Extent to which array genotyping and imputation with large reference panels approximate deep whole-genome sequencing.

Understanding the genetic basis of human diseases and traits is dependent on the identification and accurate genotyping of genetic variants. Deep whole-genome sequencing (WGS), the gold standard technology for SNP and indel identification and genotyping, remains very expensive for most large studies. Here, we quantify the extent to which array genotyping followed by genotype imputation can approximate WGS in studies of individuals of African, Hispanic/Latino, and European ancestry in the US and of Finnish ancestry in Finland (a population isolate). For each study, we performed genotype imputation by using the genetic variants present on the Illumina Core, OmniExpress, MEGA, and Omni 2.5M arrays with the 1000G, HRC, and TOPMed imputation reference panels. Using the Omni 2.5M array and the TOPMed panel, ≥90% of bi-allelic single-nucleotide variants (SNVs) are well imputed (r2 > 0.8) down to minor-allele frequencies (MAFs) of 0.14% in African, 0.11% in Hispanic/Latino, 0.35% in European, and 0.85% in Finnish ancestries. There was little difference in TOPMed-based imputation quality among the arrays with >700k variants. Individual-level imputation quality varied widely between and within the three US studies. Imputation quality also varied across genomic regions, producing regions where even common (MAF > 5%) variants were consistently not well imputed across ancestries. The extent to which array genotyping and imputation can approximate WGS therefore depends on reference panel, genotype array, sample ancestry, and genomic location. Imputation quality by variant or genomic region can be queried with our new tool, RsqBrowser, now deployed on the Michigan Imputation Server.

Reactivation of maternal SNORD116 cluster via SETDB1 knockdown in Prader-Willi syndrome iPSCs.

Prader-Willi syndrome (PWS), a disorder of genomic imprinting, is characterized by neonatal hypotonia, hypogonadism, small hands and feet, hyperphagia and obesity in adulthood. PWS results from the loss of paternal copies of the cluster of SNORD116 C/D box snoRNAs and their host transcript, 116HG, on human chromosome 15q11-q13. We have investigated the mechanism of repression of the maternal SNORD116 cluster and 116HG. Here, we report that the zinc-finger protein ZNF274, in association with the histone H3 lysine 9 (H3K9) methyltransferase SETDB1, is part of a complex that binds to the silent maternal but not the active paternal alleles. Knockdown of SETDB1 in PWS-specific induced pluripotent cells (iPSCs) causes a decrease in the accumulation of H3K9 trimethylation (H3K9me3) at 116HG and corresponding accumulation of the active chromatin mark histone H3 lysine 4 dimethylation (H3K4me2). We also show that upon knockdown of SETDB1 in PWS-specific iPSCs, expression of maternally silenced 116HG RNA is partially restored. SETDB1 knockdown in PWS iPSCs also disrupts DNA methylation at the PWS-IC where a decrease in 5-methylcytosine is observed in association with a concomitant increase in 5-hydroxymethylcytosine. This observation suggests that the ZNF274/SETDB1 complex bound to the SNORD116 cluster may protect the PWS-IC from DNA demethylation during early development. Our findings reveal novel epigenetic mechanisms that function to repress the maternal 15q11-q13 region.

Imprinted expression of UBE3A in non-neuronal cells from a Prader-Willi syndrome patient with an atypical deletion.

Prader-Willi syndrome (PWS) and Angelman syndrome (AS) are two neurodevelopmental disorders most often caused by deletions of the same region of paternally inherited and maternally inherited human chromosome 15q, respectively. AS is a single gene disorder, caused by the loss of function of the ubiquitin ligase E3A (UBE3A) gene, while PWS is still considered a contiguous gene disorder. Rare individuals with PWS who carry atypical microdeletions on chromosome 15q have narrowed the critical region for this disorder to a 108 kb region that includes the SNORD116 snoRNA cluster and the Imprinted in Prader-Willi (IPW) non-coding RNA. Here we report the derivation of induced pluripotent stem cells (iPSCs) from a PWS patient with an atypical microdeletion that spans the PWS critical region. We show that these iPSCs express brain-specific portions of the transcripts driven by the PWS imprinting center, including the UBE3A antisense transcript (UBE3A-ATS). Furthermore, UBE3A expression is imprinted in most of these iPSCs. These data suggest that UBE3A imprinting in neurons only requires UBE3A-ATS expression, and no other neuron-specific factors. These data also suggest that a boundary element lying within the PWS critical region prevents UBE3A-ATS expression in non-neural tissues.

