A Novel Likely Pathogenic Variant in the RUNX1 Gene as the Cause of Congenital Thrombocytopenia.

Introduction: Heterozygous pathogenic and likely pathogenic sequence variants in the RUNX1 (Runt-related Transcription Factor 1) gene are a common genetic cause of decreased platelet count and/or platelet dysfunction and an increased risk of developing myelodysplasia and acute myeloid leukemia. The majority of causative variants are substitutions, which rarely occur de novo. The aim of this case report is to present a patient with congenital thrombocytopenia caused by a deletion variant in exon 9 in the RUNX1 gene. Case report: A one-month-old male infant was admitted to the Clinical Hospital Center Rijeka because of anemia and thrombocytopenia verified in the course of an acute viral infection. During follow-up, he occasionally had petechiae and ecchymoses on the lower extremities after mild trauma, with no other symptoms. The patient had persistent slightly decreased values of platelets with normal morphology, but with pathological aggregation with adrenaline and adenosine diphosphate. Due to the unclear etiology of persistent mild thrombocytopenia, he was referred for genetic testing at the age of five. Genomic DNA was isolated from the patient's peripheral blood and whole-exome sequencing was performed using the next-generation sequencing method. A heterozygous frameshift variant, c.1160delG (NM_001754.4), was identified in exon 9. The variant is classified as likely pathogenic. Conclusion: To the best of our knowledge, the heterozygous variant c.1160delG in the RUNX1 gene was first described in our patient. Although pathogenic variants in the RUNX1 genes are very rare, persistently low platelet counts of unclear etiology should raise suspicion of an underlying genetic disorder.

Melatonin levels in human diabetic dental pulp tissue and its effects on dental pulp cells under hyperglycaemic conditions.

AIM: To investigate melatonin (MEL) levels in human dental pulp tissue (hDP) in type 2 diabetic (T2D) participants and the underlying molecular mechanisms of its effects in human dental pulp cells (hDPCs) under hyperglycaemia. METHODOLOGY: The study included 16 healthy and 16 T2D participants who underwent vital pulp extirpation for hDP and four healthy participants undergoing third molar extraction for hDPCs analyses. MTT and NRU were used as tests for cytotoxicity. The pulp tissue levels of MEL, inducible NO synthase (iNOS) and superoxide dismutase (SOD) activity, as well as iNOS, histone acetyltransferase p300 (p300) and SOD activity levels in hDPCs incubated with MEL (0.1 and 1.0 mmol L-1 ) under normoglycaemia and hyperglycaemia were measured by enzyme-linked immunosorbent assay. Comparisons between the two groups were made by unpaired t-tests or Mann-Whitney test whilst the chi-square test was used for dichotomous variables. To compare more groups, the Kruskal-Wallis test with Dunn's multiple comparison was used, whilst Spearman correlation was used to assess association between two variables. RESULTS: Melatonin was decreased (124.30 ± 21.6 vs. 240.0 ± 19.1 pg mL-1 , P < 0.01), whilst iNOS levels increased (0.92 ± 0.08 vs. 0.32 ± 0.09 ng mL-1 , P < 0.01) in hDP from T2D compared to nondiabetic participants. In hDPCs, MEL (0.1 and 1.0 mmol L-1 ) had no cytotoxicity. Incubation with 1.0 mmol L-1 of MEL (24 h) decreased hyperglycaemia-induced increases of iNOS (0.34 ± 0.01 ng mL-1 vs. 0.40 ± 0.01 ng mL-1 , P < 0.01) and p300 (11.59 ± 0.58 ng mL-1 vs. 16.12 ± 0.39 ng mL-1 , P < 0.01), and also, increased SOD activity (87.11 ± 3.10% vs. 68.56 ± 3.77%, P < 0.01) to the levels comparable to the normoglycaemic; iNOS and p300 protein expression levels showed strong positive correlation under hyperglycaemia (Spearman r = 0.8242, P < 0.001). CONCLUSION: Type 2 diabetic participants had decreased MEL in hDP. At pharmacological concentrations, MEL is not cytotoxic for hDPCs and normalizes iNOS and SOD activity levels in hyperglyceamic hDPCs suggesting its antioxidant and protective effects in human dental pulp tissue under hyperglycaemia.

Reduced muscarinic parotid secretion is underlain by impaired NO signaling in diabetic rabbits.

