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.
Hoyeraal Hreidarsson syndrome (HH) is a clinically severe variant of the telomere biology disorder dyskeratosis congenita (DC) [1]. DC is a heterogeneous inherited bone marrow failure syndrome (IBMFS) diagnosed by the presence of the classic triad of dysplastic nails, abnormal skin pigmentation, and oral leukoplakia. However, substantial clinical heterogeneity has been observed and the phenotype may include pulmonary fibrosis, liver disease, esophageal, urethral, or lacrimal duct stenosis, developmental delay, and/or other complications. Individuals with DC are at very high risk of bone marrow failure (BMF), myelodysplastic syndrome, and cancer [2]. The clinical consequences of DC manifest at variable ages and in different patterns, even within the same family. Independent of the classic triad, lymphocyte telomere lengths less than the first percentile for age are diagnostic of DC [3]. Depending on the affected gene, DC can be inherited in X-linked recessive (XLR), autosomal dominant (AD), or autosomal recessive (AR) patterns. Germline mutations in DKC1 result in XLR inheritance, mutations in TERC, TERT, RTEL1, or TINF2 result in AD inheritance, and mutations in TERT, RTEL1, CTC1, NOP10, NHP2, or WRAP53 result in AR inheritance [4]–[7] [8]; mutations in these genes account for approximately one-half of classic DC cases. Patients with HH have many of the DC features listed above; however, severe immunodeficiency [9], non-specific enteropathy, intrauterine growth retardation (IUGR), and developmental delay may be the presenting features. In addition to features of DC, the presence of cerebellar hypoplasia is often the basis for a diagnosis of HH [1]. Patients with HH have extremely short telomeres, even when compared with other DC patients [3]. Germline mutations in DKC1 (XLR), TINF2 (AD), or TERT (AR) have been shown to cause HH. The causative mutation in HH is known in less than one-half of cases. We clinically characterized individuals with HH from two different families. The affected individuals had IUGR, immunodeficiency, enteropathy, and extremely short telomeres. In both families, we discovered homozygous recessive germline mutations in Regulator of Telomere Elongation Helicase 1 (RTEL1) and characterized the telomere defect that resulted from these mutations. While RTEL1 mutations have been previously implicated in AD and AR compound heterozygous cases of DC, HH, and DC-like cases [6], [7], this report is the first instance of a homozygous DC-causative mutation in this gene.
This study demonstrates the clinical and molecular consequences of homozygous autosomal recessive mutations in RTEL1. We identified two families with children who had HH, were of AJ ancestry, and had the same homozygous RTEL1R1264H mutations. These data provide further evidence that defects in RTEL1 function can lead to clinical phenotypes consistent with the HH variant of DC [6]. Our molecular analyses indicate that the homozygous RTEL1R1264H mutation results in short, heterogeneous telomeres. Additionally, cell lines bearing this mutation produce excess extrachromosomal T-circles, but only in the presence of functioning DNA replication machinery. RTEL1 is proposed to resolve T-circles to enable proper telomeric replication; in the absence of this activity, T-loops are inappropriately resolved as a circle when encountered by the replication machinery, resulting in a shortened telomere [18]. T-circle formation in the presence of RTEL1R1264H is SLX4-dependent, similar to T-circle formation in RTEL1-deficient cells [14]. RTEL1 also aids in suppressing inappropriate recombination throughout the genome. We have shown that the RTEL1R1264H mutation results in a modest enhancement in sensitivity to DNA damage, as well as an increase in SCE, indicating that the RTEL1R1264H mutation impairs both telomeric and non-telomeric aspects of RTEL1 function. The fact that both the probands were homozygous for the identical risk haplotype suggests that there is an ancestral haplotype that is shared by parents in both families (Figure 1A and 1B). We were able to reconstruct the haplotype based on the genotypes obtained using Sanger sequencing. This haplotype was also seen without the mutation in 14/378 (TSI/GBR/FIN) samples of EUR ethnicity in the 1000 Genomes data. Together with the occurrence of the risk haplotype in the two families with AJ ethnicity, the evidence supports the interpretation that this mutation is confined to EUR populations and is most likely an AJ founder mutation. We have not extended the 34 kb haplotype further since the number of individuals with this rare recessive disorder in our study is too small to investigate the age of the mutation based on haplotypes and population history. We and others recently reported that AD nonsense RTEL1 mutations are present in HH and that an additional missense mutation in the helicase domain further exacerbates the clinical and telomere length phenotype, while the presence of only a single missense mutation in the helicase domain resulted