Long-chain polyunsaturated fatty acid status in phenylketonuric patients treated with tetrahydrobiopterin

Objectives:To evaluate LCPUFA composition in PKU patients treated with BH₄.Design and methods:Cross-sectional study of plasma and erythrocyte LCPUFA composition of 13 PKU patients treated with BH₄ compared with data from 48 PKU patients on protein-restricted diet, and 17 mild HPA patients on free diet. PUFA were analysed by gas chromatography.Results:Plasma and erythrocyte docosahexaenoic acid (DHA), and LCPUFA deficiency markers did not show significant differences in PKU patients on BH₄ compared with those with mild HPA and our reference values, but they did in comparison with PKU on protein-restricted diet (p < 0.0001). Essential fatty acids and arachidonic acid composition were not significantly different in any of the studied groups. DHA values correlate with the index of dietary control only in PKU patients on protein-restricted diet (p =   0.002).Conclusion:LCPUFA status is within the reference values in PKU patients treated with BH₄. This translates to a further advantage of BH₄ therapy.     

RAS mutation analysis in a large cohort of Chinese patients with acute myeloid leukemia

ObjectiveWe examined RAS mutational status and correlated this with presenting morphology, cytogenetics, clinical outcome and other gene aberrations in a large cohort of Chinese acute myeloid leukemia (AML) patients.Designs and methodsN-RAS and K-RAS were screened for mutations at hot-spot codons 12, 13 and 61 using high resolution melting analysis (HRMA) and direct DNA sequencing in 504 Chinese AML patients and their clinical relevance was analyzed.ResultsThe frequencies of mutations of N-RAS and K-RAS were 9.7% (49/504) and 2.9% (15/504), respectively. c.35 G > A (rs121913237: G > A; p.Gly12Asp and rs121913529: G > A; p.Gly12Asp) and c.38 G > A (rs121434596: G > A; p.Gly13Asp and rs112445441: G > A; p.Gly13Asp) were the most common base substitutions (46% in N-RAS and 60% in K-RAS, respectively). AML patients with RAS mutations presented significantly higher white blood cell count (WBC) at diagnosis than those without mutations (p < 0.001). RAS mutations were underrepresented in patients with t(15;17) (2.9%, p = 0.01), while overrepresented in cases with abn11q23 (50%, p = 0.002) and inv(16) (66.6%, p = 0.04). In the FAB subtypes M4 and M5, RAS mutations were more frequent (21.6% and 20.6%, respectively) than they were in other subtypes (7.5%, p = 0.006 and 0.005, respectively). FLT3-ITD and RAS mutation were rarely coexistent (p = 0.03). RAS mutation didn't influence overall survival (OS) either in the entire cohort or within some defined subgroups.ConclusionsRAS mutations are associated with some biologically specific subtypes of AML but don't impact clinical outcome in Chinese patients.     

The spectrum of mutations identified in Cypriot patients with phenylalanine hydroxylase deficiency detected through neonatal screening

ObjectivesThe purpose of this study was to identify the mutations responsible for phenylalanine hydroxylase deficiency in Cypriot patients detected through neonatal screening.Design and MethodsAnalysis of the PAH gene was performed by direct sequencing of the patients' genomic DNA, MLPA analysis and real-time PCR.ResultsAmong 22 independent alleles thirteen previously described mutations were detected (detection rate 100%), all in compound heterozygosity: p.Arg395Gly (18.2%), c.168 + 5 G > C (13.6%), p.EX3del (9%), c.1066-11 G > A (9%), p.Ala403Val (9%), p.Glu178Gly (9%), p.Ser70Pro (4.5%), p.Arg241His (4.5%), p.Phe55fs (4.5%), p.Arg158Gln (4.5%), p.Asp222Gly (4.5%), p.Ala300Ser (4.5%), p.Pro225Thr (4.5%). Of the ten different genotypes, three have been previously reported to be associated with a mild clinical phenotype and to respond to tetrahydrobiopterin (BH₄) administration.ConclusionsMarked genetic heterogeneity was found in Cypriot patients with hyperphenylalaninemia with two mutations accounting for 32% of the alleles. Most of the mutations detected have been found in other European and Mediterranean populations.     

