Association of a synonymous SCN1B variant affecting splicing efficiency with Benign Familial Infantile Epilepsy (BFIE).

Benign Familial Infantile Epilepsy (BFIE) is clinically characterized by clusters of brief partial seizures progressing to secondarily generalized seizures with onset at the age of 3-7 months and with favorable outcome. PRRT2 mutations are the most common cause of BFIE, and found in about 80% of BFIE families. In this study, we analyzed a large multiplex BFIE family by linkage and whole exome sequencing (WES) analyses. Genome-wide linkage analysis revealed significant evidence for linkage in the chromosomal region 19p12-q13 (LOD score 3.48). Mutation screening of positional candidate genes identified a synonymous SCN1B variant (c.492T>C, p.Tyr164Tyr) affecting splicing by the removal of a splicing silencer sequence, shown by in silico analysis, as the most likely causative mutation. In addition, the PRRT2 frameshift mutation (c.649dupC/p.Arg217Profs*8) was observed, showing incomplete, but high segregation with the phenotype. In vitro splicing assay of SCN1B expression confirmed the in silico findings showing a splicing imbalance between wild type and mutant exons. Herein, the involvement of the SCN1B gene in the etiology of BFIE, contributing to the disease phenotype as a modifier or part of an oligogenic predisposition, is shown for the first time.

Functional characterisation of the type 1 von Willebrand disease candidate VWF gene variants: p.M771I, p.L881R and p.P1413L.

BACKGROUND: Abnormalities in the biosynthetic pathway or increased clearance of plasma von Willebrand factor (VWF) are likely to contribute to decreased plasma VWF levels in inherited type 1 von Willebrand disease (VWD). Recent studies demonstrated that 65% of type 1 VWD patients have candidate VWF mutations, the majority of which are missense variants. The purpose of this study was to explore the effects of three VWF missense mutations (p.M771I, p.L881R and p.P1413L) located in different functional domains of VWF, reported as candidate mutations in type 1 VWD patients in the course of the MCMDM-1VWD study. MATERIALS AND METHODS: The focus of these studies was on the intracellular biosynthetic processing and localisation of VWF in a heterologous cell system. Molecular dynamic simulation for p.M771I and p.P1413L was also performed to analyse the conformational effects of the changes. RESULTS: As determined by immunofluorescence antibody staining and confocal microscopy of HEK293 cells, the intracellular localisation of recombinant VWF with the p.M771I variation was impaired. Transient transfection studies and phorbol myristate acetate stimulation in COS-7 cells revealed significant intracellular retention. In addition, major loss of VWF multimers was observed for only the p.M771I mutation. Molecular dynamic simulations on p.M771I mutant VWF revealed distinct structural rearrangements including a large deviation in the E' domain, and significant loss of ß-sheet secondary structure. DISCUSSION: The pathogenic effects of candidate VWF gene mutations were explored in this study. In vitro expression studies in heterologous cell systems revealed impaired secretion of VWF and a dominant negative effect on the processing of the wild-type protein for only the p.M771I mutation and none of the mutations affected the regulated secretion.

SCN1A gene sequencing in 46 Turkish epilepsy patients disclosed 12 novel mutations.

PURPOSE: The SCN1A gene is one of the most commonly mutated human epilepsy genes associated with a spectrum of phenotypes with variable degrees of severity. Despite over 1200 distinct mutations reported, it is still hard to draw clear genotype-phenotype relationships, since genetic and environmental modifiers contribute to the development of a particular disease caused by an SCN1A mutation. We aimed to initiate mutational screening of the SCN1A gene in Turkey and advance further our understanding of the relationship between the SCN1A sequence alterations and disease phenotypes such as GEFS+, DS and related epileptic encephalopathies. METHODS: Mutational analysis of the SCN1A gene was carried out in 46 patients with DS, late-onset DS, GEFS+ and unspecified EE using either direct Sanger sequencing of the coding regions and exon/intron boundaries or massively parallel sequencing. RESULTS: Nineteen point mutations, 12 of which were novel were identified, confirming the clinical diagnosis of the patients. Patients with a mutation (either truncating or missense) on linker regions had significantly later disease onset than patients with mutations in homology regions. The presence of SCN1A mutations in two clinically unclassified patients supported the association of SCN1A mutations with a wide range of phenotypes. CONCLUSION: The conventional Sanger sequencing method was successfully initiated for the detection of SCN1A point mutations in Turkey in epilepsy patients. Furthermore, a modified strategy of massively parallel pyro-sequencing was also established as a rapid and effective mutation detection method for large genes as SCN1A.

