Clinical and genetic study of ABCB4 gene-related cholestatic liver disease in China: children and adults.

BACKGROUND: ABCB4 gene-related cholestatic liver diseases have a wide spectrum of clinical and genetic variations. The correlation between genotype and clinical phenotype still unclear. This study retrospectively analyzed the clinical and pathological characteristics of 23 patients with ABCB4 gene-related cholestatic liver diseases. Next-generation sequencing was used to identify the genetic causes. RESULTS: The 23 included patients (15 children and 8 adults) were diagnosed as progressive familial intrahepatic cholestasis type 3 (PFIC3), drug-induced liver injury (DILI), cirrhosis cholestasis, cirrhosis, and mild liver fibrosis. Nineteen patients underwent liver pathological examination of the liver, exhibiting fibrosis, small bile duct hyperplasia, CK7(+), Cu(+), bile duct deletion, and cirrhosis. Thirty ABCB4 variants were identified, including 18 novel variants. CONCLUSION: ABCB4 gene-related cholestatic liver diseases have a wide spectrum of clinical and genetic variations. Biallelic ABCB4 mutation carriers tended to severe PFIC3, which mostly occurs in children; while ABCB4 non-biallelic variants can lead to milder ICP, LACP, DILI or overlapping, mostly in adults. Thus, the ABCB4 genotype has a specific correlation with the phenotype, but there are exceptions. Non-biallelic null mutations can cause severe diseases. The mechanisms underlying this genetic phenotype require further investigation.

A novel mutation in the NR3C1 gene associated with reversible glucocorticoid resistance.

OBJECTIVE: Glucocorticoid resistance is a rare endocrine disease caused by variants of the NR3C1 gene encoding the glucocorticoid receptor (GR). We identified a novel heterozygous variant (GRR569Q) in a patient with uncommon reversible glucocorticoid resistance syndrome. METHODS: We performed ex vivo functional characterization of the variant in patient fibroblasts and in vitro through transient transfection in undifferentiated HEK 293T cells to assess transcriptional activity, affinity, and nuclear translocation. We studied the impact of the variant on the tertiary structure of the ligand-binding domain through 3D modeling. RESULTS: The patient presented initially with an adrenal adenoma with mild autonomous cortisol secretion and undetectable adrenocorticotropin hormone (ACTH) levels. Six months after surgery, biological investigations showed elevated cortisol and ACTH (urinary free cortisol 114 µg/24 h, ACTH 10.9 pmol/L) without clinical symptoms, evoking glucocorticoid resistance syndrome. Functional characterization of the GRR569Q showed decreased expression of target genes (in response to 100 nM cortisol: SGK1 control +97% vs patient +20%, P < .0001) and impaired nuclear translocation in patient fibroblasts compared to control. Similar observations were made in transiently transfected cells, but higher cortisol concentrations overcame glucocorticoid resistance. GRR569Q showed lower ligand affinity (Kd GRWT: 1.73 nM vs GRR569Q: 4.61 nM). Tertiary structure modeling suggested a loss of hydrogen bonds between H3 and the H1-H3 loop. CONCLUSION: This is the first description of a reversible glucocorticoid resistance syndrome with effective negative feedback on corticotroph cells regarding increased plasma cortisol concentrations due to the development of mild autonomous cortisol secretion.

[Analysis of 9 patients with adolescence-onset methylenetetrahydrofolate reductase deficiency].

