Effects of different high temperature-pressure processing times on the sensory quality, nutrition and allergenicity of ready-to-eat clam meat.

Investigating technologies to control the allergenicity of seafood is particularly important to safeguard consumer health, but there is currently a dearth of research focused on reducing the allergenicity of clam meat. This study aimed to investigate the effects of high temperature-pressure (HTP) processing times (121 °C, 0.14 MPa; 5, 10, 15, 20 min) on the sensory quality, nutrition, and allergenicity of ready-to-eat clam meat. With the extension of HTP time, the hardness of clam meat gradually decreased, the chewiness decreased initially and then increased, and the meat became tender. HTP processing endowed clam meat with abundant esters and aldehydes. Among all the processing groups, the umami and saltiness were better at 15 min, correlating with the highest overall acceptability. Ready-to-eat clam meat contained high-protein nutritional value. Compared with raw clam meat, the tropomyosin allergenicity of clam meat treated with HTP for 15 and 20 min was significantly reduced by 51.9 % and 56.5 %, respectively (P < 0.05). However, there was no significant difference between these two groups. Appropriate HTP processing time might be an efficient condition to reduce the tropomyosin allergenicity of ready-to-eat clam meat and improve its quality, particularly for the time of 15 min. The results of this study could provide a reliable theoretical basis for the development of hypoallergenic clam foods.

B7-H3 promotes angiogenesis in rheumatoid arthritis.

OBJECTIVE: The primary pathological changes of rheumatoid arthritis (RA) include chronic synovial inflammation, bone destruction, and aggressive pannus formation on cartilage, in which angiogenesis plays a critical role. B7-H3, an important immune checkpoint molecule, represents a novel target in tumor therapy and plays a significant role in the pathogenesis of autoimmune diseases. However, its biological mechanism in RA remains unclear. METHODS: Hematoxylin-eosin (HE) staining and immunohistochemistry were used to explore the histological characteristics and expression of B7-H3, CD34, and vascular endothelial growth factor (VEGF) in patients with RA and collagen-induced arthritis (CIA) mice. ELISA was used to detect VEGF, soluble B7-H3, and disease markers in the peripheral blood of patients. A monoclonal anti-B7-H3 antibody was used to treat CIA mice by blocking B7-H3-mediated signaling. RESULTS: The ELISA and HE staining results showed a positive correlation between the expression of B7-H3 and the degree of joint cavity destruction and pannus formation. B7-H3 expression also correlated with increased expression of the vessel biomarkers CD34 and VEGF. Anti-B7-H3 effectively reduced pannus formation in CIA mice. CONCLUSION: B7-H3 modulates angiogenic activity in the joint synovium, demonstrating its therapeutic value in the context of RA.

Dysregulated PD-L2 is correlated with disease activity and inflammation in rheumatoid arthritis.

The programmed death-1 (PD-1) pathway has been shown to deliver an inhibitory signal, and aberrant expression of the PD-1 molecule and/or its ligand programmed death ligand 1 (PD-L1) has been demonstrated in human diseases, while its other ligand, programmed death ligand 2 (PD-L2), has rarely been studied. Here, we investigated the expression of PD-L2 in synovial tissue and blood from patients with rheumatoid arthritis (RA). Soluble PD-L2 and inflammatory cytokine levels in serum among healthy controls and patients with RA were compared via enzyme-linked immunosorbent assay (ELISA). Membrane PD-L2 on monocytes in blood was analyzed through flow cytometry (FCM). The different expression levels of PD-L2 between the RA and non-RA synovium were semi-quantified by immunohistochemical (IHC) staining. The soluble PD-L2 levels in serum from patients with RA were significantly lower than those in healthy subjects, correlating with active parameters (rheumatoid factor) and inflammatory cytokine secretion. The FCM results showed that patients with RA had significantly increased percentages of PD-L2-expressing CD14+ monocytes and correlated with inflammatory cytokines. PD-L2 expression on macrophages in the synovium from patients with RA was recorded by IHC staining with a higher score, and its correlation with pathological scores and clinical features was determined. Together, our results revealed aberrant expression of PD-L2 in RA, which may be a promising biomarker and therapeutic target associated with the pathogenesis of RA.

Effects of ultrasound-assisted high temperature-pressure treatment on the structure and allergenicity of tropomyosin from clam (Mactra veneriformis).

