein was localized in the cytoplasm at the ring and trophozoite, and in the parasitophorous vacuole at the schizont stage. Two large fragments of PfKbeta representing the N- and C-terminal halves were expressed in E. coli. The recombinant proteins were highly immunogenic in mice, and also found to be the target for immune response in natural infections of Plasmodium spp. Anti-sera against the protein showed a low level of anti-parasitic activity. Immunization with recombinant PfKbeta fragments was only partially protective against a heterologous challenge infection in mice. Our results show that the parasite releases a highly immunogenic, cytoplasmic protein into the host which may not contribute to the development of protective immunity.