Human cytomegalovirus DNA detection in a recurrent glioblastoma multiforme tumour, but not in whole blood: a case report and discussion about the HCMV latency and therapy perspectives.

In the current study, a 58-year-old male patient presented with recurrent glioblastoma multiforme (GBM). The patient underwent surgical resection, 4 months earlier, followed by radiotherapy and chemotherapy. During the second surgical intervention, tumour tissue and whole blood were sampled and analysed for human cytomegalovirus (HCMV) DNA, immediate early (IE) mRNA and pp65 mRNA. HCMV DNA was detected only in the recurrent tumour tissue but not in the whole blood. Neither IE mRNA nor pp65 mRNA was expressed. Our result suggests HCMV latency in the brain tumour with detectable level of viral DNA. More data are needed to understand the HCMV infection chronology in the brain tumours but our data could be important for further studies of HCMV antigens on the tumour surface and anti-GBM therapy.

Height, selected genetic markers and prostate cancer risk: results from the PRACTICAL consortium.

This corrects the article DOI: 10.1038/bjc.2017.231.

Height, selected genetic markers and prostate cancer risk: results from the PRACTICAL consortium.

BACKGROUND: Evidence on height and prostate cancer risk is mixed, however, recent studies with large data sets support a possible role for its association with the risk of aggressive prostate cancer. METHODS: We analysed data from the PRACTICAL consortium consisting of 6207 prostate cancer cases and 6016 controls and a subset of high grade cases (2480 cases). We explored height, polymorphisms in genes related to growth processes as main effects and their possible interactions. RESULTS: The results suggest that height is associated with high-grade prostate cancer risk. Men with height >180 cm are at a 22% increased risk as compared to men with height <173 cm (OR 1.22, 95% CI 1.01-1.48). Genetic variants in the growth pathway gene showed an association with prostate cancer risk. The aggregate scores of the selected variants identified a significantly increased risk of overall prostate cancer and high-grade prostate cancer by 13% and 15%, respectively, in the highest score group as compared to lowest score group. CONCLUSIONS: There was no evidence of gene-environment interaction between height and the selected candidate SNPs.Our findings suggest a role of height in high-grade prostate cancer. The effect of genetic variants in the genes related to growth is seen in all cases and high-grade prostate cancer. There is no interaction between these two exposures.

HPLC-UV and LC-MS Analyses of Acylquinic Acids in Geigeria alata (DC) Oliv. & Hiern. and their Contribution to Antioxidant and Antimicrobial Capacity.

INTRODUCTION: Geigeria alata is a traditional plant used in Sudanese folk medicine for treatment of diabetes, cough, epilepsy and intestinal complaints. OBJECTIVE: To analyze phenolic acids in Geigeria alata roots and leaves and to evaluate their antioxidant and antimicrobial activities. METHODOLOGY: Phenolic acids in the aqueous-methanol extracts were identified by LC-MS. Major compounds were isolated using low-pressure liquid chromatography. The quantitative analysis of phenolic acids was performed by a validated HPLC-UV method with limits of detection ranging from 0.04 to 0.57 µg/mL. 2,2-Diphenyl-1-picrylhydrazyl (DPPH), 2,2'-azinobis-(3-ethylbenzothiazine-6-sulphonic acid) (ABTS) and ferric reducing antioxidant power (FRAP) methods were used for antioxidant activity evaluation. In addition, the minimal inhibitory concentration and the minimal bactericidal concentration against a panel of pathogenic bacteria and fungi were determined by the broth microdilution test. RESULTS: For the first time protocatechuic, caffeic, p-coumaroylquinic, caffeoylsinapoylquinic, caffeoylferuloylquinic, three feruloylquinic, six caffeoylquinic acids, and a caffeic acid hexoside were detected in Geigeria alata roots by LC-MS. HPLC-UV analyses showed that 3,5-dicaffeoylquinic acid (25.96 ± 2.08 mg/g dry weight (DW)) was the most abundant phenolic acid in roots, while 4,5-dicaffeoylquinic acid (8.99 ± 0.56 mg/g DW) was the main compound present in leaves. 3,5-Dicaffeoylquinic acid demonstrated stronger radical scavenging activity and reducing power compared with the crude extracts and the positive control 5-caffeoylquinic acid. 3,4,5-Tricaffeoylquinic acid revealed the highest antibacterial potential against the penicillin sensitive and resistant Staphylococcus aureus strains, as well as methicillin-resistant S. aureus. CONCLUSION: The caffeoylquinic acids content of up to 6.22% in Geigeria alata roots establishes this species as a new source rich in these bioactive molecules. Copyright © 2016 John Wiley & Sons, Ltd.

