REEV: review, evaluate and explain variants.

In the era of high throughput sequencing, special software is required for the clinical evaluation of genetic variants. We developed REEV (Review, Evaluate and Explain Variants), a user-friendly platform for clinicians and researchers in the field of rare disease genetics. Supporting data was aggregated from public data sources. We compared REEV with seven other tools for clinical variant evaluation. REEV (semi-)automatically fills individual ACMG criteria facilitating variant interpretation. REEV can store disease and phenotype data related to a case to use these for phenotype similarity measures. Users can create public permanent links for individual variants that can be saved as browser bookmarks and shared. REEV may help in the fast diagnostic assessment of genetic variants in a clinical as well as in a research context. REEV (https://reev.bihealth.org/) is free and open to all users and there is no login requirement.

Loss-of-function variants affecting the STAGA complex component SUPT7L cause a developmental disorder with generalized lipodystrophy.

Generalized lipodystrophy is a feature of various hereditary disorders, often leading to a progeroid appearance. In the present study we identified a missense and a frameshift variant in a compound heterozygous state in SUPT7L in a boy with intrauterine growth retardation, generalized lipodystrophy, and additional progeroid features. SUPT7L encodes a component of the transcriptional coactivator complex STAGA. By transcriptome sequencing, we showed the predicted missense variant to cause aberrant splicing, leading to exon truncation and thereby to a complete absence of SUPT7L in dermal fibroblasts. In addition, we found altered expression of genes encoding DNA repair pathway components. This pathway was further investigated and an increased rate of DNA damage was detected in proband-derived fibroblasts and genome-edited HeLa cells. Finally, we performed transient overexpression of wildtype SUPT7L in both cellular systems, which normalizes the number of DNA damage events. Our findings suggest SUPT7L as a novel disease gene and underline the link between genome instability and progeroid phenotypes.

Protocol for chromatin accessibility profiling of human endothelial cells cultured under fluid shear stress using ATAC-seq.

Chromatin accessibility influences gene regulation and can be quantified using assay for transposase-accessible chromatin with high-throughput sequencing (ATAC-seq). Recapitulating in vivo fluid shear stress (FSS) mechano-regimes in vitro allows the study of atheroprone and atheroprotective mechanisms. In this protocol, we show how to culture and harvest endothelial cells from microfluidic channels for the preparation of ATAC-seq, highlighting optional growth factor stimulation and different FSS rates. This extends the application of ATAC-seq to the analysis of in vitro mechanically stimulated cells. For complete details on the use and execution of this protocol, please refer to Jatzlau et al.1.

A Novel Variant in the WRN Gene Detected in a Case of Early-Onset Severe Insulin Resistance Displaying Some but Not All Hallmarks of Progeroid Werner Syndrome.

OBJECTIVE: Determining the cause of severe insulin resistance and early-onset diabetes in the case of a young woman in which a wide range of differential diagnoses did not apply. RESEARCH DESIGN AND METHODS: Diagnostic workup including medical history, physical examination, specialist consultations, imaging methods, laboratory assessment, and genetic testing carried out by next-generation panel sequencing. RESULTS: After ruling out several differential diagnoses, genetic testing revealed a previously unknown homozygous variant within the canonical splice site of intron 4 in the WRN gene classified as pathogenic. Thus, although not all cardinal clinical criteria according to existing guidelines had been met, the phenotype of our patient was attributed to Werner syndrome (WS), an autosomal-recessive inherited progeroid syndrome. CONCLUSIONS: WS, although rare, must be considered as a differential diagnosis in cases of severe insulin resistance. Moreover, recognized clinical criteria of WS may not lead to diagnosis in all cases.

Whole genome sequencing in families with oligodontia.

BACKGROUND/OBJECTIVES: Tooth agenesis (TA) is among the most common malformations in humans. Although several causative mutations have been described, the genetic cause often remains elusive. Here, we test whether whole genome sequencing (WGS) could bridge this diagnostic gap. METHODS: In four families with TA, we assessed the dental phenotype using the Tooth Agenesis Code after intraoral examination and radiographic and photographic documentation. We performed WGS of index patients and subsequent segregation analysis. RESULTS: We identified two variants of uncertain significance (a potential splice variant in PTH1R, and a 2.1 kb deletion abrogating a non-coding element in FGF7) and three pathogenic variants: a novel frameshift in the final exon of PITX2, a novel deletion in PAX9, and a known nonsense variant in WNT10A. Notably, the FGF7 variant was found in the patient, also featuring the WNT10A variant. While mutations in PITX2 are known to cause Axenfeld-Rieger syndrome 1 (ARS1) predominantly featuring ocular findings, accompanied by dental malformations, we found the PITX2 frameshift in a family with predominantly dental and varying ocular findings. CONCLUSION: Severe TA predicts a genetic cause identifiable by WGS. Final exon PITX2 frameshifts can cause a predominantly dental form of ARS1.

