When a disorder of genome instability is suspected, the clinician is challenged with choosing the appropriate laboratory tests to secure a diagnosis and to provide guidance for affected patients and families. Table 110-5 lists some hallmark clinical features that may indicate the presence of such disorders and should prompt clinicians to initiate testing. Various laboratory tests for genome stability, DNA repair, and response to physical and chemical agents are listed in Table 110-6.
Cells obtained directly from patients and grown in culture medium are termed primary cultures. Dermal fibroblasts generally grow easily in culture and can generally be established from a 2- to 4-mm sterile skin punch biopsy specimen. The inner surface of the upper arm has proven to be a suitable biopsy site because this area heals easily, the resulting scar is not readily visible, the site is shielded from UV radiation, and attempts to establish cultures from specimens are generally successful. The tissue is placed in sterile culture medium (or sterile saline) with antibiotics and transported to a cell culture laboratory at room temperature.
Human cell cultures are made available for research by the National Institutes of Health-funded Human Genetic Mutant Cell Repository (401 Haddon Ave, Camden, NJ 08103; telephone: 856–966-7377; http://ccr.coriell.org).
Diagnostic Tests of Genome Instability and DNA Repair
Tests to assess genome instability and/or DNA repair capacity may be divided into tests of intact cellular function and tests of chromosome integrity and breakage in response to DNA-damaging agents. Other tests measure the mechanism of impairment of a cell function such as DNA repair or characterize or determine the expression of defective genes (see Table 110-6).
Tests of Intact Cell Function
Tests of cellular function measure the capacity of the intact cell to recover from DNA damage. These tests do not provide information regarding the specific type of damage resulting in cellular injury or the mechanism of cellular recovery, but they do form the basis for identifying cells as hypersensitive to DNA-damaging agents and are often used as simple screening tests.
Cell Counts or Thymidine Incorporation
One of simplest tests after exposure to UV radiation or X-rays is the assessment of the growth rate in mass culture by using a microscope or an automated cell counter to count the number of cells or by measuring incorporation of radioactive thymidine into newly synthesized DNA (see Tables 110-6 and 110-7).
Table 110-7 Suggested Sequence of Diagnostic Testing for Diseases of Genome Instability or DNA Repaira ||Download (.pdf)
Table 110-7 Suggested Sequence of Diagnostic Testing for Diseases of Genome Instability or DNA Repaira
Post-UV DNA repair
Xeroderma pigmentosum variant
Post-UV hypersensitivity (with caffeine)
Post-UV DNA repair (normal)
Post-UV inhibition of RNA synthesis
Post-UV DNA repair
Western blotting for level of protein
Sister chromatid exchange
Hypersensitivity to DNA cross-linking agents
Western blotting for FANCD2
Western blotting for level of protein
A test of colony-forming ability assesses the capacity of a single cell to proliferate enough to form a visible colony (Fig. 110-5).
Colony-forming ability assay of cell sensitivity. Xeroderma pigmentosum (XP) fibroblasts from two affected siblings (XP12TA and XP25TA), their father (XPH27TA), and an unaffected brother (35TA) as well as normal fibroblasts (96TA and HSTA) were treated with 254-nm ultraviolet C (UVC) radiation and colony-forming ability was determined. The XP complementation group C (XPC) fibroblast strains from the affected siblings were much more sensitive than the normal strains and showed similar post-UVC hypersensitivity. Cells from the unaffected brother and the clinically normal father, a heterozygous carrier of the XPC defect, had normal post-UVC survival. (Modified from Slor H et al: Clinical, cellular, and molecular features of an Israeli xeroderma pigmentosum family with a frameshift mutation in the XPC Gene: Sun protection prolongs life. J Invest Dermatol 115:974, 2000.)
Tests of Chromosome Integrity and Breakage
Chromosome breakage is usually assessed in primary cultures of mitogen-stimulated peripheral blood leukocytes or in long-term cultures of fibroblasts or lymphoblastoid cell lines. Cell cycle progression is stopped at metaphase by treatment of the cells with an inhibitor of mitosis such as colchicine. In this procedure, the 23 pairs of metaphase chromosomes from a single cell are spread over a discrete area of the slide and stained (usually with Giemsa stain). Preparations may be analyzed for the number of chromosomes per metaphase, the morphology of the individual chromosomes, and the attachments or rearrangements of chromosomes in relation to each other.
