A genetic test is any clinical or laboratory investigation that provides information about the likelihood that an individual is affected with a heritable disease. The majority of genetic tests are based on molecular evaluations of genomic DNA designed to identify the DNA mutations responsible for the disease.
Genetic Testing
Role of Genetic Testing in the Clinic
DNA-based genetic tests can identify individuals at risk for disease before any clinical evidence is present (presymptomatic testing). This information coupled with effective genetic counseling and clinical screening can be useful. An effective presymptomatic test needs to meet the specificity and sensitivity expectations for any clinical test. Sensitivity is the number of affected individuals that are positive for a test compared with the total number of affected individuals (including those that tested negative for the test). Specificity is the number of unaffected individuals that are negative for the test compared with the total number of unaffected individuals tested (including those that tested positive for the test) ( Fig. 1.3.1 ).
The identification of a mutation responsible for a disease through DNA-based genetic testing can establish a molecular diagnosis. For some disorders, such as juvenile open-angle glaucoma caused by mutations in MYOC, specific mutations have been correlated with severity of disease or other clinical features that are useful prognostically. A molecular diagnosis may also help guide therapy and is required before gene-based therapies can be utilized. For example, mutations in a number of different genes can cause Leber’s hereditary amaurosis, but only those patients with disease due to mutations in RPE65 will benefit from novel RPE65 -based therapies using gene replacement.
Methods for DNA-Based Genetic Testing
Although genetic testing can be performed using DNA, RNA, or protein, DNA is the easiest to work with, and most genetic tests use this as the starting material. A biological sample from the patient is needed before genetic testing can be performed. The inclusion of family members may help the evaluation, but they are not absolutely required. DNA for testing can be obtained from a number of sources, including blood samples, mouthwash samples or buccal swabs, archived pathology specimens, or from hair.
Genomic DNA sequencing is the most commonly used method to detect mutations. For many disorders, sequencing the entire responsible gene is necessary, including all exons, immediate flanking intron sequences with splice signals and 5′ and 3′ flanking regulatory regions. Some disorders are caused by a specific mutation in a gene, and genetic testing can be limited to an evaluation of a single gene. For other diseases, however, such as the inherited retinal degenerations, sequencing multiple genes may be required before a causative mutation is identified. For diseases with many causative genes, a panel test that allows for sequencing all genes at once is both more effective and more efficient. Alternatively, whole exome sequencing (WES) that captures and sequences all coding regions of the genome can also be a preferred approach for disorders with many possible genetic mutations. Genomic DNA sequencing will not usually identify large chromosomal abnormalities, including large copy number variations (deletions or insertions) or chromosomal translocations. Other techniques are necessary to detect large chromosomal abnormalities, including karyotyping and multiplex ligation-dependent probe amplification (MLPA). For diseases that are caused primarily by a limited set of mutations (for example, the three mutations that commonly cause Leber’s hereditary optic neuropathy (LHON), specific tests such as allele-specific polymerase chain reaction (PCR) amplification or TaqMan assays can be used and can be more efficient than sequencing the entire gene ( Table 1.3.1 ).
