Matej Šapina

Objectives: The aim of this study was to examine the impact of the pentraxin 3 (PTX3) serum level and angiotensin-converting enzyme (ACE) gene insertion/deletion (I/D) polymorphism on the severity of radiographic pulmonary infiltrates and the clinical outcomes of COVID-19. Methods: The severity of COVID-19 pulmonary infiltrates was evaluated within a week of admission by analyzing chest X-rays (CXR) using the modified Brixia (MBrixa) scoring system. The insertion (I)/deletion (D) polymorphism of the ACE gene and the serum levels of PTX3 were determined for all patients included in the study. Results: This study included 80 patients. Using a cut-off serum level of PTX3 ≥ 2.765 ng/mL, the ROC analysis (AUC 0.871, 95% CI 0.787–0.954, p < 0.001) showed a sensitivity of 85.7% and specificity of 78.8% in predicting severe MBrixa scores. Compared to ACE I/I polymorphism, D/D polymorphism significantly increased the risk of severe CXR infiltrates, OR 7.7 (95% CI: 1.9–30.1), and p = 0.002. Significant independent predictors of severe CXR infiltrates include hypertension (OR 7.71), PTX3 (OR 1.20), and ACE D/D polymorphism (OR 18.72). Hypertension (OR 6.91), PTX3 (OR 1.47), and ACE I/I polymorphism (OR 0.09) are significant predictors of poor outcomes. Conclusion: PTX3 and ACE D/D polymorphism are significant predictors of the severity of COVID-19 pneumonia. PTX3 is a significant predictor of death.

Endothelial cell injury is a hallmark of IgA vasculitis (IgAV), possibly associated with various factors, including oxidative stress. Certain single nucleotide polymorphisms (SNPs) of glutathione S-transferases (GST) genes have been shown to increase susceptibility to oxidative stress. The objective of our study was to evaluate the gene polymorphisms of GSTM1, GSTT1, GSTP1, and GSTA1 in patients with IgAV. DNA was extracted from the blood of 124 children with IgAV and 168 age-matched healthy controls. A higher frequency of the GSTM1 null genotype was observed in patients with gastrointestinal (GI) system involvement compared to those without GI system involvement (51.5% vs. 28.6%, p = 0.011). Additionally, the GSTM1 null genotype was less prevalent (30.8% vs. 69.2%, p = 0.032), while the GSTP1 Val/Val genotype was significantly more prevalent in patients who developed urogenital complications (scrotal swelling) during the course of the disease (60% vs. 40%, p = 0.039). This study is the first to suggest an association between GSTM1 and GSTP1 polymorphisms and various phenotypes observed during the clinical course of IgAV in the pediatric population. However, it was performed on a national and likely single ethnic cohort, too small for definitive conclusions, so larger studies are needed to confirm this association.

Studies concerning the genetic background of IgA vasculitis (IgAV), a small-vessel vasculitis occurring predominantly in childhood, have confirmed that the HLA-DRB1 gene showed a strong association with disease susceptibility. The objective was to investigate human leukocyte antigen (HLA) polymorphisms among Croatian patients with IgAV and their influence on disease susceptibility and clinical heterogeneity. Thus, 130 children with IgAV and 202 unrelated healthy individuals were enrolled in the study. Genomic DNA was extracted from whole peripheral blood, and HLA-A, -B, -DRB1 and -DQB1 gene polymorphism analysis was performed. HLA-A*03 (21.4% vs. 12.38%, p = 0.0092), HLA-B*37 (2.9% vs. 0.2%, p = 0.0054) and HLA-DRB1*12 (3.1% vs. 0.7%, p = 0.0216) alleles were significantly more frequent in IgAV patients than in controls. High-resolution typing revealed significantly higher frequency of HLA-DRB1*10:01 and -DRB1*11:03 among IgAV patients with gastrointestinal manifestations of the disease in comparison to controls (p = 0.0021 and p = 0.0301, respectively), while HLA-DRB1*14:01P occurred significantly more often in the group of patients who developed nephritis during the course of the disease (17.5% vs. 4.5%, p = 0.0006). Our results demonstrated that there is an association of HLA-A*03, HLA-B*37 and HLA-DRB1*12 alleles with susceptibility to IgAV in the examined Croatian pediatric population. Studies which aim to determine the HLA profile may contribute to the elucidation of the genetic background of autoimmune diseases, including IgAV.

