Investigation of the diagnostic potential of Whole-Genome-Sequencing by the example of 69 cases with congenital limb malformations

Since the sequencing of the first human genome in 2001, Whole-Genome-Sequencing (WGS) has made great progress. It is now possible to apply the method in everyday clinical diagnostics. However, there are established approaches for the identification of mutations, which, due to years of application, have a high status in genetic diagnostics. To what extent WGS represents a complement or even an alternative to these methods, and which diagnostic results are possible by considering the genome in its completeness, will be investigated in the present work. Sixty-nine cases with malformations of at least one limb received WGS, excluding those who had already received a molecularly confirmed genetic diagnosis. The data of all were processed using VarFish (version v0.17.2) and SODAR (System for Omics Data Analysis and Retrieval) and then analyzed manually. Single nucleotide variants (SNVs) and structural variants (SVs) in the protein-coding and non-protein-coding re-gions of the genome were examined. Subsequently, GLI3 and HMGB1 were investigated for possible genotype-phenotype correlations using findings from the WGS. Overall, we identified pathogenic variants in twelve cases (17.4%). SNVs appeared in four patients in each of the genes FGFR1, FGFR2, GLI3, and BHLHA9. The variant in FGFR1 had already been described in the literature, the other three were detected for the first time. In one case, a variant in HMGB1 was identified as pathogenic. Further-more, SNVs were detected in two potentially disease-causing genes (SEMA3D, ALDH1A). In addition, UBA2 variants were found in two ectrodactyly cases in the cohort, as well as another case known to the Institute of Medical Genetics and Human Genetics. This supports the current hypothesis of UBA2 as a disease gene. In addition to SNVs, two complex SVs were detected. One inversion, which is flanked by a deletion, was found in a patient with ectrodactyly at the locus of split hand foot malformation (SHFM) 3. The other variant is a complex translocation between 7q36.3 and 9p24.1 in a patient with bilateral mirror-image polydactyly of the hands and feet. Furthermore, a pathogenic alanine repeat expansion in HOXD13 was identified in a patient with synpolydactyly. No pathogenic variants could be found in the non-protein-coding region of the genome. In GLI3 and HMGB1, new findings regarding the geno-type-phenotype correlations at the corresponding loci were obtained. Thus, of sixty-nine cases in which conventional methods did not previously yield a genetic diagnosis, WGS enabled the establishment of a diagnosis in twelve cases (17.4%). In addition, WGS provided the basis for a closer look at the loci of GLI3 and HMGB1, with final-exon frameshifts of HMGB1 being identified as the cause of brachyphalangia-polydactyly-tibia-hypo/aplasia syndrome. Future improvements in sequencing methods, as well as in processing and evaluation software, are expected to further increase the diagnostic yield of WGS. However, it is already proving to be a potent tool for everyday clinical genetics, as well as various research approaches. As a single comprehensive test, it allows the identification of multiple variants of several types, including small but complex structural variants that are not detected by