GENETIC ANALYSIS APPLIED TO REPRODUCTIVE SYNDROMES AND CANCER
Dr. Dorward’s laboratory utilizes a forward genetics approach (classical genetics) in the laboratory mouse (Mus musculus) to identify DNA polymorphisms that contribute to reproductive disorders (Androgen Insensitivity Syndrome, AIS) and pediatric ovarian cancer susceptibility (granulosa cell tumours). Forward genetics is a process whereby a trait (phenotype) of interest is mapped to the relevant sequences in the DNA (genotype) that are responsible for variation of the trait. This method is advantageous in that no preconceived hypotheses for gene action are required; rather, new information can be obtained for the biological functions of DNA coding sequences, non-coding sequences or the epigenetic state (non-sequence related changes) of genes that are linked to a phenotype of interest. Combined with reverse genetics approaches in genetically engineered strains of mice (gene knock-outs, knock-downs and transgenics), public access to the complete genome sequence and powerful informatics tools, this phenotype-driven strategy is an important component of a systems biology approach to modeling disease traits in a mammalian organism. Ultimately, new genetic discoveries in the mouse will be the focus of translational investigations to explore the biology of human disease.
Granulosa Cell Tumourigenesis:
Female mice of the SWR/Bm inbred mouse strain spontaneously develop granulosa cell (GC) tumours at an early stage of ovarian maturation, and represent a model for juvenile-onset GC tumours that appear in infants and young girls. GC tumours are rare in the human population generally, although they appear at both ends of the female reproductive spectrum with both pre-pubertal and peri-menopausal onset. The rarity of this tumour has precluded genetic etiology data, such that identification of the tumour susceptibilty genes in the mouse model will provide new leads for investigation in human cases. GC tumour initiation in susceptible female mice is a complex trait involving multiple genes. The three most influential granulosa cell tumour (Gct) loci are mapped to distal Chr 4 (Gct1), and two distinct regions of mouse Chr X (Gct4, Gct6). Positional cloning efforts to identify the tumour susceptibility genes at each of this loci are at various stages of genetic resolution.
Chromosome interval map for the Gct1 tumour susceptibility locus showing the peak DNA marker D4Mit232 with a highly significant (p<0.01) Logarithm of odds ratio (LOD) score for trait linkage.
Parent-of-Origin Effects (Epigenetics):
It has been observed that the X-linked Gct loci show an unusual parent-of-origin effect for ovarian tumourigenesis, whereby paternal inheritance of the susceptibility alleles greatly increases GC tumour frequency in the next generation of daughters, in comparison to the lesser impact of maternal inheritance of the same alleles. This finding supports two experimental hypotheses that both involve the concept of “epigenetics”, (a term for changes in gene expression regulated by changes in the chromatin and not the DNA sequence)1: a) that the process of Chr X inactivation (a gene dosage compensation mechanism in mammalian females) is skewed in the daughters of this breeding cross and/or, b) the GC tumour susceptibility loci themselves are imprinted (show parent-of-origin restricted gene expression). SWR mice that carry an X-linked green fluorescent protein (GFP) transgene have been developed to investigate these hypotheses.
1 Epigenetics. 2007. Allis CD, Jenuwein T and Reinberg D, Eds. CSHL Press.
SWR strain newborn mice homozygous for an X-linked GFP transgene (female) or hemizygous (male).
Androgen Insensitivity Syndrome:
Androgen insensitivity syndrome is clinically defined as the under-masculinization of individuals with a normal XY sex chromosome karyotype. This syndrome shows a broad clinical spectrum ranging from a completely female appearance to a primarily male appearance, and is sub-categorized as complete (CAIS), partial (PAIS, Reifenstein syndrome), and mild (MAIS). AIS is primarily linked to mutations in the androgen receptor (AR) gene, the steroid-binding nuclear transcription factor that mediates sexual development in males. Perturbation of androgen signaling also has an impact on the normal development of bone and the brain, as shown in human patients with CAIS and the tfm (testicular feminized) mouse that models CAIS. An ENU mutagenesis screen at The Jackson Laboratory has produced a new mouse model for PAIS; characterization of the causative genetic mutation is underway.
Photodynamic Detection of Mammary Cancer:
Epithelial-derived tumours share the biochemical property of increased production and retention of protoporphyrin IX (PpIX), the immediate biochemical precursor of heme, following administration of the rate-limiting substrate 5-aminolevulinic acid (5-ALA). PpIX is a naturally fluorescent molecule that emits red light under blue light excitation, and thus tumour-selective loading of PpIX following 5-ALA administration is under consideration as a photodetection and photodiagnostic tool in anatomical areas where light administration and detection are endoscopically feasible, such as the colon, oral cavity and bladder. PpIX loading following oral administration of 5-ALA has also been shown as a sensitive photodetection method for visualizing human breast tumour margins during surgery, and the histological identification of lymph node metastases (2,3). In collaboration with Dr. Heinrich Walt of University Hospital Zurich and Cancer Network Zurich, a follow up survey of mammary tumours from several transgenic mouse strains revealed a similar pattern of rapid PpIX loading following a single iv injection of 5-ALA. Like human patients, PpIX fluorescence was useful for identification of metastases and surprisingly, very early stages of ductal carcinoma in situ. Future efforts on this project will consider the significance of PpIX biochemistry to tumour development, and the opportunities afforded by this generalized tumour-marker to design non-invasive imaging strategies that are useful for early detection of solid tumours.
2 Ladner, DP et al. Br J Cancer. 2001 Jan 5;84(1):33-7
3 Frei, KA et al. Br J Cancer. 2004 Feb 23;90(4):805-9
Photographic image of PpIX-stimulated red fluorescence in a metastatic tumour of the lung.