Matryoshka genetics: identification of disease-associated mutations in the human genome

Abstract

This thesis discusses the identification of pathogenic mutations within the human genome, specifically in the context of clinical medicine. It is principally a publication-based thesis, with each of the chapters focusing on the use of a specific molecular technique or combination of techniques to address a particular clinical question, or clinical need. Indeed clinical need is the thread that binds the various chapters together. The work was predominantly carried out at LabPLUS, the tertiary referral medical laboratory attached to Auckland City Hospital. The techniques discussed range from targeted mutation analysis using amplification refractory mutation system polymerase chain reaction (ARMS-PCR), Sanger-based full gene sequencing, multi-gene panel next-generation sequencing, and deletion/duplication analysis by array comparative genomic hybridisation (aCGH). The assays are variously applied to single gene and chromosomal disorders, and in some instances several techniques are applied together in order to provide more comprehensive analysis, or to guide the use of further testing. The clinical contexts for the analyses discussed span prenatal to post-mortem diagnosis, and involve a range of clinical specialities, including cardiology, paediatric metabolic medicine, clinical genetics, neurology, psychiatry, and endocrinology. The Matryoshka, or Russian nesting doll, analogy refers primarily to the way that each assay takes a snapshot of the genome at a variable degree of resolution, depending on the type of mutation targeted – ranging from whole genome copy number analysis (the largest doll), through multi-gene sequencing panels of a range of sizes, to ARMS-PCR to detect one specific mutation in one specific gene (the smallest doll). Each chapter is able to function as a stand-alone, but together they represent the current state of diagnostic genetic testing in clinical practice. The intention is to illustrate the principle tools available, the indications for, and limitations/potential pitfalls of these tools. Of particular concern is the utility of diagnostic genetic testing within routine medical practice, and how such testing is best integrated into the clinical paradigm. An awareness of the differences between genetic testing in the research as opposed to the clinical environment is crucial, and several chapters discuss the validation process of translating assays from the research to the diagnostic laboratory. The importance of collaboration between laboratory and clinical staff in order to ensure that testing is appropriately targeted is reflected in the multi-disciplinary authorship of the publications included. Over the course of the thesis, the landscape of diagnostic genetic testing has changed markedly. Much of this change involves the introduction of techniques that blur the boundaries of more conventional analysis - whole genome assays that have a resolution approaching that of more targeted techniques. The concluding discussion focuses on the implications of these changes and how they may have an impact on genomic medicine in the future.