conventional methods.Seit der Sequenzierung des ersten menschlichen Genoms 2001 hat die Ganz-Genom-Analyse (Whole-Genome-Sequencing, WGS) große Fortschritte gemacht. Mittlerweile ist es möglich, die Methode im klinisch-diagnostischen Alltag anzuwenden. Allerdings existieren etablierte Verfahren zur Identifikation von Mutationen, welche durch jahrelange Anwendung einen hohen Stellenwert in der genetischen Diagnostik einnehmen. Inwiefern WGS eine Ergänzung oder gar Alternative zu diesen Verfahren darstellt, und welches diagnostische Potential die Methode besitzt, soll in der vorliegenden Arbeit untersucht werden. In 69 Fällen mit angeborenen Fehlbildungen von mindestens einer Extremität, bei denen die genetische Routinediagnostik keine pathogene Variante identifizieren konnte, wurde ein WGS durchgeführt. Die Daten aller Patient*innen wurden mit Hilfe von VarFish (Version v0.17.2) und SODAR (System for Omics Data Analysis and Retrieval) prozessiert und danach manuell ausgewertet. Dabei erfolgte die Betrachtung von Einzelnukleotidvarianten (Single Nucleotide Variants, SNVs) und Strukturvarianten (SVs) im proteinkodierenden wie nicht-proteinkodierenden Bereich des Genoms. Im Anschluss wurden GLI3 und HMGB1, hinsichtlich möglicher Genotyp-Phänotyp-Korrelationen untersucht, wobei Erkenntnisse aus dem WGS als Grundlage genutzt wurden. Insgesamt konnten pathogene Varianten in 12 Fällen (17.4%) identifiziert werden. SNVs zeigten sich in vier Patienten jeweils in den Genen FGFR1, FGFR2, GLI3 und BHLHA9. Die Variante in FGFR1 war bereits in der Literatur beschrieben worden, die anderen drei wurden erstmals nachgewiesen. In einem weiteren Fall konnte eine Variante in HMGB1 als pathogen identifiziert werden. Weiterhin konnten SNVs in zwei potenziell krankheits-auslösenden Genen (SEMA3D, ALDH1A) aufgefunden werden. Darüber hinaus fanden sich UBA2-Varianten in zwei Ektrodaktylie-Fällen der Kohorte sowie einem weiteren, dem Institut für Medizinische Genetik und Humangenetik bekannten, Fall. Dies unterstützt die aktuelle These von UBA2 als Krankheitsgen. Neben den SNVs konnten zwei komplexe SVs gefunden werden. Weiterhin konnte eine pathogene Alanin-Repeat-Expansion in HOXD13 identifiziert werden. Es konnten keine pathogenen Varianten im nicht-protein-kodierenden Bereich des Genoms gefunden werden. Bei GLI3 und HMGB1 wurden neue Erkenntnisse bezüglich der Genotyp-Phänotyp-Korrelationen an den entsprechenden Loci gewonnen. Insgesamt konnte bei 12 (17,4%) von 69 Fällen, bei denen herkömmliche Verfahren bisher keine molekular-genetische Diagnose erbrachten, durch das WGS die Etablierung einer Diagnose ermöglicht werden. Darüber hinaus, lieferte WGS die Grundlage für die genauere Betrachtung der Loci von GLI3 und HMGB1, wobei final-exon frameshifts von HMGB1 als Ursache des Brachyphalangie-Polydaktylie-Tibia-Hypo/Aplasie-Syndroms identifiziert werden konnten. Durch künftige Verbesserung von Sequenzierungsmethoden sowie prozessierender und auswertender Software ist zu erwarten, dass die diagnostische Rate des WGS weiter ansteigen wird. Allerdings zeigt es sich bereits heute als potentes Verfahren für den klinisch-genetischen Alltag sowie verschiedene Forschungsansätze. Es ermöglicht als ein einziger umfassender Test die Identifikation von Varianten unterschiedlichen Typs, inklusiver kleiner, aber komplexer Strukturvarianten, die durch herkömmliche Verfahren nicht erfasst werden