Steroid-resistant nephrotic syndrome caused by novel WT1 mutation inherited from a mosaic parent.

Steroid-resistant nephrotic syndrome (SRNS) is a severe childhood disorder frequently progressing toward renal failure. Among its genetic causes are mutations in the Wilms tumor gene, WT1, which codes for a transcription factor with key role for the embryonic development of the genitourinary tract as well as for maintaining podocyte differentiation and slit diaphragm structure in adults. Defects in WT1 are associated with sporadic cases of both syndromic and isolated SRNS. We report here a novel WT1 mutation associated with SRNS in a female patient, which leads to a Cys428Ser substitution on protein level, affecting one of the cysteine residues responsible for zinc binding in the second zinc finger domain. Surprisingly, the mutation identified in the patient was found to be inherited from the healthy mosaic mother. The presence of mosaicism was confirmed using quantitative polymerase chain reaction (PCR) high-resolution melting. The clinical implications of this finding for the family are discussed.

Spectrum and frequencies of BRCA1/2 mutations in Bulgarian high risk breast cancer patients.

BACKGROUND: About 3885 women are diagnosed with breast cancer and 1285 die from the disease each year in Bulgaria. However no genetic testing to identify the mutations in high-risk families has been provided so far. METHODS: We evaluated 200 Bulgarian women with primary invasive breast cancer and with personal/ family history of breast cancer for the presence of unequivocally damaging germline mutations in BRCA1/2 using Sanger sequencing. RESULTS: Of the 200 patients, 39 (19.5 %) carried a disease predisposing mutation, including 28 (14 %) with a BRCA1 mutation and 11 (5.5 %) with a BRCA2 mutation. At BRCA1, 6 different mutations were identified, including 2 frameshifts, 1 nonsense and 1 missense that had been previously reported (c.5030_5033delCTAA, c.5263_5264insC, c.4603G > T, c.181 T > G), and 2 frameshifts, which were novel to this study (c.464delA, c.5397_5403delCCCTTGG). At BRCA2, 7 different frameshift mutations were identified, including 5 previously reported (5851_5854delAGTT, c.5946delT, c.5718_5719delCT, c.7910_7914delCCTTT,c.9098_9099insA) and 2 novel (c.8532_8533delAA, c.9682delA). A BRCA1 mutation was found in 18.4 % of women diagnosed with breast cancer at/or under the age of 40 compared to 11.2 % of women diagnosed at a later age; a BRCA2 mutation was found in 4 % of women diagnosed at/or under the age of 40 compared to 6.5 % of women diagnosed at a later age. A mutation was present in 26.8 % patients with a positive family history and in 14.4 % of women with a negative family history. The most prevalent mutation observed in 22 patients (11 %) was BRCA1 c.5263_5264insC, a known Slavic mutation with founder effect in Eastern European and AJ communities. Other recurrent mutations were BRCA2 c.9098-9099insA (2 %), BRCA1 c.181T > G (1 %) and BRCA2 c.5851_5854delAGTT (1 %). Notably, BRCA1 c.5263_5264insC represented 56 % of all mutations identified in this series. Of the 22 patients with BRCA1 c.5263_5264insC, 9 were diagnosed with early onset breast cancer, 11 with TNBCs, 4 with bilateral breast cancer, and 6 with both breast and ovarian cancer. CONCLUSIONS: This is the first comprehensive study of the BRCA1/2 mutation spectrum in Bulgaria and will assist the establishment of efficient protocols for genetic testing and individualized risk assessment for Bulgarian breast/ovarian cancer patients and healthy individuals at a high-risk.

