Diagnostic exome sequencing of syndromic epilepsy patients in clinical practice.

Although genetic revolution of recent years has vastly expanded a list of genes implicated in epilepsies, complex architecture of epilepsy genetics is still largely unknown, consequently, universally accepted workflows for epilepsy genetic testing in a clinical practice are missing. We present a comprehensive NGS-based diagnostic approach addressing both the clinical and genetic heterogeneity of disorders involving epilepsy or seizures. A bioinformatic panel of 862 epilepsy- or seizure-associated genes was applied to Mendeliome (4813 genes) or whole-exome sequencing data as a first stage, while the second stage included untargeted variant interpretation. Eighty-six consecutive patients with epilepsy or seizures associated with neurodevelopmental disorders and/or congenital malformations were investigated. Of the 86 probands, 42 harbored pathogenic and likely pathogenic variants, giving a diagnostic yield of 49%. Two patients were diagnosed with pathogenic copy number variations and 2 had causative mitochondrial DNA variants. Eleven patients (13%) were diagnosed with diseases with specific treatments. Besides, genomic approach in diagnostics had multiple additional benefits due to mostly non-specific, overlapping, not full-blown phenotypes and abilities to diagnose novel and ultra rare epilepsy-associated diseases. Likely pathogenic variants were identified in SOX5 gene, not previously associated with epilepsy, and UBA5, a recently associated with epilepsy gene.

Biochemical, pathological and oncological relevance of Gb3Cer receptor.

Glycosphingolipids are amphipathic molecules composed of hydrophilic oligosaccharide chain and a hydrophobic ceramide part, located primarily in the membrane microdomains of animal cells. Their oligosaccharide chains make them excellent candidates for the cell surface recognition molecules. Natural glycosphingolipid, globotriaosylceramide (Gal α1-4, Gal ß1-4, Glc ß1-1, ceramide), is also called CD77 and its expression was previously associated with proliferating centroblasts undergoing somatic hypermutation, but it has been demonstrate that globotriaosylceramide is not a reliable marker to discriminate human centroblasts from centrocytes. Globotriaosylceramide constitutes rare P k blood group antigen on erythrocytes, and it is also known as Burkitt's lymphoma antigen. On endothelial cells, globotriaosylceramide plays as the receptor for bacterial toxins of the Shiga family, also called verotoxins. Precise biological function and significance of globotriaosylceramide expression on endothelial cells remains to be the subject of many studies and it is believed globotriaosylceramide represents an example of a glycolipid antigen able to transduce a signal leading to apoptosis. In past decade, cancer researches put a great afford in determining new therapeutic agents such as bacterial toxins against tumor malignancies. Reports have demonstrated that verotoxin-1 induces apoptosis in solid tumor cell lines expressing globotriaosylceramide such as astrocytoma, renal cell carcinoma, colon cancer and breast cancer due to verotoxin-1 high specificity and apoptosis-inducing properties, and therefore, it is suggested to be an anticancer agent. Verotoxins have been investigated weather they could reduce treatment side-effects and toxicity to normal tissues and become a new oncological tool in cancer labeling.

Dose dependent effects of standardized nose-horned viper (Vipera ammodytes ammodytes) venom on parameters of cardiac function in isolated rat heart.

Direct, dose dependent effects of the nose-horned vipers (Vipera ammodytes ammodytes) venom on various parameters of cardiac action in isolated rat hearts were examined. Biochemical (protein content, SDS polyacrylamide gel electrophoresis) and biological (minimum haemorrhagic and necrotizing dose and lethal dose (LD(50))) characterization of the venom was performed before testing. The hearts were infused with venom doses of 30, 90 and 150 microg/mL for 10 min followed by 30 min of wash out period. Left ventricular pressure, coronary flow, heart rate, atrioventricular conduction, myocardial oxygen consumption, incidence and duration of arrhythmias were measured and relative cardiac efficiency was calculated. Cardiac CPK, LDH, AST and troponin I were measured as biochemical markers of myocardial damage. The venom caused dose dependent electrophysiological instability and depression of contractility and coronary flow. Effects on the heart rate were biphasic; transient increase followed by significant slowing of the frequency. Relative cardiac efficiency decreased as oxygen consumption remained high relative to the heart rate-contractility product, indicating purposeless expenditure of oxygen and energy. Effects by the dose of 30 microg/mL were highly reversible while the dose of 90 mug/mL caused damages that were mostly irreversible. The dose of 150 mug/mL induced irreversible asystolic cardiac arrest.

