overactivity suspected: try to suppress it
Dynamic tests can be split into two categories: provocative ones to interrogate suspected inadequate function; or suppression tests, taking advantage of negative feedback to investigate potential overactivity (Box 4.2). For instance, ACTH is injected to see if cortisol secretion rises in suspected adrenocortical inadequacy (Addison disease; Chapter 6); whereas dexamethasone, a potent synthetic glucocorticoid, is given to see if pituitary ACTH and consequently cortisol secretion is appropriately diminished. If it is not, it implies that the adrenal cortex is overactive (Cushing syndrome; Chapter 6).
Cell and molecular biology as diagnostic tools
Karyotype
Karyotype refers to the number and microscopic appearance of chromosomes arrested at metaphase (Chapter 2). The word also describes the complement of chromosomes within an individual’s cells, i.e. the normal karyotype for females is 46,XX and for males is 46,XY. A karyogram is the reorganized depiction of metaphase chromosomes as pairs in ascending number order. An abnormal total number of chromosomes is called aneuploidy (common in malignant tumours). More detail comes from Giemsa (G) staining of metaphase chromosomes, where each chromosome can be identified by its particular staining pattern, called ‘G‐banding’.
Ascertaining the karyotype can be useful in congenital endocrinopathy, such as genital ambiguity (i.e. is it 46,XX or 46,XY?), or if there is concern over Turner syndrome (45,XO) or Klinefelter syndrome (47,XXY) (Chapter 7). G‐banding allows experienced cytogeneticists to resolve chromosomal deletions, duplications or translocations (when fragments are swapped between two chromosomes) to within a few megabases. Sometimes, there is evidence of mosaicism when cells from the same person show more than one karyotype. This implies that something went wrong downstream of the first cell division such that some cell lineages have a normal karyotype while others are abnormal.
Fluorescence in situ hybridization
When a syndrome is suspected, for which the causative gene or locus (genomic position) is known, fluorescence in situ hybridization (FISH) can allow assessment of duplications, deletions or translocations on a smaller scale. For instance, a locus for congenital hypoparathyroidism, as part of DiGeorge syndrome, exists on the long arm of chromosome 22 (22q). FISH utilizes the principle that complementary DNA sequences will hybridize together by hydrogen bonding. Stretches of DNA from the region of interest are fluorescently labelled and hybridized to the patient’s DNA. The fluorescence is visible as a dot on each sister chromatid of each relevant chromosome (Figure 4.4). Therefore, normal autosomal copy number is viewed as two pairs of two dots; one pair indicates a deletion; and three pairs indicate either duplication or potentially a translocation breakpoint (where the probe detects sequence either side of the breakpoint on different chromosomes). As technologies based on genome‐wide microarray and, especially, next‐generation sequencing have become more prevalent (see below), FISH is now less often used as the main methodology but its description is still useful to explain diagnosis based on DNA hybridization and fluorescence detection.
Figure 4.4 Fluorescent in situ hybridization in a patient with congenital hypoparathyroidism due to DiGeorge syndrome causing hypocalcaemia and congenital heart disease. Metaphase chromosomes were hybridized with a fluorescent probe from chromosome 22q11. The two bright dots indicate hybridization on the sister chromatids of the normal chromosome 22. The arrow points to the other chromosome 22 that lacks signal, indicating a deletion.
Images kindly provided by Professor David Wilson, University of Southampton.
Genome‐wide microarray‐based technology
Applying the principles of FISH on a genome‐wide scale in a microarray format is called ‘array comparative genomic hybridization’ (array CGH). Short stretches of the genome are printed as thousands of microscopic spots on a glass slide (the ‘microarray’). The patient’s genomic DNA is fluorescently labelled and hybridized to the spots on the slide. According to the strength of the fluorescent signal, microdeletions or duplications anywhere in the whole genome can be detected in one experiment with a resolution of several kilobases.
Single nucleotide polymorphism (SNP) arrays are being used similarly. Spread across the entire genome, there are millions of subtle 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