Mosaic dominant TUBB4A mutation in an inbred family with complicated hereditary spastic paraplegia.

BACKGROUND: Mutations in TUBB4A have been associated with a spectrum of neurological conditions, ranging from the severe hypomyelination with atrophy of the basal ganglia and cerebellum syndrome to the clinically milder dystonia type 4. The presence of movement abnormalities was considered the common hallmark of these disorders. METHODS: Clinical, neurological, and neuroimaging examinations, followed by whole exome sequencing and mutation analysis, were performed in a highly consanguineous pedigree with five affected children. RESULTS: We identified a novel c.568C>T (p.H190Y) TUBB4A mutation that originated de novo in the asymptomatic mother. The affected subjects presented with an early-onset, slowly progressive spastic paraparesis of the lower limbs, ataxia, and brain hypomyelination, in the absence of dystonia or rigidity. CONCLUSIONS: Our study adds complicated hereditary spastic paraplegia to the clinical spectrum of TUBB4A-associated neurological disorders. We establish genotype-phenotype correlations with mutations located in the same region in the tertiary structure of the protein.

Acenocoumarol Pharmacogenetic Dosing Algorithms and Their Application in Two Bulgarian Patients with Low Anticoagulant Requirements.

BACKGROUND: The anticoagulant therapy with acenocoumarol is generally associated with a high risk of bleeding and thromboembolic events. PURPOSE: We applied eight already existing acenocoumarol dosing algorithms to Bulgarian patients with low acenocoumarol dose requirements and investigated which of these algorithms would predict most precisely the dose anticoagulant. MATERIALS AND METHODS: Two patients with Bulgarian origin were referred to the outpatient clinical laboratory of "St. Ekaterina" University Hospital for Cardiovascular Surgery and Cardiology, Sofia, Bulgaria. After obtaining written informed consent, both patients were genotyped for polymorphisms in genes for Cytochrome P450 2C9 (CYP2C9), Vitamin K epoxide reductase (VKORC1), Apolipoprotein E (APOE), and Cytochrome P450 4F2 (CYP4F2). RESULTS: All applied acenocoumarol dosing algorithms predicted relatively similar doses of coumarin anticoagulant in both patients. However, van Schie et al.'s algorithm allowed more accurate calculation of the optimal dose in our patients with extremely low acenocoumarol requirements. Genotyping of selected polymorphic variants in CYP2C9 and VKORC1 showed that both patients were compound heterozygotes for CYP2C9 (CYP2C9*2/*3) and homozygotes for both variants in VKORC1 (VKORC1 1173 T/T, and VKORC1-1639 A/A). This combination of genotypes suggested high sensitivity to acenocoumarol leading to the low anticoagulant dose requirements (0.25 and 1 mg/day, respectively) needed to reach the target International Normalized Ratio of 2.5-3.5. CONCLUSIONS: The genotyping of polymorphic variants in VKORC1 and CYP2C9, together with clinical and demographic parameters, can serve for more precise definition of the individual starting and maintenance doses of coumarin derivatives in each patient.

Gender-Specific Effect of CYP2C8*3 on the Risk of Essential Hypertension in Bulgarian Patients.

