Early Symptoms and Treatment Outcomes in Neuronal Ceroid Lipofuscinosis Type 2: Croatian Experience.

BACKGROUND: Late infantile neuronal ceroid lipofuscinosis type 2 (CLN2) is a rare neurodegenerative disease that generally appears in children between 2 and 4 years old, leading to seizures and a progressive loss of language and motor functions. As the disease progresses, affected individuals typically experience blindness and ultimately pass away in late childhood. Treatment with intracerebroventricular cerliponase alfa has been shown to slow the deterioration of motor and language functions compared to the natural progression of the disease. We aim to highlight the early symptoms of CLN2 which help with early diagnosis and timely treatment initiation in children with specific medical indications, as well as identify medical contraindications for enzyme replacement therapy. METHODS: We describe five Croatian patients and one Bosnia and Herzegovinian patient with CLN2 disease, analyzing the clinical characteristics, neuroimaging findings, electroencephalogram results, genetic analysis, treatment indications and contraindications, and disease progression. RESULTS: All six patients presented with seizures: focal seizures (n = 1), myoclonic-atonic seizures (n = 1), febrile seizures (n = 2), and tonic-clonic seizures (n = 2), along with language delay (n = 6). Despite this, one patient refused treatment, two were initially included in the clinical trial and then continued treatment, one did not indicate starting treatment, and three continued treatment. One patient, after 4.5 years of treatment, no longer had medical indications for the therapy, which was discontinued. The other two patients who received treatment had a significant slowing of disease progression. CONCLUSIONS: The early onset of seizures between ages 2 and 4, alongside delayed language development, is a defining characteristic of CLN2 disease. Enzyme replacement therapy using cerliponase alfa represents the initial treatment for neuronal ceroid lipofuscinosis type 2, targeting the underlying cause of the disease. It effectively delays the progression of language and motor decline in patients diagnosed with this condition.

Copy number variation analysis identifies MIR9-3 and MIR1299 as novel miRNA candidate genes for CAKUT.

BACKGROUND: Congenital anomalies of the kidney and urinary tract (CAKUT) represent a frequent cause of pediatric kidney failure. CNVs, as a major class of genomic variations, can also affect miRNA regions. Common CNV corresponding miRNAs (cCNV-miRNAs) are functional variants regulating crucial processes which could affect urinary system development. Thus, we hypothesize that cCNV-miRNAs are associated with CAKUT occurrence and its expressivity. METHODS: The extraction and filtering of common CNVs, identified in control samples deposited in publicly available databases gnomAD v2.1 and dbVar, were coupled with mapping of miRNA sequences using UCSC Genome Browser. After verification of the mapped miRNAs using referent miRBase V22.1, prioritization of cCNV-miRNA candidates has been performed using bioinformatic annotation and literature research. Genotyping of miRNA gene copy numbers for MIR9-3, MIR511, and MIR1299, was conducted on 221 CAKUT patients and 192 controls using TaqMan™ technology. RESULTS: We observed significantly different MIR9-3 and MIR1299 gene copy number distribution between CAKUT patients and controls (Chi-square, P = 0.006 and P = 0.0002, respectively), while difference of MIR511 copy number distribution showed nominal significance (Chi-square, P = 0.027). The counts of less and more than two of MIR1299 copy numbers were more frequent within CAKUT patients compared to controls (P = 0.01 and P = 0.008, respectively) and also in cohort of patients with anomalies of the urinary tract compared to controls (P = 0.016 and P = 0.003, respectively). CONCLUSIONS: Copy number variations of miRNA genes represent a novel avenue in clarification of the inheritance complexity in CAKUT and provide potential evidence about the association of common genetic variation with CAKUT phenotypes.

A preliminary study of the miRNA restitution effect on CNV-induced miRNA downregulation in CAKUT.

