Direct inspection of nucleic acids—often called "molecular genetics" or "DNA diagnosis"—is an important tool in a number of clinical areas, including oncology, infectious disease, forensics, and the general study of pathophysiology. A major impact has been in the diagnosis of mendelian disorders. Molecular testing is available for more than 3000 separate hereditary conditions. Once a particular gene is shown to be defective in a given condition, the nature of the mutation in a patient can be determined by sequencing the nucleotides of the coding exons and the splice sites. One of a variety of techniques can then be used to determine whether that same mutation is present in other patients with the same disorder. Genetic heterogeneity is so extensive that most mendelian conditions are associated with numerous mutations at one locus—or often multiple loci—that produce the same phenotype. Mutations at several hundred different genes cause vitreoretinal disorders, such as retinitis pigmentosa, and changes in several dozen genes cause familial hypertrophic cardiomyopathy. This fact complicates DNA diagnosis of patients and screening for carriers of defects in specific genes.
A few conditions are associated with relatively few mutations or with only one highly prevalent mutation. For example, all sickle cell disease is caused by exactly the same change of glutamate to valine at position 6 of beta-globin, and that substitution in turn is due to a change of one nucleotide at the sixth codon in the beta-globin gene. But such uniformity is the exception. In cystic fibrosis, about 70% of heterozygotes of northern European ancestry have an identical deletion of three nucleotides that causes loss of a phenylalanine residue from a chloride transport protein; however, the remaining 30% of mutations of that protein are diverse (several thousand have been discovered), so that no simple screening test will detect all carriers of cystic fibrosis.
Reviews of the current technical status of DNA analysis appear regularly in the medical literature. Polymerase chain reaction (PCR) studies revolutionized many aspects of molecular biology, and DNA diagnosis came to involve this technique. If the sequences of the 10–20 nucleotides at the ends of a region of DNA of interest (such as a portion of a gene) are known, then "primers" complementary to these sequences can be synthesized. When even a minute amount of DNA from a patient (eg, from a few leukocytes, buccal mucosal cells, or hair bulbs) is combined with the primers in a reaction mixture that replicates DNA—and after several dozen cycles are then performed—the region of DNA between the primers will be amplified exponentially. For example, the presence of early HIV infection can be detected after PCR amplification of a portion of the viral genome.
The speed of sequencing DNA has accelerated tremendously, and its cost has plummeted due to the arrival of "next-generation sequencing." The "Holy Grail" of DNA analysis was defined over a decade ago as the "$1000 human genome," and that benchmark was reached in 2014. Thus, it is already feasible for a researcher to sequence just the coding sequences (whole exome sequencing) or the entire 6.4 billion nucleotide base pairs of any given individual (whole genome sequencing). It is now less expensive to sequence a person's entire exome than to sequence selectively all of the genes known to be involved in some conditions, eg, hypertrophic cardiomyopathy. Storing and interpreting the mass of data that emerge from sequencing an entire genome are daunting and expensive tasks but likely to be surmountable. The entire sequences of tens of thousands of individuals have been analyzed, and a few that are published are freely available for perusal on the Internet. Most of these individuals have agreed to have the clinical implications of their genomes publicly analyzed. The international effort to sequence the entire genomes of at least 1000 individuals of diverse ethnic groups was exceeded in 2011. The United Kingdom and the United States have established projects to sequence genomes of 100,000 and 1 million volunteers, respectively, and correlate genetic variation with health status.
When the sequence of a patient's particular gene is found to be different from 'normal,' this can be interpreted as benign, likely benign, likely pathogenic or pathogenic. Any one of these interpretations can be useful clinically. However, another category of interpretations is the variant of unknown significance for which no decision can be made at that time. This is frustrating for the patient, the person who ordered the test, and the laboratory. Often the variant of unknown significance will be reinterpreted, sometime years later, and it is important to transmit this information to the patient.
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INDICATIONS FOR DNA DIAGNOSIS
The basic requirement for the use of nucleic acids in the diagnosis of specific hereditary conditions is that a probe be available for the gene in question. The probe may be a piece of the actual gene, a sequence close to the gene, or just a few nucleotides at the actual mutation. The closer the probe is to the actual mutation, the more accurate and the more useful will be the information derived. DNA diagnosis involves one of two general approaches: (1) direct detection of the mutation or (2) linkage analysis, whereby the presence of a mutation is inferred from the nature of a probe DNA sequence remote from the mutation. In the latter approach, as the probe moves farther from the mutation, the chances increase that recombination will have separated the two sequences and confused the interpretation of the data.
