The case of coronavirus disease (COVID-19) has become a global crisis. In addition, more variants of the virus have been discovered with easier transmission and more harmful effects. Thus, understanding the risk factors associated with the susceptibility and severity of COVID-19 is critical for disease control. This review article aims to describe the risk factors associated with the severity of COVID-19. This study uses the article review method from research results obtained through searching the journal sites Google Scholar, PubMed, ProQuest and ScientDirect in the 2020–2021 period. To find articles that match the inclusion criteria we used the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) strategy. A total of nine studies met the inclusion criteria for this review. These nine studies were assessed for quality, data extraction and synthesis. Risk factors that contribute to the severity of COVID-19 are age, gender, chronic comorbidities, cardiovascular disease, diabetes, hypertension, kidney failure, cancer and a history of smoking. New findings, unvaccinated patients are at higher risk of severity. Risk factors associated with the severity of COVID-19 include a person’s individual characteristics, co-morbidities, smoking history and unvaccinated. Today the world is shocked by a new type of pneumonia which the World Health Organization (WHO) named severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) with coronavirus disease 2019 (COVID-19) as the disease. Although the pandemic has been running for more than 2 years, the new cases of COVID-19 are still not over. Based on global distribution data by WHO, the update on 8 January 2022, from 204 affected countries has reached 281,638,005 confirmed positive people and 5,152,602 people died. Meanwhile, according to the Indonesian COVID-19 Handling Task Force, data reached 4,265,666 positive people, 4,115,747 people recovered and 144,127 people died. From this very large number, it indicates that today’s pandemic is still very worrying, especially with so many variants appearing.Various variants of the COVID-19 virus that have mutated such as the Alpha, Beta, Gamma and Delta variants have made this pandemic not end soon  the IndoGiven the increasing number of COVID-19 cases and the discovery of new variants, understanding the risk factors associated with the susceptibility and severity of COVID-19 becomes very important for disease control.In this article review following the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) .In this article review, the search for articles uses the search engines Google Scholar, PubMed, ProQuest and ScientDirect. Articles published in March 2020 to December 2021. When conducting searches, researchers used the keywords ‘risk factor’, OR ‘mortality’, OR ‘comorbidity’, OR ‘severe’ OR ‘COVID-19’. Articles used are national and international in Indonesian or English.The inclusion criteria in this study are: i) articles whose research respondents are confirmed COVID-19 patients; ii) the results of the article on the analysis of risk factors associated with the severity of COVID-19; iii) using a quasi-experimental study design or a systematic review or a randomised control or trial, a qualitative study or a cross-sectional study; iv) published in 2020 or 2021 and v) English.Study selection following the PRISMA guidelines . An artiA total of nine studies were selected and reviewed based on inclusion criteria. The guideline used for this article review is PRISMA as shown in Of the nine studies that were used as article reviews, six of them were conducted in Asia and three was conducted in Europe, namely from London, USA, China and Malaysia. The number of samples in each article varies between 174 and 1,456,462 respondents. The studies included confirmed positive COVID-19 patients with adult age 18 years old in five studies and included all ages in four studies. The total number of respondents in the article review is 1,986,871 respondents.The results of an article review show that the risk factor associated with the severity of COVID-19 patients is old age, but based on new findings, unvaccinated patients are at higher risk of severe. Male sex is also a contributing factor, as shown in Based on research journals  it showsArticle reviews of eight research journals show seven of them are related to the severity of COVID-19 , 4. AccoHowever, based on data from COVID-19.go.id, the findings of June 2021 currently show that children are at an age that is vulnerable to get infected with COVID-19 with a percentage of 12.6%, meaning one in eight children is infected. Positive cases of COVID-19 children aged 1 year old–5 years old were 2.9%, while school-age/adolescents aged 6 years old–18 years old were 9.7%. The mortality rate for children aged 1 year old–5 years old was 0.6%, aged 6 years old – 18 years was 0.6%. According to IDAI (Indonesian Pediatric Society) in  is a comorbid disease with the greatest risk according to the odds ratio in the seventh research journal with an OR of 14.06 . This shPeople who suffer from cardiovascular disease will be treated with drugs that contain ACE2 and angiotensin receptor blockers (ARBs) to protect the lungs. Meanwhile, SARS-CoV2 or coronavirus uses the ACE2 protein to enter cells. ACE2 is a membrane protein that has a physiological function, which is the protection of the lung, however, it becomes the entry point for viruses into the body that will cause cardiovascular toxicity. Several cardiovascular complications after being infected with COVID-19 include arrhythmia, myocarditis, acute coronary syndrome, venous thromboembolism, cardiogenic shock and heart failure .P < 0.0001.In a cohort study  involvinP < 0.0001  of 3.68–1.82, meaning that patients with diabetes have 2.68–1.82 times greater risk of worsening the condition of COVID-19 if they are infected than those who do not . This is< 0.0001 .People with diabetes mellitus with COVID-19 will increase the secretion of hyperglycemic hormones such as catecholamines and glucocorticoids by producing glucose elevations in the blood, abnormal glucose variability and complications of diabetes   .From a study of eight journals, there is one journal that provides data that chronic kidney failure has a risk factor for the severity of COVID-19 patients. Supported by an article  which stIn the article study of nine journals with the relation between cancer comorbidities and the severity of COVID-19 patients, the third research journal, data show that patients with cancer are more likely to have severe COVID-19 than patients without cancer. This is in line with data  that colBased on the journals that we referenced, cancer is not an independent risk factor for the severity of patients with COVID-19 as well as if the patient underwent radiotherapy. Here’s the explanation. According to research  cancer pP = 0.03). This study also found a higher rate of admission in ICU  and more severe symptoms . In subgroup analyses, patients with lung cancer or haematological malignancies and patients receiving immunotherapy, chemotherapy or undergoingsurgery presented an excess risk of severe forms of COVID-19. However, no statistically significant relationship with severe forms of COVID-19 was observed in patients treated by radiotherapy.Supported by retrospective study  based onConfirmed by retrospective cohort study  almost 2The smoking history factor is found in the first, fourth and eighth research journals. This is in line with the study  which stI 2 = 30.1%), USA  and other areas . Subgroup analysis based on median age groups showed a statistically significant association among those whose age ≥ 40 years old ( P < 0.001) but not in the age group of < 40 years old.According to research journals  the poolBased on research  smoking According to Dr. James McCarthy , the hosIndividual characteristic factors, among others, older age > 60 years old and male sex have a greater risk of severity compared to productive age and female sex. Meanwhile, the new finding is that children aged 18 years old have continuous symptoms (old COVID-19) if the child is confirmed positive. In addition, several comorbidities that can increase the severity of COVID-19 patients are COPD, cardiovascular disease, diabetes mellitus, hypertension, chronic kidney failure, cancer and a history of smoking. Recent findings that play a role are patients not vaccinated.


