A transcription factor atlas of directed differentiation.

Transcription factors (TFs) regulate gene programs, thereby controlling diverse cellular processes and cell states. To comprehensively understand TFs and the programs they control, we created a barcoded library of all annotated human TF splice isoforms (>3,500) and applied it to build a TF Atlas charting expression profiles of human embryonic stem cells (hESCs) overexpressing each TF at single-cell resolution. We mapped TF-induced expression profiles to reference cell types and validated candidate TFs for generation of diverse cell types, spanning all three germ layers and trophoblasts. Targeted screens with subsets of the library allowed us to create a tailored cellular disease model and integrate mRNA expression and chromatin accessibility data to identify downstream regulators. Finally, we characterized the effects of combinatorial TF overexpression by developing and validating a strategy for predicting combinations of TFs that produce target expression profiles matching reference cell types to accelerate cellular engineering efforts.

Integrated loss- and gain-of-function screens define a core network governing human embryonic stem cell behavior.

Understanding the genetic control of human embryonic stem cell function is foundational for developmental biology and regenerative medicine. Here we describe an integrated genome-scale loss- and gain-of-function screening approach to identify genetic networks governing embryonic stem cell proliferation and differentiation into the three germ layers. We identified a deep link between pluripotency maintenance and survival by showing that genetic alterations that cause pluripotency dissolution simultaneously increase apoptosis resistance. We discovered that the chromatin-modifying complex SAGA and in particular its subunit TADA2B are central regulators of pluripotency, survival, growth, and lineage specification. Joint analysis of all screens revealed that genetic alterations that broadly inhibit differentiation across multiple germ layers drive proliferation and survival under pluripotency-maintaining conditions and coincide with known cancer drivers. Our results show the power of integrated multilayer genetic screening for the robust mapping of complex genetic networks.

Haploid mouse embryonic stem cells: rapid genetic screening and germline transmission.

Most animal genomes are diploid, and mammalian development depends on specific adaptations that have evolved secondary to diploidy. Genomic imprinting and dosage compensation restrict haploid development to early embryos. Recently, haploid mammalian development has been reinvestigated since the establishment of haploid embryonic stem cells (ESCs) from mouse embryos. Haploid cells possess one copy of each gene, facilitating the generation of loss-of-function mutations in a single step. Recessive mutations can then be assessed in forward genetic screens. Applications of haploid mammalian cell systems in screens have been illustrated in several recent publications. Haploid ESCs are characterized by a wide developmental potential and can contribute to chimeric embryos and mice. Different strategies for introducing genetic modifications from haploid ESCs into the mouse germline have been further developed. Haploid ESCs therefore introduce new possibilities in mammalian genetics and could offer an unprecedented tool for genome exploration in the future.

A mutation in F-actin polymerization factor suppresses the distal arthrogryposis type 5 PIEZO2 pathogenic variant in Caenorhabditis elegans.

The mechanosensitive PIEZO channel family has been linked to over 26 disorders and diseases. Although progress has been made in understanding these channels at the structural and functional levels, the underlying mechanisms of PIEZO-associated diseases remain elusive. In this study, we engineered four PIEZO-based disease models using CRISPR/Cas9 gene editing. We performed an unbiased chemical mutagen-based genetic suppressor screen to identify putative suppressors of a conserved gain-of-function variant pezo-1[R2405P] that in human PIEZO2 causes distal arthrogryposis type 5 (DA5; p. R2718P). Electrophysiological analyses indicate that pezo-1(R2405P) is a gain-of-function allele. Using genomic mapping and whole-genome sequencing approaches, we identified a candidate suppressor allele in the C. elegans gene gex-3. This gene is an ortholog of human NCKAP1 (NCK-associated protein 1), a subunit of the Wiskott-Aldrich syndrome protein (WASP)-verprolin homologous protein (WAVE/SCAR) complex, which regulates F-actin polymerization. Depletion of gex-3 by RNAi, or with the suppressor allele gex-3(av259[L353F]), significantly increased brood size and ovulation rate, as well as alleviating the crushed oocyte phenotype of the pezo-1(R2405P) mutant. Expression of GEX-3 in the soma is required to rescue the brood size defects in pezo-1(R2405P) animals. Actin organization and orientation were disrupted and distorted in the pezo-1 mutants. Mutation of gex-3(L353F) partially alleviated these defects. The identification of gex-3 as a suppressor of the pathogenic variant pezo-1(R2405P) suggests that the PIEZO coordinates with the cytoskeleton regulator to maintain the F-actin network and provides insight into the molecular mechanisms of DA5 and other PIEZO-associated diseases.