DNA diagnosis is finding frequent application in presymptomatic detection of individuals with age-dependent disorders such as Huntington disease and adult polycystic kidney disease, screening for carriers of autosomal recessive conditions such as cystic fibrosis and thalassemias, screening for female heterozygotes of X-linked conditions such as Duchenne muscular dystrophy and hemophilia A and B, and prenatal diagnosis. For some conditions in which serious complications occur in adolescence or early adulthood—such as von Hippel–Lindau syndrome, hereditary hemorrhagic telangiectasia, and familial polyposis coli—genetic testing at an early age can identify those relatives who need frequent clinical monitoring and prophylactic management; almost as important, relatives who test negative for the mutation can be spared the inconvenience, cost, and risk of clinical tests. In all instances of DNA testing, primary care providers and specialists alike must be mindful that substantive ethical, psychological, legal, and social issues remain unresolved. For example, some conditions for which hereditary susceptibility can be readily defined (such as Alzheimer disease, Huntington disease, and many cancers) have no effective therapy at this time. For these same conditions, health insurance and life insurance providers may be especially interested in learning who among their current or prospective customers is at higher risk. Many states have enacted legislation to protect people identified as having a heightened genetic risk of disease. The federal Genetic Information Nondiscrimination Act (GINA) prohibits unfair discrimination in health insurance and employment. The protections are limited to those individuals who are detected as having a genetic susceptibility to a disease that is not evident clinically. Importantly, in the United States the Affordable Care Act provides protection for all individuals with preexisting conditions.
A number of commercial firms offer targeted or extensive genotyping to anyone who wants to submit a saliva specimen and pay a fee. Some of the reasons suggested for doing this include identification of ancestral background, relationship certification and most commonly, detection of genetic susceptibilities to disease. The latter are almost entirely based on GWAS that have associated specific SNPs with an increased (or decreased) likelihood of developing a particular common disease. In almost all such GWAS-based analyses, the association with disease is highly statistically significant but of remarkably little predictive value. In other words, the relative risks of developing a disease based on having one of these markers is typically in the range of 1.2–1.4. Moreover, virtually no research has been done to examine the clinical utility of being identified as having one of these risk markers. For example, is someone with the 9p21-linked SNP that has no known biologic function but is associated with a slightly greater risk of developing an atherosclerosis-related condition more likely to alter their lifestyle, change their diet, or stop smoking? There are also direct-to-consumer marketing efforts for specific genetic tests that must be ordered by an individual's clinician, such as those suggesting that any woman with a family history of breast cancer consider being tested for mutations in BRCA1 and BRCA2.
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LOGISTICS OF DNA DIAGNOSIS
Lymphocytes are a ready source of DNA; 10 mL of whole blood yields up to 0.5 mg of DNA, enough for dozens of analyses, each of which requires only 5 mcg. Once isolated, the DNA sample can be divided into aliquots and frozen. Alternatively, lymphocytes can be transformed with viruses into lymphoblasts; these cells are immortal, can be frozen, and—whenever DNA is required—can be thawed, propagated, and their DNA isolated. These stored specimens provide access to a person's genome long after the individual dies. This is such an important advantage that many clinical genetics centers and commercial laboratories "bank" DNA from patients and informative relatives even if the samples cannot be put to use immediately. The specimens may later prove invaluable to relatives or to other patients being evaluated. DNA in some instances has become more reliable than the medical records. However, for routine clinical purposes, the analysis is often focused on one or a few genes (such as in a family study, in which only one specific nucleotide change is addressed). The amount of DNA needed is quite small—a few hair bulbs or sperm are adequate. The simplest source is saliva (20–30 mL) or a scraping of the buccal mucosa.
Fetal DNA can be isolated from amniotic cells, from trophoblastic cells taken by chorionic villus sampling, or from either cell type grown in culture. Samples need to be processed promptly but can be shipped by overnight mail and must not be frozen.
Fetal DNA fragments also circulate in maternal blood. Analysis of these fragments allows highly sensitive and specific diagnosis of fetal trisomy in a much less invasive manner than through amniocentesis.
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