Mutations In TCAB1 Cause Dyskeratosis Congenita

Abstract 197

Dyskeratosis congenita (DC) is a multisystem disorder characterized by the diagnostic triad of nail dysplasia, abnormal skin pigmentation, and oral leukoplakia. Patients with DC are at very high risk of bone marrow failure, cancer, pulmonary fibrosis, and other complications. All patients with DC have very short telomeres; approximately 60% have a mutation in one of 6 telomere biology genes (DCK1, TERC, TERT, TINF2, NOP10 or NHP2). Telomerase (TERT) is the reverse transcriptase which, with its RNA component TERC, extends telomeric repeats to offset the telomere shortening that occurs with DNA replication. Mutations in the catalytic core component of TERT (autosomal dominant or recessive) or TERC (autosomal dominant) cause DC and related telomere biology disorders. The function of the telomerase enzyme in vivo requires additional components including dyskerin (DKC1), which is mutated in patients with X-linked recessive DC. Dyskerin binds the H/ACA sequence within TERC and is required for the biogenesis of telomerase, as well as the biogenesis and function of other RNP complexes, including small nucleolar RNAs (snoRNAs) and small Cajal body RNAs (scaRNAs), involved in the modification of ribosomal RNAs and splicing RNAs, respectively. We recently identified TCAB1 (gene names WDR79, WRAP53) as a novel telomerase component through biochemical purification of telomerase complexes. TCAB1 is a WD40-repeat containing protein that binds the CAB box sequence within TERC. It is a constituent of the active telomerase holoenzyme and inhibition of TCAB1 prevents telomerase from localizing to Cajal bodies where RNA-protein complexes are assembled and modified.

Since TCAB1 is required for telomerase trafficking, we evaluated mutations in TCAB1 as a potential cause of DC in 16 mutation-negative patients who were participants in the NCI's prospective Inherited Bone Marrow Failure Syndromes (IBMFS) study. Nine patients had classic DC, defined by the diagnostic triad, or the combination of 1 of the triad plus BMF and 2 other DC-related complications, and leukocyte telomeres <1^st percentile for age. An additional 7 DC-like patients (lacking the diagnostic triad) with BMF and telomeres <1^st percentile were also evaluated. We performed bi-directional sequence analysis of all 10 exons of TCAB1; PCR and sequence analysis were independently repeated 3 times and reviewed by individuals blinded to patient status. Two unrelated patients with classic DC and very short telomeres had different compound heterozygous mutations in TCAB1. Their parents were not consanguineous and had normal telomere lengths. Each healthy parent harbored a single heterozygous mutation in TCAB1 consistent with autosomal recessive inheritance. These mutations were not present in 380 healthy controls. Each of the four disease-associated TCAB1 alleles showed a defect in accumulation and localization within the Cajal body. Mutant TCAB1 alleles were stably expressed in HeLa cells, but showed markedly reduced accumulation in Cajal bodies by immunofluorescence compared with wild-type TCAB1. Analysis of endogenous TCAB1 expression in TCAB1-mutant, EBV-immortalized lymphoblasts similarly showed a marked impairment of TCAB1 accumulation in Cajal bodies. To model the effects of impaired TCAB1 function, we depleted TCAB1 using siRNA in HeLa cells. TCAB1 depletion prevented TERC from localizing in Cajal bodies, and instead caused TERC to accumulate in nucleoli, as judged by RNA fluorescence in situ hybridization for TERC RNA. Analysis of TERC localization in TCAB1-mutant DC patient lymphoblasts similarly showed that TERC aberrantly accumulated in nucleoli, rather than in Cajal bodies (p<0.001). The overall levels of TERT RNA were unaltered in TCAB1-mutant lymphoblasts by Northern blot. Our data show that compound heterozygosity for TCAB1 mutations leads to a marked mislocalization of telomerase and can cause classic DC. Our findings identify telomerase mislocalization as a novel cause of disease and provide a framework for investigating telomerase trafficking in DC and other disease states.