Steven M Powell 1 Introduction

Over the past decade, the genes that underlie the development of many human diseases have been identified and the diseases causing mutations within these genes have been unveiled. Many genetic alterations responsible for a variety of human disorders have been characterized. These alterations range from simple Mendelian inherited syndromes to more complex traits such as cancers that involve multiple genetic and environmental factors. Identification and characterization of disease-causing mutations has practical as well as biological implications. As our understanding of these alterations advances, the potential for developing molecular genetic markers with clinical applications increases. This improved understanding also opens new avenues for advances in diagnostic testing, prognostication, and design of preventative strategies or therapeutic interventions. Indeed, direct genetic testing for an inherited colorectal cancer predisposition syndromes, Familial Adenomatous Polyposis (FAP) is currently available to the medical community with appropriate genetic counseling (1).

This chapter describes the application of the in vitro synthesis (IVS) protein assay, which is a sensitive and rapid method for detecting truncating gene mutations (1,2). The importance of mutational analyses that can be applied routinely in clinical practice is highlighted by the IVS protein assay's current use to FAP presymptomatically. This assay may also potentially aid in the diagnosis and management of many other diseases that involve truncating genetic mutations (see Table 1).

We may soon be entering into an era where mutational analysis and detection become the limiting steps in our diagnosis and care of patients. For instance, we may know the gene(s) involved in a disease, but not have the

From: Methods in Molecular Medicine, vol. 50: Colorectal Cancer: Methods and Protocols Edited by: S. M. Powell © Humana Press Inc., Totowa, NJ

Table 1

Applications of the IVS Protein Assay


Familial adenomatous polyposis syndromes APC Emerging

Hereditary nonpolyposis colon DNA repair genes (MMR)0

Neurofibromatosis type 1 NF1

Hereditary breast/ovarian cancer BCRA1

Duchenne muscular Dystrophin

"MMR = mismatch repair genes that include: hMSH2, hM:LH1, hPMSl, hPMS2.

ability to conveniently test those individuals who may have or are at risk for having the disease for causative alterations in the responsible gene(s). Currently, identifying the appropriate clinical setting for genetic testing is of paramount importance. The group of patients and relatives for whom genetic testing will be beneficial is presently being defined as we better understand genotype to phenotype relationships and the penetrance of pathologic traits.

Many conventional techniques of mutational analysis, such as direct nucle-otide sequencing, ribonuclease protection assays, or other chemical cleavage of nucleotide mismatch methods (i.e., hydroxylamine and osmium tetroxide) that can identify genetic mutations sensitively are labor intensive and usually reserved for the research setting (reviewed in ref. 3). Other methods of detecting gene mutations, such as single-strand conformation polymorphism (SSCP) analysis (4), denaturing gradient gel electrophoresis (DGGE) analysis (5), or heteroduplex analysis, may require only a few steps but are limited in their sensitivity of mutation detection. The narrow range of a gene's size that can be analyzed by these methods at any one time also restricts their use for mutation identification, specifically limited are those assays that involve altered hetero-duplex migration on gel electrophoresis analysis. Moreover, some methods such as allele-specific amplification (ASA), allele-specific hybridization (ASH), ligation amplification reactions (LAR) (6), or restriction site amplifi


Neurofibromatosis type 2 Von Hippel-Lindau Retinoblastoma Becker muscular dystrophy ß-Thalassemia Hemophilia B Cystic fibrosis Osteogenesis Imperfecta Werner's syndrome


Dystrophin ß-Globin Factor IX CFTR


cations are relatively simple to perform, but they are designed to detect only a specific nucleotide change. This specificity limits their usefulness for screening genes that tend to have multiple types of mutations occurring at different locations in the gene.

Additionally, genes can be altered in the noncoding region with important functional effects. For example, changes that occur in the promoter or enhancer regions of a gene or alterations that change methylation patterns might result in abnormal gene expression. Moreover, gross allelic or chromosomal deletions, amplifications, or rearrangements are known to occur at gene loci that result in the loss, disruption, or increased expression of its product. High-resolution cytogenetic analyses such as those involving fluorescent in situ hybridization (FISH) and Southern blot-based restriction fragment length polymorphism (RFLP) analysis can facilitate the detection of these alterations (7); however, only a few genes presently allow routine identification of such alterations. Thus, none of these conventional methods of mutation detection are readily applicable for routine screening of large genes with a wide distribution and spectrum of mutations. Therefore, one can see how efficient mutational analysis has become a pressing issue.

The APC gene was isolated in 1991 (8,9) and so named Adenomatous Polyposis Coli when it was found to be altered in the germline of FAP patients and cosegregated with this disease (10,11). FAP is a clinically well-described highly penetrant autosomal dominant trait that has been reported for over a century (reviewed in ref. 12). Affected individuals develop hundreds to thousands of colorectal adenomatous polyps, some of which inevitably progress to colorectal carcinomas unless they are removed surgically.

