and Genome-Based Diagnostics



Molecular and Genome-Based Diagnostics



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.



Gene Panels and “Clinical Whole Exomes”



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.

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Nov 27, 2016 | Posted by in GENERAL & FAMILY MEDICINE | Comments Off on and Genome-Based Diagnostics
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