Abstract
Understanding the aetiology of genetic diseases is important for furthering the fields of genetics, biochemistry and medicine. This project utilises microarray and Next Generation Sequencing technology to elucidate the genetic causes of rare Mendelian disorders. There were three levels of exploration to demonstrate the technology. The first stage focused on well established recessive inheritance with either a known or unknown genetic aetiology, the second stage explored a sporadic skeletal syndrome with numerous individuals and some heterogeneity, and the third was an autosomal dominant skeletal dysplasia with incomplete penetrance.
Two autosomal recessive diseases were used to highlight the power of this technology in situations where the affected families are highly informative, using more traditional genome-wide linkage approaches. A complex form of Hereditary Spastic Paraplegia was found to be recessively inherited in a New Zealand family. A genome-wide linkage approach, followed by candidate gene analysis was able to identify a new mutation in this family in an established disease gene, FA2H. The second disease investigated was van Maldergem Syndrome, a newly delineated pleiotropic condition with no known genetic cause or candidate genes. A sub selection of cases likely to be segregating the disease in an autosomal recessive manner (on account of parental consanguinity and sibling recurrence) was used to obtain a single linked interval for this disease. Targeted Next Generation Sequencing was used in this family to screen every gene within the linked region. A single gene was identified as a candidate in the sequenced cases. However, some cases of van Maldergem Syndrome suggested locus heterogeneity for this disorder, on account of having no identifiable mutation at the initial locus. Exome sequencing of those cases, with candidate gene analysis, identified a second locus for van Maldergem syndrome. Both genes have a well-established role as a receptor-ligand pair in Drosophila development, but studies in mammalian development are limited.
A cohort of individuals with the autosomal dominant disease Hajdu-Cheney Syndrome was investigated, after the discovery of the causative locus, exon 34 of NOTCH2, by collaborators using an exome sequencing approach. Exome sequencing was applied to this disease as the extant families were too small for linkage analysis. The closely allied condition, Serpentine Fibular Polycystic Kidney Syndrome was proven to be allelic to Hajdu-Cheney Syndrome through identification of exon 34 NOTCH2 mutations. Additionally, a NOTCH2-negative case was investigated by array comparative genomic hybridization and a duplication involving a regulator of endochondral bone development was identified. The link between this duplicated gene and NOTCH2 is only tentative, but this discovery may encourage further exploration of the relationship between the two genes.
The final sections of this thesis explore the largely sporadic skeletal dysplasia, osteofibrous dysplasia. One large New Zealand family with autosomal dominant osteofibrous dysplasia was analysed using genome-wide linkage analysis. Non-penetrance was clearly an issue in this family, which meant there were two candidate regions identified by linkage analysis under different penetrance models. Targeted exome sequencing of the genes within the candidate regions revealed a mutation in a gene encoding a receptor tyrosine kinase that had previously been implicated in human cancers. The mutation caused an in-frame alternative splice event that resulted in the exclusion of a protein domain involved in the down-regulation of the receptor. Interestingly, this splice form was observed to occur physiologically during mouse and zebrafish development. The use of morpholinos in the zebrafish to alter the ratio of full length to spliced receptor resulted in global developmental defects, indicating that tight regulation of this splice form is vital during early development.
The techniques explored in this thesis highlight the technological evolution of disease gene identification, and exemplify the challenges inherent in studying rare Mendelian diseases. The new biology revealed by the mutations discovered in this thesis will provide a platform for further understanding of human development.