Impact of glucocorticoid receptor gene (NR3C1) polymorphisms in Turkish patients with metabolic syndrome.

BACKGROUND: The metabolic syndrome (MetS) is characterized by a cluster of metabolic factors, including insulin resistance and type-2 diabetes, abdominal obesity, dyslipidemia, hypertension and microalbuminuria. Impaired glucocorticoid receptor (GR) activity also plays an important role in the etiology of MetS. The objective of our study is to evaluate the effects of GR gene polymorphisms (BclI, N363S, TthIII1 and ER22/23EK) in Turkish patients with MetS. MATERIALS AND METHODS: Seventy subjects with MetS and 185 healthy controls were enrolled in the study. PCR-RFLP analysis was used for genotyping. Results for each polymorphism have been verified by allele-specific oligonucleotide analysis. RESULTS: BclI GG genotype was significantly associated with an increased risk of MetS (p = 0.02). Also, only in women, the G allele carriers were significantly associated with higher C-peptide. T allele carriers of TthIII1 polymorphism were significantly associated with higher C-peptide, triglyceride, insulin and C-reactive protein (CRP, p value 0.048, 0.022, 0.005 and 0.022, respectively), and lower fasting blood glucose (FBG, p = 0.02). The combined carriers of BclI polymorphism G allele and TthIII1 polymorphism T allele were significantly associated with higher diastolic blood pressure in all patients, and lower FBG and postprandial blood glucose in only men. All the ER22/23EK polymorphisms coexisted with polymorphic variant of TthIII1 (p = 0.0058). CONCLUSION: The presence of homozygote polymorphic variant of BclI might be good predictive markers for the disease susceptibility. The BclI and the TthIII1 polymorphism are associated with sex-specific clinical parameters. Our findings also suggest that the combination of BclI and TthIII1 polymorphisms may play a protective role in blood glucose.

A common VWF exon 28 haplotype in the Turkish population.

An increasing number of mutations and polymorphisms have been identified in the von Willebrand factor (VWF) gene of patients with von Willebrand disease (VWD). Most of the sequence alterations are within exon 28, duplicated in the VWF pseudogene on chromosome 22. Genetic recombination causing the gene conversion between the VWF gene and its pseudogene is associated with multiple substitutions in the VWF gene and with VWD. In the present study, VWF gene exon 28 was analyzed in 33 patients with VWD by DNA sequencing. A total of 73% of the patients were heterozygous for p.D1472H, p.V1485L, p.1500A, p.1501F, p.L1503P, and p.S1506L single-nucleotide polymorphisms. Family analysis revealed that the gene conversion occurred between the VWF gene and its pseudogene in 3 patients. Case-control association analysis by Haploview 4.2 did not show an association between the haplotype and VWD. In conclusion, a common exon 28 haplotype in the Turkish population, which might have arisen from the gene conversion events in the founder population, was identified.

Four novel and two recurrent NHLRC1 (EPM2B) and EPM2A gene mutations leading to Lafora disease in six Turkish families.

Lafora disease (LD) is a type of autosomal recessive, progressive myoclonus epilepsy resulting mostly from mutations in the EPM2A and NHLRC1 genes. Mutational analysis in both genes was initiated with the aim of establishing LD DNA diagnosis in Turkey. Four novel NHLRC1 (p.G131X, p.P69S and p.D82H) and EPM2A (p.V7A) and two recurrent NHLRC1 (p.D146N) and EPM2A (p.R241X) mutations were identified in six families. The delineation of causative mutations in patients provided early disease diagnosis for other family members and contributed to the knowledge of LD pathogenesis.

Factor 8 (F8) gene mutation profile of Turkish hemophilia A patients with inhibitors.