Fused filament fabrication: Comparison of methods for determining the interfacial strength of single welded tracks

The mechanical properties of plastic-based additively manufactured specimens have been widely discussed. However, there is still no standard that can be used to determine properties such as the interfacial strength of adjacent tracks and also to exclude the influence of varying manufacturing conditions. In this paper, a proposal is made to determine the interfacial strength using specimens with only one track within a layer. For this purpose, so-called single-wall specimens of polylactide were characterised under tensile load and the interfacial area between the adjacent layers was determined using three methods. It turned out that the determination of the interfacial area via the fracture surface is the most accurate method for determining the interfacial strength. The measured interfacial strengths were compared with the bulk material strength and it was found that the bulk material strength can be achieved under optimal conditions in the FFF process. It was also observed that with increasing nozzle temperature, the simultaneous printing of specimens influences the interfacial strength. To conclude, this method allows to measure the interfacial strength without superimposing the influence of voids. However, for example, the interfacial strength within a layer cannot be determined

GLI3 variants causing isolated polysyndactyly are not restricted to the protein's C-terminal third

Loss of function variants of GLI3 are associated with a variety of forms of polysyndactyly: Pallister-Hall syndrome (PHS), Greig-Cephalopolysyndactyly syndrome (GCPS), and isolated polysyndactyly (IPD). Variants affecting the N-terminal and C-terminal thirds of the GLI3 protein have been associated with GCPS, those within the central third with PHS. Cases of IPD have been attributed to variants affecting the C-terminal third of the GLI3 protein. In this study, we further investigate these genotype-phenotype correlations. Sequencing of GLI3 was performed in patients with clinical findings suggestive of a GLI3-associated syndrome. Additionally, we searched the literature for reported cases of either manifestation with mutations in the GLI3 gene. Here, we report 48 novel cases from 16 families with polysyndactyly in whom we found causative variants in GLI3 and a review on 314 previously reported GLI3 variants. No differences in location of variants causing either GCPS or IPD were found. Review of published data confirmed the association of PHS and variants affecting the GLI3 protein's central third. We conclude that the observed manifestations of GLI3 variants as GCPS or IPD display different phenotypic severities of the same disorder and propose a binary division of GLI3-associated disorders in either PHS or GCPS/polysyndactyly

Efficiency of Computer-Aided Facial Phenotyping (DeepGestalt) in Individuals With and Without a Genetic Syndrome: Diagnostic Accuracy Study

Background: Collectively, an estimated 5% of the population have a genetic disease. Many of them feature characteristics that can be detected by facial phenotyping. Face2Gene CLINIC is an online app for facial phenotyping of patients with genetic syndromes. DeepGestalt, the neural network driving Face2Gene, automatically prioritizes syndrome suggestions based on ordinary patient photographs, potentially improving the diagnostic process. Hitherto, studies on DeepGestalt’s quality highlighted its sensitivity in syndromic patients. However, determining the accuracy of a diagnostic methodology also requires testing of negative controls. Objective: The aim of this study was to evaluate DeepGestalt's accuracy with photos of individuals with and without a genetic syndrome. Moreover, we aimed to propose a machine learning–based framework for the automated differentiation of DeepGestalt’s output on such images. Methods: Frontal facial images of individuals with a diagnosis of a genetic syndrome (established clinically or molecularly) from a convenience sample were reanalyzed. Each photo was matched by age, sex, and ethnicity to a picture featuring an individual without a genetic syndrome. Absence of a facial gestalt suggestive of a genetic syndrome was determined by physicians working in medical genetics. Photos were selected from online reports or were taken by us for the purpose of this study. Facial phenotype was analyzed by DeepGestalt version 19.1.7, accessed via Face2Gene CLINIC. Furthermore, we designed linear support vector machines (SVMs) using Python 3.7 to automatically differentiate between the 2 classes of photographs based on DeepGestalt's result lists. Results: We included photos of 323 patients diagnosed with 17 different genetic syndromes and matched those with an equal number of facial images without a genetic syndrome, analyzing a total of 646 pictures. We confirm DeepGestalt’s high sensitivity (top 10 sensitivity: 295/323, 91%). DeepGestalt’s syndrome suggestions in individuals without a craniofacially dysmorphic syndrome followed a nonrandom distribution. A total of 17 syndromes appeared in the top 30 suggestions of more than 50% of nondysmorphic images. DeepGestalt’s top scores differed between the syndromic and control images (area under the receiver operating characteristic [AUROC] curve 0.72, 95% CI 0.68-0.76; P<.001). A linear SVM running on DeepGestalt’s result vectors showed stronger differences (AUROC 0.89, 95% CI 0.87-0.92; P<.001). Conclusions: DeepGestalt fairly separates images of individuals with and without a genetic syndrome. This separation can be significantly improved by SVMs running on top of DeepGestalt, thus supporting the diagnostic process of patients with a genetic syndrome. Our findings facilitate the critical interpretation of DeepGestalt’s results and may help enhance it and similar computer-aided facial phenotyping tools

INQUÉRITO EPIDEMIOLÓGICO DE SUPOSTO FOCO DE FEBRE MACULOSA

O presente estudo objetivou avaliar a suspeita de ocorrência da febre maculosa (FM) em Quirinópolis – GO, por meio de inquérito sorológico em amostras de humanos, cães e equinos e identificação de carrapatos vetores. Em 2007, foram realizadas reações de imunofluorescência indireta (RIFI) para Rickettsia spp. em amostras de sangue colhidas de 28 pessoas do grupo de convívio de um caso suspeito (uma criança de três anos que apresentou sinais de riquetsiose, após exposição a carrapatos). Também foram colhidas amostras de sangue em cinco cães e 42 equinos, havendo soro reagentes somente entre os equinos (28,6%). Não foi constatada a presença do carrapato Amblyomma cajennense. Sete indivíduos foram reagentes para Rickettsia spp., a maioria desses eram parentes paternos do caso suspeito. Em 2008, na mesma região, foram realizadas RIFI em amostras de 30 humanos sem sinais clínicos de FM e constatou-se 50% de soro reagentes para Rickettsia spp. Adicionalmente, colheram-se amostras de sangue de 24 cães errantes, havendo um cão soropositivo. A evidência sorológica do contato entre bactérias do gênero Rickettsia com habitantes humanos e animais de Quirinópolis indica que a FM ou outra riquetsiose possa estar ocorrendo no Estado de Goiás. Palavras-chave: A. cajennense; anticorpos; cães; cavalos; humanos; Rickettsia

