High- throughput mutation discovery

Our lab's research is mainly focused on mutation discovery and characterization of mouse models for musculoskeletal diseases using high-throughput technology. The completion of the genome sequence brings us several major advantages that we can use in positional cloning. First, all of the coding sequences of a chromosomal region of interest can be identified. Secondly, information on the introns, 5'UTR, and 3'UTR will be available, making it possible for the gene to be analyzed thoroughly throughout the coding region and the regulatory region. Thirdly, with the availability of all the sequences of a chromosomal region, nucleotide organization, gene ordering, gene expression patterns, and chromosomal structure can be analyzed. At the same time, high-throughput technologies for mutation analysis and gene expression profile analysis have also rapidly developed to meet these new needs. All of these developments will greatly improve our search for candidate genes in positional cloning.

By using a new strategy which combines genetic mapping, genome resources, and high-throughput technology, we have identified mutated genes from three spontaneous mutation models in the last two years. Our work suggests that positional cloning no longer requires years of team effort by several laboratories for identifying one gene, but is now a work by one laboratory in one year for several genes. In this NIH supported study, we employ the massive screening of the targeted spontaneous mutants in JAX, and we expect to identify mutated genes from more than 10 spontaneous mutations.

Mapping of QTLs that regulate bone quality

Using nanoindentation technology to identify quantitative trait loci (QTLs) that regulate bone quality represents a novel approach to improving our understanding of molecular mechanisms that control bone matrix properties. Nanoindentation is a nano-scale test and therefore gives the best precision of measurement, which classic methodologies do not achieve. The force required to press a sharp diamond indenter into a material is measured as a function of indentation depth. As depth resolution is on the scale of nanometers (hence the name of the instrument), it is possible to conduct indentation experiments even on thin films. Two quantities that can be readily extracted from nanoindentation measurements are the material's modulus, or stiffness, and its hardness, which can be correlated to yield strength. It is, therefore, a very powerful tool for the measurement of local mechanical properties, which represent bone quality.

While it has been shown that bone mineral density (BMD) is highly heritable, bone mineral distribution and architecture are also under strong genetic influence. However, in the past, the difficulty of measuring these properties has been one of the major obstacles in identification of QTLs. Fortunately, now we are able to use the nanoindentation technique to conduct such measurements accurately.

We are currently mapping QTLs for tibia nanoindentation properties across the whole genome. For this work we have already acquired a set fully genotyped F2 progeny from a cross between B6 and C3H that are known to differ significantly in many bone and connective tissue properties. We then will use RI strains to confirm and fine map QTLs regulating bone nanoindentation properties. Furthermore, due to the difficulty of the technique, tibiae have never been used in mapping QTLs of bone. With the accurate detection power of nanoindentation, we have been able to determine the difference in bone quality of tibiae. We have found that the bone quality of tibiae is significantly different between B6 and C3H. Once the QTLs are identified, we will be able to compare them with the QTLs from femurs as well.

Identification of pathogenic pathways involved in spontaneous arthritis in IL-1ra deficient mice

Inflammatory arthritis is a relatively common condition that frequently has severe consequences. In spite of substantial effort, the basic pathogenesis of most kinds of human inflammatory arthritis remains incompletely understood. In collaborating with Dr. John Stuart, we have been working on the genetic pathways of arthritis suitability based on spontaneous arthritis in IL-1ra deficient mice. IL-1 has been implicated as a key regulator of joint destruction in rheumatoid arthritis. Mice homozygous for IL-1ra deficiency develop inflammation of the hind limbs beginning at about 8 weeks of age. Because of the different genomic backgrounds, not all strains of mice are susceptible. BALB/c are highly susceptible with an incidence approaching 100% by 12 weeks of age, whereas both C57BL and DBA/1 mice are resistant.

To better understand the pathogenesis of arthritis in IL-1ra deficient mice, we are constructing a network of pathways of arthritis susceptibility with a 2-pronged approach. First, we are analyzing the gene expression profile of susceptible and resistant mice. The gene expression profiles generated using microarray will provide candidate genes in the molecular pathway in the arthritis susceptibility. Second, we are breeding susceptible and resistant mice to obtain an F2 generation. The F2 population will be used for the QTL mapping to determine the genetic loci that regulate the supportability of arthritis in IL-1ra deficient mice. By combining these 2 powerful techniques, we hope to gain insight into pathogenetic mechanisms and construct a network underlying the development of inflammatory arthritis.

Study of molecular mechanism of motor control using the wdl mouse model

We are studying the molecular mechanism of motor control using a unique mouse model named the waddles (wdl) mouse. The wdl mutation was found at The Jackson Laboratory in 1995 in the C57BLKS strain. The wdl phenotype is very similar to that of a previous model, waddler, which was discovered in 1959. It is an autosomal recessive mutant. Genetic mapping at JAX placed the wdl mutation into the same locus of waddler, although neither mutant gene had been identified until our recent discovery. According to observations from JAX, the wdl phenotype is characterized by a " wobbly side-to-side gait, which is noticeable by two weeks of age and remains phenotypically similar throughout life. " Some, but not all, of the homozygous mutant mice are slightly smaller than normal littermates. Homozygotes do not clasp their hind legs when picked up by the tail. Both mutant and control mice swim in a straight line when placed in water. Our observations show that wdl mice exhibit both ataxia and dystonia. They frequently exhibit elevated tail postures and intermittent Straub tail. The pelvis of wdl mice is elevated, especially during runs. The trunks of wdl mice are abnormally elevated during locomotion due to appendicular dystonia. Abnormal muscular co-contraction limits motion at the elbow and knee joints and this is associated with a " bouncy " quality to the gait of wdl mice. The hindlimbs are affected to a greater degree than the forelimbs. Surprisingly, pathological examinations of wdl mice at JAX have been unremarkable except for one isolated case of hydrocephalus. In addition, vision and hearing were also found to be normal

Linkage mapping indicated that the phenotype of wdl mice was caused by a single mutation on chromosome 4. The location of this mutation has been narrowed to a chromosomal position with flanking markers 1.9 cM on one side and 0.8 cM on the other.

By employing an integrated genomics approach that combines classical positional cloning strategy with genome resources and high throughput mutation detection technologies, we have identified a mutation responsible for the disease phenotype in wdl mice. In our current study, we are performing a comprehensive characterization of this mutation by examining its pathological and genetic bases and the altered molecular networks caused by the mutation in the regulation of motor control.

Study of molecular basis of the spontaneous fracture mouse

The sfx mouse was discovered in a BALB/c strain segregating for severe combined anemia and thrombocytopenia (scat).   It is a new model for bone growth failure and fracture. Initial studies showed that sfx mice have reduced bone mass, abnormalities of bone architecture and a disposition to fracture spontaneously at a young age. Detailed examination of the sfx mice suggested that the mutation affects other tissues as well.
Linkage mapping indicated that the phenotype of sfx mice was caused by a single mutation on chromosome 14. The location of this mutation has been narrowed to a chromosomal position with flanking markers 2 cM on one side and 10 cM on the other.