Sister Chromatid Exchange
During DNA replication, chromatids occasionally exchange positions along the arms of a chromosome. This sister chromatid exchange (SCE) may be detected by permitting the cells to grow through two cycles of replication in medium containing the nucleic acid analog bromodeoxyuridine (BrUdR) (Fig. 110-6). SCEs are thought to be related to DNA recombination repair, although their precise significance is not understood.
A. Sister chromatid exchange (SCE) assay of chromosome integrity. After the first cycle of replication, the DNA of the newly synthesized strand is labeled with bromodeoxyuridine (BrUdR), whereas the older strand is unlabeled. Such chromosomes appear uniformly dark with Giemsa stain. After a second cycle of replication in BrUdR-containing medium, one arm of a chromosome will contain two labeled chromatids, whereas the other will contain one labeled and one unlabeled chromatid. The doubly substituted arm will stain lightly, whereas the singly substituted arm will stain darkly. If an SCE occurred during replication, a portion of each chromosome arm will be doubly substituted and the remainder singly substituted with BrUdR. B. Undamaged normal cultured peripheral blood lymphocytes have approximately ten SCEs per metaphase. C. Cultured peripheral blood lymphocytes from a patient with Bloom syndrome have a manifold increase in SCEs. (From Chaganti RS, Schonberg S, German J: A manyfold increase in sister chromatid exchanges in Bloom's syndrome lymphocytes. Proc Natl Acad Sci U S A 71:4508, 1974, with permission.)
Shortened telomeres characterize cells from patients with dyskeratosis congenita. Several methods can be used to measure telomere length, including terminal restriction fragment (TRF) measurement on Southern blots, fluorescence in situ hybridization (FISH) with immunostaining, quantitative polymerase chain reaction (PCR), single telomere length analysis, and flow-cytometry with FISH (flow-FISH).31 Those tests are usually done on freshly isolated white blood cells, not cultured cells.
Unscheduled DNA Synthesis
One of the most commonly used tests of NER is unscheduled DNA synthesis. This test has been used to measure DNA repair in intact human skin, in cultured epidermal or dermal cells, and in blood cells, and for prenatal diagnosis using amniotic fluid cells. Unscheduled DNA synthesis testing measures the repair-associated DNA synthesis in G1 or G2 cells (which usually do not synthesize DNA). Cells are treated with UV radiation or another DNA-damaging agent and then incubated in medium containing radioactive thymidine. During the process of NER, the damage is removed and the radioactive thymidine is incorporated into the repaired region. The cells are treated with fixative, coated with autoradiographic (photographic) emulsion, and kept in the dark for an appropriate interval, and then the emulsion is developed. UV radiation of normal fibroblasts results in a large increase in the number of grains seen over all the nuclei. In marked contrast, irradiation of the XP fibroblasts that cannot repair DNA damage results in very few grains over the nuclei.
After exposure to UV radiation, normal cells temporarily reduce RNA synthesis from active genes. Synthesis resumes when the damage is repaired. This resumption of RNA synthesis is delayed in cells from patients with CS and some forms of XP.
The host cell reactivation assay relies on the fact that plasmids do not have the ability to repair damage to their DNA but depend on cellular repair systems. Thus, when transfecting plasmids with DNA damage into host cells, the DNA repair enzymes of the host cells need to repair the damage before the plasmids’ genes can be expressed. Therefore, damaged plasmids would be expected to be expressed at a higher level in cells with normal repair capacity. A nonreplicating plasmid that contains the gene for the firefly enzyme luciferase, constructed to permit expression in human cells,32 is widely used; generation of light provides a quantitative endpoint for its repair (Fig. 110-7).
Plasmid host cell reactivation assay with assignment to xeroderma pigmentosum (XP) complementation group. A. The plasmid is damaged by ultraviolet (UV) radiation and introduced into cultured human cells by a transfection technique. The cells’ DNA repair enzymes repair the damage in a manner similar to the repair of cellular DNA. Repaired DNA will then function to transcribe the plasmid-encoded luciferase gene in the human cell. The amount of luciferase activity within the host cells therefore reflects the efficiency of the cellular DNA repair system. This assay can also be used to determine the complementation group by cotransfecting UV-treated plasmid plus plasmids expressing wild-type XP complementary DNA (cDNA). B. The results of plasmid host cell reactivation experiments and complementation group assignment with DNA excision repair-deficient XP46DC cells. UV-treated plasmid showed low expression in the XP65BE cells that was increased only by cotransfection with a plasmid expressing the wild-type XPC cDNA. This result indicates that XP65BE cells are in XP complementation group C (XPC). rLU = relative light units; pCMVluc and pLUC = plasmids containing gene for luciferase; pXPA–pXPG = plasmids expressing xeroderma pigmentosum complementation groups A–G.