Fluid shear stress-modulated chromatin accessibility reveals the mechano-dependency of endothelial SMAD1/5-mediated gene transcription.

Bone morphogenetic protein (BMP) signaling and fluid shear stress (FSS) mediate complementary functions in vascular homeostasis and disease development. It remains to be shown whether altered chromatin accessibility downstream of BMP and FSS offers a crosstalk level to explain changes in SMAD-dependent transcription. Here, we employed ATAC-seq to analyze arterial endothelial cells stimulated with BMP9 and/or FSS. We found that BMP9-sensitive regions harbor non-palindromic GC-rich SMAD-binding elements (GGCTCC) and 69.7% of these regions become BMP-insensitive in the presence of FSS. While GATA and KLF transcription factor (TF) motifs are unique to BMP9- and FSS-sensitive regions, respectively, SOX motifs are common to both. Finally, we show that both SOX(13/18) and GATA(2/3/6) family members are directly upregulated by SMAD1/5. These findings highlight the mechano-dependency of SMAD-signaling by a sequential mechanism of first elevated pioneer TF expression, allowing subsequent chromatin opening to eventually providing accessibility to novel SMAD binding sites.

AML with complex karyotype: extreme genomic complexity revealed by combined long-read sequencing and Hi-C technology.

Acute myeloid leukemia with complex karyotype (CK-AML) is associated with poor prognosis, which is only in part explained by underlying TP53 mutations. Especially in the presence of complex chromosomal rearrangements, such as chromothripsis, the outcome of CK-AML is dismal. However, this degree of complexity of genomic rearrangements contributes to the leukemogenic phenotype and treatment resistance of CK-AML remains largely unknown. Applying an integrative workflow for the detection of structural variants (SVs) based on Oxford Nanopore (ONT) genomic DNA long-read sequencing (gDNA-LRS) and high-throughput chromosome confirmation capture (Hi-C) in a well-defined cohort of CK-AML identified regions with an extreme density of SVs. These rearrangements consisted to a large degree of focal amplifications enriched in the proximity of mammalian-wide interspersed repeat elements, which often result in oncogenic fusion transcripts, such as USP7::MVD, or the deregulation of oncogenic driver genes as confirmed by RNA-seq and ONT direct complementary DNA sequencing. We termed this novel phenomenon chromocataclysm. Thus, our integrative SV detection workflow combing gDNA-LRS and Hi-C enables to unravel complex genomic rearrangements at a very high resolution in regions hard to analyze by conventional sequencing technology, thereby providing an important tool to identify novel important drivers underlying cancer with complex karyotypic changes.

Doxorubicin Changes the Spatial Organization of the Genome around Active Promoters.

In this study, we delve into the impact of genotoxic anticancer drug treatment on the chromatin structure of human cells, with a particular focus on the effects of doxorubicin. Using Hi-C, ChIP-seq, and RNA-seq, we explore the changes in chromatin architecture brought about by doxorubicin and ICRF193. Our results indicate that physiologically relevant doses of doxorubicin lead to a local reduction in Hi-C interactions in certain genomic regions that contain active promoters, with changes in chromatin architecture occurring independently of Top2 inhibition, cell cycle arrest, and differential gene expression. Inside the regions with decreased interactions, we detected redistribution of RAD21 around the peaks of H3K27 acetylation. Our study also revealed a common structural pattern in the regions with altered architecture, characterized by two large domains separated from each other. Additionally, doxorubicin was found to increase CTCF binding in H3K27 acetylated regions. Furthermore, we discovered that Top2-dependent chemotherapy causes changes in the distance decay of Hi-C contacts, which are driven by direct and indirect inhibitors. Our proposed model suggests that doxorubicin-induced DSBs cause cohesin redistribution, which leads to increased insulation on actively transcribed TAD boundaries. Our findings underscore the significant impact of genotoxic anticancer treatment on the chromatin structure of the human genome.

HOXD13-associated synpolydactyly: Extending and validating the genotypic and phenotypic spectrum with 38 new and 49 published families.

