Stevens-Johnson syndrome (SJS) and toxic epidermal necrolysis (TEN) are severe cutaneous adverse drug reactions with high mortality rates. Its sequelae, such as blindness, persist even after recovery. Patients with SJS/TEN should be accurately diagnosed and receive appropriate treatment as soon as possible. Therefore, identifying the factors for severity prediction is necessary. We aimed to clarify the clinical parameters and biological markers that can predict acute severe ocular complications (SOCs) in SJS/TEN. This retrospective cross-sectional study enrolled 47 patients with SJS/TEN who were divided into two groups according to ocular severity at acute onset: non-severe ocular complications group (n = 27) and severe ocular complications group (n = 20). Multivariate logistic regression analysis revealed that disease severity (body surface area detachment ≥ 10%) was a predictive factor for acute SOCs, and older age (≥ 60 years) was marginally significantly predictive of SOCs. Serum biomarker levels of S100A8/A9 and granulysin were marginally significant and tended to increase in the SOC group. Therefore, during the early acute stage, focusing on disease severity, patient age, and serum inflammatory biomarkers (S100A8/A9 and granulysin) might help predict SOC progression in patients with SJS/TEN who need prompt and aggressive ocular management to prevent severe ocular sequelae. The incidence of SJS and TEN is approximately 0.4 to 1 case per 1 million persons and 1 to 6 cases per 1 million persons, respectively2. Although rare, SJS/TEN is significant because of its high mortality and morbidity rates4.Stevens-Johnson syndrome (SJS) and toxic epidermal necrolysis (TEN) are severe cutaneous adverse reactions (SCARs) characterized by blistering mucocutaneous lesions leading to necrosis and sloughing of the skin epidermis and mucosa, mostly including ocular, oral, and genital membranes. SJS and TEN (SJS/TEN) can be considered as a spectrum of increasing severity and mortality and are distinguished based on the involvement of the body surface area (BSA). SJS is a less severe form, with skin sloughing of less than 10% of BSA, whereas TEN is more severe, with sloughing of more than 30% of BSA. When the involvement is greater than 10% but less than 30% of the body surface, it is referred to as the SJS/TEN overlap syndrome3. Several clinical parameters, including age, malignancy, tachycardia, initial BSA of epidermal detachment, serum urea, serum glucose, and bicarbonate, have been used for evaluation. Other predictors of mortality in SJS/TEN are age, pre-existing comorbidities, hematological malignancy, septicemia, pneumonia, tuberculosis, and renal failure5. Among the various etiological factors for SJS/TEN, drug reactions are the most common; allopurinol, antibiotics, antipsychotics, nonsteroidal anti-inflammatory drugs (NSAIDs), and antiepileptic medications are the most frequently associated3. Other causes such as infection from herpes simplex virus, mycoplasma, and other unidentified etiologies may be responsible for up to 15% of cases6.The score of toxic epidermal necrolysis (SCORTEN), is a standard scoring system for assessing SJS/TEN severity7. Acute ocular complications develop in 43–81% of patients hospitalized for SJS/TEN. Clinical features range from self-limited conjunctival hyperemia to extensive sloughing of the ocular surface and lid margins2. Chronic ocular sequelae occur in 35%–63% of cases9. Previous studies have shown that acute severe ocular complications (SOCs) are strong predictors of chronic ocular sequelae14. Delayed or inadequate ocular treatment in the acute stage can result in irreversible chronic SOCs, such as severe dry eye, trichiasis, conjunctival invasion into the cornea, and conjunctival scarring, resulting in visual disturbances and substantially lower overall health-related quality of life15. Treatments for chronic SOCs remain challenging and are prone to failure, thereby emphasizing the importance of intensive management of SOCs in the acute phase to prevent chronic SOCs.Ocular complications are the major, severe, long-term sequelae following SJS/TEN. They do not always correlate with the severity of the systemic disease17. This immune-mediated cytotoxic reaction against keratinocytes leads to massive apoptosis of the keratinocytes and/or necroptosis and causes an acute inflammatory vesiculobullous reaction of the skin18. In the eye, the early vacuolation of basal keratinocytes with the absence of a lymphocytic infiltrate offers inflammatory cytokines an important role in the pathogenesis of acute ocular inflammation19.Currently, SJS/TEN is considered a delayed T cell-mediated hypersensitivity reaction. The pathological mechanisms underlying SJS/TEN are not well understood. Cytotoxic T lymphocytes (CD8 + T cells) and natural killer (NK) cell immune-mediated reactions are the major immunologic component20. Several biomarkers, such as IL-6, IL-8, IL-15, IL-10, IL-13, IL-17, TNF-α, IFN-γ, and granulysin (GYLN), are increased in the skin lesions, peripheral blood mononuclear cells, and plasma of patients with SJS/TEN26. Some of them, such as GYLN and IL-15, can reflect the severity of SJS/TEN26, but their association with ocular involvement has not been widely explored.A severe cytokine storm occurs on the ocular surface during the SJS/TEN onset28. Moreover, none of the studies have evaluated the correlation between biological markers and SOCs. The purpose of this study was to determine the clinical parameters and serum biomarkers that can predict acute SOCs in patients with SJS/TEN. Prompt identification of the high-risk group will guide clinicians in early intervention and thereby prevent long-term devastating ocular conditions.Most previous studies have focused on predicting factors for chronic SOCs in SJS/TEN. In contrast, there have been very few studies on the prognostic value of acute SOCsThe patient characteristics are summarized in Table Among all causative drugs Table , antibioP = 0.019), age ≥ 60 years , SCORTEN ≥ 3 , and antibiotics  were associated with SOCs (ocular severity grade 0–2 vs grade 3) as the candidate predictive factors. Multivariate logistic regression analysis revealed that BSA was a significant predictive factor for acute SOCs . However, we found that age ≥ 60 years was a marginally significant predictive factor .Clinical factors related to acute SOCs are listed in Table The geometric means and the geometric mean ratios (GMR) of serum biomarker levels between healthy controls (HC) and patients with SJS/TEN with different ocular severities are shown in Table P values < 0.001, including IP-10, IL-6, and IL-17A. Two biomarkers (SCF and S100A8/A9) had P values < 0.01, and five biomarkers  had P values < 0.05.Compared to the serum from the HC group, the SJS/TEN group serum showed significant upregulation of 11 factors. Three biomarkers had Five of the 11 biomarkers had geometric mean ratios (GMR) > 2. The geometric mean of the IP-10 serum concentration in the SJS/TEN group was 7.2 times higher than that in the HC group , followed by IL-6, S100A8/A9, PDGF-AA, and PDGF-BB .P = 0.067, 95% CI 0.95–4.61) and GYLN  were marginally significant and tended to increase in the severe groups.We have analyzed different groups of SJS/TEN to compare both non-severe n = 24) and severe n = 16) ocular complications , antibiotics as causative drugs, and heightened SCORTEN were associated with acute SOCs. Among these factors, only BSA (detachment ≥ 10%) was identified as a predictive factor for acute SOCs in the multivariate analysis. Yip33. They hypothesized that not only younger age but also medication for common cold and viral infections causing cold-like symptoms play an essential role in the development of SOCs33. In contrast, we found that older age and antibiotic use were associated with acute SOCs in our study; cold medications (CM) were not a risk factor for SOCs. The explanation for the disparity between the present study and the aforementioned Japanese studies might be the difference in rational drug use, which cold remedies are used less often in our country. Genetic diversity may also play an important role. Moreover, the criteria for identifying causative drugs differed between studies. Although Thailand and Japan are both located in East Asia, associations between HLA genotype and cold medicine-related SJS/TEN with SOCs in Thailand have been shown to differ from Japanese patients34. Besides, the incidence of CM-related SJS/TEN in our study was relatively low ; therefore, our sample size might have been inadequate to obtain statistical significance.Previous studies have shown that younger age (< 50 years) is associated with acute SOCs29. SCORTEN tends to use clinical parameters that reflect overall patients’ systemic conditions to predict the severity and morbidity. Since the ocular surface has a unique and privileged immune response, the discordance between ocular and systemic manifestations might explain why this score was not directly related to the severity of ocular manifestations in acute SJS/TEN.Similar to earlier reports, we did not find an association between SCORTEN and SOCs37. Currently, no data are available on the potential function of S100A8/A9 in SJS/TEN. S100A8/A9 is expressed in monocytes, which are present in the epidermis of SJS/TEN skin lesions38, and can also be produced by epidermal keratinocytes39. While previous studies have implicated a role for S100A8 and S100A9 in causing accelerated inflammation41, opposite effects were reported in models of type IV-c hypersensitivity reaction42. S100A8/A9 inhibits dendritic cell maturation and antigen-presenting ability, leading to decreased T-cell activation and reduced intensity of immune responses in contact dermatitis42. Due to contradictory evidence on whether S100A8/A9 amplifies inflammation or exhibits an anti-inflammatory effect, the role of S100A8/A9 expression in the pathogenesis of SJS/TEN needs to be further elucidated.Different inflammatory mediators and cell types have been proposed to regulate the immunopathology of SJS/TEN. In our study, S100A8/A9 levels were upregulated in patients with SJS/TEN compared to those in the control group and tended to increase in patients with severe ocular involvement. The S100A8/A9 complex is a critical alarmin that is upregulated in numerous inflammatory diseases such as rheumatoid arthritis, chronic inflammatory bowel disease, psoriasis, systemic lupus erythematosus, and atopic dermatitis43, and S100A8/A9 could alter the skin barrier proteins in atopic dermatitis36. In the present study, the elevation of serum levels of S100A8/A9 possibly arose from epithelial damage to the skin in SJS/TEN. Further studies are required to investigate S100A8/A9 expression and function in SJS/TEN skin lesions.S100A8 and S100A9 are found to be upregulated in the epidermis of the skin during the active epidermal regeneration process45. Moreover, there is evidence that elevated serum S100A8/A9 levels correlate with clinically active joint and eye inflammation in autoimmune uveitis46. As primary ocular manifestations in acute SJS/TEN are intense ocular inflammation and epithelial sloughing, S100A8/A9 probably plays a role in ocular inflammation during the acute phase of SJS/TEN.In the eye, S100A8/A9 enhances inflammation in the ocular surface by promoting PMN infiltration and the expression of IL-1b, IL-6, and TNF-α, as an inflammatory amplifier48. IL-6 levels were also found to be higher in tears from SJS/TEN patients, although no correlation with the severity of ocular involvement was