Variants in the ß-globin Locus are Associated with Pneumonia in African American Children.

In African American adults, the strongest genetic predictor of pneumonia appears to be the A allele of rs334, a variant in the ß-globin gene which in homozygous form causes sickle cell disease (SCD). No comparable studies have been done in African American children. We performed genome-wide association analyses of 482 African American children with documented pneumonia and 2048 African American controls using genotypes imputed from two reference panels: 1000 Genomes (1KG) (which contains rs334) and TOPMed (does not contain rs334). Using 1KG imputed genotypes, the most significant variant was rs334 (A allele (OR = 2.76 (2.21-3.74), p=5.9x10-19); using TOPMed imputed genotypes the most significant variant was rs2226952, found in the ß-globin locus control region (G allele (OR =2.14 (1.78-2.57), p = 5.1x10-16). After conditioning on rs334, the most strongly associated variant in the ß-globin locus was rs33930165, (allele T, 1KG: OR=4.09 (2.29-7.29), p=1.7x10-6; TOPMed: OR=3.58 (2.18-5.90), p=4.7x10-7), which as a compound heterozygote with rs334 A allele can cause SCD. To compare the power of different sample sets we developed a way to estimate the power of sample sets with different sample sizes, genotype arrays and imputation platforms. Our results suggest that in African American children the strongest genetic determinants of pneumonia are those that increase the risk of SCD.

Investigation of SARS-CoV-2 Infection among Companion Animals in Households with Confirmed Human COVID-19 Cases.

We aimed to characterize SARS-CoV-2 infection in companion animals living in households with COVID-19-positive people and understand the dynamics surrounding how these animals become infected. Public health investigators contacted households with at least one confirmed, symptomatic person with COVID-19 for study recruitment. Blood, nasal, and rectal swab specimens were collected from pet dogs and cats and a questionnaire was completed. Specimens were tested for SARS-CoV-2 by RT-PCR, and for neutralizing antibodies; genomic sequencing was performed on viral-positive samples. A total of 36.4% of 110 pets enrolled had evidence of infection with SARS-CoV-2. Pets were more likely to test positive if the pet was immunocompromised, and if more than one person in the home was positive for COVID-19. Among 12 multi-pet households where at least one pet was positive, 10 had at least one other pet test positive. Whole-genome sequencing revealed the genomes of viral lineages circulating in the community during the time of sample collection. Our findings suggest a high likelihood of viral transmission in households with multiple pets and when pets had very close interactions with symptomatic humans. Further surveillance studies are needed to characterize how new variants impact animals and to understand opportunities for infection and spillover in susceptible species.

Concise review: Genomic stability of human induced pluripotent stem cells.

The usefulness of human induced pluripotent stem cells (hiPSCs) in research and therapeutic applications highly relies on their genomic integrity and stability. Many laboratories including ours have addressed this concern by comparing genomic (at both karyotypic and subkaryotypic levels) and epigenomic abnormalities of hiPSC lines (derived via either DNA- or non-DNA-based methods), as well as human embryonic stem cell lines during long-term culture. A variety of methods have been used for this purpose, such as karyotyping and fluorescent in situ hybridization to detect karyotypic abnormalities, array-based comparative genomic hybridization to detect copy number variations (CNVs), single-nucleotide polymorphism-based microarrays to detect both CNVs and loss of heterozygosity, analysis of integration sites in the genome, and whole genome sequencing for protein-coding exome and DNA methylome profiling. Here, we summarize the progresses in this dynamically evolving field and also discuss how the findings apply to the study and application of hiPSCs.

