P-704 Genomic DNA damage in individuals with sex determination defects and germ cell cancer
Authors: Krivega M , Zimmer J , Rehnitz J , Frank-Herrmann P , Strowitzki T 
Published: June 2023

Study question

Does genomic instability contribute to common mechanisms underlying variable defects of gonadal differentiation and susceptibility to germ cells cancer?

Summary answer

Genomic DNA damage and associated phenotypes in leukocytes and gonadal tissue indicate systemic dependence on DNA repair mechanisms, connected to germ cell tumorigenesis when defective.

What is known already

Variabilities related to dysfunctional sex chromosomes are associated to Differences of Sex Development (DSD), characterized by dysgenic gonads, deregulated gender specification and failed germ cell maturation. This causes infertility and elevated risk of germ cell tumors (GCT). DSD was described in individuals with Swyer syndrome (46,XY, females), complete or partial androgen insensitivity syndrome (CAIS, PAIS, 46,XY, females), Turner (45,X0, females) and Klinefelter (47,XXY, males) syndromes, characterized by malignant tumor development. Unbalanced karyotype is associated with genome-wide changes, including proliferation delay, defects in proteostasis, DNA damage and activation of innate immune response, that contribute to genome instability phenotypes.

Study design, size, duration

Individuals with DSD were enrolled into study during routine examination in our clinic. Samples from men with testicular germ cell tumors (TGCT) were used as positive controls for malignancy. The samples were collected with written patients’ informed consent. For this study we analyzed 40 individuals with DSD and 19 samples with GCT. Control group contained samples from fertile men and women of appropriate age interval.

Participants/materials, setting, methods

Study groups included individuals with Swyer (n = 7), Turner (n = 11), Klinefelter (n = 6), CAIS (n = 9) syndromes and men with TGCT (n = 14). Blood was collected in EDTA and DNA, RNA and protein were isolated from leukocytes. Individuals with Swyer and CAIS, that underwent gonadectomy, formed additional group with histologically proven GCT (DSD-GCT, n = 6). Samples were analyzed with quantitative real-time PCR (qRT-PCR), immunoblotting, mass spectrometry, immunofluorescence.

Main results and the role of chance

We described an increase of DNA damage compared to unaffected controls via γH2AX, labeling double strand brakes in DNA, in leukocytes (immunoblotting: Swyer p = 0,0438; DSD-GCT p = 0,0185; CAIS NS; Turner p = 0,0117; Klinefelter p = 0,0068; TGCT p = 0,0207) and gonads (immunofluorescence, γH2AX+ puncta per cell: Swyer n = 75, p &lt; 0,0001; DSD-GCT n = 46, p &lt; 0,0001; CAIS n = 33, NS; TGCT n = 40, p &lt; 0,0001, n-number of cells analyzed). We associated malignancy in our study groups with DNA damage associated phenotypes by evaluating innate immune response activation via IFNβ and ISG15 RNA levels (DSD-GCT p = 0,0350 and p = 0,0411; TGCT p = 0,0007 and p = 0,0437) and inhibition of autophagy via decrease of LC3 and accumulation of P62 proteins (DSD-GCT p &lt; 0,0001 and p = 0,0453; TGCT p = 0,036 and p = 0,0219). This was supported by whole proteome analysis. Compromised genomic DNA integrity suggested an involvement of DNA repair pathways, illustrated by upregulation of deltaTP53 (Swyer p = 0,0104; DSD-GCT p = 0,0478; CAIS p = 0,0046; Klinefelter p = 0,0426; TGCT p = 0,006). TP53 exhibited significant increase in missense mutations in sequences, encoding transactivation domains, which compromised protein stability, particularly, in samples with GCT. We also illustrated that leukocytes of individuals with DSD-GCT restore DNA damage upon direct DNA repair mechanisms activation by Enoxacin (p = 0,0056) or autophagy inhibition by Bafilomycin A1 (p = 0,0034). T test was used for statistical analysis.

Limitations, reasons for caution

This study is based on a limited number of samples available from the individuals with the rare genetic syndromes, for some observed only in 1 out of 10,000 clinical cases. Therefore, development of in vitro study models will promote the research in this area.

Wider implications of the findings

This study elucidated possibilities for prophylactic treatments of DSD-individuals as well as new diagnostic approaches of GCT. It additionally emphasized the importance to address genome integrity in infertility research.

Trial registration number

NA