In cancer and autoimmune diseases, immunoglobulins with a specific molecular signature that could potentially be used as diagnostic or prognostic markers are released into body fluids. An immunomics approach based on this phenomenon relies on the ability to identify the specific amino acid sequences of the complementarity-determining regions (CDR) of these immunoglobulins, which in turn depends on the level of accuracy, resolution, and sensitivity that can be achieved by advanced mass spectrometry. Reproducible isolation and sequencing of antibody fragments (e.g., Fab) by high-resolution mass spectrometry (MS) from seven healthy donors revealed 43 217 MS signals: 225 could be associated with CDR1 peptides, 513 with CDR2 peptides, and 19 with CDR3 peptides. Seventeen percent of the 43 217 MS signals did not overlap between the seven donors. The Fab isolation method used is reproducible and fast, with a high yield. It provides only one Fab sample fraction for subsequent characterization by high-resolution MS. In 17% and 4% of these seven healthy donors, qualitative (presence/absence) and quantitative (intensity) differences in Fab fragments could be demonstrated, respectively. From these results, we conclude that the identification of a CDR signature as biomarker for autoimmune diseases and cancer without prior knowledge of the antigen is feasible.

Nonsyndromic orofacial clefts (OFC) are common birth defects caused by certain genes interacting with environmental factors. Mutations and association studies indicate that the homeobox gene MSX1 plays a role in human clefting. In a Dutch case-control triad study (mother, father, and child), we investigated interactions between MSX1 and the parents' periconceptional lifestyle in relation to the risk of OFC in their oVspring. We studied 181 case-and 132 control mothers, 155 case-and 121 control fathers, and 176 case-and 146 control children, in which there were 107 case triads and 66 control triads. Univariable and multivariable logistic regression analyses were applied, and odds ratios (OR), 95% conWdence intervals (CI) were calculated. Allele 4 of the CA marker in the MSX1 gene, consisting of nine CA repeats, was the most common allele found in both the case and control triads. SigniWcant interactions were observed between allele 4 homozygosity of the child with maternal smoking (OR 2.7, 95% CI 1.1-6.6) and with smoking by both parents (OR 4.9, 95% CI 1.4-18.0). Allele 4 homozygosity in the mother and smoking showed a risk estimate of OR 3.2 (95% CI 1.1-9.0). If allele 4 homozygous mothers did not take daily folic acid supplements in the recommended periconceptional period, this also increased the risk of OFC for their oVspring (OR 2.8, 95% CI 1.1-6.7). Our Wndings show that, in the Dutch population, periconceptional smoking by both parents interacts with a speciWc allelic variant of MSX1 to signiWcantly increase OFC risk for their oVspring. Possible underlying mechanisms are discussed.

Late diagnosis of lung cancer is still the main reason for high mortality rates in lung cancer. Lung cancer is a heterogeneous disease which induces an immune response to different tumor antigens. Several methods for searching autoantibodies have been described that are based on known purified antigen panels. The aim of our study is to find evidence that parts of the antigen-binding-domain of antibodies are shared among lung cancer patients. This was investigated by a novel approach based on sequencing antigen-binding-fragments (Fab) of immunoglobulins using proteomic techniques without the need of previously known antigen panels. From serum of 93 participants of the NELSON trial IgG was isolated and subsequently digested into Fab and Fc. Fab was purified from the digested mixture by SDS-PAGE. The Fab containing gel-bands were excised, tryptic digested and measured on a nano-LC-Orbitrap-Mass-spectrometry system. Multivariate analysis of the mass spectrometry data by linear canonical discriminant analysis combined with stepwise logistic regression resulted in a 12-antibody-peptide model which was able to distinguish lung cancer patients from controls in a high risk population with a sensitivity of 84% and specificity of 90%. With our Fab-purification combined Orbitrap-mass-spectrometry approach, we found peptides from the variable-parts of antibodies which are shared among lung cancer patients.

Endocardial cushion defects (ECDs) of the cardiac outflow tract are among the most common congenital heart disease phenotypes. VEGF is essential for endocardial cushion formation and derangements in VEGF synthesis lead to ECD. Three functional single nucleotide polymorphisms (SNPs) in the VEGF gene Ϫ2578 CϾA, Ϫ1154 GϾA, and Ϫ634 GϾC play a role in cardiogenesis. In a Dutch case-control family study of triads, 190 case and 317 control children with both parents, we investigated linkage and association between these VEGF SNPs and ECD. Allele frequencies for the three VEGF SNPs were comparable between ECD children and controls. However, VEGF alleles Ϫ2578 C and Ϫ1154 G were transmitted more frequently to children with ECD (p ϭ 0.003 and p ϭ 0.002), in particular perimembranous ventricular septal defects (p ϭ 0.012 and p ϭ 0.006). The Ϫ2578A/Ϫ1154A/Ϫ634G haplotype was associated with a reduced risk of ECD (OR 0.7; 95% CI, 0.6 -1.0) and was significantly less transmitted to children with ECD (p ϭ 0.002). In a Dutch population, we show that the VEGF 2578 C, Ϫ1154 G alleles, and the AAG haplotype are associated with ECD. Possible VEGF gene-environment interactions exposures are discussed. (Pediatr Res 67: 23-28, 2010) C ongenital heart diseases are the most frequent major congenital malformations with a worldwide birth prevalence rate of over one million per year (1). Because of the high infant mortality and morbidity rates, the causes of these malformations should be identified and targeted for preventive strategies in the future (2). Most congenital heart diseases have a complex origin, in which interactions between subtle genetic variations and periconception exposures play a role (3). Vasculogenesis and heart development are dependent on the genetic constitution of the embryo derived from both parents, and the maternal genetically controlled nutritional, endocrine and metabolic environment (4). During cardiac development, the outflow tract is divided into the ascending aorta and the pulmonary trunk and aligned to the left and right ventricle, respectively. The formation of the endocardial cushions in the atrioventricular canal and outflow tract takes place by epithelial-mesenchymal transformation, which is a key process in the development of the heart septa and valves. Numerous lines of research have implicated a strict spatiotemporal expression pattern of the VEGF gene in the control of endocardial cushion development (5). In addition to its tight regulation during cardiogenesis, VEGF gene expression is also regulated by environmental exposures. A nice example is that hypoxia induces VEGF gene expression and seems to contribute as such to the generation of major malformations in the atrioventricular-canal and outflow tract in mice (6). Hyperglycemia inhibits epithelial-mesenchymal transformation by reducing VEGF gene expression (7). Furthermore, hyperhomocysteinemia, which increases the risk of human congenital heart disease, was shown to up-regulate VEGF mRNA in a human endothelial cell line (8). Intere...