Immunohistochemical analysis of hepatic ganglioside distribution following a partial hepatectomy and exposure to different hyperbaric oxygen treatments.

Ganglioside GM3(Neu5Ac) expression is highly increased in liver 54h following 15% partial hepatectomy in pre-operatively oxygenated rats. GM3(Neu5Gc), GM2, GalNAc-GM1b and gangliosides of the neolacto-series are less affected. GM3(Neu5Ac) is a potent inhibitor of epidermal growth factor signaling. Since GM3(Neu5Ac) growth inhibitory effect depends on its cellular localization, the aim of this study was to detect ganglioside cellular localization during liver regeneration. The experiment was performed using the same rat model which previously showed increased ganglioside expression and more efficient liver regeneration. Frozen sections of liver were analyzed using confocal microscopy after labeling for binding of five ganglioside-specific antibodies, with or without hepatocyte membrane permeabilization. Ganglioside precursors, ceramide (Cer), monohexaosylceramide and lactosylceramide (LacCer) were determined by high-performance thin-layer chromatography. Apoptosis was assessed by fluorescein-dUTP end-labeling of fragmented DNA. Liver of pre-operative oxygenated rats showed high perinuclear labeling of GM3(Neu5Ac) which was absent in post-operative oxygenated and control animals. In the same group, Cer content was lower, monohexaosylceramide and LacCer were absent, and content of apoptotic cells was significantly the lowest, compared to other groups examined (F=20.36, p=0.0001). These findings indicate that ganglioside GM3(Neu5Ac) may be involved in mediation of beneficial effects of pre-operatively oxygenation during the liver regeneration.

Haplotype and AGG interspersion analysis of FMR1 alleles in a Croatian population: no founder effect detected in patients with fragile X syndrome.

Several studies have suggested that fragile X syndrome (FRAXA), the most common inherited form of mental retardation, originated from a limited number of founder chromosomes. The aim of this study is to assess the genetic origin of fragile X syndrome in a Croatian population. We performed a haplotype analysis of the polymorphic loci DXS548 and FRAXAC1 in 18 unrelated fragile X and 56 control chromosomes. The AGG interspersion pattern of the FMR1 CGG repeat region was analyzed by sequencing. This is the first report on haplotype and AGG interspersion analysis of the fragile X syndrome gene in a Croatian population-the only eastern European population of Slavic origin analyzed so far. Our findings are intriguing, because they show a distinct distribution of the DXS548 and FRAXAC1 alleles in our fragile X population compared to other European fragile X populations. The DXS548/FRAXAC1 haplotype 194/154 (7-3), which is common among normal populations, was found to be the most frequent haplotype in our fragile X population as well. The AGG interspersion analysis indicated that AGG loss rather than haplotype may determine FMR1 allele instability. Our results suggest that no common ancestral X chromosome is associated with fragile X syndrome in the Croatian population studied. Further analysis of the origin of fragile X syndrome among other Slavic populations will be necessary to better define its eastern European distribution.

Platelet function analysis in children with Schönlein-Henoch syndrome.

BACKGROUND: Schönlein-Henoch syndrome (SHS) or anaphylactic purpura in childhood is the result of pathologic and immunologic responses to different antigens. These antigens could induce the formation of immune complexes responsible for vasculitis and their precipitation on the endothelium of small blood vessels. Purpuric bruises, hematuria, hematemesis, melena, or hematochesis may suggest coagulation disturbances. Increasing bleeding tendency may suggest platelet function disturbance. To examine the qualitative function of platelets in children with SHS, we decided to analyze its aggregation function. METHODS: Using the Born method of testing, we analyzed platelet aggregation in 24 children with SHS. RESULTS: Based on the aggregograms examined, we observed that most patients had abnormal aggregation curves, in which platelets demonstrated a block of release of the endogenous ADP, with or without disaggregation. CONCLUSIONS: One clinical symptom of SHS appearing in most patients is a mild or increased tendency toward bleeding. On measuring induced aggregation of platelets in children with SHS, we observed that the qualitative function of platelets was disturbed.

