Tumor-Induced Generation of Splenic Erythroblast-like Ter-Cells Promotes Tumor Progression.

Identifying tumor-induced leukocyte subsets and their derived circulating factors has been instrumental in understanding cancer as a systemic disease. Nevertheless, how primary tumor-induced non-leukocyte populations in distal organs contribute to systemic spread remains poorly defined. Here, we report one population of tumor-inducible, erythroblast-like cells (Ter-cells) deriving from megakaryocyte-erythroid progenitor cells with a unique Ter-119+CD45-CD71+ phenotype. Ter-cells are enriched in the enlarged spleen of hosts bearing advanced tumors and facilitate tumor progression by secreting neurotrophic factor artemin into the blood. Transforming growth factor ß (TGF-ß) and Smad3 activation are important in Ter-cell generation. In vivo blockade of Ter-cell-derived artemin inhibits hepatocellular carcinoma (HCC) growth, and artemin deficiency abolishes Ter-cells' tumor-promoting ability. We confirm the presence of splenic artemin-positive Ter-cells in human HCC patients and show that significantly elevated serum artemin correlates with poor prognosis. We propose that Ter-cells and the secreted artemin play important roles in cancer progression with prognostic and therapeutic implications.

Ret is a multifunctional coreceptor that integrates diffusible- and contact-axon guidance signals.

Growing axons encounter multiple guidance cues, but it is unclear how separate signals are resolved and integrated into coherent instructions for growth cone navigation. We report that glycosylphosphatidylinositol (GPI)-anchored ephrin-As function as "reverse" signaling receptors for motor axons when contacted by transmembrane EphAs present in the dorsal limb. Ephrin-A receptors are thought to depend on transmembrane coreceptors for transmitting signals intracellularly. We show that the receptor tyrosine kinase Ret is required for motor axon attraction mediated by ephrin-A reverse signaling. Ret also mediates GPI-anchored GFRα1 signaling in response to GDNF, a diffusible chemoattractant in the limb, indicating that Ret is a multifunctional coreceptor for guidance molecules. Axons respond synergistically to coactivation by GDNF and EphA ligands, and these cooperative interactions are gated by GFRα1 levels. Our studies uncover a hierarchical GPI-receptor signaling network that is constructed from combinatorial components and integrated through Ret using ligand coincidence detection.

GDF15 mediates the effects of metformin on body weight and energy balance.

Metformin, the world's most prescribed anti-diabetic drug, is also effective in preventing type 2 diabetes in people at high risk1,2. More than 60% of this effect is attributable to the ability of metformin to lower body weight in a sustained manner3. The molecular mechanisms by which metformin lowers body weight are unknown. Here we show-in two independent randomized controlled clinical trials-that metformin increases circulating levels of the peptide hormone growth/differentiation factor 15 (GDF15), which has been shown to reduce food intake and lower body weight through a brain-stem-restricted receptor. In wild-type mice, oral metformin increased circulating GDF15, with GDF15 expression increasing predominantly in the distal intestine and the kidney. Metformin prevented weight gain in response to a high-fat diet in wild-type mice but not in mice lacking GDF15 or its receptor GDNF family receptor α-like (GFRAL). In obese mice on a high-fat diet, the effects of metformin to reduce body weight were reversed by a GFRAL-antagonist antibody. Metformin had effects on both energy intake and energy expenditure that were dependent on GDF15, but retained its ability to lower circulating glucose levels in the absence of GDF15 activity. In summary, metformin elevates circulating levels of GDF15, which is necessary to obtain its beneficial effects on energy balance and body weight, major contributors to its action as a chemopreventive agent.

Camptothecin effectively treats obesity in mice through GDF15 induction.

