COVID-19-Associated Cognitive Biases on Pneumonia Differential Diagnosis.

The coronavirus disease 2019 (COVID-19) pandemic favors cognitive biases such as anchoring and availability biases. The first refers to overvaluing some of the initial information and establishing a diagnosis too early, with resistance to future adjustments. The latter happens when diagnoses more frequently considered are regarded as more common in reality. This case, in which the correct diagnosis was delayed due to these biases, highlights the need to remain aware of them as a means toward timely diagnosis and therapeutic success of pneumonia cases. An 84-year-old woman presented with a mild non-productive cough for two months and fever. She had a history of breast carcinoma treated with radiotherapy in the previous year. Computerized tomography (CT) showed extensive bilateral consolidation foci with ground-glass-opacification areas and bilateral pleural effusion, CO-RADS 3. COVID-19 with bacterial superinfection was suspected and levofloxacin was initiated. Nasopharyngeal swab polymerase chain reaction (PCR) was carried out three times, always negative for SARS-CoV-2. As the patient remained with fever and cough, the antibiotic was escalated to piperacillin/tazobactam and then to meropenem/vancomycin. She underwent bronchofibroscopy and alveolar lavage, with negative SARS-CoV-2 PCR. The re-evaluation CT scan maintained bilateral consolidations, with an aerial bronchogram. The biopsy of pulmonary consolidation allowed the diagnosis of radiation-induced organizing pneumonia. Prednisolone was initiated and achieved clinical remission and radiological improvement. This case highlights the need to remain aware of cognitive biases both when COVID-19 is suspected or ruled out and to consider other diagnoses when there is a lack of therapeutic response.

Oligodendrocyte development and myelin sheath formation are regulated by the antagonistic interaction between the Rag-Ragulator complex and TFEB.

Myelination by oligodendrocytes is critical for fast axonal conduction and for the support and survival of neurons in the central nervous system. Recent studies have emphasized that myelination is plastic and that new myelin is formed throughout life. Nonetheless, the mechanisms that regulate the number, length, and location of myelin sheaths formed by individual oligodendrocytes are incompletely understood. Previous work showed that the lysosomal transcription factor TFEB represses myelination by oligodendrocytes and that the RagA GTPase inhibits TFEB, but the step or steps of myelination in which TFEB plays a role have remained unclear. Here, we show that TFEB regulates oligodendrocyte differentiation and also controls the length of myelin sheaths formed by individual oligodendrocytes. In the dorsal spinal cord of tfeb mutants, individual oligodendrocytes produce myelin sheaths that are longer than those produced by wildtype cells. Transmission electron microscopy shows that there are more myelinated axons in the dorsal spinal cord of tfeb mutants than in wildtype animals, but no significant change in axon diameter. In contrast to tfeb mutants, oligodendrocytes in rraga mutants produce shorter myelin sheaths. The sheath length in rraga; tfeb double mutants is not significantly different from wildtype, consistent with the antagonistic interaction between RagA and TFEB. Finally, we find that the GTPase activating protein Flcn and the RagCa and RagCb GTPases are also necessary for myelination by oligodendrocytes. These findings demonstrate that TFEB coordinates myelin sheath length and number during myelin formation in the central nervous system.

Being a Young Carer in Portugal: The Impact of Caring on Adolescents' Life Satisfaction.

Caring for an ill or disabled relative can present significant challenges that may exceed the personal resources of the caregiver. Young carers (YCs) often take on this role, providing support to family members or friends, which can have far-reaching effects on various aspects of their lives. This study involved 235 adolescents, 106 YCs, and 129 non-carers (NCs), who completed questionnaires assessing life satisfaction, satisfaction with social support, family functioning, academic functioning, and caregiving activities. Tests of group differences (MANOVA and MANCOVA controlling for age) showed YCs had more caregiving activities than NCs (as expected) and, critically, significantly lower life satisfaction. Hierarchical regressions with the YCS subsample showed academic functioning, social support, and the negative impact of caregiving were associated with life satisfaction, and that the negative influence of caregiving was linked to family functioning and the quantity of caregiving activities. For NCs, academic functioning, satisfaction with social support, and family functioning were associated with life satisfaction. In conclusion, caregiving in adolescents appears to be linked to lower life satisfaction, but this effect is determined by their social support, academic functioning, and negative impact of caring, which in turn depends on their family functioning and amount of caring activities.

