Читать книгу Essential Endocrinology and Diabetes - Richard I. G. Holt - Страница 99

With the discovery of more and more disease‐causing genes for monogenic disorders (i.e. where a single gene is at fault), genetic testing has expanded rapidly into clinical endocrinology and diabetes. Increasingly precise prediction is possible from correlating genotype (i.e. the gene and the position of the mutation within that gene) and phenotype (i.e. the clinical appearance and course of the patient). For instance, in type 2 multiple endocrine neoplasia (MEN2; Chapter 10) certain mutations in the RET proto‐oncogene have never been associated with phaeochromocytoma, normally one of its commonest features. In contrast, other RET mutations predict medullary carcinoma of the thyroid at a very young age, thus instructing when earlier total thyroidectomy is needed. Genetically defining certain forms of monogenic diabetes is now dictating choice of therapy (Case history 11.3).

Polymerase chain reaction (PCR) sequencing has been the mainstay of identifying mutations in user‐defined specific genes (Figure 4.5). Using DNA isolated from the patient’s white blood cells, PCR amplifies the exons of the gene of interest in a reaction catalyzed by bacterial DNA polymerases that withstand high temperature (>90 °C). These enzymes originate from microorganisms that replicate in hot springs. A second modified PCR, the sequencing reaction, provides the base pair sequence of the DNA, demonstrating whether or not the gene is mutated.

Since sequencing the human genome in 2003 technology has advanced enormously, greatly bringing down cost. What was once achieved by cutting‐edge multi‐million pound international research consortia is now possible within an individual laboratory in a matter of days for a few hundred pounds, dollars or euros. In addition to the ethical implications of holding data on an individual’s entire genome, the bioinformatics required for analysis is massive. Nevertheless, via next‐generation sequencing, defining a patient’s entire genome is fast becoming a diagnostic reality. In its current, most prevalent form, all exons from all genes are covered in whole‐exome sequencing (WES). Based on current research, it is to be expected that this will transition rapidly to whole‐genome sequencing (WGS) that includes the 98.5% which is non‐coding and capture all the critical regulatory information in promoters and enhancers.