Inactivation of RB1, CDKN2A, and TP53 have distinct effects on genomic stability at side-by-side comparison in karyotypically normal cells.

Chromosomal instability is a common feature in malignant tumors. Previous studies have indicated that inactivation of the classical tumor suppressor genes RB1, CDKN2A, and TP53 may contribute to chromosomal aberrations in cancer by disrupting different aspects of the cell cycle and DNA damage checkpoint machinery. We performed a side-by-side comparison of how inactivation of each of these genes affected chromosomal stability in vitro. Using CRISPR-Cas9 technology, RB1, CDKN2A, and TP53 were independently knocked out in karyotypically normal immortalized cells, after which these cells were followed over time. Bulk RNA sequencing revealed a distinct phenotype with upregulation of pathways related to cell cycle control and proliferation in all three knockouts. Surprisingly, the RB1 and CDKN2A knocked out cell lines did not harbor more copy number aberrations than wild-type cells, despite culturing for months. The TP53-knocked out cells, in contrast, showed a massive amount of copy number alterations and saltatory evolution through whole genome duplication. This side-by-side comparison indicated that the effects on chromosomal stability from inactivation of RB1 and CDKN2A are negligible compared to inactivation of TP53, under the same conditions in a nonstressful environment, even though partly overlapping regulatory pathways are affected. Our data suggest that loss of RB1 and CDKN2A alone is not enough to trigger surviving detectable aneuploid clones while inactivation of TP53 on its own caused massive CIN leading to saltatory clonal evolution in vitro and clonal selection.

Tracing the evolution of aneuploid cancers by multiregional sequencing with CRUST.

Clonal deconvolution of mutational landscapes is crucial to understand the evolutionary dynamics of cancer. Two limiting factors for clonal deconvolution that have remained unresolved are variation in purity and chromosomal copy number across different samples of the same tumor. We developed a semi-supervised algorithm that tracks variant calls through multi-sample spatiotemporal tumor data. While normalizing allele frequencies based on purity, it also adjusts for copy number changes at clonal deconvolution. Absent à priori copy number data, it renders in silico copy number estimations from bulk sequences. Using published and simulated tumor sequences, we reliably segregated clonal/subclonal variants even at a low sequencing depth (~50×). Given at least one pure tumor sample (>70% purity), we could normalize and deconvolve paired samples down to a purity of 40%. This renders a reliable clonal reconstruction well adapted to multi-regionally sampled solid tumors, which are often aneuploid and contaminated by non-cancer cells.

Differential Activation of Immune Effector Processes in Mature Compared to Immature Sacrococcygeal Teratomas.

Objective: This study aims to characterize the molecular signatures of sacrococcygeal teratomas (SCTs). Methods: Three SCTs were analyzed with whole genome genotyping. RNA sequencing of 10 SCTs dominated by mature, immature and neuroglial elements was analyzed. Expression in SCT-samples with different elements were compared to each other and to a reference group of malignant pediatric tumors. Macrophages, T- and B-lymphocytes were detected by immunohistochemistry. Results: No chromosomal imbalances were detected. SCTs showed overexpression of genes involved in neurosignaling, DNA-binding molecules and pathways of early germ cells. Genes associated with immune effector processes were overexpressed in mature compared to immature SCTs, and immune cell infiltration was found predominantly around mature epithelial elements. Conclusion: The broad repertoire of histological elements in SCTs reflects differences in transcriptional regulation rather than differences in gene copy numbers. A paucity of immune response in immature SCTs may be a factor contributing to their uninhibited growth.

A dynamic mutational landscape associated with an inter-regionally diverse immune response in malignant rhabdoid tumour.