Elevated circulating levels of growth differentiation factor 15 (GDF15) have been shown to reduce food intake and lower body weight through activation of hindbrain receptor glial-derived neurotrophic factor (GDNF) receptor alpha-like (GFRAL) in rodents and nonhuman primates, thus endogenous induction of this peptide holds promise for obesity treatment. Here, through in silico drug-screening methods, we found that small molecule Camptothecin (CPT), a previously identified drug with potential antitumor activity, is a GDF15 inducer. Oral CPT administration increases circulating GDF15 levels in diet-induced obese (DIO) mice and genetic ob/ob mice, with elevated Gdf15 expression predominantly in the liver through activation of integrated stress response. In line with GDF15's anorectic effect, CPT suppresses food intake, thereby reducing body weight, blood glucose, and hepatic fat content in obese mice. Conversely, CPT loses these beneficial effects when Gdf15 is inhibited by a neutralizing antibody or AAV8-mediated liver-specific knockdown. Similarly, CPT failed to reduce food intake and body weight in GDF15's specific receptor GFRAL-deficient mice despite high levels of GDF15. Together, these results indicate that CPT is a promising anti-obesity agent through activation of GDF15-GFRAL pathway.

Plzf regulates germline progenitor self-renewal by opposing mTORC1.

Hyperactivity of mTORC1, a key mediator of cell growth, leads to stem cell depletion, although the underlying mechanisms are poorly defined. Using spermatogonial progenitor cells (SPCs) as a model system, we show that mTORC1 impairs stem cell maintenance by a negative feedback from mTORC1 to receptors required to transduce niche-derived signals. We find that SPCs lacking Plzf, a transcription factor essential for SPC maintenance, have enhanced mTORC1 activity. Aberrant mTORC1 activation in Plzf(-/-) SPCs inhibits their response to GDNF, a growth factor critical for SPC self-renewal, via negative feedback at the level of the GDNF receptor. Plzf opposes mTORC1 activity by inducing expression of the mTORC1 inhibitor Redd1. Thus, we identify the mTORC1-Plzf functional interaction as a critical rheostat for maintenance of the spermatogonial pool and propose a model whereby negative feedback from mTORC1 to the GDNF receptor balances SPC growth with self-renewal.

The GDF15-GFRAL Pathway in Health and Metabolic Disease: Friend or Foe?

GDF15 is a cell activation and stress response cytokine of the glial cell line-derived neurotrophic factor family within the TGF-ß superfamily. It acts through a recently identified orphan member of the GFRα family called GFRAL and signals through the Ret coreceptor. Cell stress and disease lead to elevated GDF15 serum levels, causing anorexia, weight loss, and alterations to metabolism, largely by actions on regions of the hindbrain. These changes restore homeostasis and, in the case of obesity, cause a reduction in adiposity. In some diseases, such as advanced cancer, serum GDF15 levels can rise by as much as 10-100-fold, leading to an anorexia-cachexia syndrome, which is often fatal. This review discusses how GDF15 regulates appetite and metabolism, the role it plays in resistance to obesity, and how this impacts diseases such as diabetes, nonalcoholic fatty liver disease, and anorexia-cachexia syndrome. It also discusses potential therapeutic applications of targeting the GDF15-GFRAL pathway and lastly suggests some potential unifying hypotheses for its biological role.

Adult medial habenula neurons require GDNF receptor GFRα1 for synaptic stability and function.