Preimplantation genetic testing: A remarkable history of pioneering, technical challenges, innovations, and ethical considerations.

Preimplantation genetic testing (PGT) has emerged as a powerful companion to assisted reproduction technologies. The origins and history of PGT are reviewed here, along with descriptions of advances in molecular assays and sampling methods, their capabilities, and their applications in preventing genetic diseases and enhancing pregnancy outcomes. Additionally, the potential for increasing accuracy and genome coverage is considered, as well as some of the emerging ethical and legislative considerations related to the expanding capabilities of PGT.

The spectrum of phenylalanine hydroxylase variants and genotype-phenotype correlation in phenylketonuria patients in Gansu, China.

BACKGROUND: Phenylketonuria (PKU) is a common, congenital, autosomal recessive, metabolic disorder caused by Phenylalanine hydroxylase (PAH) variants. METHODS: 967 PKU patients from Gansu, China were genotyped by Sanger sequencing, multiplex ligation-dependent probe amplification, and whole exome sequencing. We analyzed the variants of PAH exons, their flanking sequences, and introns. RESULTS: The detection of deep intronic variants in PAH gene can significantly improve the genetic diagnostic rate of PKU. The distribution of PAH variants among PKU subtypes may be related to the unique genetic background in Gansu, China. CONCLUSION: The identification of PAH hotspot variants will aid the development of large-scale neonatal genetic screening for PKU. The five new PAH variants found in this study further expand the spectrum of PAH variants. Genotype-phenotype correlation analysis may help predict the prognosis of PKU patients and enable precise treatment regimens to be developed.

Germline and somatic testing for ovarian Cancer: An SGO clinical practice statement.

Germline and somatic genetic testing have become critical components of care for people with ovarian cancer. The identification of germline and somatic pathogenic variants as well as homologous recombination deficiency can contribute to the prediction of treatment response, prognostic outcome, and suitability for targeted agents (e.g. poly (ADP-ribose) polymerase (PARP) inhibitors). Furthermore, identifying germline pathogenic variants can prompt cascade genetic testing for at-risk relatives. Despite the clinical benefits and consensus recommendations from several organizations calling for universal genetic testing in ovarian cancer, only about one third of patients complete germline or somatic genetic testing. The members of the Society of Gynecologic Oncology (SGO) Clinical Practice Committee have composed this statement to provide an overview of germline and somatic genetic testing for patients with epithelial ovarian cancer, focusing on available testing modalities and options for care delivery.

Strategies for obstetricians and gynecologists to advance reproductive autonomy in a post-Roe landscape.

The monumental reversal of Roe vs Wade dramatically impacted the landscape of reproductive healthcare access in the United States. The decision most significantly affects communities that historically have been and continue to be marginalized by systemic racism, classism, and ableism within the medical system. To minimize the harm of restrictive policies that have proliferated since the Supreme Court overturned Roe, it is incumbent on obstetrician-gynecologists to modify practice patterns to meet the pressing reproductive health needs of their patients and communities. Change will require cross-discipline advocacy focused on advancing equity and supporting the framework of reproductive justice. Now, more than ever, obstetrician-gynecologists have a critical responsibility to implement new approaches to service delivery and education that will expand access to evidence-based, respectful, and person-centered family planning and early pregnancy care regardless of their practice location or subspecialty.

The psychosocial impact of prostate cancer screening for BRCA1 and BRCA2 carriers.

OBJECTIVES: To report the long-term outcomes from a longitudinal psychosocial study that forms part of the 'Identification of Men with a genetic predisposition to ProstAte Cancer: Targeted Screening in men at higher genetic risk and controls' (IMPACT) study. The IMPACT study is a multi-national study of targeted prostate cancer (PrCa) screening in individuals with a known germline pathogenic variant (GPV) in either the BReast CAncer gene 1 (BRCA1) or the BReast CAncer gene 2 (BRCA2). SUBJECTS AND METHODS: Participants enrolled in the IMPACT study were invited to complete a psychosocial questionnaire prior to each annual screening visit for a minimum of 5 years. The questionnaire included questions on sociodemographics and the following measures: Hospital Anxiety and Depression Scale, Impact of Event Scale, 36-item Short-Form Health Survey, Memorial Anxiety Scale for PrCa, Cancer Worry Scale, risk perception and knowledge. RESULTS: A total of 760 participants completed questionnaires: 207 participants with GPV in BRCA1, 265 with GPV in BRCA2 and 288 controls (non-carriers from families with a known GPV). We found no evidence of clinically concerning levels of general or cancer-specific distress or poor health-related quality of life in the cohort as a whole. Individuals in the control group had significantly less worry about PrCa compared with the carriers; however, all mean scores were low and within reported general population norms, where available. BRCA2 carriers with previously high prostate-specific antigen (PSA) levels experience a small but significant increase in PrCa anxiety (P = 0.01) and PSA-specific anxiety (P < 0.001). Cancer risk perceptions reflected information provided during genetic counselling and participants had good levels of knowledge, although this declined over time. CONCLUSION: This is the first study to report the longitudinal psychosocial impact of a targeted PrCa screening programme for BRCA1 and BRCA2 carriers. The results reassure that an annual PSA-based screening programme does not have an adverse impact on psychosocial health or health-related quality of life in these higher-risk individuals. These results are important as more PrCa screening is targeted to higher-risk groups.