Peppermint essential oil (Mentha piperita L.) increases time to exhaustion in runners.

PURPOSE: This study aimed to evaluate the capacity of peppermint essential oil to improve the physical performance of runners in running protocol until exhaustion. METHODS: In a clinical, randomized, double-blind, cross-over and controlled study, fourteen male recreational runners (37.1 ± 2.0 years; 24 ± 1.1 kg/m2; 53.1 ± 1.7 mL kg min) performed two runs to exhaustion at 70% of VO2max, after intake of 500 mL of water added with 0.05 mL of peppermint essential oil (PEO) or placebo (PLA), plus 400 mL of the drink during the initial part of the exercise. Records were made of body temperature (BT), thermal sensation (TS), thermal comfort (TC), subjective perception of effort (SPE), sweat rate (SR), and urine volume and density. RESULTS: Time to exhaustion was 109.9 ± 6.9 min in PEO and 98.5 ± 6.2 min in PLA (p = 0.009; effect size: 0.826). No significant changes were observed in the values of BT, TS, TC, SPE, SR, lost body mass, and urine volume and density (p > 0.05). CONCLUSION: Peppermint essential oil added to water before and during a race significantly increases the time to exhaustion of recreational runners but without altering BT, TS, TC, or hydration status, so the mechanisms involved were not clarified in this study. BRAZILIAN REGISTRY OF CLINICAL TRIALS (REBEC): RBR-75zt25z.

Systematically characterizing the roles of E3-ligase family members in inflammatory responses with massively parallel Perturb-seq.

E3 ligases regulate key processes, but many of their roles remain unknown. Using Perturb-seq, we interrogated the function of 1,130 E3 ligases, partners and substrates in the inflammatory response in primary dendritic cells (DCs). Dozens impacted the balance of DC1, DC2, migratory DC and macrophage states and a gradient of DC maturation. Family members grouped into co-functional modules that were enriched for physical interactions and impacted specific programs through substrate transcription factors. E3s and their adaptors co-regulated the same processes, but partnered with different substrate recognition adaptors to impact distinct aspects of the DC life cycle. Genetic interactions were more prevalent within than between modules, and a deep learning model, comßVAE, predicts the outcome of new combinations by leveraging modularity. The E3 regulatory network was associated with heritable variation and aberrant gene expression in immune cells in human inflammatory diseases. Our study provides a general approach to dissect gene function.

O papel dos RNAs longos não codificantes na insuficiência cardíaca: uma revisão de literatura / The role of non-coding long RNAs in heart failure: a literature review / El papel de las RNAs largas no codificantes en la insuficiencia cardiaca: una revisión de la literatura

A Insuficiência Cardíaca (IC) é uma das principais causas de internação hospitalar no mundo e tem um elevado grau de morbidade e mortalidade, sendo um grave problema de saúde pública. Os lncRNAs (RNAs longo não codificantes), têm funções regulatórias transcricionais e/ou pós transcricionais bem complexas e que ainda não são totalmente claras, mas que podem exercer influência sobre as doenças cardiovasculares, dentre elas a IC. Assim o estudo teve como objetivo identificar na literatura o papel dos lncRNAs na patogênese da IC por meio de uma revisão integrativa com busca sistemática. Foram considerados elegíveis para leitura e composição do estudo 33 artigos e os principais papéis dos lncRNA na IC foram relatados como possíveis marcadores biológicos para diagnóstico e prognóstico da doença devido a sua expressividade na corrente sanguínea. Além disso, os lncRNAs podem estar relacionados à capacidade funcional uma vez que o aumento ou diminuição de sua expressão promove redução da apoptose de células endoteliais, melhora a disfunção cardíaca, distúrbios de contratilidade e dos canais de cálcio em pacientes com IC. Portanto, os lncRNAs parecem estar envolvidos na patogênese e/ou fisiopatologia da IC, podendo ser utilizados como biomarcadores genéticos com sensibilidade e especificidade semelhantes ou superiores aos empregados atualmente no diagnóstico e prognóstico da IC.