Malignant rhabdoid tumour (MRT) is a childhood neoplasm of high malignancy characterised by biallelic mutation and/or loss of the epigenetic master regulator SMARCB1, accompanied by no or few other oncogenic drivers. In spite of their generally low mutational burden, an intratumoural T-cell response has been reported in a subset of MRTs, indicating that immune checkpoint inhibition may be considered a viable therapy option for some patients. We assess here the evolution over time and space of predicted neoantigens and indicators of immune checkpoint status in two MRT patients who progressed under treatment. Both patients showed an accumulation of novel clonal and subclonal mutations, including predicted neoantigens, in metastases compared to their inferred ancestral clones in the primary tumours. The first patient had peritoneal metastases from an MRT of the liver. Clonal deconvolution revealed polyclonal seeding from the primary tumour to a single metastatic site, followed by a local subclonal burst of mutations. The second patient had a renal MRT with multiple pulmonary metastases, each of which could be traced back to a single genetically unique founder cell, with formation of novel subclones in two metastases. Both patients showed a regionally heterogeneous landscape of predicted neoantigens and of tumour-infiltrating lymphocytes expressing CD8 and PD1. In both patients, some tumour regions fulfilled established criteria for PD-L1 positivity (> 1% of tumour cells), while others did not. This suggests that even in a tumour type like MRT, with a single driver mutation, there can be heterogeneity in neoantigen repertoire, immune response, and biomarkers for checkpoint blockade among sampled locations. This must be taken into account when assessing progressed MRT patients for checkpoint inhibition therapy. © 2020 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of Pathological Society of Great Britain and Ireland.

A Frequency-Multiplexed Coherent Electro-optic Memory in Rare Earth Doped Nanoparticles.

Quantum memories for light are essential components in quantum technologies like long-distance quantum communication and distributed quantum computing. Recent studies have shown that long optical and spin coherence lifetimes can be observed in rare earth doped nanoparticles, opening exciting possibilities over bulk materials, e.g., for enhancing coupling to light and other quantum systems, and material design. Here, we report on coherent light storage in Eu3+:Y2O3 nanoparticles using the Stark echo modulation memory (SEMM) quantum protocol. We first measure a nearly constant Stark coefficient of 50 kHz/(V/cm) across a bandwidth of 15 GHz, which is promising for broadband operation. Storage of light is then demonstrated with an effective coherence lifetime of 5 µs. Pulses with two different frequencies are also stored, confirming frequency-multiplexing capability, and are used to demonstrate the memory high phase fidelity. These results open the way to nanoscale optical quantum memories with increased efficiency, bandwidth, and processing capabilities.

The Iroquois homeobox proteins IRX3 and IRX5 have distinct roles in Wilms tumour development and human nephrogenesis.

Wilms tumour is a paediatric malignancy with features of halted kidney development. Here, we demonstrate that the Iroquois homeobox genes IRX3 and IRX5 are essential for mammalian nephrogenesis and govern the differentiation of Wilms tumour. Knock-out Irx3- /Irx5- mice showed a strongly reduced embryonic nephron formation. In human foetal kidney and Wilms tumour, IRX5 expression was already activated in early proliferative blastema, whereas IRX3 protein levels peaked at tubular differentiation. Accordingly, an orthotopic xenograft mouse model of Wilms tumour showed that IRX3-/- cells formed bulky renal tumours dominated by immature mesenchyme and active canonical WNT/ß-catenin-signalling. In contrast, IRX5-/- cells displayed activation of Hippo and non-canonical WNT-signalling and generated small tumours with abundant tubulogenesis. Our findings suggest that promotion of IRX3 signalling or inhibition of IRX5 signalling could be a route towards differentiation therapy for Wilms tumour, in which WNT5A is a candidate molecule for enforced tubular maturation. © 2018 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of Pathological Society of Great Britain and Ireland.

Clonal evolution through genetic bottlenecks and telomere attrition: Potential threats to in vitro data reproducibility.

Tissue cultures of immortalized human cells, also known as established cell lines, are broadly accessible and cost-efficient tools for biomedical research. We here review potential genetic sources of systematic error in cell line experiments due to clonal evolution in vitro. In particular, the authors highlight alterations in telomere function over prolonged culture and population bottlenecks, respectively, as two commonly overlooked phenomena that can result in significant alterations in cell line genotypes over just one or a few passages in vitro. These alterations may include changes in mutation status of oncogenes and large scale chromosomal imbalances. We introduce a simple list of factors to be avoided in order to reduce the risk of data misinterpretation due to clonal evolution, including unacknowledged in vitro selection pressures, prolonged culture per se, harsh population size reductions, experiments at early phases after establishment, and the employment of cell lines not sufficiently analyzed by high resolution genetic techniques.

Convergent evolution of 11p allelic loss in multifocal Wilms tumors arising in WT1 mutation carriers.

