Essential Endocrinology and Diabetes. Richard I. G. Holt
variations (polymorphisms) at specific nucleotides between different individuals. On SNP arrays, the spots on the glass slide represent the different sequences at each SNP. As an individual’s paired chromosomes come one from each parent, this means that at any one SNP, there are often two different sequences (one from the mother, one from the father; this is called heterozygosity). Across stretches of DNA, SNP arrays can identify regions showing ‘loss of heterozygosity’ (i.e. there is no variation in the signal), which is indicative of deletion of either the maternal or paternal copy, or altered ratio of signals indicative of duplication.
Diagnosing mutations in single genes by polymerase chain reaction and sequencing
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.
Imaging in endocrinology
Ultrasound
Ultrasound travels as sound waves beyond the range of human hearing and, according to the surface encountered, is reflected back towards the emitting source (the ultrasound probe). Different tissues have different reflective properties. By knowing the speed of the waves and the time between emission and detection, the distance between the reflective surfaces and the source can be calculated. These data allow a two‐dimensional image to be generated (Figure 4.6). The major advantage of ultrasound is its simplicity, safety and non‐invasiveness. Machines are portable. It is helpful as an initial imaging investigation of many endocrine organs. For instance, the thyroid has a characteristic appearance in Graves disease because of its increased vascularity (Chapter 8). The ovaries can be delineated transabdominally, or with specific consent, transvaginally, when the shorter distance between probe and ovary and fewer reflective surfaces create higher resolution images (Figure 4.6).
Computed tomography and magnetic resonance imaging
Computed tomography (CT) and magnetic resonance imaging (MRI) provide excellent depiction of the body’s internal organs and tissues. The principle of CT is the same as for X‐ray. X‐rays pass differently through the various organs and tissues of the body. For instance, bone is not penetrated very well so a plain X‐ray image is obtained as if the skeleton has cast a shadow. In CT scanning, the patient lies on a table that slides through a motorized ring, which rotates and emits X‐rays. Data are acquired on penetration from different angles (i.e. as if multiple plain X‐rays had been taken), which are then constructed by computer into a single transverse ‘slice’ through the body (Figure 4.7). The brain is encased by the skull, hence its imaging by CT is limited.
In comparison to CT, MRI does not rely on X‐rays and is particularly useful at imaging intracranial structures, such as the pituitary (Figure 4.8). It is also very useful for screening purposes when a patient will need life‐long monitoring, e.g. to assess tumour formation in MEN. Repeat CT would provide a large cumulative radiation dose, itself a risk factor for tumour formation, which is avoided by MRI. The key components of MRI are magnets. At their centre is a hollow tube, into which the patient passes on a horizontal table. Once inside the tube, the patient is in a very strong magnetic field (this is the reason why MRI is dangerous to people with metallic implants such as pacemakers or aneurysm clips). Within the magnetic field, some of the body’s hydrogen atoms resonate after absorbing energy from a pulse of radio waves. Once the pulse ends, the resonating atoms give up energy as they return to their original state. These emission data are collected and differ slightly for different tissues, allowing the construction of high‐definition images. By altering time (T) constants, different images can be generated. For instance, in T1‐weighted images, cerebrospinal fluid (CSF) appears dark (Figure 4.8a), whereas in T2‐weighted images, CSF appears white (Figure 4.8b).
Figure 4.5 The basic principles of the polymerase chain reaction (PCR). PCR allows the amplification of a user‐defined stretch of genomic DNA. In diagnostic genetics, this is commonly an exonic sequence where a mutation is suspected to underlie the patient phenotype. (a) Starting DNA. (b) The double helix is separated into two single strands by heating to ∼94 °C. (c) Cooling from this high temperature allows binding of user‐designed short stretches of DNA (primers) that are complementary to the opposite strands at each end of the region to be amplified. (d) DNA polymerase catalyzes the addition of deoxynucleotide residues according to the complementary base pairs of the template strand. (e) Once complete, two double‐stranded sequences arise from the original target DNA. Another cycle then recommences at (a) with double the amount of template, making the increase in DNA exponential. Having amplified large amounts of the desired DNA sequence, a modified PCR reaction and analysis sequences the DNA to discover the presence or absence of a mutation.
Contrast agents are useful for both CT and MRI scanning (Figure