Heart Failure (HF) is one of the main causes of hospitalization worldwide and has a high degree of morbidity and mortality being considered a public health pro- blem. lncRNAs (non-coding long RNAs) have very complex transcriptional and/or post- transcriptional regulatory functions that are still not entirely clear but may influence car- diovascular diseases, including HF. Thus, the study aimed to identify in the literature the role of lncRNAs in the pathogenesis of HF through an integrative review with a systema- tic search. A total of 33 articles were considered eligible for reading and composition of the study. The roles of lncRNA in HF were reported as possible biological markers for the diagnosis and prognosis of the disease due to its expressiveness in the bloodstream. In addition, lncRNAs may be related to functional capacity since the increase or decrease in their expression promotes a reduction in endothelial cell apoptosis, and improves car- diac dysfunction, contractility, and calcium channel disorders in patients with HF. The- refore, lncRNAs seem to be involved in the pathogenesis and/or pathophysiology of HF and can be used as genetic biomarkers with sensitivity and specificity similar or superior to those currently employed in the diagnosis and prognosis of HF.

La Insuficiencia Cardiaca (IC) es una de las principales causas de hospita- lización en el mundo y tiene un alto grado de morbimortalidad considerándose un pro- blema de salud pública. Los lncRNAs (ARN largos no codificantes) tienen funciones re- guladoras transcripcionales y/o post-transcripcionales muy complejas que aún no están del todo claras pero que pueden influir en las enfermedades cardiovasculares, incluida la IC. Así pues, el estudio se propuso identificar en la literatura el papel de los lncRNAs en la patogénesis de la IC mediante una revisión integradora con una búsqueda sistemática. Un total de 33 artículos fueron considerados elegibles para su lectura y composición del estudio. Las funciones de los lncRNA en la IC se señalaron como posibles marcadores biológicos para el diagnóstico y pronóstico de la enfermedad debido a su expresividad en el torrente sanguíneo. Además, los lncRNAs pueden estar relacionados con la capacidad funcional, ya que el aumento o disminución de su expresión promueve una reducción de la apoptosis de las células endoteliales y mejora la disfunción cardiaca, la contractilidad y los trastornos de los canales de calcio en pacientes con IC. Por tanto, los lncRNAs parecen estar implicados en la patogénesis y/o fisiopatología de la IC y pueden ser utili- zados como biomarcadores genéticos con sensibilidad y spe-cificidad similares o superi- ores a los empleados actualmente en el diagnóstico y pronóstico de la IC.

De novo variants in FRMD5 are associated with developmental delay, intellectual disability, ataxia, and abnormalities of eye movement.

Proteins containing the FERM (four-point-one, ezrin, radixin, and moesin) domain link the plasma membrane with cytoskeletal structures at specific cellular locations and have been implicated in the localization of cell-membrane-associated proteins and/or phosphoinositides. FERM domain-containing protein 5 (FRMD5) localizes at cell adherens junctions and stabilizes cell-cell contacts. To date, variants in FRMD5 have not been associated with a Mendelian disease in OMIM. Here, we describe eight probands with rare heterozygous missense variants in FRMD5 who present with developmental delay, intellectual disability, ataxia, seizures, and abnormalities of eye movement. The variants are de novo in all for whom parental testing was available (six out of eight probands), and human genetic datasets suggest that FRMD5 is intolerant to loss of function (LoF). We found that the fly ortholog of FRMD5, CG5022 (dFrmd), is expressed in the larval and adult central nervous systems where it is present in neurons but not in glia. dFrmd LoF mutant flies are viable but are extremely sensitive to heat shock, which induces severe seizures. The mutants also exhibit defective responses to light. The human FRMD5 reference (Ref) cDNA rescues the fly dFrmd LoF phenotypes. In contrast, all the FRMD5 variants tested in this study (c.340T>C, c.1051A>G, c.1053C>G, c.1054T>C, c.1045A>C, and c.1637A>G) behave as partial LoF variants. In addition, our results indicate that two variants that were tested have dominant-negative effects. In summary, the evidence supports that the observed variants in FRMD5 cause neurological symptoms in humans.

Hereditary spastic paraparesis presenting as cerebral palsy due to ADD3 variant with mechanistic insight provided by a Drosophila γ-adducin model.