Wilms tumors in patients with constitutional WT1 mutations are examples of Knudson's tumor suppressor paradigm, with somatic inactivation of the second allele occurring through 11p loss of heterozygosity. The time point of this second hit has remained unknown. We analyzed seven Wilms tumors from two patients with constitutional WT1 mutations by whole exome sequencing and genomic array. All tumors exhibited wild type WT1 loss through uniparental isodisomy. Each tumor had a unique genomic breakpoint in 11p, typically accompanied by a private activating mutation of CTNNB1. Hence, convergent evolution rather than field carcinogenesis underlies multifocal tumors in WT1 mutation carriers.

The extracellular matrix - the under-recognized element in lung disease?

The lung is composed of airways and lung parenchyma, and the extracellular matrix (ECM) contains the main building blocks of both components. The ECM provides physical support and stability to the lung, and as such it has in the past been regarded as an inert structure. More recent research has provided novel insights revealing that the ECM is also a bioactive environment that orchestrates the cellular responses in its environs. Changes in the ECM in the airway or parenchymal tissues are now recognized in the pathological profiles of many respiratory diseases, including asthma, chronic obstructive pulmonary disease (COPD), and idiopathic pulmonary fibrosis (IPF). Only recently have we begun to investigate whether these ECM changes result from the disease process, or whether they constitute a driving factor that orchestrates the pathological outcomes. This review summarizes our current knowledge of the alterations in the ECM in asthma, COPD, and IPF, and the contributions of these alterations to the pathologies. Emerging data suggest that alterations in the composition, folding or rigidity of ECM proteins may alter the functional responses of cells within their environs, and in so doing change the pathological outcomes. These characteristics highlight potential avenues for targeting lung pathologies in the future. This may ultimately contribute to a better understanding of chronic lung diseases, and novel approaches for finding therapeutic solutions. © 2016 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of Pathological Society of Great Britain and Ireland.

BCOR internal tandem duplication and YWHAE-NUTM2B/E fusion are mutually exclusive events in clear cell sarcoma of the kidney.

Clear cell sarcoma of the kidney (CCSK) is the second most common pediatric renal tumor. Two recurrent genetic aberrations have been described in CCSK. One is a fusion of YWHAE and NUTM2B/E, the other is an internal tandem duplication (ITD) in the BCOR gene. Here it is shown that YWHAE-NUTM2B/E fusion and the BCOR ITD are mutually exclusive events and activated different downstream signaling systems. This has important diagnostic implications and opens up for further mechanistic studies of CCSK pathogenesis.

ROS-induced endothelial stress contributes to pulmonary fibrosis through pericytes and Wnt signaling.

Pulmonary fibrosis is a grave diagnosis with insidious progression, generally considered as a consequence of aberrant epithelial wound healing and excessive scarring. This process is commonly modeled in animals by local bleomycin administration, resulting in peribronchial inflammation and subsequent fibrosis. We have previously described initiation and early development of distal pulmonary fibrosis following repeated subcutaneous bleomycin injections (systemic administration). The aim of this study was to identify mechanisms for the development of pulmonary fibrosis, which we hypothesize is related to endothelial stress and activation. Bleomycin was administered subcutaneously 3 times/week during 0.33-4w, and parenchymal alterations were studied. In addition, we used microvascular endothelial cells to investigate effects of bleomycin in vitro. Our results confirmed that systemic administration of bleomycin exerts oxidative stress indicated by an increase in Sod1 at 0.33, 1, and 4w (P<0.05). Endothelial cells were activated (increased CD106 expression) from 1w and onwards (P<0.05), and p21 expression was increased 2-3 times throughout the study (P<0.05) as were the number of ß-catenin-positive nuclei (P<0.001). Wnt3a was increased at 0.33, 1, and 4w (P<0.01) and Wnt5a from 1w and onwards (P<0.001). The present study suggests that bleomycin-induced reactive oxygen species (ROS) causes DNA stress affecting the endothelial niche, initiating repair processes including Wnt signaling. The repeated systemic administrations disrupt a normally fine-tuned balance in the Wnt signaling. In addition, pericyte differentiation was affected, which may have significant effects on fibrosis due to their ability to differentiate into myofibroblasts. We conclude that the endothelial niche may have an important role in the development of pulmonary fibrosis and warrants further investigations.

Versican in inflammation and tissue remodeling: the impact on lung disorders.