Update on the practice of premarital screening for sickle cell traits in Africa: a systematic review and meta-analysis.

BACKGROUND: Screening for sickle cell traits before marriage or producing children is one of the outstanding preventive measures for sickle cell disease (SCD).The disease is a collection of inherited blood disorders that impact millions globally, with a predominant 75% occurrence in the sub-Saharan region. With increasing burden of SCD on the continent amidst a cost effective prevention method, no study has systematically reviewed or presented meta-analytic uptake or practice of premarital sickle cell trait screening. METHODS: This review systematically explored the uptake or practice of premarital genotype screening in Africa. We searched PubMed and Scopus databases for African studies on premarital screening for sickle cell traits. RESULTS: Our results indicate that the pooled uptake of premarital sickle cell trait screening in Africa is 47.82% (95% CI: [46.53-49.11]; I2: 98.95% [98.74-99.13]). Our review observed, a significant relationship between the awareness of sickle cell disease and the uptake of genotype screening; F(1, 13) = 12.04, p = 0.004). The model explained approximately 48.08% of the variation in genotype screening (R² = 0.4808) and predicted a 0.729 increase in the likelihood of genotype screening uptake for every unit rise in sickle cell disease awareness (ß = 0.729, p = 0.004). Additionally, Pearson correlation (r = 0.6934) indicated a moderately strong positive correlation between the two variables. CONCLUSION: With over 75% of the global burden of sickle cell disease domiciled in Africa, the continent cannot overlook the cost of hemoglobinopathies. The uptake of sickle cell traits screening is suboptimal across the continent. To achieve the mandate of sustainable development goal number (3); to end preventable deaths of newborns and children under 5 years of age by 2030, there is need to intensify campaigns on premarital genetic screening through education and other health promotion tools.

Expanded carrier screening for inherited genetic disease using exome and genome sequencing.

The goal of this study was to assess the feasibility of using exome (ES) and genome sequencing (GS) in guiding preconception genetic screening (PCGS) for couples who are planning to conceive by creating a workflow for identifying risk alleles for autosomal recessive (AR) and X-linked (XL) disorders without the constraints of a predetermined, targeted gene panel. There were several limitations and challenges related to reporting and the technical aspects of ES and GS, which are listed in the discussion. We selected 150 couples from a cohort of families (trios) enrolled in a research protocol where the goal was to define the genetic etiology of disease in an affected child. Pre-existing, de-identified parental sequencing data were analyzed to define variants that would place the couple at risk of having a child affected by an AR or XL disorder. We identified 17 families who would be selected for counseling about risk alleles. We noted that only 3 of these at-risk couples would be identified if we limited ourselves to the current ACMG-recommended expanded carrier screening gene panel. ES and GS successfully identified couples who are at risk of having a child with a rare AR or XL disorder that would have been missed by the current recommended guidelines. Current limitations of this approach include ethical concerns, difficulties in reporting results including variant calling due to the rare nature of some of the variants, determining which disorders to report, as well as technical difficulties in detecting certain variants such as repeat expansions.

Preimplantation genetic testing in the current era, a review.

BACKGROUND: Preimplantation genetic testing (PGT), also referred to as preimplantation genetic diagnosis (PGD), is an advanced reproductive technology used during in vitro fertilization (IVF) cycles to identify genetic abnormalities in embryos prior to their implantation. PGT is used to screen embryos for chromosomal abnormalities, monogenic disorders, and structural rearrangements. DEVELOPMENT OF PGT: Over the past few decades, PGT has undergone tremendous development, resulting in three primary forms: PGT-A, PGT-M, and PGT-SR. PGT-A is utilized for screening embryos for aneuploidies, PGT-M is used to detect disorders caused by a single gene, and PGT-SR is used to detect chromosomal abnormalities caused by structural rearrangements in the genome. PURPOSE OF REVIEW: In this review, we thoroughly summarized and reviewed PGT and discussed its pros and cons down to the minutest aspects. Additionally, recent studies that highlight the advancements of PGT in the current era, including their future perspectives, were reviewed. CONCLUSIONS: This comprehensive review aims to provide new insights into the understanding of techniques used in PGT, thereby contributing to the field of reproductive genetics.