Optic neuritis, a characteristic feature of multiple sclerosis (MS), involves the inflammation of the optic nerve and the degeneration of retinal ganglion cells (RGCs). Although previous studies suggest that retinal blood flow alterations occur during optic neuritis, the precise location, the degree of impairment, and the underlying mechanisms remain unclear. In this study, we utilized two emerging non-invasive imaging techniques, laser speckle flowgraphy (LSFG) and optical coherence tomography angiography (OCTA), to investigate retinal vascular changes in a mouse model of MS, known as experimental autoimmune encephalomyelitis (EAE). We associated these changes with leukostasis, RGC injury, and the overall progression of EAE. LSFG imaging revealed a progressive reduction in retinal blood flow velocity and increased vascular resistance near the optic nerve head in the EAE model, indicating impaired ocular blood flow. OCTA imaging demonstrated significant decreases in vessel density, number of junctions, and total vessel length in the intermediate and deep capillary plexus of the EAE mice. Furthermore, our analysis of leukostasis revealed a significant increase in adherent leukocytes in the retinal vasculature of the EAE mice, suggesting the occurrence of vascular inflammation in the early development of EAE pathology. The abovechanges preceded or were accompanied by the characteristic hallmarks of optic neuritis, such as RGC loss and reduced visual acuity. Overall, our study sheds light on the intricate relationship between retinal vascular alterations and the progression of optic neuritis as well as MS clinical score. It also highlights the potential for the development of image-based biomarkers for the diagnosis and monitoring of optic neuritis as well as MS, particularly in response to emerging treatments. Multiple sclerosis (MS) is a progressive autoimmune disorder marked by the demyelination of the central nervous system (CNS). It primarily presents in young adulthood and is estimated to affect 2.8 million people worldwide . MS seveON is characterized by inflammation of the optic nerve . It was In addition to the use of OCTA for the non-invasive monitoring and quantification of MS-induced alterations in retinal vascular features, laser speckle flowgraphy (LSFG) has emerged as a valuable non-invasive tool to monitor changes in retinal blood flow under various pathological conditions ,16,17,18The current understanding of retinal vascular changes in animal models accompanying ON is limited. To address this, we conducted a longitudinal evaluation of retinal vascular alterations in the mouse model of experimental autoimmune encephalomyelitis (EAE), a well-established model for studying ON . UtiliziIn our current pre-clinical study, which received approval from the Institutional Animal Care and Use Committee at the University of Texas Medical Branch, we utilized C57BL/6J wild type (WT) mice (Stock No: 000664) and C57BL/6-EGFP (Stock No: 006567) from the Jackson Laboratory . The mice were bred in the animal care facility at the University of Texas Medical Branch. All of the experimental procedures and animal usage strictly adhered to the guidelines provided by the Association for Research in Vision and Ophthalmology Statement for the Use of Animals in Ophthalmic and Vision Research.n = 7) and control (n = 5) mice was obtained at 0, 7, 10, 14, 21, and 28 days post-immunization (dpi), in accordance with the manual of the MOG 35-55/CFA EMULSION PTX kit and based on changes in the motor functions of the tail, legs, and neck. Rapid increases in the clinical score usually occur between 12 and 18 dpi [We chose to exclusively use female mice for this study, because C57BL/6J males do not display severe clinical symptoms and reach the maximum clinical score . At 13 w7 cells) into WT recipient mice via tail vein. After 4 weeks, the EAE model was induced in WT recipient mice. Right before or 7 days after EAE induction, SLO imaging was performed. Briefly, the mice were injected intraperitoneally with 100 mg/kg ketamine and 10 mg/kg xylazine for anesthesia, and their eyes were dilated with topical tropicamide and phenylephrine. The mice were then placed on a heated platform, and the Heidelberg Spectralis HRA system  was used to record in vivo movement of GFP-tagged leukocytes in the retinas. The frame rate was set to 5 frames/second with a 55° field of view, and each image consisted of 100 frames. Leukocytes were counted for each retina .BM transplantation was performed as previously described ,23. Brien = 6 Control, n = 6 EAE) were dissected for the quantification of the total number of adherent leukocytes.Retinal leukostasis was performed as previously described ,24,25. In = 9–10 Control, n = 14–16 EAE). Prior to LSFG imaging, the anesthesia of the mice was achieved with an intraperitoneal injection, consisting of 100 mg/kg ketamine and 10 mg/kg xylazine. The eyes were dilated with one drop of phenylephrine and one drop of tropicamide. GenTeal® Tears Eye Gel  was lightly applied to the mouse corneas, a circular glass slide was placed on top of the gel, and the mice were then positioned on the LSFG imaging platform. Images were acquired with the optic nerve head (ONH) in the center over a 4 s (118 frames) period at a rate of 30 frames per second to produce a 3.2 × 2.5 mm2 composite area. At each timepoint, each eye was measured three consecutive times without changing positions between each scan. The average of these values was used for subsequent statistical analyses.The retinas of the EAE and control mice were imaged using an LSFG-Micro System  at 7, 10, 14, and 28 dpi   . Recorden = 14 Control, n = 16 EAE) at 14 dpi. Prior to imaging, the anesthesia of the mice was achieved with 3.8% isoflurane using a Digital Low-Flow Anesthesia System , and phenylephrine and tropicamide were administered to their eyes to induce mydriasis. Next, the mice were wrapped in cotton gauze to inhibit their movement and maintain their body temperature. They were subsequently placed on a heating pad on the imaging platform with a continuous supply of isoflurane. GenTeal® Tears Eye Gel was applied to the eye that was not actively being imaged to prevent corneal clouding. OCTA images were acquired using the Heidelberg HRA + OCT Spectralis , as previously described [OCTA scans were performed for each retina  ,27. The n = 9 Control, n = 14, EAE) was measured based on an innate visual–motor reflex using a plexiglass testing chamber and the software OptoMotry [The visual acuity of the mice  were mounted, and images were captured using confocal microscopy .At 28 dpi, the mouse eyeballs were fixed in 4% PFA at 4 °C overnight. Following dissection, the retinas were washed with PBS, blocked, and then permeabilized, using PBS that contained 5% donkey serum and 0.3% Triton-X-100 , for a period of 3 h. Next, they were incubated with an antibody against RBPMS , a marker for RGCs, at 4 °C overnight. The next day, the retinas were incubated with an Alexa Fluor 594-conjugated secondary antibody  at 4 °C for 4 h. In the end, the retinas . The alpha value was designated at 0.05, such that a p < 0.05 was considered statistically different.The mean and the standard error of the mean (SEM) for both groups were reported. To compare the EAE and control mice, the student’s First, we sought to establish the EAE model in the mice. After disease induction, the mice were monitored for changes in body weight and EAE clinical scores, which were evaluated based on the appearance of clinical signs of EAE at 0, 7, 10, 14, 21, and 28 dpi A,B. WhilOCTA is a dye-free, non-invasive, and cost-effective imaging modality that generates high resolution images of the retinal vasculature. Currently, it is widely used in clinical settings to examine vascular alterations in retinopathies. OCTA distinguishes vasculature from background tissue based on the movement of red blood cells, thus only detecting vasculature that is both structurally present and functionally intact ,30. At 1To further characterize retinal vascular alterations in EAE, we utilized the LSFG-Micro System, another non-invasive and dye-free imaging technology, to quantitatively assess ocular blood flow near the optic nerve head . The maiIn addition to the MBR, waveform analysis was also conducted to assess the BS, BOM, Max MBR, Min MBR, and the Max–Min MBR, as described in Finally, the total retinal flow index divided by vessel width (TRFI/w) was calculated as the retinal blood flow volume in the top four highest velocity arteries C. A trenVisual acuity assessment is one method used to monitor disease progression and assess functional impairment in MS patients ,32,33. TTo understand the potential mechanisms underlying reduced blood flow in EAE, we focused on vascular inflammation, as extensive leukocyte recruitment with subsequent adhesion to the vascular endothelium could block blood flow ,35. AddiUsing non-invasive imaging modality, LSFG, we provide evidence that retinal blood flow velocity is significantly reduced in a mouse EAE model, which is frequently used to study the mechanisms of optic neuritis. We found that profoundly impaired vessel perfusion occurs at very early stages in EAE as demonstrated by decreased MBR in the tissue area. Initially, blood perfusion within small retinal vasculature progressively decreased, followed by alterations in large retinal vessels accompanying changes in the clinical score and body weight as indicators of disease progression. While there are no other studies that have used LSFG to characterize retinal blood flow velocity in EAE, our results are in line with animal studies that demonstrate hypoxia and hypoperfusion of the inflamed spinal cord occur during EAE ,38,39. CThe mechanism underlying this pathognomonic decrease in blood flow velocity has yet to be understood, but it is widely known that inflammation could cause reduced blood flow, vascular blockage, and vasoconstriction . We obse+/K+-ATPase, promotes the extrusion of Na+ into the extracellular space, following electrical stimulation under physiologic circumstances. In cases of ATP depletion, the excessive accumulation of intracellular sodium from a loss of the Na+/K+-ATPase promotes sodium–calcium exchanger activity, resulting in calcium excitotoxicity with subsequent cell death [Our results also suggest that vascular impairment occurs prior to RGC dysfunction, as evidenced by a significant decrease in MT beginning at 7 dpi vs. visual acuity decline beginning at 10 dpi. It is still unclear whether vascular alterations could precipitate or contribute to retinal neurodegeneration in optic neuritis in MS. Nonetheless, significant research points towards vascular hypoperfusion playing a role in neuronal compromise through a hypoxia-induced mechanism. A decreased availability of oxygen leads to the decreased mitochondrial consumption of oxygen, with a subsequent depletion of ATP and loss of ATP-dependent metabolic processes ,54. One ll death . This hall death , lamotrill death , and carll death  to treatll death . Anotherll death . These sAs the number one cause of non-traumatic disabling disease in young adults , it is iIn summary, we provide original evidence that the retina undergoes significant hypoperfusion during the course of optic neuritis in a mouse EAE model. These pathologic alterations in the retina of the EAE mice clears contradictions in clinical studies and supports clinical studies showing decreased retinal vessel density in optic neuritis, similar to vascular changes observed in the brain and spinal cord of MS patients ,66,67,68