The medial habenula (mHb) is an understudied small brain nucleus linking forebrain and midbrain structures controlling anxiety and fear behaviors. The mechanisms that maintain the structural and functional integrity of mHb neurons and their synapses remain unknown. Using spatiotemporally controlled Cre-mediated recombination in adult mice, we found that the glial cell-derived neurotrophic factor receptor alpha 1 (GFRα1) is required in adult mHb neurons for synaptic stability and function. mHb neurons express some of the highest levels of GFRα1 in the mouse brain, and acute ablation of GFRα1 results in loss of septohabenular and habenulointerpeduncular glutamatergic synapses, with the remaining synapses displaying reduced numbers of presynaptic vesicles. Chemo- and optogenetic studies in mice lacking GFRα1 revealed impaired circuit connectivity, reduced AMPA receptor postsynaptic currents, and abnormally low rectification index (R.I.) of AMPARs, suggesting reduced Ca2+ permeability. Further biochemical and proximity ligation assay (PLA) studies defined the presence of GluA1/GluA2 (Ca2+ impermeable) as well as GluA1/GluA4 (Ca2+ permeable) AMPAR complexes in mHb neurons, as well as clear differences in the levels and association of AMPAR subunits with mHb neurons lacking GFRα1. Finally, acute loss of GFRα1 in adult mHb neurons reduced anxiety-like behavior and potentiated context-based fear responses, phenocopying the effects of lesions to septal projections to the mHb. These results uncover an unexpected function for GFRα1 in the maintenance and function of adult glutamatergic synapses and reveal a potential new mechanism for regulating synaptic plasticity in the septohabenulointerpeduncular pathway and attuning of anxiety and fear behaviors.

ARTN-GFRA3 axis induces epithelial-mesenchymal transition phenotypes, migration, and invasion of gastric cancer cells via KRAS signaling.

Neural invasion underlies the local spread of gastric cancer and is associated with poor prognosis. This process has been receiving increasing attention in recent years. However, the relationship between neural invasion and the malignant phenotypes of gastric cancer cells, as well as the molecular mechanism involved in this process, remain unclear. In this study, bioinformatics analysis was performed using a dataset obtained from The Cancer Genome Atlas-Stomach Adenocarcinoma. The results revealed that high expression of GDNF family receptor alpha 3 (GFRA3) was associated with a poor prognosis of patients with gastric cancer. GFRA3 is a receptor for artemin (ARTN), a glial cell line-derived neurotrophic factor (GDNF). This association was indicated by short overall/disease-free survival, as well as the presence of high-stage and high-grade disease. Gene set enrichment analysis showed that two cancer-associated pathways, namely KRAS signaling and epithelial-mesenchymal transition (EMT), were activated when GFRA3 was highly expressed in gastric cancer. Further studies confirmed that GFRA3 activated KRAS downstream signaling phosphatidylinositol 3 kinase/protein kinase B (PI3K/AKT) or extracellular signal-regulated kinase (ERK) and induced EMT markers, as well as promoted the migration and invasion of gastric cancer cells. As a ligand of GFRA3, ARTN induced the EMT, migration, and invasion of gastric cancer cells via GFRA3. Notably, the effects of the ARTN-GFRA3 axis were attenuated by treatment with a KRAS inhibitor. The present findings indicated that, during the neural invasion of gastric cancer, ARTN-mediated activation of GFRA3 induces EMT phenotypes, migration, and invasion of gastric cancer cells via KRAS signaling.

GFRAL-expressing neurons suppress food intake via aversive pathways.

The TGFß cytokine family member, GDF-15, reduces food intake and body weight and represents a potential treatment for obesity. Because the brainstem-restricted expression pattern of its receptor, GDNF Family Receptor α-like (GFRAL), presents an exciting opportunity to understand mechanisms of action for area postrema neurons in food intake; we generated GfralCre and conditional GfralCreERT mice to visualize and manipulate GFRAL neurons. We found infection or pathophysiologic states (rather than meal ingestion) stimulate GFRAL neurons. TRAP-Seq analysis of GFRAL neurons revealed their expression of a wide range of neurotransmitters and neuropeptides. Artificially activating GfralCre -expressing neurons inhibited feeding, decreased gastric emptying, and promoted a conditioned taste aversion (CTA). GFRAL neurons most strongly innervate the parabrachial nucleus (PBN), where they target CGRP-expressing (CGRPPBN) neurons. Silencing CGRPPBN neurons abrogated the aversive and anorexic effects of GDF-15. These findings suggest that GFRAL neurons link non-meal-associated pathophysiologic signals to suppress nutrient uptake and absorption.

