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Current Applications of Genetic Technology in Predisposition Testing and Microsatellite Instability Assays

From the Departments of Epidemiology, Gastrointestinal Medical Oncology and Digestive Diseases, and Molecular and Cellular Oncology, The University of Texas M.D. Anderson Cancer Center, Houston, TX.

Address reprint requests to Marsha L. Frazier, MD, Department of Epidemiology, The University of Texas M.D. Anderson Cancer Center, 1515 Holcombe Blvd, Houston, TX 77030; email mlfrazier{at}notes .mdacc.tmc.edu.

	INTRODUCTION

TOP INTRODUCTION THE BASICS STRATEGY FOR GENETIC TESTING REFERENCES

 
IT IS POSSIBLE TO test selected subjects for germline mutations in genes causing familial adenomatous polyposis (FAP),¹ hereditary nonpolyposis colorectal cancer (HNPCC),^2-8 Peutz-Jeghers syndrome,^9,10 and juvenile polyposis.^11-13 Because the genes that are mutated in familial colorectal cancer syndromes can be mutated at a variety of different locations, assays for mutation detection are not simple. Many different approaches to mutation detection have been described in the literature, some of which are also described here. Specific strategies for testing are also discussed.

	THE BASICS

TOP INTRODUCTION THE BASICS STRATEGY FOR GENETIC TESTING REFERENCES

 
Isolation of DNA and Polymerase Chain Reaction (PCR)
DNA or RNA for genetic testing is almost always isolated from peripheral-blood leukocytes. This requires that the blood be drawn in tubes containing some sort of anticoagulant. The preferred anticoagulants are either citrate or EDTA. The cells are lysed followed by removal of the other cellular components and precipitation of the DNA or RNA in ethanol. One of the drawbacks of this approach is that the blood must be rapidly transported to the laboratory where the testing will be performed before the nucleic acids begin to degrade. Recent developments in filter paper technology show promise in obviating this problem. Special filter papers have been developed that allow convenient transport and room temperature storage of blood specimens and the subsequent purification of genomic DNA. The DNA can be used for PCR for DNA analysis. Additionally, DNA stored on the filter paper is more amenable to automated DNA extraction and PCR, because the DNA remains bound to the paper through processing. Once blood has been spotted to the filter paper, the paper is processed to remove other cellular components, and then a small punch of the paper can be removed and placed in a test tube in which PCR can be performed. DNA can also be extracted from buccal swabs, but the yield of DNA from this procedure is not sufficient for identifying a mutation in a patient if the families’ mutation has not yet been identified.

If microsatellite testing is to be performed (as discussed below), then DNA must also be extracted from tumor tissue. This may be fresh tissue, but generally archival tissue is used so that the tumor tissue can be dissected away from any surrounding normal tissue.

A first step for most genetic testing approaches is to amplify the target DNA or cDNA sequence that is to be tested for mutations using PCR. PCR requires nucleic acid primers of around 20 base pairs in length that flank the target sequence to be amplified.

Microsatellite Instability (MSI) Testing
Microsatellites are short tandem repeats of DNA that are distributed throughout the genome, and tumors with MSI tend to accumulate errors at a much higher rate than other sequences in the genome because of the defective DNA repair.^2,14-17 MSI can be detected by examining microsatellite repeat fragments, as the numbers of repeats in some of the microsatellites can change due to the defective DNA repair.^2,16,17 MSI also occurs in a subset of sporadic colorectal carcinomas.^16-18 However, the efficient identification of tumors with MSI is important because it provides a valuable criterion for identifying patients whose DNA should undergo the work-intensive process of mutation screening for mismatch repair genes.

A panel of five microsatellite markers (BAT 25, BAT 26, D5S346, D2S123, and D17S250) were identified at a recent National Cancer Institute (NCI) workshop as being efficient markers for detection of MSI.¹⁹ These markers are detected using PCR. Although MSI is known to occur in both colorectal carcinomas and extracolonic malignancies, the recommendations for the use of these markers pertain only to colorectal carcinoma. The workshop defined three groups of patients whose tumors fit the category of either MSI–high frequency (MSI-H), MSI–low frequency (MSI-L), or microsatellite stable (MSS). These were based on the findings of Thibodeau et al,¹⁶ who studied 508 patients using 11 microsatellites. Although there were no significant differences between the MSI-L and MSS cases, the MSI-H cases were distinct from the other two groups in that they were found predominantly in the proximal colon, occurred more frequently in females, tended to be lower in stage, and tended to be diploid. At the NCI workshop, tumors were defined as possessing MSI-H if two or more of five markers are unstable. If greater than five markers are used to identify these particular tumor phenotypes, then it was recommended that the criteria be modified to reflect the percentage of markers demonstrating instability. Therefore, MSI-H would be defined as having MSI in more than 30% to 40% of the markers tested, and MSI-L would exhibit MSI in less than 30% to 40% of the markers. If none of the markers display MSI, then the tumor would be MSS, although the number of markers that would need to be tested before concluding that a tumor is MSS has not been established and therefore these tumors would be grouped with the MSI-L category. To be certain that a tumor is MSI-L versus MSI-H, it was recommended that if only one of the five markers tested were positive, then a second panel of five markers be tested. Specific recommendations for these markers were not made.