Tropomyosin (TM) is the major allergen in clams. This study aimed to evaluate the effects of ultrasound-assisted high temperature-pressure treatment on the structure and allergenicity of TM from clams. The results showed that the combined treatment significantly affected the structure of TM-converting the α-helix to ß-sheet and random coil, and decreasing the sulfhydryl group content, surface hydrophobicity, and particle size. These structural changes caused the unfolding of the protein, disrupting and modifying the allergenic epitopes. The significant reduction in the allergenicity of TM was approximately 68.1% when treated with combined processing (P < 0.05). Notably, an increase in the content of the relevant amino acids and a smaller particle size accelerated the penetration of the enzyme into the protein matrix, resulting in strengthening the gastrointestinal digestibility of TM. These results prove that ultrasound-assisted high temperature-pressure treatment has great potential in reducing allergenicity, benefiting the development of hypoallergenic clam products.

miR-196a-mediated downregulation of p27kip1 protein promotes prostate cancer proliferation and relates to biochemical recurrence after radical prostatectomy.

BACKGROUND: Dysregulation of microRNAs has performed vital gene regulatory functions in the genesis, progression, and prognosis of multiple malignant tumors. This study aimed to elucidate the regulatory mechanism of miR-196a in prostate cancer (PCa) and explore its clinical significance. METHODS: Quantitative real-time polymerase chain reaction was implemented to examine miR-196a and p27kip1 messenger RNA expression in PCa. Cell proliferation was evaluated via Cell Counting Kit-8, colony formation, and nude mouse tumorigenicity assays. Luciferase reporter assay was applied to identify target genes. p27kip1 protein expression in PCa was investigated using Western blot analysis and immunohistochemistry. RESULTS: There was a dramatic upregulation of miR-196a in PCa. Upregulated miR-196a was related to worse Gleason score (GS), later pathological stage, and poor biochemical recurrence (BCR)-free survival. In vivo and in vitro experiments exhibited that miR-196a promoted PCa proliferation and expedited G1/S-phase progression through the downregulation of p27kip1 protein. Additionally, p27kip1 protein was distinctly downregulated in PCa. Low p27kip1 protein expression had a strong correlation with increased GS and was an independent predictor of BCR after radical prostatectomy (RP). CONCLUSIONS: Excessive expression of miR-196a and subsequent downregulation of p27kip1 protein play essential roles in promoting PCa proliferation and leading to BCR after RP. miR-196a and its target p27kip1 may become novel molecular biomarkers and therapeutic targets for PCa.

Interleukin-6 from Adipose-Derived Stem Cells Promotes Tissue Repair by the Increase of Cell Proliferation and Hair Follicles in Ischemia/Reperfusion-Treated Skin Flaps.

The most common postoperative complication after reconstructive surgery is flap necrosis. Adipose-derived stem cells (ADSCs) and their secretomes are reported to mediate skin repair. This study was designed to investigate whether conditioned media from ADSCs (ADSC-CM) protects ischemia/reperfusion- (I/R-) induced injury in skin flaps by promoting cell proliferation and increasing the number of hair follicles. The mouse flap model of ischemia was ligating the long thoracic vessels for 3 h, followed by blood reperfusion. ADSC-CM was administered to the flaps, and their survival was observed on postoperative day 5. ADSC-CM treatment led to a significant increase in cell proliferation and the number of hair follicles. IL-6 levels in the lysate and CM from ADSCs were significantly higher than those from Hs68 fibroblasts. Furthermore, a strong decrease in cell proliferation and the number of hair follicles was observed after treatment with IL-6-neutralizing antibodies or si-IL-6-ADSC. In addition, ADSC transplantation increased flap repair, cell proliferation, and hair follicle number in I/R injury of IL-6-knockout mice. In conclusion, IL-6 secreted from ADSCs promotes the survival of I/R-induced flaps by increasing cell proliferation and the number of hair follicles. ADSCs represent a promising therapy for preventing skin flap necrosis following reconstructive and plastic surgery.

Reduced DAXX Expression Is Associated with Reduced CD24 Expression in Colorectal Cancer.