Single-Cell Analysis and Tracking of Antigen-Specific T Cells: Integrating Paired Chain AIRR-Seq and Transcriptome Sequencing: A Method by the AIRR Community.

Single-cell adaptive immune receptor repertoire sequencing (scAIRR-seq) offers the possibility to access the nucleotide sequences of paired receptor chains from T-cell receptors (TCR) or B-cell receptors (BCR ). Here we describe two protocols and the downstream bioinformatic approaches that facilitate the integrated analysis of paired T-cell receptor (TR ) alpha/beta (TRA /TRB ) AIRR-seq, RNA sequencing (RNAseq), immunophenotyping, and antigen-binding information. To illustrate the methodologies with a use case, we describe how to identify, characterize, and track SARS-CoV-2-specific T cells over multiple time points following infection with the virus. The first method allows the analysis of pools of memory CD8+ cells, identifying expansions and contractions of clones of interest. The second method allows the study of rare or antigen-specific cells and allows studying their changes over time.

Inhibition of caspase-mediated anoikis is critical for basic fibroblast growth factor-sustained culture of human pluripotent stem cells.

Apoptosis and proliferation are two dynamically and tightly regulated processes that together maintain the homeostasis of renewable tissues. Anoikis is a subtype of apoptosis induced by detachment of adherent cells from the extracellular matrix. By using the defined mTeSR1 medium and collecting freshly detached cells, we found here that human pluripotent stem (PS) cells including embryonic stem (ES) cells and induced pluripotent stem cells are subject to constant anoikis in culture, which is escalated in the absence of basic fibroblast growth factor (bFGF). Withdrawal of bFGF also promotes apoptosis and differentiation of the remaining adherent cells without affecting their cell cycle progression. Insulin-like growth factor 2 (IGF2) has previously been reported to act downstream of FGF signaling to support self-renewal of human ES cells. However, we found that IGF2 cannot substitute bFGF in the TeSR1-supported culture, although endogenous IGF signaling is required to sustain self-renewal of human ES cells. On the other hand, all of the bFGF withdrawal effects observed here can be markedly prevented by the caspase inhibitor z-VAD-FMK. We further demonstrated that the bFGF-repressed anoikis is dependent on activation of ERK and AKT and associated with inhibition of Bcl-2-interacting mediator of cell death and the caspase-ROCK1-myosin signaling. Anoikis is independent of pre-detachment apoptosis and differentiation of the cells. Because previous studies of human PS cells have been focused on attached cells, our findings revealed a neglected role of bFGF in sustaining self-renewal of human PS cells: preventing them from anoikis via inhibition of caspase activation.

Determinants of pluripotency: from avian, rodents, to primates.

Since mouse embryonic stem (ES) cells was first derived in 1981, the ability of this unprecedented cell type to self-renew and differentiate without limit has revolutionized the discovery tools that are used to study gene functions and development. Furthermore, they have inspired others to hunt for similar cells from other species. The derivation of human ES cells in 1998 has accelerated these discoveries and has also widely provoked public interest, due to both the scientific significance of these cells for human tissue regeneration and the ethical disputes over the use of donated early human embryos. However, this is no longer a barrier, with the recent discovery of methods that can convert differentiated somatic cells into ES-like cells or induced pluripotent stem (iPS) cells, by using defined reprogramming factors. This review attempts to summarize the progresses in the derivation of ES cells (as well as other embryo-derived pluripotent cells) and iPS cells from various species. We will focus on the molecular and biological features of the cells, as well as the different determinants identified thus far to sustain their pluripotency.

New alleles of SIR2 define cell-cycle-specific silencing functions.