Versican is a proteoglycan that has many different roles in tissue homeostasis and inflammation. The biochemical structure comprises four different types of the core protein with attached glycosaminoglycans (GAGs) that can be sulfated to various extents and has the capacity to regulate differentiation of different cell types, migration, cell adhesion, proliferation, tissue stabilization and inflammation. Versican's regulatory properties are of importance during both homeostasis and changes that lead to disease progression. The GAGs that are attached to the core protein are of the chondroitin sulfate/dermatan sulfate type and are known to be important in inflammation through interactions with cytokines and growth factors. For a more complex understanding of versican, it is of importance to study the tissue niche, where the wound healing process in both healthy and diseased conditions take place. In previous studies, our group has identified changes in the amount of the multifaceted versican in chronic lung disorders such as asthma, chronic obstructive pulmonary disease, and bronchiolitis obliterans syndrome, which could be a result of pathologic, transforming growth factor ß driven, on-going remodeling processes. Reversely, the context of versican in its niche is of great importance since versican has been reported to have a beneficial role in other contexts, e.g. emphysema. Here we explore the vast mechanisms of versican in healthy lung and in lung disorders.

Hindering and buffering factors for parental sleep in neonatal care. A phenomenographic study.

AIMS AND OBJECTIVES: To explore and describe how parents of preterm and/or sick infants in neonatal care perceive their sleep. BACKGROUND: Parents experience many stressful situations when their newborn infant is preterm and/or sick. This affects bonding. By developing more family-centred care units with single-family rooms, parents are given the opportunity to stay and care for their newborn infant(s) 24 hours a day. Lack of sleep may affect new parents' ability to cope with the many challenges they face on a daily basis. DESIGN: A phenomenographic study with an inductive and exploratory design. METHODS: Semi-structured interviews were conducted with twelve parents of infants in neonatal care between January-March 2012. To describe variations in perception of the phenomenon, data were analysed using phenomenography. FINDINGS: Four descriptive categories were identified within the phenomenon sleep in parents of preterm and/or sick infants in neonatal care: impact of stress on sleep; how the environment affects sleep; keeping the family together improves sleep; and, how parents manage and prevent tiredness. CONCLUSION: Anxiety, uncertainty and powerlessness have a negative influence on sleep. This can be decreased by continuous information, guidance and practical support. Skin-to-skin care was perceived as a stress-reducing factor that improved relaxation and sleep and should be encouraged by the nurse. The parents also mentioned the importance of being together. Having a private place where they could relax and take care of themselves and their newborn infant improved sleep. It was also desirable to involve older siblings in order to decrease feelings of loneliness, sadness and isolation. RELEVANCE FOR CLINICAL PRACTICE: Improved parental sleep in neonatal care may help the families cope with the situation and facilitate problem-solving, emotional regulation and the transition to parenthood.

Clear cell sarcoma of the kidney demonstrates an embryonic signature indicative of a primitive nephrogenic origin.

Clear cell sarcoma of the kidney (CCSK) is a tumor affecting children with a median age of 3 years at diagnosis. The cell of origin of CCSK is unknown and data on the molecular changes giving rise to CCSK is scarce. This has hindered the identification of positive diagnostic markers and development of molecularly targeted treatment protocols for CCSK. We have characterized a panel of CCSK to gain information regarding its molecular profile and possible origin. High-resolution genomic analysis with single nucleotide polymorphism array of 37 tumors did not reveal any clues to the mechanisms behind tumor development as remarkably few genetic imbalances were found. Gene expression analysis revealed a highly characteristic gene signature, enriched for pathways involved in embryonic development, including kidney formation. The presence of markers for two different developmental lineages in the embryonic kidney was therefore investigated in the tumor cells. FOXD1 which identifies cells giving rise to stromal elements, and CITED1, a marker for cells primed for nephrogenic epithelial differentiation, were both highly expressed in CCSK. In addition, the early embryonic marker OSR1 was expressed at higher levels in CCSK than in Wilms tumor, normal fetal kidney or adult kidney. As this marker discriminates the intermediate mesoderm from other mesodermal structures, our study could suggest that CCSK arises from a mesodermal cell type that retains the capacity to initiate differentiation towards both nephrons and stroma, but remains locked in a primitive state.

Seroconversion for infectious pathogens among UK military personnel deployed to Afghanistan, 2008-2011.