observed49. Studies have demonstrated that GLYN is probably a key mediator in keratinocyte apoptosis in SJS/TEN, and corneal epithelium could produce GYLN in the presence of TNF-α in an ex vivo report26. However, the fact that GYLN levels were marginally significantly higher in those with SOCs compared to those with non-severe reactions in our study might be due to the limited sample size, or the roles of GYLN in SOCs may be, in fact, not as important as that of S100A8/A9 in SJS/TEN. The functions of PDGFs in the pathogenesis of SJS/TEN remain to be explored.Our study demonstrated the elevation of several mediators in the serum of patients with SJS/TEN compared to those in HC. These results confirm the previous finding that IP-10 and IL-6 levels were elevated in the serum of patients with SJS/TENAfter physicians confirmed the diagnosis of SJS/TEN, all patients were registered with the ThaiSCAR. This systematic registration not only provides better holistic care for patients but also helps physicians to collect comprehensive data for each individual. This study identified predictive factors for SOCs in SJS/TEN patients and found that BSA and older age were correlated with SOCs. In addition, we analyzed the correlation between inflammatory cytokines and SOCs. Previous studies have examined local specimens, including tears and conjunctival swabs, but because of the complexity involved in collecting local specimens and the lack of a standard protocol for handling samples, we decided to interpret biomarkers from serum. We found that S100A8/A9 and GYLN levels tended to increase in the SOC group. To our knowledge, this is the first report indicating that S100A8/A9 is upregulated and might be involved in the pathogenesis of SJS/TEN.This study had a few limitations, including its retrospective design. Second, the sample size was limited due to the rarity of the disease, with a relatively small number of patients being treated at one center. Finally, all identified causes of SJS/TEN in the ThaiSCAR registry were medications; infections or other unidentified factors that could be potential etiologies of SJS/TEN causing SOCs were not included. Future large-scale, multicenter cohort studies, and the analysis of tear biomarkers or impression cytology evaluation of conjunctiva with their temporary changes are needed to verify the risk factors associated with SOCs in SJS/TEN.In summary, these identified factors consisting of clinical manifestations and upregulated biomarkers would be beneficial for developing screening protocols or universal tools that will help physicians recognize high-risk patients, leading to prompt management and prevention of chronic severe ocular sequelae.From the clinical factor analysis, disease severity (BSA detachment ≥ 10%) and older age were predictive factors for acute SOCs in SJS/TEN. From the serum biomarker analysis, the levels of S100A8/A9, IP-10, IL-6, PDGF-AA, and PDGF-BB were increased in the SJS/TEN group; they may be potential markers that could differentiate between HC and those with SJS/TEN. The S100A8/A9 and GYLN levels tended to increase in the severe ocular involvement group, suggesting that S100A8/A9 and GYLN are involved in the pathogenesis of SOCs and serve as helpful biomarkers to predict acute SOCs in SJS/TEN.Taken together, two clinical factors, namely BSA and older age, and increased levels of serum biomarkers, namely S100A8/A9 and GYLN, may guide clinicians in identifying high-risk groups that could develop acute SOCs. Prompt management in these patients will minimize the chance of developing chronic ocular sequelae, leading to vision loss.1 and the suspected culprit drugs were assessed according to the algorithm of drug causality for epidermal necrolysis (ALDEN)50.This study followed the tenets of the Declaration of Helsinki and was approved by the Institutional Review Ethics Committee of the Faculty of Medicine, Chulalongkorn University. This cross-sectional study was conducted at King Chulalongkorn Memorial Hospital (KCMH), Bangkok, Thailand, between July 2020 and January 2021. Forty-seven patients diagnosed with SJS/TEN at KCMH were enrolled in this study. These patients were part of the Thailand Severe Cutaneous Adverse Reactions (ThaiSCAR) cohort registered at ClinicalTrials.gov (NCT02574988). The study protocol was approved by the Institutional Review Board of the Faculty of Medicine, Chulalongkorn University . Informed consent was obtained from all participants. The classification criteria of Bastuji-Garin et al. were used to describe SJS, TEN, and SJS/TEN overlap syndromeAccording to the ThaiSCAR protocol, SJS/TEN patients were admitted after the onset of symptoms. Patients’ serum was then collected, and referrals were made to the ophthalmologist. The ocular complications were evaluated primarily within 24 h of admission.We retrospectively reviewed the medical records and electronic database of patients with SJS/TEN. The inclusion criteria were as follows: (1) patients with fully accessible medical records of the acute phase for evaluation of prognostic factors (2) no history of other ocular surface disorders or ocular surgeries, and (3) age > 18 years. For simplicity, patients with SJS/TEN overlap were assigned to the TEN group.51.When the acute ocular severity differed between the eyes, the eye with greater severity was chosen for evaluation. If both eyes were symmetrically severe, the right eye was selected. The acute ocular severity score was defined as previously described by Sotozono et al.27  and SJS/TEN patients with SOC . The disease severity , age, sex, laboratory results, SCORTEN, mucosal involvement, causative drugs, flu-like symptoms, underlying diseases, initial visual acuitiy, sepsis complications, and HIV infection in the acute phase were analyzed.The serum samples of patients with SJS/TEN were cryopreserved from a ThaiSCAR prospective study.51. Undiluted samples were stored at − 80 °C until biomarker measurement. Patients in this study underwent serum collection and ocular examination within a maximum of 3 days following admission.Serum (30 ml) was collected from patients registered in the ThaiSCAR database while admitted to KCMH during the acute phase of SJS/TEN, within 8–12 days of symptom onsetBecause sepsis appears to be a possible confounder, seven patients with sepsis were excluded. Serum from 40 patients with SJS/TEN and 18 HC was analyzed to explore the relationship between serum biomarkers in SJS/TEN and HC. A non-significant difference was noted in the mean age between the SJS/TEN and HC groups. After thawing, the samples were analyzed using a bead-based multiplex immunoassay. Panels of 42 biomarkers were used to identify biomarkers with prognostic potential for SJS/TEN. All samples were analyzed in duplicate. Biomarkers with a GMR of more than two were considered clinically significant.To identify the biological markers related to acute SOCs, we analyzed and compared the data between SJS/TEN patients with non-SOC (n = 24) and SOC (n = 16).To quantify soluble analytes simultaneously in plasma samples, we do the Human Th17 Cytokine Panel 7-plex , the Human Growth Factor Panel 13-plex (Angiopoietin-2 (Ang-2), EGF, EPO, FGF-basic, G-CSF, GM-CSF, HGF, M-CSF, PDGF-AA, PDGF-BB, SCF, TGF-α and VEGF), Human Vascular Inflammation Panel 13-plex , Lipocalin A (NGAL), C-Reactive Protein (CRP), MMP-2, Osteopontin (OPN), Myeloperoxidase (MPO), Serum Amyloid A (SAA), IGFBP-4, ICAM-1 (CD54), VCAM-1 (CD106), MMP-9, and Cystatin C), and the custom Human panel 9-plex  were used. . 25 μl of assay buffer was added into each well. Then, 25 μl of diluted standard or plasma were added in standard or sample wells. After that, 25 μl of mixed beads and 25 μl of detection antibodies were added into each well and incubated for 2 h at room temperature on an orbital plate shaker. After incubation, 25 μl of streptavidin-PE solution was added and then incubated for 30 min at room temperature on an orbital plate shaker. The plates were centrifuged at 1,000 rpm for 5 min. After decanting liquid and wash more one time with wash buffer, all wells added 150 μl of wash buffer and shaked for 2–3 min prior to analyze by flow cytometry .P value were less than 0.2 in univariate analysis. The biomarker levels were log-transformed and described as geometric means (GM) with the percent coefficient of variation (%CV). Differences between groups was presented as geometric mean ratios (GMR). Normal distribution was determined using histograms and the Shapiro–Wilk test for normality after logarithmic transformation. Linear regression analysis was used to evaluate the differences in logarithmic mean values of the biomarker concentrations among various groups . Mann–Whitney test was used for comparison between HC and SJS/TEN groups and between SJS/TEN with non-severe and severe ocular complications groups. Statistical significance was defined as P < 0.05. Scatter plot graphs were created by GraphPad Software. All analyses were performed by STATA Statistical software version 15.1 .Data were analyzed as means and standard deviations for continuous variables and counts and percentages for categorical variables. Univariate and multivariate logistic regression analyses were used to investigate prognostic factors related to SOCs. Multivariate logistic regression analysis was performed by stepwise backward elimination approach with variables whose


Recent guidelines have provided recommendations for the care of patients with chronic hypoparathyroidism. Very little is known about actual physicians’ practices or their adherence to such guidelines.To describe the physicians’ practice patterns and their compliance with international guidelines.The cohort studies included were Épi-Hypo  and ePatients .Internet-based cohorts involving all settings at a nationwide level (France). Participants were (i) physicians treating patients with chronic hypoparathyroidism and patients with chronic hypoparathyroidism either participating in the (ii) Épi-Hypo study (Épi-Hypo 2019 patients), or (iii) Hypoparathyroidism France, the national representative association (ePatients).The physicians’ specialties were mainly endocrinology (61%), nephrology (28%), family medicine (2.5%), pediatrics (2.5%), rheumatology (2%), or miscellaneous (4%) and 45% were practicing in public universities. The median number of pharmaceutical drug classes prescribed was three per patient. The combination of active vitamin D and calcium salt was given to 59 and 58% of ePatients and Épi-Hypo 2019 patients, respectively. Eighty-five percent of ePatients and 87% of physicians reported monitoring plasma calcium concentrations at a steady state at least twice a year. In 32 and 26% of cases, respectively, ePatients and physicians reported being fully in accordance with international guidelines that recommend targeting symptoms, plasma calcium and phosphate values, and urine calcium excretion.The care of patients with chronic hypoparathyroidism involves physicians with very different practices, so guidelines should include and target other specialists as well as endocrinologists. Full adherence to the guidelines is low in France. Hypoparathyroidism is a rare condition caused by undetectable or inappropriately low secretion of parathyroid hormone (PTH) that is insufficient for maintaining the plasma calcium concentration (PCa) within the normal range . The mosSymptoms  are nonspecific, and hypoparathyroidism can be asymptomatic. The diagnosis can be straightforward when symptoms occur within hours to days after neck surgery; however, diagnosis is sometimes delayed for years, especially in children. Diagnosing hypoparathyroidism is crucial due to its renal , ocular (cataract), and neurological  effects and its resultant negative impact on the quality of life.In contrast to other hormone-deficiency syndromes commonly treated by hormone replacement, the standard of care of hypoparathyroidism is based on oral calcium supplementation  and active vitamin D analog (active vitamin D) administration , 3. In 2Due to the huge phenotypic variability of hypoparathyroidism, physicians involved in the care and follow-up of patients range from pediatricians to endocrinologists and work in very different settings and from family medicine offices to tertiary care hospitals. To increase awareness of the guidelines, it is important to target the right audience , but verHypoparathyroidisme France association to its members. We collected ‘ePatients data’ during November 2019 and excluded those who also participated in Épi-Hypo. Finally, we also used data collected by investigators from the medical records of patients participating in Épi-Hypo who had at least one follow-up visit in 2019 (referred to as ‘Épi-Hypo 2019 patients’) to obtain data from the same period of time as that of the ePatients.