ABO maternal-child discordance: Evidence of variable allelic expression and considerations for investigation.

BACKGROUND: We report a case of apparent mother-child ABO group noninheritance. A Caucasian mother initially typed as group O and her infant group AB. Investigation ruled out preanalytical causes such as mislabeled samples and in vitro fertilization. MATERIALS AND METHODS: Red blood cells were characterized by routine serologic testing. Genomic data were analyzed by targeted polymerase chain reaction-restriction fragment length polymorphism and Sanger sequencing. Transferase structures were modeled using PyMOL molecular visualization software. RESULTS: Serologic testing initially demonstrated the mother was group O, father group AB, and infant group AB. Further testing of the maternal sample with anti-A,B demonstrated weak A expression. Molecular testing revealed the maternal sample had an ABO*O.01.01 allele in trans to an A allele, ABO*AW.29 (c.311T>A, p.Ile104Asn), determined by gene sequencing. The sample from the infant carried the same ABO*AW.29 allele in trans to a B allele, ABO*B.01. CONCLUSION: ABO genotyping revealed an A transferase encoded by ABO*AW.29, with apparent variable activity. Although A antigen expression is well known to be weak in newborns, it was robust on the red blood cells (RBCs) of the AB infant and undetectable with anti-A on the mother. Variable expression of weak subgroups may reflect competition or enhancement by a codominant allele, as well as glycan chain maturation on red cells. Previous examples in group AB mothers with Aweak infants suggested that the decreased expression is primarily due to glycan immaturity. To our knowledge, this is the first reported case of the ABO*AW.29 allele presenting with weak A expression in a group Aweak mother and robust A expression in a group AB infant, suggesting the in trans allele is an important factor in determining transferase activity and may override age-related effects.

Diagnostic utility of glycosyltransferase mRNA expression in gastric cancer.

OBJECTIVE/BACKGROUND: Posttranslational modification of proteins, including glycosylation, is known to differ between normal and tumor cells. Altered glycosyltransferase levels have been observed in tumor tissues and their role in tumor metastasis and invasion has been implicated. In this study the role of altered glycosyltransferase messenger RNA (mRNA) levels in serum of gastric cancer patients as early markers of gastric cancer was evaluated. METHODS: In this case control study the expression profile of ppGalNAc-T6, GlcNAcT-V, ST3Gal I, ST3 Gal IV, and ST6GalNAc-I in normal healthy control and gastric cancer patients was compared. Serum was isolated from blood samples of gastric cancer patients (n = 200) and controls (n = 200). Following RNA extraction, reverse transcription was carried out and transcript levels of glycosyltransferases were determined using real-time quantitative polymerase chain reaction and normalized against glyceraldehyde-3-phosphate dehydrogenase (GAPDH) expression. The amount of target gene, normalized to an endogenous reference gene relative to calibrator was calculated by using ΔΔCT method. Transcript levels in the serum samples of gastric cancer patients were compared with those of controls; also the same was correlated within sex and different stages of disease. RESULTS: The mRNA expression of ppGalNAc-T6 and ST6GalNAc-I was significantly higher in serum samples of gastric cancer patients on comparison with controls (p = .008), however, there was no significant difference in mRNA expression of GlcNAcT-V, ST3Gal I, and ST3 Gal IV in serum samples of gastric cancer patients and controls (p = .097). In addition, no significant association of mRNA expression of these glycosyltransferases was found within sex and stages in this study. CONCLUSION: This study revealed the potential of ppGalNAc-T6 and ST6GalNAc-I mRNA transcript levels in serum as markers of gastric cancer. Further studies on the wider range of glycosyltransferases in various cancers are needed to establish signature mRNA batteries as minimally invasive markers of gastric cancer.

Effects of plasma glycosyltransferase on the ABO(H) blood group antigens of human von Willebrand factor.

Von Willebrand factor (VWF) is one of the plasma protein carrying ABO(H) blood group antigens, but the combining process of these antigens is not clear. In the present study, we examined whether plasma glycosyltransferase affects the blood group antigens on VWF. VWF expressing H-antigen (H-VWF) from blood group O and bovine serum albumin conjugated with H-antigen (H-BSA) were incubated with recombinant α1-3-N-acetylgalactosaminyltransferase (rA-transferase) and A-plasma with or without an additional UDP-GalNAc. Transformed antigens were detected by western blotting and ELISA, using an anti-A antibody. Both H-VWF and H-BSA acquired the A-antigen after incubation with rA-transferase and UDP-GalNAc. Incubation with A-plasma very weakly converted the H-antigen on BSA and VWF to A-antigen only in the presence of supplemented UDP-GalNAc. This conversion was enhanced on desialylation of H-VWF. These results indicate that sugar chains of plasma VWF can be modified by the external glycosyltransferase, but that plasma glycosyltransferase has no effect on the blood group antigens of VWF due to its low activity and the lack of donor sugars. Further, sialic acid residues of VWF may exert a protective effect against post-translational glycosylation. Our results clearly exclude the possibility that blood group antigens of VWF are constructed extracellularly in plasma.