Military personnel are at high risk of contracting vector-borne and zoonotic infections, particularly during overseas deployments, when they may be exposed to endemic or emerging infections not prevalent in their native countries. We conducted seroprevalence testing of 467 UK military personnel deployed to Helmand Province, Afghanistan, during 2008-2011 and found that up to 3.1% showed seroconversion for infection with Rickettsia spp., Coxiella burnetii, sandfly fever virus, or hantavirus; none showed seroconversion for infection with Crimean-Congo hemorrhagic fever virus. Most seroconversions occurred in personnel who did not report illness, except for those with hantavirus (70% symptomatic). These results indicate that many exposures to infectious pathogens, and potentially infections resulting from those exposures, may go unreported. Our findings reinforce the need for continued surveillance of military personnel and for education of health care providers to help recognize and prevent illnesses and transmission of pathogens during and after overseas deployments.

Fibrosis reduces severity of acute-on-chronic pancreatitis in humans.

BACKGROUND & AIMS: Acute pancreatitis (AP) and chronic pancreatitis (CP) share etiologies, but AP can be more severe and is associated with a higher rate of mortality. We investigated features of CP that protect against severe disease. The amount of intrapancreatic fat (IPF) is increased in obese patients and fibrosis is increased in patients with CP, so we studied whether fibrosis or fat regulate severity of AP attacks in patients with CP. METHODS: We reviewed records from the University of Pittsburgh Medical Center/Presbyterian Hospital Autopsy Database (1998-2008) for patients with a diagnosis of AP (n = 23), CP (n = 35), or both (AP-on-CP; n = 15). Pancreatic histology samples from these patients and 50 randomly selected controls (no pancreatic disease) were analyzed, and IPF data were correlated with computed tomography data. An adipocyte and acinar cell Transwell coculture system, with or without collagen type I, was used to study the effects of fibrosis on acinar-adipocyte interactions. We studied the effects of nonesterified fatty acids (NEFAs) and adipokines on acinar cells in culture. RESULTS: Levels of IPF were significantly higher in nonobese patients with CP than in nonobese controls. In patients with CP or AP-on-CP, areas of IPF were surrounded by significantly more fibrosis than in controls or patients with AP. Fat necrosis-associated peri-fat acinar necrosis (PFAN, indicated by NEFA spillage) contributed to most of the necrosis observed in samples from patients with AP; however, findings of peri-fat acinar necrosis and total necrosis were significantly lower in samples from patients with CP or AP-on-CP. Fibrosis appeared to wall off the fat necrosis and limit peri-fat acinar necrosis, reducing acinar necrosis. In vitro, collagen I limited the lipolytic flux between acinar cells and adipocytes and prevented increases in adipokines in the acinar compartment. This was associated with reduced acinar cell necrosis. However, NEFAs, but not adipokines, caused acinar cell necrosis. CONCLUSIONS: Based on analysis of pancreatic samples from patients with CP, AP, or AP-on-CP and in vitro studies, fibrosis reduces the severity of acute exacerbations of CP by reducing lipolytic flux between adipocytes and acinar cells.

Mechanism of transfer of functional microRNAs between mouse dendritic cells via exosomes.

Dendritic cells (DCs) are the most potent APCs. Whereas immature DCs down-regulate T-cell responses to induce/maintain immunologic tolerance, mature DCs promote immunity. To amplify their functions, DCs communicate with neighboring DCs through soluble mediators, cell-to-cell contact, and vesicle exchange. Transfer of nanovesicles (< 100 nm) derived from the endocytic pathway (termed exosomes) represents a novel mechanism of DC-to-DC communication. The facts that exosomes contain exosome-shuttle miRNAs and DC functions can be regulated by exogenous miRNAs, suggest that DC-to-DC interactions could be mediated through exosome-shuttle miRNAs, a hypothesis that remains to be tested. Importantly, the mechanism of transfer of exosome-shuttle miRNAs from the exosome lumen to the cytosol of target cells is unknown. Here, we demonstrate that DCs release exosomes with different miRNAs depending on the maturation of the DCs. By visualizing spontaneous transfer of exosomes between DCs, we demonstrate that exosomes fused with the target DCs, the latter followed by release of the exosome content into the DC cytosol. Importantly, exosome-shuttle miRNAs are functional, because they repress target mRNAs of acceptor DCs. Our findings unveil a mechanism of transfer of exosome-shuttle miRNAs between DCs and its role as a means of communication and posttranscriptional regulation between DCs.

Specific subsets of mesenchymal stroma cells to treat lung disorders--finding the Holy Grail.