Mental health problems, behavior changes, and addictive issues have been consistently documented among healthcare workers during the pandemic. The objective of this study was to investigate the levels of anger and aggression in relation to psychological resilience and alcohol abuse among healthcare workers during the first wave of the COVID-19 pandemic. A total of 120 physicians and 123 nurses completed an online survey of the Dimensions of Anger Reactions-5 (DAR-5), the Brief Aggression Questionnaire (BAQ), the Brief Resilience Scale (BRS), and the Alcohol Screening questionnaire CAGE which is an acronym for the focus of the questions . Demographic and professional data were also recorded. A total of 53 men and 190 women participated in the study. Almost one-third of the participants had a positive score on the DAR-5 scale and one out of ten respondents presented with current problematic alcohol use. Male participants demonstrated lower scores on the DAR-5 scale compared to females. Individuals with current problematic alcohol use displayed higher scores on the BAQ compared to those without alcohol use disorders. Regression analysis revealed that 16.4% of the variance in the BAQ scores can be attributed to scores on the DAR-5, 5.9% to the BRS scores, 2.1% to the CAGE scores, 1.7% to gender, and 1.2% to years of work experience. Mediation analysis highlighted the role of psychological resilience as a negative mediator in the DAR-5 and BAQ relationship. Professional experience and alcohol abuse emerged as positive and negative risk factors contributing to aggression and psychological resilience. The findings hold practical implications for implementing interventions to strengthen resilience in order to compensate for aggressive tendencies and discourage addictive issues. The COVID-19 pandemic and other previous epidemics have primarily and disproportionately exerted their deleterious effects upon healthcare professionals, who are the first line of defense committed to handling the health emergency situation ,3,4,5. DLiterature suggests that exposure to stress, especially from fatal, catastrophic events, elevates the risk of subsequent alcohol consumption and alcohol use disorders ,11,12. DIn the meantime, symptoms of anger and confrontation have been reported in more than half of participants in a national cross-sectional survey in the United Kingdom during the first pandemic wave . Anger, Hopefully, feelings of anger do not necessarily result in aggressive behavior. Aggression as a concept refers to a pattern of hostile, harmful, or destructive behaviors, usually meant to inflict deleterious repercussions. Recent aggression models, such as the General Aggression Model, argue that aggression is derived from combined biological, situational, and individual factors, in particular personal traits, prior life experiences and environmental inputs . WorkplaStudies conducted among healthcare workers, after the onset of the COVID-19 pandemic, evidenced an escalation in alcohol consumption, presumably as a result of maladaptive attempts to relieve stress and overcome the increased psychological difficulties resulting from the pandemic ,47,48. PPsychological resilience is expected to provide protection in traumatic and distressing situations and earlier research supported that resilience has been proven to effectively protect healthcare staff from psychological distress, anxiety, depression, and burnout ,60. DuriThe effect of resilience in the relation between occupational stress and anger in first responder populations in emergency situations has already been studied and as suggested in previous research resilience to challenging workplace situations is negatively related with state and trait anger ,65. AlsoStudies confirm significant negative associations between aggression and psychological resilience and argue that spirituality and self-control may have a positive impact on reducing aggression ,72,73. FThe conceptual framework for our study was that enhanced psychological resilience may effectively mitigate anger and counteract aggression, even in adverse circumstances, but the question is if its protective role is compromised by certain risk factors such as alcohol abuse. The above hypothetical question underlies the main purpose of the study which is to have the opportunity to concurrently investigate the protective role of psychological resilience and the impact of alcohol abuse on aggression and their possible emerging interactions. Secondary objectives of the present study are to:1. Evaluate the levels of anger, aggression, psychological resilience, and alcohol abuse among healthcare professionals during the first wave of the COVID-19 pandemic. 2. Explore for other demographic and work-related factors potentially contributing to aggression and psychological resilience. The scientific significance of the study becomes apparent considering the limited research on healthcare workers’ aggression towards others in conjunction with the investigation of potentially modifiable risk and protective factors such as alcohol abuse and psychological resilience. This was a cross-sectional correlational design study, conducted from 15 June to 30 June 2020, through web-based self-report surveys administered via email. The authors designed the survey draft, and its final version was approved by consensus. The email addresses of the participants were retrieved through links to the websites of Greek healthcare workers from their scientific and professional associations. The first page of the electronic survey provided information about its scientific purpose and its voluntary nature. Also, the first page of the electronic questionnaire included a consent form to participate where we ensured that participants gave online informed consent by responding to the question: “Do you agree to participate in this study?” which was the first question in the online questionnaire. Participants with positive responses could then participate in our study. The study sample consisted of medical and nursing staff who agreed to respond to the email as a convenience sample and no measures were taken to increase the response rate, apart from the reassurance of data privacy. Τhe invitation was sent as an electronic message also contained an anonymous link allowing access to the online survey platform. The study protocol has been approved by the Clinical Research Ethics Committee of the “Sotiria” General Hospital (Number 12253/7-5-20). This study was conducted in accordance with the ethical principles as defined by the Declaration of Helsinki, the International Committee of Medical Journal Editors, and the General Data Protection Regulation (GDPR-2016/679) of the European Union.The study was conducted during the gradual de-escalation of the restrictive measures implemented by the Greek authorities. The lockdown in Greece was imposed from 23 March until 4 May 2020 and was characterized as one of the most restrictive in Europe, attempting to control the infection and the mortality rate. In a pilot study of ours , during A total of 150 doctors and 250 nurses were invited to participate in the study, 243 of whom (120 doctors and 123 nurses) responded to the invitation. Sample adequacy was calculated using the G-Power Version 3.1 software . With a Participants were invited, before completing the questionnaires, to state their demographic and professional data including age, gender, professional status and working experience in years.The Dimensions of Anger Reactions-5 (DAR-5) is a 5-item self-administered scale designed to measure the experience of anger over the past four weeks, by answering on a 5-point Likert scale grading from 1  to 5 . Total scores vary from 5 to 25, with higher scores reflecting more anger experiences. A cut-off value of 12 classifies respondents as high or low scorers . The DARThe Brief Aggression Questionnaire (BAQ) is a 12 item self-report measure of trait aggression. Participants are prompt to rate on a scale from 1 (strongly agree) to 5 (strongly disagree), the extent to which statements describing behaviors and emotions, are characteristic of themselves. The BAQ measures aggression on the dimensions of physical aggression, verbal aggression, anger, and hostility. The BAQ has been postulated as a valid and reliable tool, with good test–retest reliability and convergent validity with other behavioral measures of aggression ,93,94. CThe Brief Resilience Scale (BRS) is a 6-item self-report measure of resilience, reflecting the competence to overcome stress and adversity. Responses are rated on a 5-point Likert scale from 1 (Strongly Disagree) to 5 (Strongly Agree), but half of the items are reversed scored. Scores range from 6–30, with higher scores suggesting a higher level of resilience. The total score needs to be divided by the total number of questions answered and scores between 1.00 and 2.99 represent low resilience, between 3.00 and 4.30 normal resilience and between 4.31 and 5.00 high resilience. The original BRS demonstrated good levels of internal consistency and test–retest reliability, and adequate factorial, convergent and discriminant validity . BRS wasThe CAGE questionnaire is a brief and popular screening instrument for detecting alcohol abuse and dependence that is easily applied in clinical practice . It const-test and analysis of variance (ANOVA) were employed. The assumption testing for equality of error variances using Levene’s Test was performed before the analyses. Independent sample t-tests assessed for differences in gender and differences between healthcare workers as to their profession and alcohol use. The effect size was calculated with Hedges’ g-value from the results of the independent samples t-test, considering that values less than 0.2 suggest a small effect size, less than 0.5 a medium effect size, and less than 0.8 a large effect size. The results of the ANOVA test were estimated with the eta squared value (ηp2), counting ηp2 = 0.01 as a small effect size, ηp2 = 0.06 as a medium effect size, and ηp2 ≥ 0.14 as a large effect size. To investigate if there is an interaction effect between the two independent variables  in terms of a continuous dependent variable (aggression), whilst adjusting for continuous covariates that are thought to influence this interaction effect, a two-way analysis of covariance (ANCOVA) was employed. The assumption testing for a two-way ANCOVA  was carried out before the analysis. Pearson correlation was conducted to assess the direction and strength of the relationship between variables and the values of the correlation coefficients were interpreted as weak from ±0.10 to ±0.29, moderate from ±0.30 to ±0.49, and strong from ±0.50 to ±1.00. Linear regression models were built to explore if the related variables were significant predictors of the dependent variable, aggression. The assumption testing for regression analysis  was carried out performing a graphical review of the variables, residuals and collinearity statistics, probability–probability (PP) plots, and scatterplots. A simple mediation analysis using the Hayes SPSS Process Macro was performed to investigate the role of resilience in the relationship between anger and aggression, setting anger as the predictor variable, aggression as the outcome variable and resilience as the mediator variable for the analysis. Statistical significance level was set at p < 0.05 and p values were two-tailed. Analyses were performed using IBM SPSS Statistics 23 . Mediation analysis was conducted using the Hayes SPSS Process Macro [All variables in the analysis were computed with descriptive statistics. To enable statistical analysis of the data and to make comparisons easier, some independent variables were grouped. The variable alcohol abuse/dependence was recoded as two categories for the associations: low-risk alcohol abuse when scores were below two on the CAGE questionnaire and risky alcohol abuse when scores were above or equal to two. Likewise, the resilience variable was also recoded into three categories, for the same purpose: low, moderate, and high levels of resilience. The Fisher’s exact test was utilized to establish the differences between qualitative data. In order to assess differences between variables, Student’s ss Macro . IBM SPSp = 0.164), 58.4% as normal , and 23.5% as high resilient , concluding that gender differences in levels of resilience did not reach statistical significance.The study included a total of 243 participants (53 men and 190 women). As to the profession, 120 of them belonged to the medical and 123 to the nursing staff. Means and standard deviations for general characteristics of participants and all outcome variables as to gender are presented in t-test p < 0.05, Hedges’ g: 0.30), , , (p > 0.05).Female participants evidenced higher scores on the DAR-5 scale compared with their male counterparts , . Also, f: 0.43), , whereas: 0.66), . Levene’p < 0.05, Hedges’ g: 0.66 and 5.83 ± 2.74 versus 4.24 ± 1.95, p < 0.01, Hedges’ g: 0.78), (t-test p < 0.01). Levene’s test showed that the variances were equal (p > 0.05).Healthcare workers with alcohol use