Cerebral palsy (CP) causes neurological disability in early childhood. Hypoxic-ischaemic injury plays a major role in its aetiology, nevertheless, genetic and epigenetic factors may contribute to the clinical presentation. Mutations in ADD3 (encoding γ-adducin) gene have been described in a monogenic form of spastic quadriplegic cerebral palsy (OMIM 601568). We studied a 16-year-old male with spastic diplegia. Several investigations including neurometabolic testing, brain and spine magnetic resonance imaging (MRI) and CGH-Array were normal. Further, clinical genetics assessment and whole exome sequencing (WES) gave the diagnosis. We generated an animal model using Drosophila to study the effects of γ-adducin loss and gain of function. WES revealed a biallelic variant in the ADD3 gene, NM_016824.5(ADD3): c.1100G > A, p.(Gly367Asp). Mutations in this gene have been described as an ultra-rare autosomal recessive, which is a known form of inherited cerebral palsy. Molecular modelling suggests that this mutation leads to a loss of structural integrity of γ-adducin and is therefore expected to result in a decreased level of functional protein. Pan-neuronal over-expression or knock-down of the Drosophila ortholog of ADD3 called hts caused a reduction of life span and impaired locomotion thereby phenocopying aspects of the human disease. Our animal experiments present a starting point to understand the biological processes underpinning the clinical phenotype and pathogenic mechanisms, to gain insights into potential future methods for treating or preventing ADD3 related spastic quadriplegic cerebral palsy.

A lysosomal regulatory circuit essential for the development and function of microglia.

As the primary phagocytic cells of the central nervous system, microglia exquisitely regulate their lysosomal activity to facilitate brain development and homeostasis. However, mechanisms that coordinate lysosomal activity with microglia development, chemotaxis, and function remain unclear. Here, we show that embryonic macrophages require the lysosomal guanosine triphosphatase (GTPase) RagA and the GTPase-activating protein Folliculin to colonize the brain in zebrafish. We demonstrate that embryonic macrophages in rraga mutants show increased expression of lysosomal genes but display significant down-regulation of immune- and chemotaxis-related genes. Furthermore, we find that RagA and Folliculin repress the key lysosomal transcription factor Tfeb and its homologs Tfe3a and Tfe3b in the macrophage lineage. Using RNA sequencing, we establish that Tfeb and Tfe3 are required for activation of lysosomal target genes under conditions of stress but not for basal expression of lysosomal pathways. Collectively, our data define a lysosomal regulatory circuit essential for macrophage development and function in vivo.

Expanding ACTA2 genotypes with corresponding phenotypes overlapping with smooth muscle dysfunction syndrome.

Pathogenic variants in ACTA2, encoding smooth muscle α-actin, predispose to thoracic aortic aneurysms and dissections. ACTA2 variants altering arginine 179 predispose to a more severe, multisystemic disease termed smooth muscle dysfunction syndrome (SMDS; OMIM 613834). Vascular complications of SMDS include patent ductus arteriosus (PDA) or aortopulmonary window, early-onset thoracic aortic disease (TAD), moyamoya-like cerebrovascular disease, and primary pulmonary hypertension. Patients also have dysfunction of other smooth muscle-dependent systems, including congenital mydriasis, hypotonic bladder, and gut hypoperistalsis. Here, we describe five patients with novel heterozygous ACTA2 missense variants, p.Arg179Gly, p.Met46Arg, p.Thr204Ile, p.Arg39Cys, and p.Ile66Asn, who have clinical complications that align or overlap with SMDS. Patients with the ACTA2 p.Arg179Gly and p.Thr204Ile variants display classic features of SMDS. The patient with the ACTA2 p.Met46Arg variant exhibits exclusively vascular complications of SMDS, including early-onset TAD, PDA, and moyamoya-like cerebrovascular disease. The patient with the ACTA2 p.Ile66Asn variant has an unusual vascular complication, a large fusiform internal carotid artery aneurysm. The patient with the ACTA2 p.Arg39Cys variant has pulmonary, gastrointestinal, and genitourinary complications of SMDS but no vascular manifestations. Identifying pathogenic ACTA2 variants associated with features of SMDS is critical for aggressive surveillance and management of vascular and nonvascular complications and delineating the molecular pathogenesis of SMDS.