Unlocking Glioblastoma Vulnerabilities with CRISPR-Based Genetic Screening.

Glioblastoma (GBM) is the most common malignant brain tumor in adults. Despite advancements in treatment, the prognosis for patients with GBM remains poor due to its aggressive nature and resistance to therapy. CRISPR-based genetic screening has emerged as a powerful tool for identifying genes crucial for tumor progression and treatment resistance, offering promising targets for tumor therapy. In this review, we provide an overview of the recent advancements in CRISPR-based genetic screening approaches and their applications in GBM. We highlight how these approaches have been used to uncover the genetic determinants of GBM progression and responsiveness to various therapies. Furthermore, we discuss the ongoing challenges and future directions of CRISPR-based screening methods in advancing GBM research.

The Role of Exceptionalism in the Evolution of Bioethical Regulation.

The paper aims to present a critical analysis of the phenomenon and notion of exceptionalism in bioethics. The authors demonstrate that exceptionalism pertains to phenomena that are not (yet) entirely familiar to us and could potentially bear risks regarding their regulation. After an overview of the state of the art, we briefly describe the origins and evolution of the concept, compared to exception and exclusion. In the second step, they look at the overall development debates on genetic exceptionalism, compared to other bioethical debates on exceptionalism, before presenting a detailed analysis of a specific case of early regulation of genetic screening. In the last part, the authors explain the historical background behind the connection between exceptionalism and exclusion in these debates. Their main conclusion is that while the initial stage of the discussion is shaped by the concept of exceptionalism and awareness of risks of exclusion, the later development centers around exceptions that are needed in detailed regulatory procedures.

Ocular adnexal sebaceous carcinoma in a patient with Li-Fraumeni syndrome.

Li-Fraumeni syndrome (LFS) is caused by a pathogenic germline variant at the TP53 locus and is associated with an increased predisposition to a variety of cancers. The neoplasms most frequently associated with LFS are sarcomas, breast cancer, brain tumors, and adrenocortical carcinomas. In this case report, we present a 43-year-old male diagnosed with an ocular adnexal sebaceous carcinoma of the right upper eyelid who was confirmed to have LFS with subsequent genetic testing. The mutational profile of both the patient's genetic screen and tumor sequencing were congruent, demonstrating the same pathogenic loss-of-function TP53 variant. This case report highlights the importance of pursuing genetic testing in patients with a history of multiple tumor types, particularly those with uncommon diagnoses. In this case, confirmation of LFS had important implications for personalized patient care, including identification of contraindicated treatment interventions and the imaging modalities necessary for vigilant follow-up screening.

Incidence, Origin, and Predictive Model for the Detection and Clinical Management of Segmental Aneuploidies in Human Embryos.

Despite next-generation sequencing, which now allows for the accurate detection of segmental aneuploidies from in vitro fertilization embryo biopsies, the origin and characteristics of these aneuploidies are still relatively unknown. Using a multifocal biopsy approach (four trophectoderms [TEs] and one inner cell mass [ICM] analyzed per blastocyst; n = 390), we determine the origin of the aneuploidy and the diagnostic predictive value of segmental aneuploidy detection in TE biopsies toward the ICM's chromosomal constitution. Contrary to the prevalent meiotic origin of whole-chromosome aneuploidies, we show that sub-chromosomal abnormalities in human blastocysts arise from mitotic errors in around 70% of cases. As a consequence, the positive-predictive value toward ICM configuration was significantly lower for segmental as compared to whole-chromosome aneuploidies (70.8% versus 97.18%, respectively). In order to enhance the clinical utility of reporting segmental findings in clinical TE biopsies, we have developed and clinically verified a risk stratification model based on a second TE biopsy confirmation and segmental length; this model can significantly improve the prediction of aneuploidy risk in the ICM in over 86% of clinical cases enrolled. In conclusion, we provide evidence of the predominant mitotic origin of segmental aneuploidies in preimplantation embryos and develop a risk stratification model that can help post-test genetic counseling and that facilitates the decision-making process on clinical utilization of these embryos.

A high-efficient and naked-eye visible CRISPR/Cas9 system in Arabidopsis.