BACKGROUND: The majority of CAKUT-associated CNVs overlap at least one miRNA gene, thus affecting the cellular levels of the corresponding miRNA. We aimed to investigate the potency of restitution of CNV-affected miRNA levels to remediate the dysregulated expression of target genes involved in kidney physiology and development in vitro. METHODS: Heterozygous MIR484 knockout HEK293 and homozygous MIR185 knockout HEK293 cell lines were used as models depicting the deletion of the frequently affected miRNA genes by CAKUT-associated CNVs. After treatment with the corresponding miRNA mimics, the levels of the target genes have been compared to the non-targeting control treatment. For both investigated miRNAs, MDM2 and PKD1 were evaluated as common targets, while additional 3 genes were investigated as targets of each individual miRNA (NOTCH3, FIS1 and APAF1 as hsa-miR-484 targets and RHOA, ATF6 and CDC42 as hsa-miR-185-5p targets). RESULTS: Restitution of the corresponding miRNA levels in both knockout cell lines has induced a change in the mRNA levels of certain candidate target genes, thus confirming the potential to alleviate the CNV effect on miRNA expression. Intriguingly, HEK293 WT treatment with investigated miRNA mimics has triggered a more pronounced effect, thus suggesting the importance of miRNA interplay in different genomic contexts. CONCLUSIONS: Dysregulation of multiple mRNA targets mediated by CNV-affected miRNAs could represent the underlying mechanism behind the unresolved CAKUT occurrence and phenotypic variability observed in CAKUT patients. Characterizing miRNAs located in CNVs and their potential to become molecular targets could eventually help in understanding and improving the management of CAKUT.

Identification and functional interpretation of miRNAs affected by rare CNVs in CAKUT.

Rare copy number variants (CNVs) are among the most common genomic disorders underlying CAKUT. miRNAs located in rare CNVs represent well-founded functional variants for human CAKUT research. The study aimed to identify and functionally interpret miRNAs most frequently affected by rare CNVs in CAKUT and to estimate the overall burden of rare CNVs on miRNA genes in CAKUT. The additional aim of this study was to experimentally confirm the effect of a rare CNV in CAKUT on candidate miRNA's expression and the subsequent change in mRNA levels of selected target genes. A database of CAKUT-associated rare CNV regions, created by literature mining, was used for mapping of the miRNA precursors. miRNAs and miRNA families, most frequently affected by rare CAKUT-associated CNVs, have been subjected to bioinformatic analysis. CNV burden analysis was performed to identify chromosomes with over/underrepresentation of miRNA genes in rare CNVs associated with CAKUT. A functional study was performed on HEK293 MIR484+/- KO and HEK293 WT cell lines, followed by the analysis of relative miRNA and mRNA target gene levels. 80% of CAKUT patients with underlying rare CNV had at least one miRNA gene overlapping the identified CNV. Network analysis of the most frequently affected miRNAs has revealed the dominant regulation of the two miRNAs, hsa-miR-484 and hsa-miR-185-5p. Additionally, miR-548 family members have shown substantial enrichment in rare CNVs in CAKUT. An over/underrepresentation of miRNA genes in rare CNVs associated with CAKUT was observed in multiple chromosomes, such as chr16, chr20, and chr21. A significant 0.37 fold downregulation of hsa-miR-484, followed by a notable upregulation of MDM2 and APAF1 and downregulation of NOTCH3 was detected in HEK293 MIR484+/- KO compared to HEK293 WT cell lines, supporting the study hypothesis. miRNA genes are frequently affected by rare CNVs in CAKUT patients. Understanding the potential of CNV-affected miRNAs to participate in CAKUT as genetic drivers represent a crucial implication for the development of novel therapeutic approaches.

Epidemiologic Characteristics of Children with Diabetic Ketoacidosis Treated in a Pediatric Intensive Care Unit in a 10-Year-Period: Single Centre Experience in Croatia.

