Biology subjects

Conrad, D.

Publications and source records attributed to Conrad, D..

12 recordsLinked to original sources

bioRxiv2024

Deletion of an evolutionarily conserved TAD boundary compromises spermatogenesis in mice

Spermatogenesis is a complex process that can be disrupted by genetic and epigenetic changes, potentially leading to male infertility. Recent research has rapidly increased the number of protein coding mutations causally linked to impaired spermatogenesis in humans and mice. However, the role of non-coding mutations remains largely unexplored. As a case study to evaluate the effects of non-coding mutations on spermatogenesis, we first identified an evolutionarily conserved topologically associated domain (TAD) boundary near two genes with important roles in mammalian testis function: Dmrtb1 and Lrp8. We then used CRISPR-Cas9 to generate a mouse line where 26kb of the boundary was removed including a strong and evolutionarily conserved CTCF binding site. ChIP-seq and Hi-C experiments confirmed the removal of the CTCF site and a resulting increase in the DNA-DNA interactions across the domain boundary. Mutant mice displayed significant changes in testis gene expression, abnormal testis histology, a 35% drop in the estimated efficiency of spermatogenesis and a 28% decrease in daily sperm production compared to littermate controls. Despite these quantitative changes in testis function, mutant mice show no significant changes in fertility. This suggests that non-coding deletions affecting testis gene regulation may have smaller effects on fertility compared to coding mutations of the same genes. Our results demonstrate that disruption of a TAD boundary can have a negative impact on sperm production and highlight the importance of considering non-coding mutations in the analysis of patients with male infertility.

genetics↗

bioRxiv2023

AXDND1 is required to balance spermatogonial commitment and for sperm tail formation in mice and humans

Dynein complexes are large, multi-unit assemblies involved in many biological processes including male fertility via their critical roles in protein transport and axoneme motility. Previously we identified a pathogenic variant in the dynein gene AXDND1 in an infertile man. Subsequently we identified an additional four potentially compound heterozygous variants of unknown significance in AXDND1 in two additional infertile men. We thus tested the role of AXDND1 in mammalian male fertility by generating a knockout mouse model. Axdnd1-/- males were sterile at all ages but could undergo one round of histologically complete spermatogenesis. Subsequently, a progressive imbalance of spermatogonial commitment to spermatogenesis over self-renewal occurred, ultimately leading to catastrophic germ cell loss, loss of blood-testis barrier patency and immune cell infiltration. Sperm produced during the first wave of spermatogenesis were immotile due to abnormal axoneme structure, including the presence of ectopic vesicles and abnormalities in outer dense fibres and microtubule doublet structures. Sperm output was additionally compromised by a severe spermiation defect and abnormal sperm individualisation. Collectively, our data highlight the essential roles of AXDND1 as a regulator of spermatogonial commitment to spermatogenesis and during the processes of spermiogenesis where it is essential for sperm tail development, release and motility.

cell biology↗

bioRxiv2023

The Human Infertility Single-cell Testis Atlas (HISTA): An interactive molecular scRNA-Seq reference of the human testis.

BackgroundThe Human Infertility Single-cell Testis Atlas (HISTA) is an interactive web tool and a reference for navigating the transcriptome of the human testis. It was developed using joint analyses of scRNA-Seq datasets derived from a dozen donors, including healthy adult controls, juveniles, and several infertility cases. HISTA is very different than other websites of testis scRNA-seq data, providing visualization and hypothesis testing tools on a batch-removed and integrated dataset of 23429 genes measured across 26093 cells using. ObjectiveThe main goal of this manuscript is to describe HISTA in detail and highlight its unique and novel features. MethodsTherefore, we used HISTA as a guide for its application and demonstrated HISTAs translational capacity to follow up on two observations of biological relevance. ResultsOur first analytical vignette identifies novel groupings of tightly regulated long non-coding RNA (lncRNA) molecules throughout spermatogenesis, suggesting specific functional genomics of these groupings. This analysis also found highly controlled expression of pairs of sense and antisense transcripts, suggesting conjoined regulatory mechanisms. In the next investigative vignette, we examined gene patterns in undifferentiated spermatogonia (USgs). We found the NANOS family of genes function as key drivers of transcriptomic signatures involved in human spermatogonial self-renewal programming; for the first time, demonstrating the relationship of NANOS1/2/3 transcripts in humans with scRNA-seq. Discussion and ConclusionsUsing HISTA, we found new observations that contribute to unraveling the mechanisms behind transcriptional regulation and maintenance germ cells across spermatogenesis. Furthermore, our findings provide guidance on future validation studies and experimental direction. Overall, HISTA continues to be utilized in testis-related research, and thus is updated regularly with new analytical methods, visualizations, and data. We aim to have it serve as a research environment for a broad range of investigators looking to explore the testis tissue and male infertility. Availability and ImplementationHISTA is available as an interactive web tool: https://conradlab.shinyapps.io/HISTA Source code and documentation for HISTA are provided on GitHub: https://github.com/eisascience/HISTA