The presence of an activating mutation of the Wnt/ß-catenin signaling pathway is found in ~90% of colorectal cancer (CRC) cases. Death domain-associated protein (DAXX), a nuclear protein, interacts with ß-catenin in CRC cells. We investigated DAXX expression in 106 matched sample pairs of CRC and adjacent normal tissue by Western blotting. This study evaluated DAXX expression and its clinical implications in CRC. The results revealed that DAXX expression was significantly lower in the patients with the positive serum carcinoembryonic antigen (CEA) screening results compared to the patients with negative CEA screening levels (p < 0.001). It has been reported that CD24 is a Wnt target in CRC cells. Here, we further revealed that DAXX expression was significantly correlated with CD24 expression (rho = 0.360, p < 0.001) in 106 patients. Consistent with this, in the CEA-positive subgroup, of which the carcinomas expressed DAXX at low levels, they were significantly correlated with CD24 expression (rho = 0.461, p < 0.005). Therefore, reduced DAXX expression is associated with reduced CD24 expression in CRC. Notably, in the Hct116 cells, DAXX knockdown using short-hairpin RNA against DAXX (shDAXX) not only caused significant cell proliferation, but also promoted metastasis. The DAXX-knockdown cells also demonstrated significantly decreased CD24 expression, however the intracellular localization of CD24 did not change. Thus, DAXX might be considered as a potential regulator of CD24 or ß-catenin expression, which might be correlated with proliferative and metastatic potential of CRC.

High Glucose Concentrations Negatively Regulate the IGF1R/Src/ERK Axis through the MicroRNA-9 in Colorectal Cancer.

Studies have revealed that people with hyperglycemia have a high risk of colorectal cancer (CRC). Hyperglycemia may be responsible for supplying energy to CRC cells. However, the potential molecular mechanism for this association remains unclear. Furthermore, microRNA-9 (miR-9) has a tumor-suppressive function in CRC. Aberrant reduced expression of miR-9 is involved in the development and progression of malignancy caused by a high glucose (HG) concentration. In this study, we used an HG concentration to activate miR-9 downregulation in CRC cells. Our results indicated that miR-9 decreased the insulin-like growth factor-1 receptor (IGF1R)/Src signaling pathway and downstream cyclin B1 and N-cadherin but upregulated E-cadherin. The HG concentration not only promoted cell proliferation, increased the G1 population, and modulated epithelial-to-mesenchymal transition (EMT) protein expression and morphology but also promoted the cell migration and invasion ability of SW480 (low metastatic potential) and SW620 (high metastatic potential) cells. In addition, low glucose concentrations could reverse the effect of the HG concentration in SW480 and SW620 cells. In conclusion, our results provide new evidence for multiple signaling pathways being regulated through hyperglycemia in CRC. We propose that blood sugar control may serve as a potential strategy for the clinical management of CRC.

Curcumin accelerates cutaneous wound healing via multiple biological actions: The involvement of TNF-α, MMP-9, α-SMA, and collagen.

Curcumin, a constituent of the turmeric plant, has antitumor, anti-inflammatory, and antioxidative effects, but its effects on wound healing are unclear. We created back wounds in 72 mice and treated them with or without topical curcumin (0.2 mg/mL) in Pluronic F127 gel (20%) daily for 3, 5, 7, 9, and 12 days. Healing in wounds was evaluated from gross appearance, microscopically by haematoxylin and eosin staining, by immunohistochemistry for tumour necrosis factor alpha and alpha smooth muscle actin, and by polymerase chain reaction amplification of mRNA expression levels. Treatment caused fast wound closure with well-formed granulation tissue dominated by collagen deposition and regenerating epithelium. Curcumin increased the levels of tumour necrosis factor alpha mRNA and protein in the early phase of healing, which then decreased significantly. However, these levels remained high in controls. Levels of collagen were significantly higher in curcumin-treated wounds. Immunohistochemical staining for alpha smooth muscle actin was increased in curcumin-treated mice on days 7 and 12. Curcumin treatment significantly suppressed matrix metallopeptidase-9 and stimulated alpha smooth muscle levels in tumour necrosis factor alpha-treated fibroblasts via nuclear factor kappa B signalling. Thus, topical curcumin accelerated wound healing in mice by regulating the levels of various cytokines.

Adipose-Derived Stem Cells Protect Skin Flaps against Ischemia/Reperfusion Injury via IL-6 Expression.