OBJECTIVES: The influence of experimental diabetes (alloxan, 100 mg kg(-1) ) was studied on rabbit parotid gland function. MATERIAL AND METHODS: Carbachol-induced parotid secretion in vivo, and in vitro quantification of inducible nitric oxide synthase (iNOS) mRNA expression, by real-time RT-PCR, and activity of superoxide dismutase (SOD) and total antioxidant capacity (TAC) in commercial colorimetric assays were measured in parotid glands of non-diabetic and diabetic rabbits. RESULTS: Carbachol-induced dose-dependent increase in parotid secretion significantly reduced in diabetic rabbits. Functional studies in the presence of muscarinic receptor and nitric oxide synthase (NOS) antagonists revealed that in M3 receptor-mediated carbachol secretion, nitric oxide, deriving mainly from neuronal NOS (nNOS) in control, and iNOS in diabetic rabbits, was involved. Also, upregulation of iNOS mRNA expression and enhanced SOD activity and TAC were detected in diabetic glands. CONCLUSIONS: Our data suggest that decreased M3 receptor-mediated parotid secretion in diabetic rabbits appears to be due to alterations in NO signaling, mainly due to iNOS induction, accompanied by elevated antioxidant response.

The effects of anti-hypertensives and type 2 diabetes on salivary flow and total antioxidant capacity.

OBJECTIVE: The present cross-sectional study aimed to determine the effect of first-line anti-hypertensive drugs (enalapril, metoprolol, and combinations of enalapril with metoprolol and/or hydrochlorothiazide) on salivary gland function and salivary total antioxidant capacity (TAC) in hypertensive patients with/without diabetes mellitus (DM) type 2. MATERIALS AND METHODS: Salivary gland function was measured as xerostomia (interview) and unstimulated whole saliva flow rate (UWSFR) in 447 subjects (387 hypertensive and 60 healthy). Salivary TAC was evaluated by spectrophotometric assay. RESULTS: Enalapril is not xerogenic, while metoprolol and drug combinations are. In the presence of DM type 2, all drugs, except metoprolol, had pronounced xerogenic effect. Binary logistic regression analysis found enalapril to be significantly associated with decreased risk of xerogenic effect development, while DM type 2 with increased risk. In the presence of enalapril in hypertensive patients with/without DM type 2 salivary TAC was similar to that in healthy subjects, while for metoprolol was reduced. CONCLUSIONS: Enalapril is not xerogenic but is antioxidant, which moderately reduces the risk of xerogenic effect development even in the presence of DM type 2. However, metoprolol and drug combinations exhibit xerogenic effect. In DM type 2, xerogenic effect of all drugs was pronounced except of metoprolol.

Lidocaine+clonidine for maxillary infiltration anaesthesia: parameters of anaesthesia and vascular effects.

The local anaesthetic and haemodynamic parameters achieved by lidocaine with clonidine or epinephrine, administered for maxillary infiltration anaesthesia, were studied in 40 patients (American Society of Anesthesiologists, physical status 1) who underwent upper third molar surgery. All patients received 2 ml of 2% lidocaine with clonidine (15 microg/ml; n=20) or epinephrine (12.5 microg/ml; n=20) in a randomized, double-blind fashion. Vascular effects were evaluated on the isolated human infraorbital arteries. The parameters of maxillary infiltration anaesthsia produced by a combination of lidocaine+clonidine were similar to those obtained with lidocaine+epinephrine. In both groups, haemodynamic parameters exhibited similar variations, with the exception of a significant reduction in heart rate and systolic blood pressure in the lidocaine+clonidine group and significant increase in heart rate in the lidocaine+epinephrine group, 10 min after surgery. Clonidine (10(-7), 10(-6) and 10(-5)M) produced an endothelium-independent vasocontractile effect on the isolated human infraorbital arteries. The results of this study indicate for the first time in dental anaesthesia that the lidocaine+clonidine combination could be a useful and safe alternative to lidocaine+epinephrine for intraoral infiltration anaesthesia.

Responses of the human submandibular artery to ACh and VIP.