Partial loss-of-function variant in neuregulin 1 identified in family with heritable peripheral neuropathy.

Neuregulin 1 signals are essential for the development and function of Schwann cells, which form the myelin sheath on peripheral axons. Disruption of myelin in the peripheral nervous system can lead to peripheral neuropathy, which is characterized by reduced axonal conduction velocity and sensorimotor deficits. Charcot-Marie-Tooth disease is a group of heritable peripheral neuropathies that may be caused by variants in nearly 100 genes. Despite the evidence that Neuregulin 1 is essential for many aspects of Schwann cell development, previous studies have not reported variants in the neuregulin 1 gene (NRG1) in patients with peripheral neuropathy. We have identified a rare missense variant in NRG1 that is homozygous in a patient with sensory and motor deficits consistent with mixed axonal and de-myelinating peripheral neuropathy. Our in vivo functional studies in zebrafish indicate that the patient variant partially reduces NRG1 function. This study tentatively suggests that variants at the NRG1 locus may cause peripheral neuropathy and that NRG1 should be investigated in families with peripheral neuropathy of unknown cause.

Recessive PRDM13 mutations cause fatal perinatal brainstem dysfunction with cerebellar hypoplasia and disrupt Purkinje cell differentiation.

Pontocerebellar hypoplasias (PCHs) are congenital disorders characterized by hypoplasia or early atrophy of the cerebellum and brainstem, leading to a very limited motor and cognitive development. Although over 20 genes have been shown to be mutated in PCHs, a large proportion of affected individuals remains undiagnosed. We describe four families with children presenting with severe neonatal brainstem dysfunction and pronounced deficits in cognitive and motor development associated with four different bi-allelic mutations in PRDM13, including homozygous truncating variants in the most severely affected individuals. Brain MRI and fetopathological examination revealed a PCH-like phenotype, associated with major hypoplasia of inferior olive nuclei and dysplasia of the dentate nucleus. Notably, histopathological examinations highlighted a sparse and disorganized Purkinje cell layer in the cerebellum. PRDM13 encodes a transcriptional repressor known to be critical for neuronal subtypes specification in the mouse retina and spinal cord but had not been implicated, so far, in hindbrain development. snRNA-seq data mining and in situ hybridization in humans show that PRDM13 is expressed at early stages in the progenitors of the cerebellar ventricular zone, which gives rise to cerebellar GABAergic neurons, including Purkinje cells. We also show that loss of function of prdm13 in zebrafish leads to a reduction in Purkinje cells numbers and a complete absence of the inferior olive nuclei. Altogether our data identified bi-allelic mutations in PRDM13 as causing a olivopontocerebellar hypoplasia syndrome and suggest that early deregulations of the transcriptional control of neuronal fate specification could contribute to a significant number of cases.

Portuguese adolescents' cognitive well-being and basic psychological needs during the COVID-19 outbreak: A longitudinal study.

Confinements and social distancing measures during COVID-19 pandemic were particularly challenging to adolescents, impacting significantly their life and routines. Following a longitudinal design, this study sought to compare adolescents' cognitive well-being-satisfaction with life, social support, and quality of life-before (T1) and during (T2) the COVID-19 pandemic. Additionally, it aimed to clarify the predictive value of the three dimensions of the cognitive well-being to the satisfaction of basic psychological needs of adolescents at school at T2. One thousand ninety-nine Portuguese adolescents participated, showing generally increased scores in satisfaction with life, social support, and quality of life at T2. Even so, girls revealed lower changes in cognitive well-being components compared with boys, between T1 and T2. In addition, satisfaction with life and quality of life were predictive of satisfaction of basic psychological needs at T2. This work highlights the relevance of cognitive well-being as a dispositional dimension in determining the satisfaction of basic psychological needs in adolescence, during a worldwide catastrophic event.