Activation of the hypothalamic-pituitary-adrenal axis by exogenous and endogenous GDF15.

An acute increase in the circulating concentration of glucocorticoid hormones is essential for the survival of severe somatic stresses. Circulating concentrations of GDF15, a hormone that acts in the brain to reduce food intake, are frequently elevated in stressful states. We now report that GDF15 potently activates the hypothalamic-pituitary-adrenal (HPA) axis in mice and rats. A blocking antibody to the GDNF-family receptor α-like receptor completely prevented the corticosterone response to GDF15 administration. In wild-type mice exposed to a range of stressful stimuli, circulating levels of both corticosterone and GDF15 rose acutely. In the case of Escherichia coli or lipopolysaccharide injections, the vigorous proinflammatory cytokine response elicited was sufficient to produce a near-maximal HPA response, regardless of the presence or absence of GDF15. In contrast, the activation of the HPA axis seen in wild-type mice in response to the administration of genotoxic or endoplasmic reticulum toxins, which do not provoke a marked rise in cytokines, was absent in Gdf15-/- mice. In conclusion, consistent with its proposed role as a sentinel hormone, endogenous GDF15 is required for the activation of the protective HPA response to toxins that do not induce a substantial cytokine response. In the context of efforts to develop GDF15 as an antiobesity therapeutic, these findings identify a biomarker of target engagement and a previously unrecognized pharmacodynamic effect, which will require monitoring in human studies.

Overview of growth differentiation factor 15 in metabolic syndrome.

Growth and differentiation factor 15 (GDF15) is a member of the transforming growth factor-ß (TGF-ß) superfamily. GDF15 has been linked with several metabolic syndrome pathologies such as obesity and cardiovascular diseases. GDF15 is considered to be a metabolic regulator, although its precise mechanisms of action remain to be determined. Glial cell-derived neurotrophic factor family receptor alpha-like (GRAL), located in the hindbrain, has been identified as the receptor for GDF15 and signals through the coreceptor receptor tyrosine kinase (RET). Administration of GDF15 analogues in preclinical studies using various animal models has consistently been shown to induce weight loss through a reduction in food intake. GDF15, therefore, represents an attractive target to combat the current global obesity epidemic. In this article, we review current knowledge on GDF15 and its involvement in metabolic syndrome.

The GDF15-GFRAL axis mediates chemotherapy-induced fatigue in mice.

Cancer-related fatigue is defined as a distressing persistent subjective sense of physical, emotional, and/or cognitive tiredness or exhaustion related to cancer or cancer treatment that is not proportional to recent activity and that interferes with usual functioning. This form of fatigue is highly prevalent during cancer treatment and in some patients, it can persist for years after treatment has ended. An understanding of the mechanisms that drive cancer-related fatigue is still lacking, which hampers the identification of effective treatment options. Various chemotherapeutic agents including cisplatin are known to induce mitochondrial dysfunction and this effect is known to mediate chemotherapy-induced peripheral neuropathy and cognitive dysfunction. Mitochondrial dysfunction results in the release of mitokines that act locally and at distance to promote metabolic and behavioral adjustments to this form of cellular stress. One of these mitokines, growth differentiation factor 15 (GDF15) and its receptor, glial cell line-derived neurotrophic factor family receptor α-like (GFRAL), have received special attention in oncology as activation of GFRAL mediates the anorexic response that is responsible for cancer anorexia. The present study was initiated to determine whether GDF15 and GFRAL are involved in cisplatin-induced fatigue. We first tested the ability of cisplatin to increase circulating GDF15 in mice before assessing whether GDF15 can induce behavioral fatigue measured by decreased wheel running in healthy mice and increase behavioral fatigue induced by cisplatin. Mice administered a long acting form of GDF15, mGDF15-fc, decreased their voluntary wheel running activity. When the same treatment was administered to mice receiving cisplatin, it increased the amplitude and duration of cisplatin-induced decrease in wheel running. To determine whether endogenous GDF15 mediates the behavioral fatigue induced by cisplatin, we then administered a neutralizing monoclonal antibody to GFRAL to mice injected with cisplatin. The GFRAL neutralizing antibody mostly prevented cisplatin-induced decrease in wheel running and accelerated recovery. Taken together these findings demonstrate for the first time the role of the GDF15/GFRAL axis in cisplatin-induced behaviors and indicate that this axis could be a promising therapeutic target for the treatment of cancer-related fatigue.