We conducted a case-control study to determine the contribution of polymorphisms in CYP2C8 (CYP2C8*3) and CYP2J2 (CYP2J2*7) to increased risk of coronary artery disease and essential hypertension in Bulgarians. The current analysis included 192 unrelated hypertensive patients, 261 patients with angiographically documented CAD (153 with myocardial infarction and 108 without myocardial infarction), and 496 population controls. The CYP2C8*3 and CYP2J2*7 polymorphisms were genotyped by TaqMan SNP Genotyping Assay. PLINK version 1.07 was used for the statistical analysis. No overall association was observed for the studied polymorphisms with coronary artery disease and essential hypertension. The frequency of -50T mutant allele of CYP2J2*7 was significantly higher in male with coronary artery disease without history of myocardial infarction (OR 2.16 95% CI 1.04-4.48 p = 0.035) compared to population control group, but this association did not survive after Bonferroni correction (p adj = 0.07). A significant association of CYP2C8*3 allele with increased risk of essential hypertension has found in men (OR 2.12 95% CI 1.18-3.81 p = 0.015) and this relationship remained significant after adjustment for multiple comparisons (p adj = 0.03). This is the first study showing significant gene-sex interaction for CYP2C8*3 with twofold increase in the relative risk of essential hypertension and a similar tendency for CYP2J2*7 associated with coronary artery disease without myocardial infarction in Bulgarian males. The association is not seen in females and in the whole group of patients. This result could be partly explained by the effect of estrogens on the vascular tone of coronary arteries and CYP2C8 gene expression.

In vivo investigation of homocysteine metabolism to polyamines by high-resolution accurate mass spectrometry and stable isotope labeling.

Polyamines are essential polycations, playing important roles in mammalian physiology. Theoretically, the involvement of homocysteine in polyamine synthesis via S-adenosylmethionine is possible; however, to our knowledge, it has not been established experimentally. Here, we propose an original approach for investigation of homocysteine metabolites in an animal model. The method is based on the combination of isotope-labeled homocysteine supplementation and high-resolution accurate mass spectrometry analysis. Structural identity of the isotope-labeled metabolites was confirmed by accurate mass measurements of molecular and fragment ions and comparison of the retention times and tandem mass spectrometry fragmentation patterns. Isotope-labeled methionine, spermidine, and spermine were detected in all investigated plasma and tissue samples. The induction of moderate hyperhomocysteinemia leads to an alteration in polyamine levels in a different manner. The involvement of homocysteine in polyamine synthesis and modulation of polyamine levels could contribute to a better understanding of the mechanisms connected with homocysteine toxicity.

The rs266729 single-nucleotide polymorphism in the adiponectin gene shows association with gestational diabetes.

AIM: Gestational diabetes mellitus (GDM) is a condition of impaired glucose tolerance during pregnancy in women without previous diagnosis of diabetes. It is associated with serious complications for both mother and child in the pre- and postnatal period. Moreover, women with GDM are at an increased risk of developing type 2 diabetes. Adiponectin is an important factor involved in the regulation of both carbohydrate and lipid metabolism. Polymorphisms in its gene (ADIPOQ) are known to affect the individual's predisposition to metabolic syndrome and type 2 diabetes. The aim of the current study was to investigate the possible association between three common single-nucleotide polymorphisms in ADIPOQ and gestational diabetes. METHODS: A total of 394 individuals were recruited to the study-130 pregnant women with GDM, 130 pregnant women without glucose intolerance and 134 female population controls. All subjects were genotyped for rs266729, rs2241766 and rs1501299 in the ADIPOQ gene. RESULTS: A significant association with the disease was observed for rs266729 (p = 0.0037). The rare G allele was found to be over-represented among controls (pregnant, population and pooled). While no association was found for rs2241766 and rs1501299, a GTG haplotype formed by the three polymorphisms was found to be more common among controls (0.004). CONCLUSION: The adiponectin promoter polymorphism rs266729 is associated with gestational diabetes. The minor G allele appears to confer protection against pregnancy-related diabetes mellitus. This effect is probably due to the influence of the variant on the adiponectin transcription regulation during gestation.

Introducing exome sequencing as part of the diagnostic algorithm for pediatric nephrology patients in Bulgaria - a single-center experience.

