Unlike in other autoimmune liver diseases such as autoimmune hepatitis and primary biliary cholangitis, the role and nature of autoantigenic targets in primary sclerosing cholangitis (PSC), a progressive, chronic, immune-mediated, life threatening, genetically predisposed, cholestatic liver illness, is poorly elucidated. Although anti-neutrophil cytoplasmic antibodies (ANCA) have been associated with the occurrence of PSC, their corresponding targets have not yet been identified entirely. Genome-wide association studies revealed a significant number of immune-related and even disease-modifying susceptibility loci for PSC. However, these loci did not allow discerning a clear autoimmune pattern nor do the therapy options and the male gender preponderance in PSC support a pathogenic role of autoimmune responses. Nevertheless, PSC is characterized by the co-occurrence of inflammatory bowel diseases (IBD) demonstrating autoimmune responses. The identification of novel autoantigenic targets in IBD such as the major zymogen granule membrane glycoprotein 2 (GP2) or the appearance of proteinase 3 (PR3) autoantibodies (autoAbs) have refocused the interest on a putative association of loss of tolerance with the IBD phenotype and consequently with the PSC phenotype. Not surprisingly, the report of an association between GP2 IgA autoAbs and disease severity in patients with PSC gave a new impetus to autoAb research for autoimmune liver diseases. It might usher in a new era of serological research in this field. The mucosal loss of tolerance against the microbiota-sensing GP2 modulating innate and adaptive intestinal immunity and its putative role in the pathogenesis of PSC will be elaborated in this review. Furthermore, other potential PSC-related autoantigenic targets such as the neutrophil PR3 will be discussed. GP2 IgA may represent a group of new pathogenic antibodies, which share characteristics of both type 2 and 3 of antibody-mediated hypersensitive reactions according to Coombs and Gell. Primary sclerosing cholangitis (PSC) is a chronic immune-mediated, life threatening, genetically predisposed liver disease with a largely unknown pathogenesis . The prePrimary sclerosing cholangitis is characterized by cholestasis due to inflammatory and fibrotic changes in large bile ducts. The disease has a progressive course eventually resulting in biliary fibrosis and liver cirrhosis in a proportion of cases finally. Moreover, patients with PSC have an increased risk to develop hepatobiliary (most frequently cholangiocarcinoma [CCA]) and extrahepatic neoplasia independent of the duration and activity of the disease. To date there is no causative treatment available and liver transplantation remains the only curative therapy , 4.The impact of autoimmune responses in the pathophysiology of PSC is still largely unknown. Neither therapeutic options nor male gender preponderance in PSC are indicative for a significant pathogenic role of autoimmune responses. Indeed, the administration of immunosuppressive drugs for PSC patients is at least controversial and rarely recommended , 6. OnlyCellular immune responses might be involved in the pathophysiology of PSC . HoweverThe currently known susceptibility loci do also not allow to discern a clear autoimmune pattern, though the association with distinct HLA haplotypes suggests an involvement of acquired immune responses . In partOf interest, PSC is associated with the co-occurrence of inflammatory bowel diseases (IBD) which are known to be affected by autoimmune responses , 14. Up The two main clinical entities of IBD are Crohn’s disease (CD) and UC, both relapsing systemic inflammatory illnesses , 16, 17.Primary sclerosing cholangitis patients with concomitant UC demonstrated an elevated risk of liver disease progression . ConversAkin to PSC, there is no causal treatment for IBD and the illness may progress with repeated flare-ups to therapy or digestive failure requiring surgical intervention , 24. MorTo date, 23 susceptibility loci have been identified by genome-wide association studies (GWAS) for PSC . In contGiven the close association of PSC with IBD, the discovery of distinct autoantigenic targets in IBD and the use of the corresponding autoantibodies (autoAbs) for IBD serology renewed the interest in humoral autoimmune responses in PSC , 17. DesThe occurrence of autoAbs in IBD was first shown for UC by revealing autoreactivity against intestinal cellular antigens in the late 1950s –34. LateIn CD, the first report on humoral autoimmunity referred to the occurrence of autoAbs against buccal mucosa cells  and, latIn PSC, humoral autoimmunity in the form of autoAbs to several targets including biliary epithelial targets was reported , 46. HowUntil lately, there have been only few reports that the above mentioned autoAbs in patients with IBD and PSC are correlated with clinical parameters or even the phenotype of the disease. Consequently, they have not widely been employed in clinical routine. Thus, the identification of the respective autoantigenic targets could help in shedding light on the role of humoral autoimmunity in IBD and PSC. This would furthermore enable the development of clinically useful tools for the diagnosis thereof.Autoimmune processes have been considered to play an active role in disease development and to modulate inflammatory processes in CD . TherefoOnly 25 years after the first report on PAb in patients with CD and numerous unsuccessful attempts by several research groups, the corresponding molecular autoantigenic targets could be discovered , 53–57. Altogether, the identification of GP2 and CUZD1 as autoantigenic targets in CD ushered in a new era in IBD serology and triggered an impressive number of clinical studies investigating the potential role of the respective autoAbs in the differential diagnosis of IBD , 64.Saccharomyces cerevisiae (ASCA) in CD serology, autoAbs to GP2 demonstrated a remarkable specificity allowing even the discrimination of intestinal diseases with similar clinical symptoms such as intestinal tuberculosis and Behcet’s disease [Already two meta-analyses encompassing 17 and 15 serological studies have been reported to date , 66. For disease . This is disease .The moderate sensitivity of GP2 autoAb appears to limit its use as diagnostic marker for CD . HoweverSimilar studies for autoAbs to CUZD1 supporting an association with disease phenotypes  have been scarce or have not shown a significant correlation –78. PappMycobacterium avium induced paratuberculosis [Remarkably, GP2 autoAb occurrence could be linked with de novo CD in patients suffering from severe UC with pouchitis after colectomy and ileal pouch anal anastomosis (IPAA) , 80. Thirculosis –83. GP2 In contrast to CD, ANCA to unknown neutrophil targets were already reported in the 1980s as serological markers of PSC and UC , 37. HowTeegen et al.  have repMoreover, the identification of a colon specific 40 kDa murine protein linked to UC could be reproduced in humans, but the sequence analysis of this target failed to match it with a particular molecule .Hence, the independent reports of autoAbs to proteinase 3 (PR3), a neutrophil target, by sensitive bead-based chemiluminescence and fluorescence techniques in patients with UC provided a new diagnostic option for the differential diagnosis of IBD –91. DespNevertheless, this intriguing finding warrants further clinical evaluation to elucidate the putative autoantigenic role of PR3 in UC and its possible link with the pathophysiology in GPA. It provides further evidence, however, for a potential role for neutrophils in the pathophysiology of IBD as does the report of DNA-dependent lactoferrin autoAbs. Due to the high prevalence of concomitant UC in patients with PSC, the question of a potential role of PR3 as an autoantigenic target in PSC began to appear on the horizon.At the beginning of the millennium, IgG autoAbs recognizing biliary epithelial cells have been shown to be specific for PSC and in this context combine adaptive and innate immune responses , 93. HowOf note, all other relevant PSC-specific autoAbs appear to recognize non-biliary targets . Thus, tAkin to UC, atypical ANCA or more precisely p-ANNA have been one of the most debated diagnostic markers in PSC , 94. AftThus, the report on PR3 autoAbs detected by a sensitive chemiluminescence assay in patients with PSC and its correlation with elevated liver enzymes, particularly with alkaline phosphatase, renewed the interest in potential ANCA targets in PSC . As outlBesides the association of PR3 autoAb with the PSC phenotype, another groundbreaking finding came into the spotlight of autoimmune research in PSC. The first report in the year 2015 on the association of GP2 autoAb with concomitant PSC in patients with UC triggered several studies on the role of loss of tolerance to GP2 in PSC . This fiIn total, autoAbs to four isoforms of GP2 have been reported in patients with IBD , 103. ThIn summary, GP2 seems to function as a unique autoantigenic target in CD and in PSC. Given the close link with disease severity and carcinogenesis, IgA to GP2 and its isoforms have the potential to become accepted as pathognomonic and predictive for PSC. It remains to be shown what additional diagnostic or even prognostic role PR3 autoAbs can play in the serology of PSC.The novel autoantigenic target in PSC, GP2, is not organ-specific nor is the PR3-AutoAb. In this context it is interesting to note that the much debated controversy on the expression of CD-specific autoantigenic targets in extraintestinal organs  and not at the site of inflammation in the gut could only be overcome for GP2 , 105. ElIn earlier studies, GP2 was