Comparison of intergrowth-21st and Fenton curves for evaluation of premature newborns / Comparação das curvas de intergrowth-21st e de Fenton para avaliação de recém-nascidos prematuros

Abstract Objectives: to compare the intrauterine and postnatal growth of preterm infants according to the Intergrowth-21st and Fenton curves. Methods: study carried out in a maternity hospital, reference in high-risk pregnancy, with preterm infants born in 2018 who were hospitalized in the neonatal units of the institution. Preterm newborns weighed at least twice after birth were included in the sample and those that were syndromic, malformed or presented fluid retention were excluded. Proportions and means were compared using Pearson's chi-square and Student's t tests for paired samples, respectively. The McNemar test was used to compare categorical variables and the Kappa test to verify the degree of agreement between birth weight classifications obtained by the curves. Results: one hundred and fifty three infants with a median gestational age of 34.4 weeks were included. The incidences of the categories of nutritional status at birth did not differ between the curves. There was perfect agreement between the curves, except when newborns born under 33 weeks of gestational age were evaluated, in which case the agreement was substantial. About 21% of the babies classified as small for gestational age (SGA) by Intergrowth-21st were adequate for gestational age (AGA) according to Fenton and, on average, 20% of cases that had postnatal growth restriction (PNGR) according to Fenton standards were categorized as adequate weight by Intergrowth-21st. Postnatal weight classifications obtained by the evaluated curves had perfect agreement. Conclusions: the differences in theclassifications found between the charts reveal the importance of choosing the growth curve for monitoring preterm infants since behaviors based on their diagnoses can impact the life of this population.

Resumo Objetivos: comparar o crescimento intrauterino e pós-natal de prematuros segundo as curvas de Intergrowth-21st e Fenton. Métodos: estudo realizado em uma maternidade de referência em gestação de alto risco com prematuros nascidos em 2018 que ficaram internados nas unidades neonatais da instituição. Foram incluídos os pré-termos pesados em pelo menos dois momentos após o nascimento e excluídos aqueles sindrômicos, malformados ou com retenção hídrica. As proporções e médias foram comparadas a partir dos testes qui-quadrado de Pearson e t de student para amostras emparelhadas, respectivamente. Já o teste de McNemar foi utilizado para comparar as variáveis categóricas e teste Kappa para verificar o grau de concordância entre as classificações de peso ao nascer obtidos pelas curvas. Resultados: foram incluídos 153 lactentes com idade gestacional mediana de 34,4 semanas. As incidências das categorias de estado nutricional ao nascer não diferiram entre as curvas. Houve concordância perfeita entre as mesmas, exceto quando se avaliou os nascidos com menos de 33 semanas, onde a concordância foi substancial. Cerca de 21% dos bebês classificados como pequenos para a idade gestacional (PIG) por Intergrowth-21st foram adequados para idade gestacional (AIG) segundo Fenton e, em média, 20% dos casos que tiveram restrição de crescimento pós-natal (RCPN) de acordo aos padrões de Fenton foram categorizados com peso adequado por Intergrowth-21st. As classificações de peso pós-natal obtidas pelas curvas avaliadas tiveram concordância perfeita. Conclusões: as diferenças de classificação encontradas revelam a importância da escolha da curva de crescimento para monitorização de prematuros visto que, condutas baseadas em seus diagnósticos, podem impactar na vida dessa população.

Recurrent de novo missense variants across multiple histone H4 genes underlie a neurodevelopmental syndrome.

Chromatin is essentially an array of nucleosomes, each of which consists of the DNA double-stranded fiber wrapped around a histone octamer. This organization supports cellular processes such as DNA replication, DNA transcription, and DNA repair in all eukaryotes. Human histone H4 is encoded by fourteen canonical histone H4 genes, all differing at the nucleotide level but encoding an invariant protein. Here, we present a cohort of 29 subjects with de novo missense variants in six H4 genes (H4C3, H4C4, H4C5, H4C6, H4C9, and H4C11) identified by whole-exome sequencing and matchmaking. All individuals present with neurodevelopmental features of intellectual disability and motor and/or gross developmental delay, while non-neurological features are more variable. Ten amino acids are affected, six recurrently, and are all located within the H4 core or C-terminal tail. These variants cluster to specific regions of the core H4 globular domain, where protein-protein interactions occur with either other histone subunits or histone chaperones. Functional consequences of the identified variants were evaluated in zebrafish embryos, which displayed abnormal general development, defective head organs, and reduced body axis length, providing compelling evidence for the causality of the reported disorder(s). While multiple developmental syndromes have been linked to chromatin-associated factors, missense-bearing histone variants (e.g., H3 oncohistones) are only recently emerging as a major cause of pathogenicity. Our findings establish a broader involvement of H4 variants in developmental syndromes.

