Type 1 von Willebrand Disease in a Pediatric Patient Caused by a Novel Heterozygous Deletion of Exons 1 to 6 of the von Willebrand Factor Gene: A Case Report.

A 6-year-old boy was referred to a hematologist due to excessive mucocutaneous bleeding. Diagnostic assessment for von Willebrand disease (VWD) was indicated and included both coagulation and genetic testing. Laboratory testing revealed proportionally decreased von Willebrand factor (VWF) glycoprotein Ib-binding activity (23.6%) compared to VWF antigen (24.7%), similarly decreased VWF collagen-binding activity (24.2%), and normally distributed VWF multimers, with decreased intensity of all fractions. Diagnosis of type 1 VWD was established. Genetic analysis by means of next-generation sequencing (NGS) of VWF and coagulation factor VIII genes did not identify any causative mutations. Additionally, multiplex ligation-dependent probe amplification (MLPA) of VWF gene exons revealed a heterozygous deletion of exons 1 to 6, which is reported in type 1 VWD for the first time. Application of MLPA was crucial for revealing the genetic basis of type 1 VWD in this case, which would have remained undetected if only NGS was used.

Activation of NLRP3 inflammasome in stable chronic obstructive pulmonary disease.

Nucleotide-binding oligomerization domain-like receptor family pyrin domain-containing 3 (NLRP3) inflammasome activation plays an important role in chronic obstructive pulmonary disease (COPD) pathogenesis and might be involved in ongoing chronic inflammation. This study aimed to determine interleukin-1beta (IL-1ß) plasma concentration as well as IL1B, NLRP3 and caspase-1 (CASP1) gene expression in the Croatian COPD patients. 109 patients with stable COPD and age- and sex-matched 95 controls were included in the study. Plasma IL-1ß concentration was measured by Luminex technology, and gene expression analysis was performed using TaqMan assays. It was shown that COPD patients had increased concentration of IL-1ß and enhanced gene expression of IL1B, NLRP3 and CASP1 compared to controls. There was no difference in IL-1ß or IL1B, NLRP3 and CASP1 in patients with COPD regarding airflow obstruction severity and smoking history. Finally, the diagnostic potential of the determined parameters was evaluated, and it was found that IL-1ß correctly classified 89% of cases in the combination with common inflammatory biomarkers, white blood cell count and fibrinogen, showing a potential in COPD prediction. In conclusion, up-regulation of IL1B, NLRP3, CASP1 and increased IL-1ß concentration suggest the activation of NLRP3 inflammasome in the systemic compartment of patients with stable COPD.

Next-generation sequencing of von Willebrand factor and coagulation factor VIII genes: a cross-sectional study in Croatian adult patients diagnosed with von Willebrand disease.

AIM: To identify the von Willebrand factor (VWF) gene variant status in Croatian adult patients diagnosed with von Willebrand disease (VWD), provide differential diagnosis of VWD subtypes, and identify patients with mild hemophilia A (HA) who were earlier misdiagnosed as VWD. METHODS: Coagulation testing included determination of VWF gain-of-function mutant glycoprotein Ib binding activity (VWF:GPIbM), VWF antigen, VWF collagen-binding activity, and multimeric analysis. Genetic analysis of VWF and FVIII genes was performed with next-generation sequencing (NGS). RESULTS: The study enrolled 50 patients (72% women; median age 37 years, range 18-75) from 44 unrelated families. Fourteen patients were heterozygous for VWF gene variants compatible with type-1 VWD. Twelve had variants associated with type 2, of whom seven were classified as type 2A, four as type 2B, and one as type 2N. Six type-3 VWD patients were either homozygotes for null variants or combined heterozygotes. Eleven variants within the VWF gene were novel. Three female patients had variants within the FVIII gene, and were re-classified as mild-HA carriers, of whom one had causative novel variants both within VWF and FVIII genes. Fifteen patients remained without a defined genetic cause of their disorder, of whom five had VWF:GPIbM levels below 50%. CONCLUSION: Croatian adult patients with VWD have considerable genetic heterogeneity. NGS of both VWF and FVIII genes provided accurate differential diagnosis of VWD subtypes and distinction of VWD from mild HA.

Association of polymorphisms in genes encoding prothrombotic and cardiovascular risk factors with disease severity in COVID-19 patients: A pilot study.