Glycosyltransferases as marker genes for the quantitative polymerase chain reaction-based detection of circulating tumour cells from blood samples of patients with breast cancer undergoing adjuvant therapy.

Altered glycosylation is a predominant feature of tumour cells; it serves for cell adhesion and detachment, respectively, and facilitates the immune escape of these cells. Therefore changes in the expression of glycosyltransferase genes could help to identify circulating tumour cells (CTCs) in the blood samples of cancer patients using a quantitative polymerase chain reaction (PCR) approach. Blood samples of healthy donors were inoculated with certain numbers of established breast cancer cell line cells, thus creating a model system. These samples were analysed by quantitative PCR for the expression of six different glycosyltransferase genes. The three genes with the best results in the model system were consecutively applied to samples from adjuvant breast cancer patients and of healthy donors. FUT3 and GALNT6 showed the highest increase in relative expression, while GALNT6 and ST3GAL3 were the first to reach statistically significant different ∆CT-values comparing the sample with and without addition of tumour cells. These three genes were applied to patient samples, but did not show any significant results that may suggest the presence of CTCs in the blood. Although the relative expression of some of the glycosyltransferase genes exhibited reasonable results in the model system, their application to breast cancer patient samples will have to be further improved, e.g. by co-analysis of patient blood samples by gold-standard methods.

Pleiotropic effect of common variants at ABO Glycosyltranferase locus in 9q32 on plasma levels of pancreatic lipase and angiotensin converting enzyme.

For forty-three clinical test values presumably associated to common complex human diseases, we carried out a genome-wide association study using 600K SNPs in a general Japanese population of 1,639 individuals (1,252 after quality control procedures) drawn from a regional cohort, followed by a replication study for statistically significant SNPs (p = 1.95 × 10(-9)-8.34 × 10(-39)) using an independent population of 1,671 from another cohort. In this single two-stage study, we newly found strong and robust associations of common variants at the ABO histo-blood glycosyltransferase locus in 9q32 with the plasma levels of pancreatic lipase (P-LIP), in addition to successful confirmation of the known ABO association of angiotensin converting enzyme (ACE) independent of the ACE1 gene in 17q23.2 with the ACE level. Our results are compatible with the previously reported association between the ABO gene and pancreatic cancer, and show that the effect of these common variants at the ABO locus on the P-LIP and ACE levels is largely opposing and pleiotropic.

ITIH3 polymorphism may confer susceptibility to psychiatric disorders by altering the expression levels of GLT8D1.

A recent genome-wide analysis indicated that a polymorphism (rs2535629) of ITIH3 showed the strongest association signal with susceptibility to psychiatric disorders in Caucasian populations. The aim of the study was to replicate the association of rs2535629 with schizophrenia and major depressive disorder (MDD) in Japanese subjects. A total of 611 patients with schizophrenia, 868 with MDD, and 1193 healthy controls were successfully genotyped for rs2535629. A significant difference in allele distribution was found between patients with schizophrenia and controls (odds ratio [OR] = 1.21, 95% confidence interval [CI]: 1.05-1.39, P = 0.0077). A similar trend was found for patients with MDD (OR = 1.11, 95% CI: 0.98-1.26, P = 0.092). The allele distribution in the combined patient group (schizophrenia and MDD) was significantly different from that of the control group (OR = 1.15, 95% CI: 1.03-1.28, P = 0.011). Gene expression microarray analysis of whole blood samples in 39 MDD patients and 40 healthy controls showed that rs2535629 has a strong influence on the expression levels of ITIH4 and GLT8D1. The expression levels of GLT8D1 were significantly higher in patients with MDD than in controls (P = 0.021). To our knowledge, the present study showed for the first time the association of rs2535629 with psychiatric disorders in an Asian population. Our findings suggest that rs2535629 influences the susceptibility to psychiatric disorders by affecting the expression level of GLT8D1.

Loci associated with N-glycosylation of human immunoglobulin G show pleiotropy with autoimmune diseases and haematological cancers.