High resolution melting analysis facilitates mutation screening of ETFDH gene: Applications in riboflavin-responsive multiple acyl-CoA dehydrogenase deficiency

BackgroundMultiple acyl-CoA dehydrogenase deficiency (MADD) or gluaric aciduria type II is an autosomal recessive disease caused by defects in mitochondrial electron transfer system and metabolism of fatty acid. Recently, ETFDH mutations were reported to be major causes of riboflavin-responsive MADD. The present study is aimed at screening ETFDH mutations.MethodsHigh resolution melting (HRM) analysis was performed to screen ETFDH mutations. Genomic DNA was extracted from peripheral blood samples of the 9 patients with MADD and normal controls. Total 13 exons of ETFDH were screened by HRM analysis. The results were subsequently confirmed by direct DNA sequencing.ResultsThis diagnostic strategy proved to be feasible in detecting 3 known (c.250G > A, c380T > A, c.524G > T) and 1 novel (c.1831G > A) ETFDH mutations. Each mutation could be readily and accurately identified in the difference plot curves. We estimated the carrier frequency of the hotspot mutation, c.250G > A, in the Taiwanese population to be 1:125 (0.8%).ConclusionsHRM analysis can be successfully applied to screen ETFDH mutations. Since riboflavin-responsive MADD is often treatable, especially with mutations in ETFDH, identifying ETFDH mutations is crucial for these patients.     

Molecular analysis of Cypriot patients with Glutaric aciduria type I: Identification of two novel mutations

ObjectivesThe purpose of this study was to identify the mutations in the glutaryl-CoA dehydrogenase gene (GCDH) in ten Cypriot patients with Glutaric aciduria type I (GAI).Design and methodsMolecular analysis of the GCDH gene was performed by direct sequencing of the patients' genomic DNA. In silico tools were applied to predict the effect of the novel variants on the structure and function of the protein.ResultsAll disease alleles were characterized (mutation detection rate 100%). Five missense mutations were identified: c.192G > T (p.Glu64Asp) and c.803G > T (p.Gly268Val), which are novel, and three previously described mutations, c.1123T > C (p.Cys375Arg), c.1204C > T (p.Arg402Trp) and c.1286C > T (p.Thr429Met).ConclusionsTwo novel mutations, p.Glu64Asp and p.Gly268Val, account for the majority of disease alleles (76.5%) in Cypriot patients with Glutaric aciduria type I. A founder effect for the p.Glu64Asp and the p.Gly268Val can be suggested based on the place of origin of the carriers of these mutations. Identification of the causative mutations of GAI in Cypriot patients will facilitate carrier detection as well as post- and pre-natal diagnosis.     

A novel method for determining functional LDL receptor activity in familial hypercholesterolemia: Application of the CD3/CD28 assay in lymphocytes

BackgroundThe objective of this study was to develop a new and simple method for measuring low-density lipoprotein receptor (LDLR) activity using peripheral lymphocytes enabling us to clinically diagnose familial hypercholesterolemia (FH) and ascertain the involved mutations (such as K790X mutation), that might not be clearly detected in the conventional method.MethodsOur method comprised the following 2 features: first, we used anti-CD3/CD28 beads to stimulate T-lymphocytes to obtain a uniform fraction of lymphocytes and maximum up-regulation of LDLR. Second, we excluded the possibility of overestimation of lymphocyte signals bound only to its surface, by adding heparin to the cultured lymphocytes used for the LDLR assay.ResultsBased on the genetic mutation, the FH subjects were divided into 2 groups, K790X, (n = 20) and P664L, (n = 5), and their LDLR activities was measured by this method, which was found to be 55.3 ± 8.9% and 63.9 ± 13.8%, respectively, of that of the control group (n = 15). In comparison, the LDLR activity was 86.1 ± 11.6% (K790X) and 73.3 ± 6.3% (P664L) of that of the control group when measured by the conventional method, indicating that impairment of LDLR function in FH K790X subjects was much more clearly differentiated with our method than with the conventional method (paired t-test, p < 0.0001). The levels of LDLR expression also showed similar tendencies, that is, 89.4 ± 13.2% (K790X) and 76.9 ± 17.4% (P664L) of that of the control group when measured by the conventional method, and 78.1 ± 9.7% (K790X) and 70.3 ± 26.5% (P664L) when measured by our new method. In addition, we confirmed that there was little influence of statin treatment on LDLR activity among the study subjects when our method was used.ConclusionThese results demonstrate that our new method is applicable for measuring LDLR activity, even in subjects with an internally defective allele, and that T-lymphocytes of the FH K790X mutation possess characteristics of that allele.     