FAP patients harbored multiple types of nucleotide changes widely distributed throughout APC's relatively large coding region with some trend toward concentrating in its mid-portion (the 5' end of the last large exon 15). Conventional genetic screening methods were applied in early research-based studies of the coding region of the APC gene. They could detect mutations in the range of 30-60% of patients with FAP depending on the technique used (13-15). The variegated nucleotide changes and wide distribution of APC gene mutations presented a formidable obstacle in the development of a rapid mutational assay for this gene by conventional approaches.

It was observed that the overwhelming majority of these APC gene mutations would result in a truncated gene product when expressed because of small insertions or deletions producing frameshifts and subsequent premature stop codons, nonsense point mutations, or splice site alterations (reviewed in ref. 16). Thus, it was surmised that the examination of an individual's APC protein would identify the majority of APC mutations. A novel assay was developed to examine APC's gene based on IVS of its protein from a PCR-amplified prod uct (2,3). In this assay, an individual's gene or mRNA transcript, amplified by polymerase chain reaction (PCR) or reverse transcription and polymerase chain reaction (RT-PCR) serves as a nucleotide surrogate template of the APC gene for rapid in vitro transcription and translation. The protein synthesized in vitro is then analyzed electrophoretically for its size. This method of mutational detection was shown to sensitively identify the germline truncating APC mutations in 82% of 62 unique FAP kindreds.

The IVS protein assay originated in an effort to efficiently identify truncating APC mutations. This test was first validated in the analysis of sporadic colorectal tumors containing known truncating APC gene mutations. The accuracy of identifying APC mutations in this manner was illustrated by the clearly visible mutant protein bands in these samples. The sensitivity of this assay was demonstrated in the detection of APC mutations in tiny dysplastic colonic polyps and aberrant crypt lesions (17).

The strength of the IVS protein test lies in its ability to rapidly identify truncating gene mutations irrespective of their origin or nature at the nucleotide level. Truncation of a gene's product is a drastic alteration that is expected generally to have critical effects on the protein's normal function in a cell. Therefore, the ability to identify only these truncating kinds of alterations, while avoiding numerous inconsequential polymorphisms or rare variant changes, is a significant advantage offered by the IVS protein assay in muta-tional screening.

A variety of mutations at the genetic level such as nonsense point mutations, frameshifts, or alterations producing splice abnormalities that result in a truncated gene product, can be detected sensitively all at once by this method. Additionally, the IVS protein assay can be used to analyze relatively long gene segments. This is especially advantageous for large genes having a widespread distribution of mutations. The ability to generate cDNA from mRNA transcripts by RT-PCR reactions for use as a template in this assay facilitates rapid screening of multiple exons and long regions of coding sequence at one time as well as of the splicing pattern of a particular gene.

Limitations of the IVS protein assay include its inability to identify nontruncating genetic mutations such as missense point mutations. Gross allelic loss, insertion, or rearrangement, which may prohibit the amplification of a genetic locus, also would not be detected by the PCR-based IVS protein assay. Furthermore, alterations in noncoding regions such as those that may occur in the promoter or intron regions and affect gene product expression would not be detected by the IVS protein assay.

Finally, the IVS protein assay would not detect epigenetic alterations, such as methylation changes and imprinting abnormalities, that might affect gene expression. Therefore, additional more broad analyses, such as the allele-spe-

cific expression (ASK) assay (2), Southern blot analysis, or Western blot analysis, are needed when these types of mutations are sought. Interestingly, a novel strategy, termed monoallelic mutation analysis (MAMA), which is based on somatic cell hybridization technology, was recently reported to identify germline mutations sensitively and specifically (18).

This assay was readily applied to FAP patients' blood samples in the original quest to identify APC mutations efficiently and sensitively for clinical use. This assay lends itself to routine use by utilization of supplies and equipment that are commonly available in most molecular biology laboratories. Moreover, RNA and especially DNA can be extracted by standard means from routinely available clinical samples such as blood and stored stably for analysis at convenient times.

At-risk family members are commonly the greatest beneficiaries of using the IVS protein assay to make a molecular diagnosis of FAP patients (see Fig. 1). Once a causative APC mutation is identified with this assay, one can employ the test presymptomatically to determine with virtually 100% accuracy whether or not a family member has inherited the specific genetic abnormality and the resultant risk of neoplasia associated with this disease. Presymptomatic direct genetic testing greatly aids in the clinical management of FAP kindred members and allows more directed screening for cancer development. Genetic counseling is a prerequisite for this type of testing to convey information appropriately to these patients (19).