The present study aimed to assess the association of 16 polymorphisms in genes encoding prothrombotic and cardiovascular risk factors with COVID-19 disease severity: FV G1691A, FV H1299R, FII G20210A, MTHFR C677T, MTHFR A1298, factor XIII V34L, PAI-1 4G/5G, EPCR haplotypes (A1/A2/A3), eNOS -786 T > C, eNOS G894T, LTA C804A, ACE I/D, ITGB3 PIA1/A2, ITGA2B Baka/b, ß-Fbg -455 G > A and ApoB R3500Q. The study included 30 patients with severe COVID-19 and 49 non-severe COVID-19 patients. All studied polymorphisms except ITGA2B Baka/b were determined using multilocus genotyping assays CVD StripAssays (ViennaLab Diagnostics), while ITGA2B was genotyped using a real-time PCR method based on TaqMan technology. A higher frequency of carriers of at least one ITGB3 PIA2 allele was found in severe COVID-19 patients (p = 0.009). The distribution of genotypes was significantly different for ß-Fbg -455 G > A (p = 0.042), with only three homozygous AA genotypes found among severe COVID-19 patients. The association with an increased risk for severe COVID-19 was found for ITGB3, with carriers of at least one ITGB3 PIA2 allele having a 3.5-fold greater risk of severe COVID-19 (p = 0.011). Genotype distribution differences were obtained for the combinations of FV H1299R and FXIII V34L (p = 0.026), ITGB3 PIA1/A2 and ITGA2B Baka/b (p = 0.024), and ACE I/D and PAI-1 4G/5G (p = 0.046). ITGB3 polymorphism emerged as an independent risk factor for severe COVID-19 and homozygosity for ß-Fbg -455 G > A mutation could contribute to disease severity. The combined effect of polymorphisms in genes encoding prothrombotic and cardiovascular risk factors could further contribute to disease severity.

Reevaluation of von Willebrand disease diagnosis in a Croatian paediatric cohort combining bleeding scores, phenotypic laboratory assays and next generation sequencing: a pilot study.

INTRODUCTION: This study reevaluated von Willebrand disease (vWD) diagnosis in a Croatian paediatric cohort by combining bleeding scores (BS), phenotypic laboratory testing, and next-generation sequencing (NGS). MATERIALS AND METHODS: A total of 25 children (11 males and 14 females, median age 10 years, from 2 to 17) previously diagnosed with vWD were included. BS were calculated using an online bleeding assessment tool. Phenotypic laboratory analyses included platelet count, platelet function analyser closure times, prothrombin time, activated partial thromboplastin time, von Willebrand factor antigen (vWF:Ag), vWF gain-of-function mutant glycoprotein Ib binding activity (vWF:GPIbM), vWF collagen binding activity (vWF:CBA), factor VIII activity (FVIII:C) and multimeric analysis. Next-generation sequencing covered regions of both vWF and FVIII genes and was performed on MiSeq (Illumina, San Diego, USA). RESULTS: Disease-associated variants identified in 15 patients comprised 11 distinct heterozygous vWF gene variants in 13 patients and one novel FVIII gene variant (p.Glu2085Lys) in two male siblings. Four vWF variants were novel (p.Gln499Pro, p.Asp1277Tyr, p.Asp1277His, p.Lys1491Glu). Three patients without distinctive variants had vWF:GPIbM between 30 and 50%. Patients with identified vWF gene variants had statistically significant lower values of vWF:GPIbM (P = 0.002), vWF:Ag (P = 0.007), vWF:CBA (P < 0.001) and FVIII:C (P = 0.002), compared to those without. Correlations between BS and phenotypic laboratory test results were not statistically significant for either of the tests. CONCLUSION: The applied diagnostic approach confirmed the diagnosis of vWD in 13 patients and mild haemophilia A in two. Limited utility of BS in the paediatric population was evidenced.

Increased HSP70 and TLR2 Gene Expression and Association of HSP70 rs6457452 Single Nucleotide Polymorphism with the Risk of Chronic Obstructive Pulmonary Disease in the Croatian Population.