identified as the major pancreatic zymogen granule membrane protein with an assumed but non-confirmed role in zymogen granula formation –112. ThuE. coli which can target human PP via long polar fimbriae and point mutations in their FimH amino acid sequences were linked with CD [In this context, GP2’s modulating role of innate and adaptive immune responses by sensing microbiota and regulating intestinal anti-microbial immune responses is quite remarkable –126. The with CD –129. Int with CD , 62. Fur with CD , 130. ThWith regard to liver autoimmunity, loss of tolerance to similar surface receptors such as the hepatocyte-specific asialoglycoprotein receptor interacting also with potential pathogens or their components has been demonstrated in patients with AIH and PSC recently , 132. AlGiven the association of both GP2 IgG and IgA to the fibrostenosing subtype and the severity of CD, it is quite remarkable that only IgA to GP2 has been linked with the clinical phenotype and severity in PSC. Of note, dimeric GP2 IgA like most of the IgA secreted by mucosal plasma cells could be actively transported by epithelial cells onto mucosal surfaces as has been shown for GP2 IgA in pouchitis patients with de novo CD . Thus, GFaecalibacterium prausnitzii was linked with an elevated risk of postoperative recurrence of ileal CD [Streptococcus is considered to play a putative role in the progression of PSC.There is growing evidence to suggest that the biliary tract is not a sterile environment as previously thought , 137. A ileal CD . Butyratileal CD . The aetileal CD . HoweverFinally, an elevated secretion and re-absorption of secretory IgA was reported in patients with PSC . The majHence, it is tempting to speculate that GP2 as microbiota-sensing receptor and the ensuing mucosal loss of tolerance are not only involved in the pathophysiology of CD, but also in that of PSC Fig. . It woulIn CD, GP2 IgA aside from GP2 IgG has been linked with fibrotic changes in several studies whereas only GP2 IgA was associated with severity in PSC Table . ConcentThe close association of GP2 IgA with tumorigenesis in PSC is another intriguing point regarding a putative pathophysiological role of GP2 IgA. The two main precursor lesions of cholangiocarcinoma are biliary intraepithelial neoplasia and intraductal papillary neoplasm of the bile duct .In general, the intestinal mucosa harbours the largest population of immunoglobulin-secreting plasma cells in humans, shedding daily several grams of IgA. This exceeds the production of all other immunoglobulin subtypes in the human body . IgA-posDoes secretory GP2 IgA enhance the up-take of pathogenic microbes which in turn further trigger the generation of plasma cells secreting IgA autoAbs?Is GP2 secreted into the bile by exocrine glands?Is there a specific microbe in the bile which can interact with GP2?Are GP2 IgA-positive plasma cells abundant in the biliary mucosa in patients with PSC?Do GP2 IgA-positive plasma cells have an immunosuppressive phenotype fostering neoplasia and impacting the immune surveillance of tumor cells?Thus, in terms of a putative pathogenic role of the mucosal loss of tolerance to GP2 in PSC, the above mentioned findings give rise to the following questions:The putative pathogenic role of PSC-specific (auto)Abs has not yet been addressed by appropriate studies. There is an urgent need to shed a light on the yet unresolved pathophysiological role of autoimmunity in PSC, associated IBD and finally carcinogenesis to provide more effective diagnostic and therapeutic strategies. We assume that mucosal autoimmunity to GP2 could be a promising candidate to demonstrate active involvement of autoimmune responses based on an intestinal or biliary dysbiosis in the pathophysiology of PSC. Glycoprotein 2 appears to be a unique autoantigenic target in PSC. Thus, GP2 has been shown (i) to be discharged by mucus glands into the digestive tract, (ii) to interact with microbiota by binding FimH-positive microbes, (iii) to be expressed as specific receptor on M cells of the FAE and (iv) to be an immunomodulating factor of innate and acquired immune responses. Consequently, the mucosal loss of tolerance to GP2 in form of GP2 IgA is a potential pathognomonic marker of PSC with predictive value that may improve the diagnosis and prognosis of the illness.


Pancreatic cancer refers to a group of malignancies of which pancreatic ductal adenocarcinoma (PDAC) is the most common form. It is an aggressive tumour with few treatment options and very poor outcomes. There is an unmet need for novel targeted therapies for PDAC. In this regard, a better understanding of PDAC biology and in particular new pathways that contribute to disease progression would help identify novel targets for therapeutic intervention. Growing evidence implicates the unfolded protein response (UPR) in many cancers, and this article explores the evidence that supports a role for the UPR in PDAC.Pancreatic ductal adenocarcinoma (PDAC) is the most common form of pancreatic cancer and one of the leading causes of cancer-associated deaths in the world. It is characterised by dismal response rates to conventional therapies. A major challenge in treatment strategies for PDAC is the presence of a dense stroma that surrounds the tumour cells, shielding them from treatment. This unique tumour microenvironment is fuelled by paracrine signalling between pancreatic cancer cells and supporting stromal cell types including the pancreatic stellate cells (PSC). While our molecular understanding of PDAC is improving, there remains a vital need to develop effective, targeted treatments. The unfolded protein response (UPR) is an elaborate signalling network that governs the cellular response to perturbed protein homeostasis in the endoplasmic reticulum (ER) lumen. There is growing evidence that the UPR is constitutively active in PDAC and may contribute to the disease progression and the acquisition of resistance to therapy. Given the importance of the tumour microenvironment and cytokine signalling in PDAC, and an emerging role for the UPR in shaping the tumour microenvironment and in the regulation of cytokines in other cancer types, this review explores the importance of the UPR in PDAC biology and its potential as a therapeutic target in this disease. Pancreatic cancer refers to a group of malignancies that originate from the pancreatic tissue. The majority of these malignancies arise from the glandular exocrine tissue, potentially from both ductal or acinar cells, and are referred to as pancreatic ductal adenocarcinoma (PDAC). PDAC is by far the most common form of pancreatic cancer, representing more than 90% of all pancreatic cancer cases . AlthougConventional treatments such as chemotherapy, surgery and radiation do not significantly improve survival rates for PDAC patients. Although resection and adjuvant chemotherapy is the only potential curative treatment approach, only 10–20% of patients present with resectable PDAC, and the five-year survival rate for these patients is only 15–25% . The majHistorically, the standard-of-care chemotherapy for PDAC was gemcitabine. However, first line therapies now tend to be FOLFIRINOX  or a combination of gemcitabine plus nanoparticle albumin-bound (nab)-paclitaxel .KRAS, CDKN2A/p16, TP53 and SMAD4 and activation of their associated downstream signalling pathways appear to play a key role in the disease [G12C, as well as the development of inhibitors of Src homology-2 domain-containing protein tyrosine phosphatase-2 (SHP2), a non-receptor protein tyrosine phosphatase that modulates RAS activity [While the molecular pathways involved in the development, progression and metastasis of PDAC are not fully elucidated, mutations in genes such as  disease . KRAS mu disease . These m disease . While oactivity ,11, haveThe poor prognosis associated with PDAC is largely due to both intrinsic and acquired chemoresistance. Components of the tumour microenvironment (TME), also known as the tumour stroma, and associated desmoplasia are major contributing factors to chemoresistance in PDAC. Desmoplasia, a key pathological characteristic of PDAC, occurs as a result of a dramatic increase in the proliferation of alpha-smooth muscle actin-positive fibroblasts and is accompanied by increased deposition of extracellular matrix components (ECM). In this environment, extensive extracellular matrix deposition alongside rapidly dividing tumour cells that often outgrow vascular supply leads to oxygen and nutrient deprived regions of tumour tissue. These environmental stresses cause PDAC cells, as well as cells within the stromal tissue, to rely on adaptive mechanisms to survive and thrive.The unfolded protein response (UPR), a well-characterised cellular stress response pathway, is activated in many cancers, due to several factors which may include oncogenic stress, metabolic reprogramming as well as the harsh conditions in the TME. The UPR is an area of intense investigation for therapeutic intervention in cancer. In this review article, we will describe PDAC and its unique TME/stroma and discuss the emerging roles of the UPR in PDAC progression and how the UPR may regulate PDAC-stromal crosstalk and contribute to chemoresistance.The TME can occupy up to 90% of the entire tumour mass and comprises both cellular and acellular components . High stPSCs are the most prominent cell type in the PDAC stroma and can constitute about 50% of the total tumour mass. They are periacinar star-shaped cells that were first isolated from the pancreas in 1998 and characterised as having striking similarities to hepatic stellate cells (HSCs) ,15. PSCsAlcohol consumption, smoking, environmental