Endothelial vasodilatory substances may play a central role in the local regulation of vascular tone. We hypothesized that these substances can mediate endothelium-dependent vasodilatory responses to acetylcholine (ACh) and vasoactive intestinal peptide (VIP) in the human submandibular artery. We evaluated the contributions of endothelial vasodilatory substances to vessel relaxation in response to ACh and VIP, using different inhibitors of endothelial vasodilation, the nitric oxide synthase inhibitor, the cyclo-oxygenase inhibitor, indomethacin, the potassium channel blocker, and 4-aminopyridine. ACh and VIP caused an endothelium- and concentration-dependent relaxation in this artery. ACh relaxation was completely blocked after the concomitant addition of N(G)-nitro-L-arginine and indomethacin. The vasorelaxant effect of ACh was not influenced by 4-aminopyridine. VIP relaxation was almost completely abolished by 4-aminopyridine, and was partly inhibited by N(G)-nitro-L-arginine, but was not affected by indomethacin. Thus, in the human submandibular artery, ACh and VIP produced endothelium-dependent vasodilation with different underlying mechanisms: release of nitric oxide (NO) and cyclo-oxygenase products for ACh, and release of NO and endothelium-derived hyperpolarizing factor for VIP.

[Intra-abdominelle Lymphangiome]. / Intra-abdominal lymphangioma - Case Report.

Intra-abdominal lymphangiomas are very rare. We report the unusual case of a 4-year-old boy, who presented with acute abdominal symptoms due to intracystic hemorrhage following a blunt abdominal trauma. After the diagnostic management, the radical resection was done with a good outcome.

[Familial adenomatous colonic polyposis]. / Obiteljska adenomatozna polipoza crijeva.

We described two patients (brother and sister) with familial adenomatous polyposis of the colon. It is an inherited disease with autosomal dominant pattern of inheritance. The incidence is 1:8.000, with usual onset of polyps development late in the first decade of life or during adolescence, and malignant alteration up to the fourth decade of life. APC gene located on long arm of chromosome 5 is responsible for occurrence of the disease that presents with onset of multiple adenomatous polyps in the colon (from some of them to 1000). The treatment includes chemoprevention by sulindac or aspirin that prevents or reverse process of carcinogenesis. Surgical approach is preventive colectomy up to 20 (25) years of life. APC gene mutation (deletion at codon 1309-1311) was proven by DNA analysis from blood and polyp in both patients. There was no evidence of mutations of genes p53 and K-ras. Preventive colectomy is planned as soon as possible.

Mutator phenotype in human hematopoietic neoplasms and its association with deletions disabling DNA repair genes and bcl-2 rearrangements.

As mice carrying mutations of the DNA mismatch repair genes MSH2 and MSH6 often develop lymphoid neoplasms, we addressed the prevalence of the replication error (RER(+)) phenotype, a manifestation of an underlying defect of DNA mismatch repair genes, in human lymphoid tumors. We compared microsatellite instability (MSI) at 10 loci in 37 lymphoid tumors, including 16 acute lymphoid leukemias (ALL) and 21 non-Hodgkin's lymphomas (NHL), and in 29 acute myeloid leukemias (AML). Significant differences in MSI prevalence between AMLs and ALLs emerged, and MSI occurrence was more frequent in the NHLs versus AMLs. Indeed, only 3 of 29 (10%) AMLs exhibited MSI, thus confirming its paucity in myeloid tumors, while 10 of 37 (27%) lymphoid tumors, 6 ALLs and 4 NHLs, disclosed an RER(+) phenotype. In 1 ALL patient, the same molecular alterations were observed in correspondence with a relapse, but were not detected during remission over a 14-month follow-up; in another ALL patient, findings correlated with impending clinical relapse. These results suggest that the study of MSI in lymphoid tumors might provide a useful molecular tool to monitor disease progression in a subset of ALLs. To correlate MSI with other known genetic abnormalities, we investigated the status of the proto-oncogene, bcl-2, in the lymphoma patients and found that 4 of 4 NHL patients with MSI carried bcl-2 rearrangements, thus linking genomic instability to enhanced cell survival in NHL; moreover, no p53 mutations were found in these patients. Finally, we addressed the putative cause of MSI in hematopoietic tumors by searching for both mutations and deletions affecting DNA repair genes. A limited genetic analysis did not show any tumor-specific mutation in MLH1 exons 9 and 16 and in MSH2 exons 5 and 13. However, loss of heterozygosity (LOH) of markers closely linked to mismatch repair genes MLH1, MSH2, and PMS2 was demonstrated in 4 of 6 ALLs and 1 of 3 AMLs with MSI. These observations indicate that chromosomal deletions might represent a mechanism of inactivation of DNA repair genes in acute leukemia.