Table of Contents- Lesson 1) ( Next) (Glossary)

A decade or so ago, testing for genetic diseases was limited to determining the accumulation of precursor proteins or secondary metabolites, the deficiency of an end product, or the absence of enzyme activity. By and large, genes that code for enzymatic proteins were amenable to testing (PKU, galactosemia), whereas genes responsible for structural proteins were not (the skeletal dysplasias). Another limitation was the need to sample and test the appropriate tissue. PKU, for example, was not diagnosable prenatally because the phenylalanine hydroxylase enzyme is not expressed in amniotic fluid cells (fetal skin cells) since it is primarily a liver enzyme.

Since all cells in the body have the same DNA composition, technically all genetic disorders should be diagnosable using any tissue. With this in mind, researchers have been working to locate and sequence all of the genes in the human genome. The only limitation now in our ability to test for genetic disorders is the availability of DNA probes or markers for specific disorders.

As soon as the gene coding for a specific abnormality has been identified or isolated, DNA testing becomes possible. DNA is usually isolated from the white blood cells and has to be cut into smaller pieces to be analyzed. This is accomplished by using restriction enzymes (naturally occurring enzymes from bacteria) which cut the DNA at specific sites with the appropriate sequence of bases. For instance, EcoRI (a restriction enzyme from E. coli) will cut DNA wherever the sequence GAATTC appears. Exposure to this enzyme results in the DNA being chopped into millions of fragments of varying size, called restriction fragments.

Once cut, the DNA is loaded into a well on one end of a slab of gel. The fragments are then separated according to size by electrophoresis. As electric current passes through the gel, the fragments move according to size. The bigger fragments stay close to the origin, and the smaller fragments move farther down the length of the gel. The DNA is then denatured (by exposure to alkaline solutions) to render the DNA single-stranded (instead of the natural double-stranded form). Since the gel is difficult to handle, the DNA is transferred to a nitrous cellulose paper to create a Southern blot (named after the researcher who developed the procedure). The DNA probe which is radioactively labeled (or fluorescent labeled) is then applied to the Southern blot. Since the probe is also single-stranded, it will seek the single-stranded DNA fragments that are complementary, and undergo hybridization. The excess probe is washed out and only the bound probe will remain on the Southern blot paper. This is then laid on an x-ray film. The radioactive probe will leave bands on the x-ray film. Depending on the type of probe used, there could be hundreds of bands (much like bar codes) or only a few bands present on the x-ray film. By having several wells on one end of the gel, several samples can be loaded, and DNA fragments in corresponding lanes can be analyzed concurrently. By running control samples, with known DNA fragment sizes, on the same gel with patient samples, it is possible to identify changes in the size of a DNA fragment and, therefore, a change in a specific gene. Since each step takes about a day and since samples are batched, the procedure ordinarily takes one to two weeks to complete.

Although there are several permutations as to how the test is run, the basic steps are as follows: the DNA is isolated, cut into smaller pieces, spread on the gel and then exposed to a probe to identify the DNA fragment of interest. The suspected diagnosis determines the choice of restriction enzymes and probes that are used.

Fig. 1.20. Basic steps of DNA testing

Fragile X syndrome is an X-linked trait. Males with fragile X syndrome are mentally retarded. Females with fragile X have a 30% chance of being mentally deficient (based on lyonization). As previously noted in the section on nontraditional inheritance, the triplet repeat CGG on the front part of the FMR-1 gene is repeated less than 50 times in normal individuals, 50 to 200 times in mentally normal carrier females or transmitting males (premutation), and over 200 to 1,000 times in mentally retarded fragile X males and females (full mutation). CGG codes for the amino acid glutamine. It is not quite clear why lengthening of this triplet repeat leads to mental retardation. Using the procedure noted above, this direct DNA test can differentiate affected males, normal transmitting males, affected females and normal carrier females.

Cases 1 and 3 have the full mutation (F) X chromosome, cases 2 and 4 have the premutation (P) X chromosome, and cases 5 and 6 have the normal (N) X chromosome. Since males have one X, they have only one bar. Since females have 2 Xs, one is active (P or N), the other is inactive (NM-normal methylated).