bioinformatics↗

bioRxiv2023

A naturally occurring variant of MBD4 causes maternal germline hypermutation in primates

As part of an ongoing genome sequencing project at the Oregon National Primate Research Center, we identified a rhesus macaque with a rare homozygous frameshift mutation in the gene Methyl-CpG binding domain 4 (MBD4). MBD4 is responsible for the repair of C>T deamination mutations at CpG locations and has been linked to somatic hypermutation and cancer predisposition in humans. We show here that MBD4-associated hypermutation also affects the germline: the 6 offspring of the MBD4-null dam have a 4-6 fold increase in de novo mutation burden. This excess burden was predominantly C>T mutations at CpG locations consistent with MBD4 loss-of-function in the dam. There was also a significant excess of C>T at CpA sites, indicating an important, underappreciated role for MBD4 to repair deamination in CpA contexts. The MBD4-null dam developed sustained eosinophilia later in life, but we saw no other signs of neoplastic processes associated with MBD4 loss-of-function in humans, nor any obvious disease in the hypermutated offspring. This work provides what is likely the first evidence for a genetic factor causing hypermutation in the maternal germline of a mammal, and adds to the very small list of naturally occurring variants known to modulate germline mutation rates in mammals.

genetics↗

bioRxiv2022

Consensus Label Propagation with Graph Convolutional Networks for Single-Cell RNA Sequencing Cell Type Annotation

MotivationSingle-cell RNA sequencing (scRNA-seq) data, annotated by cell type, is useful in a variety of downstream biological applications, such as profiling gene expression at the single-cell level. However, manually assigning these annotations with known marker genes is both time-consuming and subjective. ResultsWe present a Graph Convolutional Network (GCN) based approach to automate the annotation process. Our process builds upon existing labeling approaches, using state-of-the-art tools to find cells with highly confident label assignments through consensus and spreading these confident labels with a semi-supervised GCN. Using simulated data and two scRNA-seq data sets from different tissues, we show that our method improves accuracy over a simple consensus algorithm and the average of the underlying tools. We also compare our method to a non-parametric neighbor majority approach, showing comparable results. We then demonstrate that our GCN method allows for feature interpretation, identifying important genes for cell type classification. We present our completed pipeline, written in PyTorch, as an end-to-end tool for automating and interpreting the classification of scRNA-seq data. AvailabilityOur code for conducting the experiments in this paper and using our model is available at https://github.com/lewinsohndp/scSHARP Contactd_lewinsohn@coloradocollege.edu Supplementary informationSupplementary data are available at Journal Name online.

bioinformatics↗

bioRxiv2022

SATINN: An automated neural network-based classification of testicular sections allows for high-throughput histopathology of mouse mutants

MotivationThe mammalian testis is a complex organ with a hierarchical organization that changes smoothly and stereotypically over time in normal adults. While testis histology is already an invaluable tool for identifying and describing developmental differences in evolution and disease, methods for standardized, digital image analysis of testis are needed to expand the utility of this approach. ResultsWe developed SATINN (Software for Analysis of Testis Images with Neural Networks), a multi-level framework for automated analysis of multiplexed immunofluorescence images from mouse testis. This approach uses a convolutional neural network (CNN) to classify nuclei from seminiferous tubules into 7 distinct cell types with an accuracy of 94.2%. These cell classifications are then used in a second-level tubule CNN, which places seminiferous tubules into one of 7 distinct tubule stages with 90.4% accuracy. We further describe numerous cell- and tubule-level statistics that can be derived from wildtype testis. Finally, we demonstrate how the classifiers and derived statistics can be used to rapidly and precisely describe pathology by applying our methods to image data from two mutant mouse lines. Our results demonstrate the feasibility and potential of using computer-assisted analysis for testis histology, an area poised to evolve rapidly on the back of emerging, spatially-resolved genomic and proteomic technologies. Availability and implementationScripts to apply the methods described here are available from http://github.com/conradlab/SATINN.

