Key Points An international panel established the first ever diagnostic criteria for iMCD based on review of 244 clinical cases and 88 tissue samples. The criteria require multicentric lymphadenopathy with defined histopathology, ≥2 clinical/laboratory changes, and exclusion of iMCD mimics.

Multicentric Castleman disease (MCD) describes a heterogeneous group of disorders involving systemic inflammation, characteristic lymph node histopathology, and multi-organ dysfunction because of pathologic hypercytokinemia. Whereas Human Herpes Virus-8 (HHV-8) drives the hypercytokinemia in a cohort of immunocompromised patients, the etiology of HHV-8-negative MCD is idiopathic (iMCD). Recently, a limited series of iMCD cases in Japan sharing a constellation of clinical features, including thrombocytopenia (T), anasarca (A), fever (F), reticulin fibrosis (R), and organomegaly (O) has been described as TAFRO syndrome. Herein, we report clinicopathological findings on 25 patients (14 males and 11 females; 23 Japanese-born and two US-born), the largest TAFRO syndrome case series, including the first report of cases from the USA. The median age of onset was 50 years old (range: 23-72). The frequency of each feature was as follows: thrombocytopenia (21/25), anasarca (24/25), fever (21/25), organomegaly (25/25), and reticulin fibrosis (13/16). These patients frequently demonstrated abdominal pain, elevated serum alkaline phosphatase levels, and acute kidney failure. Surprisingly, none of the cases demonstrated marked hypergammoglobulinemia, which is frequently reported in iMCD. Lymph node biopsies revealed atrophic germinal centers with enlarged nuclei of endothelial cells and proliferation of endothelial venules in interfollicular zone. 23 of 25 cases were treated initially with corticosteroids; 12 patients responded poorly and required further therapy. Three patients died during the observation period (median: 9 months) because of disease progression or infections. TAFRO syndrome is a unique subtype of iMCD that demonstrates characteristic clinicopathological findings. Further study to clarify prognosis, pathophysiology, and appropriate treatment is needed.

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AID/APOBEC family cytosine deaminases, known to function in diverse cellular processes from antibody diversification to mRNA editing, have also been implicated in DNA demethylation, an important process for transcriptional activation. While oxidation-dependent pathways for demethylation have been described, pathways involving deamination of either 5-methylcytosine (mC) or 5-hydroxymethylcytosine (hmC) have emerged as alternatives. Here, we have addressed the biochemical plausibility of deamination-coupled demethylation. We found that purified AID/APOBECs have substantially reduced activity on mC relative to cytosine, their canonical substrate, and no detectable deamination of hmC. This finding was explained by the reactivity of a series of modified substrates, where steric bulk was increasingly detrimental to deamination. Further, upon AID/APOBEC overexpression, the deamination product of hmC was undetectable in genomic DNA, while oxidation intermediates remained detectable. Our results indicate that the steric requirements for cytosine deamination are one intrinsic barrier to the proposed function of deaminases in DNA demethylation.

Multicentric Castleman's disease (MCD) describes a heterogeneous group of disorders involving proliferation of morphologically benign lymphocytes due to excessive proinflammatory hypercytokinemia, most notably of interleukin-6. Patients demonstrate intense episodes of systemic inflammatory symptoms, polyclonal lymphocyte and plasma cell proliferation, autoimmune manifestations, and organ system impairment. Human herpes virus-8 (HHV-8) drives the hypercytokinemia in all HIV-positive patients and some HIV-negative patients. There is also a group of HIV-negative and HHV-8-negative patients with unknown etiology and pathophysiology, which we propose referring to as idiopathic MCD (iMCD). Here, we synthesize what is known about iMCD pathogenesis, present a new subclassification system, and propose a model of iMCD pathogenesis. MCD should be subdivided into HHV-8-associated MCD and HHV-8-negative MCD or iMCD. The lymphocyte proliferation, histopathology, and systemic features in iMCD are secondary to hypercytokinemia, which can occur with several other diseases. We propose that 1 or more of the following 3 candidate processes may drive iMCD hypercytokinemia: systemic inflammatory disease mechanisms via autoantibodies or inflammatory gene mutations, paraneoplastic syndrome mechanisms via ectopic cytokine secretion, and/or a non-HHV-8 virus. Urgent priorities include elucidating the process driving iMCD hypercytokinemia, identifying the hypercytokine-secreting cell, developing consensus criteria for diagnosis, and building a patient registry to track cases.

Histone deacetylases (HDACs) are believed to regulate gene transcription by catalyzing deacetylation reactions. HDAC3 depletion in mouse liver upregulates lipogenic genes and results in severe hepatosteatosis. Here we show that pharmacologic HDAC inhibition in primary hepatocytes causes histone hyperacetylation but does not upregulate expression of HDAC3 target genes. Meanwhile, deacetylase-dead HDAC3 mutants can rescue hepatosteatosis and repress lipogenic genes expression in HDAC3-depleted mouse liver, demonstrating that histone acetylation is insufficient to activate gene transcription. Mutations abolishing interactions with the nuclear receptor corepressor (NCOR or SMRT) render HDAC3 nonfunctional in vivo. Additionally, liver-specific knockout of NCOR, but not SMRT, causes metabolic and transcriptomal alterations resembling those of mice without hepatic HDAC3, demonstrating that interaction with NCOR is essential for deacetylase-independent function of HDAC3. These findings highlight non-enzymatic roles of a major HDAC in transcriptional regulation in vivo and warrant reconsideration of the mechanism of action of HDAC inhibitors.

A multitude of functions have evolved around cytosine within DNA, endowing the base with physiological significance beyond simple information storage. This versatility arises from enzymes that chemically modify cytosine to expand the potential of the genome. Some modifications alter coding sequences, such as deamination of cytosine by AID/APOBEC enzymes to generate immunologic or virologic diversity. Other modifications are critical to epigenetic control, altering gene expression or cellular identity. Of these, cytosine methylation is well understood, in contrast to recently discovered modifications, such as oxidation by TET enzymes to 5-hydroxymethylcytosine. Further complexity results from cytosine demethylation, an enigmatic process that impacts cellular pluripotency. Recent insights help us to propose an integrated DNA demethylation model, accounting for contributions from cytosine oxidation, deamination and base excision repair. Taken together, this rich medley of alterations renders cytosine a genomic “wild card”, whose context-dependent functions make the base far more than a static letter in the code of life.

Here we present APOBEC-Coupled Epigenetic Sequencing (ACE-Seq), a bisulfite-free method for localizing 5-hydroxymethylcytosine (5hmC) at single-base resolution with low DNA input. The method builds upon the observation that AID/APOBEC family DNA deaminase enzymes can potently discriminate between cytosine modification states, and exploits the non-destructive nature of enzymatic, rather than chemical, deamination. ACE-Seq yields high-confidence 5hmC profiles with at least 1000-fold less DNA input than conventional methods. Applying ACE-Seq to generate a base-resolution map of 5hmC in tissue-derived cortical excitatory neurons, we find that 5hmC is almost entirely confined to CG dinucleotides. The map permits cytosine, 5-methylcytosine (5mC) and 5hmC to be parsed and reveals genomic features that diverge from global patterns, including enhancers and imprinting control regions with high and low 5hmC/5mC ratios, respectively. Enzymatic deamination overcomes many challenges posed by bisulfite-based methods and expands the scope of epigenome profiling to include scarce samples and open new lines of inquiry regarding the role of cytosine modifications in genome biology.