stresses such as hypoxia, hyperfusion, oxidative stress and excessive cellular signalling by growth factors and/or cytokines can all cause damage to the pancreas . During While the primary function of aPSCs in PDAC tumorigenesis is to produce ECM components and form the physical desmoplasia, there is growing recognition of the important role of PSC–tumour cell interactions in promoting all aspects of PDAC pathogenesis. There is compelling evidence for a dynamic cross-talk between PSCs and cancer cells ,19, whicMost solid tumours exhibit altered metabolic pathways in the form of the Warburg or reverse Warburg effects. However, this is often insufficient for tumour growth, and further metabolic reprogramming is required. It has been shown that PSC paracrine signalling increases pancreatic cancer cell proliferation and can regulate cancer cell metabolism, thereby aiding tumorigenesis ,25. ParaMost PDAC tumours develop from malignant lesions termed pancreatic intraepithelial neoplasias (PanINs) . It has Proteomic profiling of aPSCs revealed there were 641 proteins secreted by these cells. The same profiling revealed 46 proteins that were unique to the secretome of qPSCs . AmongstRegulation of PSC activation and pro-tumorigenic processes associated with PSC activation is currently a relatively unexplored avenue of therapeutic interrogation in PDAC. It is tempting to suggest that targeting these cells may provide a substantial benefit to PDAC patients as a way of suppressing oncogenic signalling in the TME. Given that the UPR is known to play an important role in regulation of secretion, and the impact that this regulation can have on multiple cell processes, understanding its role in PSC phenotype may offer novel insight into PDAC progression.The ER is an important organelle with key roles in proteostasis, lipid metabolism and drug detoxification. Perturbation of ER homeostasis results in a condition termed “ER stress” that is characterized by a build-up of unfolded proteins in the ER lumen . This acThe UPR is controlled by three ER-anchored receptors, namely, inositol-requiring enzyme 1 (IRE1), protein kinase RNA-like ER kinase (PERK) and activating transcription factor 6 (ATF6). Glucose-related protein 78 ), an ER chaperone protein present in the ER lumen, blocks activation of IRE1, PERK and ATF6 by binding to their ER luminal domains. Build-up of misfolded proteins in the ER lumen, which occurs under ER stress conditions, promotes GRP78 dissociation resulting in the activation of IRE1, PERK and ATF6 and their downstream signalling pathways as described in XBP1s) [Following dissociation of GRP78, IRE1 oligomerises and trans-autophoshorylates enabling its RNase activity ,47. The XBP1s) . When trXBP1s) . Our undXBP1s) . The IREXBP1s) . In addiXBP1s) .Upon GRP78 dissociation, PERK dimerises, undergoes trans-autophosphorylation of its cytosolic domain and phosphorylates its primary substrate, eukaryotic translation initiation factor 2A (eIF2α). This leads to attenuation of global mRNA translation thus reducing the load of nascent proteins entering the ER . In contXBP1 mRNA [Release of GRP78 causes a conformational change in ATF6 revealing two Golgi localisation signals . Upon trBP1 mRNA .PDAC tumours and the cells comprising their surrounding microenvironments are reported to exhibit basal ER stress due to their hypoxic, nutrient-deprived state . Thus, iThe most commonly researched UPR component in pancreatic cancer is GRP78. It is important to point out that GRP78 expression is not itself a marker of UPR activity due to its constitutive expression in normal cells. However, elevated levels of GRP78 are commonly observed under conditions of ER stress, and its expression is increased upon activation of the UPR. Increased expression of GRP78 has been correlated to poor disease prognosis and progression of multiple cancer types including prostate cancer , ovarianGRP78, suggests its influence may be broad-reaching with changes noted in key signalling pathways including cell-cycle, apoptosis and actin-cytoskeleton regulation [Elevated expression of GRP78 is a feature of PDAC cells when compared to normal pancreatic ductal cells , althouggulation . PDAC cegulation . In the gulation .G12D/+; p53f/+Pdx1-Cre; Kras (PKC) mouse model of PDAC, high GRP78 was observed in the pancreata of 2 month old mice that had developed tumours by 3 months old. Interestingly, in the same genetic background, mice pancreata bearing an additional f/+Grp78 allele exhibited reduced pancreatic tumourigenesis [A role for GRP78 in murine models of PDAC has also been observed. In a igenesis .GRP78 reduces efflux by ATP-binding cassette transporters [GRP78 can also impact chemoresistance, and some have suggested that a combination therapy that includes an anti-GRP78 agent may be beneficial in the treatment of pancreatic cancer ,73. One sporters . In thissporters .Despite these studies, there remains significant questions regarding the precise role of GRP78 in PDAC. High levels of GRP78 are present in PDAC, and these studies indicate that reducing GRP78 has anti-tumour effects. However, these observations seem paradoxical when UPR activation in the context of altered GRP78 expression is taken into account. While it is likely that the levels of GRP78 reflect perturbations in proteostasis in PDAC, there remains many unknowns regarding the precise role of this protein in PDAC as well as other cancers. Precisely why GRP78 is increased in PDAC has not been extensively explored. High levels of expression could simply reflect enhanced ER homeostasis that is common in many cancer cells. Alternatively, high GRP78 could be a consequence of rapid proliferation of the cells, which would lead to high levels of protein synthesis. There remains much to learn regarding the molecular mechanisms governing its expression and regulation in PDAC.While IRE1 ,75 and PSimilar to other cancer types , a role for IRE1-dependent signaling in maintaining proliferative capacity of PDAC cells has been reported ,79,80. Sfl/fl mice, XBP1 loss resulted in apoptosis of acinar cells demonstrating the importance of this gene in acinar cells [As well as ductal cells, enzyme producing acinar cells have also been suggested as PDAC cells of origin . Acinar ar cells . PERK waar cells . Taking Limited efficacy and acquired resistance to chemotherapy are significant issues in treatment of PDAC. Gemcitabine resistance is commonly reported and has led to a surge in research for combinatory treatments that can overcome chemoresistance. While gemcitabine can be an effective chemotherapeutic, it is also thought to contribute to relapse of a more aggressive tumour type. Gemcitabine is known to induce PDAC epithelial to mesenchymal transition, enhance survival of cells that persist after treatment ,89,90, iA possible mechanism of chemoresistance may utilise pro-survival signalling of the UPR such that cancer cells can evade chemotherapy-induced apoptosis. It was suggested that a cancer stem cell-like population isolated from a PANC1 cell line displayed divergent UPR responses after treatment with gemcitabine, where phospho-PERK and ATF6N were increased, and phospho-IRE1 was decreased . InteresAs previously discussed, PSCs are a key cell type within the pancreas, and growing evidence suggests that they promote PDAC progression. PSCs may also impact responsiveness of PDAC to chemotherapy . The rolAlthough knowledge of UPR activation in PSCs is lacking, it is informative to consider the role of the UPR in HSCs. PSCs share many functional and phenotypical characteristics with HSCs. Transcriptional analysis reveals that HSCs and PSCs share very similar genetic profiles . In HSCsKB activation for example). This may be an important consideration in the context of pancreatic cancer.The unique PDAC microenvironment promotes tumour development and is a major source of cytokines that promote oncogenesis. It has been hypothesized that the UPR represents a potential crossroads between ER stress and inflammatory signalling . UPR canThe role and mechanics of UPR-mediated cytokine regulation in inflammatory and disease conditions is extensively reviewed elsewhere . A recenThe concept of transmissible ER stress (TERS) is quickly gaining traction in the UPR field. This concept is based on the observation that cells experiencing ER stress secrete some unknown factor(s) capable of inducing ER-stress response/UPR in the nearby recipient cells. Originally identified in myeloid cells , TERS haPancreatic cancer is predicted to rise from the fourth to second highest mortality rate for cancer-related deaths in developed countries in the next decade . There iTargeting UPR in PDAC may be highly beneficial. Although the UPR is considered to be an integrated pathway requiring coordinated activation of IRE1, PERK and ATF6, independent activation of these stress sensors under certain conditions highlights the complexity of the pathway ,93. DeveA key aspect of PDAC biology lies in its unique TME. In this setting, UPR activation likely affects both autocrine and paracrine interactions, and UPR targeted therapies could offer a novel approach to modulate these PDAC signals. As the dense stroma surrounding pancreatic tumours creates a physical barrier against current treatments (largely chemotherapeutics), targeting the interactions that foster the expansion and growth of this barrier will enhance the efficiency of already available treatments. If the UPR does in fact regulate pancreatic cytokines and growth factors, promoting a tumour microenvironment that supports PDAC progression, it represents an exciting prospect for future studies in the field of pancreatic cancer research.