Flap necrosis is the most frequent postoperative complication encountered in reconstructive surgery. We elucidated whether adipose-derived stem cells (ADSCs) and their derivatives might induce neovascularization and protect skin flaps during ischemia/reperfusion (I/R) injury. Flaps were subjected to 3 hours of ischemia by ligating long thoracic vessels and then to blood reperfusion. Qtracker-labeled ADSCs, ADSCs in conditioned medium (ADSC-CM), or ADSC exosomes (ADSC-Exo) were injected into the flaps. These treatments led to significantly increased flap survival and capillary density compared with I/R on postoperative day 5. IL-6 levels in the cell lysates or in conditioned medium were significantly higher in ADSCs than in Hs68 fibroblasts. ADSC-CM and ADSC-Exo increased tube formation. This result was corroborated by a strong decrease in skin repair after adding IL-6-neutralizing antibodies or small interfering RNA for IL-6 ADSCs. ADSC transplantation also increased flap recovery in I/R injury of IL-6-knockout mice. IL-6 was secreted from ADSCs through signal transducer and activator of transcription phosphorylation, and then IL-6 stimulated angiogenesis and enhanced recovery after I/R injury by the classic signaling pathway. The mechanism of skin recovery includes the direct differentiation of ADSCs into endothelial cells and the indirect effect of IL-6 released from ADSCs. ADSC-CM and ADSC-Exo could be used as off-the-shelf products for this therapy.

A Comprehensive Functional Analysis of NTRK1 Missense Mutations Causing Hereditary Sensory and Autonomic Neuropathy Type IV (HSAN IV).

Hereditary sensory and autonomic neuropathy type IV (HSAN IV) is an autosomal recessive disorder characterized by a complete lack of pain perception and anhidrosis. Here, we studied a cohort of seven patients with HSAN IV and describe a comprehensive functional analysis of seven novel NTRK1 missense mutations, c.1550G >A, c.1565G >A, c.1970T >C, c.2096T >C, c.2254T >A, c.2288G >C, and c.2311C >T, corresponding to p.G517E, p.G522E, p.L657P, p.I699T, p.C752S, p.C763S, and p.R771C, all of which were predicted pathogenic by in silico analysis. The results allowed us to assess the pathogenicity of each mutation and to gain novel insights into tropomyosin receptor kinase A (TRKA) downstream signaling. Each mutation was systematically analyzed for TRKA glycosylation states, intracellular and cell membrane expression patterns, nerve growth factor stimulated TRKA autophosphorylation, TRKA-Y496 phosphorylation, PLCγ activity, and neurite outgrowth. We showed a diverse range of functional effects: one mutation appeared fully functional, another had partial activity in all assays, one mutation affected only the PLCγ pathway and four mutations were proved null in all assays. Thus, we conclude that complete abolition of TRKA kinase activity is not the only pathogenic mechanism underlying HSAN IV. By corollary, the assessment of the clinical pathogenicity of HSAN IV mutations is more complex than initially predicted and requires a multifaceted approach.

A microtubule inhibitor, ABT-751, induces autophagy and delays apoptosis in Huh-7 cells.

The objective was to investigate the upstream mechanisms of apoptosis which were triggered by a novel anti-microtubule drug, ABT-751, in hepatocellular carcinoma-derived Huh-7 cells. Effects of ABT-751 were evaluated by immunocytochemistry, flow cytometric, alkaline comet, soft agar, immunoblotting, CytoID, green fluorescent protein-microtubule associated protein 1 light chain 3 beta detection, plasmid transfection, nuclear/cytosol fractionation, coimmunoprecipitation, quantitative reverse transcription-polymerase chain reaction, small-hairpin RNA interference and mitochondria/cytosol fractionation assays. Results showed that ABT-751 caused dysregulation of microtubule, collapse of mitochondrial membrane potential, generation of reactive oxygen species (ROS), DNA damage, G2/M cell cycle arrest, inhibition of anchorage-independent cell growth and apoptosis in Huh-7 cells. ABT-751 also induced early autophagy via upregulation of nuclear TP53 and downregulation of the AKT serine/threonine kinase (AKT)/mechanistic target of rapamycin (MTOR) pathway. Through modulation of the expression levels of DNA damage checkpoint proteins and G2/M cell cycle regulators, ABT-751 induced G2/M cell cycle arrest. Subsequently, ABT-751 triggered apoptosis with marked downregulation of B-cell CLL/lymphoma 2, upregulation of mitochondrial BCL2 antagonist/killer 1 and BCL2 like 11 protein levels, and cleavages of caspase 8 (CASP8), CASP9, CASP3 and DNA fragmentation factor subunit alpha proteins. Suppression of ROS significantly decreased ABT-751-induced autophagic and apoptotic cells. Pharmacological inhibition of autophagy significantly increased the percentages of ABT-751-induced apoptotic cells. The autophagy induced by ABT-751 plays a protective role to postpone apoptosis by exerting adaptive responses following microtubule damage, ROS and/or impaired mitochondria.

