Deficiency in COG5 causes a moderate form of congenital disorders of glycosylation

The conserved oligomeric Golgi (COG) complex is a tethering factor composed of eight subunits that is involved in the retrograde transport of intra-Golgi components. Deficient biosynthesis of COG subunits leads to alterations of protein trafficking along the secretory pathway and thereby to severe diseases in humans. Since the COG complex affects the localization of several Golgi glycosyltransferase enzymes, COG deficiency also leads to defective protein glycosylation, thereby explaining the classification of COG deficiencies as forms of congenital disorders of glycosylation (CDG). To date, mutations in COG1, COG4, COG7 and COG8 genes have been associated with diseases, which range from severe multi-organ disorders to moderate forms of neurological impairment. In the present study, we describe a new type of COG deficiency related to a splicing mutation in the COG5 gene. Sequence analysis in the patient identified a homozygous intronic substitution (c.1669-15T>C) leading to exon skipping and severely reduced expression of the COG5 protein. This defect was associated with a mild psychomotor retardation with delayed motor and language development. Analysis of different serum glycoproteins revealed a CDG phenotype with typical undersialylation of N- and O-glycans. Retrograde Golgi-to-endoplasmic reticulum trafficking was markedly delayed in the patient's fibroblast upon brefeldin-A treatment, which is a hallmark of COG deficiency. This trafficking delay could be restored to normal values by expressing a wild-type COG5 cDNA in the patient cells. This case demonstrates that COG deficiency and thereby CDG must be taken into consideration even in children presenting mild neurological impairment

Advances in hemoglobinopathy detection and identification

Hemoglobin disorders consist of two different groups, the structural hemoglobin variants and the thalassemias. The structural hemoglobin variants typically are based on the point mutations in the alpha- or beta-globin chain that results in a single-amino acid substitution in the corresponding globin chain, whereas thalassemias are caused by quantitative reduction in globin chain synthesis. Various techniques are applied for the laboratory investigation of these diseases, among them mass spectrometry (MS) for the detection and identification of structural hemoglobin variants and array techniques for the thalassemias. In this review, we present in the first part the most important mass spectrometric techniques applied in hemoglobin variant detection and identification and discuss several important aspects of this analysis technique in hematology. In the second part, the DNA analysis techniques used in hemoglobin analysis, such as reverse hybridization or microarray-based comparative genomic hybridization (CGH) techniques, are briefly discussed

High levels of anti-inflammatory and pro-resolving lipid mediators lipoxins and resolvins and declining docosahexaenoic acid levels in human milk during the first month of lactation

BACKGROUND: The fatty acid mixture of human milk is ideal for the newborn but little is known about its composition in the first few weeks of lactation. Of special interest are the levels of long-chain PUFAs (LCPUFAs), since these are essential for the newborn's development. Additionally, the LCPUFAs arachidonic acid (AA), eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) are precursors for lipid mediators which regulate inflammation. METHODS: We determined the composition of 94 human milk samples from 30 mothers over the first month of lactation for fatty acids using GC-MS and quantified lipid mediators using HPLC-MS/MS. RESULTS: Over the four weeks period, DHA levels decreased, while levels of γC18:3 and αC18:3 steadily increased. Intriguingly, we found high concentrations of lipid mediators and their hydroxy fatty acid precursors in human milk, including pro-inflammatory leukotriene B4 (LTB4) and anti-inflammatory and pro-resolving lipoxin A4 (LXA4), resolvin D1 (RvD1) and resolvin E1 (RvE1). Lipid mediator levels were stable with the exception of two direct precursors. CONCLUSIONS: Elevated levels of DHA right after birth might represent higher requirements of the newborn and the high content of anti-inflammatory and pro-resolving lipid mediators and their precursors may indicate their role in neonatal immunity and may be one of the reasons for the advantage of human milk over infant formula

Neonatal cyanosis due to a new (G)γ-globin variant causing low oxygen affinity: Hb F-Sarajevo [(G)γ102(G4)Asn→Thr, AAC>ACC]

