Identification of robust cellular programs using reproducible LDA that impact sex-specific disease progression in different genotypes of a mouse model of AD.

The gene expression profiles of distinct cell types reflect complex genomic interactions among multiple simultaneous biological processes within each cell that can be altered by disease progression as well as genetic background. The identification of these active cellular programs is an open challenge in the analysis of single-cell RNA-seq data. Latent Dirichlet Allocation (LDA) is a generative method used to identify recurring patterns in counts data, commonly referred to as topics that can be used to interpret the state of each cell. However, LDA's interpretability is hindered by several key factors including the hyperparameter selection of the number of topics as well as the variability in topic definitions due to random initialization. We developed Topyfic, a Reproducible LDA (rLDA) package, to accurately infer the identity and activity of cellular programs in single-cell data, providing insights into the relative contributions of each program in individual cells. We apply Topyfic to brain single-cell and single-nucleus datasets of two 5xFAD mouse models of Alzheimer's disease crossed with C57BL6/J or CAST/EiJ mice to identify distinct cell types and states in different cell types such as microglia. We find that 8-month 5xFAD/Cast F1 males show higher level of microglial activation than matching 5xFAD/BL6 F1 males, whereas female mice show similar levels of microglial activation. We show that regulatory genes such as TFs, microRNA host genes, and chromatin regulatory genes alone capture cell types and cell states. Our study highlights how topic modeling with a limited vocabulary of regulatory genes can identify gene expression programs in single-cell data in order to quantify similar and divergent cell states in distinct genotypes.

The ENCODE4 long-read RNA-seq collection reveals distinct classes of transcript structure diversity.

The majority of mammalian genes encode multiple transcript isoforms that result from differential promoter use, changes in exonic splicing, and alternative 3' end choice. Detecting and quantifying transcript isoforms across tissues, cell types, and species has been extremely challenging because transcripts are much longer than the short reads normally used for RNA-seq. By contrast, long-read RNA-seq (LR-RNA-seq) gives the complete structure of most transcripts. We sequenced 264 LR-RNA-seq PacBio libraries totaling over 1 billion circular consensus reads (CCS) for 81 unique human and mouse samples. We detect at least one full-length transcript from 87.7% of annotated human protein coding genes and a total of 200,000 full-length transcripts, 40% of which have novel exon junction chains. To capture and compute on the three sources of transcript structure diversity, we introduce a gene and transcript annotation framework that uses triplets representing the transcript start site, exon junction chain, and transcript end site of each transcript. Using triplets in a simplex representation demonstrates how promoter selection, splice pattern, and 3' processing are deployed across human tissues, with nearly half of multi-transcript protein coding genes showing a clear bias toward one of the three diversity mechanisms. Evaluated across samples, the predominantly expressed transcript changes for 74% of protein coding genes. In evolution, the human and mouse transcriptomes are globally similar in types of transcript structure diversity, yet among individual orthologous gene pairs, more than half (57.8%) show substantial differences in mechanism of diversification in matching tissues. This initial large-scale survey of human and mouse long-read transcriptomes provides a foundation for further analyses of alternative transcript usage, and is complemented by short-read and microRNA data on the same samples and by epigenome data elsewhere in the ENCODE4 collection.

Micronuclei in Kif18a mutant mice form stable micronuclear envelopes and do not promote tumorigenesis.

Micronuclei, whole or fragmented chromosomes spatially separated from the main nucleus, are associated with genomic instability and have been identified as drivers of tumorigenesis. Paradoxically, Kif18a mutant mice produce micronuclei due to asynchronous segregation of unaligned chromosomes in vivo but do not develop spontaneous tumors. We report here that micronuclei in Kif18a mutant mice form stable nuclear envelopes. Challenging Kif18a mutant mice via deletion of the Trp53 gene led to formation of thymic lymphoma with elevated levels of micronuclei. However, loss of Kif18a had modest or no effect on survival of Trp53 homozygotes and heterozygotes, respectively. Micronuclei in cultured KIF18A KO cells form stable nuclear envelopes characterized by increased recruitment of nuclear envelope components and successful expansion of decondensing chromatin compared with those induced by nocodazole washout or radiation. Lagging chromosomes were also positioned closer to the main chromatin masses in KIF18A KO cells. These data suggest that not all micronuclei actively promote tumorigenesis.