Hemodialysis vascular access thrombosis: The role of factor V Leiden, prothrombin gene mutation and ABO blood groups

BackgroundVascular access thrombosis increases morbidity in hemodialysis (HD) patients. The aim of this study was to investigate the association between HD vascular access thrombosis and mutations in the prothrombin and factor V Leiden (FV) genes and ABO blood system.MethodsThis cross-sectional study included 195 patients with end stage renal disease (ESRD) on HD for more than six months. HD patients were allocated into two groups according to the occurrence (cases, N = 46) or not (controls, N = 149) of previous vascular access thrombosis. FV and prothrombin gene mutations were investigated by polymerase chain reaction and ABO blood group phenotyping was performed by the indirect technique. Univariate analysis detected the variables with a trend to be associated with thrombosis and was followed by multivariate analysis to define independent predictors of vascular access thrombosis.ResultsFV Leiden mutation and ABO blood group were not associated with vascular access thrombosis, whereas G20210A mutation in the prothrombin gene was significantly higher in patients with vascular access thrombosis and independently associated with this complication (OR = 12.0; CI 95% = 1.8–83.5; p = 0.012).ConclusionsG20210A mutation emerges as an important genetic factor predisposing to vascular access thrombosis. The definition of risk factors for thrombosis will certainly enable a rational approach for HD patients.     

Application of allele-specific quantitative PCR using genomic DNA to monitor minimal residual disease based on mutant gene levels following allogeneic hematopoietic stem cell transplantation in patients with hematological malignancies: comparison of mutant levels with autologous DNA percentage by short tandem repeat-PCR

BackgroundQuantitative evaluation of minimal residual disease (MRD) following hematopoietic stem cell transplantation (HSCT) is indispensable for patients with hematological malignancies. In addition to established MRD markers such as immunoglobulin and T-cell receptor gene rearrangements, fusion genes, or aberrantly expressed genes, single nucleotide mutations are considered one of the MRD markers that reflect the malignant cell clone.MethodsWe compared the quantity of mutant genes by allele-specific quantitative polymerase chain reaction (AS-qPCR) for single nucleotide mutations (TP53 410T > A and PTPN11 1508G > A) with the percentage of autologous DNA by short tandem repeat (STR)-PCR.ResultsFollowing HSCT, the quantity of mutant genes detected by AS-qPCR correlated with the percentage of autologous DNA assessed by the STR-PCR. Moreover, mutant DNAs were detected at a quantifiable level before relapse, whereas the percentage of autologous DNA was less than 5%, that is, complete chimerism.ConclusionsThe AS-qPCR approach for single nucleotide mutations was accurate and highly sensitive for monitoring pre-transplantation as well as post-transplantation MRD. AS-qPCR for single nucleotide mutation is suitable for monitoring MRD in patients who lack previously established MRD markers.     

Development of novel microarray methodology for the study of mutations in the SERPINA1 and ADRB2 genes—Their association with Obstructive Pulmonary Disease and Disseminated Bronchiectasis in Greek patients