A baby girl, born at term, presented with severe cyanosis and received oxygen supplementation. Consecutive arterial blood gas analysis showed a pronounced right shift of the saturation curve, suggesting the presence of a hemoglobin (Hb) variant. A new (G)γ-globin variant was detected, namely HBG2:c.308G, which we have named Hb F-Sarajevo, the city from where the baby's parents originate. This A to C transversion exists in cis to the common (A)γ(T) and the resulting mutant Hb molecule exhibits very low oxygen affinity and cooperativity. Its analogue in the β-globin gene is Hb Kansas [β102(G4)Asn→Thr, AAC>ACC]

Sugar-sweetened beverages with moderate amounts of fructose, but not sucrose, induce Fatty Acid synthesis in healthy young men: a randomized crossover study

Context: The impact of sugar-sweetened beverages (SSB) on lipid metabolism when consumed in moderate amounts by normal weight subjects is debated. Objective: The objective of the study was to investigate the effect of different types of sugars in SSB on fatty acid metabolism (ie, fatty acid synthesis and oxidation) in healthy young men. Design: Thirty-four normal-weight men were studied in a randomized crossover study. Four isocaloric 3-week interventions with SSB were performed in random order: medium fructose (MF; 40 g/d); high fructose (HF; 80 g/d), high sucrose (HS; 80 g/d), and high glucose (HG; 80g/d). Fasting total plasma fatty acid composition was measured after each intervention. Acylcarnitines were measured in the fasting state and after a euglycemic hyperinsulinemic clamp in nine subjects. Results: The relative abundance of palmitate (16:0) and the molar fatty acid ratio of palmitate to linoleic acid (16:0 to18:2) as markers of fatty acid synthesis were increased after HF [relative abundance of palmitate: 22.97% ± 5.51% (percentage of total fatty acids by weight ±SD)] and MF (26.1% ± 1.7%) compared with HS (19.40% ± 2.91%, P < .001), HG (19.43% ±3.12 %, P < .001), or baseline (19.40% ± 2.79%, P < .001). After HS and HG, the relative abundance of palmitate was equal to baseline. Fasting palmitoylcarnitine was significantly increased after HF and HS (HF and HS vs. HG: P = .005), decreasing after inhibition of lipolysis by insulin in the clamp. Conclusions: When consumed in moderate amounts, fructose but not sucrose or glucose in SSB increases fatty acid synthesis (palmitate), whereas fasting long-chain acylcarnitines are increased after both fructose and sucrose, indicating an impaired β-oxidation flux

Quantification of phenylalanine hydroxylase activity by isotope-dilution liquid chromatography-electrospray ionization tandem mass spectrometry

BACKGROUND: Residual phenylalanine hydroxylase (PAH) activity is the key determinant for the phenotype severity in phenylketonuria (PKU) patients and correlates with the patient's genotype. Activity of in vitro expressed mutant PAH may predict the patient's phenotype and response to tetrahydrobiopterin (BH(4)), the cofactor of PAH. METHODS: A robust LC-ESI-MSMS PAH assay for the quantification of phenylalanine and tyrosine was developed. We measured PAH activity a) of the PAH mutations p.Y417C, p.I65T, p.R261Q, p.E280A, p.R158Q, p.R408W, and p.E390G expressed in eukaryotic COS-1 cells; b) in different cell lines (e.g. Huh-7, Hep3B); and c) in liver, brain, and kidney tissue from wild-type and PKU mice. RESULTS: The PAH assay was linear for phenylalanine and tyrosine (r(2)≥0.99), with a detection limit of 105 nmol/L for Phe and 398 nmol/L for Tyr. Intra-assay and inter-assay coefficients of variation were <5.3% and <6.2%, respectively, for the p.R158Q variant in lower tyrosine range. Recovery of tyrosine was 100%. Compared to the wild-type enzyme, the highest PAH activity at standard conditions (1 mmol/L L-Phe; 200 μmol/L BH(4)) was found for the mutant p.Y417C (76%), followed by p.E390G (54%), p.R261Q (43%), p.I65T (33%), p.E280A (15%), p.R158Q (5%), and p.R408W (2%). A relative high PAH activity was found in kidney (33% of the liver activity), but none in brain. CONCLUSIONS: This novel method is highly sensitive, specific, reproducible, and efficient, allowing the quantification of PAH activity in different cells or tissue extracts using minimum amounts of samples under standardized condition