Since its emergence in 1993, the IVS protein assay has also been used to identify truncating genetic mutations in other genes, most notably the DNA mismatch repair genes, the Duchennes muscular dystrophy gene, BRCA1, and NF1. HNPCC is a cancer predisposition syndrome inherited as an autosomal dominant trait with fairly high penetrance which is associated with colorectal and other cancer development (reviewed in ref. 20). This disease was recently demonstrated to result from alterations in DNA mismatch repair genes (21-25). The IVS protein assay was used initially to screen the candidate genes in HNPCC patients for deleterious mutations and revealed germline truncating alterations of varied genetic origins in four different genes, namely hMSH2, hMLHl, hPMSI, and hPMS2, which reflect the heterogeneity of this disease.

The clinical utility of the IVS protein test in identifying alterations in DNA repair genes is just beginning to be established (26,27). The spectrum of mutations in the DNA repair genes in HNPCC patients suggests that more than half of those identified are truncating in nature and would be amenable to detection by the IVS protein assay. A clinically useful genetic test to identify an HNPCC kindred's causative mutation would have important implications for presymptomatic screening of at-risk family members similar to those described for FAP. An additional subgroup of patients that might benefit from the use of

Affected FAP kindred member

Perform the genetic blood test (IVS-protein assay) to identify an APC mutation

(over 80% sensitive)

If negative - continue with conventional' screening of all at risk members

If positive - perform the specific genetic blood test on all at risk members

If positive - perform the specific genetic blood test on all at risk members

If positive - directed surveillance with conventional* screening measures:

If negative - Repeat genetic blood test at n or 12 years.

polyps are noted. • Screen for extraintestinal features as beginning at 11 or 12 years. • Plan colectomy when adenomatous

• Repeated sigmoidoscopy exams

If remains negative, continue with routine general population screening with additional sigmoidoscopy exams at 18,20, and 25 years.


• Once colonic polyposis established, periodic screening for duodenal and other tumors as indicated,

Fig. 1. Algorithm for the management of FAP kindreds. These management guidelines of FAP kindreds incorporate presymptomatic direct genetic testing for APC mutations. The conventional measures of screening for members of FAP kindreds at risk may vary in frequency (e.g., sigmoidoscopic exams usually performed annually until approx 40 yr of age or until significant adenomatous polyposis is noted). Endoscopic surveillance exams once colectomy has been performed is dependent on the surgical procedure performed, severity of polyposis, and amount of remaining colon mucosa left at risk (e.g., sigmoidoscopic exams every 6 mo if the rectum is intact vs annual exams after ileoanal anastomosis procedures). Extraintestina screening examinations advocated by some physicians inclucle fundoscopic exams and radiologic exams of the skull, mandible, and teeth. Once colonic adenomatous polyposis is established, surveillance for duodenal polyposis is considered every 1-3 yr, although cost to benefit ratios are not well established. Surveillance for other extraintestinal tumors, such as brain, thyroid, and soft tissues (e.g., desmoids), must then be considered, especially in kindreds already manifesting these features (e.g., Gardner's or Turcot's syndrome). Adapted from ref. 44 with permission.

the IVS protein assay to identify DNA repair gene mutations are those individuals who display microsatellite instability in their colon tumor and are diagnosed with colorectal cancer at <35 yr of age. A study found that 5 of 12 such subjects, who were examined for DNA mismatch repair gene abnormalities, harbored germline truncating alterations in hMSH2 or hMLHl (28).

Other genes that potentially lend themselves to clinically applicable mutational screening by the IVS protein assay include: the neurofibromatosis 2 gene (NF2) (29,30), the von Hippel- Lindau gene (31), Duchenne and Becker muscular dystrophy gene (32), BCM1 (33) collagen genes (e.g., COLlAI or COLIA2, which cause osteogenesis imperfecta when altered [34]), the retinoblastoma gene (35), the beta-thalassemia gene (36), and the hemophilia B gene (37)(see Table 1). All of these genes have a significant proportion (many greater than 50%) of truncating intragenic mutations in the patients examined so far. These mutations appear to be detectable by the IVS protein test. Over 50% of the various cystic fibrosis mutations that have been characterized, other than the common phenylalanine deletion at codon 508, appear to be detectable by the IVS protein assay as well (38-40).

The Neurofibromatosis l gene (NF1), Duchenne muscular dystrophy gene, and BCRA1 have been screened for mutations using the methodology of the IVS protein test with successful identification of truncating mutations (41-43). Many of these genes are quite large with widespread genetic changes that could not be screened easily for mutations by conventional approaches, as they are too laborious or cumbersome for routine clinical use.

Of course, before one would decide to perform a genetic test, such as the IVS protein assay, to identify a causative mutation clinically, a benefit would have to be gained in doing so (e.g., enabling more directed screening measures or allowing earlier preventive or therapeutic interventions to be given). Studies are also needed to determine which individuals would be the best to screen and who would gain the most from these direct mutational tests. Sensitivity and cost-to-benefit ratio analyses are needed to help address these issues.

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