Objective: To explore the diagnosis and treatment of adolescence-onset methylenetetrahydrofolate reductase (MTHFR) deficiency. Methods: This was a retrospective case study. Nine patients with adolescence-onset MTHFR deficiency were diagnosed at Peking University First Hospital from January 2016 to December 2022, and followed up for more than 1 year. Their general information, clinical manifestations, laboratory tests, cranial images, MTHFR gene variants, diagnosis, treatment, and outcome were analyzed retrospectively. Results: The 9 patients came from 8 families. They had symptoms at age of 8.0 years to 17.0 years and diagnosed at 9.0 years to 17.5 years. Eight were male and 1 was female. Two patients were brothers, the elder brother developed abnormal gait at 17.0 years; and the younger brother was then diagnosed at 15.0 years of age and treated at the asymptomatic stage, who was 18.0 years old with normal condition during this study. The main manifestations of the 8 symptomatic patients included progressive dyskinesia and spastic paralysis of the lower limbs, with or without intellectual decline, cognitive impairment and behavioral abnormalities. Totally, 15 variants of MTHFR gene were identified in the 9 patients, including 8 novel variants. Five patients had brain image abnormalities. Increased plasma total homocysteine level (65-221 µmol/L) was found in all patients, and decreased to 20-70 µmol/L after treatment with betaine and calcium folinate. Besides, the 8 symptomatic patients had their behavior and cognitive problems significantly improved, with a legacy of lower limb motor disorders. Conclusions: Late-onset MTHFR deficiency can occur in adolescence. The diagnosis is usually delayed because of non-specific clinical symptoms. The test of blood total homocysteine could be used as a selective screening test. Eight novel varients of MTHFR gene were identified. Timely treatment can improve clinical condition significantly, and pre-symptomatic treatment may prevent brain damage.

Phosphoserine aminotransferase deficiency diagnosed by whole-exome sequencing and LC-MS/MS reanalysis: A case report and review of literature.

BACKGROUND: Phosphoserine aminotransferase deficiency (PSATD) is an autosomal recessive disorder associated with hypertonia, psychomotor retardation, and acquired microcephaly. Patients with PSATD have low concentrations of serine in plasma and cerebrospinal fluid. METHODS: We reported a 2-year-old female child with developmental delay, dyskinesia, and microcephaly. LC-MS/MS was used to detect amino acid concentration in the blood and whole-exome sequencing (WES) was used to identify the variants. PolyPhen-2 web server and PyMol were used to predict the pathogenicity and changes in the 3D model molecular structure of protein caused by variants. RESULTS: WES demonstrated compound heterozygous variants in PSAT1, which is associated with PSATD, with a paternal likely pathogenic variant (c.235G>A, Gly79Arg) and a maternal likely pathogenic variant (c.43G>C, Ala15Pro). Reduced serine concentration in LC-MS/MS further confirmed the diagnosis of PSATD in this patient. CONCLUSIONS: Our findings demonstrate the importance of WES combined with LC-MS/MS reanalysis in the diagnosis of genetic diseases and expand the PSAT1 variant spectrum in PSATD. Moreover, we summarize all the cases caused by PSAT1 variants in the literature. This case provides a vital reference for the diagnosis of future cases.

Molecular Insights of Cholestasis in MDR2 Knockout Murine Liver Organoids.

MDR3 (multidrug resistance 3) deficiency in humans (MDR2 in mice) causes progressive familial intrahepatic cholestasis type 3 (PFIC3). PFIC3 is a lethal disease characterized by an early onset of intrahepatic cholestasis progressing to liver cirrhosis, a preneoplastic condition, putting individuals at risk of hepatocellular carcinoma (HCC). Hepatocyte-like organoids from MDR2-deficient mice (MDR2KO) were used in this work to study the molecular alterations caused by the deficiency of this transporter. Proteomic analysis by mass spectrometry allowed characterization of 279 proteins that were differentially expressed in MDR2KO compared with wild-type organoids. Functional enrichment analysis indicated alterations in three main cellular functions: (1) interaction with the extracellular matrix, (2) remodeling intermediary metabolism, and (3) cell proliferation and differentiation. The affected cellular processes were validated by orthogonal molecular biology techniques. Our results point to molecular mechanisms associated with PFIC3 that may drive the progression to liver cirrhosis and HCC and suggest proteins and cellular processes that could be targeted for the development of early detection strategies for these severe liver diseases.

Formation of memory assemblies through the DNA-sensing TLR9 pathway.

