RNA Synthesis   

 inhibition assay measures how rapidly UV damaged cells regain the ability to make RNA at a normal rate. CS cells have a defect in rapid repair of UV damaged genes, which are needed to make RNA. The picture below shows normal (C5RO, CRL1876) and CS (KK9VD) cells with and without UV. The further to the left the peak of RNA synthesis for the individual square, the less RNA is being made. UV causes little or no change in the RNA synthesis of the two normal cell cultures, indicating a normal test response. In contrast, UV irradiation of KK9VD cells causes a dramatic shift to the left, indicating abnormally slow repair of UV damaged genes, a defect normally encountered in CS cells.

Unscheduled DNA Synthesis   

 (UDS) test for DNA repair measures how the cell can removed UV damaged DNA and replace it with normal DNA. As shown in the next picture, the test involves generating tiny black dots (silver grains) overlying the cell nucleus, with the number of dots increasing with the cell's ability to DNA repair. The left side of the picture shows irradiated (UV exposed) normal cells, with many black dots; while the right side shows unirradiated cells with undamaged DNA that does not require repair. With abnormal UDS, the grain density on the left side would have been much less. UDS is abnormal in most cases of xeroderma pigmentosum (XP), but almost always is normal in Cockaynes syndrome. However, there are unusual cases of CS in which the disease is caused by an unusual mutation (allele) in a gene that can cause XP. In such cases, UDS is abnormal.

Complementation testing   

means identifying the gene that is abnormal in the CS patient. IF UDS is normal, as in almost all cases, then this almost certainly means there is damage in either the CS-A gene (about 1/4 of cases), or the CS-B gene (about 3/4 of cases). There have been about 20 cases of CS in which UDS is abnormal, and the defect was instead in one of three genes causing XP (XP-B, D, or G gene). In about half of these cases, the patients were not spared the heavy sun induced freckling and predisposition to skin cancer that is characteristic of XP but not of CS. Complementation testing is of little practical value to patients at present, since almost all have either a CS group A orCS group B gene defect and either has about the same overall effect on the patient's health. Knowing the affected gene could become in the future if protein or gene therapy became available, however.

Complementation testing may be performed by injecting the patient's cells with different CS genes to see which gene can make the cells behave more normally (microinjection method). Injection of the same gene as is damaged in the patient results in more normal cellular behavior. Thus, if injection of ERCC6 (CS group B gene) improves the cell's DNA repair, the patient presumably has CS group B. It also may be performed using the traditional somatic cell hybridization method, in which the patient's cells are fused (made to form a single cell) with cells known to be defective in a panel of different CS genes, and then observing which hybrid (fused) cells behaved normally and not like CS cells. Normal behavior is seen only if the fused cells are affected in different genes. Thus, if the patient's cells behave normally when fused with CS group A cells but not when fused with CS group B cells, then the patient is in CS group B.

The next picture shows Complementation Testing with XP Cells, in which there are two different cell types having different sized beads and a central hybrid cell with both bead sizes and multiple nuclei; note that the hybrid cell has more black dots over its nuclei than the neighboring unfused cells, indicating that it is showing more normal repair as a result of the fusion. CS cells would behave similarly if studied in the RNA synthesis inhibition assay instead.

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