INTRODUCTION: In pediatric kidney patients, where clinical presentation is often not fully developed and renal biopsy too risky or inconclusive, it may be difficult to establish the underlying pathology. In cases such as these, genetic diagnosis may be used to guide the treatment, prognosis and counselling. Given the large number of genes involved in kidney disease, introducing next generation sequencing with extended gene panels as part of the diagnostic algorithm presents a viable solution. METHODS: A cohort of 87 consecutive independent cases (83 children and 4 terminated pregnancies) with renal disease were recruited. Exome sequencing with MiSeq or NovaSeq 6 000 (Illumina) platforms and analysis of extended gene panels was used for genetic testing. RESULTS: Depending on the presenting pathology, the cases were grouped as patients with glomerular disease, ciliopathies, congenital anomalies, renal electrolyte imbalances and chronic/acute kidney disease. The overall diagnostic yield was app. 42% (37 out of 87) with most disease-causing mutations found in COL4A3, COL4A4, COL4A5 and PKHD1 genes. A change or clarification of preliminary diagnosis, or adjustment of initial treatment plan based on the results from the genetic testing was made for app. one third of the children with meaningful genetic findings (11 out 37). DISCUSSION: Our results prove the value of targeted exome sequencing as non-invasive, versatile and reliable diagnostic tool for pediatric renal disease patients. Providing genetic diagnosis will help for better understanding of disease etiology and will give basis for optimal clinical management and insightful genetic counseling.

Clinical and genetic variability among Bulgarian patients with autosomal recessive spastic ataxia of Charlevoix-Saguenay.

BACKGROUND: Autosomal recessive spastic ataxia ofCharlevoix-Saguenay (ARSACS) is a rare neurodegenerative disorder characterizedby early-onset cerebellar ataxia, peripheral sensorimotor neuropathy, and lowerlimb spasticity. We present clinical andgenetic data of the first Bulgarian patients diagnosed with ARSACS by wholeexome sequencing (WES). METHODS: Variant filtering was performed usinglocally established pipeline and the selected variants were analysed by Sangersequencing. All patients underwent clinical examination and testingincluding the standard rating scales for spastic paraplegia and ataxia. RESULTS: Five different SACS gene variants, three of which novel, have been identified inpatients from three different ethnic groups. In addition to the classicalclinical triad, brain MRI revealed cerebellar atrophy, linear pontineT2-hypointensities, and hyperintense rim lateral tothalamus combined with retinal nerve fiber layer thickening on opticcoherence tomography (OCT). CONCLUSION: We expand the mutation, geographic, and phenotypic spectrum of ARSACS, adding Bulgaria to the world map of the disease, and drawing attention to the fact that it is still misdiagnosed. We demonstrated that brain MRI and OCT are necessary clinical tests for ARSACS diagnosis, even if one of the cardinal clinical features is lacking.

CK2 is acting upstream of MEK3/6 as a part of the signal control of ERK1/2 and p38 MAPK during keratinocytes autocrine differentiation.

Protein kinase CK2 (formerly termed "casein kinase II") is a ubiquitously in mammalian cells distributed Ser/Thr kinase, with global role in cell regulation. Although, the involvement of CK2 in cell signalling is vast-investigated, virtually nothing is known about its contribution to signal control of keratinocytes differentiation. Here we show that, in autocrine differentiating keratinocytes, inhibition of the CK2 activity induced by 4,5,6,7-tetrabromobenzotriazole (TBB) causes reciprocal changes in the activities of major signal transduction regulators of keratinocytes differentiation, i.e. ERK1/2 and p38 MAPK, without affecting their protein levels. The ERK1/2 activity is strongly suppressed, while the activity of p38 is increased. We have also found that the activity of upstream and specific for p38 MAPK kinase MEK3/6 is also stimulated by TBB. These original results clearly demonstrate the participation of CK2 in the signal transduction pathway controlling MEK3/6, p38 MAPK, and ERK1/2 in the used model system.

The effect of sample size on polygenic hazard models for prostate cancer.

