UV-sensitive Syndrome - Diagnostic Tests
Diagnostic Tests
Patients with symptoms diagnostic for UVSS show an abnormal cellular response to UV light. Alterations typically observed in UVSS cells are inability to recover normal RNA and DNA synthesis levels at late times after irradiation (recovery of post-UV DNA/RNA synthesis, RRS and RDS), substantial sensitivity to the killing effects of UV light but normal levels of UV-induced DNA repair synthesis (unscheduled DNA synthesis, UDS). XP/CS patients show the cellular response to UV typical of XP, that is reduced UDS, RRS and RDS levels and reduced post-UV survival. The DNA repair parameters are listed in Table 1.
|
Group |
Post-UV survivala |
Post-UV UDSb |
Post-UV RRSc |
Post-UV RDSd |
|
CS-A |
3-5x |
N |
D |
D |
|
CS-B |
3-5x |
N |
D |
D |
|
UVSS-A |
3-5x |
N |
D |
D |
a Post-UV survival is measured by methods such as colony forming ability, growth inhibition, or 3H-thymidine pulse several days after UV exposure. Sensitivity factors are based on comparison of patient and normal values from the same experiment.
b Post-UV unscheduled DNA synthesis (UDS) is measured by autoradiography or scintillation counting 3 h after UV. N= normal.
c Post-UV recovery of RNA synthesis (RRS) is measured by autoradiography or scintillation counting 16–24 h after UV. D= decreased.
d Post-UV recovery of DNA synthesis (RDS) is measured 16–24 h after UV by use of scintillation counting. D= decreased.
Definition of the gene responsible for the repair defect in UVSS patients is performed by complementation analysis based on somatic cell hybridization. The complementation assay is carried out by fusing fibroblasts of the patient under evaluation with fibroblasts mutated in either the CSA, CSB or UVSSA gene and by analyzing RRS levels at late times after irradiation in heterodikaryons. The restoration after fusion of normal RRS levels allows the classification of patients into different complementation groups (i.e., they are defective in different genes) whereas the maintenance of impaired RRS levels indicates that the patients are in the same complementation group (i.e., they are defective in the same gene). In parallel to the classical complementation assay, genetic analysis may be carried out also by analyzing the level of RRS in patient’s cells after microinjection of plasmid vectors expressing the UVSS genes or after infection with recombinant lentivirus or adenovirus vectors (Jia et al., Nature Protocols 10:12-24, 2015).
Identification of the defective gene is followed by sequence analysis to detect the type and location of the causing mutations. Sequence analysis of the affected gene is usually carried out at the cDNA and genomic DNA levels. A new diagnostic approach based on the direct search for mutations in a panel of relevant genes has been recently developed by the use of next generation sequencing technology (Calmels et al., Orphanet Journal of Rare Diseases 22:11-26, 2016).
Data provided so far by mutational analysis in UVSS patients are summarized in Table 2.
Table 2. Mutational analysis in UVSS patients.
|
Gene |
Number of cases |
Recurrent mutations |
Mutated productsa |
|
CSA/ERCC8 |
1 |
- |
single aa substitution |
|
CSB/ERCC6 |
2 |
- |
truncated products |
|
UVSSA/KIAA1530 |
4 |
- |
truncated products |
a Truncated products include all truncations originating from either stop codons, frameshifts, splice abnormalities or genomic DNA deletions. In brackets changes identified in single or rare cases.
Diagnostic Tools
Model Questionnaire
The model questionnaire is the same used for Cockayne syndrome patients.
Cell Biology Tools
Detailed protocols for evaluation of UDS, RRS and viability after UV irradiation and for complementation analysis are reported in the corresponding section in Cockayne syndrome card.