| Method | Indication | Example |
|---|---|---|
| Single gene DNA sequencing | Different mutations distributed throughout a single gene are known to cause the inherited condition | Sequencing OPA1 in patients with autosomal dominant optic neuropathy |
| Multiple gene DNA sequencing | Mutations in multiple genes are known to cause the condition | Inherited retinal degenerations |
| Multiplex ligation-dependent probe amplification (MLPA) | Detects deletions and duplications in genes known to cause the condition and that may be missed by sequence-based approaches | MLPA testing for PAX6 deletions in patients with aniridia |
| TaqMan assay or allele-specific assay | Detects a single DNA base pair change and is used if a small set of mutations are primarily the cause of the condition | Three mutations commonly cause Leber’s hereditary optic neuropathy (LHON) |
| Karyotype | Detects large chromosomal rearrangements including deletions, duplications, and translocations | Down syndrome |
Current Recommendations for Genetic Testing for Ophthalmic Diseases
Currently, genetic testing is indicated for patients with clinical evidence of a disorder whose causative genes have been identified and for which the identification of the genetic mutation contributing to the disease has sufficient specificity and sensitivity that testing will be clinically useful. Serious failures of a diagnostic test are false positives (individuals without the disease who test positively) and false negatives (individuals with the disease who test negatively). Although genes have been identified for some common complex disorders such as age-related macular degeneration, primary open-angle glaucoma, and exfoliation syndrome, in general, testing for these mutations is not sufficiently sensitive and specific that the test results are clinically meaningful. For example, over 90% of patients with exfoliation syndrome carry one of two missense changes in LOXL1; however, up to 80% of normal individuals also carry these same DNA sequence variants. Clearly the identification of these missense mutations alone is not clinically useful. Examples of genetic tests that are useful include RPE65 for Leber’s hereditary amaurosis, PAX6 for aniridia, MYOC for early onset primary open-angle glaucoma, and OPA1 for optic neuropathy, as well as many other genes that are known to cause inherited ocular conditions.
CLIA Laboratories
Laboratories in the United States offering genetic testing must comply with regulations under the Clinical Laboratory Improvement Amendments of 1988 (CLIA). The Centers for Medicare and Medicaid Services administers CLIA and requires that laboratories meet certain standards related to personnel qualifications, quality control procedures, and proficiency testing programs in order to receive certification. This regulatory system was put in place to encourage safe, accurate, and accessible genetic tests. In addition to ensuring that consumers have access to genetic tests that are safe, accurate, and informative, these policies encourage the development of genetic tests, genetic technologies, and the industry that produces these products. A number of CLIA-certified laboratories performing genetic testing for eye diseases exist in the United States. For a list of CLIA-certified laboratories participating in the National Eye Institute (NEI)-sponsored eyeGENE network, see the NEI website at http://www.nei.nih.gov.easyaccess1.lib.cuhk.edu.hk . CLIA-certified laboratories offering genetic testing can also be found at GeneTests: https://www.genetests.org/ .
Genetic Reports
A genetic test report is a sensitive document that is the main form of communication between the CLIA laboratory and the physician requesting the genetic test. Genetic test reports may be shared with the patient and with genetic counselors. The report should include (1) the type of genetic test performed (i.e., sequencing or other methodology), (2) the gene or genes that were evaluated, (3) the results of the testing, (4) information about the pathogenicity of the sequence variants, (5) recommendations for clinical follow-up based on the results of testing, and (6) literature references providing additional information about the genes and mutations responsible for the disease. The report should be written clearly and have appropriate contact information.
Novel DNA sequence changes are frequently found as a result of genomic DNA sequencing. New DNA sequence changes (variants) may be benign polymorphisms or causative mutations. Additional studies must be done before the sequence change can be designated as disease causing. Demonstrating that the mutant protein has an abnormal function or evaluation of the mutant gene in an animal model would be an ideal test of pathogenicity, but these approaches are time consuming and may not be possible. Current approaches to evaluate the pathogenicity of a novel DNA sequence variant are based on (1) population data, (2) computational and predictive data from in silico estimates for pathogenicity such as SIFT and PolyPhen-2, (3) functional data, and (4) segregation data for families.
Role of Genetic Testing in the Clinic
DNA-based genetic tests can identify individuals at risk for disease before any clinical evidence is present (presymptomatic testing). This information coupled with effective genetic counseling and clinical screening can be useful. An effective presymptomatic test needs to meet the specificity and sensitivity expectations for any clinical test. Sensitivity is the number of affected individuals that are positive for a test compared with the total number of affected individuals (including those that tested negative for the test). Specificity is the number of unaffected individuals that are negative for the test compared with the total number of unaffected individuals tested (including those that tested positive for the test) ( Fig. 1.3.1 ).