Etoposide as the basic and interferon-alpha as the maintenance therapy for Langerhans cell histiocytosis: a RTC.

The treatment of patients who suffer from a disseminated form of Langerhans cell histiocytosis (LCH) is still controversial. So far, few larger randomized studies have been performed. The authors present 3 patients with a disseminated form of LCH--4 months, 9 months, and 2 years old, respectively. The lesional Langerhans cells in each patient showed positive immunohistochemical reaction to S-100 protein and the presence of Birbeck granules was confirmed by electron microscopy. All the patients were treated with etoposide (VP-16), 200 mg/m2 for 3 consecutive days, with 15 cycles at intervals of 3 weeks between each cycle, followed by maintenance therapy with IFN-alpha. All 3 patients reached complete stabile remission. The patients were young, at high risk, with multiple-organ involvement of LCH, and two of them had obvious signs of organ dysfunction at presentation, suggesting a poor prognosis. All remain disease-free several years after therapy. The results suggest that INF-alpha may prevent recurrences in high-risk patients.

Trends in myocardial infarction in Middle Dalmatia during the war in Croatia.

The aim of this study was to determine the pattern of myocardial infarction (MI) incidence with regard to age, gender, infarction site, and the most important risk factors. All 3,454 patients hospitalized in coronary care units of Clinical Hospital Split between 1989 and 1997 were analyzed. In the 3-year period preceding the war, from 1989 to 1991, 1,024 patients were hospitalized because of MI. During the 3 years of full war activities, from 1992 to 1994, there were 1,257 patients (significantly more; p < 0.05). And in the 3-year period after the war, from 1995 to 1997, there were 1,173 patients. In the war period, there were 151 (12%) patients younger than 45 years of age (p < 0.05); of that number, 143 (95%) were men (significantly more than in the other two periods; p < 0.05) and 8 (5%) were women. In the period preceding the war, there were 66 (6.5%) patients younger than 45 years: 60 (91%) men and 6 (9%) women. In the period after the war, those numbers were 88 (7.5%), 81 (92%), and 7 (8%), respectively. The patients younger than 45 years (305) more often had MI of an inferior than an anterior site (49% vs. 28%; p < 0.001), whereas there was no difference in patients older than 45 years (36% vs. 37%; p > 0.05). The patients older than 45 years had significantly greater hospital mortality (21% vs. 4%; p < 0.001) and were more likely to have hypertension (51% vs. 15%; p < 0.001) as well as hypercholesterolemia (54% vs. 14%; p < 0.001). Smokers prevailed among those younger than 45 years (75% vs. 51%; p < 0.001). The number of hospitalized patients with MI was greatest during the war period. It included a significant increase in the incidence in men younger than 45 years (12% vs. 7%; p < 0.05), with smoking as the most important risk factor, especially for infarctions of inferior sites.

Correlation between symptomatology and site of acute myocardial infarction.

OBJECTIVE: We determined the occurrence of presenting symptoms in patients with different sites of acute myocardial infarction after controlling for age and conventional risk factors. METHODS: Hospital-based study of patients hospitalized because of first anterior (n=731), inferior (n=719) and lateral (n=96) infarction in Clinical Hospital Split between 1990 and 1994. Data form about presenting symptoms and clinical profile was completed for each patient. RESULTS: Anterior infarctions were more often presented by headache (adjusted odds ratio (OR)=1.67, 95% CI=1.06-2.62), weakness (OR=1.60, 95% CI=1.31-1.96), dyspnea (OR=1.40, 95% CI=1.14-1.72), cough (OR=2.24, 95% CI=1.59-3.16), vertigo (OR=2.04, 95% CI=1.40-2.99) and tinnitus (OR=2.09, 95%CI=1.06-4.14). Inferior infarctions were more often associated with epigastric (OR=1.71, 95%CI=1.30-2.24), neck (OR=1.47, 95% CI=1.10-1.98) and jaw pain (OR=2.16, 95% CI=1.42-3.27), sweating (OR=1.56, 95% CI=1.27-1.92), nausea (OR=2.01, 95%CI=l.64-2.46), vomiting (OR=1.55, 95% CI=1.22-1.97), belching (OR=1.57, 95% CI=1.21-2.03) and hiccups (OR=2.88, 95%CI=1.53-5.42). Patients with lateral infarctions were more likely to complain of left arm (OR=1.80, 95% CI=1.07-3.05), left shoulder (OR=1.82, 95% CI=1.19-2.79) and back pain (OR=2.40, 95% CI=1.28-4.46). Pain was less frequently reported by hypercholesterolemic (P=l.4x10(-7)), patients over 70 years (P=0.002), women (P=0.0007) and those with non-triggered infarction (P=0.0009), whereas those over 70 (P=1.7x10(-6)) and men (P=0.0003) were less likely to report other relevant symptoms. CONCLUSIONS: Our study suggests a linkage between different infarction sites and specific groups of symptoms. Furthermore, coronary patients should give their full attention to non-specific symptoms and any kind of discomfort.