disorders scored higher on the BAQ scale and its physical aggression subscale compared to participants without alcohol abuse , . As to pF = 5.296, p < 0.01, ηp2= 0.042), the BAQ scores  = 4.892, p < 0.001, ηp2 = 0.068), and on working experience  = 5.296, p = 0.012, ηp2 = 0.036). A Tukey post hoc test identified statistically significant differences among normal  and high resilient groups , compared to the low resilience group (11.59 ± 3.526) as to the DAR-5 scores, among normal  and high resilience groups , compared to the low resilience group (27.39 ± 7.434) as to the BAQ scores and among normal  and high resilience groups , compared to the low resilience group (10.93 ± 8.689) as to working experience. There were no statistically significant differences among the normal and high resilience groups (p = 0.862) as for the DAR-5 scores, the BAQ scores (p = 0.878), and working experience (p = 0.596). Levene’s test showed that the variances were equal (p > 0.05).Meanwhile, participants’ level of resilience revealed significant differences, as determined by one-way ANOVA, the DAR-5 scores  = 0.015, p = 0.985, partial η2 = 0.010,  = 0.891, p = 0.412, partial η2 =0.008), , .p < 0.001) and negative between BRS scores with both DAR-5 and BAQ scores (p < 0.001). Age and work experience correlated positively with BRS scores (p < 0.001) and negatively with BAQ scores (p < 0.05). Scores on the CAGE questionnaire correlated positively with the BAQ scores (p < 0.05), . Scores on the hostility subscale of the BAQ were not related to CAGE scores (p > 0.05). Also, significant negative correlations were revealed among BRS and three out of the four BAQ subscales; hostility, verbal aggression, and anger . Scores on the physical aggression subscale of the BAQ were not related to BRS scores (p > 0.05). Significant positive correlations were identified between DAR-5 and BAQ scores (< 0.05),  and threp = 0.235). The regression analysis revealed that ‘scores on DAR-5′, ‘scores on BRS’, and ‘scores on CAGE’, gender and working experience were all significant predictors of ‘scores on the BAQ’, each explaining 16.4%, 5.9%, 2.1%, 1.7%, and 1.2% of the variance  from the general characteristics of participants , CAGE, DAR-5, and the BRS scores (independent variables). The assumptions of multiple regression analysis were satisfied. The independence of the residuals was checked with the Durbin–Watson test value being 2.291 . The absvariance . Particip < 0.001). Hence, resilience partially mediated the relationship between anger and aggression and the model explains 13.55% of the variance in the outcome variable aggression . Furthermore, the direct effect of anger on aggression in the presence of the mediator resilience was also found significant (b = 0.7211, gression . AdditioDuring the first pandemic wave in this study one third of healthcare workers expressed high anger emotions, primarily prevalent in the female population and one out of ten respondents exhibited problematic alcohol use. Gender-specific types of aggression were identified with males displaying more physical aggression and females preferably reporting anger and hostility. Participants with current problematic alcohol use scored higher in physical and total aggression compared to those without alcohol use disorders. Psychological resilience presented negative associations with both anger and aggression and partially mediated the relationship between anger and aggression. Professional experience and alcohol abuse emerged as positive and negative risk factors contributing to aggression and psychological resilience.Negative associations were identified between psychological resilience with the three of the aggression subscales, namely hostility, anger, and verbal aggression, which implies that increasing the level of resilience reduces the level of aggression. Among the three correlated with resilience dimensions of aggression, hostility had the strongest negative association with respect to the rest of the other subscales; results that are also supported by other research studies ,102. As expected, alcohol abuse presented positive associations with aggression, having the strongest correlation with physical aggression, but the only domain among the four aggressive domains that did not correlate with alcohol abuse was hostility, the cognitive domain of aggression. In our sample psychological resilience and alcohol abuse operate in different ways, primarily affecting distinct aggression domains, and no interaction was identified between these two constructs in terms of aggression. The fact that alcohol abuse impairs cognitive functions, specifically executive functions, information processing, and attentional control, among other individual differences, leading to alcohol-related aggression while psychological resilience requires integrity of self-regulation processes and intact prefrontal cortical control to achieve its salutary benefits, by taming aggressive urges, justifies the aforementioned results ,104,105.Research suggests that work-related variables, like professional experience, is positively associated with resilience and studies evidence that the less experienced nurses have lower resilience compared with the higher experienced ones ,106,107.Work experience, among other factors, predicted workplace aggression in healthcare workers attributed to the fact that the more experienced healthcare workers have eventually acquired skills enabling them to foresee, appraise, and resolve conflicts in a more competent way than their less experienced counterparts ,116,117.Workplace aggression in healthcare is an immerging growing phenomenon as if the pandemic kindled an outrage against healthcare professionals. Previous research argued that being exposed to workplace aggression undoubtedly evokes emotional responses like anger, despair, or fear and the more harm-causing incidents of workplace aggression have a higher impact on female employees . A revieAnother alarming trend is problematic alcohol use among physicians, which appears to have been increasing during the pandemic ,127. A rIt has been argued that the pandemic created the optimal setting for testing psychological resilience in the presence of worldwide challenges heavily impacting mental health. A recent review examining resilience among frontline healthcare workers during the pandemic evidenced modest resilience scores . AccordiResilience has an important role in the resolution of psychological difficulties such as aggression, depression, or anxiety, caused by early adversities and allows people to deal with emotional and behavioral reactions and, therefore, reduces the likelihood for displaying aggressive behaviors when confronted with any distressing situation . Levels Studies highlight that people who are inclined to risk-taking behaviors, such as alcohol and substance use, aggressive, and other disruptive behaviors, have maladaptive coping styles and are not capable of managing life stressors with resilience . In otheLikewise, investigating the association between resilience and various psychosocial factors, it was reported that resilience has a negative correlation with anger expression and was also implicated in emotion regulation and impulse control ,142. ThiIn our sample, male respondents demonstrated higher levels of self-reported physical aggression compared with females and female participants exhibited higher scores in self-reported levels of anger and hostility, in agreement with other research . There iLiterature suggests that there are differences in aggression according to gender, with men displaying direct and women indirect aggression . BiologiUpon provocation, the gender effect on aggression may be blunted. Angry rumination triggered by interpersonal provocation reduces self-control and raises aggression . Angry rThrough diverse proposed mechanisms, mindfulness may counteract angry rumination and decrease aggression, anger, and hostility . Left unAnother example of psychotherapeutic interventions, from a cognitive–behavioral approach, suggests that stress-related mental disorders like depression, anxiety disorder, and substance abuse, can be viewed as the outcome of dysfunctional thinking patterns. When people are exposed to stressful situations, they often exhibit maladaptive behavioral reactions and suffer from negative emotions because of dysfunctional cognitions. This is consistent with other theories of stress and resilience, suggesting that it is the way the stressor is perceived that drives stress reactions. Consequently, modifying cognitive processes to more adaptive thought patterns is likely to adjust both behavioral and emotional responses to stress. Questioning maladaptive thinking patterns and introducing novel strategies eventually promotes the resilience factors of cognitive flexibility and adaptive coping . Cognitive–behavioral models of addiction and relapse treatment emphasize the role of adverse emotional experiences as determinants of alcohol use and relapse, and research suggests that being able to communicate negative emotional states, a feature of psychological resilience, prevents alcohol use disorders . ResiliePrimary, secondary, and tertiary are the three levels of addressing healthcare workers’ resilience; with primary interventions targeting coping strategies and facilitating communication skills, while secondary interventions screening for burnout and taking care of those at risk should be implemented . FinallyAnother critical issue to attend to is workplace aggression. Occupational protocols and administrative strategies are needed that target safety issues, monitoring, and referral of events, as well as workforce training on the prevention and management of workplace aggression . It is sProviding healthcare workers with appropriate training to improve self-control in stressful situations and upgrade their social competencies will diminish aggressive incidents, while enabling feedback and support from their supervisors will increase their sense of security and boost their self-confidence ,181. MeaFor alcohol use disorders among healthcare workers, it is important that both diagnostic tests and therapeutic interventions are implemented as an appropriate approach to this important health problem . These pFinally, the workplace should be monitored for stress problems on an ongoing basis, in order to early detect and address these problems, but also to promote a healthy work environment and reduce the harmful aspects of work . The resIn our study, we examined the role of psychological resilience on anger and aggression in relation to alcohol abuse among healthcare professionals, but apart from psychological resilience, there are other identified protective factors such as family and social support, along with negative risk factors such as anxiety, depression, and burnout prevalent among healthcare professionals, particularly among those allocated on the front line of the pandemic. To draw effective individualized psychological interventions all these factors, not taken into account in our study, should be considered. Other limitations in the present study include the limited sample size which impedes the drawing of safe epidemiological conclusions; the convenience sampling method possibly reflecting selection bias; the gender disproportionality of participants, which may have affected the generalizability of the results; employing self-administered questionnaires which raises concerns about the objectivity of the estimates; the cross-sectional design of the study which precludes causal inferences and online data collection excluding healthcare professionals without internet access and further influencing the generalizability of the results. The present study confirmed the protective role of psychological resilience neutralizing anger experiences and averting aggressive tendencies among healthcare professionals during the first pandemic wave. Further, professional experience and alcohol abuse were identified as positive and negative risk factors, correspondingly, impacting aggression and psychological resilience and explaining part of the variability in aggression. Implementing interventions to address workplace aggression and alcohol use disorders among healthcare workers and enhance resilience to counteract the harmful aspects of work is of outmost importance.