Factor VIII (FVIII) replacement therapy is ineffective in hemophilia A patients who develop alloantibodies (inhibitors) against FVIII. The type of factor 8 (F8) gene mutation, genes in the major histocompatibility complex loci, and also polymorphisms in IL-10 and tumor necrosis factor-alpha are the major predisposing factors for inhibitor formation. The present study was initiated to reveal the F8 gene mutation profile of 30 severely affected high-responder patients with inhibitor levels of more than 5 Bethesda U (BU)/ml and four low-responder patients with inhibitors less than 5 BU/ml. Southern blot and PCR analysis were performed to detect intron 22 and intron 1 inversions, respectively. Point mutations were screened by DNA sequence analysis of all coding regions, intron/exon boundaries, promoter and 3' UTR regions of the F8 gene. The prevalent mutation was the intron 22 inversion among the high-responder patients followed by large deletions, small deletions, and nonsense mutations. Only one missense and one splicing error mutation was seen. Among the low-responder patients, three single nucleotide deletions and one intron 22 inversion were found. All mutation types detected were in agreement with the severe hemophilia A phenotype, most likely leading to a deficiency of and predisposition to the development of alloantibodies against FVIII. It is seen that Turkish hemophilia A patients with major molecular defects have a higher possibility of developing inhibitors.

A novel missense mutation (N258S) in the KCNQ2 gene in a Turkish family afflicted with benign familial neonatal convulsions (BFNC).

Benign familial neonatal convulsions (BFNC) is a rare monogenic subtype of idiopathic epilepsy exhibiting autosomal dominant mode of inheritance. The disease is caused by mutations in the two homologous genes KCNQ2 and KCNQ3 that encode the subunits of the voltage-gated potassium channel. Most KCNQ2 mutations are found in the pore region and the cytoplasmic C domain. These mutations are either deletions/insertions that result in frameshift or truncation of the protein product, splice-site variants or missense mutations. This study reveals a novel missense mutation (N258S) in the KCNQ2 gene between the S5 domain and the pore of the potassium channel in two BFNC patients in a Turkish family. The absence of the mutation both in the healthy members of the family and in a control group, and the lack of any other change in the KCNQ2 gene of the patients indicate that N258S substitution is a pathogenic mutation leading to epileptic seizures in this family.

Molecular pathology of haemophilia B in Turkish patients: identification of a large deletion and 33 independent point mutations.

Heterogeneous mutations in the coagulation factor IX (FIX) gene result in a bleeding tendency known as haemophilia B. The haemophilia B mutation database has a total of 2353 patient entries, including 10 of the estimated 1000 Turkish patients. In this study, a more comprehensive analysis of the molecular pathology of haemophilia B in Turkey revealed one large deletion and 33 point mutations in the FIX gene of 34 unrelated patients. Haplotype analysis using six polymorphic sites showed that the mutations identified in a total of 45 patients occurred on 13 different haplotypes and that each mutation was family specific.

R506Q (FV Leiden) and R485K Mutations in the Factor V Gene: Incidence in Deep Venous Thrombosis and Hemophilia A Patients.

R506Q (FV Leiden) mutation in exon 10 of the factor V (FV) gene is highly prevalent in European populations and it has been suggested that the coinheritance of FV Leiden mutation may be an important modifier of hemophilia A phenotype. One other substitution R485K in the same exon, with no functional consequences in vitro, is significantly higher in Thailand and has been associated with thrombophilia. In order to see if any correlation exists between R506Q and hemophilia phenotype and between R485K and thrombosis in Turkish patients, DGGE analysis of exon 10 of the FV gene is carried out among deep venous thrombosis (DVT) and hemophilia A patients. Our results indicate that the allelic frequency of the R485K polymorphism is similar to the frequency detected in Europe, and apparently, is not associated with an increased risk of thrombosis in the Turkish population. It is also not possible to show a modifier effect of FV Leiden on hemophilia A phenotype among the limited number of patients included in this study.

Molecular pathology of haemophilia A in Turkish patients: identification of 36 independent mutations.