Non-homeostatic body weight regulation through a brainstem-restricted receptor for GDF15.

Under homeostatic conditions, animals use well-defined hypothalamic neural circuits to help maintain stable body weight, by integrating metabolic and hormonal signals from the periphery to balance food consumption and energy expenditure. In stressed or disease conditions, however, animals use alternative neuronal pathways to adapt to the metabolic challenges of altered energy demand. Recent studies have identified brain areas outside the hypothalamus that are activated under these 'non-homeostatic' conditions, but the molecular nature of the peripheral signals and brain-localized receptors that activate these circuits remains elusive. Here we identify glial cell-derived neurotrophic factor (GDNF) receptor alpha-like (GFRAL) as a brainstem-restricted receptor for growth and differentiation factor 15 (GDF15). GDF15 regulates food intake, energy expenditure and body weight in response to metabolic and toxin-induced stresses; we show that Gfral knockout mice are hyperphagic under stressed conditions and are resistant to chemotherapy-induced anorexia and body weight loss. GDF15 activates GFRAL-expressing neurons localized exclusively in the area postrema and nucleus tractus solitarius of the mouse brainstem. It then triggers the activation of neurons localized within the parabrachial nucleus and central amygdala, which constitute part of the 'emergency circuit' that shapes feeding responses to stressful conditions. GDF15 levels increase in response to tissue stress and injury, and elevated levels are associated with body weight loss in numerous chronic human diseases. By isolating GFRAL as the receptor for GDF15-induced anorexia and weight loss, we identify a mechanistic basis for the non-homeostatic regulation of neural circuitry by a peripheral signal associated with tissue damage and stress. These findings provide opportunities to develop therapeutic agents for the treatment of disorders with altered energy demand.

Hypomethylation of GDNF family receptor alpha 1 promotes epithelial-mesenchymal transition and predicts metastasis of colorectal cancer.

Tumor metastasis is the major cause of poor prognosis and mortality in colorectal cancer (CRC). However, early diagnosis of highly metastatic CRC is currently difficult. In the present study, we screened for a novel biomarker, GDNF family receptor alpha 1 (GFRA1) based on the expression and methylation data in CRC patients from The Cancer Genome Altlas (TCGA), followed by further analysis of the correlation between the GFRA1 expression, methylation, and prognosis of patients. Our results show DNA hypomethylation-mediated upregulation of GFRA1 in invasive CRC, and it was found to be correlated with poor prognosis of CRC patients. Furthermore, GFRA1 methylation-modified sequences were found to have potential as methylation diagnostic markers of highly metastatic CRC. The targeted demethylation of GFRA1 by dCas9-TET1CD and gRNA promoted CRC metastasis in vivo and in vitro. Mechanistically, demethylation of GFRA1 induces epithelial-mesenchymal transition (EMT) by promoting AKT phosphorylation and increasing c-Jun expression in CRC cells. Collectively, our findings indicate that GFRA1 hypomethylation can promote CRC invasion via inducing EMT, and thus, GFRA1 methylation can be used as a biomarker for the early diagnosis of highly metastasis CRC.

A dual role for peripheral GDNF signaling in nociception and cardiovascular reflexes in the mouse.