Accumulating studies, both in animals and human clinical trials with mesenchymal stroma cells (MSC) support the hypothesis of therapeutic effects of these cells in various disorders. However, despite success in immune-mediated disorders such as Crohns' disease, lung disorders such as chronic obstructive pulmonary disease (COPD) and idiopathic pulmonary disease (IPF) treated with MSC have so far not yielded a revolutionary effect on clinical symptoms. Promising data on immunomodulatory effects in COPD have kept nourishing the research into finding specific traits of MSC beneficial in disease. A heterogeneous population of injected cells might drown a potential therapeutic role of a specific group of MSC. Thus careful analysis of MSC regarding their molecular capabilities such as delivering specific therapeutic vesicles to the environment, or plain cytokine/chemokine fingerprinting might prove useful in augmenting therapies against lung diseases.

Preclinical Efficacy of a PSMA-Targeted Actinium-225 Conjugate (225Ac-Macropa-Pelgifatamab): A Targeted Alpha Therapy for Prostate Cancer.

PURPOSE: Initially, prostate cancer responds to hormone therapy, but eventually resistance develops. Beta emitter-based prostate-specific membrane antigen (PSMA)-targeted radionuclide therapy is approved for the treatment of metastatic castration-resistant prostate cancer. Here we introduce a targeted alpha therapy (TAT) consisting of the PSMA antibody pelgifatamab covalently linked to a macropa chelator and labeled with actinium-225 and compare its efficacy and tolerability with other TATs. EXPERIMENTAL DESIGN: The in vitro characteristics and in vivo biodistribution, antitumor efficacy, and tolerability of 225Ac-macropa-pelgifatamab (225Ac-pelgi) and other TATs were investigated in cell line- and patient-derived prostate cancer xenograft models. The antitumor efficacy of 225Ac-pelgi was also investigated in combination with the androgen receptor inhibitor darolutamide. RESULTS: Actinium-225-labeling of 225Ac-pelgi was efficient already at room temperature. Potent in vitro cytotoxicity was seen in PSMA-expressing (LNCaP, MDA-PCa-2b, and C4-2) but not in PSMA-negative (PC-3 and DU-145) cell lines. High tumor accumulation was seen for both 225Ac-pelgi and 225Ac-DOTA-pelgi in the MDA-PCa-2b xenograft model. In the C4-2 xenograft model, 225Ac-pelgi showed enhanced antitumor efficacy with a T/Cvolume (treatment/control) ratio of 0.10 compared with 225Ac-DOTA-pelgi, 225Ac-DOTA-J591, and 227Th-HOPO-pelgifatamab (227Th-pelgi; all at 300 kBq/kg) with T/Cvolume ratios of 0.37, 0.39, and 0.33, respectively. 225Ac-pelgi was less myelosuppressive than 227Th-pelgi. 225Ac-pelgi showed dose-dependent treatment efficacy in the patient-derived KuCaP-1 model and strong combination potential with darolutamide in both cell line- (22Rv1) and patient-derived (ST1273) xenograft models. CONCLUSIONS: These results provide a strong rationale to investigate 225Ac-pelgi in patients with prostate cancer. A clinical phase I study has been initiated (NCT06052306).

GJC2 missense mutations cause human lymphedema.

Lymphedema is the clinical manifestation of defects in lymphatic structure or function. Mutations identified in genes regulating lymphatic development result in inherited lymphedema. No mutations have yet been identified in genes mediating lymphatic function that result in inherited lymphedema. Survey microarray studies comparing lymphatic and blood endothelial cells identified expression of several connexins in lymphatic endothelial cells. Additionally, gap junctions are implicated in maintaining lymphatic flow. By sequencing GJA1, GJA4, and GJC2 in a group of families with dominantly inherited lymphedema, we identified six probands with unique missense mutations in GJC2 (encoding connexin [Cx] 47). Two larger families cosegregate lymphedema and GJC2 mutation (LOD score = 6.5). We hypothesize that missense mutations in GJC2 alter gap junction function and disrupt lymphatic flow. Until now, GJC2 mutations were only thought to cause dysmyelination, with primary expression of Cx47 limited to the central nervous system. The identification of GJC2 mutations as a cause of primary lymphedema raises the possibility of novel gap-junction-modifying agents as potential therapy for some forms of lymphedema.