Changes in the expression of selenoproteins in mesial temporal lobe epilepsy patients.

Selenoproteins are enzymes containing selenium in their structure and are involved in cellular processes such as defense against oxidative stress and cell survival. The aim of this study is to investigate the expression of four selenoproteins (GPX1, TRXR1, SELP and SELW) in the hippocampus of intractable mesial temporal lobe epilepsy (MTLE) patients who underwent curative surgery. The selenoproteins is investigated at the mRNA level via RT-PCR and in situ hybridization and by immunostaining at the protein level. The expression of SELW exhibited a relative induction of more than tenfold, and immunostaining findings provided evidence that this upregulation is confined to neurons. GPX1 was also upregulated 2.3-fold, and TRXR1 was downregulated between 70 and 20% in MTLE patients. The profound induction of SELW has been accompanied by GPX1 and displayed a strong correlation with BCL2 expression, suggesting a protective role for these selenoproteins, and may be an indicator of a defense mechanism in surviving neurons.

Statin potentiates human platelet eNOS activity without enhancing eNOS mRNA and protein levels.

BACKGROUND/AIMS: Experimental studies suggest an enhanced endothelial and platelet nitric oxide (NO) generation after statin treatment, possibly due to increased endothelial NO synthase (eNOS) activity and protein levels. In parallel with experimental research, statins were shown to increase the forearm blood flow independently of serum cholesterol in humans. However, it was not possible to correlate blood flow changes with eNOS levels in these studies due to limitations in obtaining arterial samples. Hence, we investigated changes in eNOS activity, mRNA and protein levels after statin treatment in human platelets, which are readily accessible unlike arteries. METHODS: In vitro bleeding times were measured in 22 patients by stimulating platelets with collagen-epinephrine or collagen-ADP. To assess platelet eNOS activity, the bleeding times were also determined after incubating platelets with L-arginine. The measurements were repeated following 14 days of pravastatin (40 mg/day) treatment. Platelet-rich plasma was collected before and after statin treatment to evaluate eNOS mRNA (semiquantitative RT-PCR) and protein levels (Western blotting). RESULTS: The basal bleeding time was prolonged by 24 +/- 3% (mean +/- SE) when the samples were incubated with 500 microM of L-arginine. The NOS inhibitor L-N(5)-(I-iminoethyl)ornithine reversed this effect, suggesting that it was mediated by NO. After statin treatment, the NO-mediated prolongation of the bleeding time with 500 microM of L-arginine was significantly potentiated (to 44 +/- 10%). Despite enhanced eNOS activity, there was no significant change in platelet eNOS mRNA and protein levels after statin treatment. CONCLUSION: These data demonstrate that platelet eNOS activity is potentiated after statin treatment in humans in parallel with experimental studies.

Report of an international survey of molecular genetic testing laboratories.

OBJECTIVE: To collect data on the practices of molecular genetic testing (MGT) laboratories for the development of national and international policies for quality assurance (QA). METHODS: A web-based survey of MGT laboratory directors (n = 827; response rate 63%) in 18 countries on 3 continents. QA and reporting indices were developed and calculated for each responding laboratory. RESULTS: Laboratory setting varied among and within countries, as did qualifications of the directors. Respondents in every country indicated that their laboratory receives specimens from outside their national borders (64%, n = 529). Pair-wise comparisons of the QA index revealed a significant association with the director having formal training in molecular genetics (p < 0.005), affiliation with a genetics unit (p = 0.003), accreditation of the laboratory (p < 0.005) and participation in proficiency testing (p < 0.005). Research labs had a lower mean report score compared to all other settings (p < 0.05) as did laboratories accessioning <150 samples per year. CONCLUSION: MGT is provided under widely varying conditions and regulatory frameworks. The data provided here may be a useful guide for policy action at both governmental and professional levels.

The role of TAP1 and TAP2 gene polymorphism in idiopathic bronchiectasis in children.