Introduction IgA vasculitis (IgAV) is a disease in which genetic background also plays an important role. Some small studies have indicated the importance of variants in various non-HLA genes in the manifestation of different disease phenotypes. Objectives The aim of this research was to investigate single nucleotide polymorphisms (SNPs) of genes HMGB1 and AGER encoding for high mobility group box-1 (HMGB1) and receptor for advanced glycation endproducts (RAGE), both acting as mediators of inflammation, in the susceptibility and clinical features of patients with IgAV. Methods HMGB1 and RAGE gene polymorphisms were genotyped using a real-time polymerase chain reaction. The presence and frequency of polymorphisms in HMGB1 (rs2249825, rs1045411, rs1060348, rs1412125 and rs41369348) and RAGE (rs1800625, rs1800624, rs2070600 and rs3134940) were determined. Clinical data were collected from database of IgAV patients from two Croatian University Centers for pediatric rheumatology. Results 81 pediatric IgAV patients were included, of whom 45 were boys and 36 girls, as well as 150 age- and sex-matched healthy controls without any history of autoimmune disease. The median (range) age of IgAV patients was 6.25 (4.60-8.20) years, and among them 71.6% had joint involvement, 29.62% had gastrointestinal manifestations, while 27.16% developed nephritis. The purpuric rash which extended from lower extremities to the trunk, upper extremities and face (generalized rash) was present in 43.20% of patients and 27.16% had at least one relapse. Among the analyzed polymorphisms, only in the rs1412125 there was a deviation from the Hardy Weinberger equilibrium. There was no statistically significant association of the analyzed polymorphisms with the IgAV susceptibility, compared to healthy controls. Polymorphism rs2070600 was significantly related with the development of nephritis in IgAV, while rs1412125 was associated with gastrointestinal involvement. The IgAV patients carrying the T allele (rs2070600) of the AGER had significantly increased risk of nephritis development compared with the IgAV patients with homozygous CC genotype in dominant (OR 4.05, CI 1.09-15.03, p = 0.037) and additive genetic models (OR 3.95, CI 1.16-13.47, p = 0.049). The minor C allele (rs1412125) of the HMGB1 was found to significantly increase the risk of gastrointestinal involvement in overdominant model with an allelic odd ratio of 2.78 (CI 1.04-7.43, p = 0.04). Conclusion Although neither of analyzed HMGB1 and RAGE polymorphisms was not associated with IgAV susceptibility, our results indicated that these polymorphisms may be involved in the pathogenesis of IgAV with possible effect on different disease phenotypes. SUPPORT: Croatian Science Foundation project IP-2019-04-8822.