Fig. 1.21. Fragile X syndrome Southern blot analysis. To determine size and methylation status of the FMR-1 CGG repeat, the enzymes EcoRI and Eagl and the probe StB12.3 are used. The 2.8 kb fragment is the normal X with 6-50 CGG repeats. The premutation X has 50-200 CGG repeats, and the full mutation X has over 200-1,000 CGG repeats. The 5.2 kb fragment is the methylated inactive normal X chromosome in females.

Breast cancer and ovarian cancer are common adult onset conditions. A subset of breast/ovarian cancer, 10%, behaves in an autosomal dominant fashion. There is a major gene for breast/ovarian cancer that has been identified on the long arm of chromosome 17 (17q21) called BRCA1, and another gene for breast cancer on the long arm of chromosome 13 called BRCA2. Considering the time and expense involved, DNA screening for breast cancer is only offered to families at high risk. Among the factors that are taken into consideration are a family history of breast/ovarian cancer in first or second degree relatives, premenopausal onset and a history of more than one primary tumor.

Following pedigree analysis, the appropriate family members are chosen for testing. This usually consists of affected sisters of the index patient, both parents and other affected members on the mother's (or father's) side of the family. It is important to remember that males can carry and transmit the breast cancer genes.

DNA can be isolated from blood, old slides, or paraffin blocks from biopsies or tumors. These specimens are tested using probes for "markers" within the gene. The purpose is to show that the affected sisters, the affected parent (or a normal transmitting parent) and the affected aunts all carry the same markers. The shared set of markers will identify the abnormal gene or chromosome segregating in the family. The other set of markers will identify the normal chromosome inherited from the normal parent. Realize that this is an indirect DNA test. Having established linkage of a set of markers to the abnormal gene, one can then begin to test the unaffected at-risk members of the family.

Fig. 1.22. Breast cancer pedigree. Breast cancer patients V-4,V-5, V-8 and IV-11 share the same markers 155, 111 and 152 (black solid bar). The sisters in generation V inherited the gene through their father and grandfather. Their transmitting father IV-9 carries the same markers as his affected female second cousin IV-11. Having established linkage of these markers to the BRCA-1 gene, this test was then used for presymptomatic diagnosis of their three unaffected sisters and a brother (not shown on the pedigree).

Markers are anonymous segments of DNA. They do not form part of the gene. They are segments of DNA that are repeated in tandem (microsatellite, minisatellite, or variable number of tandem repeats (VNTR)). These segments of DNA are randomly distributed throughout the genome in all the chromosomes and as yet they have no known function. Since these DNA sequences are technically nonfunctional, they can increase or decrease in number, be present or absent, or undergo change without any clinical consequences, and thus, are highly variable among members of a family or individuals in the population. These markers are used as signposts to mark the abnormal gene or the abnormal chromosome.

Cystic fibrosis is a relatively common condition with an incidence of 1 in 2,500. It is common in Caucasians, uncommon in African Americans and rare in Asians. Cystic fibrosis is an autosomal recessive trait, and parents of an affected child are presumed carriers. The CF gene has been mapped to chromosome 7q31.2. The most common CF gene mutation is F508. The sign means change, F is the symbol for phenylalanine, and 508 is the position of the codon that has mutated. The test for the CF gene is a direct DNA mutation analysis using PCR (polymerase chain reaction) and an ASO probe (allele specific oligonucleotide).

In PCR, DNA isolated from the patient is mixed with appropriate bases (adenine, guanine, cytosine, thymine-the building blocks of DNA), a set of primers that flank the gene and Taq polymerase (an enzyme that copies DNA and comes from the bacteria Thermus aquaticus). The mixture is cycled through a series of reactions by changing the temperature from hot to cold. In the process of heating, the DNA separates into single strands; in the process of cooling, the DNA is reconstituted into double strands by the annealing of bases from the mixture with the single strands of DNA. The heating and cooling cycle is repeated 20 to 30 times, increasing the desired segment of DNA geometrically to millions of copies.