Haemophilia A is an X-linked recessive bleeding disorder caused by heterogeneous mutations in the factor VIII gene. In an attempt to reveal the molecular pathology of Turkish haemophilia A patients, the coding sequence of the gene, excluding a large portion of exon 14, was amplified from genomic DNA and subjected to denaturing gradient gel electrophoresis prior to DNA sequencing. Fifty-nine haemophilia A patients were included in the study with severe, moderate and mild phenotypes observed in 24, 15 and 16 patients, respectively. Factor VIII activity and clinical phenotypes were not available for four patients. A total of 36 independent mutations were found, with a mutation detection efficacy of 61%. The mutations that were reported for the first time include 20 point mutations, one 8-bp insertion (TCAAGATA) in exon 4 and one large deletion greater than 2.8 kb involving exon 14. The novel point mutations were composed of three nonsense (Ser681Ter, Cys2021Ter and Gln2113Ter), one splicing error (IVS-1G-->A), 15 missense mutations (Lys48Asn; Leu-98Phe; Thr118Ala; Cys248Tyr; Glu456Lys; Asp560Ala; Tyr664Cys; Phe679Leu; Gly691Trp; Asp1769His; Val1857Leu; Gly2026Gln; Arg2163Pro; Asp2288Ala; and Arg2304Leu) and a T deletion in exon 25 that caused a frameshift followed by a stop codon. All missense mutations except Val1857Leu, which maintained a conserved nonpolar R group, occurred at amino acids conserved among four species and were most probably pathogenic. In addition, two sequence changes (IVS3-9C-->T) and (Leu2230Leu) were also detected in patients carrying Val1857Leu and Phe679Leu missense mutations, respectively. Identification of mutation origins in eight sporadic cases revealed an equal sex ratio of mutations.

Synchronicity of frequently sampled thyrotropin (TSH) and leptin concentrations in healthy adults and leptin-deficient subjects: evidence for possible partial TSH regulation by leptin in humans.

Leptin signals the status of energy reserves to the brain. Leptin stimulates biosynthesis of TRH in vitro and influences the activity of the hypothalamic-pituitary-thyroid axis in vivo in rodents. Because blood levels of both leptin and TSH display diurnal variation with a distinct nocturnal rise, we sought to determine whether a relationship exists between fluctuations in circulating leptin and TSH. We measured serum leptin and TSH levels every 7 min for 24 h in five healthy men and found that both leptin and TSH levels are highly organized and pulsatile. A similar pattern of leptin and TSH rhythms was observed, with TSH and leptin levels reaching a nadir in late morning and a peak in the early morning hours. Importantly, cosinor analysis on the absolute leptin and TSH levels revealed a statistically significant fit for a 24-h period and the two hormones showed similar probabilities of rhythm and superimposable peak values. Furthermore, this study shows a strong positive Pearson correlation between the 24-h patterns of variability of leptin and TSH in healthy subjects. Finally, the ultradian fluctuations in leptin levels showed pattern synchrony with those of TSH as determined by cross-correlation analysis, by cross-approximate enthropy and Bayessian analysis applied independently. To further explore whether these associations could reflect an underlying regulation of TSH secretion by leptin, we also studied frequently sampled leptin and TSH levels in four brothers, members of a family with leptin deficiency (one normal homozygote, two heterozygotes, and one leptin-deficient homozygote). Leptin levels of the homozygous leptin-deficient subject are detectable but bioinactive, and the rhythm of his TSH is disorganized. 24-h pattern of leptin and TSH variability in the heterozygous subjects, although significantly correlated, showed a weaker correlation compared with the strong correlation in the normal subjects. These data are consistent with the possibility that leptin may regulate TSH pulsatility and circadian rhythmicity, but interventional studies are needed to definitively prove whether leptin regulates the minute-to-minute oscillations and ultradian rhythm of TSH levels.

Vitamin d-receptor gene polymorphisms and vertebral bone density in men with idiopathic hypogonadotrophic hypogonadism.

