Identifying phenotypic expansions for congenital diaphragmatic hernia plus (CDH+) using DECIPHER data.

Congenital diaphragmatic hernia (CDH) can occur in isolation or in conjunction with other birth defects (CDH+). A molecular etiology can only be identified in a subset of CDH cases. This is due, in part, to an incomplete understanding of the genes that contribute to diaphragm development. Here, we used clinical and molecular data from 36 individuals with CDH+ who are cataloged in the DECIPHER database to identify genes that may play a role in diaphragm development and to discover new phenotypic expansions. Among this group, we identified individuals who carried putatively deleterious sequence or copy number variants affecting CREBBP, SMARCA4, UBA2, and USP9X. The role of these genes in diaphragm development was supported by their expression in the developing mouse diaphragm, their similarity to known CDH genes using data from a previously published and validated machine learning algorithm, and/or the presence of CDH in other individuals with their associated genetic disorders. Our results demonstrate how data from DECIPHER, and other public databases, can be used to identify new phenotypic expansions and suggest that CREBBP, SMARCA4, UBA2, and USP9X play a role in diaphragm development.

A new case of SMABF2 diagnosed in stillbirth expands the prenatal presentation and mutational spectrum of ASCC1.

Spinal muscular atrophy with congenital bone fractures 2 (SMABF2) is a rare autosomal recessive neuromuscular disorder characterized by arthrogryposis multiplex congenita and prenatal fractures of the long bones, with poor prognosis. The most affected patients present with biallelic loss-of-function nucleotide variants in ASCC1 gene, coding a subunit of the transcriptional coactivator ASC-1 complex, although the exact pathogenesis is yet unknown. This work describes the first case of SMABF2 in a stillbirth with documented evolution of the disease in the prenatal period. A microdeletion copy number variant (CNV) of about 64 Kb, involving four exons of ASCC1, was firstly detected by microarray analysis, requested for arthrogryposis and hydrops. Subsequent exome analysis disclosed a nucleotide variant of the same gene [c.1027C>T; (p. Arg343*)], resulting in the introduction of a premature termination codon. This stillbirth represents the first case of ASCC1 compound heterozygosity, due to an exonic microdeletion and a nucleotide variant, expanding the mutational spectrum of this gene. It also provides further evidence that exonic CNVs are an underestimated cause of disease-alleles and that the integrated use of the last generation genetic analysis tools, together with careful clinical evaluations, are fundamental for the characterization of rare diseases even in the prenatal setting.

Fennel allergy is a lipid-transfer protein (LTP)-related food hypersensitivity associated with peach allergy.

Fennel allergy has been rarely reported, and the association with peach allergy has never been described. Our aim was to (i) study the correlation between symptom severity of peach and fennel and (ii) identify fennel allergens and the role of rPru p 3 antibodies in severe reactions to fennel. In 148 patients with peach allergy, we investigated 58 patients with symptoms and IgE antibodies positive to fennel. IgE to rPru p 1, 3, and 4 and rBet v 1, 2, and 4 were measured by immunoblotting, and the N-terminal amino acid sequences and relevant allergens were determined. We found significant association between severe reactions to fennel and peach (p = 0.0009). A major allergen was ~9 kDa lipid-transfer protein (LTP), cross-reactive with Pru p 3, a 15 kDa protein identified as a pathogenesis-related protein 1 of the Bet v 1 family. In conclusion, peach and fennel severe allergic symptoms are significantly related, and LTP is a major fennel allergen. Fennel should be included in the LTP syndrome.

Autoantibody signature in human ductal pancreatic adenocarcinoma.

Pancreatic ductal adenocarcinoma (PDAC) is a highly aggressive malignancy characterized by rapid progression, invasiveness, and resistance to treatment. It is the fourth leading cause of cancer death with a 2% 5-year survival rate. Biomarkers for its early detection are lacking. This study was designed to use a proteomics-based approach as a means of identifying antigens that elicit a humoral response in PDAC patients. Antibodies against PDAC-associated antigens are useful for early cancer diagnosis and therapy. Proteins from PDAC cell lines were separated by 2-DE, and the serum IgG reactivity of 70 PDAC patients, 40 healthy subjects (HS), 30 non-PDAC tumor patients, and 15 chronic pancreatitis (CP) patients was tested by Western blot analysis. Spots specifically recognized by PDAC sera and revealed by mass spectrometry corresponded to metabolic enzymes or cytoskeletal proteins. Most were up-regulated in PDAC tissues. Thus, it seems that metabolic enzymes and cytoskeletal proteins are specific targets of the humoral response during PDAC. The results of further studies of these serological-defined antigens could be of diagnostic and therapeutic significance in PDAC.

Combined cup loading, bis(2-hydroxyethyl) disulfide, and protein precipitation protocols to improve the alkaline proteome of Lactobacillus hilgardii.

