Consistent with reduced ALAS1 expression, we observed a slight decrease ofFPN, FT-LandFT-HmRNA levels in patient compared to control LCLs (Fig 2F). == Fig 2 . of the cytosolic heme pool required to sustain metabolic activity of different cell types. Mutations inFLVCR1have previously been linked to vision impairment and posterior column ataxia in humans, but not to HSAN. Using fibroblasts and lymphoblastoid cell lines from patients with sensory neurodegeneration, we here show that the FLVCR1-mutations reduce heme export activity, enhance oxidative stress and increase sensitivity to programmed cell death. Our data link heme metabolism to sensory neuron maintenance and suggest that intracellular heme overload causes early-onset degeneration of pain-sensing neurons in humans. == Author Summary == Hereditary Sensory and Autonomic Neuropathy (HSAN) is a genetic disorder mainly characterized by the impairment of sensory neurons, which transmit information about sensations such as pain, temperature and touch. Therefore , unintentional self-injury, leading to ulcers and eventually amputations are common in affected individuals. Although mutations in several genes were previously associated with sensory neurodegeneration and pain insensitivity, the etiology of many cases remains unknown. We here identify mutations in the heme exporter protein FLVCR1 in patients with congenital inability to experience pain. We showed that FLVCR1 mutations results in reduced heme export activity, enhanced oxidative stress and increased sensitivity to programmed cell death. These data assign a surprising role intended for heme to sensory neuron maintenance. == Introduction == Neurodegenerative disorders affecting peripheral sensory neurons lead to loss of pain perception as disease hallmark. The absence of protective behaviors towards noxious (+)-Phenserine stimuli causes unintentional self-injuries and chronic ulcerations. Soft tissue infections and osteomyelitis, often requiring amputations, are common and complicate this disorder [1, 2]. Autonomic dysfunction and motor deficits may be additional features of sensory and autonomic neuropathies (HSANs). Prominent loss of large and small myelinated fibers distinguishes sensory neuropathies from clinically similarly presenting channelopathy-associated pain insensitivity (CIP)[3]. Proteins which are involved in sensory neurodegeneration affect distinct molecular pathways: sphingolipid-metabolism, membrane-shaping of organelles, regulation of ion channels, endoplasmic reticulum turnover and axonal trafficking[1, 48]. However , the molecular mechanisms underlying sensory neurodegeneration are still incompletely understood and disease-causing mutations remain to be recognized in a substantial number of patients. Rapid progress in next-generation sequencing (NGS) technology has transformed the field of medical genomics leading to the identification of novel disease-genes[9, 10]. In this study, next generation sequencing was performed in patients with HSAN but without mutations in the known genes associated with the disorder. Causative mutations were found inFLVCR1(Feline Leukemia Virus subgroup C Receptor 1), a gene that has previously been associated to Posterior Column Ataxia and Retinitis Pigmentosa (PCARP)[1116]. FLVCR1 is an ubiquitously expressed heme exporter[17, 18], member of the Major Facilitator Superfamily (MFS) transporters[19]. Two different isoforms have been described. FLVCR1a resides in the plasma membrane and is responsible for heme detoxification in several cell types, such as erythroid progenitors, endothelial cells, hepatocytes, lymphocytes and intestinal cells[18, 2025]. FLVCR1b is located on mitochondria (+)-Phenserine and is involved in the transport of newly synthesized heme from mitochondria to the cytosol[18]. The expression of FLVCR1a and FLVCR1b is needed to control the size of the cytoplasmic free-heme pool, Igf2 which is essential for proper metabolic functions[21, 23]. Heme is an essential co-factor involved in multiple biological processes: oxygen transport and storage, electron transfer, drug and steroid metabolism, signal transduction and microRNA processing[26]. However , (+)-Phenserine excess free-heme is highly toxic due to its ability to promote oxidative stress, proteasome inhibition and mitochondrial dysfunction, that ultimately (+)-Phenserine lead to cell death[2628]. For this reason, the intracellular free-heme pool is finely regulated at multiple levels[26] and FLVCR1a-mediated heme export contributes to this process[21, 22, 26]. Our data show that primary fibroblasts and lymphoblastoid cell lines from patients with sensory neurodegeneration have reduced heme export activity due to FLVCR1-mutations. This results in enhanced oxidative stress and increased sensitivity to programmed cell death. These data add heme metabolism to the molecular pathways implicated in sensory neuron maintenance and pain processing. == Results == == Mutations in theFLVCR1gene cause sensory neurodegeneration with loss of pain perception in humans == We studied trios with an affected child and healthy parents with the diagnosis of hereditary sensory and autonomic neuropathy (HSAN) (Fig 1andTable 1). In an Italian boy with early-onset pain insensitivity (patient 1) and non-consanguineous parents whole-exome sequencing recognized 559 variants with low prevalence ( <0, 01%) in dbSNP, the 1000-Genomes project, the Exome Variant Server or the ExAC browser. Variants were prioritized forde novomutations and compound-heterozygous / homozygous variants (S1 Table)..