in a less clinically severe phenotype [6], [7]. [8] The current study provides important insight into the function of the C-terminal end of the human RTEL1 protein. RTEL1 deficiency confers embryonic lethality in mice [19], suggesting that the R1264H allele is hypomorphic. As is the case for the two families described here, hypomorphs are usually recessive; for example, AR partial loss-of-function mutations in FANCD2 cause Fanconi anemia and AR LIG4 mutations result in Ligase IV syndrome [20], [21]. Furthermore, this mutation is distal to the RTEL1 helicase domain, and is thus unlikely to directly affect enzymatic activity. Nevertheless, the phenotypic impact of RTEL1R1264H at the cellular level was pronounced. The RTEL1R1264H mutation falls within exon 34, which encodes a predicted C4C4 RING domain of RTEL1, lying downstream of a putative PIP box. Many RING domain-containing proteins are E3 ubiquitin ligases that interact with E2 ubiquitin-conjugating enzymes through their RING domains. BRCA1, MDM2, and Parkin are all examples of RING domain-containing proteins that are involved in human disease [22]. The putative RTEL1 RING domain is distant from the helicase domain, suggesting that the RTEL1R1264H mutation may affect the RING domain while leaving the helicase activity intact. Given the severity of the clinical and cellular phenotypes of this mutation, the data suggest that this domain exerts a significant influence on the biological function of RTEL1. Further analysis of this domain to define the mechanism (s) of its influence is ongoing. These findings, together with the recent report that non-coding SNPs in RTEL1 have been found to be associated with susceptibility to high-grade glioma [23]–[25], broadly implicate the RTEL1 locus in human cancer susceptibility. Given the cellular phenotypes of DC/HH and those reported here, the clinical features of DC are likely sequelae of defects in maintenance and functions of the telomere. We have demonstrated that the RTEL1R1264H mutation affects both the telomeric and non-telomeric functions of RTEL1. Individually, proteins involved in either telomere maintenance or DNA repair can result in immunodeficiency when perturbed: DC is an example of the former, and Bloom syndrome of the latter. The patients described here exhibit severe immunodeficiency, which may be the result of a mutation affecting both of these pathways. However, future studies are required to better understand this observation.
This research was approved by the Institutional Review Boards (IRB) of the National Cancer Institute and Memorial Sloan Kettering Cancer Center. All participants or their parents signed IRB-approved informed consent forms. Patient NCI-318 and her family were participants in an IRB-approved longitudinal cohort study at the National Cancer Institute (NCI) entitled “Etiologic Investigation of Cancer Susceptibility in Inherited Bone Marrow Failure Syndromes” (NCI 02-C-0052, ClinicalTrials. gov Identifier: NCT00027274). In this study, patients and their family members complete questionnaires and undergo thorough clinical evaluations at the NIH Clinical Center [2]. Telomere length was measured by flow cytometry with fluorescent in situ hybridization (flow FISH) in leukocytes [26]. THE MSKCC proband was ascertained on IRB-approved protocol 95-091 entitled “Collection of Hematopoietic Progenitor Cell and/or Blood Samples From Patients For Research Studies. ” Other family members consented to germline testing in the Clinical genetics Service, as well as MSKCC 93-102 “Ascertainment of Peripheral Blood or Saliva Samples for Genetic Epidemiology Studies of Familial Cancers, ” as well as a specific consent for the novel homologous recombination gene described in this report. Whole exome sequencing for family NCI-318 was performed at the NCI' s Cancer Genomics Research Laboratory as previously described [6]. Reads were aligned to the hg19 reference genome using Novoalign software version 2. 07. 14 (http: //www. novocraft. com), Picard software version 1. 67 (http: //picard. sourceforge. net/) and the Genome Analysis Toolkit (GATK, http: //www. broadinstitute. org/gatk/) [27]. Variant discovery, genotype calling, and annotation were performed as described [6] using data from the UCSC GoldenPath database (http: //hgdownload. cse. ucsc. edu/goldenPath/hg19/database/), the ESP6500 dataset from the Exome Variant Server, NHLBI Exome Sequencing Project (ESP), Seattle, WA (http: //evs. gs. washington. edu/EVS/) (accessed August 2012), the Institute of Systems Biology KAVIAR (Known VARiants) database (http: //db. systemsbiology. net/kaviar/) [28], the National Center for Biotechnology Information dbSNP database (http: //www. ncbi. nlm. nih. gov/projects/SNP/) [29] build 137, and the 1000 Genomes (http: //www. 1000genomes. org/) [12]. Variants were also annotated for their presence in an in-house database consisting of over 700 whole exomes that were sequenced in parallel with our DC families. Variants within each family were filtered and categorized as indicated in Table S1. Validation of