Genome sequencing in families with congenital limb malformations

The extensive clinical and genetic heterogeneity of congenital limb malformation calls for comprehensive genome-wide analysis of genetic variation. Genome sequencing (GS) has the potential to identify all genetic variants. Here we aim to determine the diagnostic potential of GS as a comprehensive one-test-for-all strategy in a cohort of undiagnosed patients with congenital limb malformations. We collected 69 cases (64 trios, 1 duo, 5 singletons) with congenital limb malformations with no molecular diagnosis after standard clinical genetic testing and performed genome sequencing. We also developed a framework to identify potential noncoding pathogenic variants. We identified likely pathogenic/disease-associated variants in 12 cases (17.4%) including four in known disease genes, and one repeat expansion in HOXD13. In three unrelated cases with ectrodactyly, we identified likely pathogenic variants in UBA2, establishing it as a novel disease gene. In addition, we found two complex structural variants (3%). We also identified likely causative variants in three novel high confidence candidate genes. We were not able to identify any noncoding variants. GS is a powerful strategy to identify all types of genomic variants associated with congenital limb malformation, including repeat expansions and complex structural variants missed by standard diagnostic approaches. In this cohort, no causative noncoding SNVs could be identified. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1007/s00439-021-02295-y

Aberrant phase separation and nucleolar dysfunction in rare genetic diseases

Thousands of genetic variants in protein-coding genes have been linked to disease. However, the functional impact of most variants is unknown as they occur within intrinsically disordered protein regions that have poorly defined functions1-3. Intrinsically disordered regions can mediate phase separation and the formation of biomolecular condensates, such as the nucleolus4,5. This suggests that mutations in disordered proteins may alter condensate properties and function6-8. Here we show that a subset of disease-associated variants in disordered regions alter phase separation, cause mispartitioning into the nucleolus and disrupt nucleolar function. We discover de novo frameshift variants in HMGB1 that cause brachyphalangy, polydactyly and tibial aplasia syndrome, a rare complex malformation syndrome. The frameshifts replace the intrinsically disordered acidic tail of HMGB1 with an arginine-rich basic tail. The mutant tail alters HMGB1 phase separation, enhances its partitioning into the nucleolus and causes nucleolar dysfunction. We built a catalogue of more than 200,000 variants in disordered carboxy-terminal tails and identified more than 600 frameshifts that create arginine-rich basic tails in transcription factors and other proteins. For 12 out of the 13 disease-associated variants tested, the mutation enhanced partitioning into the nucleolus, and several variants altered rRNA biogenesis. These data identify the cause of a rare complex syndrome and suggest that a large number of genetic variants may dysregulate nucleoli and other biomolecular condensates in humans.© 2023. The Author(s)

Hurricanes and Climate: The U.S. CLIVAR Working Group on Hurricanes

While a quantitative climate theory of tropical cyclone formation remains elusive, considerable progress has been made recently in our ability to simulate tropical cyclone climatologies and to understand the relationship between climate and tropical cyclone formation. Climate models are now able to simulate a realistic rate of global tropical cyclone formation, although simulation of the Atlantic tropical cyclone climatology remains challenging unless horizontal resolutions finer than 50 km are employed. This article summarizes published research from the idealized experiments of the Hurricane Working Group of U.S. Climate and Ocean: Variability, Predictability and Change (CLIVAR). This work, combined with results from other model simulations, has strengthened relationships between tropical cyclone formation rates and climate variables such as midtropospheric vertical velocity, with decreased climatological vertical velocities leading to decreased tropical cyclone formation. Systematic differences are shown between experiments in which only sea surface temperature is increased compared with experiments where only atmospheric carbon dioxide is increased. Experiments where only carbon dioxide is increased are more likely to demonstrate a decrease in tropical cyclone numbers, similar to the decreases simulated by many climate models for a future, warmer climate. Experiments where the two effects are combined also show decreases in numbers, but these tend to be less for models that demonstrate a strong tropical cyclone response to increased sea surface temperatures. Further experiments are proposed that may improve our understanding of the relationship between climate and tropical cyclone formation, including experiments with two-way interaction between the ocean and the atmosphere and variations in atmospheric aerosols