Hypertension and type 2 diabetes (T2D) are the most prevalent noncommunicable diseases in Mexico and worldwide. According to international practice management guidelines, the principal chronic management therapy is daily oral medication.  We aim to describe the trends of antihypertensive, antidiabetic, and nonsteroidal anti-inflammatory (NSAID) drugs use among the Mexican adult population from 2004–2018.  We analyzed data from the Health Workers Cohort Study (HWCS) for males and females aged >18 years. We calculated the prevalence of chronic diseases and utilization for every kind of antihypertensive, antidiabetic, and NSAIDs (measured by self-reported utilization) at baseline and two follow-ups . Trends were analyzed using Fisher's exact test.  Hypertension prevalence increased from 19.8 to 30.3%, higher than T2D prevalence from 7.0 to 12.8% through fourteen years of follow-up. Like the self-reported dual therapy, the proportion of patients using beta-blockers and angiotensin II receptor blockers increased. Regarding T2D, the prevalence of metformin utilization increased to 83.9%. The utilization of common NSAIDs, mainly for muscular pain, remained around 13 to 16%.  Our findings showed a changing prevalence of drug utilization for hypertension and T2D between 2004 and 2018 and consistent use of NSAIDs in the adult Mexican population. Chronic diseases in adults such as hypertension , type 2 Mexican current clinical practice guidelines agree with international guidelines for hypertension  and recoHowever, since the mid-2000s, blood pressure control and glycemic management treatment have considerably changed in medical institutions around the world –9, partiFurthermore, half of the adult population reported having suffered a musculoskeletal condition in recent years , besidesGiven these changes in the pharmacotherapy approaches, it is essential to examine the temporal trends to provide information about care patterns, drug disuse described as noneffective, frequent side effects caused or determinant of chronic health status in response to changes in drug utilization, and identify potential improvement areas, like the adoption of recent specific drug indications adequate to each patient on daily practice medicine for avoiding polypharmacy in adults.We aimed to investigate prevalence trends in the use of antihypertensive and antidiabetic drugs in diagnosed participants and NSAIDs in all adult participants using data from the Health Workers Cohort Study.We used data from the Health Workers Cohort Study (HWCS) . The HWCWe analyzed data from the three assessments of the HWCS participants aged ≥18 years and older; from the baseline assessment, we included 9,522 adult participants; the second assessment of the cohort included 2,075 participants, and the third assessment included 1,299 participants.The participants completed an extensive self-administered questionnaire in the presence of a health expert for clarification, providing detailed information about their demographics, health status, and lifestyle. Available data included the birth date, education, marital and employment status, family medical history, prior chronic illness diagnoses, medication regular utilization, diet, physical activity, smoking and alcohol consumption, social support, and quality of life , 21. ParThe commonly prescribed medications used to treat hypertension and T2D regularly were the exposure to interest. The questionnaire applied to the entire sample (same questionnaire in the three assessments) included a single-item question aiming to evaluate the common drug utilization prevalence, where the question was: “Which drugs do you take? ” and the response options included none, acetaminophen, NSAIDs, thiazide diuretics, beta-blockers, calcium channel blockers, and others antihypertensive drugs, acetylsalicylic acid, hypolipemic, hormonal, anxiolytics, antidiabetic (metformin), antidepressants drugs, among others. Antihypertensive drugs were classified into five major categories: ACE-I , ARBs , CCB , TD , and beta-blockers , the example drugs in parenthesis were the most specific drug used for each category. We only had metformin, glibenclamide, and sitagliptin information regarding antidiabetic drugs.2) was calculated as weight in kilograms divided by the square of height in meters.After answering the questionnaire, participants were scheduled for an appointment within three months to take anthropometric and laboratory measurements. Body weight (kg) was measured with calibrated electronic scales (Tanita BC-533®) with a precision of 0.2 kg, with the participant wearing minimum clothing and no shoes. Height (mt) was measured using a conventional stadiometer (SECA 213®) with a precision of 1 mm, with the participants standing barefoot with their shoulders in a normal position. Body mass index  and medical history, the prevalence of hypertension, T2D, and musculoskeletal disorders, among other comorbidities, for the three assessments, and percentages comparison to baseline assessment were conducted by Fisher's exact test.P value <0.05 was considered statistically significant. Stata 15 and StataCorp LP  statistical package were used for statistical analyses.For the conducted prevalence trends analysis, we included participants with self-reported hypertension diagnosed by a physician or antihypertensive drug use, those with self-reported T2D diagnosed by a physician or antidiabetic drugs, and those with self-reported NSAID use. Over the three assessments, we estimated the prevalence and 95% interval confidence of using antihypertensives, antidiabetics, and NSAID drugs. Comparison to baseline assessment was conducted by Fisher's exact test for each difference in antihypertensive, antidiabetic, and NSAID drug proportions . A two-sp < 0.0001). The prevalence of obesity increased from 19.4% to 26% from 2004 to 2017 (p < 0.0001). The education degree increased, and nurses' participation decreased across the study population. Between comorbidities, only renal chronic disease prevalence remained stable.p < 0.0001), respectively; therefore, the patients with hypertension diagnosis were included in this analysis.From 9,522, 2,975, and 1,299 participants of the HWCS in 2004, 2010, and 2017, respectively, the change in self-reported hypertension prevalence was from 1,882 (19.8%) to 725 (24.4%) and 394 (30.3%)   and then remained stable for the following years (Beta-blockers and ARB II utilization increased during the fourteen years (26.6% to 74.6% ctively) ; meanwhing years . Monotheng years .p < 0.0001), respectively. Monotherapy is the most prevalent treatment election within both diseases' hypertension and T2D .The change in self-reported T2D prevalence was 668 (7.0%), 325 (10.9%), and 167 (12.8%) in participants of the HWCS in 2004, 2010, and 2017 (p < 0.001).The use of any glucose-lowering medication increased by 27.3 (percentage points) from 2004 to 2018 . Until the third assessment data, the introduction of newer agents as sitagliptin (DPP-4 inhibitor) was evident in the institutional treatment , then remained stable until 2017. The regular use of prescription analgesics increased from 2004 (45.6%) to 2017 (49.3%). Of the various analgesic classes, we showed a decrease in acetaminophen use, from 2.9% in 2006 to 2.1% in 2010 (p = 0.01).The overall percentage of people who reported musculoskeletal pain decreased from 2004 to 2010  (p =0.05) and acetaminophen  percentage use from 2004 to 2010 were statistically significant  slightly increased; then from 2010 to 2018, decreased from 17% to 14% in all participants ( =0.006) . The decnificant .Among those regular NSAID users, the prevalence of reported self-medication practice decreased from 13.3% in 2006 to 8.24% in 2018.Our study showed changes in the prevalence trends in utilizing antihypertensive, antidiabetic, and NSAID through 14 years of study follow-up in HWCS adult participants. Independent of their health condition, we indeed observed an increase in common utilization of drugs in participants with a chronic disease.In this study, the punctual prevalence of hypertension in the period study  increased from 20% to 30%, following continuous results from the National Health and Nutrition Surveys of Mexico . The utiThe proportion of hypertensive patients treated with polytherapy was higher in 2013 than in 2006 and 2018 assessments , higher than reported by Chinese (24%) , lower tMoreover, the burden of type 2 diabetes is still growing in Mexico, and the prevalence of T2D has constantly increased from 2000 to 2018, from 7.4% to 17%, with corresponding state-level variance , 32. OurNevertheless, we would like to discuss some crucial aspects of the health systems in Mexico. There are three major health systems: IMSS, which provides health care to 51% of the Mexican population; the Minister of Health; and the Institute of Security of Social Services for Employers . Public healthcare institutions purchase medications for their services and do not charge patients per product or event, but the availability and medication supply have been affected in the last few years –38. AlsoIn Mexico and other countries, the use and prescription of older classes of drugs such as sulfonylureas (glibenclamide specific) as monotherapy remained for decades –42, untiFurthermore, in recent years, we observed that the use of newer second-line glucose-lowering medications, like DPP4-inhibitor (sitagliptin and linagliptin), which were included in 2015 to the national formulary coverage by the health institutional lists, among other classes like SGLT2 