Blood pressure variability (BPV) is a complex physiological phenomenon and it is known that increased BPV exacerbates the progression of hypertension and correlates with adverse cardiovascular outcomes. However, there is paucity of data regarding factors influencing BPV in pediatric population. This study aimed to analyze blood pressure (BP) pattern and assess blood pressure variability and its possible determinants in untreated children with essential hypertension. The study group included 132 children, average age 15 y (7- 18 y), 84 boys and 48 girls with essential hypertension and no use of antihypertensive therapy. Anthropometric parameters, serum glucose level (sG), serum uric acid (sUA), serum creatinine (sCr), total cholesterol (TCh), HDL cholesterol (HDL), LDL cholesterol (LDL) and triglycerides (TG) were measured in all participants. Office blood pressure (BP) and ambulatory blood pressure (AMBP) was measured according to recent European Society of Hypertension (ESH) guidelines. BPV was defined as value of the standard deviation of blood pressure for day and night time periods. In addition to the classical statistical analysis, an unsupervised machine learning approach using the expected maximization algorithm was implemented to find groups of patients with similar characteristics. The average age was 14.98 ± 2.1 years. Comparing sex-specific BP parameters, boys had higher daytime and nighttime systolic BP, although the difference was not statistically significant (p = 0.067 and p = 0.583). Girls had significantly higher daytime and nighttime diastolic BP values (p=0.017; p=0.027). There was no difference in BPV according to gender while boys have higher values of sCr, sG, and sUA despite similar body mass index (BMI) [27.18±5.27 kg/m2 (males) vs 27.94±5.27 kg/m2 (females), p= 0.427]. A statistically significant positive correlation was found between the Z-score for BMI and daytime systolic BPV, nighttime systolic BP and total cholesterol, nighttime diastolic BPV and sUA (r= 0.19, r= 0.28 and r = 0.26, respectively). Within the male population two clusters were found. The subjects in cluster 2 had higher daytime and nighttime systolic and diastolic BP values, higher total cholesterol and triglycerides and significantly higher night time systolic and diastolic BPV. Understanding potential determinants of BPV in hypertensive children could help identify patients with increased cardiovascular risk earlier. Our results suggest that clustering of metabolic factors influence BPV in untreated children with essential hypertension which could also be gender specific for male subjects.

To evaluate characteristics of patients with severe skin manifestations of Henoch Schoenlein purpura (HSP) in five tertiary centres in Croatia in time period from 2009 until 2019. Retrospective statistical chart analysis of clinical symptoms and laboratory parameters of patients with HSP was done. Severe skin manifestations were defined as haemorrhagic bullous, ulcerated or necrotic lesions. During observed time period 583 patients (303 boys and 280 girls) were admitted to hospital due to HSP. Prevalence of severe skin manifestations was 2.41% (14 patients, CI 1.32 – 4.003%). Severe skin manifestations occurred more often in boys than in girls with a difference on a margin of statistical significance (11 boys, 3 girls, p=0.057). Patients with severe skin manifestations were more prone to relapses than those with milder skin forms (severe 28.57%, milder 18.66%), with most notable difference in patients with 2 relapses of disease (severe 14.29%, milder 3.17%, p=0.029). We were able to isolate infectious trigger of disease more often in patients with milder form of disease than in those with more severe (milder 38.42%, severe 7.69%, p=0.036). Patients with severe skin manifestations had more often gastrointestinal system affected than those with milder skin forms (severe 70%, milder 36.03%, p=0.043). There was no statistically significant difference between those two groups in other organs involvement (kidneys, joints, central nervous system, lung). Regarding distribution of skin rash, patients with severe skin manifestations more often had a rash on a trunk, with a difference on a margin of statistical significance (severe 85.71%, milder 57.19%, p=0.051). There was no statistical difference between two groups in most of other clinical and laboratory parameters. Only blood leukocyte levels were near to statistical significance with higher levels in patients with severe skin manifestations (12.1*109/L (SD 10-17.62):10.71*109/L (SD 8.45-13.42), p=0.059). Patients with severe skin manifestations of HSP were more prone to relapses, and had gastrointestinal symptoms more often affected than those with milder forms, but less often had infectious trigger isolated. There was no significant statistical difference in other clinical symptoms and laboratory parameters. We couldn’t set predictive model for disease outcome based on these data. Small number of patients with severe manifestations and retrospective data analysis could be the reason. Perhaps better structured prospective trial would be more appropriate in that sense. SUPPORT: Croatian Science Foundation project IP-2019-04-8822

