Disruption of neural crest enhancer landscapes as an etiological mechanism for human neurocristopathies

The embryonic development of the human facial features is a highly complex mechanism which requires very exact spatial and temporal regulation of gene expression during neural crest (NC) development. NC cells (NCC) are a transient embryonic cell type with wide differentiation potential that contributes to the formation and morphogenesis of multiple tissues and organs, including many parts of the face. Just like any other cell type, NCC possess a characteristic set of enhancers that, by controlling the expression of specific genes, define cellular identity. Impairment of this regulation can lead to craniofacial malformations, such as orofacial cleft (OFC), which are frequently referred to as neurocristopathies and that represent a heavy burden on both the affected individuals and society. Understanding how genetic or structural disruption of enhancer activity during NC development can lead to human neurocristopathies is the central goal of this work. In the long term, the gained knowledge should serve to enable early detection and show potential therapeutic approaches. Here we investigate the pathomechanism of both syndromic (i.e. Branchiooculofacial Syndrome (BOFS)) and non-syndromic (i.e. OFC) neurocristopathies, by combining in vitro and in vivo NC developmental models with genetic engineering approaches and multiple genomic methods. First, we describe a unique patient with BOFS, who, in contrast to previously reported cases, does not present a heterozygous mutations within TFAP2A, a NC master regulator. Instead, the patient carries a de novo heterozygous 89 Mb inversion in which one of the breakpoints is located 40 kb downstream of TFAP2A. We first showed that this inversion separates TFAP2A from enhancers that are located within the same large topologically associating domain (TAD) and that are essential for TFAP2A expression in NCC. Importantly, using patient-specific human induced pluripotent stem cells (hiPSC) and a robust in vitro differentiation system towards NCC, we then showed that the inversion causes a loss of physical interactions between the inverted TFAP2A allele and its cognate enhancers, leading to TFAP2A monoallelic and haploinsufficient expression in human NCC. Overall, this first part provides a powerful approach to investigate the pathological mechanisms of structural variants predicted to disrupt 3D genome organization of gene regulatory landscapes and that, due to various reasons (i.e. limited access to relevant patient material, differences in gene dosage sensitivity between mice and humans, difficulties in recapitulating certain structural variants), cannot be properly evaluated in vivo. Second, we combined previously generated hNCC enhancer maps with OFC risk-loci identified through genome-wide association studies (GWAS) and, as a result, we revealed a highly conserved enhancer (i.e. Enh2p24.2) as a potential candidate harboring genetic variants involved in OFC. GWAS link common single nucleotide polymorphisms (SNPs) with quantitative traits and complex disorders. However, most disease-associated SNPs occur in non-coding regions of the human genome and consequently, the etiological relevance of these genetic variants cannot be easily connected to a gene. Nevertheless, accumulating evidences suggest that these disease-associated SNPs may contribute to human disease susceptibility by altering enhancers. Interestingly, SNPs associated with OFC are overrepresented in NCC enhancers. Therefore, we hypothesize that SNPs associated with OFC contribute to the etiology of the disorder by altering NCC enhancers and, consequently, the expression of relevant genes. Using Enh2p24.2 as a bait in circularized chromosome conformation capture sequencing (4C-seq) experiments, we identified two distally located genes, MYCN and DDX1, as its potential targets. Using in vitro and in vivo NCC developmental models, we then demonstrated that both genes are essential for normal facial development. While MYCN was not a surprising candidate to be involved in the etiology of OFC, the identification of DDX1 as a novel regulator of facial development might provide new insights into the molecular processes (e.g. transcription-coupled DNA repair) implicated in OFC and, potentially, other human neurocristopathies (e.g. neuroblastoma)

Экспериментальное обоснование эффективности газовых инфракрасных излучателей как элемента системы отопления производственных помещений

В рамках работы экспериментально установлены температурные поля поверхностей и температуры воздуха внутри производственного помещения при работе газового инфракрасного излучателя. В результате экспериментального исследования определено влияние расположения газового инфракрасного излучателя и типа поверхности пола на интенсивность теплопереноса в помещении. Разработана методика проведения эксперимента. Получены температурные поля для двух типов поверхностей пола (бетон + бетон, бетон + керамика) и проведен анализ распределений по пространственным координатам X и Y. Для установления распределения тепловой энергии излучателем, непосредственно затрачиваемой на нагрев воздуха рабочей зоны сформулирована математическая модель с описанием физической и геометрической модели.As part of the work, the temperature fields of surfaces and air temperatures inside the production room were experimentally established during operation of a gas infrared emitter. As a result of an experimental study, the influence of the location of the gas infrared emitter and the type of floor surface on the intensity of heat transfer in the room is determined. An experimental technique has been developed. Temperature fields were obtained for two types of floor surfaces (concrete + concrete, concrete + ceramics), and the distribution of spatial coordinates X and Y was analyzed. To establish the distribution of thermal energy by the emitter, directly spent on heating the air of the working area, a mathematical model is formulated with a description of the physical and geometric model

Enhancer-associated H3K4 methylation safeguards in vitro germline competence.