We determined the effect of sample size on performance of polygenic hazard score (PHS) models in prostate cancer. Age and genotypes were obtained for 40,861 men from the PRACTICAL consortium. The dataset included 201,590 SNPs per subject, and was split into training and testing sets. Established-SNP models considered 65 SNPs that had been previously associated with prostate cancer. Discovery-SNP models used stepwise selection to identify new SNPs. The performance of each PHS model was calculated for random sizes of the training set. The performance of a representative Established-SNP model was estimated for random sizes of the testing set. Mean HR98/50 (hazard ratio of top 2% to average in test set) of the Established-SNP model increased from 1.73 [95% CI: 1.69-1.77] to 2.41 [2.40-2.43] when the number of training samples was increased from 1 thousand to 30 thousand. Corresponding HR98/50 of the Discovery-SNP model increased from 1.05 [0.93-1.18] to 2.19 [2.16-2.23]. HR98/50 of a representative Established-SNP model using testing set sample sizes of 0.6 thousand and 6 thousand observations were 1.78 [1.70-1.85] and 1.73 [1.71-1.76], respectively. We estimate that a study population of 20 thousand men is required to develop Discovery-SNP PHS models while 10 thousand men should be sufficient for Established-SNP models.

A Genetic Risk Score to Personalize Prostate Cancer Screening, Applied to Population Data.

BACKGROUND: A polygenic hazard score (PHS), the weighted sum of 54 SNP genotypes, was previously validated for association with clinically significant prostate cancer and for improved prostate cancer screening accuracy. Here, we assess the potential impact of PHS-informed screening. METHODS: United Kingdom population incidence data (Cancer Research United Kingdom) and data from the Cluster Randomized Trial of PSA Testing for Prostate Cancer were combined to estimate age-specific clinically significant prostate cancer incidence (Gleason score ≥7, stage T3-T4, PSA ≥10, or nodal/distant metastases). Using HRs estimated from the ProtecT prostate cancer trial, age-specific incidence rates were calculated for various PHS risk percentiles. Risk-equivalent age, when someone with a given PHS percentile has prostate cancer risk equivalent to an average 50-year-old man (50-year-standard risk), was derived from PHS and incidence data. Positive predictive value (PPV) of PSA testing for clinically significant prostate cancer was calculated using PHS-adjusted age groups. RESULTS: The expected age at diagnosis of clinically significant prostate cancer differs by 19 years between the 1st and 99th PHS percentiles: men with PHS in the 1st and 99th percentiles reach the 50-year-standard risk level at ages 60 and 41, respectively. PPV of PSA was higher for men with higher PHS-adjusted age. CONCLUSIONS: PHS provides individualized estimates of risk-equivalent age for clinically significant prostate cancer. Screening initiation could be adjusted by a man's PHS. IMPACT: Personalized genetic risk assessments could inform prostate cancer screening decisions.

Evaluation of recombinant caspase specificity by competitive substrates.

The specificity of 10 recombinant caspases was investigated using a set of competitive substrates. The caspase activity was determined by high-performance liquid chromatography using highly fluorescent peptides containing 2-aminoacridone (AMAC) as reporting group. The sequences of the used substrates were designed according to literature data for being specific for 10 of the caspases. The described approach allows the concentration changes of several substrates to be monitored simultaneously in a single sample. Because the substrates are in competitive conditions, the preferences of particular caspases to given peptide sequences are most clearly demonstrated. In the studied competitive assay conditions, all tested caspases except caspase 2 exhibit activity toward more than one substrate. None of the used peptide sequences was found to be highly specific for a defined caspase. The results obtained indicate that there is well-expressed group specificity among the caspases.

Peptide substrate for caspase-3 with 2-aminoacridone as reporting group.

Synthesis and properties of a new fluorescent/fluorogenic substrate Ac-DEVD-AMAC for caspase-3 are reported. The substrate is obtained by conventional Fmoc-based solid phase peptide synthesis and its properties are investigated with regard to fluorescence, sensitivity, applicability and kinetic constants. A non-traditional approach to assay the proteases activity using 2-aminoacridone labeled peptides is proposed. This approach utilizes the decrease of fluorescence intensity of a sample as a measure for the enzyme activity.