Cockayne Syndrome - Diagnostic Tests

Diagnostic Tests

About 50% of the patients with symptoms diagnostic for CS show an abnormal cellular response to UV light. Alterations typically observed in CS 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.

 

Table 1. Typical results of cellular functional assays for the distinct groups of CS and XP/CS.

Group

Post-UV survivala

Post-UV UDSb

Post-UV

RRSc

Post-UV

RDSd

CS

 

 

 

 

CS-A

3-5x

N

D

D

CS-B

3-5x

N

D

D

XP/CS

 

 

 

 

XP-B

7-8x

5%

D

D

XP-D

7-9x

40-50%

D

D

XP-G

7-10x

2-10%

D

D

ERCC-1

5x

15-20%

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. Percentages compared to normal donors analyzed in parallel are reported, 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 CS 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 or CSB 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 CS genes or after infection with recombinant lentivirus or adenovirus vectors (Jia et al., Nature Protocols 10:12-24, 2015).

Different from CS cases, patients showing clinical features of CS in association with those of XP (XP/CS) show reduced UDS (Table 1). In these cases, genetic analysis relies on complementation assays developed for XP defects.
 

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 CS and XP/CS patients are summarized in Table 2.

 

 

Table 2. Mutational analysis in CS and XP/CS patients.

 

Gene

Number of cases

Recurrent mutations

Mutated productsa

CS

 

 

 

CSA/ERCC8

84

Yes

truncated products (single aa substitutions)

CSB/ERCC6

172

Yes

truncated products (single aa substitutions)

XP/CS

 

 

 

XPB/ERCC3

5

-

single aa substitutions, truncated products

XPD/ERCC2

10

Yes

single aa substitutions (truncated products)

XPF/ERCC4

2

-

single aa substitutions, truncated products

XPG/ERCC5

22

No

truncated products (single aa substitutions)

ERCC1

3

-

single aa substitutions, 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

Here you will find the downloadable version of the Cockayne syndrome patient form. This document is currently used to collect patient details before sending the biological sample to the diagnostic laboratory, in order to ease cellular and/or molecular investigations.

 

Cell Biology Tools

UV irradiation

Medium is removed and the cells are exposed to UVC irradiation (254 nm) using a Philips TUV 15 Watt lamp.

Unscheduled DNA synthesis (UDS) by the autoradiographic technique

UDS is analyzed in proliferating fibroblasts by seeding the cells in dishes containing a coverslip. After 2 days of incubation at 37°C, the cells are UV irradiated (10 and 20 J/m2) and incubated again in medium containing 10 mCi/ml 3H-thymidine (3H-TdR, specific activity 25 Ci/mmol) and fixed 3 h later. Autoradiography is performed with Ilford emulsion; after 2-4 days at 4°C, the slides are developed and stained with May-Grunwald and Giemsa solutions. S-phase nuclei are heavily labelled and easily distinguishable from non-S phase nuclei undergoing repair synthesis. Repairing nuclei are lightly labelled, and UDS is measured by counting the number of grains on at least 50 non-S-phase nuclei.

Recovery of RNA synthesis (RRS) by the autoradiographic technique

Fibroblasts (3-4x104 cells per 30-mm dish containing a coverslip) are seeded in complete medium, which is replaced the following day with medium containing 0.5% fetal calf serum (FCS). After 5 days of incubation at 37°C, the cells are UV irradiated (20 J/m2), incubated again in medium containing 0.5% FCS for a further 24 h, and then labelled for 1 h with 10 mCi/ml 3H-Uridine (3H-Urd, specific activity 25-30Ci/mmol). One dish is treated in parallel but without UV irradiation. Autoradiography is performed with Ilford emulsion; after 2-4 days at 4°C, the slides are developed and stained with May-Grunwald and Giemsa solutions. RRS is measured by counting the number of grains on at least 50 nuclei.

Viability in stationary phase fibroblasts

Sensitivity to UV irradiation is analyzed in stationary phase fibroblasts by plating cells (1.5x105 cells per 60-mm dish or 5x104 cells per 30-mm dish) in complete medium, which is replaced the following day with medium containing 0.5% fetal calf serum (FCS) to bring the cells into a non-proliferating state. After 7 days of incubation at 37°C, the cells are UV irradiated (multiple doses from 5 to 30 J/m2) and incubated again in medium containing 0.5% FCS; 14 days later, when dead cells have detached from the plates, the adhering (i.e., viable) cells are fixed and counted.

Cell hybridization and complementation assay*

The fibroblasts used as partners in the fusion experiments are labeled with latex beads of different sizes (0.72 and 2.0 micrometers) by growing the cells for 3 days in medium containing beads. The cells are then trypsinized, mixed in a 1: 1 ratio, centrifuged, fused using polyethylene glycol (PEG-4000, Merck), diluted in medium containing 3% fetal calf serum (FCS), and seeded in dishes containing a coverslip.  After 48 h incubation at 37°C, cells are irradiated with a UV dose of 20 Jm2, incubated again at 37°C in medium with 3% FCS for a further 24 h, and then labelled for 1 h with 10 mCi/ml 3H-Urd. Slides are processed for autoradiography as described above. The number of grains over nuclei in 25 homodikaryons (identified as binuclear cells containing beads of one size) and in 25 heterodikaryons (identified as binuclear cells containing beads of different sizes) are counted. Two cell strains are classified in the same complementation group if the number of grains present in the heterodikaryons is similar to that in the homodikaryons of the fusion partners.

 

* For CS patients in whom cellular analysis demonstrated the presence of reduced UDS, the complementation assay is carried out using this alteration as a cellular parameter (see the corresponding section in XP for details).