Group III/IV muscle afferents transduce nociceptive signals and modulate exercise pressor reflexes (EPRs). However, the mechanisms governing afferent responsiveness to dually modulate these processes are not well characterized. We and others have shown that ischemic injury can induce both nociception-related behaviors and exacerbated EPRs in the same mice. This correlated with primary muscle afferent sensitization and increased expression of glial cell line-derived neurotrophic factor (GDNF) in injured muscle and increased expression of GDNF family receptor α1 (GFRα1) in dorsal root ganglia (DRG). Here, we report that increased GDNF/GFRα1 signaling to sensory neurons from ischemia/reperfusion-affected muscle directly modulated nociceptive-like behaviors and increased exercise-mediated reflexes and group III/IV muscle afferent sensitization. This appeared to have taken effect through increased cyclic adenosine monophosphate (cAMP) response element binding (CREB)/CREB binding protein-mediated expression of the purinergic receptor P2X5 in the DRGs. Muscle GDNF signaling to neurons may, therefore, play an important dual role in nociception and sympathetic reflexes and could provide a therapeutic target for treating complications from ischemic injuries.

The Role of Promyelocytic Leukemia Zinc Finger (PLZF) and Glial-Derived Neurotrophic Factor Family Receptor Alpha 1 (GFRα1) in the Cryopreservation of Spermatogonia Stem Cells.

The cryopreservation of spermatogonia stem cells (SSCs) has been widely used as an alternative treatment for infertility. However, cryopreservation itself induces cryoinjury due to oxidative and osmotic stress, leading to reduction in the survival rate and functionality of SSCs. Glial-derived neurotrophic factor family receptor alpha 1 (GFRα1) and promyelocytic leukemia zinc finger (PLZF) are expressed during the self-renewal and differentiation of SSCs, making them key tools for identifying the functionality of SSCs. To the best of our knowledge, the involvement of GFRα1 and PLZF in determining the functionality of SSCs after cryopreservation with therapeutic intervention is limited. Therefore, the purpose of this review is to determine the role of GFRα1 and PLZF as biomarkers for evaluating the functionality of SSCs in cryopreservation with therapeutic intervention. Therapeutic intervention, such as the use of antioxidants, and enhancement in cryopreservation protocols, such as cell encapsulation, cryoprotectant agents (CPA), and equilibrium of time and temperature increase the expression of GFRα1 and PLZF, resulting in maintaining the functionality of SSCs. In conclusion, GFRα1 and PLZF have the potential as biomarkers in cryopreservation with therapeutic intervention of SSCs to ensure the functionality of the stem cells.

Expression of Glial-Cell-Line-Derived Neurotrophic Factor Family Ligands in Human Intervertebral Discs.

Glial-cell-line-derived neurotrophic factor (GDNF) family ligands (GFLs) contribute to the sensitization of primary afferents and are involved in the pathogenesis of inflammatory pain. The purpose of this preliminary study was to examine the expression of other GFLs (neurturin (NRTN), artemin (ARTN), persephin (PSPN)) and receptors in human IVD cells and tissues exhibiting early and advanced stages of degeneration. Human IVD cells were cultured as a monolayer after isolation from the nucleus pulposus (NP) and anulus fibrosus (AF) tissues. The mRNA expression of NRTN, ARTN, PSPN, and their receptors (GFRA2-GFRA4) was quantified using real-time PCR. Protein expression was evaluated using immunohistochemistry and Western blotting. The expression of NRTN, ARTN, PSPN, and their co-receptors (GFRA2-GFRA4) was identified in human IVD cells at both mRNA and protein levels. A trend was noted wherein the mRNA expression of ARTN, PSPN, and GFRA2 was upregulated by IL-1ß treatment in a dose-dependent manner. The percentages of immunopositive cells in the advanced degenerate stage of ARTN, PSPN, and GFRA2 were significantly higher than those in the early degenerate stage. Their expression was enhanced in advanced tissue degeneration, which suggests that GFLs (ARTN and PSPN) may be involved in the pathogenesis of discogenic pain.