As hippocampal neurons respond to diverse types of information1, a subset assembles into microcircuits representing a memory2. Those neurons typically undergo energy-intensive molecular adaptations, occasionally resulting in transient DNA damage3-5. Here we found discrete clusters of excitatory hippocampal CA1 neurons with persistent double-stranded DNA (dsDNA) breaks, nuclear envelope ruptures and perinuclear release of histone and dsDNA fragments hours after learning. Following these early events, some neurons acquired an inflammatory phenotype involving activation of TLR9 signalling and accumulation of centrosomal DNA damage repair complexes6. Neuron-specific knockdown of Tlr9 impaired memory while blunting contextual fear conditioning-induced changes of gene expression in specific clusters of excitatory CA1 neurons. Notably, TLR9 had an essential role in centrosome function, including DNA damage repair, ciliogenesis and build-up of perineuronal nets. We demonstrate a novel cascade of learning-induced molecular events in discrete neuronal clusters undergoing dsDNA damage and TLR9-mediated repair, resulting in their recruitment to memory circuits. With compromised TLR9 function, this fundamental memory mechanism becomes a gateway to genomic instability and cognitive impairments implicated in accelerated senescence, psychiatric disorders and neurodegenerative disorders. Maintaining the integrity of TLR9 inflammatory signalling thus emerges as a promising preventive strategy for neurocognitive deficits.

On the genetic basis of tail-loss evolution in humans and apes.

The loss of the tail is among the most notable anatomical changes to have occurred along the evolutionary lineage leading to humans and to the 'anthropomorphous apes'1-3, with a proposed role in contributing to human bipedalism4-6. Yet, the genetic mechanism that facilitated tail-loss evolution in hominoids remains unknown. Here we present evidence that an individual insertion of an Alu element in the genome of the hominoid ancestor may have contributed to tail-loss evolution. We demonstrate that this Alu element-inserted into an intron of the TBXT gene7-9-pairs with a neighbouring ancestral Alu element encoded in the reverse genomic orientation and leads to a hominoid-specific alternative splicing event. To study the effect of this splicing event, we generated multiple mouse models that express both full-length and exon-skipped isoforms of Tbxt, mimicking the expression pattern of its hominoid orthologue TBXT. Mice expressing both Tbxt isoforms exhibit a complete absence of the tail or a shortened tail depending on the relative abundance of Tbxt isoforms expressed at the embryonic tail bud. These results support the notion that the exon-skipped transcript is sufficient to induce a tail-loss phenotype. Moreover, mice expressing the exon-skipped Tbxt isoform develop neural tube defects, a condition that affects approximately 1 in 1,000 neonates in humans10. Thus, tail-loss evolution may have been associated with an adaptive cost of the potential for neural tube defects, which continue to affect human health today.

Diagnosis and management of pyruvate kinase deficiency: international expert guidelines.

Pyruvate kinase (PK) deficiency is the most common cause of chronic congenital non-spherocytic haemolytic anaemia worldwide, with an estimated prevalence of one in 100 000 to one in 300 000 people. PK deficiency results in chronic haemolytic anaemia, with wide ranging and serious consequences affecting health, quality of life, and mortality. The goal of the International Guidelines for the Diagnosis and Management of Pyruvate Kinase Deficiency was to develop evidence-based guidelines for the clinical care of patients with PK deficiency. These clinical guidelines were developed by use of GRADE methodology and the AGREE II framework. Experts were invited after consideration of area of expertise, scholarly contributions in PK deficiency, and country of practice for global representation. The expert panel included 29 expert physicians (including adult and paediatric haematologists and other subspecialists), geneticists, laboratory specialists, nurses, a guidelines methodologist, patients with PK deficiency, and caregivers from ten countries. Five key topic areas were identified, the panel prioritised key questions, and a systematic literature search was done to generate evidence summaries that were used in the development of draft recommendations. The expert panel then met in person to finalise and vote on recommendations according to a structured consensus procedure. Agreement of greater than or equal to 67% among the expert panel was required for inclusion of a recommendation in the final guideline. The expert panel agreed on 31 total recommendations across five key topics: diagnosis and genetics, monitoring and management of chronic complications, standard management of anaemia, targeted and advanced therapies, and special populations. These new guidelines should facilitate best practices and evidence-based PK deficiency care into clinical practice.

Decreased TET2/5-hmC reduces the integrity of the epidermal barrier via epigenetic dysregulation of filaggrin in psoriatic lesions.