Bronchiectasis is characterized by permanent changes in the structure and function of the airways. Its cause cannot be identified in some cases. A genetic disease can predispose to bronchiectasis in our country, where consanguinity of parents is common. Transporter associated with antigen presentation (TAP) deficiency syndrome is characterized by recurrent bacterial lower respiratory tract infections, which cause bronchiectasis. Our aim was to document the relationship between idiopathic bronchiectasis and TAP gene polymorphisms. Forty-four patients with idiopathic bronchiectasis and 100 healthy individuals as the control group were included. DNA was extracted and gene polymorphisms for TAP1 and TAP2 were studied. When compared to healthy controls, in the patient group, Ile/Ile genotype was decreased and Ile/Val genotype was increased in TAP1-333 polymorphism analysis; Asp/Asp and Gly/Gly genotypes were decreased and Asp/Gly frequency was increased in TAP1-637 polymorphism analysis; Ile/Val genotype was increased and Ile/Ile genotype was decreased in TAP2-379 polymorphism analysis; and Thr/Thr genotype frequency was decreased and Thr/Ala and Ala/Ala genotypes were increased in TAP2-665 polymorphism analysis. No statistically significant difference between patient and control groups was noted only in TAP2-565 polymorphism analysis. These results indicate that TAP gene polymorphisms may have had a role in the development of bronchiectasis in our patient group. Therefore, TAP deficiency syndrome should be considered in children with idiopathic diagnosis, since early diagnosis of the disease will improve life quality and survival.

Application of medical genetics in Turkey.

Turkey is among the most populous countries of the world, and has a young population structure. The rate of consanguinity has been approximately 20-25% for the last 25 years. Various studies have shown that high consanguinity can be a contributing factor to the high incidence of some rare autosomal recessive diseases. Hemoglobinopathies are an important health problem, and Turkey also has one of the highest incidences of phenylketonuria in the world. Training and education in medical genetics, established as a specialty since 1972, play an important role in the setting of genetic services and meeting public health problems. Prenatal and preimplantation diagnosis is available for a variety of fetal diseases.

Identification of two novel PNPLA1 mutations in Turkish families with autosomal recessive congenital ichthyosis.

Dökmeci-Emre S, Taskiran ZE, Yüzbasioglu A, Önal G, Akarsu AN, Karaduman A, Özgüç M. Identification of two novel PNPLA1 mutations in Turkish families with autosomal recessive congenital ichthyosis. Turk J Pediatr 2017; 59: 475-482. Autosomal recessive congenital ichthyosis (ARCI) is a group of inherited keratinization disorders that are characterized by abnormal epidermal keratinization. ARCI patients generally represent serious symptoms including collodion baby phenotype accompanied by dehydration, heat loss, electrolytic imbalance, and sepsis. ARCI shows high degree of clinical and genetic heterogeneity. To date, nine genes were shown to be responsible for ARCI phenotype. One of these genes, patatin-like phospholipase domain containing protein-1 (PNPLA1) was suggested to be involved in the synthesis of ω-O-acylceramides related to epidermal cornified lipid envelope organization. In addition to previously reported PNPLA1 mutations, we report two novel PNPLA1 mutations including one novel missense mutation c.335C > A (p.Ser112Tyr) and one novel deletion mutation c.733_735delTAC (p.Tyr245del) in Turkish ARCI patients from unrelated consanguineous families. We also report previously reported missense mutation c.514G > A (p.Asp172Asn) in Turkish ARCI patients. Novel PNPLA1 mutations were shown to be located in the catalytic patatin domain of PNPLA1 gene. Identification of novel mutations in PNPLA1 gene expands the mutational spectrum in the causative gene. Increase in the total number of cases has high diagnostic value in terms of genotype-phenotype correlation in ARCI patients.

Liposome-mediated intraperitoneal interleukin 10 gene transfer increases survival in cecal litigation and puncture model of sepsis.