BACKGROUND: Optimal peak bone mass is closely related to sufficient and appropriately timed androgen release. However, attainment of peak bone mass in men, as in women, is under genetic control, as well as being subject to hormonal and mutational effects. With increasing recognition of osteoporosis and related fractures in men, it is of interest to consider whether there is relationship between bone density and vitamin D receptor (VDR) polymorphisms, as described in women. MATERIAL AND METHODS: To assess the influence of allelic variation in the VDR gene on vertebral bone density in men with idiopathic hypogonadrotrophic hypogonadism (IHH), 27 patients (mean age 21.4 +/- 0.4 yrs) and 25 age-and-BMI matched healthy males (mean age 21.2 +/- 0.3) were genotyped using three restriction enzymes (Apa I, Bsm I, and Taq I). Vertebral bone mineral density was measured using dual energy X-ray absorptiometry (DEXA). RESULTS: As expected, vertebral bone density was reduced significantly in patients with IHH (p < 0.001). Despite weak evidence for an association between Apa I polymorphism and vertebral bone density in the IHH group (r = 0.454, p = 0.017 and r2 = 0.20), VDR genotype was not associated with vertebral bone density in either group. When analyzing homozygous haplotypes, the probability of carrying the favorable BAt haplotype was greater in the control group (OR = 2.000 vs. 0.500). CONCLUSION: We conclude that VDR genotype has no influence on vertebral bone density in men with IHH. Thus, allelic variation in the VDR cannot help define those at increased risk for osteoporosis and related fractures among such patients.

Plasma melatonin concentration before and during testosterone replacement in Klinefelter's syndrome: relation to hepatic indolamine metabolism and sympathoadrenal activity.

The mechanisms leading to alterations in plasma melatonin (MT) levels with testosterone replacement in Klinefelter's syndrome (KS) remain elusive. We investigated early morning plasma MT levels, urinary 6-sulfatoxymelatonin (6-SM) levels, and urinary catecholamine levels before and 6 months after testosterone treatment in 31 patients with KS and 20 healthy males to demonstrate whether alterations in plasma MT levels in such patients are due to subtle changes in sympathoadrenal activity and/or alterations in the hepatic indolamine metabolism influenced by testosterone replacement. The plasma MT level was measured by RIA. The sensitivity of the test was 10.7 pmol/L. The 6-SM level was measured by enzyme-linked immunosorbent assay. Urinary catecholamines were determined by high performance liquid chromatography. The pretreatment mean plasma MT level was insignificantly higher in the patient group than in the control group (72.57 +/- 74.82 vs. 42.37 +/- 29.02 pmol/L; z = -1.218; P = 0.223). The pretreatment urinary 6-SM and norepinephrine (NE) levels were significantly lower and, the epinephrine (E) and dopamine levels were insignificantly lower in the patient group than those in the control group [6-SM, 76.54 +/- 31.92 vs. 125.49 +/- 50.16 nmol/L (z = -3.727; P < 0.001); NE, 120.79 +/- 58.33 vs. 178.84 +/- 81.61 nmol/day (z = -2.585; P = 0.01); E, 31.27 +/- 27.42 vs. 34.65 +/- 28.33 nmol/day (z = -0.39; P: = 0.692); dopamine, 1577.02 +/- 863.02 vs. 1812.32 +/- 677.59 nmol/day (z = -1.03, P = 0.308)]. After testosterone replacement, plasma MT levels were significantly decreased (72.57 +/- 74.82 vs. 24.73 +/- 23.61 pmol/L; z = -4.29; P < 0.001), and urinary 6-SM, NE, E, and dopamine levels were significantly increased [6-SM, 25.04 +/- 10.44 vs. 40.05 +/- 17.65 ng/mL (z = -4.78; P < 0.001); NE, 120.78 +/- 58.33 vs. 154.08 +/- 61.35 nmol/day (z = -4.27; P < 0.001); E, 31.27 +/- 27.42 vs. 40.74 +/- 30.04 nmol/day (z = -4.22; P < 0.001); dopamine, 1577.02 +/- 863.02 vs. 2162.67 +/- 823.15 (z = -6.127; P < 0.001)]. There was no relation between plasma MT levels, urinary 6-SM, and catecholamine levels and levels of gonadotropins or gonadal steroids either before or after treatment. We demonstrate that in untreated KS, plasma MT levels tend to be higher than those in normal controls, whereas those of the melatonin metabolite 6-SM and those of NE in urine tend to be lower. After testosterone treatment, however, plasma MT levels fall significantly, whereas urinary levels of 6-SM and NE rise. Our data show that the effect of testosterone is mediated by enhanced metabolism of melatonin, not by any effect on net sympathetic outflow, and that the increase in plasma melatonin in untreated KS patients also results from an alteration in the rate of melatonin metabolism and not from increased net sympathetic activity.

