Integrin alpha 6 homozygous splice-site mutation causes a new form of junctional epidermolysis bullosa in Charolais cattle.

BACKGROUND: Inherited epidermolysis bullosa (EB) is a group of painful and life-threatening genetic disorders that are characterized by mechanically induced blistering of the skin and mucous membranes. Congenital skin fragility resembling EB was recently reported in three Charolais calves born in two distinct herds from unaffected parents. Phenotypic and genetic analyses were carried out to describe this condition and its molecular etiology. RESULTS: Genealogical, pathological and histological investigations confirmed the diagnosis of recessive EB. However, the affected calves showed milder clinical signs compared to another form of EB, which was previously reported in the same breed and is caused by a homozygous deletion of the ITGB4 gene. Homozygosity mapping followed by analysis of the whole-genome sequences of two cases and 5031 control individuals enabled us to prioritize a splice donor site of ITGA6 (c.2160 + 1G > T; Chr2 g.24112740C > A) as the most compelling candidate variant. This substitution showed a perfect genotype-phenotype correlation in the two affected pedigrees and was found to segregate only in Charolais, and at a very low frequency (f = 1.6 × 10-4) after genotyping 186,154 animals from 15 breeds. Finally, RT-PCR analyses revealed increased retention of introns 14 and 15 of the ITGA6 gene in a heterozygous mutant cow compared with a matched control. The mutant mRNA is predicted to cause a frameshift (ITGA6 p.I657Mfs1) that affects the assembly of the integrin α6ß4 dimer and its correct anchoring to the cell membrane. This dimer is a key component of the hemidesmosome anchoring complex, which ensures the attachment of basal epithelial cells to the basal membrane. Based on these elements, we arrived at a diagnosis of junctional EB. CONCLUSIONS: We report a rare example of partial phenocopies observed in the same breed and due to mutations that affect two members of the same protein dimer, and provide the first evidence of an ITGA6 mutation that causes EB in livestock species.

Development and evaluation of a core genome multilocus sequence typing scheme for Paenibacillus larvae, the deadly American foulbrood pathogen of honeybees.

Paenibacillus larvae is the causative agent of the fatal American foulbrood disease in honeybees (Apis mellifera). Strain identification is vital for preventing the spread of the disease. To date, the most accessible and robust scheme to identify strains is the multilocus sequence typing (MLST) method. However, this approach has limited resolution, especially for epidemiological studies. As the cost of whole-genome sequencing has decreased and as it becomes increasingly available to most laboratories, an extended MLST based on the core genome (cgMLST) presents a valuable tool for high-resolution investigations. In this study, we present a standardized, robust cgMLST scheme for P. larvae typing using whole-genome sequencing. A total of 333 genomes were used to identify, validate and evaluate 2419 core genes. The cgMLST allowed fine-scale differentiation between samples that had the same profile using traditional MLST and allowed for the characterization of strains impossible by MLST. The scheme was successfully used to trace a localized Swedish outbreak, where a cluster of 38 isolates was linked to a country-wide beekeeping operation. cgMLST greatly enhances the power of a traditional typing scheme, while preserving the same stability and standardization for sharing results and methods across different laboratories.

Mitotic bookmarking redundancy by nuclear receptors in pluripotent cells.

Mitotic bookmarking transcription factors (TFs) are thought to mediate rapid and accurate reactivation after mitotic gene silencing. However, the loss of individual bookmarking TFs often leads to the deregulation of only a small proportion of their mitotic targets, raising doubts on the biological significance and importance of their bookmarking function. Here we used targeted proteomics of the mitotic bookmarking TF ESRRB, an orphan nuclear receptor, to discover a large redundancy in mitotic binding among members of the protein super-family of nuclear receptors. Focusing on the nuclear receptor NR5A2, which together with ESRRB is essential in maintaining pluripotency in mouse embryonic stem cells, we demonstrate conjoint bookmarking activity of both factors on promoters and enhancers of a large fraction of active genes, particularly those most efficiently reactivated in G1. Upon fast and simultaneous degradation of both factors during mitotic exit, hundreds of mitotic targets of ESRRB/NR5A2, including key players of the pluripotency network, display attenuated transcriptional reactivation. We propose that redundancy in mitotic bookmarking TFs, especially nuclear receptors, confers robustness to the reestablishment of gene regulatory networks after mitosis.

Analysis of the impact of DGAT1 p.M435L and p.K232A variants on pre-mRNA splicing in a full-length gene assay.

DGAT1 is playing a major role in fat metabolism and triacylglyceride synthesis. Only two DGAT1 loss-of-function variants altering milk production traits in cattle have been reported to date, namely p.M435L and p.K232A. The p.M435L variant is a rare alteration and has been associated with skipping of exon 16 which results in a non-functional truncated protein, and the p.K232A-containing haplotype has been associated with modifications of the splicing rate of several DGAT1 introns. In particular, the direct causality of the p.K232A variant in decreasing the splicing rate of the intron 7 junction was validated using a minigene assay in MAC-T cells. As both these DGAT1 variants were shown to be spliceogenic, we developed a full-length gene assay (FLGA) to re-analyse p.M435L and p.K232A variants in HEK293T and MAC-T cells. Qualitative RT-PCR analysis of cells transfected with the full-length DGAT1 expression construct carrying the p.M435L variant highlighted complete skipping of exon 16. The same analysis performed using the construct carrying the p.K232A variant showed moderate differences compared to the wild-type construct, suggesting a possible effect of this variant on the splicing of intron 7. Finally, quantitative RT-PCR analyses of cells transfected with the p.K232A-carrying construct did not show any significant modification on the splicing rate of introns 1, 2 and 7. In conclusion, the DGAT1 FLGA confirmed the p.M435L impact previously observed in vivo, but invalidated the hypothesis whereby the p.K232A variant strongly decreased the splicing rate of intron 7.

Allergen Risk Assessment for Specific Allergy to Small Ruminant's Milk: Development of Sensitive Immunoassays to Detect Goat's and Sheep's Milk Contaminations in Dairy Food Matrices.

Despite a high level of sequence identity between cow's, goat's, and sheep's milk (CM, GM, and SM, respectively) proteins, some patients tolerant to CM are allergic to GM and SM. In most cases, this specificity is due to the presence of IgE antibodies that bind only to caprine and ovine caseins. The patients may then develop severe allergic reactions after ingestion of CM products contaminated with low amounts of GM or SM. We thus aimed to develop an assay able to detect traces of caprine/ovine ß-caseins in different food matrices, irrespective of the presence of the bovine homolog. We produced monoclonal antibodies (mAbs) specific to caprine caseins in mice tolerized to the bovine whole casein then sensitized to the caprine whole casein. In order to develop a two-site immunometric assay, we selected mAbs that could discriminate the caprine ß-casein from its bovine homolog. Characteristics and performances of two tests were determined with various dairy products. Results were analyzed in relation with the IgE-immunoreactivity of the food matrices, thanks to sera from CM, GM/SM allergic patients. Our two-site immunometric assays demonstrated a high sensitivity with a detection limit of 1.6-3.2 ng/mL of caprine and ovine ß-caseins. The tests were able to detect contaminations of GM in CM at the ppm level. Heat-treatment, ripening and coagulation processes, usually applied to dairy products that exhibit a very high IgE-immunoreactivity, did not impair the test sensitivity. These quantitative assays could then be useful for the risk assessment of food products potentially contaminated with GM and SM in order to prevent adverse reactions in patients specifically allergic to these milks.