bioinformatics↗

bioRxiv2021

A de novo paradigm for male infertility

IntroductionDe novo mutations (DNMs) are known to play a prominent role in sporadic disorders with reduced fitness1. We hypothesize that DNMs play an important role in male infertility and explain a significant fraction of the genetic causes of this understudied disorder. To test this hypothesis, we performed trio-based exome-sequencing in a unique cohort of 185 infertile males and their unaffected parents. Following a systematic analysis, 29 of 145 rare protein altering DNMs were classified as possibly causative of the male infertility phenotype. We observed a significant enrichment of Loss-of-Function (LoF) DNMs in LoF-intolerant genes (p-value=1.00x10-5) as well as predicted pathogenic missense DNMs in missense-intolerant genes (p-value=5.01x10-4). One DNM gene identified, RBM5, is an essential regulator of male germ cell pre-mRNA splicing2. In a follow-up study, 5 rare pathogenic missense mutations affecting this gene were observed in a cohort of 2,279 infertile patients, with no such mutations found in a cohort of 5,784 fertile men (p-value=0.009). Our results provide the first evidence for the role of DNMs in severe male infertility and point to many new candidate genes affecting fertility.

genetics↗

bioRxiv2019

Mutation of CFAP57 causes primary ciliary dyskinesia by disrupting theasymmetric targeting of a subset of ciliary inner dynein arms

Primary ciliary dyskinesia (PCD) is characterized by chronic airway disease, male infertility, and randomization of the left/right body axis, and is caused by defects of motile cilia and sperm flagella. We screened a cohort of affected individuals that lack an obvious TEM structural phenotype for pathogenic variants using whole exome capture and next generation sequencing. The population sampling probability (PSAP) algorithm identified one subject with a homozygous nonsense variant [(c.1762C>T) p.(Arg588*) exon 11] in the uncharacterized CFAP57 gene. In normal human nasal epithelial cells, CFAP57 localizes throughout the ciliary axoneme. Analysis of cells from the PCD patient shows a loss of CFAP57, reduced beat frequency, and an alteration in the ciliary waveform. Knockdown of CFAP57 in human tracheobronchial epithelial cells (hTECs) recapitulates these findings. Phylogenetic analysis showed that CFAP57 is conserved in organisms that assemble motile cilia, and CFAP57 is allelic with the BOP2 gene identified previously in Chlamydomonas. Two independent, insertional fap57 Chlamydomonas mutant strains show reduced swimming velocity and altered waveforms. Tandem mass spectroscopy showed that CFAP57 is missing, and the \"g\" inner dyneins (DHC7 and DHC3) and the \"d\" inner dynein (DHC2) are reduced. Our data demonstrate that the FAP57 protein is required for the asymmetric assembly of inner dyneins on only a subset of the microtubule doublets, and this asymmetry is essential for the generation of an effective axonemal waveform. Together, our data identifies mutations in CFAP57 as a cause of PCD with a specific defect in the inner dynein arm assembly process.\n\nSignificanceMotile cilia are found throughout eukaryotic organisms and performs essential functions. Primary ciliary dyskinesia (PCD) is a rare disease that affects the function of motile cilia. By applying a novel population sampling probability algorithm (PSAP) that uses large population sequencing databases and pathogenicity prediction algorithms, we identified a variant in an uncharacterized gene, CFAP57. This is the first reported example of PCD caused by a mutation that affects only a subset of the inner dynein arms, which are needed to generate the waveform. CFAP57 identifies an address for specific dynein arms. These findings demonstrate the effectiveness of the PSAP algorithm, expand our understanding of the positioning of dynein arms, and identify mutations in CFAP57 as a cause of PCD.