PURPOSE: HOXD13 is an important regulator of limb development. Pathogenic variants in HOXD13 cause synpolydactyly type 1 (SPD1). How different types and positions of HOXD13 variants contribute to genotype-phenotype correlations, penetrance, and expressivity of SPD1 remains elusive. Here, we present a novel cohort and a literature review to elucidate HOXD13 phenotype-genotype correlations. METHODS: Patients with limb anomalies suggestive of SPD1 were selected for analysis of HOXD13 by Sanger sequencing, repeat length analysis, and next-generation sequencing. Literature was reviewed for HOXD13 heterozygotes. Variants were annotated for phenotypic data. Severity was calculated, and cluster and decision-tree analyses were performed. RESULTS: We identified 98 affected members of 38 families featuring 11 different (likely) causative variants and 4 variants of uncertain significance. The most frequent (25/38) were alanine repeat expansions. Phenotypes ranged from unaffected heterozygotes to severe osseous synpolydactyly, with intra- and inter-familial heterogeneity and asymmetry. A literature review provided 160 evaluable affected members of 49 families with SPD1. Computer-aided analysis only corroborated a positive correlation between alanine repeat length and phenotype severity. CONCLUSION: Our findings support that HOXD13-protein condensation in addition to haploinsufficiency is the molecular pathomechanism of SPD1. Our data may, also, facilitate the interpretation of synpolydactyly radiographs by future automated tools.

Disruption of regulatory domains and novel transcripts as disease-causing mechanisms.

Deletions, duplications, insertions, inversions, and translocations, collectively called structural variations (SVs), affect more base pairs of the genome than any other sequence variant. The recent technological advancements in genome sequencing have enabled the discovery of tens of thousands of SVs per human genome. These SVs primarily affect non-coding DNA sequences, but the difficulties in interpreting their impact limit our understanding of human disease etiology. The functional annotation of non-coding DNA sequences and methodologies to characterize their three-dimensional (3D) organization in the nucleus have greatly expanded our understanding of the basic mechanisms underlying gene regulation, thereby improving the interpretation of SVs for their pathogenic impact. Here, we discuss the various mechanisms by which SVs can result in altered gene regulation and how these mechanisms can result in rare genetic disorders. Beyond changing gene expression, SVs can produce novel gene-intergenic fusion transcripts at the SV breakpoints.

Parallel sequencing of extrachromosomal circular DNAs and transcriptomes in single cancer cells.

Extrachromosomal DNAs (ecDNAs) are common in cancer, but many questions about their origin, structural dynamics and impact on intratumor heterogeneity are still unresolved. Here we describe single-cell extrachromosomal circular DNA and transcriptome sequencing (scEC&T-seq), a method for parallel sequencing of circular DNAs and full-length mRNA from single cells. By applying scEC&T-seq to cancer cells, we describe intercellular differences in ecDNA content while investigating their structural heterogeneity and transcriptional impact. Oncogene-containing ecDNAs were clonally present in cancer cells and drove intercellular oncogene expression differences. In contrast, other small circular DNAs were exclusive to individual cells, indicating differences in their selection and propagation. Intercellular differences in ecDNA structure pointed to circular recombination as a mechanism of ecDNA evolution. These results demonstrate scEC&T-seq as an approach to systematically characterize both small and large circular DNA in cancer cells, which will facilitate the analysis of these DNA elements in cancer and beyond.

Enhancer hijacking at the ARHGAP36 locus is associated with connective tissue to bone transformation.

Heterotopic ossification is a disorder caused by abnormal mineralization of soft tissues in which signaling pathways such as BMP, TGFß and WNT are known key players in driving ectopic bone formation. Identifying novel genes and pathways related to the mineralization process are important steps for future gene therapy in bone disorders. In this study, we detect an inter-chromosomal insertional duplication in a female proband disrupting a topologically associating domain and causing an ultra-rare progressive form of heterotopic ossification. This structural variant lead to enhancer hijacking and misexpression of ARHGAP36 in fibroblasts, validated here by orthogonal in vitro studies. In addition, ARHGAP36 overexpression inhibits TGFß, and activates hedgehog signaling and genes/proteins related to extracellular matrix production. Our work on the genetic cause of this heterotopic ossification case has revealed that ARHGAP36 plays a role in bone formation and metabolism, outlining first details of this gene contributing to bone-formation and -disease.

3D genome mapping identifies subgroup-specific chromosome conformations and tumor-dependency genes in ependymoma.

Ependymoma is a tumor of the brain or spinal cord. The two most common and aggressive molecular groups of ependymoma are the supratentorial ZFTA-fusion associated and the posterior fossa ependymoma group A. In both groups, tumors occur mainly in young children and frequently recur after treatment. Although molecular mechanisms underlying these diseases have recently been uncovered, they remain difficult to target and innovative therapeutic approaches are urgently needed. Here, we use genome-wide chromosome conformation capture (Hi-C), complemented with CTCF and H3K27ac ChIP-seq, as well as gene expression and DNA methylation analysis in primary and relapsed ependymoma tumors, to identify chromosomal conformations and regulatory mechanisms associated with aberrant gene expression. In particular, we observe the formation of new topologically associating domains ('neo-TADs') caused by structural variants, group-specific 3D chromatin loops, and the replacement of CTCF insulators by DNA hyper-methylation. Through inhibition experiments, we validate that genes implicated by these 3D genome conformations are essential for the survival of patient-derived ependymoma models in a group-specific manner. Thus, this study extends our ability to reveal tumor-dependency genes by 3D genome conformations even in tumors that lack targetable genetic alterations.