inhibitors and GLP-1 receptor analogs , their aOn the other hand, more than 50% of the cohort participants reported some musculoskeletal disorders and pain as the primary symptom, very similar to those shown by a Finnish cohort , accordiThe prescribed NSAIDs increased from 2004 to 2018 in our cohort (45.6% to 49.3%), higher than reported in the US . HoweverFor this study, 14 years of data were available, all collected rigorously and systematically by trained personnel who used standardized protocols. The list of drugs reported by the participants agreed with the national formulary coverage available at the IMSS.Some limitations of this study need to be emphasized; the number of participants enrolled in each assessment decreased because of the focus on worksite available to following and monetary resources; these could lead to selection bias with overrepresentation of participants with some chronic disease diagnosis, limiting the external validity of the prevalence showed; also, we could not correlate drug self-report use with specific physician prescription of medical record history of participants; furthermore, we did not collect information on precise daily dose and medication adherence; drug utilization was self-reported, and patients' health literacy could impact their recognition of the drugs which can lead to underestimation of the drugs' utilization and misclassification. Access to these essential drugs depends on institutional pharmacy supply and entire filled prescriptions. However, these drugs are free to buy in Mexico, and many generic presentations are available.The present study showed that the proportion of participants with hypertension using beta-blockers and ARB II increased. In contrast, TD and CCB utilization decreased during the follow-up time. As shown in other countries, the primary drug used as monotherapy for T2D has wholly changed. The use of NSAID in the study population remained constant across the three assessments without considering comorbidities, polytherapy, and age. In Mexico, given the fractional structure of the health system plus private medical attention, different studies made by some institutions like ours contribute to the knowledge of the pharmacoepidemiology of commonly used drugs for chronic diseases in the Mexican adult population; the integration of all this information could support changes in public policies for monitoring and regulation of drugs utilization.


Van Maldergem syndrome (VMLDS) is a recessive disease which affects multiple organs including the face, ear, and limb extremities. It can be caused by pathogenic variants in either the gene DCHS1 or FAT4. Diagnosis of VMLDS is complicated, especially regarding its similarity of symptoms to Hennekam syndrome, another disorder caused by FAT4 variants. Reported patients are two infantile siblings with multiple congenital anomalies, who deceased without clinical diagnosis. Whole exome sequencing was exploited for expanded carrier screening (ECS) of their parents, which revealed a novel splicing variant in the gene FAT4, NM_024582.6: c.7018+1G>A. In silico analysis of the variant indicates loss of canonical donor splice site of intron 6. This variant is classified as pathogenic based on ACMG criteria. Reverse phenotyping of patients resulted in likely diagnosis of VMLDS2. This study reaffirms the possibility of using ECS, leading to the genetic diagnosis of a rare disease with complicated clinical features. Van Maldergem syndrome (VMLDS/VMS) and Hennekam lymphangiectasia–lymphedema syndrome (HKLLS/HS) are two rare autosomal recessive disorders with highly similar features which were described around three decades ago. Overlapping characteristics of both syndromes include dysmorphic facial features such as epicanthal folds, blepharophimosis, hypertelorism, flat nasal bridge, microstomia besides to microtia, genital involvement, camptodactyly, syndactyly, and developmental delay. However, there are distinctive signs, presented differentially in each syndrome, as lymphangiectasia and lymphedema, which are associated with HKLLS and abnormal findings in brain magnetic resonance imaging (MRI) such as large anterior fontanelles, neonatal feeding, and breath difficulties, which are associated with VMLDS , 2, 3, 4FAT4 gene, also known as FAT–J, encodes a member of the cadherin protein family in humans and its pathogenic variants were revealed as causality in patients with VMLDS or HKLLS [DCHS1 in VMLDS and CCBE1, ADAMTS3 and FBXL7 in HKLLS, have been identified as well. These syndromes have been numbered according to the related causative genes in the OMIM database (https://www.omim.org/), suggesting that VMLDS2 (MIM number: # 615546) and HKLLS2 (MIM number: # 616006) are caused by the gene FAT4 [or HKLLS , 7. VMLDene FAT4 , 9, 10. FAT4 followed by revaluation of phenotypic characteristics of the patients, which showed comparability with VMLDS2. Therefore, this finding was considered as the underlying cause of the disease.Genetic carrier testing, a method for identifying pathogenic variants in asymptomatic heterozygote parents, emerged in 1970 for hemoglobinopathies. To date, owing to the potency of next-generation sequencing (NGS) methods, it has evolved to expanded carrier screening (ECS), encompassing a broad range of diseases with variable number of genes . Here, wFigure 1 shows the pedigree of patients. Regarding evidence indicative to a possible genetic defect, six months after the death of the second child, parents were referred for genetic counseling. Written informed consent form for genetic analysis and publication of clinical and genetic data was obtained from the parents.They were two male siblings with a similar set of malformations and their asymptomatic parents were first cousins. All prenatal screening laboratory tests were normal for both patients and they were born via cesarean section.Proband 1: He was the first child of a healthy 22-year-old mother and a 26-year-old father, with normal prenatal sonography. After his birth, MCA was observed, including tracheomalacia with tachypnea, leading to tracheostomy in the early weeks after birth. He had facial characteristics of prominent eyes, epicanthal folds, micrognathia, microstomia, and bulbous nose together with short neck, low set ears, microtia with aural atresia and conductive hearing impairment, and clubfoot. No more prenatal or neonatal information is available for this patient.Proband 2: He was the mother's second delivery at age 24. Several minor abnormalities were found in his fetal sonographies: Nuchal translucency (NT) was 3 mm at gestational age of 12 6/7 weeks. Due to this finding, chromosome analysis of amniotic fluid was performed at gestational age of 15 1/7 weeks. It showed a normal male karyotype 46, XY, which was confirmed by his peripheral blood karyotyping after birth. Prenatal ultrasound at gestational age of 17 3/7 weeks showed breech presentation, normal fetal abdomen, and echogenic bowel. At gestational age of 21 weeks, in addition to echogenic bowel, lateral ventricles were found to be at the upper limit of normal range (9.6 mm). No bowel obstruction or brain abnormality could be found by ultrasound imaging. His birth weight was 2500 g with an Apgar score of 9. Similar to proband 1, after birth he had anomalies of prominent eyes, epicanthal folds, micrognathia, microstomia, and bulbous nose with short neck, low set ears, microtia with aural atresia, and conductive hearing impairment, but limb abnormality was in the form of camptodactyly. In addition, minor cardiac abnormalities were present, including patent foramen ovale (PFO) and mild physiologic tricuspid regurgitation (TR) still in normal ranges. Ultrasound sonography revealed two corticomedullary cysts in his right kidney.Due to tracheostomy of both of them in the early weeks of life, it is not possible to rule out/in feeding difficulties. The only abnormal finding in laboratory tests of proband 1 was a high level of C-reactive protein (CRP) without evidence of infectious disease. They had respiratory distress and were under ventilator for weeks. Both the patients deceased in the second month of life due to hypoxemia and cardiac arrest.Carrier screening of parents was carried out using WES. In brief, Twist Human Core Exome Plus Kit  was used for exome enrichment and the library was sequenced on Illumina platform . Mean on-target coverages were 131X and 167X for the mother and father, respectively.http://www.1000genomes.org), gnomAD (https://gnomad.broadinstitute.org/), ExAC (data available in gnomAD browser), and our local database Iranome (http://www.iranome.ir/). Its damaging effect on gene product was assessed using online prediction tools MutationTaster (http://www.mutationtaster.org/) and Combined Annotation Dependent Depletion (CADD) (https://cadd.gs.washington.edu/) [https://www.fruitfly.org/seq_tools/splice.html), SD-score (https://www.med.nagoya-u.ac.jp/neurogenetics/SD_Score/sd_score.html) and NetGen2 v2.4 (http://www.cbs.dtu.dk/services/NetGene2/) were applied for further analysis of spliceogenic effects of the variant [In silico pathogenicity assessments: Allele frequency of the variant was investigated in population databases of 1000genome project (on.edu/) , 13. Web variant , 16, 17.This study was in conformity with the Helsinki Declaration and ethical approval was not required for this study in accordance with the local/national guidelines. Written informed consent was obtained from the parent of patients for publication of the details of their medical case.FAT4 which was