Heat shock protein 70 (Hsp70) engages Toll-like receptors (TLR) 2 and 4 when found in the extracellular compartment and contributes to inflammation in chronic obstructive pulmonary disease (COPD). Since there is growing evidence for the genetic risk factors for COPD, the gene expression of HSP70, TLR2 and TLR4 was determined, as well as the association between HSP70, TLR2 and TLR4 single nucleotide polymorphisms, (SNPs) and COPD. The gene expression was assessed in peripheral blood cells of 137 COPD patients and 95 controls by a quantitative polymerase chain reaction (qPCR), while a total of nine SNPs were genotyped by TaqMan allelic discrimination real-time PCR. HSP70 and TLR2 gene expression was increased in COPD patients compared to the controls, regardless of the disease severity and smoking status of participants. The rs6457452 SNP of HSP70 was associated with COPD, indicating the protective role of the T allele (OR = 0.46, 95% CI = 0.24-0.89, p = 0.022). Furthermore, COPD C/T heterozygotes showed a decreased HSP70 mRNA level compared to COPD C/C homozygotes. In conclusion, HSP70 and TLR2 may have a role in the pathogenesis of COPD, and the HSP70 rs6457452 variant might influence the genetic susceptibility to COPD in the Croatian population.

Development and validation of a rapid method for genotyping three P-selectin gene polymorphisms based on high resolution melting analysis.

BACKGROUND: High resolution melting (HRM) analysis is one of the newer, reliable, and sensitive genotyping techniques, which offers considerable time and cost savings. P-selectin is an adhesion molecule that has a role in the initial phases of leukocyte adhesion to stimulated platelets and endothelial cells in inflammation. Multiple polymorphisms in P-selectin gene (SELP) that affect the protein sequence have been described. The aim of this study was to design, optimize, and validate a simple and rapid in-house HRM-based method for genotyping the NM_003005.3:c.992G>A (c.992G>A), NM_003005.3:c.1918G>T (c.1918G>T), and NM_003005.3:c.2266A>C (c.2266A>C) SELP polymorphisms. METHODS: Initial genotyping of three SELP polymorphisms was performed by applying polymerase chain reaction (PCR) with sequence-specific primers (SSP), which was used as a reference method for determination of analytical sensitivity. PCR-HRM was performed with primers for c.2266A>C reported in the literature. Primers for the remaining two polymorphisms were designed using Primer-BLAST. Precision testing was performed using three samples with different genotypes. For accuracy, analytical sensitivity and specificity testing, 20 wild type, 10 heterozygous, and 10 homozygous samples were chosen per polymorphism. Results were expressed as percentage of concordance with the acceptability criterion ≥95%. RESULTS: Agreement of results was 100% for all validation parameters except for analytical sensitivity for c.1918G>T and c.2266A>C, with agreement of 90%. Repeated analysis using both methods revealed an error in initial genotyping and correct genotyping by PCR-HRM, which was confirmed by Sanger sequencing. CONCLUSION: The validation confirmed PCR-HRM as a precise, accurate, and specific method for genotyping the c.992G>A, c.1918G>T, and c.2266A>C SELP polymorphisms.

PON1 gene polymorphisms in patients with chronic obstructive pulmonary disease.

AIMS: Chronic obstructive pulmonary disease (COPD) is characterised with oxidative stress. Paraoxonase 1 (PON1) is an enzyme, coded by PON1 gene, with distinctive antiatherogenic and antioxidative roles. We aimed to investigate the frequencies of Q192R, L55M and -108C>T polymorphisms and association of those polymorphisms with paraoxonase and arylesterase activities in patients with COPD. METHODS: PON1 genotype was determined by PCR-restriction fragment length polymorphism method. PON1 activity was measured by paraoxon and phenylacetate. RESULTS: Only -108C>T polymorphism resulted in significantly different distribution of genotypes and alleles, with higher frequency of TT genotype and T allele in patients compared with control subjects. Moreover, T allele (OR 2.29 (95% CI 1.54 to 3.41); p<0.001) as well as TT genotype (OR 5.00 (95% CI 2.19 to 11.43); p<0.001) showed an association with the disease. -108C>T polymorphism was suggested as a significant diagnostic predictor for the disease (OR (95% CI) 2.65 (1.53 to 4.59), p=0.001), with an area under the receiver operating characteristic curve of 0.90 (95% CI 0.84 to 0.93) and with 83.90% of correctly classified cases. CONCLUSIONS: Higher frequency of TT genotype and T allele could contribute to the observed reduction of PON1 activity in patients with COPD. T allele and TT genotype are associated with COPD, and the PON1-108C>T polymorphism could be a potential predictor of the disease.

Association of Polymorphisms in Coagulation Factor Genes and Enzymes of Homocysteine Metabolism With Arterial Ischemic Stroke in Children.