Combinatorial effects on gene expression at the Lbx1/Fgf8 locus resolve split-hand/foot malformation type 3.

Split-Hand/Foot Malformation type 3 (SHFM3) is a congenital limb malformation associated with tandem duplications at the LBX1/FGF8 locus. Yet, the disease patho-mechanism remains unsolved. Here we investigate the functional consequences of SHFM3-associated rearrangements on chromatin conformation and gene expression in vivo in transgenic mice. We show that the Lbx1/Fgf8 locus consists of two separate, but interacting, regulatory domains. Re-engineering of a SHFM3-associated duplication and a newly reported inversion in mice results in restructuring of the chromatin architecture. This leads to ectopic activation of the Lbx1 and Btrc genes in the apical ectodermal ridge (AER) in an Fgf8-like pattern induced by AER-specific enhancers of Fgf8. We provide evidence that the SHFM3 phenotype is the result of a combinatorial effect on gene misexpression in the developing limb. Our results reveal insights into the molecular mechanism underlying SHFM3 and provide conceptual framework for how genomic rearrangements can cause gene misexpression and disease.

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.

Nosology of genetic skeletal disorders: 2023 revision.

The "Nosology of genetic skeletal disorders" has undergone its 11th revision and now contains 771 entries associated with 552 genes reflecting advances in molecular delineation of new disorders thanks to advances in DNA sequencing technology. The most significant change as compared to previous versions is the adoption of the dyadic naming system, systematically associating a phenotypic entity with the gene it arises from. We consider this a significant step forward as dyadic naming is more informative and less prone to errors than the traditional use of list numberings and eponyms. Despite the adoption of dyadic naming, efforts have been made to maintain strong ties to the MIM catalog and its historical data. As with the previous versions, the list of disorders and genes in the Nosology may be useful in considering the differential diagnosis in the clinic, directing bioinformatic analysis of next-generation sequencing results, and providing a basis for novel advances in biology and medicine.

Failure of digit tip regeneration in the absence of Lmx1b suggests Lmx1b functions disparate from dorsoventral polarity.

Mammalian digit tip regeneration is linked to the presence of nail tissue, but a nail-explicit model is missing. Here, we report that nail-less double-ventral digits of ΔLARM1/2 mutants that lack limb-specific Lmx1b enhancers fail to regenerate. To separate the nail's effect from the lack of dorsoventral (DV) polarity, we also interrogate double-dorsal double-nail digits and show that they regenerate. Thus, DV polarity is not a prerequisite for regeneration, and the nail requirement is supported. Transcriptomic comparison between wild-type and non-regenerative ΔLARM1/2 mutant blastemas reveals differential upregulation of vascularization and connective tissue functional signatures in wild type versus upregulation of inflammation in the mutant. These results, together with the finding of Lmx1b expression in the postnatal dorsal dermis underneath the nail and uniformly in the regenerative blastema, open the possibility of additional Lmx1b roles in digit tip regeneration, in addition to the indirect effect of mediating the formation of the nail.

Cell adhesion and immune response, two main functions altered in the transcriptome of seasonally regressed testes of two mammalian species.

In species with seasonal breeding, male specimens undergo substantial testicular regression during the nonbreeding period of the year. However, the molecular mechanisms that control this biological process are largely unknown. Here, we report a transcriptomic analysis on the Iberian mole, Talpa occidentalis, in which the desquamation of live, nonapoptotic germ cells is the major cellular event responsible for testis regression. By comparing testes at different reproductive states (active, regressing, and inactive), we demonstrate that the molecular pathways controlling the cell adhesion function in the seminiferous epithelium, such as the MAPK, ERK, and TGF-ß signaling, are altered during the regression process. In addition, inactive testes display a global upregulation of genes associated with immune response, indicating a selective loss of the "immune privilege" that normally operates in sexually active testes. Interspecies comparative analyses using analogous data from the Mediterranean pine vole, a rodent species where testis regression is controlled by halting meiosis entry, revealed a common gene expression signature in the regressed testes of these two evolutionary distant species. Our study advances in the knowledge of the molecular mechanisms associated to gonadal seasonal breeding, highlighting the existence of a conserved transcriptional program of testis involution across mammalian clades.