ObjectivesThe aim of our study was to determine the genetic risk conferred by SNPs in the SERPINA1 and ADRB2 for development of Chronic Obstructive Pulmonary Disease (COPD) and Disseminated Bronchiectasis (DB), while at the same time validating the NanoChip technology. This was a case–control study consisting of 112 COPD, 62 DB patients and 2 control groups (106 smokers without COPD: healthy smokers control group and 205 general population subjects).Design and methodsThe novel methodology of the Nanogen NanoChip® 400 (NC400 Nanogen www.nanogen.com) was employed for genotyping five mutations/SNPs in the SERPINA1 and 2 in the ADRB2 gene.ResultsFor the SERPINA1 gene a statistically significant difference in the frequency was found for heterozygotes for p.V213A between DB patients and healthy smokers (44.1% vs. 26.4% respectively; p = 0.035) and for heterozygotes for c.1237G>A between DB patients and general population subjects (10.2% vs. 25.4% respectively; p = 0.023). There was a clustering of ADRB2 p.Gly16 homozygotes in patients with severe COPD (24/44, 54.5% with FEV₁ values <35% of predicted).ConclusionsThe SERPINA1 p.V213A polymorphism was found associated with DB risk while the ADRB2 p.G16R is a risk factor for severe COPD in smokers.     

Normal range of serum Amphiregulin in healthy adult human females

ObjectivesPrior to large studies in breast cancer patients, we have sought to establish the normal range of a potential serum biomarker, Amphiregulin, in healthy women and to determine whether sampling during the menstrual cycle influences the detected Amphiregulin levels.Design and methodsSerum Amphiregulin levels were quantified using a commercially available ELISA in 85 normal female donors.ResultsThe range of circulating Amphiregulin was 0–4467 pg/mL. The majority of women had no detectable circulating Amphiregulin (n = 54), and only five women had levels exceeding 500 pg/mL. Serum Amphiregulin levels did not vary significantly during the menstrual cycle (n = 7 women).ConclusionsDetection of circulating Amphiregulin in a significant minority of healthy women suggests that it may not have the specificity necessary for a population screening tool; however its potential utility for monitoring response to treatment or disease progression should be examined in breast cancer cases.     

Paraoxonase activity in athletes with depleted iron stores and iron-deficient erythropoiesis

ObjectiveThe aim of this study was to evaluate how conditions that precede anaemia (iron store depletion and iron-deficient erythropoiesis) affect human serum paraoxonase PON1 activity.Design and methodsBased on haemoglobin, transferrin saturation and serum ferritin values 119 athletes were divided into three groups: with iron depletion, with deficient erythropoiesis and controls. The following parameters were measured: paraoxonase activity towards paraoxon (POase) and diazoxon (DZOase), lipid hydroperoxides (LOOH), the pro-oxidant-antioxidant balance (PAB), red blood cells (RBC) and lipid status.ResultsSignificant differences were found between athletes with different stages of iron deficiency and controls with respect to PON 1 activity and oxidative stress status parameters (Wilks' Lambda = 0.712, F = 5.241, p < 0.001, η² = 0.156). There was no significant difference between the PON1 192 Q and R polymorphism distribution in the two groups of athletes with different stages of iron deficiency and controls (χ² = 1.086; p = 0.896). PON1 activity was positively correlated with RBCs, haemoglobin, transferrin saturation (p < 0.001) and ferritin (p = 0.037) and negatively correlated with LOOH (p = 0.044) in all three study groups.ConclusionsDeficient erythropoiesis in athletes contributes to impaired PON1 activity. In contrast, iron depletion, regardless of increased oxidative stress, does not affect PON1 activity.     

Paraoxonase/arylesterase ratio, PON1 192Q/R polymorphism and PON1 status are associated with increased risk of ischemic stroke

Objectives:In recent years, importance of enzyme activity measurements, in addition to genotyping, in epidemiological studies relating paraoxonase 1 (PON1) and vascular disease was emphasized. This is the first report evaluating paraoxonase and arylesterase activities as risk factors for ischemic stroke. In addition, PON1 192Gln(Q)/Arg(R) and 55Leu(L)/Met(M) polymorphisms were also analyzed.Design and methods:The study population was comprised of 108 ischemic stroke patients and 78 controls. Enzyme activities were determined by spectrophotometric assays and for genotyping, standard PCR protocols followed by restriction enzyme digestions were used.Results:The prevalence of the PON1 192RR genotype was increased among stroke patients (16.7%) as compared to controls (9.0%, P = 0.129). Paraoxonase and arylesterase activities and PON1 activity ratio (paraoxonase/arylesterase) were found to be lower in patients than in controls. Logistic regression analysis revealed PON1 activity ratio (odds ratio, OR = 0.697, 95% CI, 0.541 to 0.898, P = 0.005), PON1 192RR genotype (OR = 3.434, 95% CI, 1.159 to 10.178, P = 0.026) and PON1 status (PON1 activity ratio combined with PON1 192RR genotype; OR = 1.406, 95% CI, 1.038 to 1.905, P = 0.028) as significant predictors of stroke.Conclusions:This study identified PON1 activity ratio, PON1 192RR genotype and PON1 status as important risk factors for ischemic stroke.     