Furthermore, phthalates can pass through the placental barrier and affect the developing fetus. Thus, phthalates have ubiquitous presence in food and environment with potential adverse health effects in humans. This review focusses on studies conducted in the field of toxicogenomics of phthalates and discusses possible transgenerational and multigenerational effects caused by phthalate exposure during any point of the life-cycle.Phthalates are esters of phthalic acid which are used in cosmetics and other daily personal care products. They are also used in polyvinyl chloride (PVC) plastics to increase durability and plasticity. Phthalates are not present in plastics by covalent bonds and thus can easily leach into the environment and enter the human body by dermal absorption, ingestion, or inhalation. Several  Phthalates or diesters of phthalic acid are a group of ubiquitous synthetic compounds commonly found in a variety of consumer products like plasticizers since the 1930s  originate from the inner cell mass at the blastocyst stage of a preimplantation embryo and can differentiate into the three germ layers of the embryo: the ectoderm, endoderm, and the mesoderm  family—Tet1, Tet2, and Tet3  and Illumina Human Methylation 850K BeadChip (EPIC). The HM450K array measures DNA methylation levels at over ~450,000 CpG dinucleotides throughout the genome and has been replaced in 2016 by the EPIC array that measures over >850,000 CpG dinucleotides and and overlaps with about 90% of the sites represented by HM450K chip  studies is using embryonic stem cells (ESCs) in so-called embryonic stem cell tests (ESTs). ESCs can make organismal decisions and ultimately give rise to the three main lineages of the embryo—the ectoderm, mesoderm, and endoderm. Human embryonic stem cell (huESC) models serve as n = 48) and elevated periconceptional mono-2-ethylhexyl phthalate (MEHP) exposure (mean: 23.4 ng/mL)  affected the developmental competency, and exposure to 10−4 MBP resulted in the delay of progression from embryo to blastocyst  and embryonic stem cell tests (ESTs) , DEP, and DEHP decreased the cell expansion with DBP being the most cytotoxic , transmembrane mitochondrial potential (ΔΨm) and caspase −8,−9,−3 activity were determined. It was seen that phthalates particularly DBP and BBP increased levels of Ca 2+ and reduced ΔΨm of the PBMCs. Phthalates also increased the activity of the caspases, the most significant being caspase−9  residing in Salinas Valley, California , though correlations of individual CpG sites with low variance of methylation were modest (median r = 0.24). There was also a subset of CpG sites that had large differences in the mean methylation beta-estimates between the two platforms  hypothesis, which posits that A recent study investigated how phthalates impair human placental function by epigenetic regulation of critical placental genes  methylation and low birth weight. Placental LINE-1 methylation might serve as a biomarker for environmental exposure causing adverse fetal growth as fetal programming is regulated by appropriate methylation patterning  in placenta and prenatal exposure to HMW phthalate metabolites. Abnormal IGF2/H19 methylation in placenta suggests the fact that the developing fetus may be exposed to an adverse intrauterine environment  and maternally expressed H19 both on chromosome 11 are two reciprocally critical imprinted genes and play significant roles in fetal and embryonic growth. Inverse associations were observed between IGF2) which are important in embryonic growth and development. These associations were very predominant in fetal growth restriction (FGR) newborns as opposed to normal neonates   and whole blood samples for 9-year olds were examined to investigate the relationship between in utero may impact DNA methylation in cord blood. These changes in DNA methylation may be candidates for biomarkers used to ascertain maternal exposure to phthalates during pregnancy and potential candidates for studying the underlying mechanisms of the long-term effects of phthalates and also the ways phthalates may impact health throughout the life course  by exerting strong antiandrogenic , and progesterone (P4), play important roles in regulating the menstrual cycle, pregnancy, and embryogenesis in humans and other species , sex determining region Y-box 2 (SOX2), and NANOG homeobox genes at both transcriptional and translational levels. The cells growing in control culture media without any hormones assumed the form of tightly packed cells growing in a monolayer with clean and defined edges and showed no signs of differentiation. These cells also expressed several markers specific for undifferentiated ES cells including POU5F1, SOX2, and NANOG  that mimic the natural hormones found in the human body and thus interfere or impair normal hormonal activity . E2 or P4 treatment increases the protein expression levels of N-cadherin, Snail and Slug, which are highly expressed in mesenchymal cells and decreases E-cadherin expression, which is highly expressed in epithelial cells. Phthalates exert weak estrogenic effects  found the effects of hormones on EMT and pluripotency of ES cells were restored to control levels. These findings indicate that E2 and P4 regulation of EMT and pluripotency of human ES cells are mediated by their receptors  stimulate hematopoietic stem progenitor cells to proliferate. These cells also proliferate in response to gonadal sex hormones like androgen, estrogen, and progesterone   is defined as anogenital distance (AGD) divided by weight at examination [AGI = AGD/weight (mm/kg)]. The rationale for measuring AGD is that males have a shorter AGD than females and consequently, changes in this distance are a measure of feminization or masculinization of the reproductive organs. In a study that aimed to correlate prenatal exposure to phthalates in amniotic fluid, maternal urine, and health of newborns in humans, it was found that DEHP is primarily used to soften PVC plastic in medical apparatus like blood bags, or bags used for intravenous administration of nutrients, drugs, and fluids. DEHP can leach out from the plastic that is used to make all of these bags to enter the patient's circulation during procedures like transfusion, heart bypass surgery, or the administration of intravenous fluids Latini, . Based oandrogen receptor (AR), TNFα, and IL-4 causing asthma in children  and glypican 4 (Gpc4) in subcutaneous and visceral adipose tissues respectively. Tbx15 and Gpc4 are known to be developmental genes which play a role in obesity and body fat distribution in mice  as opposed to control samples. H19 methylation was positively associated with skinfold thickness in girls. There were sex-specific differences in exposure outcomes- among boys, adiposity was inversely associated with second trimester and adolescent MBzP  was evaluated  causing increase in body mass in early life. This observation also suggested that in utero exposure to DEP and DEHP was positively associated with body mass change of the newborns during first 3 months after birth  are single-stranded, non-coding RNA molecules (sncRNAs) which are evolutionary-conserved and are involved in the regulation of gene expression at the posttranscriptional level Ambros, . miRNAs One hypothesis is that the long-term reproductive defects associated with phthalate exposure is exerted through the action of non-coding miRNAs  during pregnancy , mono-2-ethyl-5-hydroxyhexyl phthalate (MEHHP), mono-2-ethyl-5-carboxypentyl phthalate (MECPP), and DEHP -miRNAs in their follicular fluid. The urine samples were collected from participants during ovarian stimulation and the day oocyte was retrieved  was established by the National Toxicology Program (NTP) in 1998  is a curated database that aids in understanding the effects of various environmental chemicals on human health. Biocurators at CTD use information from the literature to manually curate chemical-gene interactions, chemical-disease relationships and gene-disease relationships and construct chemical-gene-disease networks  along with BPA have 1,232 and 265 interactions with unique genes/proteins, respectively , the National Toxicology Program (NTP) typically include three doses while testing chemicals such as environmental endocrine disruptors (EEDs) like phthalates for the purposes of non-clinical risk assessment: (i) No-Observable-Adverse-Effect-Level (NOAEL): the highest dose/exposure given to an organism found by experiment or observation that has no observed toxic or adverse effect on traditional toxicological endpoints compared to an appropriate control (ii) No-Observable-Effect-Level (NOEL): the highest dose or exposure level that produces no observable effect in the animals tested when compared with its appropriate control (iii) Lowest-Observed-Adverse-Effect Level (LOAEL): the lowest concentration of a substance that causes toxic or biochemical effects in animal studies  downregulation of receptor and receptor desensitization  (v) receptor competition, in that the mixture of endogenous hormones and EDCs creates an environment that gives rise to NMDRCs , and the lowest observed adverse effect level (LOAEL) was 300 ppm or 14 mg/kg/day  followed by fertilization, gestational development of the embryo, birth, postnatal growth followed by puberty leading to sexual maturity and the ability to reproduce. Though the terms “developmental” and “reproductive” toxicities are two separate entities, there is substantial overlap between them Shelby, . ToxicolMost of the evidences we currently have for transgenerational epigenetic inheritance is in animals. In humans, because of our long lifespan and diverse genetics, it is complicated to conduct studies for 3 to 4 generations  transient which can be removed and (ii) permanent which is heritable  has adopted strict regulations around use of phthalates and other EDCs- it is an interesting natural experiment that could be used to investigate how these diseases change in Europe. The impact of phthalates varies from one population to population based on their food habits and lifestyles. The concentration of phthalate metabolites in human body widely varies based on demographics (Benjamin et al., An emerging view in the field of epidemiologic research is that intrauterine growth period of the fetus is a critical window of susceptibility during which environmental toxicants can affect the developmental trajectories and cause epigenetic information to be transmitted between generations (Morkve Knudsen et al., The various mechanisms which play an important role in passing on information from one generation to another are DNA methylation, histone modification, or changes in non-coding RNA (Heard and Martienssen, Where can research improve to develop a better understanding of the biological mechanisms underpinning phthalate exposures and human disease? Environmental epidemiology needs to focus on accurate characterization of phthalate exposure by using multiple samples across time to best capture exposure status. Epigenetic studies of phthalate exposures should consider how mixtures of phthalates