Characterization of mouse Bmp5 regulatory injury element in zebrafish wound models.

Many key signaling molecules used to build tissues during embryonic development are re-activated at injury sites to stimulate tissue regeneration and repair. Bone morphogenetic proteins provide a classic example, but the mechanisms that lead to reactivation of BMPs following injury are still unknown. Previous studies have mapped a large "injury response element" (IRE) in the mouse Bmp5 gene that drives gene expression following bone fractures and other types of injury. Here we show that the large mouse IRE region is also activated in both zebrafish tail resection and mechanosensory hair cell injury models. Using the ability to test multiple constructs and image temporal and spatial dynamics following injury responses, we have narrowed the original size of the mouse IRE region by over 100 fold and identified a small 142 bp minimal enhancer that is rapidly induced in both mesenchymal and epithelial tissues after injury. These studies identify a small sequence that responds to evolutionarily conserved local signals in wounded tissues and suggest candidate pathways that contribute to BMP reactivation after injury.

Diagnostic yield of rare skeletal dysplasia conditions in the radiogenomics era.

BACKGROUND: Skeletal dysplasia (SD) conditions are rare genetic diseases of the skeleton, encompassing a heterogeneous group of over 400 disorders, and represent approximately 5% of all congenital anomalies. Developments in genetic and treatment technologies are leading to unparalleled therapeutic advances; thus, it is more important than ever to molecularly confirm SD conditions. Data on 'rates-of-molecular yields' in SD conditions, through exome sequencing approaches, is limited. Figures of 39% and 52.5% have been reported in the USA (n = 54) and South Korea (n = 185) respectively. METHODS: We discuss a single-centre (in the UK) experience of whole-exome sequencing (WES) in a cohort of 15 paediatric patients (aged 5 months to 12 years) with SD disorders previously molecularly unconfirmed. Our cohort included patients with known clinical diagnoses and undiagnosed skeletal syndromes. Extensive phenotyping and expert radiological review by a panel of international SD radiology experts, coupled with a complex bioinformatics pipeline, allowed for both gene-targeted and gene-agnostic approaches. RESULTS: Significant variants leading to a likely or confirmed diagnosis were identified in 53.3% (n = 8/15) of patients; 46.7% (n = 7/15) having a definite molecular diagnosis and 6.7% (n = 1/15) having a likely molecular diagnosis. We discuss this in the context of a rare disease in general and specifically SD presentations. Of patients with known diagnoses pre-WES (n = 10), molecular confirmation occurred in 7/10 cases, as opposed to 1/5 where a diagnosis was unknown pre-test. Thus, diagnostic return is greatest where the diagnosis is known pre-test. For WGS (whole genome sequencing, the next iteration of WES), careful case selection (ideally of known diagnoses pre-test) will yield highest returns. CONCLUSIONS: Our results highlight the cost-effective use of WES-targeted bioinformatic analysis as a diagnostic tool for SD, particularly patients with presumed SD, where detailed phenotyping is essential. Thorough co-ordinated clinical evaluation between clinical, radiological, and molecular teams is essential for improved yield and clinical care. WES (and WGS) yields will increase with time, allowing faster diagnoses, avoiding needless investigations, ensuring individualised patient care and patient reassurance. Further diagnoses will lead to increased information on natural history/mechanistic details, and likely increased therapies and clinical trials.

Genotype-phenotype correlations and novel molecular insights into the DHX30-associated neurodevelopmental disorders.

