Research & Discovery
Complementation group assignment
by David Busch, Ph.D., M.D.
There are four laboratory tests that are particularly likely to be used in study of XP patients’ cells, of which the first two currently are in use in my laboratory, and the third is in the process of being introduced and is the main subject of this discussion.
The first method is the UV survival curve study, which measures how UV sensitive are the cultured cells i.e., how much UV is required to kill a given percentage of cells. XP patients may have anywhere from normal UV sensitivity (unusual) to up to about 10x normal UV sensitivity. Measuring their UV sensitivity this way provides one way of describing the severity of their defect.
The second method is the unscheduled DNA synthesis (UDS) assay, which measures proficiency of repair of UV induced DNA damage; this is the method that was provided by Dr. James Cleaver when he discovered the defect present in XP. Except for the occasionally encountered XP variants, who have the skin lesions of XP but normal UDS, XP patients may have anywhere from O% to about 50% of normal UDS. It is possible to have close to normal UV sensitivity with low UDS, or relatively high UDS but extreme UV sensitivity, so that UDS is another way of measuring the degree of the defect that is independent from UV sensitivity. The UDS assay is the most likely method to use when prenatal diagnosis is done, because results can be obtained quickly using relatively few cells,
The third method, complementation group assignment, determines what gene is affected in the patient. Seven different genes (XP group A through G genes) have been found to be affected in different XP patients. The severity and nature of the XP patient’s problems tend to correlate with what gene is affected, although it is important to realize that exceptions can occur due to differences in how the gene is mutated (see next paragraph). In general, XP group C, E, and F patients (and also XP variants) are spared the neurological problems that affect about a fifth of XP patients, while neurological problems may or may not occur in groups A, D, and G. Thus, if one is told one is in group C (the commonest group), one can generally rest easy from the standpoint of worrying about neurological problems; while if one is told one is in group A one is likely to worry a lot more, although there still is uncertainty about whether neurological problems will develop. Probably many would prefer to not be told their complementation group. Individuals in different complementation groups tend to have different degrees of deficiency in UV sensitivity and in UDS, so that it is possible usually to make an educated guess about what gene is affected if the first two tests have been done, realizing that this is only a guess and not reliable. For example, Katie Mahar’s lab tests indicate that she probably is in group C, and this certainly is consistent with her complete lack of any neurological problems, so that when we try to find out her complementation group we will first see if she is in group C before looking into other groups. However, it still is possible that she could be in another group, since her initial lab tests are no substitute for a complementation test. Different techniques exist for performing complementation group assignment, but generally they involve determining whether performing a laboratory procedure results in an improvement in the cell’s UDS. In my laboratory, the preferred method is DNA microinjection, in which the nucleus of a cell is injected with the normal version of an XP gene to see if the gene increases UDS, in which case the gene is proved to be same gene as is affected in the patient. Another popular method is somatic cell hybridization, in which the patient’s cell is fused with a known type of XP cell so that two cells are used to make one hybrid cell, and then the UDS test is performed on the hybrid cell. In that case, UDS will increase if the known XP cell is affected in a different gene than the patient’s cell. Knowing the complementation group will become very important in the future when gene therapy becomes available, since it will indicate what gene needs to be administered in the therapy.
The fourth method, allele identification, is used to determine in what way the patient’s affected gene is abnormal. There are thousands of ways (alleles) in which a gene can have an abnormal structure that could result in XP; and the way the gene is abnormal could greatly affect UV sensitivity, UDS level, and even the chances of developing neurological problems. Thus, XP group A patients tend to be the most UV sensitive, to have the lowest UDS levels, and to have the worst neurological problems with the earliest onset. However, if a certain region of the XP group A gene is affected, there may be no neurological problems, or at least greatly delayed problems. Information about the severity and progression of cases of XP is not available for all alleles, so it is possible that a patient could learn what is the allele but may have no way of knowing the health significance of the allele; indeed the same patient may eventually provide that information to researchers as the person ages and the allele’s significance becomes clearer. Thus, there is currently a need for a database in which clinical features are correlated with allele present; and it will take years to accumulate such a database for all naturally occurring XP gene alleles.