Épi-Hypo (NCT02838927) is a cohort study that aimed to describe the natural history of chronic hypoparathyroidism in France. Data were collected online via a secure website. Physicians had to practice in France and provide care to one or more patient(s) with chronic hypoparathyroidism. They were solicited through national professional societies. At the time of registration, physicians were requested to fill out an online questionnaire  aimed toChronic hypoparathyroidism was defined as per the latest guidelines , 5, 7: thttp://www.rstudio.com/). Venn diagrams were designed using a publicly available website (http://bioinformatics.psb.ugent.be). We considered a P -value <0.05 to be significant in all cases.As no assumption could be made on the Gaussian distribution of most data (Shapiro–Wilk test), all data are reported as their median (IQR) or as numbers and proportions (%) for quantitative and qualitative data, respectively. Values were compared by Mann–Whitney or Kruskal–Wallis tests (with Dunn’s multiple comparison test) when more than two groups were compared, or chi-square tests where appropriate using RStudio: Integrated Development Environment for R  physicians who completed their questionnaire . Most ofvs.57%), whereas more nephrologists were involved in the care and follow-up of Épi-Hypo 2019 patients (42% vs.10%). ePatients were more frequently followed in a for-profit office than Épi-Hypo 2019 patients (51% vs 8%); physicians working in a public university hospital followed 38% of ePatients and 75% of Épi-Hypo 2019 patients.We then analyzed the two cohorts of patients: first, the data of 107 Épi-Hypo 2019 patients; secondly, the data of 110 patients who participated in the ePatients survey. ePatients were more frequently female (91% vs 71%) and were slightly younger (45 vs 51 years old) than Épi-Hypo 2019 patients , which ivs.55.1%) and teriparatide (2.7% vs.15.0%) were less frequently reported by ePatients than Épi-Hypo 2019 patients, whereas magnesium supplements were more frequently reported in ePatients than in Épi-Hypo 2019 patients (40% vs 23%). The median number of pharmacological classes per patient was 3 for both ePatients and Épi-Hypo 2019 patients. The combination of pharmacological classes was similar in both cohorts of patients (P  = 0.12). Overall, the treatment of hypoparathyroidism in the ePatients and Épi-Hypo 2019 cohorts was qualitatively similar, except for magnesium supplements, native vitamin D, and teriparatide (PTH(1–34)).The most frequently prescribed drug classes were active vitamin D, with a predominance of alfacalcidol over calcitriol and Ca salts . The perpatients ; all of P  < 0.001 for trend) from what was reported by physicians  and ePatients . International guidelines recommend monitoring PCa at least twice a year at steady state. Therefore, 87% of physicians and 85% of ePatients report proceeding accordingly.Our purpose was to outline the monitoring of patients with chronic hypoparathyroidism while on ‘steady state’, excluding periods of rapid fluctuations in PCa . Forty-nScreening for nephrolithiasis and nephrocalcinosis was carried out in 49% of ePatients, in 49% of Épi-Hypo 2019 patients, and was requested by 59% of physicians . Of them2/L2 . Here, we studied five specific indicators: clinical symptoms, PCa value, plasma phosphate concentration (PPi) value, calcium–phosphate product value, and urine calcium excretion (UCa) value. The absence of symptoms was an objective for 113 physicians (96%). Moreover, 112 (95%) reported that the PCa value was an indicator, and among them, 91 (81%) had a target interval in the low normal range  reported that they were aware of the indicators used by their physician. Overall, 99% of them reported that symptoms, PCa, PPi, and/or UCa were used as indicators by their physician. Eighty ePatients (89%) reported that the PCa value was an indicator and 42 of them (53%) reported that the low-normal range of PCa was the target . SymptomP  < 0.001) from the objectives of ePatients and physicians, among whom 81 and 53% reported targeting this specific range  patients had a PCa measured during this period; out of them, 33 (33%) had a value between 2.0 and 2.2 mM. This proportion highly differed  than previously reported  and active vitamin D were the most frequently prescribed therapeutics. We showed a lower percentage of patients prescribed Ca salts  endocrinologists are part of a broad network of physicians taking care of patients with chronic hypoparathyroidism, (ii) medications vary a lot among them, especially off-label (teriparatide) and over-the-counter  ones, and (iii) compliance with international guidelines regarding follow-up seems low and should be increased.N G received consulting fees from Shire, a Takeda company, which has never been involved in any part of the present study. P K received consulting fees from Shire, a Takeda company, and Kyowa Kirin, which has never been involved in any part of the present study; he is an investigator in studies sponsored by Ultragenyx, Kyowa Kirin and Shire, a Takeda company, not related to the present study. G M has served as a research investigator for NPS Pharmaceuticals. E M received consulting fees from Medtronic that has never been involved in any part of the present study. Co S received consulting fees from Shire, a Takeda company, which has never been involved in any part of the present study. M-C V received travel grants and free meeting registrations from Pfizer, Amryt, Lilly, Ipsen, Novartis, and HRA. P H has served as an advisory board member, consultant, research investigator, and speaker for Shire, a Takeda company. J-P B received consulting fees from Shire, a Takeda company, which has never been involved in any part of the present study. The other authors have nothing to disclose.This work was supported by Oscar – Filière Santé Maladies Rares and the Assistance Publique – Hôpitaux de Paris (CRC 2018).


Our quantitative analysis of the Pcdh19 mutant mouse exposes subtle changes in cortical layer composition, with no major alterations of the main axonal tracts. In addition, Pcdh19 mutant animals, particularly females, display preweaning behavioral changes, including reduced anxiety and increased exploratory behavior. Importantly, our experiments also reveal an effect of the social environment on the behavior of wild-type littermates of Pcdh19 mutant mice, which show alterations when compared with wild-type animals not housed with mutants.Mutations in the X-linked cell adhesion protein PCDH19 lead to seizures, cognitive impairment, and other behavioral comorbidities when present in a mosaic pattern. Neither the molecular mechanisms underpinning this disorder nor the function of PCDH19 itself are well understood. By combining RNA  PCDH19 mutations cause epileptic encephalopathy in humans, but the underlying pathophysiology is not completely understood. Here, we provide the first quantitative analysis of the cortical neuronal types expressing Pcdh19 in the mouse and human neocortex, and of cortical layer composition in Pcdh19 mutant animals, revealing the expression of Pcdh19 in interneurons and the presence of small, but significant, changes in neuronal distribution. The findings of our behavioral analysis indicate not only reduced anxiety and increased exploratory behavior, but also an impact of the mutant genotype on the behavior of wild-type animals when housed in the same cage. This finding underscores the importance of selecting appropriate control cohorts to avoid missing relevant behavioral changes in mutant animals.PCDH19 is one of several genes located on the X chromosome known to impact neurodevelopment and behavior. Mutations in this gene were identified in patients with EIEE9 , also known as Girls Clustering Epilepsy (GCE), over a decade ago  females and in males with somatic mutations  mouse models have been developed to explore the function of PCDH19. The first, produced by Taconic Biosciences, has the first three exons of the gene replaced by a β-galactosidase and neomycin (LacZ-neo) resistance cassette  revealed the strongest Pcdh19 expression in layers II/III and V(a) in mice  littermates.Although no major abnormalities in cortical architecture have been reported in either KO mouse model, no detailed quantitative analysis has been conducted yet. Similarly, while RNA  in mice , the neuad libitum access to water and food, and controlled temperature and humidity. All experiments using mice were approved by the local ethical boards and conducted following the directions of the UK Animal Scientific Procedures Act (update 1986).Animals were housed under a 12 h light/dark cycle with Pcdh19 KO line (TF2108) was acquired from Taconic Biosciences.C57BL6/J WT animals were purchased from Charles River Laboratories, and the Pcdh19 HET females to produce litters with WT males and females, KO males, and HET females.Experimental matings for anatomic and cellular characterization, as well as for behavioral analysis were set up using wild-type males and https://portal.brain-map.org/atlases-and-data/rnaseq. Analysis and visualization were conducted using R version 3.6.3, assisted by RStudio version 1.2.1335. Raw counts were normalized to account for library size  and transformed to counts per million (CPM) using R package scater version 1.16.2. Violin plots were generated with R packages gridExtra version 2.3 and ggplot2 version 3.3.1. River plots were made with R packages gridExtra version 2.3, ggplot2 version 3.3.1, and ggforce version 0.3.2.Gene expression matrices and metadata were downloaded from Animals were perfused with PBS followed by 4% paraformaldehyde (PFA) in PBS. After perfusion, brains were extracted and postfixed in PFA 4% overnight at 4°C. For RNA ISH, brains were then cryoprotected in 30% sucrose in PBS before embedding in OCT compound (Tissue-Tek) before freezing. Samples were stored at −80°C until sectioning. Sections of 12 or 20 μm were cut with a cryostat  and stored at −80°C until use. For immunostaining, fixed brains were briefly washed in PBS and embedded in 4% low-melting point agarose. Sections of 50 μm were cut with a vibrating microtome  and stored in PBS with 0.05% sodium azide at 4°C until use.Pcdh19 has been described previously . This plasmid was then used to generate digoxigenin (DIG)-labeled sense and antisense probes.The probe to detect