Indroduction IgA vasculitis (IgAV) is the most common childhood-vasculitis in which more than 50% of children develop gastrointestinal (GI) symptoms. In 10-20% of patients serious complications such as intussusception, bowel perforation, and massive bleeding may occur. The most important complication is the development of nephritis with progression to chronic renal failure in about 3% of children. The aim of the research was to analyze clinical and biochemical parameters in patients with IgAV and GI manifestations. Methods This retrospective study included children with IgAV reviewed in five Croatian University Centers for pediatric rheumatology in the period 2009 to 2019. Results Out of 611 children with IgAV, 320 were males and 291 were females. The overall GI symptoms prevalence was 45.9% and the median (range) age at diagnosis was 6.42 (4.5-8.83) years. Among patients with GI symptoms there were 1.44 times more males (N=166) than females (N=115), which was statistically significant (p=0.003). Patients with GI symptoms had less infections before the appearance of purpura (59.8% vs. 70.9%, p=0.005) and were found to be significantly more likely to have rash distributed on the trunk (61.9% vs. 48.5%, p=0.001), and upper extremities (35.2% vs. 24.7%, p=0.006), as well as generalized rash (38.8% vs. 28.3%, p=0.008). These patients also had significantly higher values of C-reactive protein, leukocyte count, erythrocytes and platelets, hemoglobin, hematocrit and D-dimer concentrations and lower levels of IgG and IgM. In our cohort 42 out of 281 children (14.9%) had the most severe GI manifestations (intussusception and/or massive GI bleeding) with significantly higher values of 24-hour urine protein levels and D-dimer concentrations and lower total serum protein, albumin, IgG, IgM and C3 levels in comparison with children whose GI manifestations were not severe. Predictors of severe GI involvement were: relapse of the disease, generalized rash, rash extended on upper extremities, rash extended to the face, recurrent rash and renal involvement, as well as lower values of prothrombin time, fibrinogen and IgM among the laboratory parameters. Patients with GI symptoms were 1.68 times more likely to develop nephritis, and this probability was 2.58 times higher if GI symptoms occurred before other symptoms. Other predictors of nephritis were: severe and moderate GI manifestations, recurrent rash, one or more relapses of IgAV, and older age. Conclusion Older children with IgAV and severe GI manifestations in whom IgAV begun with GI symptoms had a higher risk of acute and chronic complications of the disease. SUPPORT: Croatian Science Foundation project IP-2019-04-8822

IgA vasculitis (IgAV) is the most common childhood-vasculitis in which more than 50% of children develop gastrointestinal (GI) symptoms. In 10-20% of patients serious complications such as intussusception, bowel perforation, and massive bleeding may occur. The most important complication is the development of nephritis with progression to chronic renal failure in about 3% of children. The aim of the research was to analyze clinical and biochemical parameters in patients with IgAV and GI manifestations. This retrospective study included children with IgAV reviewed in five Croatian University Centers for pediatric rheumatology in the period 2009 to 2019. Out of 611 children with IgAV, 320 were males and 291 were females. The overall GI symptoms prevalence was 45.9% and the median (range) age at diagnosis was 6.42 (4.5-8.83) years. Among patients with GI symptoms there were 1.44 times more males (N=166) than females (N=115), which was statistically significant (p=0.003). Patients with GI symptoms had less infections before the appearance of purpura (59.8% vs. 70.9%, p=0.005) and were found to be significantly more likely to have rash distributed on the trunk (61.9% vs. 48.5%, p=0.001), and upper extremities (35.2% vs. 24.7%, p=0.006), as well as generalized rash (38.8% vs. 28.3%, p=0.008). These patients also had significantly higher values of C-reactive protein, leukocyte count, erythrocytes and platelets, hemoglobin, hematocrit and D-dimer concentrations and lower levels of IgG and IgM. In our cohort 42 out of 281 children (14.9%) had the most severe GI manifestations (intussusception and/or massive GI bleeding) with significantly higher values of 24-hour urine protein levels and D-dimer concentrations and lower total serum protein, albumin, IgG, IgM and C3 levels in comparison with children whose GI manifestations were not severe. Predictors of severe GI involvement were: relapse of the disease, generalized rash, rash extended on upper extremities, rash extended to the face, recurrent rash and renal involvement, as well as lower values of prothrombin time, fibrinogen and IgM among the laboratory parameters. Patients with GI symptoms were 1.68 times more likely to develop nephritis, and this probability was 2.58 times higher if GI symptoms occurred before other symptoms. Other predictors of nephritis were: severe and moderate GI manifestations, recurrent rash, one or more relapses of IgAV, and older age. Older children with IgAV and severe GI manifestations in whom IgAV begun with GI symptoms had a higher risk of acute and chronic complications of the disease. SUPPORT: Croatian Science Foundation project IP-2019-04-8822