Despite the identification of a wide range of inherited and acquired risk factors for arterial ischemic stroke (AIS) in children, genetic risk factors are incompletely characterized and may vary among different populations. We investigated the role of individual and combined inherited prothrombotic and intermediate-risk factors in 73 children with perinatal (n = 35) and childhood AIS (n = 38) and 100 age- and sex-matched controls. Ten polymorphisms in 8 candidate genes encoding coagulation and fibrinolytic proteins (factor V [FV] Leiden, FV HR2, factor II [FII] G20210A, ß-fibrinogen [ß-FBG]-455G>A, factor XIII [FXIII]-A p.Val34Leu, plasminogen activator inhibitor 1 4G/5G), homocysteine metabolism (methylenetetrahydrofolate reductase [MTHFR] C677T, MTHFR A1298C), and intermediate-risk factors (angiotensin-converting enzyme I/D, apoE ∊2-4) were detected using a multilocus genotyping assay. Allele-specific polymerase chain reaction was used for the determination of human platelet alloantigens (HPA-1, HPA-2, HPA-3, and HPA-5). Factor V Leiden was associated with an increased risk of AIS (odds ratio [OR]: 4.72, 95% confidence interval [CI]: 1.22-18.27) and perinatal AIS (OR: 8.29, 95% CI: 1.95-35.24). Human platelet antigen-3b allele carriers had a 2-fold lower risk of AIS (OR: 0.51, 95% CI: 0.26-0.98) and perinatal AIS (OR: 0.40, 95% CI: 0.18-0.92). A 2.21-fold increased risk of childhood AIS (95% CI: 1.03-4.73) was identified in FXIII-A Leu34 allele carriers. Combined FV Leiden/FV HR2, FV Leiden/MTHFR A1298C, FV Leiden/MTHFR C677T/MTHFR A1298C, and FV Leiden/FV HR2/MTHFR A1298C heterozygosity was identified in children with AIS but not in controls, which revealed a statistically significant difference. This case-control study shows that besides already documented association between FV Leiden and AIS, other previously unreported polymorphisms (FXIII-A p.Val34Leu, HPA-3) and several genotype combinations that always include heterozygous FV Leiden can be related to AIS in Croatian population.

Bone morphogenetic protein-7 expression is down-regulated in human clear cell renal carcinoma.

BACKGROUND: Recent studies demonstrated that the expression pattern of bone morphogenetic protein-7 (BMP-7) is altered in different tumors. We determined expression of BMP-7 in human clear cell renal carcinoma (CCRC). METHODS: Samples from cancer and corresponding healthy tissue were obtained from 20 patients who underwent nephrectomy for CCRC. Expression of BMP-7 mRNA was determined by reverse transcriptase polymerase chain reaction (RT-PCR), and protein expression was analyzed by immunohistochemistry. RESULTS: RT-PCR showed strong down-regulation of BMP-7 mRNA in cancer tissue. Immunohistochemistry revealed expression of BMP-7 in normal renal tissue, with almost complete loss of BMP-7 expression in malignant cells of 6 patients (30%). After 3 years of follow-up, 5 out of 6 patients with high BMP-7 mRNA expression were alive and disease-free, compared with 9 out of 14 patients with low BMP-7 mRNA expression. CONCLUSIONS: BMP-7 mRNA and protein expression were down-regulated in CCRC. Further prospective studies are needed to characterize the role of BMP-7 in human CCRC.

Immunolocalization and mRNA expression of bone morphogenetic protein-6 in human clear cell renal carcinoma.

Bone morphogenetic proteins (BMPs) are members of the transforming growth factor-beta superfamily of proteins. Dysregulation of BMP signaling has been suggested in the carcinogenesis of different organs. We determined BMP-6 mRNA and protein expression in localized human clear cell renal carcinoma (CCRC), obtained from 20 patients who underwent nephrectomy, by the real-time polymerase chain reaction and immunohistochemistry. 15/20 patients exhibited higher BMP-6 mRNA expression in malignant than in healthy renal tissue relative to the PBGD expression (p < 0.05). Immunostaining intensity for BMP-6 in healthy renal tissue ranged from 0 to 2 (average 0.9), as well as in renal clear cell carcinoma (average 1.1). Seven of 20 (35%) healthy tissue samples failed to stain with BMP-6 antibody, compared to 2/20 (10%) tumor samples (p < 0.05). BMP-6 immunostaining was positive in 18/20 CCRC samples. Staining was localized in the cytoplasm and/or membrane of malignant cells. Malignant tissue had significantly higher BMP-6 mRNA expression than healthy tissue. There was no significant correlation between BMP-6 mRNA and protein expression with disease presentation, disease progression and patients' characteristics. Long-term follow-up of our patients is needed to determine the possible role of increased expression of BMP-6 in CCRC.