eviously . Its seqm EDTA) for 1 h at 65°C. Slices were hybridized with the denatured sense or antisense probes overnight at 65°C in a humidified chamber. The next day, slides were washed with 0.2× SSC (Thermo Fisher Scientific) and PBST, and then blocked in ISH blocking solution (10% Denhardt’s solution and 0.1% Triton X-100 in PBS) for 20 min at room temperature (RT). After blocking, brain slices were incubated in primary antibody for 1 h at RT, washed in PBST, and incubated in secondary antibody for 1 h at RT. The antibodies used are described below. Slides were then washed in PBST, equilibrated in TN buffer , and incubated for 30 min in 1:2000 HRP-coupled anti-DIG antibody . Following the incubation, tissue was rinsed in TNT (TN+ 0.5% Tween) and immersed in Cy3-Tyramide  in a 1:50 dilution dissolved in the amplification diluent. Slides were then washed, counterstained with DAPI, and mounted with DAKO Mounting Medium.Thawed sections were postfixed in 4% PFA, endogenous peroxidases were quenched with 3% hydrogen peroxidase, and slices were then acetylated in a 0.25% acetic anhydride solution. Prehybridization took place in prewarmed hybridization buffer  or 10 min  before blocking. Coronal sections of 50 μm were blocked  at RT for 1 h. The tissue was then incubated in primary antibody diluted in blocking solution overnight at 4°C. Primary antibodies used for immunostaining were as follows: anti-CUX1 (Cut Like Homeobox 1) rabbit polyclonal ; anti-CTIP2 (COUP-TF-interacting protein 2) rat monoclonal ; anti-SATB2 mouse monoclonal ; anti-RORB rabbit polyclonal ; anti-TBR1 (T-Box Brain Transcription) rabbit polyclonal ; anti-Pvalb rabbit polyclonal ; anti-CB rabbit polyclonal ; anti-CR mouse polyclonal ; anti-SST (somatostatin) rat monoclonal ; anti-L1CAM (L1 Cell Adhesion Molecule) rat monoclonal ; and anti-Neuropilin1 goat polyclonal .Antigen retrieval was performed for staining with antibodies against RORB (RAR Related Orphan Receptor B), SATB2 , parvalbumin , and calretinin (CR), with the tissue either immersed in a 10 mSlices were then rinsed in PBS and incubated with secondary antibodies coupled to fluorochromes  for 1 h at RT. Nuclei were counterstained with DAPI for 10 min, washed again in PBS, and mounted with DAKO Mounting Medium.Images were acquired using a confocal laser scanning microscope  and ZEN Black software . Image analysis was conducted with ImageJ Fiji software . For quaPcdh19 colony. Mice were habituated to the new environment by taking them to the behavioral room 30 min before the tests. Mice were handled with open hands to reduce anxiety levels and a maximum of one behavioral test was performed per day.Behavioral tests were conducted at postnatal day 21  and in young adults (P60 and over). Two different WT controls were tested: WT littermates of the mutant animals [mixed-genotype housed (MGH) mice] and animals from pure WT litters [single-genotype housed (SGH) mice]. The WT parents of the SGH animals were derived from the Open field behavioral analysis was performed on 2 consecutive days, using the first day to habituate the mice to the new environment. Mice were allowed to explore freely, in the dark, for 20 min, in an open field arena (40 × 40 cm). Spontaneous locomotion was recorded using a computer-linked video camera (The Imaging Source) located above the arena and an infrared illumination box (Tracksys) located underneath the arena. The EthoVision XT software (Noldus) was used to analyze the total distance traveled, the distance traveled in intervals of 5 min, and the time spent in the center of the arena. The center of the arena was defined as the area separated from the wall by ≥5 cm.Each mouse was left to explore freely for 5 min in a maze consisting of the following four perpendicular arms (40 × 7 cm): two open arms (1 cm high) and two closed arms (16 cm high), in a well lit room. Behavior was recorded using a computer-linked video camera (The Imaging Source) located above the maze. The total time spent in the open arms was measured using EthoVision XT software (Noldus).At P21, test pups were habituated to the arena for 3 min. Subsequently, WT females in estrus, unfamiliar to the pups, were added to the cage, and both mice were allowed to interact with each other for another 3 min in a well lit room. The interaction between the pups and the females was recorded using a computer-linked video camera (The Imaging Source) located above the arena. Videos were manually scored, and interaction was recorded when both mice were within 2 cm of each other, not including tail–tail interactions.At P60, only female mice were tested for social interaction. In this case the unknown WT females were not required to be in estrus.To determine which females were in estrus, vaginal smears were stained with Giemsa solution  and allowed to roam free for 24 h with t test for normal data, or a Mann–Whitney test if data distribution did not meet normality criteria. If the variance of the two groups differed, a Welch correction was applied. For comparison of more than two groups, ANOVA was used for normal data and a Kruskal–Wallis test if the assumption of normality was not met. If only the assumption of homogeneity of variance was not met, a Welch’s ANOVA was used. The post hoc test following ANOVA was adjusted according to Tukey’s test HSD or, in the case of the social interaction analysis, Dunnett’s test. A Kruskal–Wallis test was followed by Dunn’s correction, and Welch’s ANOVA was followed by Games–Howell correction. Statistical data are presented as the mean ± SEM for formal tests. To carry out estimation statistics for the behavioral experiments, data were introduced into the form available at www.estimationstats.com, in the section for multiple two-group arrangements to obtain the mean differences between groups and their corresponding 95% confidence intervals (CIs). The y-axis limits were set for optimal display of the raw data, and the graphs obtained were directly used in the figures of the article. Calculation of the unbiased Cohen’s d for each comparison, as well as its 95% CI, was conducted using the esci module on jamovi version 1.6 .For all experiments, individual animals were considered the experimental unit and the data obtained from each animal were averaged if more than one quantification was performed . Experimenters were blind to the genotype of the animals until all quantification or scoring was completed. Statistical analysis was performed using GraphPad Prism version 9  or R version 3.6.2 . Normality of the data was tested using the Shapiro–Wilk test, and homogeneity of variance was assessed with Levene’s test. If either assumption was violated, an appropriate nonparametric test was used. Comparisons between two groups were performed using a two-tailed independent-samples Pcdh19 expression in the adult cortex, corresponding to the upper regions of layer V (layer Va) and II/III  against several cortical markers for principal neurons and interneurons in the somatosensory cortex at P10 and P20, respectively . These data suggest that in juvenile animals Pcdh19 is expressed in both intratelencephalic and corticofugal projection neurons and reveal a previously unreported expression in subpopulations of cortical interneurons.Previous RNA ISH studies have shown two main areas of d II/III . Howeverectively A–D. At Pns RORB; A, callosns RORB; B, corticns RORB; B, and cons RORB; C. The stII and V D, as wel10.1523/ENEURO.0510-20.2021.f1-1Figure 1-1Figure 1-1, TIF file.Gene expression and distribution of Pcdh19 in cortical excitatory projection neurons of the Allen Brain Atlas Mouse Whole Cortex & Hippocampus - SMART-SEQ (2019) with 10x Smart-Seq Taxonomy (2020), represented by violin plots. The first row shows the overall expression of Pcdh19 in the combined dataset excluding hippocampal regions for simplicity. Subsequent rows show expression by cortical region. Dots indicate the median value of the population. Absence of a violin plot in a row indicates that none or fewer than three cells from that particular cortical region were mapped to the corresponding neuronal cluster. Black and red lines indicate consistent low and high expression of Pcdh19 across areas, respectively; asterisks highlight clusters with marked variation in Pcdh19 expression across cortical regions. ACA, Anterior cingulate area; AI, agranular insular area; AUD, auditory areas; GU, gustatory areas; MOp, primary motor area; MOs-FRP, secondary motor area-frontal pole, cerebral cortex; ORB, orbital area; PL-ILA, prelimbic - infralimbic areas; PTLp, posterior parietal association areas; RSP, retrosplenial area; TEa-PERI-ECT, temporal association areas-perirhinal area-ectorhinal area; VIS, visual areas; VISp, primary visual area. Download 10.1523/ENEURO.0510-20.2021.f1-2Figure 1-2Figure 1-2, TIF file.Gene expression and distribution of Pcdh19 in cortical inhibitory neurons of the Allen Brain Atlas Mouse Whole Cortex & Hippocampus - SMART-SEQ (2019) with 10x Smart-Seq Taxonomy (2020), represented by violin plots. The first row shows the overall expression of Pcdh19 in the combined dataset excluding hippocampal regions, for simplicity. Subsequent rows show expression by cortical region. Dots indicate the median value of the population. Absence of a violin plot in a row indicates that none or fewer than three cells from that particular cortical region were mapped to the corresponding neuronal cluster. Black and red lines indicate consistent low and high expression of Pcdh19 across areas, respectively; asterisks highlight clusters with marked variation in Pcdh19 expression across cortical regions. ACA, Anterior cingulate area; AI, agranular insular area; AUD, auditory areas; GU, gustatory areas; MOp, primary motor area; MOs-FRP, secondary motor area-frontal pole, cerebral cortex; ORB, orbital area; PL-ILA, prelimbic-infralimbic areas; PTLp, posterior parietal association areas; RSP, retrosplenial area; TEa-PERI-ECT, temporal association areas-perirhinal area-ectorhinal area; VIS, visual areas; VISp, primary visual area. Download Pcdh19-expressing neurons in the mouse adult somatosensory cortex. We chose the “Whole Cortex & Hippocampus - SMART-SEQ (2019) with 10×-Smart-Seq Taxonomy (2020)” dataset from the Allen Brain Atlas  that includes 76,307 single-cell transcriptomes with cluster-assigned identity isolated from a total of 21 adult cortical and hippocampal regions, including primary and secondary somatosensory cortex. The 74,973 cells for which metadata are available in this dataset are classified into 379 cell types, of which 236 are glutamatergic, 119 GABAergic, and 24 non-neuronal , and/or clusters 304–306 of near-projecting neurons, whereas those expressing Pcdh19 and SATB2, but not CTIP2, would be layer V IT neurons, matching those in clusters 190–192, 200, and 207. We also identified neurons expressing Pcdh19 and TBR1 in layer VI , with at least one of them corresponding to bipolar or multipolar cells (47_Vip). Within the Pvalb subclass, Pcdh19 is expressed by chandelier cells  and several subtypes of basket cells . Finally, within the Sst subclass, Pcdh19 expression is strongest in some subtypes of upper layer basket and Martinotti cells (Sst clusters 94 and 95), and in the long-range projecting population (61_Sst-Chodl). Again, variations in the level of Pcdh19 expression within GABAergic clusters can be seen between brain regions .In summary, our analysis demonstrates that mouse PCDH19 cause severe impairments in brain function, yet the expression profile in human cortical neurons is unclear. We therefore extended our analysis to a