The Emerging Portrait of Glial Cell Line-derived Neurotrophic Factor Family Receptor Alpha (GFRα) in Cancers.

Glial cell line-derived neurotrophic factor family receptor alpha (GFRα) members have been widely connected to the mechanisms contributing to cell growth, differentiation, cell migration and tissue maturation. Here we review GFRα biological functions and discussed the evidence indicating whether GFRα signaling complex present novel opportunities for oncogenic intervention and treatment resistance. Thus, our work systematically reviewed the emerging role of GFRα family members in cancers, and provided novel insights for further researches.

GDNF requires HIF-1α and RET activation for suppression of programmed cell death of enteric neurons by metabolic challenge.

Intestinal inflammation challenges both function and structure of the enteric nervous system (ENS). In the animal model of TNBS-induced colitis, an influx of immune cells causes early neuron death in the neuromuscular layers, followed by axonal outgrowth from surviving neurons associated with upregulation of the neurotrophin GDNF (glial cell line-derived neurotrophic factor). Inflammation could involve ischemia and metabolic inhibition leading to neuronal damage, which might be countered by a protective action of GDNF. This was examined in a primary co-culture model of rat myenteric neurons and smooth muscle, where metabolic challenge was caused by dinitrophenol (DNP), O-methyl glucose (OMG) or hypoxia. These caused the specific loss of 50% of neurons by 24 h that was blocked by GDNF both in vitro and in whole mounts. Neuroprotection was lost with RET inhibition by vandetanib or GSK3179106, which also caused neuron loss in untreated controls. Thus, both basal and upregulated GDNF levels signal via RET for neuronal survival. This includes a key role for upregulation of HIF-1α, which was detected in neurons in colitis, since the inhibitor chetomin blocked rescue by GDNF or ischemic pre-conditioning in vitro. In DNP-treated co-cultures, neuron death was not inhibited by zVAD, necrosulfonamide or GSK872, and cleaved caspase-3 or - 8 were undetectable. However, combinations of inhibitors or the RIP1kinase inhibitor Nec-1 prevented neuronal death, evidence for RIPK1-dependent necroptosis. Therefore, inflammation challenges enteric neurons via ischemia, while GDNF is neuroprotective, activating RET and HIF-1α to limit programmed cell death. This may support novel strategies to address recurrent inflammation in IBD.

Biallelic Pathogenic GFRA1 Variants Cause Autosomal Recessive Bilateral Renal Agenesis.

BACKGROUND: Congenital anomalies of the kidney and urinary tract (CAKUT) are one of the most common malformations identified in the fetal stage. Bilateral renal agenesis (BRA) represents the most severe and fatal form of CAKUT. Only three genes have been confirmed to have a causal role in humans (ITGA8, GREB1L, and FGF20). METHODS: Genome sequencing within a diagnostic setting and combined data repository analysis identified a novel gene. RESULTS: Two patients presented with BRA, detected during the prenatal period, without additional recognizable malformations. They had parental consanguinity and similarly affected, deceased siblings, suggesting autosomal recessive inheritance. Evaluation of homozygous regions in patient 1 identified a novel, nonsense variant in GFRA1 (NM_001348097.1:c.676C>T, p.[Arg226*]). We identified 184 patients in our repository with renal agenesis and analyzed their exome/genome data. Of these 184 samples, 36 were from patients who presented with isolated renal agenesis. Two of them had loss-of-function variants in GFRA1. The second patient was homozygous for a frameshift variant (NM_001348097.1:c.1294delA, p.[Thr432Profs*13]). The GFRA1 gene encodes a receptor on the Wolffian duct that regulates ureteric bud outgrowth in the development of a functional renal system, and has a putative role in the pathogenesis of Hirschsprung disease. CONCLUSIONS: These findings strongly support the causal role of GFRA1-inactivating variants for an autosomal recessive, nonsyndromic form of BRA. This knowledge will enable early genetic diagnosis and better genetic counseling for families with BRA.