affect gene expression beyond the traditional classifications of low and high molecular weight.In conclusion, future studies of phthalates and other environmental chemicals must examine potential multigenerational effects of exposures. Maternal exposures, both prior to and during pregnancy, can potentially affect the developing egg and fetus. Paternal exposures can potentially affect the sperm. More studies with model organisms, such as with zebrafish, are needed to examine the mechanisms of multigenerational inheritance of phenotypes induced by chemicals such as phthalates.SD conducted the literature review, drafted, and revised the manuscript. DH provided critical review of the manuscript and revised the manuscript. DAR provided critical review of the manuscript. DMR conceived the review, provided critical review of the manuscript, and revised the manuscript.DAR is the Chief Scientific Officer in the Reproductive Stress company, but the collaboration on this project involves no conflict of interest. The remaining authors also declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Multiple sclerosis (MS) and neuromyelitis optica spectrum disorders (NMOSD) are autoimmune central nervous system conditions with increasing incidence and prevalence. While MS is the most frequent inflammatory CNS disorder in young adults, NMOSD is a rare disease, that is pathogenetically distinct from MS, and accounts for approximately 1% of demyelinating disorders, with the relative proportion within the demyelinating CNS diseases varying widely among different races and regions. Most immunomodulatory drugs used in MS are inefficacious or even harmful in NMOSD, emphasizing the need for a timely and accurate diagnosis and distinction from MS. Despite distinct immunopathology and differences in disease course and severity there might be considerable overlap in clinical and imaging findings, posing a diagnostic challenge for managing neurologists. Differential diagnosis is facilitated by positive serology for AQP4-antibodies (AQP4-ab) in NMOSD, but might be difficult in seronegative cases. Imaging of the brain, optic nerve, retina and spinal cord is of paramount importance when managing patients with autoimmune CNS conditions. Once a diagnosis has been established, imaging techniques are often deployed at regular intervals over the disease course as surrogate measures for disease activity and progression and to surveil treatment effects. While the application of some imaging modalities for monitoring of disease course was established decades ago in MS, the situation is unclear in NMOSD where work on longitudinal imaging findings and their association with clinical disability is scant. Moreover, as long-term disability is mostly attack-related in NMOSD and does not stem from insidious progression as in MS, regular follow-up imaging might not be useful in the absence of clinical events. However, with accumulating evidence for covert tissue alteration in NMOSD and with the advent of approved immunotherapies the role of imaging in the management of NMOSD may be reconsidered. By contrast, MS management still faces the challenge of implementing imaging techniques that are capable of monitoring progressive tissue loss in clinical trials and cohort studies into treatment algorithms for individual patients. This article reviews the current status of imaging research in MS and NMOSD with an emphasis on emerging modalities that have the potential to be implemented in clinical practice. Multiple sclerosis (MS) and neuromyelitis optica spectrum disorders (NMOSD) are inflammatory, autoimmune central nervous system conditions that have shown increasing incidence and prevalence over the past decades –5. WhileFor a long time, NMOSD had been seen as a rare variant of MS; however, the seminal discovery of a highly specific serum IgG autoantibody to the astrocyte water channel aquaporin-4 (AQP4) in up to 80% of NMOSD patients and subsequent research into the role of these antibodies in disease pathogenesis and lesion formation has made clear that this is a condition distinct from MS –17. ClinImaging of the brain, optic nerve, retina, and spinal cord is a procedure of paramount importance when managing patients with inflammatory CNS conditions at first presentation to enable diagnosis and differential diagnosis –28. OnceThis article reviews the current status of imaging research in MS and NMOSD with an emphasis on emerging modalities that have the potential to be implemented in clinical practice for diagnosis, differential diagnosis, and monitoring of disease course and immunotherapies.As in previous versions of the MS diagnostic criteria, conventional MRI of the brain and spinal cord (T2/Flair/T1 post gadolinium sequences) is a cornerstone for an MS diagnosis within the 2017 revision of the McDonald criteria , 42, takRecently the so-called “central vein sign” (CVS) was proposed as a potential new biomarker for a more specific MS diagnosis, emerging from observations, mostly at ultra-high field MRI studies, that MS lesions are frequently characterized by a small intralesional vein in contrast to relevant imaging differential diagnoses, such as NMOSD, small vessel disease, inflammatory CNS vasculopathies, Susac syndrome, and others –56.*/FLAIR and co-registered SWI images, and might, therefore, become a clinically applicable imaging feature to discriminate MS from classical mimics at a high specificity  as well as the spinal cord, are typical hallmarks of MS from earliest disease stages –68 and tOther advanced MRI techniques have been recently applied to investigate pathogenetic processes associated with neurodegeneration and disease progression. Amid other emerging quantitative MRI approaches, diffusion tensor imaging (DTI), which relies on the detection of changes in the random translational motion of water molecules and thereby estimates the level of tissue degradation in the normal-appearing white matter, provides promising imaging markers to detect neuronal damage . Post moIn clinical management, the use of MRI to monitor treatment response still relies on conventional parameters, such as new or enlarging T2 lesions and gadolinium-enhancing lesions in conjunction with clinical measures , measures summarized under the term “NEDA” (no evidence of disease activity) . HoweverNumerous non-conventional and advanced imaging modalities are currently under investigation that may help improve visualization and quantification of (covert) tissue damage in the gray and white matter of the brain and the spinal cord and could be used as an imaging surrogate of remyelination and repair; among them are magnetization transfer imaging, diffusion tensor imaging, myelin water imaging, susceptibility weighted imaging, magnetic resonance spectroscopy, sodium imaging, PET imaging, ultra-high field imaging at 7 Tesla, functional imaging with resting state fMRI, T1/T2-weighted ratio calculable from conventional T1- and T2-weighted images, machine-learning based imaging, magnetic resonance elastography, and several others, none of which will probably be used in clinical practice in the near future , 118–135A recent emerging imaging tool in neuroinflammation is retinal optical coherence tomography (OCT), a technique that takes advantage of the retinal backscatter and reflection of low coherent light and enables the reconstruction of structural images of the various retinal layers with a resolution of a few microns and a very time-efficient image acquisition of only a few minutes in a cooperative patient .www.imsvisual.org) in 879 patients with various stages of MS suggests that pRNFL may be used to predict disability worsening   to the lAlso in a clinically isolated syndrome (CIS) scenario OCT may be helpful to assess the risk of further disease activity. A bicenter study from Germany grouped 89 patients with a CIS as a qualifying event into three groups according to their baseline GCIPL values in NON eyes . PatientIn 2015, new diagnostic criteria for NMOSD with and without (or with unknown) AQP4 antibodies have been proposed against the background of a broadening clinical spectrum that was recognized with the increasing number of patients tested for AQP4 antibodies . ImagingOver the past 15 years, an impressive number of imaging studies have made clear that—in contrast to earlier views—most NMOSD patients exhibit some kind of brain lesions. Lesions are not always located in areas of high AQP4 expression, and a considerable proportion (42%) may even meet Barkhof criteria for multiple sclerosis , 175. MoAccording to newer studies, the majority of NMOSD patients show some kind of brain lesions although findings considered highly suggestive and suspicious of an NMOSD diagnosis are less prevalent. Between 43 and 70% of NMOSD patients have brain lesions at onset, and up to 85% of patients meeting the 2006 Wingerchuk criteria for NMO and up to 89% of seropositive patients were reported to have brain abnormalities , 177–180In contrast to MS, cortical lesions are usually absent in NMOSD, which is supported by 3 T double inversion recovery and ultra-high field MR studies that investigated the cortex in NMO as well as by several histopathologic studies , 186 Fi. Additioin vivo and ex vivo MRI investigations.Of note, previous research shows that non-lesional tissue damage as measured by non-conventional imaging, such as DTI, may not occur in NMOSD except in the connecting tracts upstream and downstream of lesions . AlthougLongitudinally extensive myelitis lesions (LETM) spanning three or more contiguous vertebral segments have long been regarded as an imaging feature highly suggestive of NMOSD . SensitiRecently, a study in 48 NMOSD , 22 MS patients, and 24 patients with other causes of LETM from the United States assessed spinal cord imaging features that may help discriminate NMOSD from MS. Four findings were found to be most distinctive of NMOSD vs. other etiologies: bright spotty lesions, T1 dark lesions, centrally located lesions, and lesions involving more than 50% of the cord area on axial sequences .Another study in 116 NMOSD patients (98 AQP4 ab positive) found a high proportion of patients (49%) without typical NMOSD brain and spinal cord lesions and 37% meeting the 2010 McDonald criteria. Nonetheless, a combination of easily applicable criteria for brain and spinal cord images enabled distinction from matched MS patients with good sensitivity and specificity regardless of serostatus .Although the optic nerve is frequently involved in NMOSD, few studies with orbital MRI have been conducted. AQP4 ab–positive NMOSD tends to show more often posterior involvement of the optic nerve(s) including the chiasm and a more frequent intracranial and bilateral affection of the optic nerve as compared to MS . In AQP4Advanced imaging with volumetric analyses, DTI, spectroscopy, and others have increasingly been performed over the past 10 years , albeit Spinal cord atrophy and reductions of MUCCA are a consistent feature of AQP4 ab–positive NMOSD even in the absence of myelitis attacks/spinal cord lesions , 240 Fi. In one With the introduction of retinal OCT