confirmed in parents by sanger sequencing. This variant substitutes the first nucleotide, G, of intron 6 for A, altering canonical splice site (CSS).WES data analysis revealed a heterozygous intronic variant, NM_024582.6: c.7018+1G>A, in the gene Table 1 shows in silico scores of the normal and mutant CSS.The variant was absent from population databases of 1000genome, gnomAD, ExAC, our local database Iranome and, as far as we know, has not been reported before. MutationTaster predicted it as the cause of the disease. In addition, the CADD Phred-score  is 33 for this variant, which puts it among the top 0.1% most deleterious substitutions that happen throughout the human genome. We applied splice site prediction tools to analyze the mutant sequence and detect possible cryptic donor sites. All analyses indicated that the CSS is missed and suggested new donor sites; Based on criteria from the ACMG guideline, this variant is classified as pathogenic , 19. AppFAT4, the parents shared heterozygous variants of other genes, which are present in Table 2.In addition to We report two siblings with MCA, born to consanguineous parents, who deceased at the age of two months. During genetic counseling, their parents were explained about a possible familial recessive disorder and informed that in the absence of an accurate clinical diagnosis, a genotype-first diagnosis may be achieved. Therefore, WES was suggested and, due to no samples from their deceased children, it was applied for carrier screening of the parents. Also, they were informed about the advantages and disadvantages of this method. The advantages of WES in ECS, even in cases with no post-mortem samples, have been approved through previous studies. For example, in a recently published report, by this method they could identify the causal variant of a metabolic disorder in three dead siblings. However, as far as we know, in all of them there have been a definite or probable clinical diagnosis. In the current study, no preliminary clinical diagnosis or assumption were given to deceased patients, and from this point of view it could be assumed as the first report of its kind , 21, 22.FAT4, NM_024582.6: c.7018+1G>A. (Please see Table 2 for the other variants). This variant is pathogenic by applied rules PVS1 and PM2. Regarding criteria PVS1, loss of function is an established pathogenicity underlying the diseases associated with FAT4 variants: (1) According to data from Simple ClinVar database (https://simple-clinvar.broadinstitute.org/), most of the pathogenic or likely pathogenic variants in the gene FAT4 are of types which have disrupting consequence on protein function, including stopgain, frameshift, and splicing variants; (2) the OMIM database indicates several animal studies which support the idea of loss of function in FAT4-related disorders [This approach led to identification of a number of variants shared with the parents, including a novel pathogenic heterozygous variant in the gene isorders .FAT4 variants, HKLLS, and VMLDS, but the absence of lymphangiectasia or lymphedema, which are hallmarks of HKLLS, and the presence of tracheomalacia in both the siblings with small cysts in the kidney of proband 2 can be suggestive of VMLDS2 [Table 3 shows a comparison of the clinical symptoms between the patients of this study with characteristics previously described in the literature. As mentioned before, clinical variable expressivity is common across patients with FAT4 variants, which makes the classification of patients in Van Maldergem or Hennekam syndrome challenging. An interesting case is a patient, F4–1, from Alders et al.'s [Reverse phenotyping of the patients showed overlapping features of syndromes caused by f VMLDS2 . Table 3et al.'s  study, wThe Fat cadherin subfamily are highly conserved single-pass transmembrane receptors, which in humans consist of four members FAT1–4. All FAT cadherins have a large extra-cellular domain mainly consisting of cadherin repeats, which are followed by a number of EGF-like and Laminin G-like motifs . HeteropFAT atypical cadherin 4, FAT4, is located on chromosome 4q28.1 and its transcript, NM_024582.6, contains 18 exons which is translated to a protein with 4981 amino acids [Figure 2). In silico analysis of mutant sequence using splice prediction tools showed that at least two cryptic splice sites inside intron 6 can be potentially activated, of which one of them is very strong (Table 1). Although clinical evidence supports our idea of abnormal FAT4 transcription with no functional product, unfortunately DNA or tissue samples from patients are not available, and due to remote location of the parents it is not possible to conduct further RNA studies.no acids . The novDiagnosis of rare diseases with variable expressivity and overlapping features just based on clinical evaluations is difficult, especially in infantile patients. NGS, as a powerful facility, can unravel genetic defects underlying these group of disorders. Moreover, reverse phenotyping after a genetic finding may extend the clinical assessments and anticipate diagnosis of late onset features . BesidesHowever, next to these advantages, there are routine limitations associated with NGS methods, including the lack of full coverage for all genes or exons, absence of deep intronic regions in WES, and inability to investigate CNVs or chromosomal rearrangements. Moreover, finding variants with uncertain significance (VUS) can bring even more complexity to the situation. Furthermore, there are additional challenges for cases similar to ours: first, the heterozygous variant is identified in healthy adults and cannot be confirmed in a homozygous patient. Second, genotype–phenotype correlation is not straightforward and there is always concern around other possibilities which may be missed. This makes genetic counseling complicated, and any clinical intervention based on these findings must be advised more cautiously , 30FAT4 gene and resulted in diagnosis of VMLDS in two dead infantile siblings.ECS of parents is a powerful test which can reveal the genetic defect in deceased patients suspected to have a rare disease with recessive inheritance pattern. Here, exploiting this method revealed a novel pathogenic splice variant in


After adjusting for traditional confounders, the risk of MMD  increased with each increment in Neu5Ac level (per μmol/L). The area under the curve (AUC) values of the receiver operating characteristic (ROC) curves of the basic model plus Neu5Ac binary outcomes, Neu5Ac quartiles, and continuous Neu5Ac are 0.869, 0.863, and 0.873, respectively. Furthermore, including Neu5Ac in the model offers a substantial improvement in the risk reclassification and discrimination of MMD and its subtypes. A higher level of Neu5Ac was found to be associated with an increased risk of MMD and its clinical subtypes.N-acetylneuraminic acid (Neu5Ac) is a functional metabolite and has been demonstrated to be a risk factor for cardiovascular diseases. It is not clear whether Neu5Ac is associated with a higher risk of cerebrovascular disorders, especially moyamoya disease (MMD). We sought to elucidate the association between serum Neu5Ac levels and MMD in a case–control study and to create a clinical risk model. In our study, we included 360 MMD patients and 89 matched healthy controls (HCs). We collected the participants’ clinical characteristics, laboratory results, and serum Neu5Ac levels. Increased level of serum Neu5Ac was observed in the MMD patients ( Moyamoya disease (MMD) is an abnormal cerebrovascular disease identified by progressive stenosis, occlusion of the large arteries around the Willis circle, and compensatory development of certain blood vessels in the basal brain. In the course of cerebral angiography, the consequent irregular vascular network in the brain is unclear and smoky, hence the name ,2. AlthoIn recent years, variations in the composition of gut microbiota in the body and its metabolic changes have been recognized as essential factors in the occurrence and development of cardiovascular disease ,6,7. TheN-acetylneuraminic acid (Neu5Ac), one of the most common natural carbohydrates, is an essential building block of glycoproteins with many different biological functions, such as the synthesis of glycopeptides and glycolipids . In addiIn this paper, a case–control study was conducted to investigate the correlation between baseline serum Neu5Ac concentrations and the risk of MMD in Chinese patients. Additionally, regarding the clinical importance of MMD subtypes, we investigated the association of serum Neu5Ac concentrations with MMD subtypes at baseline. We speculated that multivariate analysis would be more valuable than univariate analysis for the early diagnosis of MMD. Therefore, we established a clinical prediction model to compare the efficiency of multivariate and univariate predictions of MMD and its subtypes. The calculation of the net reclassification index (NRI) and integrated discrimination improvement (IDI) showed that Neu5Ac is of great value in improving the accuracy of the model.In this study, we consecutively recruited 500 MMD patients from the Department of Neurosurgery, Beijing Tiantan Hospital, Capital Medical University, from 1 September 2020 to 31 December 2021. According to the guidelines published in Japan in 2012, MMD is diagnosed via digital subtraction angiography : (1) uni2). Neurological status was assessed using the modified Rankin Scale (mRS) at admission and divided into two groups (0–2 and 3–5). The Suzuki stage of the MMD patients was