Sebaceous gland abnormalities in fatty acyl CoA reductase 2 (Far2) null mice result in primary cicatricial alopecia.

In a large scale screen for skin, hair, and nail abnormalities in null mice generated by The Jackson Laboratory's KOMP center, homozygous mutant Far2tm2b(KOMP)Wtsi/2J (hereafter referrred to as Far2-/-) mice were found to develop focal areas of alopecia as they aged. As sebocytes matured in wildtype C57BL/NJ mice they became pale with fine, uniformly sized clear lipid containing vacuoles that were released when sebocytes disintegrated in the duct. By contrast, the Far2-/- null mice had sebocytes that were similar within the gland but become brightly eosinophilic when the cells entered the sebaceous gland duct. As sebocytes disintegrated, their contents did not readily dissipate. Scattered throughout the dermis, and often at the dermal hypodermal fat junction, were dystrophic hair follicles or ruptured follicles with a foreign body granulomatous reaction surrounding free hair shafts (trichogranuloma). The Meibomian and clitoral glands (modified sebaceous glands) of Far2-/- mice showed ducts dilated to various degrees that were associated with mild changes in the sebocytes as seen in the truncal skin. Skin surface lipidomic analysis revealed a lower level of wax esters, cholesterol esters, ceramides, and diacylglycerols compared to wildtype control mice. Similar changes were described in a number of other mouse mutations that affected the sebaceous glands resulting in primary cicatricial alopecia.

Exome sequencing reveals pathogenic mutations in 91 strains of mice with Mendelian disorders.

Spontaneously arising mouse mutations have served as the foundation for understanding gene function for more than 100 years. We have used exome sequencing in an effort to identify the causative mutations for 172 distinct, spontaneously arising mouse models of Mendelian disorders, including a broad range of clinically relevant phenotypes. To analyze the resulting data, we developed an analytics pipeline that is optimized for mouse exome data and a variation database that allows for reproducible, user-defined data mining as well as nomination of mutation candidates through knowledge-based integration of sample and variant data. Using these new tools, putative pathogenic mutations were identified for 91 (53%) of the strains in our study. Despite the increased power offered by potentially unlimited pedigrees and controlled breeding, about half of our exome cases remained unsolved. Using a combination of manual analyses of exome alignments and whole-genome sequencing, we provide evidence that a large fraction of unsolved exome cases have underlying structural mutations. This result directly informs efforts to investigate the similar proportion of apparently Mendelian human phenotypes that are recalcitrant to exome sequencing.

Spontaneous asj-2J mutant mouse as a model for generalized arterial calcification of infancy: a large deletion/insertion mutation in the Enpp1 gene.

Generalized arterial calcification of infancy (GACI), an autosomal recessive disorder caused by mutations in the ENPP1 gene, manifests with extensive mineralization of the cardiovascular system. The affected individuals in most cases die within the first year of life, and there is currently no effective treatment for this disorder. In this study, we characterized a spontaneous mutant mouse, asj-2J, as a model for GACI. These mice were identified as part of a phenotypic deviant search in a large-scale production colony of BALB/cJ mice at The Jackson Laboratory. They demonstrated a characteristic gait due to stiffening of the joints, with phenotypic similarity to a previously characterized asj ("ages with stiffened joints") mouse, caused by a missense mutation in the Enpp1 gene. Complementation testing indicated that asj-2J and asj were allelic. PCR-based mutation detection strategy revealed in asj-2J mice a large, 40,035 bp, deletion spanning from intron 1 to the 3'-untranslated region of the Enpp1 gene, coupled with a 74 bp insertion. This was accompanied with a significant reduction in the plasma PPi concentration and reduced PPi/Pi ratio. As a consequence, extensive aberrant mineralization affecting the arterial vasculature, a number of internal organs, and the dermal sheath of vibrissae, a progressive biomarker of the ectopic mineralization process, was demonstrated by a combination of micro computed tomography, histopathology with calcium-specific stains, and direct chemical assay of calcium. Comparison of the asj and asj-2J mice demonstrated that the latter ones, particularly when placed on an acceleration diet high in phosphate and low in magnesium, had more extensive mineralization. Thus, the asj-2J mouse serves as a novel model for GACI, a currently intractable disorder.