Trichothiodystrophy - Diagnostic Tests

Diagnostic Tests

The diagnosis of the photosensitive form of TTD is made by DNA repair tests such as the measurement of UV-induced DNA repair synthesis (unscheduled DNA synthesis, UDS), UV survival and the analysis of the recovery of post-UV DNA/RNA synthesis (RRS, RDS). The DNA repair parameters are listed in Table 1.

Group	Post-UV survival^a	Post-UV UDS^b	Post-UV RRS^c	Post-UV RDS^d
XP-B	7-8x	5%	D	D
XP-D	7-9x	10-60%	D	D
TTD-A	2-4x	10-25%	D	D
Non-photosensitive	N	N	N	N

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, N= normal.

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, N= normal.

d) Post-UV recovery of DNA synthesis (RDS) is measured 16–24 h after UV by use of scintillation counting. D= decreased, N= normal.

Definition of the gene responsible for the repair defect in the NER-defective form of TTD is performed by complementation analysis based on somatic cell hybridization. The classical complementation assay is carried out by fusing fibroblasts of the patient under evaluation with fibroblasts representative of XP-B, XP-D and TTD-A groups and by analyzing the capacity to perform UDS in heterodikaryons. The restoration after fusion of normal UDS levels allows the classification of patients into different complementation groups (i.e., they are defective in different genes) whereas the maintenance of impaired UDS 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 UDS in patient’s cells after microinjection of plasmid vectors expressing the wild-type genes or after infection with recombinant lentivirus 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).

No cellular tests are available for the non-photosensitive form of TTD. The presence of inactivating mutations in the TTDN1, RNF113A and GTF2E2 genes may confirm their involvement in the pathological phenotype.

Data provided so far by mutational analysis in TTD patients are summarized in Table 2.

Table 2. Mutational analysis in TTD patients.

Gene	Number of cases	Recurrent mutations	Mutated products^a
TTDA/GTF2H5	5 (4 families)	-	single aa substitutions, truncated products
XPB/ERCC3	2 (1 family)	-	single aa substitution
XPD/ERCC2	46 (43 families)	Yes	single aa substitutions (truncated products)
TTDN1/MPLKIP	30 (17 families/kindreds)	Yes	truncated products (single aa substitutions)
RNF113A	2 (1 family)	-	truncated product
GTF2E2	5 (4 families)	-	single aa substitutions

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 Trichothiodystrophy 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 UV irradiated (20 J/m²), incubated for 3 h in ³H-TdR labeled medium and processed for autoradiography as described above. The number of grains over nuclei in at least 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 group if no restoration of UDS level is observed in the heterodikaryons.