into clinical neuroimmunology, an increasing number of studies measuring retinal damage in NMOSD have been conducted over the past 10 years . Most stIn the past few years, a plethora of publications has reported on serum antibodies to myelin oligodendrocyte glycoprotein (MOG) in a subset of adult patients with an NMOSD phenotype  and beyond  using highly specific immunoassays –275. TheNeuroimaging studies with MRI in MOGAD are scant, and the few reports suggest that there is a broad overlap with AQP4 ab–positive NMOSD as to the presentation on conventional brain and spinal cord MRIs –284 althIn contrast, orbital MRI seems to exhibit distinctive features. A combined brain and optic nerve MRI study from Australia in 11 AQP4 ab–positive, 19 MOGAD, and 13 MS patients with a first ON in the investigated eye found more frequent optic nerve swelling in MOGAD and more frequent bilateral optic tract and chiasmal involvement in AQP4 ab–positive NMOSD . A predoRetinal OCT findings in MOGAD have been inconclusive. Although some studies suggest that ON in MOGAD causes less severe retinal damage in comparison to AQP4 ab–positive NMOSD , 291, otAlthough previous research in advanced neuroimaging led to a tremendous amount of new methods, parameters, and insights into MS and NMOSD diagnostic approaches and pathophysiological processes, further efforts are highly required to make these advances applicable to the clinical setting. Main short- to mid-term aims are (1) standardization of MRI and OCT parameters related to image acquisition and post-processing, (2) transfer and integration of non-conventional techniques into clinically usable procedures, and (3) validation by comparing these readily accessible techniques with current standards within the framework of large patient cohort studies and real-world research. The ultimate goal is to provide the most accurate and most cost- and time-effective markers for clinical diagnostics, therapeutic monitoring, and prognostic forecasting in individual MS and NMOSD patients.Among the multitude of potential candidates, a selection of promising markers to be introduced into the clinical setting in the near future are the central vein sign at 3 Tesla MRI that has proven to substantially increase specificity of current McDonald 2017 diagnostic criteria in the detection of MS , global Because availability of ultra-high field (7 T) scanners has gradually increased during recent years, a noticeable shift of neuroimaging research to higher field strengths will take place in the future. By use of its higher spatial resolution and benefits to imaging contrasts inherent to higher field strengths, 7 T MRI may be used to advance quantitative neuroimaging that may have reached its technical limits at 3 T , 300. MoAnother steadily expanding field of research in MS and NMOSD that will be of interest in the long-term future of neuroimaging, is the emerging application of MRI functional and structural connectome analyses. These techniques provide novel measures by assessing the integrity and functionality of the entire CNS system rather than evaluating separate regional or qualitative alterations in isolation . PatholoImaging research in autoimmune inflammatory CNS disease has made impressive progress over the past 20 years. Yet, although we are able to deploy structural and functional imaging techniques even in patients at almost subcellular resolution that have significantly contributed to our understanding of mechanisms of tissue damage in these conditions, most of these technologies still await a validated implementation in clinical practice. This, however, is an indispensable prerequisite to make use of these advances to inform treatment decisions and monitor disease activity in individual patients. Because this has remained an unmet need from our patients' perspective, this task will hopefully be tackled despite further thrilling developments in the field of neuroimaging in autoimmune neuroinflammation, for example, OCT angiography, improved post-processing and segmentation techniques, and the use of deep learning and artificial intelligence algorithms , 307. SiFP designed the concept of the manuscript, drafted and wrote the work, and critically revised the manuscript. JK assembled, prepared and interpreted imaging data and critically revised, and added to the manuscript regarding important intellectual content.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. JK received congress registration fees from Biogen, speaker honoraria from Sanofi Genzyme and Bayer Schering, and research support from Krankheitsbezogenes Kompetenznetz, Multiple Sklerose (KKNMS), not related to this work. JK is participant in the BIH-Charité Junior Clinician Scientist Program funded by the Charité-Universitätsmedizin Berlin and Berlin Institute of Health.


The seminal discovery that sequence variation in genes encoding cardiac ion channels was behind the inherited cardiac arrhythmic syndromes has led to major advances in understanding the functional biological mechanisms of cardiomyocyte depolarization and repolarization. The cost and speed with which these genes can now be sequenced have allowed for genetic testing to become a major component of clinical care and have led to important ramifications, yet interpretation of specific variants needs to be performed within the context of the clinical findings in the proband and extended family. As technology continues to advance, the promise of therapeutic manipulation of certain genetic pathways grows ever more real. In the last two decades, genetic testing for inherited cardiac arrhythmias has evolved from gene discovery to an important component of clinical care that is both affordable and returns results in a matter of only a few weeks. The seminal discovery that genetically encoded abnormalities in cardiac ion channels underlie inherited cardiac arrhythmias has allowed for both an improved understanding of the molecular biological processes fundamental to different disease states and also has helped to guide therapeutic strategies. The evolution of patient-specific models using induced pluripotent stem cells (IPSCs) and the future potential of gene therapy to directly replace or modify specific genes have kept inherited arrhythmic syndromes in the forefront of research and scientific discovery efforts. In this review, we discuss the basic principles and applications of genetic testing and potential future avenues for two of the best-understood conditions of this nature: long QT syndrome (LQTS) and catecholaminergic polymorphic ventricular tachycardia (VT) (CPVT).1 Although ordering genetic testing has become easier, choosing the right test and then interpreting the genetic results requires a high degree of specialist knowledge such that a multidisciplinary approach is advisable. The patient should be counseled in detail by an experienced genetic counselor or clinician regarding options, possible outcomes, and how the results may impact clinical management. The patient should understand that many genetic results will be of uncertain significance and should always be interpreted in conjunction with their clinical test results and not in isolation. The goal of genetic testing is twofold: (1) to identify specific variants in known disease-associated genes that further characterize the diagnosis in the proband and (2) to use this information to determine which family members are at increased risk of developing a disease and so require longitudinal cardiovascular evaluation as well as to identify relatives who do not require long-term follow-up. However, to define a variant as pathogenic or not, it needs to be systematically analyzed within the context of the familial phenotype to best define its causative impact.Genetic testing for both LQTS and CPVT is a class 1 indication.2 True nonpenetrance can make the inheritance pattern difficult to discern and should prompt careful consideration of whether this is truly the causal variant. Variable expressivity refers to individuals with the same underlying genetic predisposition to a disease who manifest different disease features and is best exemplified by the cardiac sodium channel gene SCN5A, where family members with the same mutation may have different phenotypes, such as Brugada syndrome, long QT syndrome, and conduction system disease. Penetrance and expressivity vary significantly within families due to impacts from a variety of other factors including other genetic sequence variations, environmental factors, and epigenetic phenomena3. See Table 1 for more information on terms discussed here and elsewhere.In most cases, both LQTS and CPVT are inherited in an autosomal dominant pattern, but much rarer autosomal recessive variants have also been well-documented. Penetrance is typically less than 100%; that is, the number of individuals with a genetic predisposition to develop the condition is higher than the number of individuals who display the phenotype of that condition. Importantly, the identification of disease-causing variant(s) confers an increased risk of developing a clinical phenotype but does not equate to a clinical diagnosis.4 The choice of the correct test can be complicated by overlapping phenotypes and genetic heterogeneity, where a similar phenotype can be produced by mutations in different genes. The provider should aim to identify a genetic panel that maximizes the yield while minimizing the likelihood of receiving an uncertain or ambiguous result.5Successfully identifying the key person in the family in whom to initiate genetic testing and then selecting the correct test to perform optimizes the diagnostic yield and clinical utility of the result(s). Ideally, the patient with the most severe form and/or earliest onset of disease in the family should be the testing proband. in an attempt to standardize variant analysis and aid in interpretation.6 A pathogenic result supports a clinical diagnosis and may provide prognostic or therapeutic guidance7 as well as be impactful for cascade screening in family members. A benign variant is classified based upon prevalence in the general population or insufficient evidence to associate the gene or mutation type with disease. When there is insufficient evidence to categorize a variant as benign or pathogenic, it is termed a variant of uncertain significance (VUS). With rare exceptions, a VUS cannot be used to guide management in the proband or provide predictive information for asymptomatic family members.8 Classifications are dynamic and may change over time.Perhaps the most compelling evidence to support variant pathogenicity is segregation with the phenotype across multiple family members, which can be described statistically using a logarithm of odds score. A logarithm of odds score > 3  is typically considered as very strong evidence for variant causality in a given family. However, as sufficiently large pedigrees (with more than 10 family members characterized) are rarely available, other criteria are typically used. Depending on the level of evidence available, a genetic variant can be characterized on a continuum as