SETD2 safeguards the genome against isochromosome formation.

Isochromosomes are mirror-imaged chromosomes with simultaneous duplication and deletion of genetic material which may contain two centromeres to create isodicentric chromosomes. Although isochromosomes commonly occur in cancer and developmental disorders and promote genome instability, mechanisms that prevent isochromosomes are not well understood. We show here that the tumor suppressor and methyltransferase SETD2 is essential to prevent these errors. Using cellular and cytogenetic approaches, we demonstrate that loss of SETD2 or its epigenetic mark, histone H3 lysine 36 trimethylation (H3K36me3), results in the formation of isochromosomes as well as isodicentric and acentric chromosomes. These defects arise during DNA replication and are likely due to faulty homologous recombination by RAD52. These data provide a mechanism for isochromosome generation and demonstrate that SETD2 and H3K36me3 are essential to prevent the formation of this common mutable chromatin structure known to initiate a cascade of genomic instability in cancer.

Quantification of C. neoformans Capsule Diameter.

The polysaccharide capsule of Cryptococcus neoformans is the primary virulence factor and one of the most commonly studied aspects of this pathogenic yeast. Capsule size varies widely between strains, has the ability to grow rapidly when introduced to stressful or low-nutrient conditions, and has been positively correlated with strain virulence. For these reasons, the size of the capsule is of great interest to C. neoformans researchers. Inducing the growth of the C. neoformans capsule is used during phenotypic testing to help understand the effects of different treatments on the yeast or size differences between strains. Here, we describe one of the standard methods of capsule induction and detail two accepted methods of staining: (i) India ink, a negative stain, used in conjunction with conventional light microscopy and (ii) co-staining with fluorescent dyes of both the cell wall and capsule followed by confocal microscopy. Finally, we outline how to measure capsule diameter manually and offer a protocol for automated diameter measurement of India ink-stained samples using computational image analysis.

Distinct Patterns of Clonal Evolution Drive Myelodysplastic Syndrome Progression to Secondary Acute Myeloid Leukemia.

Clonal evolution in myelodysplastic syndrome (MDS) can result in clinical progression and secondary acute myeloid leukemia (sAML). To dissect changes in clonal architecture associated with this progression, we performed single-cell genotyping of paired MDS and sAML samples from 18 patients. Analysis of single-cell genotypes revealed patient-specific clonal evolution and enabled the assessment of single-cell mutational cooccurrence. We discovered that changes in clonal architecture proceed via distinct patterns, classified as static or dynamic, with dynamic clonal architectures having a more proliferative phenotype by blast count fold change. Proteogenomic analysis of a subset of patients confirmed that pathogenic mutations were primarily confined to primitive and mature myeloid cells, though we also identify rare but present mutations in lymphocyte subsets. Single-cell transcriptomic analysis of paired sample sets further identified gene sets and signaling pathways involved in two cases of progression. Together, these data define serial changes in the MDS clonal landscape with clinical and therapeutic implications. SIGNIFICANCE: Precise clonal trajectories in MDS progression are made possible by single-cell genomic sequencing. Here we use this technology to uncover the patterns of clonal architecture and clonal evolution that drive the transformation to secondary AML. We further define the phenotypic and transcriptional changes of disease progression at the single-cell level. See related article by Menssen et al., p. 330 (31). See related commentary by Romine and van Galen, p. 270. This article is highlighted in the In This Issue feature, p. 265.

A novel, likely pathogenic variant in UBTF-related neurodegeneration with brain atrophy is associated with a severe divergent neurodevelopmental phenotype.

BACKGROUND: A de novo, pathogenic, missense variant in UBTF, c.628G>A p.Glu210Lys, has been described as the cause of an emerging neurodegenerative disorder, Childhood-Onset Neurodegeneration with Brain Atrophy (CONDBA). The p.Glu210Lys alteration yields a positively charged stretch of three lysine residues. Functional studies confirmed this change results in a stronger interaction with negatively charged DNA and gain-of-function activity when compared to the wild-type sequence. The CONDBA phenotype reported in association with p.Glu210Lys consists of normal early-neurodevelopment followed by progressive motor, cognitive, and behavioral regression in early-to-middle childhood. METHODS AND RESULTS: The current proband presented at 9 months of age with baseline developmental delay and more extensive neuroradiological findings, including pontine hypoplasia, thalamic volume loss and signal abnormality, and hypomyelination. Like the recurrent CONDBA p.Glu210Lys variant, this novel variant, c.608A>G p.(Gln203Arg) lies within the highly conserved second HMG-box homology domain and involves the replacement of the wild-type residue with a positively charged residue, arginine. Computational structural modeling demonstrates that this amino acid substitution potentiates the interaction between UBTF and DNA, likely resulting in a gain-of-function effect for the UBTF protein, UBF. CONCLUSION: Here we present a new divergent phenotype associated with a novel, likely pathogenic, missense variant at a different position in the UBTF gene, c.608A>G p.(Gln203Arg).

The Role of Testosterone and Gibberellic Acid in the Melanization of Cryptococcus neoformans.

Cryptococcus neoformans, a spore-producing pathogenic yeast, affects immunocompromised individuals causing meningoencephalitis. Once C. neoformans is introduced via the respiratory tract, it is engulfed by macrophages and other phagocytes. One of C. neoformans's primary virulence factors is the pigment melanin, which is formed in the cell wall and protects the yeast against UV radiation and oxidizing agents produced by macrophages during phagocytosis. To better understand the observed sex bias (3:1; male:female) in C. neoformans infections, the phenotype of various virulence factors was determined in the presence of exogenous sex hormones. C. neoformans melanized faster in the presence of testosterone than it did in the presence of estradiol. Using a combination of RNA sequencing analysis and ELISA results, we identified a growth hormone, gibberellic acid (GA), produced in C. neoformans that was highly upregulated in the presence of testosterone. A variety of knockout strains of genes involved in the GA biosynthesis pathway showed significantly reduced melanization in the presence of testosterone. Additionally, inhibitors of GA also reduced melanization in the presence of testosterone. Thus, these data suggest that the gibberellic biosynthesis pathway is involved in melanization in C. neoformans, and the melanization difference observed in the presence of testosterone may be due to increased production of GA, which may partly explain the sex bias observed in C. neoformans infections.

