The biochemical/functional approach – molecular analysis of mutant function

Dr Mark Gurnell

Summer School 11-14 July 2006
The Møller Centre, Storeys Way, Cambridge, UK

The discovery of a mutation in a given gene in a specific clinical context is often a critical step on the pathway to elucidating the aetio-pathogenesis of the associated human disorder. Whilst co-segregation of a mutant allele with a disease phenotype in a large pedigree provides powerful genetic evidence of causality, in other instances a mutation may initially be identified in only one or two subjects, and the question remains as to whether it is indeed pathogenic. Screening larger cohorts of similarly affected individuals to identify other mutations, and ‘exclusion’ from disease-free control populations, are considered fundamental to establishing ‘culpability’ of the mutant allele(s).

Knowledge of, or demonstration that, the gene harbouring the mutation exhibits appropriate cell/tissue type (± temporal) expression to account for the observed disease, or that an animal model with a similar ‘genetic hit’ recapitulates the human phenotype, provide additional supporting evidence of causality. However, molecular analysis of the mutant gene is often required to ‘close the loop’ and confirm that the mutation does indeed alter the expression and/or function of the normal gene product in a manner which is likely to be detrimental, given existing knowledge of its role in normal biology. In some instances, a mutation is identified in a gene whose function is not yet known, and in these circumstances molecular characterisation of the mutant gene product can be invaluable in shedding light on the normal physiological function of the wild type protein, whilst simultaneously providing answers to key questions concerning the pathogenesis, and hence treatment, of the associated clinical disorder.

Mutations in nuclear receptor genes form the basis of a number of inherited human diseases (1), and defects have been identified using two principal lines of investigation. First, in the ‘candidate gene’ approach, nuclear receptor defects have been anticipated in cases of hormone resistance, which are characterised by a reduction in target organ responsiveness to circulating hormones. Knowledge of the genomic arrangements of the various receptor genes has enabled direct sequencing approaches to identify the molecular abnormality in many cases of hormone resistance. Evidence for the pathological role of these defects has then been provided by studies documenting either a decreased receptor number or impaired function (e.g. thyroid hormone receptor ß [TRß] in the syndrome of resistance to thyroid hormone [RTH] [1, 2]; peroxisome proliferator-activated receptor ? [PPARg] in lipodystrophic insulin resistance [1, 3, 4]). Secondly, in a ‘reverse genetic’ approach, linkage studies have localised the molecular basis of a given disease to a chromosomal locus, and positional cloning has subsequently revealed a gene encoding a nuclear receptor (e.g. hepatocyte nuclear factor 4a in maturity onset diabetes of the young type 1 [MODY1] [1, 5]).

In this talk, I will use the example of loss-of-function mutations in human PPARg to demonstrate how in vitro, in vivo and ex vivo techniques can be used to study mutant receptor function and to discriminate between different classes of mutant, thus revealing novel molecular mechanisms of human disease.

References

1. Gurnell M & Chatterjee VKK, 2004. Nuclear Receptors and Human Disease. In: Essays in Biochemistry, Ed. I McEwan, Portland Press, London, 169-189.
2. Refetoff S, Weiss RE & Usala, SJ, 1993. The syndromes of resistance to thyroid hormone. Endocr Rev, 14:348-99.
3. Barroso I, Gurnell M, Crowley VEF et al, 1999. Dominant negative mutations in human PPAR? are associated with severe insulin resistance, diabetes mellitus and hypertension. Nature, 402:880-883.
4. Gurnell M, 2005. Peroxisome proliferator-activated receptor gamma and the regulation of adipocyte function: lessons from human genetic studies. Best Pract Res Clin Endocrinol Metab, 19(4):501-23.
5. Yamagata K, Furuta H, Oda N et al, 1996. Mutations in hepatocyte nuclear factor-4a gene in maturity-onset diabetes of the young (MODY1). Nature 384, 458-60

The opinions expressed in this paper are those of the speaker and do not necessarily reflect the views of the Society

Revised: 24-Aug-2006