Applications Of Dna Sequencing In The Clinical Laboratory

In the area of clinical molecular genetics, the number of targets for sequencing-based mutation analysis is limited only by the number of genes in the human genome and the number of genes associated with human disease. Although some com

Microsatellite Instability

Fig. 20. Slab gel sequencing with radiolabeling run in mutation scanning mode. The autoradiogram shows eight patient samples, a negative control (leftmost lane in each loading) and a positive control, TGC to TAC at codon 609 or C609Y; rightmost lane in each loading). Exon 10 of the RET oncogene was amplified and sequenced using 33P-labeled ddNTPs. All of the G reactions from the 10 samples are loaded in adjacent lanes, then all of the A reactions,and so forth. Note that the presence of mutations (arrow) in the positive control and one of the patients is easily noted by inspection of the autoradiogram.

Fig. 20. Slab gel sequencing with radiolabeling run in mutation scanning mode. The autoradiogram shows eight patient samples, a negative control (leftmost lane in each loading) and a positive control, TGC to TAC at codon 609 or C609Y; rightmost lane in each loading). Exon 10 of the RET oncogene was amplified and sequenced using 33P-labeled ddNTPs. All of the G reactions from the 10 samples are loaded in adjacent lanes, then all of the A reactions,and so forth. Note that the presence of mutations (arrow) in the positive control and one of the patients is easily noted by inspection of the autoradiogram.

mercial laboratories and large academic laboratories have offered clinical sequencing for some time for targets with high allelic heterogeneity (e.g., BRCA1 sequencing at Myriad Genetics [Salt Lake City, UT] and CFTR sequencing at Ambry Genetics [Costa Mesa, CA]), other specific or scanning mutation-detection methods are more commonly utilized in the clinical laboratory. However, that situation is rapidly changing as sequencing technology becomes more common in the clinical laboratory. The great majority of DNA-sequencing applications in the clinical laboratory are simply for mutation detection. It is possible to utilize dideoxy sequencing in a "mutation scan" mode, vs a complete sequencing mode. By loading a radiolabeled dideoxy sequencing gel as shown in Fig. 20, it is possible to utilize simple pattern recognition to easily note the presence of a difference between the wild-type control and a patient (or control) bearing a sequence variation. In this case, one harnesses the very high sensitivity and specificity of dideoxy sequencing for the identification, without the detailed analysis required to "read" every A, C, G, and T.

The single test that has introduced DNA sequencing into the majority of molecular diagnostics laboratories had been HIV drug-resistance genotyping, or genotypic antiretroviral resistance testing (GART). The introduction of HAART (highly active antiretroviral therapy), which utilizes a combination of drugs targeting the HIV reverse transcriptase (RT) and protease genes, has transformed HIV infection from a sentence of certain death to a chronic disease; it is one of the most significant therapeutic advances in the past several decades. In the United States and abroad, clinical molecular diagnostics laboratories have been offering HIV viral load assays for several years.

Unfortunately, it is all too common for a patient to start a course of HARRT, have a decrease in the viral load and an increase in the CD4+ cell count, only to relapse, with increasing viral loads, at a later date. GART testing can identify specific changes in the viral genome responsible for acquired resistance to one or more antiretroviral drugs and is essential for tailoring therapy to a patient's specific virus. The evaluation of the genetic composition of HIV has progressed from an epidemio-logical tool for research into a clinically useful laboratory test that health care professionals might use to select and tailor the appropriate antiretroviral agents to manage HIV disease progression (for a recent review, see ref. 84).

Genotypic antiretroviral resistance testing by DNA sequence analysis and the identification of specific mutations of the RT and protease genes is the most common method of evaluating HIV drug resistance. Those mutations that alter the efficacy of drug binding to its active site are referred to as primary mutations. As a result of primary mutations, higher concentrations of drug are generally required to inhibit the activity of the enzyme. Although viruses with primary drug-resistance mutations can replicate in the presence of the drug, in many cases the fitness of the virus is compromised by these mutations. Secondary mutations are defined as those mutations that supplement the action of the primary mutations by increasing viral fitness. In the absence of primary mutations, secondary mutations generally lack any effect on the level of resistance demonstrated by the virus.

The GART results are usually difficult to interpret. There are several methods of assisting a practitioner with interpreting genotypic information. Tibotech-Virco (Mechelan, Belgium) offers an interpretive service, the Virtual Phenotype™, that is based on a correlative database of more than 100,000 HIV phe-notypes and genotypes. Initially, the RT and protease gene sequences of the virus are established with a DNA sequencing-based genotyping assay. The sequence from a given patient is analyzed by computer software that identifies mutations that confer resistance to any of the HAART drugs and then scans the database for similar genotypes from previous samples that might match the patterns of mutations. Once any matches are identified, the phenotypes of these samples are retrieved from the database, and for each drug, an interpretive evaluation is then provided. Although the database was developed using the

Virco genotyping system, the Virtual Phenotype is also available via secure Internet to laboratories who use other genotyping products, including procedures developed in-house by individual laboratories.

In a rules-based approach, the information provided by genotyping is presented as a categorical resistant or sensitive prediction based on a rules-based algorithm. A P-test version of online rules-based interpretation software is currently available at Stanford University's website (http://hivdb.stanford.edu).

There are several analytical platforms currently being marketed for GART testing. Two vendors offer Food and Drug Adminstration-approved systems that use Sanger sequencing on automated DNA sequence analyzers. These systems include PCR amplification reagents, DNA sequencing reagents and hardware, and software that provides a rules-based interpretation of the sequence and generates a user-friendly, easily interpreted report. These systems are available from Visible Genetics (TruGene HIV-1 Genotyping Assay; Visible Genetics, Inc., Toronto, Ontario) and Applied Biosystems (ViroSeq HIV-1 Genotyping System; Applied Biosystems, Foster City, CA). An alternative approach is the line probe assay, available an ASR (analyte specific reagent) from Innogenetics (INNO-LiPA HIV-1 RT, Innogenetics, Inc., Atlanta, GA). This system uses a large set of oligonucleotide probes complementary to known sequence variants associated with drug resistance immobilized on a nylon membrane in a reverse dot-blot format. The advantage of this system is that it has higher sensitivity to minor species, has a much shorter turnaround time, and requires significantly less technical expertise than does DNA sequencing. A disadvantage is that, as new mutations are identified, new strips that incorporate the new probes must be devised, validated, and manufactured.

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