Haemophilia is rare, inherited and severe bleeding disorder characterised with factor VIII or factor IX deficiency. The estimated glomerular filtration rate (eGFR) is one of the best-performing methods to evaluate kidney function. Glomerular filtration rate cannot be directly measured; however, it can be determined by measuring the clearance of an ideal filtration marker or estimated using predictive formulas. The aim of this preliminary study was to calculate eGFR of paediatric haemophilia patients treated in our centre and asses the correlation of eGFR calculated by creatinine-based and cystatin C-based equations. In our study, we included 36 boys with moderate or severe haemophilia. Out of a total of 36 patients, 27 had haemophilia A and 9 had haemophilia B. Their mean age was 11.2±4.31 years, with a range from 3 to 18 years. We investigated the correlation and agreement between two eGFR equations (creatinine-based ‘Bedside Schwartz’ equation and cystatin-C based equation). Along with applying correlation and linear regression tests, the Bland Altman test was performed to assess the agreement of the results. Statistically significant differences were found between the mean eGFR values (p<0.001). No significant correlation was found between the two methods (p=0.07). Bland-Altman analysis results showed higher mean eGFR values of bedside Schwartz equation compared to the cystatin-C based formula, meaning that a significant disagreement was found between those two equations. However, within the group of haemophilia B patients, statistically significant positive correlations between the two methods were found, although still a disagreement was observed Due to the observed disagreements between eGFR within haemophilia patients, further research is needed to find the optimal measure of eGFR. We suggest extending this study on a larger cohort of patients and include other possible eGFR equations.

Background/Objective: The purpose of this study was to evaluate the prevalence and severity of skin involvement in children with IgA vasculitis (IgAV) and its relationship with clinical and biochemical parameters and the risk of developing IgA vasculitis nephritis (IgAVN), the only cause of long-term morbidity and the main prognostic factor in IgAV patients. Methods: This national multicenter retrospective study included 611 patients under the age of 18 years with IgAV referred to five Croatian tertiary hospitals between 2009 and 2019. Patient data were collected from a database with systematic analysis of IgAV patients in the Croatian population. Results: Among the 611 children, 205 (33.55%) had purpura on the lower extremities, in 207 (33.88%) the rash extended on the trunk, in 149 (24.39%) it extended to the upper extremities, in 32 (5.24%) the rash was generalized, while 15 (2.47%) had the most severe skin symptoms: bullae, ulcerations, and necroses. IgAVN developed in 130 (21.28%) and persistent IgAVN (present for >3 months) in 48 (7.86%) children. Multivariate logistic regression found that presence of ulcerations and necroses (OR 3.20 [95% CI 1.03–9.91]), persistent purpura (OR 2.89 [95% CI 1.71–4.88]), and higher age (OR 1.16 [95% CI 1.09–1.23]) were significant predictors of IgAVN, whereas persistent purpura (OR 20.11 [95% CI 1.09–372.52]), male sex (OR 3.32 [95% CI 1.13–9.80]), and higher age (OR 1.15 [95% CI 1.00–1.30]) were predictors of persistent IgAVN. Among the laboratory parameters, higher serum urea (OR 1.43 [95% CI 1.03–2.00]) and reduction in activated partial thromboplastin time (OR 0.83 [95% CI 0.74–0.93]) were shown to have a significant impact on increasing the risk of persistent IgAVN. Conclusion: With increasing severity and duration of cutaneous manifestations in IgAV, the risk of developing IgAVN increases, making the prognosis worse, with a greater likelihood to need more aggressive treatment.