A recessive founder mutation in regulator of telomere elongation helicase 1, RTEL1, underlies severe immunodeficiency and features of Hoyeraal Hreidarsson syndrome

Abstract

Dyskeratosis congenita (DC) is a heterogeneous inherited bone marrow failure and cancer predisposition syndrome in which germline mutations in telomere biology genes account for approximately one-half of known families. Hoyeraal Hreidarsson syndrome (HH) is a clinically severe variant of DC in which patients also have cerebellar hypoplasia and may present with severe immunodeficiency and enteropathy. We discovered a germline autosomal recessive mutation in RTEL1, a helicase with critical telomeric functions, in two unrelated families of Ashkenazi Jewish (AJ) ancestry. The affected individuals in these families are homozygous for the same mutation, R1264H, which affects three isoforms of RTEL1. Each parent was a heterozygous carrier of one mutant allele. Patient-derived cell lines revealed evidence of telomere dysfunction, including significantly decreased telomere length, telomere length heterogeneity, and the presence of extra-chromosomal circular telomeric DNA. In addition, RTEL1 mutant cells exhibited enhanced sensitivity to the interstrand cross-linking agent mitomycin C. The molecular data and the patterns of inheritance are consistent with a hypomorphic mutation in RTEL1 as the underlying basis of the clinical and cellular phenotypes. This study further implicates RTEL1 in the etiology of DC/HH and immunodeficiency, and identifies the first known homozygous autosomal recessive disease-associated mutation in RTEL1.

Figure 1. NCI-318 and MSK-41 pedigrees with RTEL1 mutation and shared risk haplotype.

NCI-318 (A) and MSK-41 (B) pedigrees are shown. Red symbols indicate affected individuals. The pink rectangles indicate the shared haplotype between the pedigrees. Each other colored rectangle indicates a unique haplotype.

Figure 2. Telomere length is altered in individuals with RTEL1^R1264H.

(A) Primary lymphocyte telomeres in family NCI-318 were measured by flow cytometry with fluorescent in situ hybridization (FISH) . The proband is indicated by a triangle, the mother by a circle, and the father by a square. (B) Telomere FISH analysis of MSK-41 hTERT-immortalized fibroblasts revealed extreme telomere length heterogeneity. Quantitation of chromatids lacking detectable telomeric signal is shown. BJ hTERT, a normal hTERT-immortalized fibroblast line, and SaOS-2, an osteosarcoma cell line that relies on recombination-based telomere maintenance (ALT), are presented for comparison. (C) Representative metaphase spreads of MSK-41 and BJ hTERT are shown.

Figure 3. RTEL1^R1264H affects a putative conserved C4C4 domain.

As displayed on the schematic (representing ENSP00000353332), the RTEL1 mutation is at the C-terminus of the protein, distal to the helicase domain. The affected amino acid is in a putative C4C4 domain. All eight key cysteines and R1264 are conserved in human, orangutan, cattle, and mouse sequences. Higher percent identity at a given amino acid position is indicated by a deeper purple color.

Figure 4. Inhibiting DNA replication blocks T-circle formation in MSK-41 RTEL1^R1264H cells.

(A) Phi29-dependent T-circles in BJ hTERT and MSK-41. (B) Phi29-dependent T-circles in RTEL1 floxed/- MEFs ± Cre, BJ hTERT and MSK-41. (C) Phi29-dependent T-circles in BJ hTERT and MSK-41 ± aphidicolin (APD; 5 µM). (D) Dot blot of the Phi29-dependent T-circles in BJ hTERT and MSK-41 ± aphidicolin (APD; 5 µM). (E) Quantification of the fold increase in intensity of Phi29-dependent T-circles in the different cell lines subjected to the indicated treatments. Intensity mean and standard deviation were calculated over two independent experiments; statistical analysis (one-way ANOVA) was calculated with Prism (GraphPad).

Figure 5. T-circle formation in MSK-41 cells is dependent on SLX4.

(A) Two shRNAs (SLX4-1 and SLX4-2) were used to knockdown SLX4 expression. (B) T-circle formation was measured in the MSK-41 SLX4 knockdown strains relative to MSK-41 with a control shRNA. Mean and standard deviation were calculated from two independent experiments. *P<0.05, **P<0.01 by unpaired two-tailed t-test.

Figure 6. MSK-41 cells are hypersensitive to DNA damage and experience elevated levels of sister chromatid exchange.

(A) BJ hTERT (blue line) and MSK-41 cells (red line) were treated at the indicated doses of mitomycin C (MMC) for 24 hours, and colony formation was scored 14 days post-treatment. Formation of at least 50 colonies was required at each dose for the experiment to be considered valid. (B) Spontaneous (blue) and MMC-induced (red) sister chromatid exchanges were visualized by Giemsa staining; the number of exchanges per metaphase is shown. Cells were cultured in 20 µM BrdU for 40 hours, with treatment with 25 ng/mL MMC for the final 24 hours.