Constitutional mutations in RTEL1 cause severe dyskeratosis congenita

Abstract

Dyskeratosis congenita (DC) and its phenotypically severe variant, Hoyeraal-Hreidarsson syndrome (HHS), are multisystem bone-marrow-failure syndromes in which the principal pathology is defective telomere maintenance. The genetic basis of many cases of DC and HHS remains unknown. Using whole-exome sequencing, we identified biallelic mutations in RTEL1, encoding a helicase essential for telomere maintenance and regulation of homologous recombination, in an individual with familial HHS. Additional screening of RTEL1 identified biallelic mutations in 6/23 index cases with HHS but none in 102 DC or DC-like cases. All 11 mutations in ten HHS individuals from seven families segregated in an autosomal-recessive manner, and telomere lengths were significantly shorter in cases than in controls (p = 0.0003). This group had significantly higher levels of telomeric circles, produced as a consequence of incorrect processing of telomere ends, than did controls (p = 0.0148). These biallelic RTEL1 mutations are responsible for a major subgroup (∼29%) of HHS. Our studies show that cells harboring these mutations have significant defects in telomere maintenance, but not in homologous recombination, and that incorrect resolution of T-loops is a mechanism for telomere shortening and disease causation in humans. They also demonstrate the severe multisystem consequences of its dysfunction.

Figure 1

Segregation of RTEL1 Mutations in HHS-Affected Families and the Location of RTEL1 Alterations in the Different Protein Domains (A) Segregation of RTEL1 mutations causing HHS. Shown are seven families in which RTEL1 mutations segregate as an autosomal-recessive trait. Where available, the genotype of each individual is shown as a plus sign for the wild-type allele and as a minus sign for the mutated allele. The asterisk indicates that the mutation appears to have arisen de novo. (B) Conservation of altered RTEL1 amino acids. Blocks of amino acid alignment were generated with ClustalW and show the degree of conservation of the altered amino acid residues in RTEL1. Sequences are as follows: human, H. sapiens (RTEL1 [RefSeq NP_116575.3]); mouse, M. musculus (RTEL1 [RefSeq NP_001001882.3]); chicken, G. gallus (RTEL1 [RefSeq XP_417435.3]); and fruit fly, D. melanogaster (RTEL1 [RefSeq NP_572254.1]). Asterisks indicate positions that have a single fully conserved residue, colons indicate conservation between groups of strongly similar properties, and periods indicate conservation between groups of weakly similar properties. (C) Conserved functional domains predicted in the RTEL1 amino acid sequence show the relative positions of the alterations caused by the mutations observed in our subject group. Domains are as follows: I–VI, helicase domains; PIP, proliferating cell nuclear antigen (PCNA)-interacting protein domain; green bar, iron-sulfur domain; purple bar, DEXDc2-DEAD-like helicase superfamily domain; brown bar, DEAH box; orange bar, helicase C-terminal domain; and blue bar, helicase, superfamily 1 and 2, ATP-binding domain, DinG/Rad3-type. The hatched C terminus is from isoform uc021wge.1.

Figure 2

Functional Effects of RTEL1 Mutations in Individuals with HHS (A) Telomere lengths measured by monochrome multiplex quantitative PCR are expressed as a telomere-to-single-copy-gene (T/S) ratio (individuals with biallelic RTEL1 mutations are compared to controls). Red squares represent cases, and gray circles represent controls. (B) Mutations in RTEL1 affect T-circle accumulation. Linear terminal restriction fragments (TRFs) and Phi-29-dependant T-circles (TCs) were detected in genomic DNA by Southern blot analysis. (C) Graphical representation of the increase in the production of T-circles in cases (P1 and P2) and controls (C1–C3). A box plot of the median value from three different experiments shows the interquartile range (p = 0.0148, unpaired t test).