The establishment of transcriptional silencing in yeast requires cell-cycle progression, but the nature of this requirement is unknown. Sir2 is a protein deacetylase that is required for gene silencing in yeast. We have used temperature-sensitive alleles of the SIR2 gene to assess Sir2's contribution to silencing as a function of the cell cycle. When examined in vivo, these conditional alleles fall into two classes: one class exhibits a loss of silencing when raised to the nonpermissive temperature regardless of cell-cycle position, while the second class exhibits a mitosis-specific silencing defect. Alleles of the first class have a primary defect in protein deacetylase activity, while the alleles of the second class are specifically defective in Sir2-Sir4 interactions at nonpermissive temperatures. Using a SIR2 temperature-sensitive allele, we show that silencing can be established at the HML locus during progression through the G2/M-G1 interval. These results suggest that yeast heterochromatin undergoes structural transitions as a function of the cell cycle and support the existence of a critical assembly step for silent chromatin in mitosis.

Recapitulating and Correcting Marfan Syndrome in a Cellular Model.

Marfan syndrome (MFS) is a connective tissue disorder caused by mutations in FBN1 gene, which encodes a key extracellular matrix protein FIBRILLIN-1. The haplosufficiency of FBN1 has been implicated in pathogenesis of MFS with manifestations primarily in cardiovascular, muscular, and ocular tissues. Due to limitations in animal models to study the late-onset diseases, human pluripotent stem cells (PSCs) offer a homogeneic tool for dissection of cellular and molecular pathogenic mechanism for MFS in vitro. Here, we first derived induced PSCs (iPSCs) from a MFS patient with a FBN1 mutation and corrected the mutation, thereby generating an isogenic "gain-of-function" control cells for the parental MFS iPSCs. Reversely, we knocked out FBN1 in both alleles in a wild-type (WT) human embryonic stem cell (ESC) line, which served as a loss-of-function model for MFS with the WT cells as an isogenic control. Mesenchymal stem cells derived from both FBN1-mutant iPSCs and -ESCs demonstrated reduced osteogenic differentiation and microfibril formation. We further demonstrated that vascular smooth muscle cells derived from FBN1-mutant iPSCs showed less sensitivity to carbachol as demonstrated by contractility and Ca2+ influx assay, compared to the isogenic controls cells. These findings were further supported by transcriptomic anaylsis of the cells. Therefore, this study based on both gain- and loss-of-function approaches confirmed the pathogenetic role of FBN1 mutations in these MFS-related phenotypic changes.

Heterochromatin spreading at yeast telomeres occurs in M phase.

Heterochromatin regulation of gene expression exhibits epigenetic inheritance, in which some feature of the structure is retained and can reseed formation in new cells. To understand the cell-cycle events that influence heterochromatin assembly and maintenance in budding yeast, we have conducted two types of experiments. First we have examined the kinetics of heterochromatin spreading at telomeres. We have constructed a strain in which the efficient silencing of a telomere-linked URA3 gene depends on the inducible expression of the Sir3 silencing factor. Prior studies determined that S-phase passage was required for the establishment of silencing at the HM loci in yeast. We find that establishment of silencing in our strain occurs at a point coincident with mitosis and does not require S-phase passage. In addition, we find that passage through mitosis is sufficient to establish silencing at the HML locus in a strain bearing a conditional allele of SIR3. Finally, we have also assessed the stability of yeast heterochromatin in the absence of the cis-acting elements required for its establishment. We show that silencing is stable through S phase in the absence of silencers and therefore possesses the ability to self-propagate through DNA replication. However, silencing is lost in the absence of silencers during progression through M phase. These experiments point to crucial events in mitosis influencing the assembly and persistence of heterochromatin.

Role of DNMT3B in the regulation of early neural and neural crest specifiers.