Despite the large number of papers dealing with bacterial proteomes, very few include information about proteins with alkaline pI's, because of the limits inherent in 2-DE technology. Nonetheless, analyses of in silico proteomes of many prokaryotes show a bimodal distribution of their proteins based on their pI's; the most crowded areas lying between pI 4-7 and 9-11. The aim of the present research was to set up a general, simple, and standardizable 2-DE protocol suitable for studying the alkaline proteome of Lactobacillus hilgardii, a Gram-positive bacillus isolated from wine. The method has also been tested on a Gram-negative bacterium able to degrade aromatic pollutants, Acinetobacter radioresistens S13. Optimization of the method was mainly focused on improving protein extraction and IEF (pI 6-11) separation protocols. Concerning IEF, different methods for sample loading (in-gel rehydration and cup loading), and different reducing agents (DTT and bis(2-hydroxyethyl) disulfide (HED)) were tested and compared. The proposed protocol was found to resolve efficiently alkaline proteins from both of our Lactobacillus and Acinetobacter strains, in spite of their different external layers, thus, enabling a more comprehensive study of their proteomes.

Degradation of aromatic compounds by Acinetobacter radioresistens S13: growth characteristics on single substrates and mixtures.

Acinetobacter radioresistens S13 is able to grow on phenol or benzoate as the sole carbon and energy source: both these compounds are catabolized through the beta-ketoadipate pathway. Genes encoding the catabolic enzymes for degradation of aromatic compounds are localized on A. radioresistens S13 chromosome and organized in, at least, two distinct sets, one for benzoate degradation and another for phenol catabolism. In the present study, the growth and biodegradation kinetics for benzoate and phenol, and an easily metabolized substrate (acetate) were established. Benzoate was degraded slower and supports a less rapid and efficient growth than either acetate or phenol. A combined transcript-proteomic analysis of some of the major catabolic genes and their products nonetheless has shown that benzoate induces the expression of both benzoate and phenol catabolic operons. This result was confirmed by the fact that benzoate-acclimatized bacteria were rapidly able to degrade phenol too. Finally, the growth and biodegradation kinetics for different mixtures of acetate, benzoate and phenol were determined. Results indicate that a hierarchy of substrate utilization, benzoate > acetate > phenol, occurred: benzoate was the preferred substrate, despite its lower growth and biodegradation parameters. Hypotheses explaining these unusual metabolic features of A. radioresistens S13 are discussed.

Novel isoforms of Pru av 1 with diverging immunoglobulin E binding properties identified by a synergistic combination of molecular biology and proteomics.

Birch pollen-related food allergies are mainly associated to Bet v 1. Little is known about isoforms of Bet v 1 homologous in fruit of the Rosaceae family. We attempted to identify novel isoforms of Pru av 1, the major cherry allergen, at the cDNA and the protein level by a combination of molecular biology and proteomic tools. A cDNA library was screened with patients immunoglobulin E (IgE) and a specific hybridization probe. Edman sequencing, mass spectrometry (MS), and MS/MS were performed after detecting Pru av 1 on 2-D maps by immunoblotting using patients IgE and a monoclonal antibody. Partial amino acid sequences were completed with a polymerase chain reaction (PCR) strategy. The IgE-binding properties of the Pru av 1 spots were analyzed by 2-D blot inhibition. cDNA library analysis revealed a novel Pru av 1 isoform. MS and N-terminal sequencing confirmed the cDNA sequences at the protein level. A series of spots were confirmed as the already known Pru av 1. One spot, exclusively detected with patients sera, was identified as the novel isoform. A partial amino acid sequence detected with MS/MS was completed by PCR-cloning. The 2-D blot inhibition revealed epitope differences between the novel isoform and the previously published Pru av 1. Our data demonstrate that a synergistic combination of molecular biology and proteomics represents a powerful tool for reliable and comprehensive identification of allergen isoforms and variants. The newly identified isoform showed diverging IgE-binding properties and may be relevant for the diagnosis or therapy of cherry allergy.

The proteomic approach to analysis of human milk fat globule membrane.

Lactating mammary epithelial cells do not adhere to the classical rule of lipid secretion since lipids are secreted enveloped by the milk fat globule membrane (MFGM) via a budding process. Historically, biochemical studies of MFGM proteins focused on the analysis of single molecules. Today, thanks to proteomic technologies, it is now possible to comprehensively analyse the protein component of MFGM. Questions concerning: (1) protein organization within the MFGM structure; (2) correlation to the intracellular secretion pathway; and (3) display of multiple specialized functions, can now be addressed. Proteomics allows for rapid identification of the total MFGM proteins thereby providing a global functional screen of the lactating cell. In addition to their nutritional value, human MFGM proteins have important protective functions against bacteria, viruses and protozoa. Identifying the complex nature of protein-protein interactions would further enhance our present knowledge of MFGM function. A speculative molecular architecture of the human MFGM is proposed.