publicly available human dataset from the Allen Brain Atlas , obtained from several brain areas . This dataset comprises 49,417 cell nuclei  and has allowed the definition of 56 excitatory and 54 inhibitory subtypes. We applied the same strategy as with the mouse dataset, filtering for those neurons originating in the somatosensory cortex, which reduced the dataset to 5103 neurons ascribed to 12 subclasses  shows good correlation between the levels of PCDH19 expression within clusters, with only a few exceptions  than in somatosensory areas [primary somatosensory cortex lower limb region (S1lm) and primary somatosensory cortex upper limb region (S1ul)] or L1-2 VIP RPL41P3, with higher PCDH19 expression in motor areas  and Human MTG - SMART-SEQ (2018) datasets  and performed immunohistochemistry at P10, once radial migration is completed. For each marker, we determined the proportion of positive cells, as well as their distribution within 10 bins covering the whole width of the cortical plate. We analyzed males and females separately, using WT male (WT-M) controls for the KO males and WT female (WT-F) controls for the HET animals .Although no major morphologic defects have been described in t brains , a detai+ cells made up approximately one-fifth of all DAPI+ cells , and SATB2+ cells represented more than one-half of all cells . The proportion of RORB+ cells seemed lower in KO brains compared with WT-M brains , but statistical analysis revealed that this difference was not significant . CTIP2+ cells were also equally abundant among the four groups , and TBR1+ cells added up to approximately one-third of all cells .In accordance with previous reports , we foun+ neurons between the 10 bins was unchanged for males and females  = 5.81, p = 0.0239; Tukey’s test: q = 4.60, p = 0.0245 HET vs WT). For CTIP2, we found differences in bins 3 (1.7-fold increase) and 7 (1.6-fold reduction) in KO males, suggesting a redistribution of CTIP2+ neurons to higher positions in layer V  = 2.787, p = 0.0317; bin 7: WT-M = 16.74 ± 1.67%, KO-M = 10.68 ± 0.34%; independent t test with Welch correction for unequal variance, t = 3.556, p = 0.0333). HET females showed double the percentage of cells in bin 1 than their WT siblings  = 5.391, p = 0.0017; + distribution were only present in males, specifically in bin 2, with a 3.4-fold reduction  = 2.585, p = 0.0415; + cells tended to be more condensed in those animals. Finally, KO males showed a 2.4-fold increase in the percentage of TBR1+ cells in bin 1 compared with their WT counterparts  = 5.818, p = 0.0011), and HET females had a 1.4-fold reduction in the percentage of TBR1+ cells in bin 3  = 4.473, p = 0.0042) and a 1.6-fold increase in bin 5  = 3.268, p = 0.0171; The distribution of SATB2 females G–J. Howe neurons D–L. Rega neurons E. Pcdh19+ cells , which, despite no changes between genotypes within males or females, displayed a significantly lower proportion in WT females than in WT males  = 3.054, p = 0.0224). PVALB+, SST+, and CR+ accounted for <5% of DAPI+ cells each , but HET females showed a slight decrease in SST+ cells  = 2.578, p = 0.0419) and KO males a similarly small decrease in CR+ interneurons  = 2.509, p = 0.0459).To complete our analysis on cortical composition and lamination, we stained the sSC with four different interneuronal markers  in P20 brains. As before, cortical thickness showed no difference between genotypes of matched sex , CR+ , and PVALB+  interneurons  = 4.647, p = 0.0035; + interneurons in HET brains, whereas bins 2 and 3 seem to have reduced numbers, suggesting a potential redistribution of SST+ cells toward deeper layers in HET females. Changes in CR+ cell distribution were found in bin 8 of KO brains, which displayed an approximately twofold reduction over WT male brains  = 2.777, p = 0.0321; + (bin 4), CR+ (bin 8), and PVALB+ (bin 7) interneurons between WT males and females .Regarding cellular distribution in the sSC, no differences were apparent for CB females C–F. Howerneurons C–J. HET Pcdh19 mutant animals, subtle but significant differences in distribution are apparent for many of the analyzed neuronal markers.In summary, despite relative neuronal proportions and distributions being mostly normal in the sSC of Pcdh19 is expressed in cortical projection neurons that project through the corpus callosum (layer II–III and some layer V neurons), as well as in neurons projecting outside the cortex, mainly through the pyramidal tract (layer V PT neurons). Although several members of the cadherin superfamily, including δ-protocadherins 7, 10, 17, and 18, have been shown to play a role in axonal outgrowth  to improve the interpretation of results. Because estimation statistics compare the means of only two groups, we provide the average mean difference (Mdiff) and unbiased Cohen’s d of the particular comparison with their corresponding 95% confidence intervals, followed by the results of the overall comparison with ANOVA or Kruskal–Wallis test and the relevant post hoc analysis. When the means of the three groups were not deemed different by any of the two methods, we only present the common statistical inference analysis for brevity.While there are no major lamination defects in the cortex and in the main axonal tracts of the brain of  neurons . Thus, ws WTSGH; A. Indeed animals . The par10.1523/ENEURO.0510-20.2021.f6-1Figure 6-1A, Total distance travelled in the open field test by P60 males. B, Distance travelled in the open field test by P21 males, split into 5 min intervals. The data for the first 5 min are shown in C, Distance travelled in the open field test by P60 males, split into 5 min intervals. D, Time spent by males in the center of the arena during the 20 min open field test at P21 and P60. E, Time spent in the open arms of the elevated plus maze by P21 males. F, Number of beam breaks during the 24 h activity test for the males of the different conditions. Light, light phase; dark, dark phase. G, Number of beam breaks by hour in the 24 h activity test for the males of the different conditions. The time of the day is shown on the x-axis, and the gray square indicates the hours of the dark period. The numbers of tested animals were as follows: at P21: WTSGH, 26; WTMGH, 18; KO, 31; at P60: WTSGH, 24; WTMGH, 18; KO, 29. For the 24 h activity test, the numbers were as follows: WTSGH, 17; WTMGH, 10; KO, 10. Results are indicated as the mean ± SEM. *p < 0.05. Download Figure 6-1, TIF file.MGH) males traveled on average 23% more distance during the 20 min open field paradigm than WTSGH males. The unpaired Mdiff was 667.54 cm , indicating a strong effect of housing  = 5.02, p = 0.0091; post hoc Tukey’s test, WTMGH vs WTSGH: q = 4.48, p = 0.0063). In this experiment, KO animals also traveled a higher distance than WTSGH , but an effect of genotype cannot be confirmed with these data. An analysis by 5 min slots showed that the increased distance traveled by WTMGH males compared with WTSGH males was mainly because of a 47% increase in activity during the first 5 min , this difference again does not seem to reflect a real change in behavior, suggesting that increased activity might be an effect of housing in males, rather than of genotype  = 9.35, p = 0.0093; post hoc Dunn’s test, KO vs WTSGH: z = 3.01, p = 0.1711; MGH males over WTSGH males disappeared after the first 5 min and also when animals were tested again at ≥P60 , this difference is even smaller than for WTMGH males and is unlikely to reflect a real change in behavior  = 2.07, p = 0.0771). This result again points to an effect of housing on the social behavior of WTMGH males.Interestingly, we also detected a subtle difference in social behavior at P21. In this case, WTMGH males showed altered behavior at P21, with increased activity during the first 5 min of the open field test and reduced social interaction.In summary, adult KO males displayed a robust phenotype of reduced anxiety in the elevated plus maze test, and WTMGH females displaying increases of 35% and 19%, respectively, when compared with single-genotype housed controls . In adults, the increase was down to 60% and 39% . These results indicate a strong effect of genotype on reducing anxiety, as also seen for adult male KO animals.Since the increase in distance traveled during the first 5 min in the open field test does not seem to be caused by the overall hyperactivity of HET animals and their WT siblings, we again analyzed anxiety-related behaviors in these animals. There were no differences in the time spent in the center of the open field arena for any of the conditions at P21 ; unbiased Cohen’s d = −0.62 ; one-way ANOVA: F = 3.38; p = 0.0398; Dunnett’s test, WTMHG vs WTSGH: q = 2.32, p = 0.0432].As in the case of male mice, the social interaction test revealed differences between single-genotype and mixed-genotype housed WT females C,D. HoweMGH males and females presenting significant differences in the open field and social interaction tests when compared with single-genotype housed WT animals.Overall, we found significant behavioral differences between wild-type and mutant animals that were generally more pronounced in HET females than in KO males. HET females displayed consistent hyperactivity during the first 5 min of the open field test and, similar to the mutant males, a robust phenotype of decreased anxiety in the elevated plus maze, in this case both at preweaning and at adult stages. Importantly, we also uncovered an effect of housing on the behavior of WT animals, with WTPcdh19 mutations on fine cortical composition, despite the relevance of these factors to understand the pathologic mechanisms underpinning EIEE9. Here we present a detailed analysis of neuronal subtypes expressing Pcdh19 in the mouse somatosensory cortex and a comparison with human data. Our study reveals that Pcdh19/PCDH19 is not only expressed in pyramidal neurons, but also in different types of interneurons, and that, in general, higher expression is limited to specific subpopulations in both cases. Our analysis also rules out major anomalies in the main axonal tracts and provides a quantitative assessment of cortical composition and lamination. Despite the lack of major architectural defects, our data reveal subtle defects in layer composition that could contribute to the pathophysiology of EIEE9. Indeed, mutant animals display behavioral alterations in the open field test  and elevated plus maze test . Importantly, and as previously revealed with the analysis of Nlgn3 mutants  datasets that include neurons from various cortical regions (including sSC) from adult brain, which allowed us to obtain a global view of Pcdh19/PCDH19 expression across cortical areas in mouse and human.Hitherto, the characterization of the neuronal populations expressing PCDH19 has been hindered by the lack of specific antibodies that perform satisfactorily in immunohistochemistry analyses. In addition, as PCDH19 is likely distributed in both axons and dendrites , the una signals . Our ISHPcdh19 is expressed by excitatory neurons in layer V, projecting both intracortically and extracortically, as well as by certain subtypes of layer II/III projection neurons, in agreement with the ISH data. Expression in layer IV is harder to judge from the scRNAseq results, as there are no clusters representing neurons from layer IV exclusively, but several clusters in layers VI and VIb also show high Pcdh19 expression. In interneurons, the expression is widespread in the Pvalb subclass; cluster specific in the Sncg, Vip, and Sst subclasses; and very low in the Lamp5 and Pax6 clusters, except for