Clinical features for diagnosis and management of patients with PRDM12 congenital insensitivity to pain.

BACKGROUND: Congenital insensitivity to pain (CIP) is a rare extreme phenotype characterised by an inability to perceive pain present from birth due to lack of, or malfunction of, nociceptors. PRDM12 has recently been identified as a new gene that can cause CIP. The full phenotype and natural history have not yet been reported. METHODS: We have ascertained five adult patients and report their clinical features. RESULTS: Based on our findings, and those of previous patients, we describe the natural history of the PRDM12-CIP disorder, and derive diagnostic and management features to guide the clinical management of patients. CONCLUSIONS: PRDM12-CIP is a distinct and diagnosable disorder, and requires specific clinical management to minimise predictable complications.

CCDC88A mutations cause PEHO-like syndrome in humans and mouse.

Progressive encephalopathy with oedema, hypsarrhythmia and optic atrophy (PEHO) syndrome is a rare Mendelian phenotype comprising severe retardation, early onset epileptic seizures, optic nerve/cerebellar atrophy, pedal oedema, and early death. Atypical cases are often known as PEHO-like, and there is an overlap with 'early infantile epileptic encephalopathy'. PEHO is considered to be recessive, but surprisingly since initial description in 1991, no causative recessive gene(s) have been described. Hence, we report a multiplex consanguineous family with the PEHO phenotype where affected individuals had a homozygous frame-shift deletion in CCDC88A (c.2313delT, p.Leu772*ter). Analysis of cDNA extracted from patient lymphocytes unexpectedly failed to show non-sense mediated decay, and we demonstrate that the mutation produces a truncated protein lacking the crucial C-terminal half of CCDC88A (girdin). To further investigate the possible role of CCDC88A in human neurodevelopment we re-examined the behaviour and neuroanatomy of Ccdc88a knockout pups. These mice had mesial-temporal lobe epilepsy, microcephaly and corpus callosum deficiency, and by postnatal Day 21, microcephaly; the mice died at an early age. As the mouse knockout phenotype mimics the human PEHO phenotype this suggests that loss of CCDC88A is a cause of the PEHO phenotype, and that CCDC88A is essential for multiple aspects of normal human neurodevelopment.

New Mendelian Disorders of Painlessness.

Erroneous activation of the pain-sensing system, as in chronic or neuropathic pain, represents a major health burden with insufficient treatment options. However, the study of genetic disorders rendering individuals completely unable to feel pain offers hope. All causes of congenital painlessness affect nociceptors, evolutionarily conserved specialist neurons able to sense all type of tissue damage. The discovery of new genes essential for sensing pain (SCN11A, PRDM12, and CLTCL1) has provided unexpected insights into the biological mechanisms that drive distinct stages of nociception. Drugs targeting two previously discovered painlessness genes, NGF and SCN9A, are currently in late-stage clinical trials; thus, characterization of these new painlessness genes has significant potential for the generation of new classes of analgesics.

A novel disorder reveals clathrin heavy chain-22 is essential for human pain and touch development.

Congenital inability to feel pain is very rare but the identification of causative genes has yielded significant insights into pain pathways and also novel targets for pain treatment. We report a novel recessive disorder characterized by congenital insensitivity to pain, inability to feel touch, and cognitive delay. Affected individuals harboured a homozygous missense mutation in CLTCL1 encoding the CHC22 clathrin heavy chain, p.E330K, which we demonstrate to have a functional effect on the protein. We found that CLTCL1 is significantly upregulated in the developing human brain, displaying an expression pattern suggestive of an early neurodevelopmental role. Guided by the disease phenotype, we investigated the role of CHC22 in two human neural crest differentiation systems; human induced pluripotent stem cell-derived nociceptors and TRKB-dependant SH-SY5Y cells. In both there was a significant downregulation of CHC22 upon the onset of neural differentiation. Furthermore, knockdown of CHC22 induced neurite outgrowth in neural precursor cells, which was rescued by stable overexpression of small interfering RNA-resistant CHC22, but not by mutant CHC22. Similarly, overexpression of wild-type, but not mutant, CHC22 blocked neurite outgrowth in cells treated with retinoic acid. These results reveal an essential and non-redundant role for CHC22 in neural crest development and in the genesis of pain and touch sensing neurons.

