Functional Analysis of ACD Mutations in Dyskeratosis Congenita.

Abstract

Eukaryotic linear chromosomes face two problems that pose threats to genome integrity: (i) the end-protection problem, in which chromosome ends are recognized as double-stranded breaks by DNA damage responses, and (ii) the end-replication problem, in which loss of the chromosome extreme ends occurs in each cell division due to failure of DNA polymerases to replicate these termini. The shelterin complex solves both the end-protection problem and regulates access of telomerase to solve the end-replication problem. Decreased telomerase activity in stem cells causes dyskeratosis congenita (DC), an inherited bone marrow failure syndrome characterized by telomere lengths <1st percentile. Accordingly, germline mutations in telomerase subunit genes (TERC and TERT) or telomerase biogenesis genes (DKC1, NOP10, NHP2, and WRAP53) are commonly associated with DC. Mutations in telomere biology genes (TINF2, CTC1, and RTEL1) are also observed in DC. In ~30 % of DC cases, the underlying genetic cause is unknown. TPP1, encoded by the ACD gene, is a component of the shelterin complex. Like other shelterin proteins, TPP1 protects the ends of chromosomes from illicit fusion events. In addition, TPP1 is uniquely responsible for recruiting telomerase to telomeres. A group of amino acids on the surface of TPP1, termed the TEL patch, is critical for telomerase recruitment and telomerase processivity. These functions of TPP1 make it an excellent candidate for harboring causative mutations of DC. Here, I report the identification of a single-amino acid deletion in the TEL patch and a missense mutation in the TIN2-binding domain of TPP1 in a patient suffering from a severe variant of DC, Hoyeraal-Hreidarsson syndrome. I further showed that the deletion abrogates both telomerase recruitment and enzymatic processivity, whereas the missense mutation does not have a significant effect on these functions. The missense mutation, on the other hand, inhibits the interaction between TPP1 and TIN2, another shelterin component responsible for recruitment of TPP1 to telomeres. Therefore, inhibition of this interaction may exert an indirect effect on the recruitment of telomerase to telomeres due to reduced TPP1. Thus, I propose that both the single-amino acid deletion and the missense mutation contribute to the severe phenotype of the proband.