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Special Communication | Clinician's Corner

New Approaches to Molecular Diagnosis

Bruce R. Korf, MD, PhD; Heidi L. Rehm, PhD
JAMA. 2013;309(14):1511-1521. doi:10.1001/jama.2013.3239.
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Published online

Advances in understanding the molecular basis of rare and common disorders, as well as in the technology of DNA analysis, are rapidly changing the landscape of molecular genetic and genomic testing. High-resolution molecular cytogenetic analysis can now detect deletions or duplications of DNA of a few hundred thousand nucleotides, well below the resolution of the light microscope. Diagnostic testing for “single-gene” disorders can be done by targeted analysis for specific mutations, by sequencing a specific gene to scan for mutations, or by analyzing multiple genes in which mutation may lead to a similar phenotype. The advent of massively parallel next-generation sequencing facilitates the analysis of multiple genes and now is being used to sequence the coding regions of the genome (the exome) for clinical testing. Exome sequencing requires bioinformatic analysis of the thousands of variants that are identified to find one that is contributing to the pathology; there is also a possibility of incidental identification of other medically significant variants, which may complicate genetic counseling. DNA testing can also be used to identify variants that influence drug metabolism or interaction of a drug with its cellular target, allowing customization of choice of drug and dosage. Exome and genome sequencing are being applied to identify specific gene changes in cancer cells to guide therapy, to identify inherited cancer risk, and to estimate prognosis. Genomic testing may be used to identify risk factors for common disorders, although the clinical utility of such testing is unclear. Genetic and genomic tests may raise new ethical, legal, and social issues, some of which may be addressed by existing genetic nondiscrimination legislation, but which also must be addressed in the course of genetic counseling. The purpose of this article is to assist physicians in recognizing where new approaches to genetic and genomic testing may be applied clinically and in being aware of the principles of interpretation of test results.

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Figure 1. Deletions and Duplication of Multiple Genes Within a Chromosomal Segment
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Figure 2. Detection of Chromosome Deletion or Duplication by Array Comparative Genomic Hybridization
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A, Array comparative genomic hybridization (CGH) consists of mixing fragmented DNA from the patient and a reference sample labeled with different fluorescent dyes (red and green, in this case). These fragments are allowed to bind (hybridize) by base pairing to complementary genomic DNA fragments (probes) that have been immobilized on a glass chip. If there is equal binding of patient and reference DNA, yellow fluorescence results. Deletion of a DNA segment in the patient sample results in decreased binding of patient DNA relative to reference DNA and green fluorescence (loss). Duplication of a DNA segment in the patient sample results in increased binding of patient DNA relative to reference DNA and red fluorescence (gain). B, Example of array CGH analysis from a patient with a chromosome 16 microdeletion. Data markers represent probes on the array and are plotted next to the corresponding position on the chromosome ideogram (left). Black data markers are probes within an established range representing equal binding. Probes that fall outside this range are indicated as loss or gain.

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Figure 3. Variations in Types of Mutations Accounting for a Specific Phenotype and Associated Category of Genetic Testing
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Figure 4. Example of Exome Sequencing to Identify Genetic Basis of an Undiagnosed Disorder in an Affected Child
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Exome sequencing is performed on exons—the protein encoding regions of genes—that are isolated by a process of DNA fragmentation and hybridization. Using next-generation sequencing, purified exons are sequenced and compared with a standard reference human genome sequence to identify variants. To determine inheritance of a possible genetic disorder, exome sequencing can be performed on an affected child and both parents. Analysis is limited to those variants that are not known to be benign and that have potential for a damaging effect on protein function. If inheritance is recessive (biallelic), each parent carries 1 of the damaging variants as a heterozygous carrier. If inheritance is dominant (monoallelic), either parent carries the variant and would be phenotypically affected unless the mutation is nonpenetrant. Alternatively, a dominant mutation may have arisen de novo in the child, in which case neither parent carries the variant.

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