Holoprosencephaly (HPE) is the most common congenital cerebral malformation in humans, characterized by impaired forebrain cleavage and midline facial anomalies. It presents a high heterogeneity, both in clinics and genetics. We have developed a novel targeted next-generation sequencing (NGS) assay and screened a cohort of 257 HPE patients. Mutations with high confidence in their deleterious effect were identified in approximately 24% of the cases and were held for diagnosis, whereas variants of uncertain significance were identified in 10% of cases. This study provides a new classification of genes that are involved in HPE. SHH, ZIC2, and SIX3 remain the top genes in term of frequency with GLI2, and are followed by FGF8 and FGFR1. The three minor HPE genes identified by our study are DLL1, DISP1, and SUFU. Here, we demonstrate that fibroblast growth factor signaling must now be considered a major pathway involved in HPE. Interestingly, several cases of double mutations were found and argue for a polygenic inheritance of HPE. Altogether, it supports that the implementation of NGS in HPE diagnosis is required to improve genetic counseling.

Holoprosencephaly (HPE) is the most common congenital cerebral malformation, characterized by impaired forebrain cleavage and midline facial anomalies. Heterozygous mutations in 14 genes have been associated with HPE and are often inherited from an unaffected parent, underlying complex genetic bases. It is now emerging that HPE may result from a combination of multiple genetic events, rather than from a single heterozygous mutation. To explore this hypothesis, we undertook whole exome sequencing and targeted high-throughput sequencing approaches to identify mutations in HPE subjects. Here, we report two HPE families in which two mutations are implicated in the disease. In the first family presenting two foetuses with alobar and semi-lobar HPE, we found mutations in two genes involved in HPE, SHH and DISP1, inherited respectively from the father and the mother. The second reported case is a family with a 9-year-old girl presenting lobar HPE, harbouring two compound heterozygous mutations in DISP1. Together, these cases of digenic inheritance and autosomal recessive HPE suggest that in some families, several genetic events are necessary to cause HPE. This study highlights the complexity of HPE inheritance and has to be taken into account by clinicians to improve HPE genetic counselling.

Development of a new generation of vaccines is a key challenge for the control of infectious diseases affecting both humans and animals. Synthetic biology methods offer new ways to engineer bacterial chassis that can be used as vectors to present heterologous antigens and train the immune system against pathogens. Here, we describe the construction of a bacterial chassis based on the fast-growing Mycoplasma feriruminatoris, and the first steps toward its application as a live vaccine against contagious caprine pleuropneumonia (CCPP). To do so, the M. feriruminatoris genome was cloned in yeast, modified by iterative cycles of Cas9-mediated deletion of loci encoding virulence factors, and transplanted back in Mycoplasma capricolum subsp. capricolum recipient cells to produce the designed M. feriruminatoris chassis. Deleted genes encoded the glycerol transport and metabolism systems GtsABCD and GlpOKF and the Mycoplasma Ig binding protein-Mycoplasma Ig protease (MIB-MIP) immunoglobulin cleavage system. Phenotypic assays of the M. feriruminatoris chassis confirmed the corresponding loss of H 2 O 2 production and IgG cleavage activities, while growth remained unaltered. The resulting mycoplasma chassis was further evaluated as a platform for the expression of heterologous surface proteins. A genome locus encoding an inactivated MIB-MIP system from the CCPP-causative agent Mycoplasma capricolum subsp. capripneumoniae was grafted in replacement of its homolog at the original locus in the chassis genome. Both heterologous proteins were detected in the resulting strain using proteomics, confirming their expression. This study demonstrates that advanced genome engineering methods are henceforth available for the fast-growing M. feriruminatoris, facilitating the development of novel vaccines, in particular against major mycoplasma diseases.