Interleukin 10 (IL-10) has been considered to alleviate the inflammatory cytokine response in various models of sepsis. Although being regarded as a key immunomodulator molecule to be beneficial for the treatment of sepsis, recombinant IL-10 treatment is limited by efficacy and tolerability. We tested a novel approach and conducted i.p. liposomal IL-10 gene transfer 24 h before the cecal ligation and puncture in mice and observed 75% mortality at the end of the 7th day. The mortality was 100% in the group where the gene transfer was not performed. The transgene expression is observed mainly in the endothelium in all vital organs. The results demonstrate the advantageous role of de novo IL-10 synthesis in early stages of sepsis and suggest the beneficial impact of gene transfer approach to recombinant protein infusions.

Discovery of biomarkers in rare diseases: innovative approaches by predictive and personalized medicine.

There are more than 8000 rare diseases (RDs) that affect >5 % of the world's population. Many of the RDs have no effective treatment and lack of knowledge creates delayed diagnosis making management difficult. The emerging concept of the personalized medicine allows for early screening, diagnosis, and individualized treatment of human diseases. In this context, the discovery of biomarkers in RDs will be of prime importance to enable timely prevention and effective treatment. Since 80 % of RDs are of genetic origin, identification of new genes and causative mutations become valuable biomarkers. Furthermore, dynamic markers such as expressed genes, metabolites, and proteins are also very important to follow prognosis and response the therapy. Recent advances in omics technologies and their use in combination can define pathophysiological pathways that can be drug targets. Biomarker discovery and their use in diagnosis in RDs is a major pillar in RD research.

Four novel thymidine phosphorylase gene mutations in mitochondrial neurogastrointestinal encephalomyopathy syndrome (MNGIE) patients.

Mitochondrial neurogastrointestinal encephalomyopathy syndrome (MNGIE) is a rare autosomal recessive neurologic disorder characterised by multiple mitochondrial DNA deletions. In this study, five Turkish MNGIE patients are investigated for mtDNA deletions and TP gene mutations. The probands presented all the clinical criteria of the typical MNGIE phenotype; the muscle biopsy specimens also confirmed the diagnosis with ragged red fibres and cytochrome C oxidase (COX) negative fibres. The mitochondrial DNA analysis revealed no deletions in the probands' skeletal muscle samples. We have identified four novel mutations in the TP gene while one of the patients also harboured a nucleotide change, which was previously reported as a mutation.

Hypercalciuria and nephrocalcinosis in cystic fibrosis patients.

The objective of this study was to determine the frequency of nephrocalcinosis and hypercalciuria in cystic fibrosis (CF) patients, and to search possible causes of this phenomenon. Forty-three CF children (24 boys, 19 girls; mean age 64.9 months, range 5 months-18 years) were included in this study. Plasma sodium, potassium, chloride, BUN, creatinine, calcium, phosphorus, magnesium, alkaline phosphatase; spot urine sodium, potassium, chloride, creatinine, calcium, magnesium; and serum 25-hydroxyvitamin-D levels were measured in all patients. Urine samples were examined for microscopic hematuria. Fractional sodium, potassium, chloride excretion and estimated glomerular filtration rate (GFR) were calculated. All patients underwent renal ultrasonography. Hypercalciuria, nephrocalcinosis and microscopic hematuria were detected in 15 patients (34.2%), 10 patients (23.2%) and two patients (5%), respectively. There was no significant but borderline correlation between 25-hydroxyvitamin-D levels and hypercalciuria (r: 0.308, p:0.05). There were no correlations between Shwachman clinical scoring system results and hypercalciuria (r: 0.221, p: 0.148) and age and hypercalciuria (r: -0.229, p: 0.135). Patients with chronic Pseudomonas colonization showed no hypercalciuria or nephrocalcinosis. There was no difference for plasma biochemical results, renal function tests, hypercalciuria and nephrocalcinosis between CF patients who had or had not experienced pseudo Bartter's syndrome (PBS) before. There was no relation between detected CF mutations of the patients and hypercalciuria and nephrocalcinosis. These results suggested that it is a primary abnormality of calcium metabolism in the kidney.

Analysis of the modifying effects of TAP 1/2 genes on cystic fibrosis phenotype.