Lamp5 Lhx6, which shows high expression. These results demonstrate that while Pcdh19 is expressed by a variety of excitatory and inhibitory neurons, expression remains specific for particular clusters. This cluster specificity would suggest a role for PCDH19 in the establishment of neuronal circuits as a potential neuronal recognition molecule.Our analysis of a mouse dataset of whole cortex and hippocampus confirmed that PCDH19 follows a similar pattern, with expression in both excitatory and inhibitory neuronal types. Expression in human excitatory neurons of the sSC is more graded, with many more subtypes showing intermediate expression levels than in mouse, likely reflecting an averaging effect because of the smaller number of human clusters defined for that dataset. In any case, highest expression corresponds to clusters in layers III and V, in line with RNA ISH results in mice. Regarding interneurons, high PCDH19 expression can be found in subtypes of LAMP5, VIP, SST, and PVALB interneurons, which generally show a good correlation with their murine counterparts. This is a relevant finding that supports the use of mouse models to investigate some aspects of PCDH19 GCE. However, it is important to note that there are some differences as well, like the comparatively lower expression of PCDH19 in long-range projecting interneurons in humans . The functional relevance of Pcdh19/PCDH19 expression in particular neuronal subtypes will need to be established experimentally, but our results provide a framework to support those functional studies in the future, not least because of regional differences in the expression of this gene within neuronal subtypes.Human Pcdh19 KO models , which maintained it into adulthood. However, HET females also exhibited increased exploratory behavior, or maybe hypersensitivity to new environments, from a young age, as demonstrated by their consistently higher distance traveled during the first 5 min in the open field test at P21 and P60. It is important to consider that animals were placed into the open field arena four times in total, as they were tested on 2 consecutive days at both ages. Although habituation to the environment would be expected in this situation, the increased distance traveled during the first 5 min was apparent in all four trials, indicating a robust behavioral response. These results also suggest that behavioral changes in Pcdh19 heterozygous animals start early in life, validating them as a good model for a developmental condition.In line with a previous mouse study  and withPcdh19 HET females spent significantly more time in the center of the open field arena, suggesting reduced anxiety. Although our animals did not display such behavior, they were tested at approximately P60, which would be in agreement with the data from their first open field test. In addition, the results of our EPM test also indicate reduced anxiety in our animals, which could therefore represent a behavioral characteristic of Pcdh19 mutant animals. Because no specific analysis of the first 5 min was conducted in that study, it is difficult to assess whether their animals exhibited increased activity during that period. Nevertheless, the fact that WT females display the same behavioral phenotype as their HET siblings indicates an effect of the social environment that can only be detected through the inclusion of single-genotype housed WT animals. Interestingly, this effect was also present in young males, with WTMGH animals traveling a higher distance in the first 5 min of the open field test than KO or WTSGH males. However, unlike in the female population, this behavior disappeared in adulthood. Because adult male and female animals are housed separately, it is tempting to speculate that this effect of the social environment is somehow mediated by the HET females, although other causes, like a maternal effect, cannot be ruled out based on our experiments.Open field and EPM tests were also performed in the study by PCDH19 have also been linked to ASD cases . Mice carrying either a null allele for Cdkl5 or the disease-causing mutation R59X do not display behavioral seizures, but they exhibit network hyperexcitability that manifests as decreased threshold to pharmacologically induced seizures (Pcdh19 heterozygous animals (PCDH19 mutations on human connectivity.Finally, an important question is why the mutation of seizures . In the seizures , none ofseizures . Similar animals , indicat animals , this em


The levels of 12 candidate circRNAs were verified by qRT-PCR, and the quantities of 10 of these circRNAs were consistent with the data of microarray. The functions of host genes of differentially quantified circRNAs, including RNA and protein process, focal adhesion, and leukocyte transendothelial migration, were associated with the development of AIS. As a miRNA sponge, differentially quantified circRNAs had the potential to regulate pathways related to AIS, like PI3K-Akt, AMPK, and chemokine pathways. Of 198 differentially quantified circRNAs, 96 circRNAs possessing a strong translational ability could affect cellular structure and activity, like focal adhesion, tight junction, and endocytosis. Most differentially quantified circRNAs were predicted to bind to EIF4A3 and AGO2—two RNA-binding proteins (RBPs)—and to play a role in AIS. Moreover, four of ten circRNAs with verified levels by qRT-PCR  were predicted to participate in processes of AIS, including PI3K-Akt, AMPK, and chemokine pathways as well as endocytosis, and to be potentially useful as diagnostic biomarkers for AIS. In conclusion, plasma exosome-derived circRNAs were significantly differentially quantified between AIS patients and controls and participated in the occurrence and progression of AIS by sponging miRNA/RBPs or translating into proteins, indicating that circRNAs from plasma exosomes could be crucial molecules in the pathogenesis of AIS and promising candidates as diagnostic biomarkers and therapeutic targets for the condition.Stroke is one of the major causes of death and long-term disability, of which acute ischemic stroke (AIS) is the most common type. Although circular RNA (circRNA) expression profiles of AIS patients have been reported to be significantly altered in blood and peripheral blood mononuclear cells, the role of exosome-containing circRNAs after AIS is still unknown. Plasma exosomes from 10 AIS patients and 10 controls were isolated, and through microarray and bioinformatics analysis, the profile and putative function of circRNAs in the plasma exosomes were studied. A total of 198 circRNAs were differentially quantified (|log2 fold change| ≥ 1.00,  As a common vascular disease, stroke is one of the major causes of death and long-term disability, challenging the current healthcare system worldwide . Of all Exosomes, nanosized vesicles (∼30–150 nm) with lipid bilayer membranes, are secreted by various cells and naturally present in the blood . When inCircRNAs have been reported to be involved in the progression of many diseases, including Alzheimer’s disease , cardiovHowever, the profile and regulatory network of circRNAs derived from plasma exosomes of patients with AIS remain unclear. In this prospective investigation, a case-controlled study was conducted, and the circRNA profile of plasma exosomes was compared between AIS patients and controls through microarray. By bioinformatics approaches, the functions and signaling pathways of differentially quantified circRNAs were analyzed. Based on the results of microarray, 12 circRNAs with high fold changes among differentially quantified circRNAs were chosen for validation by qRT-PCR, and their roles in the pathogenesis of AIS and diagnostic values for AIS were also explored. The data of the present study shed light on the possible roles of exosome-derived circRNAs in regulating the pathogenesis of AIS and provide new potential targets for diagnosis and therapy of AIS.Ten adult patients with AIS  were selected from the First Affiliated Hospital of Chengdu Medical College in 2020. AIS was diagnosed according to the Guidelines for the Prevention of Stroke in Patients with Stroke and Transient Ischemic Attack . Ten conInclusion criteria are as follows: (1) aged 50–90 years old, (2) within 24 h after onset, and (3) have agreed to sign the informed consent. Exclusion criteria are as follows: (1) have received thrombolysis and/or thrombectomy treatment and (2) have other concomitant diseases, including acute infection, immune diseases, neurodegenerative diseases, and tumors.Blood samples from 10 AIS patients and 10 controls included in the present study were collected into EDTA tubes by venipuncture. After centrifuging at 2,000 rcf for 15 min at room temperature, a clear top layer was harvested to a labeled tube  and stored at −80°C. Plasma exosomes were isolated using the exoRNeasy Serum/Plasma Maxi kit  following the manufacturer’s instruction. The resulting exosome pellet was suspended in PBS for further RNA or protein extraction. The exosome fraction isolated from plasma was characterized by western blot, nanoparticle tracking analysis (NTA), and transmission electron microscopy (TEM).Plasma exosomes suspended in PBS were mixed with the same volume of RIPA lysis buffer, and exosome proteins were purified from each sample and separated by SDS-PAGE gel and then transferred to a polyvinylidene fluoride (PVDF) membrane. The membrane was blocked using 5% BSA and then treated with antibodies against CD63  or TSG101  at 4°C for 12 h. Horseradish peroxidase-conjugated secondary antibody  was then applied, and the blots were developed with enhanced chemiluminescence reagents.Isolated exosomes were diluted in PBS and analyzed using the ZetaView  with NTA software (ZetaView 8.04.02). Triplicate measurements were recorded for each sample. Size distribution and concentration profiles were averaged to derive the representative size distribution profiles.Plasma exosomes suspended in PBS were fixed in 2% paraformaldehyde. The fixed sample was absorbed onto formvar-coated copper grids for 5 min at room temperature. After being rinsed in distilled water, samples were stained with methyl cellulose uranyl acetate for 1 min at room temperature. Excess liquid was wicked off the grid using filter paper, and grids were stored at room temperature until imaging. Imaging was performed using Tecnai G2 Spirit BioTwin .By exoRNeasy Serum/Plasma Maxi Kit , total RNA was extracted from plasma exosomes isolated from the 10 AIS patients and 10 controls following the manufacturer’s recommendation. RNA integrity of total RNA was inspected by an Agilent Bioanalyzer 4200  (data not shown).SurePrint G3 Human ceRNA microarray  was made by Agilent Technologies and contained probes interrogating 84,569 circRNAs. Total RNAs were incubated with RNase R to eliminate linear RNAs. The enriched circRNAs were transcribed into fluorescent complementary RNA (cRNA), which then were hybridized onto the circRNA microarray. After hybridization, the signal of the circRNA microarray was scanned using the Agilent Microarray Scanner .The scanned information was extracted using the Agilent Feature Extraction software 10.7 . Raw data were normalized by quantile algorithm, R package “limma”. Differentially quantified circRNAs were identified through volcano plot filtering. Hierarchical clustering analysis was conducted to display the circRNA profile pattern between patients with AIS and controls.via TB Green®Premix Ex Taq™ II  with the BIO-RAD system  according to the instruction of the manufacturer. The reaction system was 