BACKGROUND: We aimed to define the clinical and variant spectrum and to provide novel molecular insights into the DHX30-associated neurodevelopmental disorder. METHODS: Clinical and genetic data from affected individuals were collected through Facebook-based family support group, GeneMatcher, and our network of collaborators. We investigated the impact of novel missense variants with respect to ATPase and helicase activity, stress granule (SG) formation, global translation, and their effect on embryonic development in zebrafish. SG formation was additionally analyzed in CRISPR/Cas9-mediated DHX30-deficient HEK293T and zebrafish models, along with in vivo behavioral assays. RESULTS: We identified 25 previously unreported individuals, ten of whom carry novel variants, two of which are recurrent, and provide evidence of gonadal mosaicism in one family. All 19 individuals harboring heterozygous missense variants within helicase core motifs (HCMs) have global developmental delay, intellectual disability, severe speech impairment, and gait abnormalities. These variants impair the ATPase and helicase activity of DHX30, trigger SG formation, interfere with global translation, and cause developmental defects in a zebrafish model. Notably, 4 individuals harboring heterozygous variants resulting either in haploinsufficiency or truncated proteins presented with a milder clinical course, similar to an individual harboring a de novo mosaic HCM missense variant. Functionally, we established DHX30 as an ATP-dependent RNA helicase and as an evolutionary conserved factor in SG assembly. Based on the clinical course, the variant location, and type we establish two distinct clinical subtypes. DHX30 loss-of-function variants cause a milder phenotype whereas a severe phenotype is caused by HCM missense variants that, in addition to the loss of ATPase and helicase activity, lead to a detrimental gain-of-function with respect to SG formation. Behavioral characterization of dhx30-deficient zebrafish revealed altered sleep-wake activity and social interaction, partially resembling the human phenotype. CONCLUSIONS: Our study highlights the usefulness of social media to define novel Mendelian disorders and exemplifies how functional analyses accompanied by clinical and genetic findings can define clinically distinct subtypes for ultra-rare disorders. Such approaches require close interdisciplinary collaboration between families/legal representatives of the affected individuals, clinicians, molecular genetics diagnostic laboratories, and research laboratories.

Osteocyte transcriptome mapping identifies a molecular landscape controlling skeletal homeostasis and susceptibility to skeletal disease.

Osteocytes are master regulators of the skeleton. We mapped the transcriptome of osteocytes from different skeletal sites, across age and sexes in mice to reveal genes and molecular programs that control this complex cellular-network. We define an osteocyte transcriptome signature of 1239 genes that distinguishes osteocytes from other cells. 77% have no previously known role in the skeleton and are enriched for genes regulating neuronal network formation, suggesting this programme is important in osteocyte communication. We evaluated 19 skeletal parameters in 733 knockout mouse lines and reveal 26 osteocyte transcriptome signature genes that control bone structure and function. We showed osteocyte transcriptome signature genes are enriched for human orthologs that cause monogenic skeletal disorders (P = 2.4 × 10-22) and are associated with the polygenic diseases osteoporosis (P = 1.8 × 10-13) and osteoarthritis (P = 1.6 × 10-7). Thus, we reveal the molecular landscape that regulates osteocyte network formation and function and establish the importance of osteocytes in human skeletal disease.

High diagnostic yield in skeletal ciliopathies using massively parallel genome sequencing, structural variant screening and RNA analyses.

Skeletal ciliopathies are a heterogenous group of disorders with overlapping clinical and radiographic features including bone dysplasia and internal abnormalities. To date, pathogenic variants in at least 30 genes, coding for different structural cilia proteins, are reported to cause skeletal ciliopathies. Here, we summarize genetic and phenotypic features of 34 affected individuals from 29 families with skeletal ciliopathies. Molecular diagnostic testing was performed using massively parallel sequencing (MPS) in combination with copy number variant (CNV) analyses and in silico filtering for variants in known skeletal ciliopathy genes. We identified biallelic disease-causing variants in seven genes: DYNC2H1, KIAA0753, WDR19, C2CD3, TTC21B, EVC, and EVC2. Four variants located in non-canonical splice sites of DYNC2H1, EVC, and KIAA0753 led to aberrant splicing that was shown by sequencing of cDNA. Furthermore, CNV analyses showed an intragenic deletion of DYNC2H1 in one individual and a 6.7 Mb de novo deletion on chromosome 1q24q25 in another. In five unsolved cases, MPS was performed in family setting. In one proband we identified a de novo variant in PRKACA and in another we found a homozygous intragenic deletion of IFT74, removing the first coding exon and leading to expression of a shorter message predicted to result in loss of 40 amino acids at the N-terminus. These findings establish IFT74 as a new skeletal ciliopathy gene. In conclusion, combined single nucleotide variant, CNV and cDNA analyses lead to a high yield of genetic diagnoses (90%) in a cohort of patients with skeletal ciliopathies.