Background and Objectives: The incidence of severe and moderate forms of DKA as the initial presentation of type 1 diabetes mellitus (T1D) is increasing, especially during the COVID-19 pandemic. This poses a higher risk of developing cerebral edema as a complication of diabetic ketoacidosis (DKA), as well as morbidity and mortality rates. The aim of this study was to determine the trend and clinical features of children treated in the last 10 years in the Pediatric Intensive Care Unit (PICU) due to the development of DKA. Materials and Methods: This retrospective study was performed in the PICU, Clinical Hospital Centre Rijeka, in Croatia. All children diagnosed with DKA from 2011-2020 were included in this study. Data were received from hospital medical documentation and patient paper history. The number of new cases and severity of DKA were identified and classified using recent International Society for Pediatric and Adolescent Diabetes (ISPAD) guidelines. Results: In this investigation period, 194 children with newly diagnosed T1D were admitted to our hospital: 58 of them were treated in the PICU due to DKA; 48 had newly diagnosed T1D (48/58); and ten previously diagnosed T1D (10/58). DKA as the initial presentation of T1D was diagnosed in 24.7% (48/194). Moderate or severe dehydration was present in 76% of the children at hospital admission. Polyuria, polydipsia, and Kussmaul breathing were the most common signs. Three patients (5.2%) developed cerebral edema, of whom one died. Conclusions: During the investigation period a rising trend in T1D was noted, especially in 2020. About one quarter of children with T1D presented with DKA at initial diagnosis in western Croatia, most of them with a severe form. Good education of the general population, along with the patients and families of children with diabetes, is crucial to prevent the development of DKA and thus reduce severe complications.

GLUT1 Deficiency Syndrome-Early Treatment Maintains Cognitive Development? (Literature Review and Case Report).

Glucose transporter type 1 (GLUT1) is the most important energy carrier of the brain across the blood-brain barrier, and a genetic defect of GLUT1 is known as GLUT1 deficiency syndrome (GLUT1DS). It is characterized by early infantile seizures, developmental delay, microcephaly, ataxia, and various paroxysmal neurological phenomena. In most cases, GLUT1DS is caused by heterozygous single-nucleotide variants (SNVs) in the SLC2A1 gene that provoke complete or severe impairment of the functionality and/or expression of GLUT1 in the brain. Despite the rarity of these diseases, GLUT1DS is of high clinical interest since a very effective therapy, the ketogenic diet, can improve or reverse symptoms, especially if it is started as early as possible. We present a clinical phenotype, biochemical analysis, electroencephalographic and neuropsychological features of an 11-month-old boy with myoclonic seizures, hypogammaglobulinemia, and mildly impaired gross motor development. Using sequence analysis and deletion/duplication testing, deletion of an entire coding sequence in the SLC2A1 gene was detected. Early introduction of a modified Atkins diet maintained a seizure-free period without antiseizure medications and normal cognitive development in the follow-up period. Our report summarizes the clinical features of GLUT1 syndromes and discusses the importance of early identification and molecular confirmation of GLUT1DS as a treatable metabolic disorder.

Association study of rs7799039, rs1137101 and rs8192678 gene variants with disease susceptibility/severity and corresponding LEP, LEPR and PGC1A gene expression in multiple sclerosis.

BACKGROUND: Leptin (LEP), leptin receptor (LEPR) and peroxisome proliferator-activated receptor gamma co-activator 1-alpha (PGC1A) are involved in the pathogenesis of multiple sclerosis (MS) by affecting the inflammatory response and reactive oxygen species production. LEP rs7799039 and LEPR rs1137101 genetic variants modify the serum LEP levels and PGC1A rs8192678 alters the PGC1A activity. The study objective was to explore the associations of these variants with susceptibility to MS, disease course/clinical parameters and also with peripheral blood mononuclear cell expression of the target genes and plasma LEP concentrations, in the study subjects. METHODS: The study groups included 528 patients with MS and 429 controls. TaqMan® assays were used for genotyping and gene expression quantification. The Chi-square, parametric and nonparametric tests and simple/multiple logistic regression were performed for the statistical analysis of data. RESULTS: A multiple logistic regression model including all three investigated variants, applied to patients (RRMS + SPMS) and controls, showed that PGC1A rs8192678 minor allele had an increased risk for the occurrence of disease, with OR (95%CI) = 1,32 (1,01-1,73), P = 0,04. Between-effect of gender and LEPR variant on the multiple sclerosis severity score (MSSS) was identified (P = 0,005). In male patients (relapsing-remitting and secondary progressive), LEPR minor allele carriers had increased MSSS (GG + AG vs AA, median (minimum-maximum) = 5,38 (0,64-9,88) vs 4,27 (0,78-9,63); P = 0,01, Padj = 0,03). In relapsing-remitting patients LEP rs7799039 affected the LEP gene expression (P = 0,006; Padj = 0,04). CONCLUSION: The current findings implicate an impact of investigated genetic variants on the pathogenesis of MS.