BACKGROUND: TET2 participates in tumor progression and intrinsic immune homeostasis via epigenetic regulation. TET2 has been reported to be involved in maintaining epithelial barrier homeostasis and inflammation. Abnormal epidermal barrier function and TET2 expression have been detected in psoriatic lesions. However, the mechanisms underlying the role of TET2 in psoriasis have not yet been elucidated. OBJECTIVE: To define the role of TET2 in maintaining epithelial barrier homeostasis and the exact epigenetic mechanism in the dysfunction of the epidermal barrier in psoriasis. METHODS: We analyzed human psoriatic skin lesions and datasets from the GEO database, and detected the expression of TET2/5-hmC together with barrier molecules by immunohistochemistry. We constructed epidermal-specific TET2 knockout mice to observe the effect of TET2 deficiency on epidermal barrier function via toluidine blue penetration assay. Further, we analyzed changes in the expression of epidermal barrier molecules by immunofluorescence in TET2-specific knockout mice and psoriatic model mice. RESULTS: We found that decreased expression of TET2/5-hmC correlated with dysregulated barrier molecules in human psoriatic lesions. Epidermal-specific TET2 knockout mice showed elevated transdermal water loss associated with abnormal epidermal barrier molecules. Furthermore, we observed that TET2 knockdown in keratinocytes reduced filaggrin expression via filaggrin promoter methylation. CONCLUSION: Aberrant epidermal TET2 affects the integrity of the epidermal barrier through the epigenetic dysregulation of epidermal barrier molecules, particularly filaggrin. Reduced TET2 expression is a critical factor contributing to an abnormal epidermal barrier in psoriasis.

Factors governing the transcriptome changes and chronological lifespan of fission yeast during phosphate starvation.

Starvation of Schizosaccharomyces pombe for inorganic phosphate elicits adaptive transcriptome changes in which mRNAs driving ribosome biogenesis, tRNA biogenesis, and translation are globally downregulated, while those for autophagy and phosphate mobilization are upregulated. Here, we interrogated three components of the starvation response: upregulated autophagy; the role of transcription factor Pho7 (an activator of the PHO regulon); and upregulated expression of ecl3, one of three paralogous genes (ecl1, ecl2, and ecl3) collectively implicated in cell survival during other nutrient stresses. Ablation of autophagy factor Atg1 resulted in early demise of phosphate-starved fission yeast, as did ablation of Pho7. Transcriptome profiling of phosphate-starved pho7Δ cells highlighted Pho7 as an activator of genes involved in phosphate acquisition and mobilization, not limited to the original three-gene PHO regulon, and additional starvation-induced genes (including ecl3) not connected to phosphate dynamics. Pho7-dependent gene induction during phosphate starvation tracked with the presence of Pho7 DNA-binding elements in the gene promoter regions. Fewer ribosome protein genes were downregulated in phosphate-starved pho7Δ cells versus WT, which might contribute to their shortened lifespan. An ecl3Δ mutant elicited no gene expression changes in phosphate-replete cells and had no impact on survival during phosphate starvation. By contrast, pan-ecl deletion (ecl123Δ) curtailed lifespan during chronic phosphate starvation. Phosphate-starved ecl123Δ cells experienced a more widespread downregulation of mRNAs encoding aminoacyl tRNA synthetases vis-à-vis WT or pho7Δ cells. Collectively, these results enhance our understanding of fission yeast phosphate homeostasis and survival during nutrient deprivation.

IL-10 constrains sphingolipid metabolism to limit inflammation.

Interleukin-10 (IL-10) is a key anti-inflammatory cytokine that can limit immune cell activation and cytokine production in innate immune cell types1. Loss of IL-10 signalling results in life-threatening inflammatory bowel disease in humans and mice-however, the exact mechanism by which IL-10 signalling subdues inflammation remains unclear2-5. Here we find that increased saturated very long chain (VLC) ceramides are critical for the heightened inflammatory gene expression that is a hallmark of IL-10 deficiency. Accordingly, genetic deletion of ceramide synthase 2 (encoded by Cers2), the enzyme responsible for VLC ceramide production, limited the exacerbated inflammatory gene expression programme associated with IL-10 deficiency both in vitro and in vivo. The accumulation of saturated VLC ceramides was regulated by a decrease in metabolic flux through the de novo mono-unsaturated fatty acid synthesis pathway. Restoring mono-unsaturated fatty acid availability to cells deficient in IL-10 signalling limited saturated VLC ceramide production and the associated inflammation. Mechanistically, we find that persistent inflammation mediated by VLC ceramides is largely dependent on sustained activity of REL, an immuno-modulatory transcription factor. Together, these data indicate that an IL-10-driven fatty acid desaturation programme rewires VLC ceramide accumulation and aberrant activation of REL. These studies support the idea that fatty acid homeostasis in innate immune cells serves as a key regulatory node to control pathologic inflammation and suggests that 'metabolic correction' of VLC homeostasis could be an important strategy to normalize dysregulated inflammation caused by the absence of IL-10.

