Novel Germline POT1 Variant Predisposes to Childhood Acute Myeloid Leukemia

Introduction: Current studies indicate a contribution of germline predisposition in the development of approximately 8.5% of childhood cancers (Zhang J. et al., N Engl J Med, 2015), although their apparent rate is estimated to be much higher. Understanding tumor evolution based on a predisposed cell can open unknown doors for prevention and therapy of childhood cancer e.g., leukemia. Here we present a novel rare (MAF<0.1%) germline POT1 variant (Q199*) predisposing to acute myeloid leukemia (AML). POT1 as part of the telomeric shelterin complex is known to play an important role in DNA damage protection, telomere length maintenance and chromosomal stability (Calvete O. et al., Nat. Commun., 2015). POT1 variants are associated with a broad range of cancer, including myeloid and lymphoid neoplasms in adults (Lim T.L. et al., Leukemia, 2021), but not yet described for myeloid malignancies in childhood.

Methods: Whole exome sequencing (WES) was implemented to identify germline variants. To assess the effect of POT1 p.Q199*, patient's fibroblast and stably transfected HEK293T cells were used as cell models. The variant's functional impact was experimentally tested performing yH2AX and 53BP1 immunofluorescence assays for DNA damage detection, qRT-PCR for telomere length measurement and telomere FISH to assess chromosomal instability.

Results: Utilizing WES to detect variants within shelterin complex genes we analyzed genomic data of an unselected German parent-child cohort of children with cancer (n=60, TRIO-DD), as well as a recently published parent-child pediatric cancer cohort (n=158, TRIO-D) (Wagener R. et al., Eur. J. Hum. Genet, 2021). Here, we identified a novel germline POT1 variant in a boy affected with Myelodysplastic syndrome (MDS) and secondary AML (7q-). This novel germline variant constitutes a stop-gain mutation causing a substitution of the amino acid Glutamine by a stop codon (p.Q199*).

QRT-PCR analysis within the patient's fibroblasts showed a significant (student's t-test p=0.0037) reduction of POT1 mRNA expression to ≈0.5 compared to POT1 wildtype. Western Blot analysis revealed reduced POT1 levels, confirming the loss of one POT1 allele mediated by p.Q199*. Thereupon, POT1 p.Q199* cloning and stable transfection into Hek293T cells was performed to test the variant's cooperative functionality in a controlled environment. Subsequently, POT1 p.Q199* lead to a drastically significant (student's t-test p=<0.001) increase of DNA double strand breaks in transfected Hek293T cells determined by yH2AX and 53BP1 immunofluorescence assays, which is in line with a deregulated DNA damage response and inappropriate repair by non-homologous end joining.

In addition, we detected dysregulation of telomere length maintenance. Here, relative telomere length measurement by means of qRT-PCR indicated significant (student's t-test p=0.019) telomere elongation in POT1 p.Q199* fibroblast cells. Furthermore telomere FISH on metaphase chromosomes was performed to analyse chromosomal stability. In POT1 p.Q199* Hek293T cells we identified a significant (student's t-test p=0.002) increase in telomere fragility compared to POT1 WT cells.

Conclusion: Taken together, we present the functional effects of POT1 p.Q199* leading to a significant increase of DNA damage, telomere length and chromosomal instability. Our results on functional dysregulation strengthen a potential genetic predisposition to childhood AML mediated by germline POT1 variants.