CORTICOSTEROIDS IN THE MANAGEMENT OF PEDIATRIC EPILEPSIES.

Epilepsy is one of the most common chronic diseases in children, and cannot be controlled with conventional antiepileptic drugs in 30% of cases. Therefore, in these cases, alternative approach such as corticosteroid therapy (CT) is used. The aim of this study was to analyze different types of CT used to treat drug-resistant childhood epilepsies, treated at Rijeka University Hospital Centre during a 5-year period (2016-2020). This retrospective study included 32 patients. The following parameters were analyzed: number of patients with a particular diagnosis, average age (in months) at the onset of epilepsy, average epilepsy duration (in months) prior to CT, average number of antiepileptic drugs used prior to CT, presence of changes on magnetic resonance imaging (MRI), presence of comorbidities, and types of CT. The average age at the onset of epilepsy was 14 months and average epilepsy duration prior to CT was 16 months. On average, 5 antiepileptic drugs were used prior to CT. MRI changes were present in 53.13% and comorbidities in 81.25% of study patients. Prednisone therapy was used in 28.13%, combined therapy with prednisone and methylprednisolone in 65.63%, and methylprednisolone in 6.25% of patients. Study results revealed the use of CT for particular diagnosis to differ among the centers, as well as within the same center, so it is important to highlight the importance of reaching universal guidelines for CT therapy of childhood epilepsies.

The Effects of Aronia melanocarpa Juice Consumption on the mRNA Expression Profile in Peripheral Blood Mononuclear Cells in Subjects at Cardiovascular Risk.

Foods and food products that contain polyphenols are proposed to modulate risk of cardiovascular disease. The aim of this three-arm, crossover, randomized, double-blind, placebo-controlled intervention study was to examine the impact of Aronia melanocarpa juice (AMJ), high-polyphenol (AMJ treatment, 1.17 g/100 mL polyphenols) and low-polyphenol (dAMJ treatment, 0.29 g/100 mL polyphenols) dose, on the transcriptome in peripheral blood mononuclear cells (PBMC) of 19 subjects at cardiovascular risk. Transcriptome data were obtained by microarray. Bioinformatic functional annotation analysis was performed on both the whole transcriptome datasets and the differentially expressed genes (DEGs). Expression of selected DEGs was validated by RT-qPCR. Administration of AMJ and dAMJ treatments during the two consecutive four-week treatment periods had additive effects on PBMC transcriptome profiles, with the most pronounced and specific effect noticed for AMJ in the last treatment period (TP3) of the trial. Between the high-dose and low-dose treatments in TP3, there was a multitude of overlapping DEGs and DEG-enriched biological processes and pathways, which primarily included immunomodulation and regulation of cell proliferation/death. Increased expression of TNF, IL1B, IL8, RGS1, OSM, and DUSP2 in TP3 was confirmed by RT-qPCR. The results suggest the immunomodulatory effects of prolonged habitual consumption of polyphenol-rich aronia juice in individuals at cardiovascular risk.

Expression of LEP, LEPR and PGC1A genes is altered in peripheral blood mononuclear cells of patients with relapsing-remitting multiple sclerosis.

Leptin (LEP) may contribute to the pathogenesis of multiple sclerosis (MS) by its immunomodulatory, proinflammatory and prooxidant effects. Therefore, plasma LEP levels and mRNA expression of five genes related to the LEP signaling pathway (LEP, LEP receptor (LEPR), peroxisome proliferator-activated receptor-gamma coactivator 1-alpha (PGC1A), superoxide dismutase 2, tumor necrosis factor-alpha) were investigated in relapsing-remitting MS. In patients (N = 64), compared to healthy subjects (N = 62), relative LEP mRNA levels were significantly increased (p = 0,01), while LEPR and PGC1A mRNA levels were decreased (p = 0,001 and p = 0,04, respectively). Significant positive correlation was observed between LEPR mRNA levels and clinical parameters of MS progression (EDSS, MSSS).