Genetic and acquired sucrase-isomaltase deficiency: A clinical review.

Genetic sucrase-isomaltase deficiency (GSID) is an inherited deficiency in the ability to digest sucrose and potentially starch due to mutations in the sucrase-isomaltase (SI) gene. Congenital sucrase-isomaltase deficiency is historically considered to be a rare condition affecting infants with chronic diarrhea as exposure to dietary sucrose begins. Growing evidence suggests that individuals with SI variants may present later in life, with symptoms overlapping with those of irritable bowel syndrome. The presence of SI genetic variants may, either alone or in combination, affect enzyme activity and lead to symptoms of different severity. As such, a more appropriate term for this inherited condition is GSID, with a recognition of a spectrum of severity and onset of presentation. Currently, disaccharidase assay on duodenal mucosal tissue homogenates is the gold standard in diagnosing SI deficiency. A deficiency in the SI enzyme can be present at birth (genetic) or acquired later, often in association with damage to the enteric brush-border membrane. Other noninvasive diagnostic alternatives such as sucrose breath tests may be useful but require further validation. Management of GSID is based on sucrose and potentially starch restriction tailored to the individual patients' tolerance and symptoms. As this approach may be challenging, additional treatment with commercially available sacrosidase is available. However, some patients may require continued starch restriction. Further research is needed to clarify the true prevalence of SI deficiency, the pathobiology of single SI heterozygous mutations, and to define optimal diagnostic and treatment algorithms in the pediatric population.

AAV-mediated hepatic expression of SLC30A10 and the Thr95Ile variant attenuates manganese excess and other phenotypes in Slc30a10-deficient mice.

The manganese (Mn) export protein SLC30A10 is essential for Mn excretion via the liver and intestines. Patients with SLC30A10 deficiency develop Mn excess, dystonia, liver disease, and polycythemia. Recent genome-wide association studies revealed a link between the SLC30A10 variant T95I and markers of liver disease. The in vivo relevance of this variant has yet to be investigated. Using in vitro and in vivo models, we explore the impact of the T95I variant on SLC30A10 function. While SLC30A10 I95 expressed at lower levels than T95 in transfected cell lines, both T95 and I95 variants protected cells similarly from Mn-induced toxicity. Adeno-associated virus 8-mediated expression of T95 or I95 SLC30A10 using the liver-specific thyroxine binding globulin promoter normalized liver Mn levels in mice with hepatocyte Slc30a10 deficiency. Furthermore, Adeno-associated virus-mediated expression of T95 or I95 SLC30A10 normalized red blood cell parameters and body weights and attenuated Mn levels and differential gene expression in livers and brains of mice with whole body Slc30a10 deficiency. While our in vivo data do not indicate that the T95I variant significantly compromises SLC30A10 function, it does reinforce the notion that the liver is a key site of SLC30A10 function. It also supports the idea that restoration of hepatic SLC30A10 expression is sufficient to attenuate phenotypes in SLC30A10 deficiency.

Satellite double-stranded RNA induces mesenchymal transition in pancreatic cancer by regulating alternative splicing.

Human satellite II (HSATII), composed of tandem repeats in pericentromeric regions, is aberrantly transcribed in epithelial cancers, particularly pancreatic cancer. Dysregulation of repetitive elements in cancer tissues can facilitate incidental dsRNA formation; however, it remains controversial whether dsRNAs play tumor-promoting or tumor-suppressing roles during cancer progression. Therefore, we focused on the double-stranded formation of HSATII RNA and explored its molecular function. The overexpression of double-stranded HSATII (dsHSATII) RNA promoted mesenchymal-like morphological changes and enhanced the invasiveness of pancreatic cancer cells. We identified an RNA-binding protein, spermatid perinuclear RNA-binding protein (STRBP), which preferentially binds to dsHSATII RNA rather than single-stranded HSATII RNA. The mesenchymal transition of dsHSATII-expressing cells was rescued by STRBP overexpression. Mechanistically, STRBP is involved in the alternative splicing of genes associated with epithelial-mesenchymal transition (EMT). We also confirmed that isoform switching of CLSTN1, driven by dsHSATII overexpression or STRBP depletion, induced EMT-like morphological changes. These findings reveal a novel tumor-promoting function of dsHSATII RNA, inducing EMT-like changes and cell invasiveness, thus enhancing our understanding of the biological significance of aberrant expression of satellite arrays in malignant tumors.