genetics↗

bioRxiv2019

The Molecular and Microbial Microenvironments in Chronically Diseased Lungs

To visualize the personalized distributions of pathogens, chemical environments including microbial metabolites, pharmaceuticals, and their metabolic products within and between human lungs afflicted with cystic fibrosis, we generated 3D microbiome and metabolome maps of six explanted lungs from three cystic fibrosis patients. These 3D spatial maps revealed that the chemical environments are variable between patients and within the lungs of each patient. Although the patients microbial ecosystems were defined by the dominant pathogen, their chemical diversity was not. Additionally, the chemical diversity between locales in lungs of the same individual sometimes exceeded inter-individual variation. Thus, the chemistry and microbiome of the explanted lungs appear to be not only personalized but also regiospecific. Previously undescribed analogs of microbial quinolones and antibiotic metabolites were also detected. Furthermore, mapping the chemical and microbial distributions allowed visualization of microbial community interactions, such as increased production of quorum sensing quinolones in locations where Pseudomonas was in contact with Staphylococcus and Granulicatella, consistent with in vitro observations of bacteria isolated from these patients. Visualization of microbe-metabolite associations within a host organ in early-stage CF disease in animal models will help elucidate a complex interplay between the presence of a given microbial structure, antibiotics, metabolism of antibiotics, microbial virulence factors, and host responses.\n\nImportanceMicrobial infections are now recognized to be polymicrobial and personalized in nature. A comprehensive analysis and understanding of the factors underlying the polymicrobial and personalized nature of infections remains limited, especially in the context of the host. By visualizing microbiomes and metabolomes of diseased human lungs, we describe how different the chemical environments are between hosts that are dominated by the same pathogen and how community interactions shape the chemical environment, or vice versa. We highlight that three-dimensional organ mapping are hypothesis building tools that allow us to design mechanistic studies aimed at addressing microbial responses to other microbes, the host, and pharmaceutical drugs.

microbiology↗

bioRxiv2018

Arnav: Site specific error models to identify variants in RNA

SummaryWe present Arnav (Analysis of RNA variants) a lightweight and easy-to-use statistical method for detecting mutations from RNA sequencing data. Site-specific error models allow Arnav to call variants with high specificity when the true variant allele fraction is unknown. We show the utility of Arnav by identifying variants using RNA sequencing data from the GTEx project.\n\nAvailability and ImplementationArnav is implemented in C++ and is distributed under the GPL license at https://github.com/gatoravi/arnav.\n\nContactaramu@wustl.edu

genomics↗

bioRxiv2018

Unified single-cell analysis of testis gene regulation and pathology in 5 mouse strains

By removing the confounding factor of cellular heterogeneity, single cell genomics can revolutionize the study of development and disease, but methods are needed to simplify comparison among individuals. To develop such a framework, we assayed the transcriptome in 62,600 single cells from the testes of wildtype mice, and mice with gonadal defects due to disruption of the genes Mlh3, Hormad1, Cul4a or Cnp. The resulting expression atlas of distinct cell clusters revealed novel markers and new insights into testis gene regulation. By jointly analysing mutant and wildtype cells using a model-based factor analysis method, SDA, we decomposed our data into 46 components that identify novel meiotic gene regulatory programmes, mutant-specific pathological processes, and technical effects. Moreover, we identify, de novo, DNA sequence motifs associated with each component, and show that SDA can be used to impute expression values from single cell data. Analysis of SDA components also led us to identify a rare population of macrophages within the seminiferous tubules of Mlh3-/- and Hormad1-/- testes, an area typically associated with immune privilege. We provide a web application to enable interactive exploration of testis gene expression and components at http://www.stats.ox.ac.uk/~wells/testisAtlas.html

genomics↗

bioRxiv2017

The complement system supports normal postnatal development and gonadal function in both sexes

Male and female infertility are clinically managed and classified as distinct diseases, and relatively little is known about mechanisms of gonadal function common to both sexes. We used genome-wide genetic analysis on 74,896 women and men to find rare genetic variants that modulate gonadal function in both sexes. This uncovered an association with variants disrupting CSMD1, a complement regulatory protein located on 8p23, in a genomic region with an exceptional evolution. We found that Csmd1 knockout mice display a diverse array of gonadal defects in both sexes, and in females, impaired mammary gland development that leads to increased offspring mortality. The complement pathway is significantly disrupted in Csmd1 mice, and further disruption of the complement pathway from joint inactivation of C3 leads to more extreme reproductive defects. Our results can explain a novel human genetic association with infertility and implicate the complement system in the normal development of postnatal tissues.

developmental biology↗