Phenotypic variability has been reported in cystic fibrosis (CF) patients. TAP1 and TAP2 genes are encoding "the transporter associated with antigen processing" proteins. The aim of the present study was to analyze the frequency of TAP 1/2 variants in the Turkish population and to investigate a possible modifying role of these variants in CF phenotype. Sixty-three CF patients of known genotypes and 100 healthy control subjects were analyzed. There was a significant difference in the frequencies at positions 333 and 637 of TAP 1 gene and at position 665 of TAP 2 gene between patients and controls. Comparison of TAP gene polymorphisms in 36 CF patients homozygous for AF508 mutation with control subjects revealed a significant difference at position 665 of TAP 2 gene. These findings may be useful to assess the predisposition and to predict severity of the disease. We demonstrated that TAP genes might have modifying effects on the CF phenotype.

The effect of mannose-binding protein gene polymorphisms in recurrent respiratory system infections in children and lung tuberculosis.

Mannose-binding lectin (MBL) is able to bind pathogens as an opsonin and plays an important role in the innate immunity. The aim of the present study was to determine the frequencies of the MBL gene variants in the Turkish population and to examine the presence of any association between MBL variants and development of tuberculosis (TB) in adults and recurrent respiratory tract infections in children. Two structural gene mutations in exon 1 of MBL gene (codon 54 and codon 57) were studied. The overall distribution of genotypes did not significantly differ between controls and TB patients/children with recurrent respiratory system infections. The frequency of allele B was calculated as 0.14, 0.09 and 0.06 for control, TB patients and children with recurrent respiratory system infections, respectively. It was found to be significantly lower in children with recurrent respiratory system infections than in controls (chi2: 4.68, d.f: 1, p: 0.030).

Biobanking: sample acquisition and quality assurance for 'omics' research.

After the human genome sequence has been solved using random individuals through the Human Genome Project (HGP), rapid advances in whole genome sequencing technologies with effective use at a reasonable cost, is moving the genomics research field to an era of 'personal genomes'. Biobanks in this context have played an important role by providing high quality biological samples for genomics and functional genomics research. Here we are describing biobanking and the importance of governance in biobanking activity for reliable and reproducible high throughput 'omics' data.

Genetic testing: predictive value of genotyping for diagnosis and management of disease.

This article describes predictive, preventive value of genetic tests and the implication of the use of testing for personalized treatment. This year marks the 10th anniversity of publishing of the sequence of the human genome. One important area of application of this mega project is a development of genetic tests for mutation detection in single gene disorders that has impact for pediatric age group patients and analyzing susceptibility genes as risk factors in common disorders. Types of genetic tests, new emerging technologies will enable developments of high-throughput approaches by microarrays of great application capacity as described here. As it is usual for all technologies used in health care, bioethical concerns has to be delt with. The ethical, social and governance issues associated with genetic testing are discussed.

Novel mutations of the MLC1 gene in Turkish patients.

Megalencephalic Leukoencephalopathy with Subcortical Cysts (MLC) is a rare autosomal recessive disease presenting with increased head circumference at birth or in early infancy. MLC1 (MIM 605908) mutations are responsible for this disorder. In this study, we sequenced the entire coding region of the MLC1 gene in 13 patients and detected five novel nucleotide variations in six of them. Two of the novel variations created a missense amino acid change and the other three were located in the introns and were putative splice mutations. One novel missense variation was observed in two unrelated patients from the central Black Sea region, and the data suggested a founder haplotype for this novel variation. Similarly, three unrelated patients with the previously reported p.Thr118Arg mutation shared a common haplotype. These data suggest an Anatolian origin for these two mutations. As in the previous reports, it is not possible to correlate the clinical phenotype of the patients with the mutation spectra.

TAP1 and TAP2 gene polymorphisms in childhood cystic echinococcosis.