Nuclear morphology is shaped by loop-extrusion programs.

It is well established that neutrophils adopt malleable polymorphonuclear shapes to migrate through narrow interstitial tissue spaces1-3. However, how polymorphonuclear structures are assembled remains unknown4. Here we show that in neutrophil progenitors, halting loop extrusion-a motor-powered process that generates DNA loops by pulling in chromatin5-leads to the assembly of polymorphonuclear genomes. Specifically, we found that in mononuclear neutrophil progenitors, acute depletion of the loop-extrusion loading factor nipped-B-like protein (NIPBL) induced the assembly of horseshoe, banded, ringed and hypersegmented nuclear structures and led to a reduction in nuclear volume, mirroring what is observed during the differentiation of neutrophils. Depletion of NIPBL also induced cell-cycle arrest, activated a neutrophil-specific gene program and conditioned a loss of interactions across topologically associating domains to generate a chromatin architecture that resembled that of differentiated neutrophils. Removing NIPBL resulted in enrichment for mega-loops and interchromosomal hubs that contain genes associated with neutrophil-specific enhancer repertoires and an inflammatory gene program. On the basis of these observations, we propose that in neutrophil progenitors, loop-extrusion programs produce lineage-specific chromatin architectures that permit the packing of chromosomes into geometrically confined lobular structures. Our data also provide a blueprint for the assembly of polymorphonuclear structures, and point to the possibility of engineering de novo nuclear shapes to facilitate the migration of effector cells in densely populated tumorigenic environments.

Reversal of Pulmonary Hypertension in a Human-Like Model: Therapeutic Targeting of Endothelial DHFR.

BACKGROUND: Pulmonary hypertension (PH) is a progressive disorder characterized by remodeling of the pulmonary vasculature and elevated mean pulmonary arterial pressure, resulting in right heart failure. METHODS: Here, we show that direct targeting of the endothelium to uncouple eNOS (endothelial nitric oxide synthase) with DAHP (2,4-diamino 6-hydroxypyrimidine; an inhibitor of GTP cyclohydrolase 1, the rate-limiting synthetic enzyme for the critical eNOS cofactor tetrahydrobiopterin) induces human-like, time-dependent progression of PH phenotypes in mice. RESULTS: Critical phenotypic features include progressive elevation in mean pulmonary arterial pressure, right ventricular systolic blood pressure, and right ventricle (RV)/left ventricle plus septum (LV+S) weight ratio; extensive vascular remodeling of pulmonary arterioles with increased medial thickness/perivascular collagen deposition and increased expression of PCNA (proliferative cell nuclear antigen) and alpha-actin; markedly increased total and mitochondrial superoxide production, substantially reduced tetrahydrobiopterin and nitric oxide bioavailabilities; and formation of an array of human-like vascular lesions. Intriguingly, novel in-house generated endothelial-specific dihydrofolate reductase (DHFR) transgenic mice (tg-EC-DHFR) were completely protected from the pathophysiological and molecular features of PH upon DAHP treatment or hypoxia exposure. Furthermore, DHFR overexpression with a pCMV-DHFR plasmid transfection in mice after initiation of DAHP treatment completely reversed PH phenotypes. DHFR knockout mice spontaneously developed PH at baseline and had no additional deterioration in response to hypoxia, indicating an intrinsic role of DHFR deficiency in causing PH. RNA-sequencing experiments indicated great similarity in gene regulation profiles between the DAHP model and human patients with PH. CONCLUSIONS: Taken together, these results establish a novel human-like murine model of PH that has long been lacking in the field, which can be broadly used for future mechanistic and translational studies. These data also indicate that targeting endothelial DHFR deficiency represents a novel and robust therapeutic strategy for the treatment of PH.