Glycosylation of immunoglobulin G (IgG) influences IgG effector function by modulating binding to Fc receptors. To identify genetic loci associated with IgG glycosylation, we quantitated N-linked IgG glycans using two approaches. After isolating IgG from human plasma, we performed 77 quantitative measurements of N-glycosylation using ultra-performance liquid chromatography (UPLC) in 2,247 individuals from four European discovery populations. In parallel, we measured IgG N-glycans using MALDI-TOF mass spectrometry (MS) in a replication cohort of 1,848 Europeans. Meta-analysis of genome-wide association study (GWAS) results identified 9 genome-wide significant loci (P<2.27 × 10(-9)) in the discovery analysis and two of the same loci (B4GALT1 and MGAT3) in the replication cohort. Four loci contained genes encoding glycosyltransferases (ST6GAL1, B4GALT1, FUT8, and MGAT3), while the remaining 5 contained genes that have not been previously implicated in protein glycosylation (IKZF1, IL6ST-ANKRD55, ABCF2-SMARCD3, SUV420H1, and SMARCB1-DERL3). However, most of them have been strongly associated with autoimmune and inflammatory conditions (e.g., systemic lupus erythematosus, rheumatoid arthritis, ulcerative colitis, Crohn's disease, diabetes type 1, multiple sclerosis, Graves' disease, celiac disease, nodular sclerosis) and/or haematological cancers (acute lymphoblastic leukaemia, Hodgkin lymphoma, and multiple myeloma). Follow-up functional experiments in haplodeficient Ikzf1 knock-out mice showed the same general pattern of changes in IgG glycosylation as identified in the meta-analysis. As IKZF1 was associated with multiple IgG N-glycan traits, we explored biomarker potential of affected N-glycans in 101 cases with SLE and 183 matched controls and demonstrated substantial discriminative power in a ROC-curve analysis (area under the curve = 0.842). Our study shows that it is possible to identify new loci that control glycosylation of a single plasma protein using GWAS. The results may also provide an explanation for the reported pleiotropy and antagonistic effects of loci involved in autoimmune diseases and haematological cancer.

Multiplexed surrogate analysis of glycotransferase activity in whole biospecimens.

Dysregulated glycotransferase enzymes in cancer cells produce aberrant glycans--some of which can help facilitate metastases. Within a cell, individual glycotransferases promiscuously help to construct dozens of unique glycan structures, making it difficult to comprehensively track their activity in biospecimens--especially where they are absent or inactive. Here, we describe an approach to deconstruct glycans in whole biospecimens then analytically pool together resulting monosaccharide-and-linkage-specific degradation products ("glycan nodes") that directly represent the activities of specific glycotransferases. To implement this concept, a reproducible, relative quantitation-based glycan methylation analysis methodology was developed that simultaneously captures information from N-, O-, and lipid linked glycans and is compatible with whole biofluids and homogenized tissues; in total, over 30 different glycan nodes are detectable per gas chromatography-mass spectrometry (GC-MS) run. Numerous nonliver organ cancers are known to induce the production of abnormally glycosylated serum proteins. Thus, following analytical validation, in blood plasma, the technique was applied to a group of 59 lung cancer patient plasma samples and age/gender/smoking-status-matched non-neoplastic controls from the Lung Cancer in Central and Eastern Europe (CEE) study to gauge the clinical utility of the approach toward the detection of lung cancer. Ten smoking-independent glycan node ratios were found that detect lung cancer with individual receiver operating characteristic (ROC) c-statistics ranging from 0.76 to 0.88. Two glycan nodes provided novel evidence for altered ST6Gal-I and GnT-IV glycotransferase activities in lung cancer patients. In summary, a conceptually novel approach to the analysis of glycans in unfractionated human biospecimens has been developed that, upon clinical validation for specific applications, may provide diagnostic and/or predictive information in glycan-altering diseases.

[Update regarding the role of biomarkers in early diagnosis of non-small cell bronchopulmonary cancer]. / Actualitati privind rolul markerilor tumorali in detectia precoce a neoplasmului bronhopulmonar NSCC.

Early diagnosis of lung cancer by non-invasive methods has a low sensibility: 60% of peripheral cancers could be diagnosed by computed tomography, 60% of the central ones by sputum cytology. More specific for detecting central microinvasive lesions could be bronchoscopy with autofluorescence, but this is a method with a low number of patients to be performed on, because of the specific technique. For all these reasons there are some other methods to be tried in this respect--one of them is to find one or more molecules--tumoral markers--which have to be specific in establishing the risk of developing lung cancer, to make an early diagnosis of cancer and to predict the evolution under treatment. The detecting tumoral markers in sputum, blood, bronchoalveolar lavage was not so largely explored related to the final goal--the possibility of identifying and quantifying the most specific ones for the screening of lung cancer. The present paper has as purpose to make an review of tumoral markers--"classical" markers as: CEA, NSE, TPA, beta2 microglobulina, CA 125, CA 15-3--considered not such a high sensibility and specificity for lung cancer screening versus new molecules, studied intensively as: SCC-Ag, CYFRA 21-1, ferritin, sIL-2R, CCK-BB, glycosyltransferases. Those new molecules have a higher sensibility, but also could have a higher specificity for each type of lung cancer.

