Because spontaneous mutations occur at a low frequency, chemical mutagenesis of the mouse genome with the alkylating agent N-ethyl-N-nitrosourea (ENU) increases dramatically the mouse mutants produced. ENU is the most potent mutagen in the mouse causing random point mutations by direct alkylation of nucleic acids and is emerging as a superb tool for dissecting mammalian genome-phenome links both for the rate and the nature of genetic variants it produces. Many of the mutants produced by ENU carry hypomorphic (partial loss-of-function) mutations, although also gain-of-function as well as complete loss-of-function mutants are expected. Mutations are identified by a combination of mapping and sequencing. Mapping relies on highly polymorphic markers, Simple Sequence Length Polymorphisms (SSLPs) and Single Nucleotide Polymorphisms (SNPs) of known chromosomal location that are linked to the phenotype caused by ENU mutagenesis.

The main interest in a phenotype-driven strategy is the establishment of appropriate screening procedures to assess the mutant phenotypes of interest and to obtain animal models of human diseases. In our laboratory we are screening mice for immunological, skeletal or neuromuscular phenotypes and the breeding scheme that we have established allows the detection of recessive mutations. So far, our laboratory has been focused in novel mouse models for osteopetrosis, neuromuscular disease and ataxia/seizures.

Identification of a novel loss-of-function mutation in the RANKL gene

We have recently isolated an ENU-induced mouse mutant of osteopetrosis, which is characterized by loss of tooth eruption, abnormally increased bone density, and complete absence of osteoclasts. Genetic analysis using genome-wide polymorphic markers, SSPLs and SNPs, have lead to the localization of the causal mutation in chromosome 14 at an interval including the RANKL gene. Sequencing revealed a point mutation in the RANKL coding region, which causes a single aminoacid substitution. RANKL is a central regulator of osteoclast development and function as shown by the osteopetrotic phenotype developed in RANKL deficient mice. However, RANKL expression is upregulated in many bone-related diseases such as osteoporosis, rheumatoid arthritis, or cancer metastasis in which there is an increased osteoclast activity. Targeting RANKL appears to be the most efficient and relevant approach for the treatment of bone loss.

Functional characterization of the mutated inactive protein provides a unique opportunity to elucidate interactions affecting RANKL trimerization, binding to its cellular receptor RANK or the decoy receptor OPG. This knowledge creates new possibilities for designing highly effective drugs to inhibit RANKL function.

A novel ENU-derived neuromuscular disease model

We have recently identified a family with a severe neuromuscular phenotype characterized by loss of coordination, limb weakness and severe locomotion defects which develops within the first two weeks after birth. The causal mutation has already been mapped at a chromosomal region within 5Mb. Our aim is to characterize this novel model of neuromuscular disease, to identify the mutated gene and understand the biological role of the mutated protein. This work may lead to the discovery of a new gene/pathway related to the pathogenesis of congenital muscular dystrophies.

A novel ENU-derived mouse model of ataxia/seizures

Among other ENU-derived phenotypes we have selected a family displaying severe ataxia with unsteady motion of the limbs and seizures. The causal mutation is recessive and it has been localized in a chromosomal interval of 10Mb. Ongoing studies will enable the identification of the mutated gene and the further characterization of this novel model of ataxia.

Collaborative research

In collaboration with Dr Kollias’ lab we have established a sensitized ENU mutagenesis screen in the TNF^ΔARE model of arthritis and IBD for the identification of genes that are involved in the pathogenesis of such diseases. Once identified these novel gene functions may constitute novel pharmaceutical targets for disease neutralization.