A simple and rapid analysis of triplet repeat diseases by expand long PCR.

It is now known that 15 monogenic, mostly neurological, disorders are caused by the same type of mutations that occur in trinucleotide repeat sequences in certain genes. Since they share a nonspecific and variable phenotype, the accurate diagnosis could be made only by DNA analysis. We developed an Expand Long PCR assay that provides more reliable molecular diagnosis of such disorders. Its main characteristics are robust amplification of expanded alleles, simplicity, low cost and speed. We suggest the use of Expand Long PCR for routine molecular diagnosis of triplet repeat diseases, and present such analysis of the fragile X syndrome, myotonic dystrophy and Huntington's disease.

Liver angiosarcoma and hemangiopericytoma after occupational exposure to vinyl chloride monomer.

Various malignant tumors of the liver, especially liver angiosarcoma, have been described after occupational exposure to vinyl chloride monomer. We present the case records and pathologic findings of two plastic industry workers who had been exposed to high concentrations of vinyl chloride. These workers developed hepatic neoplasms, angiosarcoma, or hemangiopericytoma. We discuss the histogenesis of these tumors; the common vascular origin and the mutual transformation of these two tumors suggest that the hemangiopericytoma may also have developed during occupational exposure to high concentrations of vinyl chloride monomer.

Gender differences in triggering of acute myocardial infarction.

The frequencies of potential triggers of acute myocardial infarction differ between men and women. There is a possibility that anti-ischemic drugs protect against trigger-related infarctions.

Dermatological indicators of coronary risk: a case-control study.

OBJECTIVE: We examined the association of dermatological signs such as baldness, thoracic hairiness, hair greying and diagonal earlobe crease with the risk of myocardial infarction in men under the age of 60 years. METHODS: A hospital-based, case-control study included 842 men admitted for the first non-fatal myocardial infarction, the controls were 712 men admitted with noncardiac diagnoses, without clinical signs of coronary disease. The relative risks were estimated as odds ratios. Logistic regression was used to control for the confounding variables. RESULTS: Baldness, thoracic hairiness and earlobe crease were approximately 40% more prevalent in cases (P<10(-6) in each case). In both cases and controls, baldness and thoracic hairiness were frequently coexistent, as well as hair greying and earlobe crease (P<10(-4) in each case). After allowing for age and other established coronary risk factors, the relative risk of myocardial infarction for fronto-parietal baldness compared with no hair loss was 1.77 (95% CI 1.27-2.45) and it was 1.83 (95 CI 1.4-2.3) for men with thick, extended thoracic hairiness. The presence of a diagonal earlobe crease yielded a relative risk of 1.37 (95% CI 1.25-1.5), while hair greying was associated with myocardial infarction only in men under the age of 50 years. CONCLUSION: It appears that baldness, thoracic hairiness and diagonal earlobe crease indicate an additional risk of myocardial infarction in men under the age of 60 years, independently of age and other established coronary risk factors.

Triggers of acute myocardial infarction regarding its site.