exome sequencing findings in the NCI-318 trio was performed by sequencing coding exons of RTEL1. Primer sequences are shown in Table S4. All samples were amplified using KAPA2 RobustHotstart Readymix (2×) (Kapa Biosystems, Johannesburg, South Africa) and the following cycling conditions: 3 min at 95°, followed by 30 cycles of 15 sec at 95°, 15 sec at 60°, 15 sec at 72°, followed by 10 min at 72°. Amplicons were purified using Agencourt' s Ampure XP beads, then libraries were constructed and barcoded using the Ion Xpress Plus Fragment Library Kit (Life Technologies, Carlsbad, CA, USA). DNA tagged beads were generated for sequencing using Life Technologies' OneTouch and run on an Ion 316 chip on the Ion PGM Sequencer (Life Technologies). The default TMAP aligner and variant caller was used to generate a variant list per sample. Targeted resequencing of DNA damage response genes was instrumental in the discovery of the RTEL1 mutation at MSKCC. Genomic enrichment via microfluidic PCR was conducted using the primer pool from Raindance Technologies [30]. Resulting libraries were prepared for sequencing using the SOLiD 4 sequencer (Life Technologies, Carlsbad). Read alignment and base-calling was done using the ABI Bioscope software with parameters optimal for targeted resequencing. Reads were filtered for mapping quality. RTEL1 contained the most biologically relevant non-synonymous exonic variant. MSK-41 was included in a panel of 24 cell lines in which targeted DNA sequencing of approximately 300 DNA damage response genes (including RTEL1) was carried out (see methods [13]). PolyPhen-2 [31] (http: //genetics. bwh. harvard. edu/pph2), SIFT [32] (http: //sift. jcvi. org), and Condel [33] (http: //bg. upf. edu/condel/home) were used to predict the severity of RTEL1 amino acid substitutions. Multiple sequence alignments were generated for homologous RTEL1 protein sequences using T-Coffee [34] (www. tcoffee. org) to evaluate conservation. Alignments were generated with NCBI Reference Sequence, GenBank or Ensembl proteins ENSP00000353332 (Homo sapiens), NP_001124929. 1 (Pongo abelii), NP_001091044. 1 (Bos taurus), and EDL07405. 1 (Mus musculus). Telomere FISH was performed as described [35]. Images were captured at 100× magnification, with precisely the same exposure time for each genotype (MSK-41 hTERT and BJ hTERT). Sensitivity (gain) is increased to saturation, and chromosome ends for which there still appears no signal are scored as signal-free ends. The heterogeneity observed in Figure 2C was reproducible over several experiments, and with different probes (data not shown). Cells were collected from 2 to 3 10 cm plates at 70% confluence for each condition. Genomic DNA extraction was performed as described [36]. DNA was double digested by AluI/HinfI restriction enzymes overnight before starting TCA assay and then Southern Blot as described [37] with minor modifications to Phi29 DNA polymerization (MBI Fermentas) with a mammalian telomeric primer and a mammalian telomeric probe for hybridization. Blot images were captured and quantified with Storm 840 scanner and ImageQuant TL software (Amersham Biosciences). Sister chromatid exchange analysis and telomere FISH were carried out as described previously [35]. Mitomycin C sensitivity assays were as described [38]. To detect SLX4 levels in the various knockdown conditions, we immunoprecipitated SLX4 (1. 5 mg protein lysate, 10 µg of antibody) with rabbit polyclonal antibody (A302-269A) followed by western blotting with polyclonal rabbit antibody A302-270A. Both antibodies were from Bethyl. T-circles were detected and quantified as previously described [14]. Immortalized conditional RTEL1F/- MEFs were as previously described [14] and were cultured in DMEM containing 10% fetal bovine serum. Cre recombination was carried out with Ad5-CMV-Cre adenovirus (Vector Biolabs) for 96 hr as described [39]. Cells were either not treated or treated with aphidicolin (5 µM) for 24 hrs.
Patients with dyskeratosis congenita (DC), a rare inherited disease, are at very high risk of developing cancer and bone marrow failure. The clinical features of DC include nail abnormalities, skin discoloration, and white spots in the mouth. Patients with Hoyeraal-Hreidarsson syndrome (HH) have symptoms of DC plus cerebellar hypoplasia, immunodeficiency, and poor prenatal growth. DC and HH are caused by defects in telomere biology; improperly maintained telomeres are thought to be a major contributor to carcinogenesis. In half the cases of DC, the causative mutation is unknown. By studying families affected by DC for whom a causative mutation has not yet been identified, we have discovered a homozygous germline mutation in RTEL1, a telomere maintenance gene that, if mutated, can result in HH. The mutations result in the inability of the RTEL1 protein to function properly at the telomere, and underscore its important role in telomere biology.
A Recessive Founder Mutation in Regulator of Telomere Elongation Helicase 1, RTEL1, Underlies Severe Immunodeficiency and Features of Hoyeraal Hreidarsson Syndrome