Abstract
Germ-line mutation rate has been regarded classically as a fundamental biological parameter, as it affects the prevalence of genetic disorders and the rate of evolution. Somatic mutation rate is also an important biological parameter, as it may influence the development and/or the course of acquired diseases, particularly of cancer. Estimates of this parameter have been previously obtained in few instances from dermal fibroblasts and lymphoblastoid cells. However, the methodology required has been laborious and did not lend itself to the analysis of large numbers of samples. We have previously shown that the X-linked gene PIG-A, since its product is required for glycosyl-phosphatidylinositol-anchored proteins to become surface bound, is a good sentinel gene for studying somatic mutations. We now show that by this approach we can accurately measure the proportion of PIG-A mutant peripheral blood granulocytes, which we call mutant frequency, ƒ. We found that the results are reproducible, with a variation coefficient (CV) of 45%. Repeat samples from 32 subjects also had a CV of 44%, indicating that ƒ is a relatively stable individual characteristic. From a study of 142 normal subjects we found that log ƒ is a normally distributed variable; ƒ variability spans a 80-fold range, from less than 1×10−6 to 37.5×10−6, with a median of 4.9×10−6. Unlike other techniques commonly employed in population studies, such as comet assay, this method can detect any kind of mutation, including point mutation, as long as it causes functional inactivation of PIG-A gene. Since the test is rapid and requires only a small sample of peripheral blood, this methodology will lend itself to investigating genetic factors that underlie the variation in the somatic mutation rate, as well as environmental factors that may affect it. It will be also possible to test whether ƒ is a determinant of the risk of cancer.
Highlights
Somatic mutations are known mostly as a source of disease [1,2], and they are often associated with exposure to mutagens [3]
The method is based on using as sentinel gene PIG-A, which encodes a component of an enzyme required for the biosynthesis of the glicosylphosphatidylinositol (GPI) molecule, which serves as anchor for many surface proteins
Somatic mutations have first become prominent in human biology with the discovery that they have a major physiological role in immunity, but we learnt promptly that hyper-mutation was a unique characteristic of few special genes confined to specific cell lineages, the B and T lymphoid cells [17]
Summary
Somatic mutations are known mostly as a source of disease [1,2], and they are often associated with exposure to mutagens [3]. In human cells m is estimated to be of the order of 1027 per gene per cell division [5,6,7]. We have developed a method to measure m in lymphoblastoid cell lines that can be obtained from a peripheral blood sample [9], and we have an estimate of the normal range [6]. The method is based on using as sentinel gene PIG-A, which encodes a component of an enzyme required for the biosynthesis of the glicosylphosphatidylinositol (GPI) molecule, which serves as anchor for many surface proteins. Since PIG-A is X-linked, a single inactivating mutation in this gene will cause deficiency of all GPIlinked proteins, and this can be conveniently demonstrated on individual cells by flow cytometry. The same approach has been applied to red cells, granulocytes and lymphocytes of humans and of laboratory animals [10,11]
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