An inherent T cell deficit in healthy males to C. neoformans infection may begin to explain the sex susceptibility in incidence of cryptococcosis.

BACKGROUND: Cryptococcus neoformans, the causative agent of cryptococcosis, causes ~ 181,000 deaths annually, with males having a higher incidence of disease than females (7M:3F). The reason for this sex bias remains unclear. We hypothesized that this disparity was due to biological differences between the male and female immune response. METHODS: Peripheral blood mononuclear cells (PBMCs) from healthy donors were isolated and infected with C. neoformans ± exogenous testosterone or 17-ß-estradiol. C. neoformans, B, T, and NK cell proliferation was quantified by flow cytometry. Cytokine analysis was conducted via protein array or ELISA. Serological testing was conducted to determine previous exposure to C. neoformans. RESULTS: C. neoformans proliferated more in male PBMCs. T cell percentages in both sexes were lower in infected versus uninfected cells. Male PBMCs had lower CD3+, CD4+, and CD8+ T cells percentages during infection compared to females. Cytokine profiles showed differences in uninfected male and female PBMCs, which subsided during infection. Only one donor was sero-negative for prior C. neoformans exposure. There was an effect of estrogen in one dataset. CONCLUSIONS: These results suggest that males show an inherent deficit in T cell response during infection, which may contribute to the increased incidence of disease in males.

An Overview of Sex Bias in C. neoformans Infections.

Cryptococcosis, a fungal disease arising from the etiologic agent Cryptococcus neoformans, sickens a quarter of a million people annually, resulting in over 180,000 deaths. Interestingly, males are affected by cryptococcosis more frequently than females, a phenomenon observed for more than a half century. This disparity is seen in both HIV− (~3M:1F) and HIV⁺ (~8M:2F) populations of cryptococcal patients. In humans, male sex is considered a pre-disposing risk factor for cryptococcosis and males suffering from the disease have more severe symptoms and poorer outcomes. There are numerous observational, clinical and epidemiological studies documenting the male disadvantage in C. neoformans but with no further explanation of cause or mechanism. Despite being commonly acknowledged, little primary research has been conducted elucidating the reasons for these differences. The research that has been conducted, however, suggests sex hormones are a likely cause. Given that the sex difference is both prevalent and accepted by many researchers in the field, it is surprising that more is not known. This review highlights the data regarding differences in sexual dimorphism in C. neoformans infections and suggests future directions to close the research gap in this area.

Size Matters: Measurement of Capsule Diameter in Cryptococcus neoformans.

The polysaccharide capsule of Cryptococcus neoformans is the primary virulence factor and one of the most commonly studied aspects of this pathogenic yeast. Capsule size can vary widely between strains, has the ability to grow rapidly when introduced to stressful or low nutrient conditions, and has been positively correlated with strain virulence. For these reasons, the size of the capsule is of great interest to C. neoformans researchers. The growth of the C. neoformans capsule is induced during phenotypic testing to help understand the effects of different treatments on the yeast or size differences between strains. Here we describe one of the standard methods of capsule induction and compare two accepted methods of staining and measuring capsule diameter: (i) India ink, a negative stain, used in conjunction with conventional light microscopy and (ii) co-staining with fluorescent dyes of both the cell wall and capsule followed by confocal microscopy. Finally, we show how measurement of capsule diameter from India ink-stained samples can be automated using computational image analysis.

Efficient replication, and evolution of Sindbis virus genomes with non-canonical 3'A/U-rich elements (NC3ARE) in neonatal mice.

Sindbis virus (SIN) is a mosquito-transmitted animal RNA virus. We previously reported that SIN genomes lacking a canonical 19 nt 3'CSE undergo novel repair processes in BHK cells to generate a library of stable atypical SIN genomes with non-canonical 3'A/U-rich elements (NC3AREs) adjacent to the 3' poly(A) tail [1]. To determine the stability and evolutionary pressures on the SIN genomes with NC3AREs to regain a 3'CSE, five representative SIN isolates and a wild type SIN were tested in newborn mice. The key findings of this study are: (a) all six SIN isolates, including those that have extensive NC3AREs in the 3'NTRs, replicate well and produce high titer viremia in newborn mice; (b) 7-9 successive passages of these isolates in newborn mice produced comparable levels of viremia; (c) while all isolates produced only small-sized plaques during primary infection in animals, both small- and large-sized plaques were generated in all other passages; (d) polymerase stuttering occurs on select 3' oligo(U) motifs to add more U residues within the NC3AREs; (e) the S3-8 isolate with an internal UAUUU motif in the 3'poly(A) tail maintains this element even after 9 passages in animals; (f) despite differences in 3'NTRs and variable tissue distribution, all SIN isolates appear to produce similar tissue pathology in infected animals. Competition experiments with wt SIN and atypical SIN isolates in BHK cells show dominance of wt SIN. As shown for BHK cells in culture, the 3'CSE of the SIN genome is not required for virus replication and genome stability in live animals. Since the NC3AREs of atypical SIN genomes are not specific to SIN replicases, alternate RNA motifs of alphavirus genome must confer specificity in template selection. These studies fulfill the need to confirm the long-term viability of atypical SIN genomes in newborn mice and offer a basis for exploring the use of atypical SIN genomes in biotechnology.