We have studied the incidence of possible triggers of the myocardial infarction regarding its site in 750 patients with anterior and 731 patients with inferior infarction. Infarctions occurred most frequently without recalling any triggering activity, especially in patients with anterior infarction (67 vs. 44%). Physical effort as the possible precipitator was also more frequent in anterior infarctions (22 vs. 16%). However, the onset of inferior infarction was more frequent during meteorological stress (9 vs. 2%), emotional stress (10 vs. 3%), after overeating (13 vs. 3%) and nicotine abuse (6 vs. 1.5%). These triggers were independent and highly significant (P < 0.02 in each case) discriminators of the site of myocardial infarction. Bimodal circadian rhythm, with primary peak between 6 and 9 h a.m. and the secondary peak between 3 and 6 p.m. was observed in patients which did not recall any triggering activity, and this was more pronounced in patients with inferior infarction. These results support the hypothesis that the influence of the vegetative tone is most pronounced in the onset of myocardial infarction of inferior wall.

Deletions in the SMN and NAIP genes in patients with spinal muscular atrophy in Croatia.

Two genes, i.e. survival motor neuron (SMN) and neuronal apoptosis inhibitory protein (NAIP) have been mapped to the SMA region of chromosome 5q13. Both genes are frequently deleted or truncated in SMA patients. We have studied 26 patients with SMA types I-III, 29 first relatives, and 14 subjects with mild adult-onset type IV. DNA deletion genotypes were determined by PCR techniques amplifying exons 7 and 8 of SMN, and exon 5 of NAIP gene which distinguish SMN and NAIP telomeric copy from a non-pathogenic gene homologue as a centromeric copy. Results revealed the homozygous deletions of exon 7 and 8 of the SMN gene and exon 5 of the NAIP gene in 3/3 infants with SMA I and in 1/20 with SMA type II. Exons 7 and 8 of the SMN gene were homozygously deleted in 10/20 and only exon 7 in 6/20 children with SMA type II. The overall percentage of deletion cases observed was 77% in children with SMA types I-III. Adult patients with type IV SMA showed no homozygous deletion of exons 7, 8 and 5 of the SMN and NAIP genes. Also, all relatives had both a telomeric and centromeric SMN and NAIP copy. Deletion analysis of SMN and NAIP genes are a significant diagnostic tool, because there are clinical entities resembling SMA which most likely have another pathogenetic background.

A Croatian case of the Schinzel-Giedion syndrome.

The Schinzel-Giedion syndrome is an infrequently described malformation syndrome, mainly characterized by a profound mental deficiency, a typical face including a midface hypoplasia, urogenital abnormalities, and minor radiographic features. Death prior to two year of age is the rule. A boy with typical features of the syndrome is described. He died at the age of 21 months. This is the first case of this syndrome reported from Croatia. The recurrence in only one of the 20 families, does not firmly sustain an autosomal recessive pattern of inheritance, although this still remains possible.

[Characteristics of patients with acute myocardial infarction treated in Split 1982-1992]. / Neke osobitosti bolesnika s akutnim infarktom miokarda lijecenih u Splitu od 1982. do 1992. godine.

Three thousand three hundred and fifty-two patients (70% males and 28% females) were hospitalized and treated in Split for acute myocardial infarction (hospital mortality 20%) between 1982-1992. The number of these admissions has been constantly rising from 226 in 1982 to 397 in 1992. Over 45 years of age were 3043 (90.8%) of the patients (70% males and 30% females), while 309 of them (9.2%) were under 45 (93% males, 7% females). The lethality rate was 21% in the first and 8% in the second subgroup. In patients over 45 inferior myocardial infarction was found in 35%, and in patients under 45 in 49%. In the older group there were 40% smokers, 42% hypertensives and 39% hypercholesterolemics, while there were 75% smokers, 35% hypertensives and 33% hypercholesterolemic patients in the younger age group. The results show that the number of patients treated for acute myocardial infarction in the Split region is constantly increasing (p < 0.001). In patients under 45 years of age there is significantly higher inferior infarction prevalence (p < 0.001); a larger percentage of males (p < 0.001) and smokers (p < 0.001), and lower percentage of hypertensives (p < 0.001) and hypercholesterolemic patients (p < 0.05) was observed, as well as lower hospital mortality rate (p < 0.001).