Congenital dyserythropoietic anemia type II (CDAII/HEMPAS): where are we now?

Congenital diserythropoietic anemias (CDA) were classified according to bone marrow changes and biochemical features 40 years ago. A consistent finding in CDA type II, the most frequent subgroup of CDAs is a relevant hypoglycosylation of erythrocyte membrane proteins. It is a matter of debate if the hypoglycosylation is the primary cause of the disorder or a phenomenon secondary to other pathomechanisms. The molecular cause of the disorder is still unknown although some enzyme deficiencies have been proposed to cause CDA II in the last 2 decades and a linkage analysis locating the CDA II gene in a 5 cM region on chromosome 20 was done in 1997. In this review biochemical and genetic data are discussed and diagnostic methods based on biochemical observations of the recent years are reviewed.

Tetragametic chimerism detected in a healthy woman with mixed-field agglutination reactions in ABO blood grouping.

BACKGROUND: The case of a healthy woman with serologic blood group AB and her biologic father showing blood group O was investigated. Further analysis, including blood, buccal swabs, and nail clippings, revealed a tetragametic chimerism. STUDY DESIGN AND METHODS: Blood grouping was performed with standard gel centrifugation test cards, ABO genotyping by sequence-specific primers (SSPs) and sequence-based typing, and HLA Class I and II typing by standard NIH cytotoxicity testing and SSP. Additionally, short-tandem-repeat (STR) and variable-number tandem-repeat (VNTR) typing was performed on blood, nail clippings, and buccal swab samples. The karyotype was analyzed by G-banded chromosomes. RESULTS: The proposita's RBCs were typed AB with a mixed-field agglutination whereas in molecular typing A, B, and O alleles were found. One paternal and two maternal haplotypes were determined by use of HLA typing. Interestingly, both paternal alleles were detected in 4 of 23 tested STR and VNTR loci only, with whole blood, nail clippings, and buccal swabs. The karyotype was identified as 46XX. The family members including the proposita's healthy twin children displayed no abnormal findings in tests performed. CONCLUSION: By investigation of DNA polymorphisms, it was possible to determine a rare case of tetragametic chimerism being the result of double parental contribution of nuclei.

Purine pathway enzymes in a cyst forming strain of Toxoplasma gondii.

The activities of purine salvage enzymes in tachyzoites from a cyst-forming strain of Toxoplasma gondii were determined using HPLC. Six enzymes were assayed both in vitro and in vivo: adenosine deaminase, guanine deaminase, purine nucleoside phosphorylase, xanthine oxidase, hypoxanthine-guanine phosphoribosyltransferase and adenine phosphoribosyltransferase. In vitro, the tachyzoites were cultured in the human myelomonocytic cell line THP-1, for 24 h to 96 h. Neither guanine deaminase nor hypoxanthine-guanine phosphoribosyltransferase activity was detected in 24 and 96 h cultures. In vivo, in controls and infected animals, the purine nucleoside phosphorylase and adenosine deaminase activities were the most important activities both in sera and cerebral tissue in comparison with the other activities. It was also noted that the infection modified the enzymatic activities of this purine salvage pathway, in particular, the guanine deaminase cerebral activity of infected mice was 20-fold lower than the value of controls. The treatment of mice with 2',3'-dideoxyinosine, a purine analog, at the dose of 100 mg.kg(-1).d for 30 days, induced an important increase of all enzymatic activities in the brains in comparison with control animals. These data suggest that one target of 2',3'-dideoxyinosine is the purine metabolism.

Immunoassays for cancer-associated carbohydrate antigens.

The advent of hybridoma technology and the availability of virtually unlimited amounts of specific high affinity monoclonal antibodies to desired antigens have ushered in a new revolution in immunoassay methodology. An ever increasing number of monoclonal antibody-based immunoassays are replacing those employing polyclonal antisera. A host of hitherto unknown tumor-associated antigens have also been identified due to the development of unique monoclonal antibodies. The exquisite specificities and affinities of monoclonals has led to the development of sensitive and reproducible immunoassays for various analytes, especially tumor-associated antigens. Most tumor-associated antigens are glycoconjugates and among these many are blood group antigens and their derivatives, found both as glycoproteins and glycolipids. The development and clinical applications of new immunoassays for cancer-associated carbohydrate antigens in the last decade has had a major positive impact on the management of cancer patients.