The incidence of cystic echinococcosis (CE) due to Echinococcus granulosus is as high as 2000-2500 patients per year in Turkey. Whether genetic characteristics of the Turkish population cause a tendency to the disease is currently unknown. We aimed at studying the role of TAP gene polymorphisms in Turkish children with cystic echinococcosis. For an overview of allelic distribution of TAP1 and TAP2 genes, genotypes of 85 patients with CE and 100 controls were studied. To determine the genotype-phenotype correlation, 81 of the patients whose clinical data were available were analyzed. For TAP1-637, Asp/Gly heterozygosity was significantly more prevalent in CE patients than in controls (20 vs. 4%, odds ratio 6.0), while Gly/Gly homozygosity was less frequent (5 vs. 14%). For TAP2-379, Ile/Val heterozygosity was significantly more prevalent in CE patients than in controls (14 vs. 1%, odds ratio 16.27), while Ile/Ile homozygosity was less frequent (13 vs. 25%). TAP1-637 and TAP2-379 polymorphisms may have a role in causing genetic tendency for CE in children. The data may reflect the genetic properties of the Turkish population or may reveal the minor role of TAP gene polymorphisms in CE.

Interleukin-10 gene transfer: prevention of multiple organ injury in a murine cecal ligation and puncture model of sepsis.

INTRODUCTION: The aim of this study was to determine the effect of immunoregulatory cytokine interleukin-10 (IL-10) gene therapy on multiple organ injury (MOI) induced by a cecal ligation and puncture (CLP) model of sepsis in mice. METHODS: Male Balb/c mice subjected to CLP were treated with either an hIL-10-carrying vector or an empty control vector. We assessed the degree of lung, liver, and kidney tissue destruction biochemically by measuring myeloperoxidase (MPO) and malondialdehyde (MDA) activity. Histologic assessments were based on neutrophil infiltration in lung and liver tissue. IL-10 protein expression was examined immunohistochemically, and ultrastructural changes in the liver were studied by transmission electron microscopy. We analyzed the expression of tumor necrosis factor-alpha (TNFalpha) mRNA by reverse transcription polymerase chain reaction 3, 8, and 24 hours after CLP in all organs. RESULTS: Organ damage was significantly reduced by hIL-10 gene transfer, which was associated at the tissue level with reduced MPO activity in the liver, lung, and kidney and decreased leukocyte sequestration and MDA formation in the lung. The liver MDA was not significantly higher in the hIL-10 gene therapy group than in the controls and seemed not to be affected by hIL-10 gene transfer. The reduced portal tract neutrophilic infiltration and preserved ultrastructure of the hepatocytes also showed that tissue function was not impaired. The lung and kidney TNFalpha mRNA expression was suppressed markedly in the hIL-10 gene therapy group, but liver TNFalpha mRNA expression varied over time. CONCLUSIONS: These findings showed that IL-10 gene therapy significantly attenuated sepsis-induced MOI.

Androgen receptor immunostaining and androgen receptor messenger ribonucleic acid expression are increased in cremaster muscles associated with undescended testis.

OBJECTIVES: To determine and compare androgen receptor (AR) immunostaining and AR messenger ribonucleic acid (mRNA) expression in cremaster muscles associated with descended or undescended testis. METHODS: Eight boys with descended testis but with inguinal hernia and 8 boys with undescended testis were evaluated. Serum follicle-stimulating hormone (FSH), luteinizing hormone (LH), testosterone, and free testosterone levels were determined, and samples of cremaster muscles were immunostained for AR. Groups were compared by unpaired t tests and Fisher's exact tests; P values of <0.05 were considered significant. Samples of cremaster muscles were obtained from another 5 boys with descended testis but with inguinal hernia and 5 boys with undescended testis. The expression of AR mRNA in those samples was determined by semiquantitative reverse transcriptase polymerase chain reaction. RESULTS: Serum FSH, LH, testosterone, and free testosterone levels were similar among groups. None of the samples from boys with descended testis showed positive staining, but 4 of 8 samples from boys with undescended testis stained positive for AR. Androgen receptor mRNA transcript levels were approximately 10 times lower in cremaster muscles of boys with descended testis compared with those in boys with undescended testis. CONCLUSIONS: Despite similar serum hormone levels, more AR expression in cremaster muscles associated with undescended testis might represent evidence of being subjected to a lesser degree of androgenic effects.