benign, likely benign, uncertain significance, likely pathogenic, or pathogenic. The criteria to support each category were defined by the American College of Medical Genetics and Genomics (ACMG) in 2015 8 In other words, a priori risk is the likelihood that a patient has a condition before the test results are known; this is usually calculated based on pretest data including phenotype, sex, ethnicity, and family history. Commercial laboratories have different pipelines for analyzing and classifying variants, creating the potential for discordant interpretation of the same variant10; therefore, the onus is on the provider to analyze critically the given evidence for the variant themselves and correlate it with the phenotype in the proband and extended family. Ultimately, detailed phenotyping of the wider family is the most powerful tool to determine pathogenicity.5 There are multiple examples of variants originally deemed pathogenic failing to match with disease phenotype in family members, discrediting them as causative variants.11Genetic testing should be used in individuals with either a clinical diagnosis or a strong clinical suspicion of a specific hereditary arrhythmic syndrome such as LQTS or CPVT. Testing an unaffected individual or a patient with a poorly defined phenotype will be less informative in general due to a low a priori risk. A low pretest probability correlates to a low positive predictive value for the test, even if the test is highly sensitive and specific.RyR2).12 Therefore, thorough phenotyping and detailed three-generation family history-taking are essential to informing the provider’s choice for the correct genetic test to order so as to avoid false negatives. Additionally, technology is rapidly advancing and novel disease genes are frequently added to panels. Retesting a previously negative individual if the sensitivity of the test used has increased since may be warranted.A negative genetic test result does not exclude a diagnosis, particularly if the proband has a clinical diagnosis. It also does not rule out the possibility that the proband’s disease is hereditary; first-degree relatives should still be clinically screened appropriately based on the proband’s diagnosis. The likelihood of an actionable result is fundamentally predicated on the accuracy of the phenotyping and the specific test or panel ordered. For example, incomplete phenotyping could misguide the provider into ordering a targeted LQTS panel after exertional syncope, when the proband actually has CPVT and harbors a pathogenic variant in the cardiac ryanodine receptor  and guide the interpretation of any identified variants. Although LQTS may occur de novo, a lack of any apparent family history is much more likely to be related to clinically quiescent disease. Disease penetrance may be as low as 25%22; hence, a normal resting electrocardiogram (ECG) does not exclude the condition in first-degree family members. Abnormal T-wave morphology and sinus bradycardia are well-recognized23 and, in both the LQTS1 and LQTS2 types, the phenotype may be unmasked with simple maneuvers such as standing and exercise.24LQTS is typically inherited in an autosomal dominant manner with highly varied penetrance; however, compound heterozygous (two variants within one gene), digenic (variants within two different genes), and homozygous  cases have all been recognized. Cases with more than one variant in KCNQ1, KCNH2, and SCN5A) that encoded ion channel proteins  responsible for transmembrane ion currents critical to cardiac depolarization and repolarization. Clinically, they are referred to as LQTS1, LQTS2, and LQTS3. Structural and functional protein abnormalities lead to either excessive sodium (INa) influx and persistent depolarization or delayed potassium (IKs and IKr) efflux and prolonged repolarization, the net effect of which is prolongation of the myocardial action potential duration and the QT interval on the surface ECG. KCNQ1, KCNH2, and SCN5A remain as the most prevalent genes and are the focus of this current review. Variants in other cardiomyocyte ion channels or assembly proteins have been implicated in monogenic LQTS but, recently, the ability of variants in the beta subunit KCNE2 to cause disease in the absence of environmental or other genetic factors has been questioned.25 The contribution of genes involved in cellular calcium homeostasis is increasingly recognized: for example, CACNA1C underlies the multisystem Timothy syndrome, while the three calmodulin genes  and triadin (TRDN) may cause highly malignant forms of LQTS.The underlying genetic defects of long QT syndrome were first described in the 1990s, identifying three genes  or MutationTaster  are computer-based algorithms used to estimate pathogenicity based on the predicted impact of the specific nucleotide and amino acids changes on protein structure and function. Many important factors should be considered when determining the potential causal relationship of any specific variant with LQTS, such as whether the clinical phenotype can be explained by the identified genotype. For example, an SCN5A variant in a 15-year-old with exertional syncope, a corrected QT (QTc) interval of 480 ms, and significant QTc prolongation in early recovery on an exercise test is unlikely to be causal, whereas a variant in KCNQ1 is much more consistent with the clinical picture. This may have increasing importance if variants in more than one gene are identified.26 Although the loss-of-function variants  are more likely to be categorized as pathogenic by the 2015 ACMG classification as compared with missense mutations, KCNQ1 may be highly tolerant to the loss of one functional allele, a factor calculated by the number of observed versus expected losses of function variants in population databases.27 Conversely, both KCNH2 and SCN5A are highly intolerant to loss of function.27Genetic testing reports include the gene; nucleotide substitution; amino acid effect; zygosity; consequence of the sequence code variation  Xenopus oocytes or human embryonic kidney cells can help to determine the consequences of genetic variation on ion channel function. However, variant location and in vitro effects are not always concordant with the associated clinical phenotype.The topological location of the variant may add further information, with specific areas showing high degrees of conservation across species and an enrichment of pathogenic variants. Cellular electrophysiology studies using transfected cell lines such as KCNQ1, A300T . If identified in a patient, a report may conclude that this variant (1) is located in the pore helix (AA 298-312) region, which shows a high degree of conservation and enrichment for pathogenic variants due to the fundamental role of the regions in channel function28; (2) reduces Iks to 15% of the wild type according to functional assessment29; and (3) has been previously associated with long QT syndrome.30 Upon review of such information, it would appear that the variant would highly likely lead to clinically severe and manifested LQTS. However, a more detailed review of the prior case would reveal that LQTS associated with A300T is apparent only in homozygous, not heterozygous, carriers,30 and the variant has been identified in healthy controls . Although the variant was predicted to be pathogenic by in silico analysis and absent from the Exome Aggregation Consortium database, the diagnosis was reversed based on detailed clinical assessment of the family and more detailed molecular modeling. Similarly, the KCNQ1 splice variant c.477+1G>A has been associated with a phenotype in homozygous but not heterozygous individuals. Although detailed functional analysis suggested an effect should be seen in heterozygote carriers, natural degradation in vivo of the abnormal messenger ribonucleic acid (RNA)  prevents production of the mutant protein and limits expression of LQTS.31To illustrate, consider a variant in SCN5A variants with severe functional consequences have been identified in neonates with severe QT prolongation and recurrent ventricular arrhythmias.32 Such variants, absent from the majority of population databases, promote early symptomatic presentation due to the severe proarrhythmic disruption of cellular depolarization. Several other SCN5A variants identified as part of the detailed sequencing of arrhythmia susceptibility genes in a sudden infant death cohort33 demonstrated a marked increase in the late sodium current (INa) when expressed in tsA201 cells consistent with a LQTS phenotype; in two, S216L and T1304M, the persistent INa was > 1%,34 but both of these variants have been identified in racially concordant population databases at mean allelic frequencies of 0.03% and 0.15%, respectively.27 Considering that the population prevalence of LQTS is 1:2,000 (0.05%), this suggests that both of these variants are too common to be considered causal for LQTS in every case, but their potential arrhythmic role under the influence of other genetic or environmental stressors is unknown. Conversely, an SCN5A variant associated with atrial standstill, conduction abnormalities, and ventricular dysfunction has been found not to disrupt channel function in Chinese hamster ovary cells but did recapitulate the phenotype in a mouse model.35De novo 36 with the former raising concern for receptor upregulation and elevated risk.37An important consideration is to what degree can or does the genotype impact clinical decision-making. Ultimately, clinical management should be governed by the phenotype, but knowledge of the underlying genetic variant can help to guide clinical management in specific circumstances. β-blockers, specifically propranolol and nadolol, remain the backbone of LQTS treatment; however, in the absence of any randomized controlled trials, it is unclear as to whether every patient identified by cascade screening with lowly penetrated forms of the disease requires treatment. Although β-blockers can be considered an “insurance policy,” they are not without side effects and, in the absence of any phenotypic expression on detailed clinical testing, their overall long-term benefit is unclear. Many patients take β-blockers intermittently or not at all,KCNQ1 R518X Swedish founder variant has been extensively studied and is associated with a relatively mild phenotype in heterozygous carriers.39 In light of this, one interesting question to consider is whether or not an asymptomatic adult male with a normal resting QT interval on repeated measurements should take a β-blocker or whether avoidance of QT-prolonging medications is sufficient in this individual. Conversely, mutations in the two cytoplasmic loops (C-loops) S2-S3 (AA171-195) and S4-S5 (AA242-262) appear to confer a significantly higher risk of life-threatening cardiac events in comparison with variants in other topographic locations, an effect significantly negated by β-blockade.40 Owing to initial concerns about the proarrhythmic effects of bradycardia and higher rate of breakthrough cardiac events,41 patients with SCN5A-mediated LQTS3 are typically treated more aggressively with