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. 2022 Feb 14;17(1):50.
doi: 10.1186/s13023-022-02199-8.

Clinical measurement of cellular DNA damage hypersensitivity in patients with DNA repair defects

Ola Hammarsten  1   2 , Anna Lyytikäinen  1 , Sofia Thunström  3   4 , Torben Ek  5 , Anders Fasth  6 , Olov Ekwall  6   7 , Sara Cajander  8 , Emilie Wahren Borgström  9   10 , C I Edvard Smith  9   11 , Pegah Johansson  12   13
Affiliations

Clinical measurement of cellular DNA damage hypersensitivity in patients with DNA repair defects

Ola Hammarsten et al. Orphanet J Rare Dis. .

Abstract

Background: DNA repair deficiency disorders are rare inherited diseases arising from pathogenic (disease-causing) variants in genes involved in DNA repair. There are no standardized diagnostic assays for the investigation of pathological significance of unknown variants in DNA repair genes. We hypothesized that our assays for measuring in vitro patient blood cell hypersensitivity to DNA-damaging agents can be used to establish the pathological significance of unknown variants in DNA repair genes. Six patients with variants in the DNA repair genes PRKDC (two siblings), DCLRE1C (two siblings), NBN, and MSH6 were included. Here, we used the cell division assay (CDA) and the γ-H2AX assay, which were both developed and clinically validated by us, to measure patient cell hypersensitivity in response to ionizing radiation, mitomycin C, cytarabine and doxorubicin.

Results: Radiation hypersensitivity was detected in the two patients with variants in the PRKDC gene (p < 0.0001 for both at 3.5 Gy), and the two patients with DCLRE1C variants (p < 0.0001 at 3.5 Gy for sibling 1 and p < 0.0001 at 1 Gy for sibling 2). The cells from the patients with the PRKDC variant were also deficient in removing γ-H2AX (p < 0.001). The cells from the patient with variants in the NBN gene were hypersensitive to mitomycin C (p = 0.0008) and deficient in both induction and removal of γ-H2AX in response to radiation.

Conclusions: The combination of the CDA and the γ-H2AX assay is useful in investigating the significance of unknown variants in some DNA repair genes.

Keywords: Cell division assay (CDA); Clinical diagnosis; DNA repair deficiency disorders; Ionizing radiation sensitivity; Mitomycin C sensitivity; γ-H2AX.

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Conflict of interest statement

The authors declare that they have no competing interests.

Figures

Fig. 1
Fig. 1
In vitro sensitivity of blood T cells from two siblings with variants in the PRKDC gene and two heathy controls using the CDA treated with the indicated doses of A IR or B MMC. The controls were assigned arbitrary numbers. Error bars indicate the standard deviation of technical replicates. C PBMC from patients and controls were treated with two doses of IR and the γ-H2AX MFI in the treated sample was corrected for the background by subtracting the γ-H2AX MFI in the non-treated samples at 24 h and plotted. Error bars indicate the standard deviation of the mean. The patient γ-H2AX MFI was compared with the controls using a two-tailed t test
Fig. 2
Fig. 2
In vitro sensitivity of blood T cells from two siblings with variants in the DCLRE1C gene and heathy controls using the CDA treated with the indicated doses of A IR, B MMC, C ARA-C, and D doxorubicin. The controls were assigned arbitrary numbers. Error bars indicate the standard deviation of technical replicates. E PBMC from patients and controls were treated with two doses of IR and the γ-H2AX MFI in the treated sample was corrected for the background by subtracting the γ-H2AX MFI in the non-treated samples at 24 h and plotted. Error bars indicate the standard deviation of the mean. The patient γ-H2AX MFI was compared with the controls using a two-tailed t test
Fig. 3
Fig. 3
In vitro sensitivity of blood T cells from a patient with homozygous variants in the NBN gene and heathy controls using the CDA treated with the indicated doses of A IR, B MMC, C ARA-C, and D Doxorubicin. The controls were assigned arbitrary numbers. Error bars indicate the standard deviation of technical replicates. E. PBMC from patients and controls were treated with two doses of IR and the γ-H2AX MFI in the treated sample was corrected for the background by subtracting the γ-H2AX MFI in the non-treated samples at 1 h and 24 h and plotted. Error bars indicate the standard deviation of the mean. Due to the lack of material, the analysis was carried out once for the patient, but 5 × 103 and 15 × 103 cells were analyzed at 1 and 24 h, respectively
Fig. 4
Fig. 4
In vitro sensitivity of blood T cells from a patient with variants in the MSH6 gene and heathy controls using the CDA treated with the indicated doses of A IR, B MMC, C ARA-C, and D TZM. The controls were assigned arbitrary numbers. Error bars indicate the standard deviation of technical replicates
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