PCR is more efficient than linkage analysis. Because of automated cyclers there is a shorter turn around time. It is also possible to use a small sample size (e.g., a hair root, drop of blood, etc.) or old samples. However, PCR requires a set of primers closely flanking the gene that is of interest and, therefore, is only possible if the DNA sequence of a gene is known.

Assuming that a family with CF is known to have the R1174 mutation, allele specific oligonucleotide (ASO) probes can be used to detect the presence of this point mutation. Two probes are used in this system. One is a short segment of DNA (18-22 bases), or oligonucleotide probe, that complements the abnormal gene with the point mutation. The other is an oligonucleotide probe that complements the normal gene. Using a dot blot procedure, these two allele specific oligonucleotides (ASO) are fixed onto a filter paper, one row normal and one row abnormal. The DNA sample from the patient is labeled and amplified by PCR and then added to the filter paper. DNA from a homozygote affected person (with two abnormal genes) will bind only to the ASO that carries the point mutation. DNA from a homozygote normal person (with two normal genes) will bind only to the ASO that is normal. The heterozygote carrier's DNA (with both the normal gene and the abnormal point mutation) will bind to both.



Among the sibs, 1 is normal, 2 is affected, 3 is a carrier. Both parents 4 and 5, are also carriers.

Fig. 1.23. Cystic fibrosis diagnosed by PCR and direct DNA testing with ASO probes

This test is used to identify CF gene carriers in a family whose mutation is known, (e.g., R1174, F508, etc.). The problem with screening the general population for CF gene mutations is the fact that there are hundreds of mutations in the CFTR (cystic fibrosis transmembrane conductance regulator) gene and different populations have different incidence rates for these mutations. For purposes of screening, it is possible to test for the 14 most common mutations. However, these mutations are present in only 90% of the CF gene carriers. For this reason CF testing is usually only offered to families at risk.

Huntington disease, an adult onset neurological disease, has been mapped to the tip of the short arm of chromosome 4 and is secondary to expansion of a triplet repeat (CAG). The normal values are 34 or less. Individuals with values of 39 or more are considered affected. The blood sample can be amplified by PCR and run through a polyacrylamide gel with the bands stained by silver staining. Two bands will invariably be present in the heterozygote, one representing the normal chromosome with a CAG repeat under 34, and the other band representing the abnormal chromosome with a repeat greater than 39.

There are numerous permutations to DNA testing. The choice of test procedure depends on the diagnosis entertained.

REASONS WHY DNA MOLECULAR TESTING MAY NOT BE AN OPTION

Based on coverage by the media, the general public has come to expect a genetic test to confirm all ailments-from rare syndromes to common chronic diseases in man. However, there is often a need to translate information from medical research to clinical application before a test can be offered to a patient. It is not surprising then that patients are often disappointed when informed that a genetic test is not possible.

The following are some reasons why genetic testing may not be available:

• The gene has not been found.
• The gene may have been localized to a chromosome segment, but has not been isolated. Since HD was localized to chromosome 4p16 in 1984, it took another 10 years to finally isolate the gene. For this reason, presymptomatic diagnosis in the late 1980s and early 1990s relied on linkage analysis utilizing markers (RFLPs-restriction fragment length polymorphisms) in extended family studies. Since the gene was identified, the direct DNA test for CAG repeat size now allows for individual diagnosis.
• The gene has not been sequenced and knowing the sequence of base pairs is essential to develop complementary DNA probes. The DNA probe is needed to seek out and label the patient's genomic DNA (on Southern blot or on the chromosome metaphase FISH slide) to detect mutations or deletions.
• The gene has not been sequenced and knowing the gene sequence, close to the beginning and at the end, is needed to create primers. Primers are used to copy the patient's genomic DNA by PCR (polymerase chain reaction). Following gel electrophoresis, the size of the PCR product will determine if the gene is normal or mutated.
• The gene has been isolated and sequenced but within the gene there are several possible mutations (allele heterogeneity). As previously noted, there are well over 100 mutations within the CF gene. A negative test does not necessarily rule out a CF mutation; a positive test verifies its presence. In CF, there are mutations common to certain ethnic groups.
• There may be several genes at different chromosome loci that are responsible for a given clinical entity (locus heterogeneity). For example, PKU can be due to an abnormality in the phenylalanine hydroxylase gene or the tetrahydrobiopterin gene. For a number of genetic disorders, the mutation in an index patient needs to be established before carrier status in a parent, sibs or a fetus (by prenatal diagnosis) can be recommended.
• If linkage analysis (rather than direct DNA analysis) is the only method available, an informative family-proband, parents, normal and affected first and second degree relatives with segregating polymorphic markers-is needed to complete the study. Family cooperation is critical.
• For susceptibility genes, the presence of the gene does not necessarily lead to the disease or illness. Linkage analysis is often the procedure used to identify individuals who carry susceptibility genes. The susceptibility genes for familial breast/ovarian cancer have been assigned to chromosomes 17q, 17p or 13q and this makes the search for the abnormal gene more tedious (genetic heterogeneity). Although the susceptibility genes are inherited in a dominant (50%) manner as a constitutional mutation that is present in all cells, an acquired somatic mutation in the counterpart normal gene is needed for the cell to become malignant. At the cell level, familial breast cancer is technically homozygous recessive. Thus, carriers of the gene have an 80% lifetime risk of developing breast cancer (rather than 100%), making it more difficult to determine which markers are traveling with the susceptibility gene. Although testing is available, most genetic and public health associations recommend limiting the test to high-risk families.
• The biologic basis of the test-sensitivity and specificity, procedural and technical considerations, statistical analysis-may provide spurious results, i.e., false positives or false negatives.
• The financial considerations in individual or family studies can be a hurdle for the laboratory and the patient.
• The social and ethical dilemmas, and the limited public health resources are unresolved issues.

Thus, DNA gene testing, although possible, may not always be doable. Nonetheless, for the more common genetic disorders confirmatory diagnosis is possible using routine laboratory tests, imaging studies, chromosomal, biochemical or DNA molecular analysis.

SUMMARY

DNA testing is accomplished by isolating DNA, cutting the DNA into fragments, separating the pieces and looking for changes in the size of the fragments. The presence of an increased number of triplet repeat sequences is diagnostic for some single gene disorders. Using specific probes it may also be possible to identify a change in a single base pair, as a small probe will only attach to a perfectly matched segment of DNA. With allele specific probes for normal and abnormal genes, normal, affected or carrier status can be established by direct DNA testing.

If the chromosomal location of a gene is known, even if the exact defect has not been established, indirect DNA testing may be possible. Indirect DNA testing involves the identification of anonymous segments of DNA that are close to the gene in question. As these segments of DNA do not code for specific proteins, it is not unusual to find a great deal of variation in these segments in the general population or among members of a family.

To do an indirect DNA study, it is necessary to collect DNA from a number of affected and normal family members. If a particular set of markers is seen only in persons with the disorder in question, it is assumed that this set of markers is linked to the disease gene. Once a marker has been identified, it is then possible to test unaffected family members and determine whether they carry this same marker, and thus the disease gene in question.

PRACTICE ACTIVITY 7

Use a T or F to show whether each statement is true or false.

1. Every Caucasian person should be offered carrier testing for the CF gene.

2. If the exact gene mutation is not known, presymptomatic testing is not possible.

PRACTICE ACTIVITY 7: ANSWERS

1. False Within the CF gene, there are hundreds of mutations that can result in the production of a nonfunctional protein. It is impractical to run tests looking for all of these mutations; therefore, CF carrier screening is usually reserved for those persons who have a family history of CF and their partners.

1. False If there are markers close to or within the gene in question, it may be possible to test affected and unaffected family members and determine which markers are traveling with or linked to the disease gene in a particular family. Once this is known, presymptomatic testing can be offered to at-risk family members.
   

Table of Contents- Lesson 1) (Next) (Glossary)