25 μl in volume and consisted of 12.5 μl of TB Green Premix Ex Taq II, 2 μl of cDNA, 1 μl of forward and reverse primers (10 μM), and 8.5 μl of RNase-free water. The optimum reaction conditions were as follows: 95°C for 30 s, followed by 40 cycles of 95°C for 5 s and 60°C for 30 s. Twelve circRNAs with high fold changes from the microarray results were chosen for validation by qRT-PCR with GAPDH as a normalization reference. The specific primers for target circRNAs are described in −ΔΔCt method (The microarray results were verified by qRT-PCR using samples from the same subjects (10 AIS patients and 10 controls). Total RNA from plasma exosomes was incubated with RNase R to eliminate linear RNAs and reverse transcribed by RevertAid First-Strand cDNA Synthesis Kit . CircRNA levels were determined by qRT-PCR t method .http://www.geneontology.org/) and Kyoto Encyclopedia of Genes and Genomes  analyses. Target miRNAs of circRNAs were predicted using miRanda (http://www.microrna.org/microrna/home.do) and TargetScan (http://www.targetscan.org/vert_72/). MiRNA response elements as regulators of mRNAs were predicted through TargetScan (http://www.targetscan.org/vert_72/) and microT-CDS (http://www.microrna.gr/microT-CDS). All circRNA–miRNA–mRNA interaction networks of differentially quantified circRNAs were visualized by Cytoscape 3.8.2. The tissue specificity of differentially quantified circRNAs was annotated via TSCD (http://gb.whu.edu.cn/TSCD/). The types and sites of RBPs binding to differentially quantified circRNAs were annotated by circinteractome (https://circinteractome.nia.nih.gov/). The probability of differentially quantified circRNAs coding for proteins was estimated by circBank (http://www.circbank.cn/), in which circRNAs with a coding probability >0.99 were analyzed.Host/target genes of differentially quantified circRNAs were illuminated by Gene Ontology  was calculated to assess the predictive value of the selected circRNAs for AIS diagnosis.2 fold change| ≥ 1.00 and p < 0.05. The p-value denotes the significance of GO and KEGG pathway analyses (p < 0.05). All statistical analyses were performed by SPSS . All data were represented as the mean ± standard deviation  and 126 downregulated  circRNAs. Hierarchical clustering of the levels between AIS patients and controls is illustrated in The profile of 84,569 human circRNAs was generated by SurePrint G3 Human ceRNA microarray using plasma exosomes from patients with AIS and controls. After fold-change filtering, 198 circRNAs were differentially quantified between patients with AIS and controls, including 72 upregulated  between AIS patients and controls, including five upregulated  and five downregulated  circRNAs . The KEGG analysis showed that metabolic , endocrine-related , and inflammatory  pathways were enriched , organelle (intracellular/intracellular membrane-bounded/membrane-bounded organelle), and membrane  . The molenriched .via miR-4269-CREB5 or miR-4533-AQP4, and hsa_circ_0041685, via miR-4267-DCTN2, played roles in the vasopressin-regulated water reabsorption. Moreover, hsa_circ_0066867 or hsa_circ_0041685 potentially regulated chemokine signaling through miR-6737-5p-CCL2 or miR-3192-5p-CXCL12, respectively.According to circRNA–miRNA–mRNA networks, by GO and KEGG analyses, several differentially quantified circRNAs verified by qRT-PCR were involved in processes and pathways related to AIS. Hsa_circ_0112036 sponging miR-24-3p and regulating the levels of STRADB/BCL2L11/CDKN1B and hsa_circ_0066867 impacting miR-182-5p-FOXO3 were predicted in the AMPK and PI3K-Akt pathways. Hsa_circ_0093708, Certain circRNAs could be translated into proteins and participate in many processes of diseases. Thus, the coding probability of the differentially quantified circRNAs was analyzed. Of all 198 differentially quantified circRNAs, 96 circRNAs had strong coding potential (coding probability > 0.99), indicating that these molecules, through translation, could contribute to the process of AIS .To further investigate the role of proteins translated from differentially quantified circRNAs with strong coding potential in AIS, GO, and KEGG pathway analyses for these proteins were performed. The results of GO analysis showed that cellular components were mainly located in the intracellular (cytoplasm and organelle) and extracellular (vesicle and exosome) compartments. The molecular functions of these proteins were related to RNA and protein binding (enzyme/cadherin/cell adhesion molecule binding), and the biological processes were mainly related to localization (protein and cell) and cellular homeostasis  . The KEGBesides sponging miRNAs, circRNAs could bind to RBPs to regulate the expression of genes and processes of diseases. Among 38 RBPs, eukaryotic initiation factor 4A-3 (EIF4A3) had the highest count of binding sites for the differentially quantified circRNAs, while Argonaute 2 (AGO2) and fragile X mental retardation protein (FMRP) ranked second and third places, respectively. Moreover, of 198 differentially quantified circRNAs, 170 circRNAs were predicted to bind to EIF4A3, 89 circRNAs to bind to AGO2, and 65 circRNAs to bind to fused in sarcoma (FUS) .p < 0.05), with the highest AUC found for hsa_circ_0112036 (AUC = 0.810)  .Stroke causes high mortality and disability rates globally . As the By microarray, 198 differentially quantified circRNAs were found between patients with AIS and controls, evidencing that the profile of circRNAs derived from plasma exosomes was affected by AIS. CircRNAs are highly conserved and characterized by a stable structure and distinct tissue-specific expression . A tissuvia feedback  and five downregulated  circRNAs. The results showed that, as competing endogenous RNAs, differentially quantified circRNAs, through misregulating RNA and DNA, would impact cellular components, including the nucleus, organelle, and membrane, and modify the function and fate of cells/tissues. AIS induces neuron apoptosis , while aignaling  and thatignaling . Therefovia several ways, like endocytosis, phagosome, focal adhesion, and tight junction. Endocytosis—a ubiquitous physiological process mediating nutrient uptake, receptor internalization, and signaling, essential events for cell growth and survival—has been reported to cause neuronal death and exacerbate brain damage during AIS (Since researchers found that certain proteins translated by circRNAs participate in the processes of human diseases , a variering AIS . RABEP1 ring AIS , atherosring AIS , and thrring AIS . Chen etring AIS . Tight jring AIS . Thus, wcis-regulatory elements and affect the expression and function of their target RNAs (via exosomes.RBPs assemble the ribonucleoprotein complexes to bind RNA sequences by interacting with specific get RNAs . Increasget RNAs  and thatget RNAs , circRNAget RNAs , and RBPget RNAs . Therefoget RNAs . AGO2 isget RNAs . Besidesget RNAs  and can get RNAs , which cCircRNAs are stable and abundant and function as effective diagnostic biomarkers of stroke, like hsa_circ_0001599  and hsa_via sponging miRNA/RBPs or encoding proteins, may be explored as biomarkers for the diagnosis of AIS, and may also be potential targets for therapeutic interventions. However, these findings are based on only 10 AIS patients and 10 controls, and more samples are necessary to assess and validate the data in the future.Taken together, the data of this study demonstrate that circRNAs derived from plasma exosomes were differentially quantified between AIS patients and controls. Importantly, these results revealed that exosomal circRNAs from plasma, especially hsa_circ_0112036, hsa_circ_0066867, hsa_circ_0093708, and hsa_circ_0041685, potentially participate in the progression of AIS


The aim of this study was to investigate the expression of liver X receptors α/β (LXR) in primary breast cancer (BC) tissues and to analyze its correlations with clinicopathological parameters including patient survival.In a well-characterized cohort of 305 primary BC, subcellular distribution of LXR was evaluated by immunohistochemistry. Correlations with clinicopathological characteristics as well as with patient outcome were analyzed.LXR was frequently localized in both nuclei and cytoplasms of BC cells, with stronger staining in nuclei. Total and nuclear LXR expression was positively correlated with ER and PR status. Overall survival analysis demonstrated that cytoplasmic LXR was significantly correlated with poor survival and appeared as an independent marker of poor prognosis, in stage I but not in stage II–III tumorsAltogether, these data suggest that cytoplasmic LXR could be defined as a prognostic marker in early stage primary BC.The online version contains supplementary material available at 10.1007/s00432-021-03670-y. Breast cancer (BC) has surpassed lung cancer as the most frequent diagnosed cancer worldwide in 2020 and is still the leading cause of cancer death among women . LXRα is highly expressed in adipose tissue, liver, adrenal glands, lungs, and gastrointestinal tract, while LXRβ is widely expressed  play a role in BC, as we and other have reported earlier  play an important role in lung and breast cancer by promoting invasion, migration, and metastasis through an LXR-dependent pathway  in a well-characterized cohort of 305 primary BC patients. We quantified LXR levels both in the nuclear and cytoplasmic compartments, and analyzed correlations with clinicopathological parameters and patient survival.n = 305, two cases from one patient with bilateral BC) in this retrospective analysis were collected at the Department of Obstetrics and Gynecology of the Ludwig-Maximilians-University Munich, Germany between 2000 and 2002. This study was approved by the Ethical Committee of the Medical Faculty, Ludwig-Maximilian-University, Munich, Germany  and informed consent was obtained from all patients. Patient data obtained from the Munich Cancer Registry were pseudonymized and samples were encoded during experiments and statistical analysis. All clinical diagnostic and therapeutic procedures were completed before this study. All tumors were evaluated according to UICC TNM classification, including tumor size (pT), lymph-node involvement (pN), and distant metastasis (cM). Tumor grade was confirmed by an experienced pathologist (Dr. D. Mayr) of the LMU Department of Pathology, according to a modification of Elston and Ellis grading proposed by Bloom and Richardson. ER, PR, HER2, and Ki67 were determined by an experienced pathologist (LMU Department of Pathology) at first diagnosis using immunohistochemistry. For ER and PR staining, tissues showing nuclear staining in more than 10% of tumor cells were considered as hormone receptor-positive. HER2 expression was analyzed with an automated staining system , according to the manufacturer’s instructions. Cases were regarded as negative for 0 or 1 + score, and positive for 3 + scores. All cases with 2 + scores needed a further evaluation, i.e., fluorescence in situ hybridization (FISH) testing. Ki67 cut-off used to differentiate luminal A from luminal B tumors  was 14% as commonly used at the time of diagnosis.All samples , determined by multiplication of the positive cell proportion score  and the staining intensity score . As previously described for RIP140, PPARγ, and THRβ1 .Receiver-operating characteristic (ROC) curve analyses were performed to calculate the optimal cut-off values between low and high LXR expression, based on the maximum differences of sensitivity and specificity. The threshold determined regarding OS was an IRS > 2.5 for nuclear LXR, > 