Rapid Identification of Family-Specific Mutations in the Factor VIII Gene by One-Step DGGE.

A one-step denaturing gradient gel electrophoresis (DGGE) strategy for the rapid detection of mutations in the factor VIII gene of haemophilia A patients is described. All coding (except the middle part of exon 14) and flanking intronic regions of the gene corresponding to approximately 6.6 kb were amplified in 27 fragments using four PCR programs. Heteroduplex formation was performed for each fragment. A common denaturant gradient gel (35-65%) was chosen that allowed the simultaneous analysis of all PCR amplified regions on a single gel and run for 3.5 h at 160 V. This method was implemented for a patient whose family was seeking carrier determinations. An abnormal pattern was detected in exon 23 and the family-specific mutation was found by subsequent DNA sequencing. One-step DGGE is a promising rapid method for the carrier detection and prenatal diagnosis in haemophilia A families when immediate results are required and when polymorphic markers fail to give information.

Adequate immune response to tetanus toxoid and failure of vitamin A and E supplementation to enhance antibody response in healthy children.

The effects of vitamin A and vitamin E supplementation on the IgG response to tetanus toxoid after primary immunization were evaluated in a prospective, randomized controlled clinical trial involving 89 healthy infants with normal serum vitamin A and E levels at 2 months of age. Before the first dose of DPT vaccine, the infants were randomly enrolled into four different study groups [Group I (n=24): 30,000 IU vitamin A for 3 days just after each three doses of primary vaccination, Group II (n=21): 150 mg oral vitamin E for only 1 day after the injections for primary immunization, Group III (n=21): vitamins A and E together in the same order, Group IV (n=23) no vitamin after DPT vaccines]. Serum tetanus antitoxin (IgG) titres were measured three times; initially at 2 months of age before the first dose of DPT, secondly at 5 months of age 1 month after primary immunization and thirdly at 16-18 months of age before the booster dose of DPT. Before the first dose of the DPT vaccine, 1 month after the third DPT injection and at 16-18 months before the booster dose of DPT, there was no significant difference in serum tetanus antitoxin levels between these four groups. A significant increase was observed in all the groups when serum tetanus antitoxin levels before (2 months) and after (5 months) primary immunization were compared. In addition, serum antibody levels against tetanus significantly decreased in the four groups before booster vaccination. Before the beginning of primary immunization, 15 infants (16.8%) had serum tetanus antitoxins (IgG) below protective level. After three doses of DPT, all the infants had protective antitoxin levels. At 16-18 months of age before booster dose, four infants (10%) also had serum tetanus antitoxins (IgG) below the protective level. No side-effects were observed except bulging fontanelle in two infants in Group I.

Plasma concentrations of granulocyte-macrophage colony-stimulating factor and interleukin-6 in septic and healthy preterms.

UNLABELLED: Plasma granulocyte-macrophage colony-stimulating factor (GM-CSF) and interleukin-6 (IL-6) concentrations were determined in 21 preterm infants with sepsis and nine healthy preterm neonates of the same postnatal age at sampling. Plasma GM-CSF levels were elevated at diagnosis in the septic preterms as compared to the healthy preterms (P = 0.01), but did not differ significantly on recovery. IL-6 levels were also elevated markedly at diagnosis (P = 0.0003), but decreased to normal on recovery as compared to the healthy preterm infants. GM-CSF levels were more prominent in septic preterms with neutropenia than those of non-neutropenic infants (P = 0.03). CONCLUSION: Preterm infants can produce high levels of granulocyte-macrophage colony-stimulating factor and interleukin-6 in response to bacterial sepsis.

Intron 22-specific long PCR for the Xba I polymorphism in the factor VIII gene.

The Xba I polymorphic site in the factor VIII gene is present in the int22h-1 region which is found in two other copies (int22h-2 and int22h-3) distal to the gene. Previously the polymorphic status of the Xba I locus was studied by either Southern blot or PCR that amplified all three copies. Here we report the use of a long PCR that specifically amplifies the intragenic site in intron 22, making use of this marker an easy and reliable assay. Moreover, about 25% of previously uninformative Turkish haemophilia A families examined with three markers proved to be informative for linkage analysis, using the Xba I polymorphism.