Detection of a novel splicing mutation causing analbuminemia in a Libyan family

Background and objectivesAnalbuminemia is a very rare autosomal recessive disorder. It is an allelic heterogeneous defect caused by a variety of mutations within the albumin gene.We describe in this report two new cases of analbuminemia in Libyans.Design and methodsThe 14 coding exons of the human serum albumin (HSA) gene and their intron–exon junctions were PCR amplified. The products were screened for mutations by Denaturing High Performance Liquid Chromatography (DHPLC). Samples with altered DHPLC profiles were sequenced.ResultsDNA sequencing revealed the presence of a novol homozygous G ? T transition in the first base of intron 11 (c.1428 + 1G>T), in both children. This mutation destroys the GT consensus donor sequence found at the 5′ end of most intervening sequences and would cause the defective pre-mRNA splicing.ConclusionMolecular diagnosis based on DHPLC and DNA sequencing represents a powerful tool to study molecular defects causing analbuminemia.     

Cytokine elevations in acute coronary syndrome and ovarian cancer: a mechanism for the up-regulation of the acute phase proteins in these different disease etiologies

Objectives:To identify if a common set of cytokines is elevated in both ovarian cancer and acute coronary syndrome (ACS).Design and methods:A cytokine array (Randox Ltd) was measured in healthy women (n = 33), women with ACS (n = 21) and ovarian cancer (n = 45).Results:Women with ACS or ovarian cancer had higher concentrations of IL-6, IL-8, VEGF, MCP-1, and EGF as compared to healthy volunteers.Discussion:Common cytokine elevations are present in both ACS and ovarian cancer.     

High resolution melting analysis for the detection of SLC25A13 gene mutations in Taiwan

BackgroundCitrin, encoded by SLC25A13 gene, is a mitochondrial solute transporter with a crucial role in urea, nucleotide and protein synthesis. SLC25A13 mutations cause two phenotypes, adult-onset type II citrullinemia and neonatal intrahepatic cholestasis caused by citrin deficiency (NICCD). This study aimed to develop a high resolution melting (HRM) analysis for SLC25A13 mutation scanning and determine the carrier rate in Taiwan.MethodsDNAs from healthy subjects (n = 479), and patients with hepatocellular carcinoma (HCC, n = 100) and NICCD (n = 5) were scanned in exons 6, 9, 11, 16, and 17 and parts of introns of SLC25A13 using HRM analysis. All mutations detected by HRM analysis were further confirmed by TaqMan method and/or direct sequencing.ResultsIn healthy subjects, seventeen carriers with mutants c.851_854del (n = 10), c.1638_1660dup, c.615 + 5G > A (n = 4), and two novel mutants, c.475C > T and c.1658G > A, were detected. The frequency of carriers was about 1/28. In patients with HCC, there were only 2 carriers with c.851_854del mutant. Patients with NICCD (n = 5) diagnosed during 2007 and 2008, harbored compound heterozygous mutations c.851_854del/c.1177 + 1G > A, c.851_854del/c.1638_1660dup (n = 2), c.851_854del/c.615 + 5G > A, and c.1638_1660dup/c.615 + 5G > A.ConclusionsHRM analysis is a simple, rapid and robust method for detecting SLC25A13 mutations in clinical laboratories. SLC25A13 mutations may not be a major contributor to the pathogenesis of HCC in Taiwan.     

