Advances over the past few years in mutation detection have provided major improvements in risk assessment, carrier detection, and prenatal diagnosis, in many cases allowing determination of the presence or absence of particular mutations with essentially 100% accuracy. Laboratory testing for direct detection of disease-causing mutations is now available for more than 3000 genes involved in well over 4000 genetic conditions. With our expanding knowledge of the genes involved in hereditary disease and the rapidly falling cost of DNA sequencing, direct detection of mutations in a patient’s or family member’s genomic DNA to make a molecular diagnosis has become standard of care for many conditions. DNA samples for analysis are available from such readily accessible tissues as a buccal scraping or blood sample, but also from tissues obtained by more invasive testing, such as chorionic villus sampling or amniocentesis (see Chapter 17). Mutation detection is most commonly performed using one of two different techniques, depending on the nature of the mutations in question. Comprehensive sequencing of polymerase chain reaction (PCR) products made from the coding regions and splice sites immediately adjacent to coding exons is effective when the mutation is a single nucleotide variant or small insertion or deletion. However, when the mutation is a large deletion involving one or more exons, attempts to sequence PCR products made from primers that fall into the deleted region is highly problematic. The sequencing will simply fail if the deletion is in an X-linked gene in a male or, even worse, can be misleading because it will yield only the sequence from the other copy of the gene on the homologous autosome. Duplications are even more challenging because they may yield a perfectly normal sequence unless the primers used for amplification happen to straddle the junction of a duplicated segment. For deletions and duplications, a variety of other methods are available that detect deletions or duplications by providing a quantitative measure of the copy number of the deleted or duplicated region. For most genetic conditions, the majority of pathogenic mutations are single nucleotide or small insertion/deletion mutations that are well detected by sequencing. One major exception is DMD, in which point mutations or small insertions or deletions account for only approximately 34% of mutations, whereas large deletions and insertions account for 60% and 6%, respectively, of the mutations in patients with DMD. In a patient with DMD, one might start with measuring the copy number of segments of DNA across the entire gene to look for deletion or duplication and, if normal, consider sequencing. For many hereditary disorders (including hereditary retinal degeneration, deafness, hereditary breast and ovarian cancer, congenital myopathy, mitochondrial disorders, familial thoracic aortic aneurysm syndrome, and hypertrophic or dilated cardiomyopathies), there is substantial locus heterogeneity, that is, a large number of genes are known to be mutated in different families with these disorders. When faced with an individual patient with one of these highly heterogeneous disorders in whom the particular gene and mutations responsible for the disorder are not known, recent advances in DNA sequencing make it possible to analyze large panels of dozens to well over 100 genes simultaneously and cost-effectively for mutations in every gene in which mutations have been seen previously to cause the disorder. In disorders for which even a large panel of relevant genes cannot be formulated for a particular phenotypically defined disorder, diagnosis might still be possible by analyzing the coding exons of every gene (i.e., by whole-exome sequencing) or by sequencing the entire genome in a search for disease-causing mutations (see Chapter 4). For example, two reported series of so-called clinical whole exome testing, one from the United States and one from Canada, showed substantial success. In a 2013 study from the United States, 250 patients with primarily undiagnosed neurological disorders underwent whole-exome sequencing and 62 (≈25%) received a diagnosis. Interestingly, among the patients receiving a diagnosis, four were likely to have had two disorders at the same time, which made a clinical diagnosis very difficult because the patients’ phenotype did not match any single known disorder. In another study in 2014 by the Canadian FORGE Consortium, approximately 1300 patients representing 264 disorders known or suspected of being hereditary, but for which the genes involved were unknown, underwent whole-exome sequencing. Mutations highly likely to explain the disorders were found in 60%; at least half of the genes had not been previously known to be involved in human disease. Of great interest in both studies was that a large number of patients carried de novo disease-causing mutations in genes not previously suspected of causing disorders. These mutations, because they are de novo, are extremely difficult to find by standard gene discovery methods as described in Chapter 10, such as linkage or association, and therefore pose particular challenges for genetic counseling and risk assessment.
Molecular and Genome-Based Diagnostics
Gene Panels and “Clinical Whole Exomes”
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