defined based on the more severe side.The clinical characteristics of the MMD patients acquired at admission included demographic data (age and sex), clinical features , medical history , and initial clinical manifestations (ischemic type or hemorrhagic type). After the patients had rested for 15 min, systolic blood pressure (SBP) and diastolic blood pressure (DBP) of the right arm were measured using a standard mercury manometer in a seated position. At the same time, heart rate was recorded using an electrocardiogram. Body mass index (BMI) was calculated as weight (kg)/height  (accessed on 15 March 2023). We performed a complete case analysis for the enrolled participants. Categorical variables were expressed as frequencies, and continuous variables were described as mean with standard deviation (SD) or median with interquartile range (IQR). Continuous data were compared using t-tests or Mann–Whitney U tests between the two groups. Kruskal–Wallis tests or one-way ANOVA were used for comparisons among multiple groups. Categorical variables were compared with Pearson’s chi-squared tests, Fisher’s exact tests, and Kruskal–Wallis tests. Kendall correlation tests were used to assess the association between clinical characteristics and Neu5Ac quartiles.Statistical analyses were carried out using SPSS (version 26.0) and R software, version 4.2.3  in MMD and its subtypes, including a Crude model, Model 1, and Model 2. Variables that were clinically meaningful and significantly different in the univariate analysis were considered for inclusion in the regression models . For thep < 0.05 was considered statistical significance.To compare the capacity of each regression model to discriminate MMD patients and subtypes of MMD patients from healthy controls, a logistic regression model was used to create receptor operating characteristic (ROC) curves for sensitivity and specificity, using the area under the ROC curve (AUC) to evaluate overall discrimination . NRI andp = 0.001; p < 0.05 for all). Furthermore, MMD patients showed lower levels of TC, HDL-C, LDL-C, ApoA, and PNR . Patients with MMD had a higher tendency to have diabetes mellitus, hypertension, and hyperlipidemia and were more inclined to be smokers and drinkers than the control subjects .Overall, 360 adult patients with MMD and 89 healthy control subjects were included in our study. Baseline variables were compared between the HCs and MMD patients . The medp = 0.015), indicating a higher risk of ischemia. Compared to the healthy control subjects, MMD patients in general (p = 0.001), ischemic-type MMD patients (p = 0.019), and hemorrhagic-type MMD patients (p < 0.001) had higher serum Neu5Ac concentrations. Furthermore, compared to ischemic-type MMD patients, hemorrhagic-type MMD patients had higher serum Neu5Ac concentrations . Furthermore, patients in the high Neu5Ac group had higher heart rates and higher levels of Cr, TC, LDL-C, and ApoB .Patients with MMD were categorized into two groups based on the median level of Neu5Ac: a low Neu5Ac group and a high Neu5Ac group. The clinical characteristics of the two groups are shown in p < 0.05 for all, p = 0.046).When all participants were stratified into quartiles based on their baseline serum Neu5Ac concentrations, participants with higher Neu5Ac levels tended to be hemorrhagic-type MMD patients and had higher levels of LY count, Cr, TC, LDL-C, and ApoB . In Model 2, Neu5Ac also increases the risk of MMD . The ROC curves reveal that the predictive accuracy of Model 2 , Model 1 , and Model 2 , Model 1 , and Model 2  show better adaptability. The AUC values of MMD overall show an improvement in the Crude model , Model 1 , and Model 2 . The AUC values of the ROC curves increase with the adjustment for cofounders in the models  . In Model 2, compared to Q1 of serum Neu5Ac level, the adjusted OR  for ischemic MMD in Q2 and Q4 is 2.493  and 3.123 , respectively. The AUCs of the ROC curves in the Crude model, Model 1, and Model 2 are 0.587, 0.754, and 0.893, respectively  and Model 1 . In Model 2, compared to Q1 of serum Neu5Ac level, the adjusted OR  for hemorrhagic MMD in Q4 is 8.442 . The AUCs of the ROC curves in the Crude model, Model 1, and Model 2 are 0.670, 0.733, and 0.867, respectively  A. We fou= 0.869) . Compareectively B. Moreovectively C.p = 0.007) and an IDI of 2.8% ; the model for ischemic MMD has an IDI of 1.8% ; and the model for hemorrhagic MMD has a categorical NRI of 19.9%  and an IDI of 6.5% . In addition, the basic model plus Neu5Ac binary outcomes for MMD has a categorical NRI of 7.0% , and the model for hemorrhagic MMD has a categorical NRI of 15.2%  and an IDI of 3.4% . Moreover, the basic model plus Neu5Ac quartiles for MMD has an IDI of 2.0% , and the model for hemorrhagic MMD has a categorical NRI of 16.6%  and an IDI of 5.6% .To explore the predictive value of serum Neu5Ac, we calculated categorical NRI and IDI for models containing serum Neu5Ac concentrations, Neu5Ac binary outcomes, and Neu5Ac quartiles on top of our previously established clinical risk model . The basIn this study, we investigated the association between baseline serum Neu5Ac and the risk of MMD in a Chinese population. Then, we compared the difference in clinical characteristics between HCs, MMD patients, and MMD subtypes. Our findings revealed that serum Neu5Ac levels were significantly higher in the MMD group and its subtypes than those in the HC group. We expounded and proved that higher Neu5Ac levels were correlated with an increased risk of MMD, especially of hemorrhagic MMD, even after adjusting for laboratory results and other basic clinical characteristics. Moreover, we created a clinical diagnostic model and examined its predictive accuracy using the ROC curve. Including Neu5Ac in the model offered substantial improvement in the risk reclassification and discrimination of MMD and its subtypes. In medical and clinical settings, serum Neu5Ac can be used as a novel biomarker for the diagnosis of MMD, and targeting Neu5Ac may serve as a novel therapeutic strategy for MMD. Furthermore, the multifactorial clinical prediction model including serum Neu5Ac concentrations developed in this study could be used to predict MMD and its subtypes. To the best of our knowledge, this is the largest and first study to date assessing the relationship between serum Neu5Ac levels and the risk of MMD.In recent years, the role of body metabolites in vascular diseases has attracted extensive attention and interest. Overall quantitative and qualitative analyses of multifarious small molecules in patients with coronary artery disease and other cardiovascular diseases have revealed changes in biological endogenous metabolites after their measurement in the internal and external environments, which has been shown to help identify disease-related disorders. A panel of metabolic markers can help facilitate the early detection and treatment of diseases ,27,28. NMMD is generally identified by stenosis at the end of the internal carotid arteries (ICAs) and the consequent dilatation of compensatory blood vessels. Histologically, it is featured by the thickening and corrugation of the elastic lamina, hypertrophy of intimal fibroblasts, and hyperplasia of smooth muscle cells involving the ICAs and proximal anterior and middle cerebral arteries . Zhang eNeu5Ac may also play a role in angiogenesis and dilatation of compensatory blood vessels in patients with MMD. During embryogenesis and wound healing, the formation of new blood vessels from existing blood vessels is called angiogenesis. Angiogenesis is stimulated by angiogenic growth factors (AGFs) released by inflammatory cells. Among them, the most crucial AGFs are the vascular endothelial growth factor (VEGF) family, including VEGF-A, B, C, D, E, and the placental growth factor . The intAlthough our study has potential benefits as the first step to illuminate the effect of Neu5Ac in MMD, our study also has limitations. First, we found that Neu5Ac is related to the risk of MMD in the Chinese population, but further verification is necessary for the broader Asian population. Second, Neu5Ac was measured at only one time point, and no dietary history was taken to estimate the effect of diet on Neu5Ac levels in the body. Third, before blood collection, no information was collected on antibiotic use or gastrointestinal symptoms. Fourth, we chose a case–control study design that considered cost and efficiency; to confirm our results, it needs to be corroborated in a larger prospective cohort. Fifth, our study included only MMD patients and did not address moyamoya syndrome or other cerebrovascular diseases. Finally, despite the adjustment for known factors, there are other traditional potential confounders that we did not use to compare the cases and controls. Future studies are required to identify the molecular mechanism that mediates the effect of Neu5Ac on the etiology of MMD and to determine whether Neu5Ac is the potential therapeutic target or the only biomarker for MMD.Taken together, our results suggest that the Neu5Ac–MMD risk association we observed may be independent. Therefore, serum Neu5Ac can be a new biomarker for diagnosing MMD, and targeting Neu5Ac in the future may be a potential therapeutic strategy for MMD. Additionally, we created a multifactorial clinical prediction model including serum Neu5Ac concentrations to predict MMD and its subtypes. Nevertheless, our results require well-designed basic and clinical trials to further explore the association of higher Neu5Ac levels with the pathogenesis of MMD.