Cluster analysis of downscaled and explicitly simulated North Atlantic tropical cyclone tracks

A realistic representation of the North Atlantic tropical cyclone tracks is crucial as it allows, for example, explaining potential changes in US landfalling systems. Here we present a tentative study, which examines the ability of recent climate models to represent North Atlantic tropical cyclone tracks. Tracks from two types of climate models are evaluated: explicit tracks are obtained from tropical cyclones simulated in regional or global climate models with moderate to high horizontal resolution (1° to 0.25°), and downscaled tracks are obtained using a downscaling technique with large-scale environmental fields from a subset of these models. For both configurations, tracks are objectively separated into four groups using a cluster technique, leading to a zonal and a meridional separation of the tracks. The meridional separation largely captures the separation between deep tropical and sub-tropical, hybrid or baroclinic cyclones, while the zonal separation segregates Gulf of Mexico and Cape Verde storms. The properties of the tracks’ seasonality, intensity and power dissipation index in each cluster are documented for both configurations. Our results show that except for the seasonality, the downscaled tracks better capture the observed characteristics of the clusters. We also use three different idealized scenarios to examine the possible future changes of tropical cyclone tracks under 1) warming sea surface temperature, 2) increasing carbon dioxide, and 3) a combination of the two. The response to each scenario is highly variable depending on the simulation considered. Finally, we examine the role of each cluster in these future changes and find no preponderant contribution of any single cluster over the others

Planck 2015 results: XXV. Diffuse low-frequency Galactic foregrounds

We discuss the Galactic foreground emission between 20 and 100 GHz based on observations by Planck and WMAP. The total intensity in this part of the spectrum is dominated by free-free and spinning dust emission, whereas the polarized intensity is dominated by synchrotron emission. The Commander component-separation tool has been used to separate the various astrophysical processes in total intensity. Comparison with radio recombination line templates verifies the recovery of the free-free emission along the Galactic plane. Comparison of the high-latitude H\u3b1 emission with our free-free map shows residuals that correlate with dust optical depth, consistent with a fraction (\ue2\u2030 30%) of H\u3b1 having been scattered by high-latitude dust. We highlight a number of diffuse spinning dust morphological features at high latitude. There is substantial spatial variation in the spinning dust spectrum, with the emission peak (in I\u3bd) ranging from below 20 GHz to more than 50 GHz. There is a strong tendency for the spinning dust component near many prominent H ii regions to have a higher peak frequency, suggesting that this increase in peak frequency is associated with dust in the photo-dissociation regions around the nebulae. The emissivity of spinning dust in these diffuse regions is of the same order as previous detections in the literature. Over the entire sky, the Commander solution finds more anomalous microwave emission (AME) than the WMAP component maps, at the expense of synchrotron and free-free emission. This can be explained by the difficulty in separating multiple broadband components with a limited number of frequency maps. Future surveys, particularly at 5-20 GHz, will greatly improve the separation by constraining the synchrotron spectrum. We combine Planck and WMAP data to make the highest signal-to-noise ratio maps yet of the intensity of the all-sky polarized synchrotron emission at frequencies above a few GHz. Most of the high-latitude polarized emission is associated with distinct large-scale loops and spurs, and we re-discuss their structure. We argue that nearly all the emission at 40deg &gt; l &gt;-90deg is part of the Loop I structure, and show that the emission extends much further in to the southern Galactic hemisphere than previously recognised, giving Loop I an ovoid rather than circular outline. However, it does not continue as far as the "Fermi bubble/microwave haze", making it less probable that these are part of the same structure. We identify a number of new faint features in the polarized sky, including a dearth of polarized synchrotron emission directly correlated with a narrow, roughly 20deg long filament seen in H\u3b1 at high Galactic latitude. Finally, we look for evidence of polarized AME, however many AME regions are significantly contaminated by polarized synchrotron emission, and we find a 2\u3c3 upper limit of 1.6% in the Perseus region