Immunohistochemistry
For HNPCC, immunostaining for the mismatch repair genes can be useful in some cases, as discussed in more detail below. Antibodies are available for hMSH2, hMLH1, PMS1, PMS2, and hMSH6. The hMSH2 and hMLH1 antibodies are the best studied, whereas those for hPMS1, hPMS2, and hMSH6 still belong in the research setting. Standard immunohistochemical techniques are used. The technique requires tissue sections that usually need to be prepared in a special way, which may vary among the different laboratories. Between the time when the tissue sections are prepared and when the immunostaining takes place, the slides should be stored at 4°C.

Single-Strand Conformational Polymorphism (SSCP)
SSCP is one of the most commonly used techniques for mutation detection. Although it is not as sensitive as nucleotide sequence analysis, it has the advantage of being rapid and less expensive. Reports on efficiencies range from 70% to 90%. We have used the technique successfully to identify more than 50 mutation carriers in either the hMSH2 or hMLH1 gene, some of which have been reported.^20,21 In SSCP, the double-stranded PCR products of the target sequence are melted by heating and the single-stranded DNA is immediately subjected to gel electrophoresis. The migration through the gel is partially related to size but also to conformation of the DNA. Mutant DNA will usually migrate differently because of conformational differences between the mutant and normal DNA.

Density-Gradient Gel Electrophoresis
Screening with density-gradient gel electrophoresis relies on the differences in the denaturing characteristics of the target sequence. Different PCR products will denature into single-stranded DNA at different concentrations of urea (which is a denaturing agent). The DNA is subjected to electrophoresis in a gradient gel that contains increasing concentrations of urea. As the DNA moves through the gel, the DNA will begin to melt when it reaches the appropriate concentration of urea to cause melting. At this point, the migration of the DNA will be retarded. When PCR products of a mutation carrier are run in parallel with PCR products of a normal control subject, the point in the gel where the DNA becomes retarded is usually different due to the differences in melting properties of the two sequences, allowing the detection of the mutation. This approach has been used by Wijnen et al^22,23 to identify several mutation in the hMSH2 and hMLH1 gene.

Protein Truncation Test (PTT)
Because the adenomatous polyposis coli (APC) gene spans more than 300 Kb of DNA and contains 15 exons, it is one of the more difficult to test. However, because most of the known mutations in the APC gene result in truncation of the protein, and because the mutated RNA is relatively stable, the PTT is very useful for detecting mutations in this gene.²⁴ This test is also sometimes calls in vitro synthesized protein assay. Exon 15 contains most of the coding sequence of the gene and is usually screened by dividing it into four segments. PCR product of each of these segments is then generated and the product is transcribed into RNA, and then the RNA is translated into protein. If a truncating mutation is present in the segment being examined, then the protein product will be shorter and this can be detected because the product will migrate faster when it is subjected to electrophoresis.

The remaining 14 exons are usually screened by isolating RNA from the patient’s peripheral-blood leukocytes. A cDNA is prepared using the RNA as template, and this cDNA is then subjected to PCR and analyzed as described above. There are some drawbacks of the PTT. One is that if truncating mutations are at the 5' or 3' ends of the segments being examined, they can be missed; in addition, if the patient being tested carries a missense mutation, this would go undetected. Furthermore, as with all PCR-based assays, certain deletion mutations can be missed if the deletion includes the primer binding site.

The PTT is used mostly for detection of mutations in the APC gene. In the case of HNPCC, the protein truncation assay can present problems for a variety of reasons, but mostly because RNA of most truncating mutant alleles for the mismatch repair genes is unstable.²¹ This phenomenon of instability is known as nonsense-mediated decay and does not occur in all genes, nor does it occur in all truncating mutations.

End-to-End Exon-Specific Nucleotide Sequence Analysis
This is the most direct approach to identifying gene mutations and has an efficiency of greater than 99%. Although nucleotide sequence analysis was once considered cost prohibitive, the costs have continuously decreased as newer automated technologies have developed. As the costs have fallen, the quality of the sequence obtained has dramatically improved with the development of better reagents for generating the PCR products to be analyzed, carrying out the sequencing reactions, and analyzing them. Primers are designed flanking each of the exons of the gene to generate a PCR product containing the entire sequence of the exon. In some cases, if the exon is very large, it may be necessary to design primers that will generate overlapping pieces of the exon using PCR. This allows analysis of the sequence within the exons and at the intron-exon boundaries where mutations interfering with RNA splicing often occur.