Insights from an advisory board: Facilitating transition of care into adulthood in brain cancer survivors with acquired pediatric growth hormone deficiency.

OBJECTIVE: Children with growth hormone deficiency (GHD) face multiple challenges that can negatively impact the transition from pediatric to adult endocrinology care. For children with GHD resulting from brain cancer or its treatment, the involvement of oncology care providers and possible disease-related comorbidities add further complexity to this transition. DESIGN: An advisory board of pediatric and adult endocrinologists was convened to help better understand the unique challenges faced by childhood cancer survivors with GHD, and discuss recommendations to optimize continuity of care as these patients proceed to adulthood. Topics included the benefits and risks of growth hormone (GH) therapy in cancer survivors, the importance of initiating GH replacement therapy early in the patient's journey and continuing into adulthood, and the obstacles that can limit an effective transition to adult care for these patients. RESULTS/CONCLUSIONS: Some identified obstacles included the need to prioritize cancer treatment over treatment for GHD, a lack of patient and oncologist knowledge about the full range of benefits provided by long-term GH administration, concerns about tumor recurrence risk in cancer survivors receiving GH treatment, and suboptimal communication and coordination (e.g., referrals) between care providers, all of which could potentially result in treatment gaps or even complete loss of follow-up during the care transition. Advisors provided recommendations for increasing education for patients and care providers and improving coordination between treatment team members, both of which are intended to help improve continuity of care to maximize the health benefits of GH administration during the critical period when childhood cancer survivors transition into adulthood.

B cells orchestrate tolerance to the neuromyelitis optica autoantigen AQP4.

Neuromyelitis optica is a paradigmatic autoimmune disease of the central nervous system, in which the water-channel protein AQP4 is the target antigen1. The immunopathology in neuromyelitis optica is largely driven by autoantibodies to AQP42. However, the T cell response that is required for the generation of these anti-AQP4 antibodies is not well understood. Here we show that B cells endogenously express AQP4 in response to activation with anti-CD40 and IL-21 and are able to present their endogenous AQP4 to T cells with an AQP4-specific T cell receptor (TCR). A population of thymic B cells emulates a CD40-stimulated B cell transcriptome, including AQP4 (in mice and humans), and efficiently purges the thymic TCR repertoire of AQP4-reactive clones. Genetic ablation of Aqp4 in B cells rescues AQP4-specific TCRs despite sufficient expression of AQP4 in medullary thymic epithelial cells, and B-cell-conditional AQP4-deficient mice are fully competent to raise AQP4-specific antibodies in productive germinal-centre responses. Thus, the negative selection of AQP4-specific thymocytes is dependent on the expression and presentation of AQP4 by thymic B cells. As AQP4 is expressed in B cells in a CD40-dependent (but not AIRE-dependent) manner, we propose that thymic B cells might tolerize against a group of germinal-centre-associated antigens, including disease-relevant autoantigens such as AQP4.

Durable and efficient gene silencing in vivo by hit-and-run epigenome editing.

Permanent epigenetic silencing using programmable editors equipped with transcriptional repressors holds great promise for the treatment of human diseases1-3. However, to unlock its full therapeutic potential, an experimental confirmation of durable epigenetic silencing after the delivery of transient delivery of editors in vivo is needed. To this end, here we targeted Pcsk9, a gene expressed in hepatocytes that is involved in cholesterol homeostasis. In vitro screening of different editor designs indicated that zinc-finger proteins were the best-performing DNA-binding platform for efficient silencing of mouse Pcsk9. A single administration of lipid nanoparticles loaded with the editors' mRNAs almost halved the circulating levels of PCSK9 for nearly one year in mice. Notably, Pcsk9 silencing and accompanying epigenetic repressive marks also persisted after forced liver regeneration, further corroborating the heritability of the newly installed epigenetic state. Improvements in construct design resulted in the development of an all-in-one configuration that we term evolved engineered transcriptional repressor (EvoETR). This design, which is characterized by a high specificity profile, further reduced the circulating levels of PCSK9 in mice with an efficiency comparable with that obtained through conventional gene editing, but without causing DNA breaks. Our study lays the foundation for the development of in vivo therapeutics that are based on epigenetic silencing.