Transcriptional regulator PRDM12 is essential for human pain perception.

Pain perception has evolved as a warning mechanism to alert organisms to tissue damage and dangerous environments. In humans, however, undesirable, excessive or chronic pain is a common and major societal burden for which available medical treatments are currently suboptimal. New therapeutic options have recently been derived from studies of individuals with congenital insensitivity to pain (CIP). Here we identified 10 different homozygous mutations in PRDM12 (encoding PRDI-BF1 and RIZ homology domain-containing protein 12) in subjects with CIP from 11 families. Prdm proteins are a family of epigenetic regulators that control neural specification and neurogenesis. We determined that Prdm12 is expressed in nociceptors and their progenitors and participates in the development of sensory neurons in Xenopus embryos. Moreover, CIP-associated mutants abrogate the histone-modifying potential associated with wild-type Prdm12. Prdm12 emerges as a key factor in the orchestration of sensory neurogenesis and may hold promise as a target for new pain therapeutics.

Identification of a receptor for neuropeptide VGF and its role in neuropathic pain.

VGF (nonacronymic) is a neuropeptide precursor that plays multiple roles in regulation of energy balance, reproduction, hippocampal synaptic plasticity, and pain. Data from a number of pain models showed significant up-regulation of VGF in sensory neurons. TLQP-21, one of the VGF-derived neuropeptides, has been shown to induce a hyperalgesic response when injected subcutaneously into the hind paw of mice. However, the precise role of VGF-derived neuropeptides in neuropathic pain and the molecular identity of the receptor for VGF-derived peptides are yet to be investigated. Here we identified gC1qR, the globular heads of the C1q receptor, as the receptor for TLQP-21 using chemical cross-linking combined with mass spectrometry analysis. TLQP-21 caused an increase in intracellular Ca(2+) levels in rat macrophages and microglia. Inoculation of TLQP-21-stimulated macrophages into rat hind paw caused mechanical hypersensitivity. The increase in intracellular Ca(2+) levels in macrophages was attenuated by either siRNA or neutralizing antibodies against gC1qR. Furthermore, application of the gC1qR-neutralizing antibody to rats with partial sciatic nerve ligation resulted in a delayed onset of nerve injury-associated mechanical hypersensitivity. These results indicate that gC1qR is the receptor for TLQP-21 and plays an important role in chronic pain through activation of macrophages. Because direct association between TLQP-21 and gC1qR is required for activation of macrophages and causes hypersensitivity, disrupting this interaction may be a useful new approach to develop novel analgesics.

Novel mutations mapping to the fourth sodium channel domain of Nav1.7 result in variable clinical manifestations of primary erythromelalgia.

We identified and clinically investigated two patients with primary erythromelalgia mutations (PEM), which are the first reported to map to the fourth domain of Nav1.7 (DIV). The identified mutations (A1746G and W1538R) were cloned and transfected to cell cultures followed by electrophysiological analysis in whole-cell configuration. The investigated patients presented with PEM, while age of onset was very different (3 vs. 61 years of age). Electrophysiological characterization revealed that the early onset A1746G mutation leads to a marked hyperpolarizing shift in voltage dependence of steady-state activation, larger window currents, faster activation kinetics (time-to-peak current) and recovery from steady-state inactivation compared to wild-type Nav1.7, indicating a pronounced gain-of-function. Furthermore, we found a hyperpolarizing shift in voltage dependence of slow inactivation, which is another feature commonly found in Nav1.7 mutations associated with PEM. In silico neuron simulation revealed reduced firing thresholds and increased repetitive firing, both indicating hyperexcitability. The late-onset W1538R mutation also revealed gain-of-function properties, although to a lesser extent. Our findings demonstrate that mutations encoding for DIV of Nav1.7 can not only be linked to congenital insensitivity to pain or paroxysmal extreme pain disorder but can also be causative of PEM, if voltage dependency of channel activation is affected. This supports the view that the degree of biophysical property changes caused by a mutation may have an impact on age of clinical manifestation of PEM. In summary, these findings extent the genotype-phenotype correlation profile for SCN9A and highlight a new region of Nav1.7 that is implicated in PEM.