Conversion of Diploidy to Haploidy
In HNPCC, FAP, Peutz-Jeghers syndrome, and juvenile polyposis, a germline mutation is present in one allele of the affected gene, whereas the other allele is normal. This makes it difficult to detect the mutation if it is a deletion that cannot be detected by PCR-based assays. Examples of this would be very large deletions or deletions spanning intron-exon boundaries where the site homologous to the PCR primer is located. A new approach to mutation detection has recently been described that allows the examination of a single chromosome at one time.²⁵ With this approach, human lymphocytes and mouse cells are fused to create hybrid cells that contain a subset of the human chromosomes. An assortment of clones resulting from the fusion can then be analyzed to determine which human chromosomes they contain. DNA polymorphisms can then be used to identify hybrids with the desired chromosomes, and comparison of the polymorphisms in the patient’s DNA with that of the hybrid can also allow one to distinguish between those cells that are haploid from those that are diploid for any given human chromosome. Once clones have been identified for each copy of the chromosomes of interest, the clone can be further studied to determine whether the gene product is normal in size and whether the levels of the expression of the gene are normal, and the monoallelic gene can be studied to determine whether the mutation can be detected.

Linkage Analysis
If DNA from multiple affected family members is available, then it is possible to rule out the likelihood of some genes being mutated. Microsatellite loci that are located near the gene of interest provide highly polymorphic markers that can be used to determine whether one of the polymorphic markers cosegregates with the disease. If it does not, then it is not likely that this locus is mutated. If a certain polymorphic marker does seem to cosegregate with the disease, then genes in this region would be candidate genes for the mutation.

	STRATEGY FOR GENETIC TESTING

TOP INTRODUCTION THE BASICS STRATEGY FOR GENETIC TESTING REFERENCES

 
HNPCC
MSI testing is the first step in identifying germline mutations in mismatch repair genes. To set the stage for clinical application of microsatellite markers, the NCI held a conference at which conferees established a set of clinical criteria (the Bethesda criteria) for microsatellite testing candidacy.²⁶ Ideally, before going through the work-intensive process of mutation testing, cases meeting the Bethesda criteria should undergo MSI testing. If classified as MSI-H, they should undergo further testing. The significance of tumors testing MSI-L has not been established, and further analysis of these tumors remains in the research setting. In our own testing of known hMSH2 and hMLH1 mutation carriers, all of them tested MSI-H, and we determined that the probability of misclassifying an MSI-H case as MSI-L is very low (P = .002 to .008).²⁷

Immunohistochemistry can be helpful in determining which gene should be screened for mutations but is not a necessary test. A negative immunostaining for hMSH2 would suggest that this should be the first gene to be tested, whereas negative immunostaining for hMLH1 could either be due to a mutation or methylation of the hMLH1 promoter region. The usefulness of immunostaining for hMSH6 and hPMS1 and hPMS2 has not been established. Also, for any of the antibodies, there can be cases where the gene is mutated, but the defective protein still reacts with an antibody.

Because mutations are most frequently detected in hMSH2 and hMLH1, these genes should be sequenced first. If no evidence for mutation is found, then hMSH6 might be the next to be tested; however, the criteria for deciding to sequence the hMSH6, hPMS1, and hPMS2 genes have not yet been clearly established.

For cases in which mutations are not detected, further study of such patients and their families remains in the research setting. If a mutation is not detected and other affected family members are available to donate blood, then linkage analysis could be performed and the family could be examined to determine whether markers located near each of the different mismatch repair genes cosegregate with the disease. Genes that fail to cosegregate with the disease can then be viewed as unlikely candidates for mutation. Cosegregation of markers with the disease phenotype would be suggestive of that gene being mutated; however, the cosegregation could also happen by chance. Although not definitive, the results of linkage analysis could point investigators in the direction in which to focus in the search for the germline mutation.

Another approach that also remains in the research setting is gene conversion from diploidy to haploidy. This technique could then be combined with several techniques such, as reverse transcriptase PCR, PTT, Western blot analysis, Southern blot analysis, and fluorescent in situ hybridization.

FAP
The standard approach for FAP testing is the PTT. If this test is negative, the next step in the process of detecting mutations for these cases falls into the research setting, where linkage analysis or conversion from diploidy to haploidy might be performed. However, because the phenotype of FAP is, in most cases, straightforward, with the typical polyposis antedating cancer by a period of years, surveillance and management may be carried out reasonably well even in the absence of genetic testing.

Peutz-Jeghers Syndrome and Juvenile Polyposis
Because the identification of the genes for these syndromes has been so recent, the most efficient methods for detecting germline mutations in these disorders have not been established. Most mutations to date have been identified by exon-specific nucleotide sequence analysis.

In conclusion, much progress has been made in the development of mutation detection technology. A variety of tests are available for mutation detection, and strategies are in place for the use of these tests for HNPCC and FAP. With much mutation data available, one of the challenges of the future is to sort out pathologic mutations from those that are simple variants. Efficient functional assays are needed to address this complicated problem.

	REFERENCES

TOP INTRODUCTION THE BASICS STRATEGY FOR GENETIC TESTING REFERENCES

 
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