Funder: Studienstiftung des Deutschen VolkesGermline specification in mammals occurs through an inductive process whereby competent cells in the post-implantation epiblast differentiate into primordial germ cells (PGC). The intrinsic factors that endow epiblast cells with the competence to respond to germline inductive signals remain unknown. Single-cell RNA sequencing across multiple stages of an in vitro PGC-like cells (PGCLC) differentiation system shows that PGCLC genes initially expressed in the naïve pluripotent stage become homogeneously dismantled in germline competent epiblast like-cells (EpiLC). In contrast, the decommissioning of enhancers associated with these germline genes is incomplete. Namely, a subset of these enhancers partly retain H3K4me1, accumulate less heterochromatic marks and remain accessible and responsive to transcriptional activators. Subsequently, as in vitro germline competence is lost, these enhancers get further decommissioned and lose their responsiveness to transcriptional activators. Importantly, using H3K4me1-deficient cells, we show that the loss of this histone modification reduces the germline competence of EpiLC and decreases PGCLC differentiation efficiency. Our work suggests that, although H3K4me1 might not be essential for enhancer function, it can facilitate the (re)activation of enhancers and the establishment of gene expression programs during specific developmental transitions

Identification of de novo variants in nonsyndromic cleft lip with/without cleft palate patients with low polygenic risk scores

Background: Nonsyndromic cleft lip with/without cleft palate (nsCL/P) is a congenital malformation of multifactorial etiology. Research has identified >40 genome-wide significant risk loci, which explain less than 40% of nsCL/P heritability. Studies show that some of the hidden heritability is explained by rare penetrant variants. Methods: To identify new candidate genes, we searched for highly penetrant de novo variants (DNVs) in 50 nsCL/P patient/parent-trios with a low polygenic risk for the phenotype (discovery). We prioritized DNV-carrying candidate genes from the discovery for resequencing in independent cohorts of 1010 nsCL/P patients of diverse ethnicities and 1574 population-matched controls (replication). Segregation analyses and rare variant association in the replication cohort, in combination with additional data (genome-wide association data, expression, protein-protein-interactions), were used for final prioritization. Conclusion: In the discovery step, 60 DNVs were identified in 60 genes, including a variant in the established nsCL/P risk gene CDH1. Re-sequencing of 32 prioritized genes led to the identification of 373 rare, likely pathogenic variants. Finally, MDN1 and PAXIP1 were prioritized as top candidates. Our findings demonstrate that DNV detection, including polygenic risk score analysis, is a powerful tool for identifying nsCL/P candidate genes, which can also be applied to other multifactorial congenital malformations.Funding information: The present study was supported by the German Research Foundation (DFG)-Grants BE 3828/8-1, LU 1944/2-1, MA 2546/5-1, and LU1944/3-1. ACKNOWLEDGMENTS: The authors thank all patients, relatives, and control individuals for their participation. We thank the German support group for individuals with cleft lip and/or palate (Wolfgang Rosenthal Gesellschaft) for assistance with recruitment.We acknowledge the invaluable assistance of all clinical, laboratory, and bioinformatic personnel. The authors thank the Next Generation Sequencing Core Facility of the Medical Faculty of the University of Bonn for sequencing the samples that were used in this study. DbGaP datasets were accessed through dbGaP accession number phs000094.v1.p1 (Supplemental Acknowledgments). Finally, the authors thank the Genome Aggregation Database (gnomAD), and all groups that provided exome and genome variant data to this resource. A full list of gnomAD contributors is provided in the gnomAD flagship paper (Karczewski et al., 2020). Open Access funding enabled and organized by Projekt DEAL

Identification of de novo variants in nonsyndromic cleft lip with/without cleft palate patients with low polygenic risk scores

[Background]: Nonsyndromic cleft lip with/without cleft palate (nsCL/P) is a congenital malformation of multifactorial etiology. Research has identified >40 genome-wide significant risk loci, which explain less than 40% of nsCL/P heritability. Studies show that some of the hidden heritability is explained by rare penetrant variants. [Methods]: To identify new candidate genes, we searched for highly penetrant de novo variants (DNVs) in 50 nsCL/P patient/parent-trios with a low polygenic risk for the phenotype (discovery). We prioritized DNV-carrying candidate genes from the discovery for resequencing in independent cohorts of 1010 nsCL/P patients of diverse ethnicities and 1574 population-matched controls (replication). Segregation analyses and rare variant association in the replication cohort, in combination with additional data (genome-wide association data, expression, protein–protein-interactions), were used for final prioritization. [Conclusion]: In the discovery step, 60 DNVs were identified in 60 genes, including a variant in the established nsCL/P risk gene CDH1. Re-sequencing of 32 prioritized genes led to the identification of 373 rare, likely pathogenic variants. Finally, MDN1 and PAXIP1 were prioritized as top candidates. Our findings demonstrate that DNV detection, including polygenic risk score analysis, is a powerful tool for identifying nsCL/P candidate genes, which can also be applied to other multifactorial congenital malformations.The present study was supported by the German Research Foundation (DFG)-Grants BE 3828/8-1, LU 1944/2-1, MA 2546/5-1, and LU1944/3-1

Injectivity conditions for STFT phase retrieval on Z\mathbb{Z}, Zd\mathbb{Z}_d and Rd\mathbb{R}^d

We study the phase retrieval problem for the short-time Fourier transform on the groups $\mathbb{Z}$, $\mathbb{Z}_d$ and $\mathbb{R}^d$. As is well-known, phase retrieval is possible, once the window's ambiguity function vanishes nowhere. However, there are only few results for windows which don't meet this condition. The goal of this paper is to establish new and complete characterizations for phase retrieval with more general windows and compare them to existing results. For a fixed window, our uniqueness conditions usually only depend on the signal's support and are therefore easily comprehensible. Additionally, we discuss sharpness of both new and existing results by looking at various examples along the way