The identification of a mutation responsible for a disease through DNA-based genetic testing can establish a molecular diagnosis. For some disorders, such as juvenile open-angle glaucoma caused by mutations in MYOC, specific mutations have been correlated with severity of disease or other clinical features that are useful prognostically. A molecular diagnosis may also help guide therapy and is required before gene-based therapies can be utilized. For example, mutations in a number of different genes can cause Leber’s hereditary amaurosis, but only those patients with disease due to mutations in RPE65 will benefit from novel RPE65 -based therapies using gene replacement.
Methods for DNA-Based Genetic Testing
Although genetic testing can be performed using DNA, RNA, or protein, DNA is the easiest to work with, and most genetic tests use this as the starting material. A biological sample from the patient is needed before genetic testing can be performed. The inclusion of family members may help the evaluation, but they are not absolutely required. DNA for testing can be obtained from a number of sources, including blood samples, mouthwash samples or buccal swabs, archived pathology specimens, or from hair.
Genomic DNA sequencing is the most commonly used method to detect mutations. For many disorders, sequencing the entire responsible gene is necessary, including all exons, immediate flanking intron sequences with splice signals and 5′ and 3′ flanking regulatory regions. Some disorders are caused by a specific mutation in a gene, and genetic testing can be limited to an evaluation of a single gene. For other diseases, however, such as the inherited retinal degenerations, sequencing multiple genes may be required before a causative mutation is identified. For diseases with many causative genes, a panel test that allows for sequencing all genes at once is both more effective and more efficient. Alternatively, whole exome sequencing (WES) that captures and sequences all coding regions of the genome can also be a preferred approach for disorders with many possible genetic mutations. Genomic DNA sequencing will not usually identify large chromosomal abnormalities, including large copy number variations (deletions or insertions) or chromosomal translocations. Other techniques are necessary to detect large chromosomal abnormalities, including karyotyping and multiplex ligation-dependent probe amplification (MLPA). For diseases that are caused primarily by a limited set of mutations (for example, the three mutations that commonly cause Leber’s hereditary optic neuropathy (LHON), specific tests such as allele-specific polymerase chain reaction (PCR) amplification or TaqMan assays can be used and can be more efficient than sequencing the entire gene ( Table 1.3.1 ).
| Method | Indication | Example |
|---|---|---|
| Single gene DNA sequencing | Different mutations distributed throughout a single gene are known to cause the inherited condition | Sequencing OPA1 in patients with autosomal dominant optic neuropathy |
| Multiple gene DNA sequencing | Mutations in multiple genes are known to cause the condition | Inherited retinal degenerations |
| Multiplex ligation-dependent probe amplification (MLPA) | Detects deletions and duplications in genes known to cause the condition and that may be missed by sequence-based approaches | MLPA testing for PAX6 deletions in patients with aniridia |
| TaqMan assay or allele-specific assay | Detects a single DNA base pair change and is used if a small set of mutations are primarily the cause of the condition | Three mutations commonly cause Leber’s hereditary optic neuropathy (LHON) |
| Karyotype | Detects large chromosomal rearrangements including deletions, duplications, and translocations | Down syndrome |
Current Recommendations for Genetic Testing for Ophthalmic Diseases
Currently, genetic testing is indicated for patients with clinical evidence of a disorder whose causative genes have been identified and for which the identification of the genetic mutation contributing to the disease has sufficient specificity and sensitivity that testing will be clinically useful. Serious failures of a diagnostic test are false positives (individuals without the disease who test positively) and false negatives (individuals with the disease who test negatively). Although genes have been identified for some common complex disorders such as age-related macular degeneration, primary open-angle glaucoma, and exfoliation syndrome, in general, testing for these mutations is not sufficiently sensitive and specific that the test results are clinically meaningful. For example, over 90% of patients with exfoliation syndrome carry one of two missense changes in LOXL1; however, up to 80% of normal individuals also carry these same DNA sequence variants. Clearly the identification of these missense mutations alone is not clinically useful. Examples of genetic tests that are useful include RPE65 for Leber’s hereditary amaurosis, PAX6 for aniridia, MYOC for early onset primary open-angle glaucoma, and OPA1 for optic neuropathy, as well as many other genes that are known to cause inherited ocular conditions.
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