5 for cytoplasmic LXR and > 8.5 for total LXR, which were used to determine the percentages of tumors expressing low or high LXR levels described in Table p value < 0.05. Kaplan–Meier curves and estimates were then provided for each group and each marker. The p value and the number of patients analyzed in each group are given for each chart.Survival times were compared by Kaplan–Meier graphics and OS differences were tested for significance using the chi-square statistics of the log-rank test. Data were assumed to be statistically significant in the case of p values below 0.05 (*), 0.01 (**), or 0.001 (***) were considered statistically significant.Statistical analyses above were performed using SPSS 25 . For all analyses, LXR expression was assessed by IHC in a cohort of 305 primary BC samples , and a positive correlation (p = 0.012) was also detected between nuclear and cytoplasmic LXR expression.Based on IRS cut-offs defined by performing an ROC-curve analysis for OS, all patients were divided into low and high expression subgroups. Based on these nuclear and cytoplasmic LXR cut-off values, an inverse distribution of BC was noticed, with a majority of samples exhibiting a high nuclear IRS (73.77% of the tumors) and a low cytoplasmic one (77.70%). Total LXR was positively correlated with nuclear and cytoplasmic , lymph-node status (pN), metastasis (cM), histology, stage, tumor grade, ER, PR, and HER2 status. As shown in Table p = 0.068).As described in Table p = 0.977), whereas in stage I subgroup .We then then stratified patients in two subgroups, according to staging (stage I vs stage II–III). As shown in Fig. oup Fig. A, patienp = 0.040) but not in the subgroup of patients with tumors presenting lymph-node invasion (p = 0.359). The correlation was even more significant in the subgroup of patient with pT1 tumors  and tumor size (pT1 vs pT2-3). The correlation of cytoplasmic LXR with patient OS was significant in lymph-node negative tumors . Altogether, these data demonstrated that cytoplasmic LXR expression is associated with poor prognosis in patients with early stage BC .Multivariate analyses were performed for the whole cohort and for the two subgroups according to staging (stage I vs stage II-III), using a Cox regression model with cytoplasmic LXR expression and other relevant clinicopathological characteristics, namely age at diagnosis, metastasis status, ER, and HER2 Table . All parp = 0.011) and 7.172 , respectively. In contrast, cytoplasmic LXR did not exhibit an independent prognostic value in the stage II–III subgroup. Similarly, cytoplasmic LXR was a strong independent prognostic marker in the pT1 subgroup (data not shown), but not in the pT2-3 subgroup, nor in the lymph-node negative or positive subgroups.Interestingly, cytoplasmic LXR was regarded as an independent prognostic marker in the whole cohort, but this correlation was much stronger in the stage I subgroup, with hazard ratio of 1.871  subgroup, which appeared stronger than that observed in the whole cohort. Therefore, subcellular distribution of LXR expression appears as a parameter that needs to be taken into account in further studies of its mechanism or prognostic value in malignant tumors. Especially for luminal (ER/PR-positive and HER2-negative) early stage BC, the main concern is to identify patients who need to receive also chemotherapy in addition to endocrine therapy  Supplemental Figure 1 Kaplan-Meier analyses of patient overall survival (OS) for RXR expression in the whole cohort and different subgroups according to lymph node status, tumor size and Staging. OS curves are presented according to RXR expression, for the whole cohort (A), for stage I (B) and stage II-III (C) subgroups of patients, for lymph node negative (D) or positive (E) subgroups and for the pT1 (F) and pT2-3 (G) subgroups. The optimal IRS cut-off values for RXR expression were determined as 3.5. The number of cases for each group are indicated in each panel.Below is the link to the electronic supplementary material.


Hypertension is a major global cause of cardiovascular disease and death with rising worldwide prevalence, particularly in low-income countries. With low awareness, poor treatment, and low control of hypertension in Africans, there is an increased number of patients with target organ damage (TOD), especially chronic kidney disease (CKD), as a consequence of hypertension. The aim of our study is to assess the prevalence of CKD from studies in Africa reporting TOD related to hypertension.  We performed a search of PubMed/MEDLINE, Web of Science, EBSCOhost, and African Journals Online (AJOL) for studies reporting on CKD as TOD in patients with hypertension. The pooled estimate of CKD was then presented by subregions, age group, eGFR equations, and urban or rural location. p < 0.0001) and in studies conducted in urban settings (p < 0.001). CKD prevalence was not significantly different by type of GFR equation or age.  We identified 1,334 articles from which 12 studies were included for quantitative analysis. The studies included 5297 participants from 6 countries . The pooled prevalence of CKD was 17.8% (95% CI 13.0–23.3%), and CKD was significantly more prevalent in West Africa (21.3% (95% CI: 16.1–27.0); CRD42018089263. This study reports a high prevalence of CKD related to hypertension with a higher prevalence in urban than rural areas. This emphasizes the role of hypertension in causing kidney damage, and the need for strategies to improve awareness, treatment, and control of hypertension in Africans. This study is registered with PROSPERO registration number  Hypertension is a prevalent worldwide public health problem and an important modifiable risk factor for cardiovascular disease (CVD) and chronic kidney disease (CKD). The prevalence of hypertension in the global adult population was estimated to be 31.1% (95% CI: 30.0%–32.2%) in 2010, representing 1.38 billion people who were affected worldwide . The Uniβ) [There are subtle differences in the pathogenesis of hypertension between populations of African ancestry and Europeans, and such differences may account for the varied course of hypertension in these populations . For insβ)  and sociβ) .Given that blood pressure (BP) is a modifiable risk factor for CKD, it is important to identify the contribution of hypertension to CKD in Africans, especially as access to treatment for CKD and kidney failure is severely limited  with extCRD42018089263. Ethics approval was not required for this study.The Preferred Reporting Items for Systematic Review and Meta-Analysis , 20 was 2) as documented by the authors using any of the standard creatinine-based formulae to estimate the glomerular filtration rate (GFR): Chronic Kidney Disease Epidemiology (CKDEPI) [Published literature between January 1995 to December 2019 was included. We selected 1995 as the start year for our search as the definitions and stages of CKD were developed subsequently. We included all studies (without language restrictions) that reported CKD  as TOD of hypertension in adults 18 years or older and included studies conducted in Africa. Hypertension was defined as systolic BP (SBP) ≥140 mmHg and/or diastolic BP (DBP) ≥90 mmHg . We repo(CKDEPI) , Modific(CKDEPI) , and Coc(CKDEPI) . We exclWe performed a search of PubMed/MEDLINE, Web of Science, EBSCOhost, and African Journals Online (AJOL). Medical subject headings (MESH), including “prevalence,” “hypertension,” blood pressure,” “hypertensive,” “hypertensive target organ damage,” “chronic kidney disease,” “kidney disease,” kidney damage,” kidney failure,” “target organ damage,” “Africa,” “sub-Saharan Africa,” ‘Africa South of Sahara,” and all African countries by names were used in the search and modified for each database, as necessary. We adapted the MEDLINE search strategy for other databases .Data extraction was done independently and in duplicates by SA and UE, and disagreement was resolved by consensus by SA and UE, and when necessary, in consultation with IGO. The following data variables were extracted from selected studies: first author's name, year of publication, country and subregion of study, race of study participants, study design, total sample size, sample size of study participants with hypertension, gender proportion, median age, mean body mass index (BMI), mean SBP, mean DBP, eGFR equation used for assessment, number, and proportion of participants with CKD.To assess the quality of methodology of the studies included for analysis, a modified 9-point rating system by Stanifer et al.  was usedSTATA 16  was used for statistical analysis.I2 statistic was used to determine the heterogeneity between studies [Q-test based on ANOVA. Comparison of prevalence of hypertension and TOD between age groups, gender, location , and region (West Africa versus East Africa versus Southern Africa) was done.The prevalence of TOD in hypertensives was performed using meta-analytic techniques. The study-specific estimates derived from the DerSimonian–Laird random-effects model  were poo studies . Subgroup value of the Egger test = 0.19 .The mean systolic blood pressure ranged from 135.9 mmHg  to 175.4I2 = 95.5%, p < 0.001)   . Four st p=0.65; . The prep=0.001) .p < 0.001, p=0.76, Supplementary p=0.95, Supplementary When assessed according to region, studies from West Africa had the highest pooled prevalence of CKD (21.3% (16.1–27.0)), while the one study from Southern Africa had the lowest (3.2% (95% CI: 1.7–5.5%)), with a significant difference in prevalence across regions . This is consistent with general population reports from previous studies showing higher CKD prevalence in West Africa [Our study also found that the West African subregion had the highest pooled prevalence of CKD (21.3% (16.1–27.0%); t Africa , 39, 47.t Africa . The APOt Africa , 50. We t Africa , 52.Furthermore, we also found a higher prevalence of CKD among the urban populations compared to rural populations. This is largely consistent with observations from studies in Africa of higher prevalence of hypertension in urban than rural settings . EpidemiFinally, our study identified nonsignificantly different CKD prevalence rates based on the different equations used for assessing CKD from the included studies. Given that the current equations in use were not developed in sub-Saharan African populations, uncertainties remain about the best equation for assessing GFR in Africans. The accuracy of current equations remains limited at an individual level, particularly due to differences in muscle mass. Other studies have also reported differences in CKD prevalence using these equations with an overestimation using the CG equation , 45. ThiThere were some limitations to our study. First, the absence of reporting serial measurements of serum creatinine and or GFR at least 3 months apart to define CKD is notable. Severe hypertension can be associated with acute kidney dysfunction; therefore, it may not be possible to distinguish such cases from CKD. Thus, the use of a single GFR estimation to assess CKD prevalence is likely to overestimate the prevalence . This haOur study shows that the prevalence of CKD is high amongst hypertensive patients in Africa, particularly for those in urban than in rural areas. Given that blood pressure is a modifiable risk factor for CKD, strategies to improve hypertension awareness, treatment, and control should be implemented in African countries. Such strategies should include strengthening NCD structures in each country and ensuring availability of cheap but effective drugs for hypertension treatment.