Rapid identification of HBB gene mutations by high-resolution melting analysis

ObjectiveThis study was undertaken to identify HBB gene mutation.Design and methodsHerein we evaluated high-resolution melting analysis in the identification of HBB mutations.ResultsWe have successfully established a diagnostic strategy for identifying HBB gene mutations including c.? 78A > G, c.? 79A > G, c.2T > G, c.79_80insT, c.84_85insC, c.123_124insT, c.125_128delTCTT, c.130 G > T, c.170G > A, c.216_217ins A and c.316–197 C > T from wild-type DNA using HRM analysis. The results of HRM analysis were confirmed by direct DNA sequencing.ConclusionsIn summary, we report that HRM analysis is an appealing technique for the identification of HBB mutations. We also believe that HRM can be used as a method for prenatal diagnosis of β-thalassemia.     

Time of sampling is crucial for measurement of cell-free plasma DNA following acute aseptic inflammation induced by exercise

ObjectivesTo determine the time-course changes of cell-free plasma DNA (cfDNA) following heavy exercise.MethodscfDNA concentration, C-reactive protein levels (hs-CRP), uric acid concentration (UA), creatine kinase activity (CK) were measured before and post-exercise (immediately post, 0.5 h, 1 h, 2 h, 3 h, 4 h, 5 h, 6 h, 8 h, 10 h, 24 h).ResultscfDNA increased (15-fold) 30-min post-exercise and normalized thereafter. hs-CRP increased (56%, p < 0.001) 1 h post-exercise, remained elevated throughout recovery (52–142%, p < 0.0001), and peaked (200% rise, p < 0.0001) at 24 h post-exercise. UA and CK increased (p < 0.05), immediately post-exercise, remained elevated throughout recovery (p < 0.0001), and peaked (p < 0.0001) at 24 h of post-exercise recovery.ConclusionscfDNA sampling timing is crucial and a potential source of error following aseptic inflammation.     

Measurement of insulin-like growth factor-1 and insulin-like growth factor binding protein-3 after delayed separation of whole blood samples

Objectives:Epidemiological studies benefit from unbiased blood specimens collected with minimal cost and effort of blood collection and storage. We evaluated the stability of IGF-1 and IGFBP-3 in whole blood samples stored at room temperature to justify delays in blood processing.Design and methods:Total IGF-1 and IGFBP-3 levels were measured in EDTA plasma (n = 12), heparin plasma (n = 12) and serum (n = 10) samples of healthy volunteers after blood processing delays up till 14 days. Stability of measured IGF-1 and IGFBP-3 levels was tested by paired t-test and a linear mixed effect model.Results:Longitudinal analysis showed that IGF-1 levels were not significantly affected by blood processing delays in EDTA tubes (p = 0.18) and IGFBP-3 levels were marginally stable (p = 0.06). In heparin plasma and serum, however, IGF-1 increased over time of delayed processing and IGFBP-3 levels tended to decrease (p < 0.01).Conclusion:Total IGF-1 and IGFBP-3 levels are stable in whole blood collected in EDTA tubes at room temperature up till 7 days, allowing a delay in blood processing to reduce costs in large multi-center studies.     

Increased urinary level of oxidized nucleosides in patients with mild-to-moderate Alzheimer's disease

Objective:Oxidative stress may play an important role in the pathogenesis of Alzheimer's disease (AD).Design and methods:To investigate the possible role of oxidative DNA damage in the pathogenesis of AD, we measured the metabolite concentrations of oxidized nucleosides (pseudouridine, 1-methyladenosine, 5-methylcytidine, 5-methyl-2′-deoxycytidine, 3-methyluridine, N², N²-dimethylguanosine, 8-hydroxy-2′-deoxyguanosine, 5-deoxyadenosine and 2-deoxyguanosine) in urine between AD (n = 36) and control subjects (n = 34) using liquid chromatography-mass spectrometry (LC-MS) without urine preparation.Results:In AD, the 3-methyluridine, 1-methyladenosine, 8-hydroxy-2′-deoxyguanosine (p < 0.05, respectively), 2-deoxyguanosine (p < 0.01) and pseudouridine, N², N²-dimethylguanosine (p < 0.001, respectively) were significantly increased when compared with the control subjects.Conclusion:The results indicate that oxidized urinary nucleosides may be useful as biomarkers for AD in early stages.