implantable cardioverter-defibrillators (ICDs). This notion was refuted by a more recent study of 391 LQTS3 patients, which found β-blockers significantly reduced the risk for cardiac events, especially in females.42 Mexiletine was recently shown to have beneficial effects in reducing both QT duration and frequency of cardiac events in LQTS3 patients, either in conjunction with β-blockade or in isolation.43The 44 Although symptoms are typically precipitated by exercise or emotion, both syncope and cardiac arrest may occur during normal activities and rest.45 First recognized in the 1960s, it was not until 1978 that Phillippe Coumel and colleagues in Paris recognized the adrenergic basis and specific ECG pattern and subsequently in 1995 reported their experience of 21 patients, proposing the descriptive term “catecholaminergic polymorphic ventricular tachycardia.”46 The hallmark ECG marker is bidirectional VT caused by triggered afterdepolarizations thought to arise in an alternating fashion from the His-Purkinje system in the right and left ventricles, giving rise to the classical appearance.47 Although the resting ECG is normal, sinus bradycardia is common48—as is atrial fibrillation,49 which may predate ventricular arrhythmias. Bidirectional VT may also be seen in Andersen-Tawil syndrome (ATS). A multisystem ion channel disorder previously referred to as LQT7, ATS is associated with a prolonged QU interval and prominent U waves on the surface ECG, a high burden of ventricular ectopy often at rest, syndactyly, characteristic dysmorphic facies, and periodic paralysis.CPVT typically presents in childhood with symptoms of palpitations, syncope, seizures, and cardiac arrest during exertion or emotional stimuli.RyR2 R420W) revealed a more subtle or absent phenotype in many. Interestingly, 50% of family members with a normal initial clinical evaluation may develop disease features, stressing the need for ongoing evaluation.50 Absence of clinical features in family members may also point to de novo disease occurrence, which may be more common than familial disease,51 the much rarer autosomal recessive CPVT,52 or germline mosaicism.53CPVT most commonly displays an autosomal dominant inheritance pattern with a high degree of penetrance, although probands typically show a more severe disease phenotype than do family members identified through cascade screening. The detailed evaluation of family members including one family with 61 individuals , calmodulin (CALM), and TRDN. Approximately 60% to 70% of patients with a definitive clinical diagnosis of CPVT will have identifiable variants in RyR2, with most located in one of the following four highly conserved, functionally important domains of the gene: domain I AA 57-466, domain II 2246-2534, domain III 3778-4201, and domain IV 4497-4959 . Most RyR2 variants associated with CPVT are missense or small insertions/deletions as opposed to truncating variants, supporting a dominant negative mechanism wherein the mutant protein prevents normal function of the wild type.52 A large inframe deletion incorporating exon 3 and associated intronic sequences has been linked with a broader phenotype of left ventricular noncompaction, conduction disease, atrial arrhythmias, exertional ventricular arrhythmias, and sudden death. The deletion  segregated with the phenotype [logarithm (base 10) of odds score: 4.5] in one family and is framed by two Alu-repeat sequences frequently associated with genomic rearrangement.54 Although the clinical phenotype and associated deletion have been identified in other unrelated cases, murine models of exon 3 deletion do not replicate the phenotype55 and the exact mechanism of how this sequence variation leads to the more diverse and complex phenotype remains unclear.CPVT is caused by genetically encoded abnormalities in cardiomyocyte proteins fundamental to cardiomyocyte calcium homeostasis, most commonly in CASQ2-mediated disease is almost exclusively related to homozygous or compound heterozygous variants involving both truncating and missense mutations.56 Heterozygote carriers are typically unaffected, but recently, a heterozygote variant was shown to segregate [logarithm (base 10) of odds score: 3.01] with an overt CPVT phenotype in a family with autosomal dominant inheritance.57 The calmodulin genes , TRDN, and trans-2,3-enoyl-CoA reductase-like protein (TECRL) may all lead to CPVT or LQTS, often with phenotypic overlap.62KCNJ2, which encodes Kir2.1, the inward rectifier potassium channel, is associated with ATS but may also produce an isolated CPVT phenotype, clinically distinguishable from other variants by the presence of ventricular arrhythmias and complex ectopy at rest.63RyR2 has almost 5,000 amino acid residues, which, in combination with the rarity of CPVT, means that many patients are found to carry novel missense variants, appropriately classified as VUSs. A de novo variant in a child with CPVT is more likely to be disease-causing with confirmed maternity and paternity and normal clinical parental evaluation. Therefore, significant care needs to be applied in interpreting RyR2 variants as disease-causing; an analysis of the Exome Sequencing Project database of 6,503 control subjects identified 41 missense variants previously associated with CPVT, which, if truly causal, would lead to a disease prevalence of 1:150, not the estimated value of 1:10,000.64 Notably, in this study, no variants were identified in the four canonical domains critical to protein function and, in comprehensively phenotyped CPVT cohorts, most variants, but not all, locate to one of these four canonical domains.51 However, variants within these domains have also been identified within larger control databases and patients undergoing whole exome sequencing for noncardiac indications,65 suggesting that, although these canonical domains may be highly enriched with true disease-causing variants, a diagnosis of CPVT cannot be made based on the topographical location of a genetic variant alone and careful evaluation in the context of the phenotype and pretest probability is required.The interpretation of variants identified in CPVT is challenging. 66 and left cardiac sympathetic denervation67 have shown beneficial effects. Furthermore, although ICDs are important components of care, interest in their use has significantly declined due to the limited effects of defibrillation in the management of arrhythmias caused by triggered activity; the well-recognized complications, including death, of ICD use in a young population; and the specific proarrhythmic complications seen in patients with CPVT.66 At present, the associated genetic variant identified in a patient with CPVT does not directly impact clinical management.Given the association of symptoms with catecholaminergic stress, β-blockers have been the mainstay of treatment, although, more recently, flecainideKCNJ2 mutation have been shown to recapitulate the phenotype identified in the patient, supporting causality for the variant in question.67 At present, such analysis is time-consuming and costly but, as gene editing technology improves, the potential for using IPSC-CMs to determine the effects of specific variants will become increasingly recognized.Technological advances in the use of cardiomyocytes derived from IPSCs (IPSC-CMs) have allowed for the creation of patient-specific models for both LQTS and CPVT, providing further insights into the therapeutic effects of different pharmacological agents and disease biology. Although such models do not completely recapitulate the in vivo biology of mature human cardiomyocytes, they have many advantages over animal models and transfected cell lines. IPSC-CMs incorporating both rare variants with large effects and other single-nucleotide polymorphisms correlate well with the molecular and arrhythmic phenotypes seen in both LQTS and CPVT, which proposes the interesting possibility of human-derived models for variant testing. Commercially available IPSC-CMs transfected with a clinically identified CASQ2 R33Q−/− CPVT, in vivo delivery of an adeno-associated virus complementary DNA CASQ2 (AAV-CASQ2) construct was performed in three-day-old and three-month-old knock-in animals. A single injection was able to prevent the development of CPVT in the younger mice and revert the phenotype in the older, symptomatic mice who had already demonstrated either polymorphic or bidirectional VT in response to epinephrine. In both groups, there was a restoration of the physiological levels of calsequestrin and associated proteins junctin and triadin, with normal protein–protein interactions, as well as the prevention or restoration of ultrastructural cellular changes. Although only 40% of myocardial cells were infected with the AAV9-CASQ2 construct in both the neonatal and the older mice, interestingly, this was sufficient to confer a significant antiarrhythmic benefit, an effect attributed to the presence of sufficient rescued cells to prevent widespread transmission of delayed afterdepolarizations.68 Subsequent studies have been able to replicate the beneficial effects of AAV-CASQ2 delivery in IPSC-CMs obtained from a patient with CPVT secondary to a homozygous CASQ2-truncating mutation.69 In animal models of RyR2-mediated CPVT, a different approach is necessary to suppress the dominant negative effects of the mutant protein. Allele-specific silencing using adeno-associated virus small interfering RNA duplexes has been shown to suppress the mutant RyR2 messenger RNA (RyR2-R4496C) with no significant effect on wild-type levels, correlating with a reduction in delayed afterdepolarizations, ventricular arrhythmias, and ultrastructural abnormalities in treated animals.70In the last three to four years, the possibility of gene therapy for inherited cardiovascular disorders has moved closer to reality, specifically by overexpression of the target protein in autosomal recessive CPVT and by silencing of the mutant allele in the dominant form. In murine models of homozygous Since the seminal discovery 30 years ago of the genetic abnormalities that underlie inherited cardiac arrhythmias, our understanding of the complexities of monogenic disorders such as LQTS and CPVT has advanced parallel to technological changes that have supported deeper investigations into the associated genetic and molecular pathways. It is increasingly clear that the relationship between rare genetic variants and associated phenotypes is far from linear, influenced by multiple factors, which thus far remain beyond the scope of day-to-day clinical practice. Consequently, care should always be taken in the interpretation of identified genetic variants, with detailed phenotypic evaluation of the wider family used to validate genetic findings and dictate clinical management. As technology continues to advance at a rapid pace, the opportunities for creating patient-specific models to better define different genetic influences on cardiomyocyte biology will become greater and the possibility of ultimately correcting the underlying genetic sequence variation of inherited cardiac disorders will grow closer.