The comet assay is a single cell-based technique that allows detection and quantitation of DNA damage, in particular DNA strand breaks that were either introduced directly by the DNA-damaging agent or by repair endonucleases at sites of other types of DNA damage. For this assay, damaged cells are embedded in agarose, lysed, and exposed to an electric field. In the electrical field, the DNA migrates out of the nucleus forming a “comet” when stained. In presence of DNA strand breaks, DNA migrates out of the nucleus faster and with that generates a longer comet. Thus, the length of the comet is proportional to the fragmentation of the nuclear DNA. This assay can be modified for detection of single-strand or double-strand DNA breaks, UV damage, or oxidative DNA damage. Cells from patients with XP have defective repair in the post-UV comet assay.
Normal DNA has tens of thousands of regions with repeats of the dinucleotide CA or other short motifs up to five nucleotides long. In normal individuals each of these microsatellites (also called simple sequence repeats or short tandem repeats) has a uniform size. However, these sizes are highly variable among different individuals and are often used for DNA “fingerprinting.” The appearance of abnormally longer or shorter simple sequence repeats in different tissues or tumors from a patient is called microsatellite instability. This can be associated with a defect in mismatch repair genes.
Characterization and Expression of Defective Genes
Real-Time Polymerase Chain Reaction
Many disease-causing mutations, including those responsible for disorders of genome instabilty disrupt the gene product's function by changing its amino acid sequence. In some genome instability genes, disease-causing mutations create premature stop codons for protein synthesis. These mutations result not only in truncated but also in low levels of messenger RNA (mRNA) for the gene product through a process called nonsense-mediated message decay. The mRNA levels can be accurately measured by the use of quantitative reverse transcriptase real-time polymerase chain reaction. For example, low XPC mRNA levels have been found in cells from most XP patients with defects in this gene.33
Some mutations result in reduced levels or size of encoded proteins, often through creation of premature stop codons.33 Reduced protein levels are most commonly detected by Western blotting. Cells are lysed and the proteins are extracted and separated by gel electrophoresis. The separated proteins are transferred to a membrane and probed with an antibody that is specific for the protein of interest. The intensity of the antibody staining reflects the amount of protein in the cells, and its location on the membrane is an indication of the size of the protein molecules. For example, undetectable or low levels of polymerase eta protein are present in cells from most XP variant patients.34
Direct sequencing of defective genes is the gold standard for determination of the presence of a mutation. The final step in confirmation of a diagnosis may be DNA sequencing to determine the disease-causing mutation. By definition, disease-causing mutations alter the function of genes. However, not every alteration in the sequence of a gene alters the function of the encoded protein. In the human genome there are millions of single nucleotide polymorphisms, or changes in one nucleotide, that are not associated with disease and may not even change the amino acid composition of the encoded protein. In recessive disorders, each clinically unaffected parent has one normal allele and one potentially disease-causing mutation in the other allele. The affected child receives an allele with a disease-causing mutation from each parent. The two mutations must be in the same gene, although they need not necessarily be identical to each other.
Considerations in Diagnostic Testing
Diagnosis of disorders of genome instability or DNA repair is often a multistep process. A suggested sequence of steps is listed in Table 110-7. As new tests are developed and new information is obtained about these disorders, testing procedures may change accordingly. In addition, decisions regarding the extent of testing performed may be made with consideration of how much the tests cost and whether additional information would alter treatment. For example, DNA sequencing is rarely required for establishment of a diagnosis of XP in a child with classical clinical features and cells that are hypersensitive to killing by UV and have defective DNA repair. But if a family has one child affected with XP and is considering having additional children, DNA sequencing might offer them the possibility of prenatal diagnosis through DNA sequencing of a trophoblast biopsy specimen.
Genetic counseling is an important component of patient management for these genetic diseases. This function may be performed by the treating physician or by a trained genetic counselor.
In the United States, only laboratories certified in accordance with the Clinical Laboratory Improvement Act (CLIA) are allowed to perform these specialized tests in the context of patient care. Tests performed in research laboratories generally have limited use in clinical practice. However, a research laboratory may identify a disease mutation in cells from a patient that could then be confirmed in a CLIA-certified laboratory and used in clinical practice. A current listing of laboratory testing facilities for these diseases may be found at the Web site http://genetests.org, funded by the National Institutes of Health.