The de novo DNA methyltransferase DNMT3B functions in establishing DNA methylation patterns during development. DNMT3B missense mutations cause immunodeficiency, centromere instability and facial anomalies (ICF) syndrome. The restriction of Dnmt3b expression to neural progenitor cells, as well as the mild cognitive defects observed in ICF patients, suggests that DNMT3B may play an important role in early neurogenesis. We performed RNAi knockdown of DNMT3B in human embryonic stem cells (hESCs) in order to investigate the mechanistic contribution of DNMT3B to DNA methylation and early neuronal differentiation. While DNMT3B was not required for early neuroepithelium specification, DNMT3B deficient neuroepithelium exhibited accelerated maturation with earlier expression, relative to normal hESCs, of mature neuronal markers (such as NEUROD1) and of early neuronal regional specifiers (such as those for the neural crest). Genome-wide analyses of DNA methylation by MethylC-seq identified novel regions of hypomethylation in the DNMT3B knockdowns along the X chromosome as well as pericentromeric regions, rather than changes to promoters of specific dysregulated genes. We observed a loss of H3K27me3 and the polycomb complex protein EZH2 at the promoters of early neural and neural crest specifier genes during differentiation of DNMT3B knockdown but not normal hESCs. Our results indicate that DNMT3B mediates large-scale methylation patterns in hESCs and that DNMT3B deficiency in the cells alters the timing of their neuronal differentiation and maturation.

H2A.Z (Htz1) controls the cell-cycle-dependent establishment of transcriptional silencing at Saccharomyces cerevisiae telomeres.

The establishment of transcriptional silencing in Saccharomyces cerevisiae requires progression through the cell cycle. We have previously found that transit through M-phase is necessary and sufficient to establish silencing at telomeres following induction of the Sir3 silencing factor. In this study we find that halting cell-cycle progression in either G(1) or at the beginning of M-phase limits the ability of Sir3 to associate with a telomere-linked reporter gene and prevents the changes in histone modifications associated with gene repression. Deletion of genes coding for the histone variant H2A.Z (Htz1 in yeast) and histone acetyltransferase Sas2 abolish the cell-cycle progression requirement for the establishment of silencing. Cells blocked in telophase (but not at metaphase) are also able to establish silencing. We show that H2A.Z binds to the promoter of our telomere-linked reporter gene and that this binding diminishes in silenced cells. Finally, we observe a specific displacement of H2A.Z from chromatin in telophase-blocked cells, regardless of the silencing status of the reporter gene. These results suggest that the requirement for M-phase in the establishment of silencing may reflect a cell-cycle regulated relaxation of heterochromatin barriers.

Human embryonic stem cell derivation, maintenance, and differentiation to trophoblast.

Since the first report of derivation of human embryonic stem cell (hESC) lines in 1998, many progresses have been achieved to reliably and efficiently derive, maintain, and differentiate this therapeutically promising cell type. This chapter introduces some basic and widely recognized methods that we use in our hESC core laboratory. Specifically, it includes methods for (1) deriving hESC lines without using enzyme and antibody to isolate the inner cell mass; (2) sustaining hESC self-renewal under feeder-dependent, feeder-conditioned, and defined conditions as well as pluripotency validation and quality control assays; and (3) inducing hESC differentiation to trophoblast with BMP4.

Specification of region-specific neurons including forebrain glutamatergic neurons from human induced pluripotent stem cells.

BACKGROUND: Directed differentiation of human induced pluripotent stem cells (hiPSC) into functional, region-specific neural cells is a key step to realizing their therapeutic promise to treat various neural disorders, which awaits detailed elucidation. METHODOLOGY/PRINCIPAL FINDINGS: We analyzed neural differentiation from various hiPSC lines generated by others and ourselves. Although heterogeneity in efficiency of neuroepithelial (NE) cell differentiation was observed among different hiPSC lines, the NE differentiation process resembles that from human embryonic stem cells (hESC) in morphology, timing, transcriptional profile, and requirement for FGF signaling. NE cells differentiated from hiPSC, like those from hESC, can also form rostral phenotypes by default, and form the midbrain or spinal progenitors upon caudalization by morphogens. The rostrocaudal neural progenitors can further mature to develop forebrain glutamatergic projection neurons, midbrain dopaminergic neurons, and spinal motor neurons, respectively. Typical ion channels and action potentials were recorded in the hiPSC-derived neurons. CONCLUSIONS/SIGNIFICANCE: Our results demonstrate that hiPSC, regardless of how they were derived, can differentiate into a spectrum of rostrocaudal neurons with functionality, which supports the considerable value of hiPSC for study and treatment of patient-specific neural disorders.