MAIN CONCLUSION: Introducing 35S-dsRED2 into the Cas9 vector which expresses naked-eye visible dsRED2 greatly facilitates the genetic screening, and the WUS promoter driving the Cas9 expression can improve editing efficiency in Arabidopsis. CRISPR/Cas9-dependent genome editing has been applied to generate random insertions and deletions, targeted insertions or replacements, and precise base changes for both fundamental studies in many plant species and crop improvement. To simplify the screening procedure for target gene-edited transformants, we introduced a CaMV 35S-driven dsRED2 cassette (35S-dsRED2) into the Cas9 vector to express the naked-eye visible protein dsRED2, which can be observed under white light, greatly facilitated the genetic screening and reduced labor intensity without using any instrument. In addition, the WUS promoter was used to drive the expression of Cas9, which successfully improved the target genes editing efficiency and enabled the homozygous mutagenesis of two genes in T1 generation in Arabidopsis. Considering the conserved function and expression pattern of WUS across the plant species, this dsRED2-WUS/Cas9 system could also be used in many crops.

An intervention strategy to improve genetic testing for dilated cardiomyopathy in a heart failure clinic.

PURPOSE: Despite its clinical implications in screening and therapy, genetic testing in dilated cardiomyopathy (DCM) is underused. This study evaluated implementing a practice intervention in a heart failure clinic to automate and streamline the process of genetic testing. METHODS: Eligible patients with DCM were compared for frequency of pretest genetic education and testing during pre- and postintervention periods. The intervention comprised automated prescheduling of a cardiovascular genomics e-consult that served as a placeholder for downstream, pretest education, testing, and post-test review of genetic results. RESULTS: Patients with DCM were more likely to undergo pretest genetic education after intervention than before intervention (33.5% vs 14.8%, P < .0001). Similarly, patients with DCM were more likely to undergo genetic testing after intervention than before intervention (27.3% vs 13.0%, P = .0006). The number of patients who were diagnosed to have likely pathogenic or pathogenic genetic variants were 2 of 21 (9.5%) and 6 of 53 (11.1%) before and after intervention, respectively, and variants were present in the following genes: FLNC, TTN, DES, LMNA, PLN, and TNNT2. CONCLUSION: An intervention strategy in a heart failure clinic to increase the rates of pretest genetic education and testing in eligible patients with DCM was feasible and efficacious and may have important implications for the management of DCM.

Barriers to completion of expanded carrier screening in an inner city population.

PURPOSE: The American College of Medical Genetics and Genomics emphasizes a "consistent and equitable approach for offering carrier screening." At our academic center, publicly insured prenatal patients underwent universal expanded carrier screening (ECS) to promote equitable care. The aim of the study was to evaluate rates, time, and barriers to complete ECS. This was defined as post-test counseling and partner testing after a patient was found heterozygous for a pathogenic variant. METHODS: In this descriptive retrospective cohort study from 2018 to 2021, patients were offered ECS, consisting of 283 recessive and X-linked genes. Heterozygotes were contacted by genetic counselors (≤5 attempts) for education and partner testing. Rates of counseling, partner testing, diagnostic procedures, follow-up times, and barriers to completion were assessed. RESULTS: During this time, 643 women underwent ECS. Of these 643 women, 462 were heterozygotes and 326 of 462 had undergone counseling. Two hundred twenty-two of 462 partners obtained testing, with a median of 32 days from patient to partner result. Approximately 21 couples were heterozygous for the same pathogenic variant. One patient pursued diagnostic testing. CONCLUSION: ECS offers useful information; however, this study highlights significant barriers to completion. There was suboptimal patient follow-up and low partner screening, perhaps from insufficient time to educate and counsel. Future directions include implementing quality measures to ensure optimal completion.

Pooled genetic screens with image-based profiling.

Spatial structure in biology, spanning molecular, organellular, cellular, tissue, and organismal scales, is encoded through a combination of genetic and epigenetic factors in individual cells. Microscopy remains the most direct approach to exploring the intricate spatial complexity defining biological systems and the structured dynamic responses of these systems to perturbations. Genetic screens with deep single-cell profiling via image features or gene expression programs have the capacity to show how biological systems work in detail by cataloging many cellular phenotypes with one experimental assay. Microscopy-based cellular profiling provides information complementary to next-generation sequencing (NGS) profiling and has only recently become compatible with large-scale genetic screens. Optical screening now offers the scale needed for systematic characterization and is poised for further scale-up. We discuss how these methodologies, together with emerging technologies for genetic perturbation and microscopy-based multiplexed molecular phenotyping, are powering new approaches to reveal genotype-phenotype relationships.