Functional Consequences of TCAB1 Mutations in Dyskeratosis Congenita

Dyskeratosis congenita (DC) is a rare telomere disease with pleiotropic manifestations and bone marrow failure (BMF) as a major cause of mortality. The genes involved in DC pathogenesis play a role in telomere maintenance but their function in other in biological systems has not been well characterized. Compound heterozygous missense mutations in TCAB1(WRAP53) were reported so far in two pedigrees with classic DC (Zhong et al, 2011). TCAB1 functions as a scaffold protein recruiting TERC to the sub-organelle Cajal bodies, thus contributing to the assembly of telomerase enzyme; among its other functions it regulates DNA double-strand break repair. Here we functionally characterize novel TCAB1 mutations and describe transcriptome and proteome profiles in patient-derived lymphoblastoid cell lines (LCL). Among a cohort of 50 DC patients we identified three pedigrees with TCAB1 mutations, BMF and telomeres below first percentile. Index 1 carried compound heterozygous mutations R155X/Y345C also present in the affected brother. Both siblings suffered from transfusion-dependent pancytopenia manifesting in the childhood and typical dyskeratotic features. Index 2 harbored a single mutation Q7TfsX27 and presented with hypocellular BMF. Although non-hematologic symptoms were initially absent, the patient suffered from mild restrictive lung disease after stem cell transplantation. Index 3 carried biallelic mutations Y345C/G435R. He was diagnosed with microcephaly, cerebellar hypoplasia, early onset BMF with immunodeficiency, consistent with Hoyeraal-Hreidarsson syndrome (HHS).

To gain insight into the functional consequences of novel mutations identified, we first assessed RNA, protein levels and localization in patient-derived LCLs and HeLa cells stably expressing wildtype (WT) and mutant TCAB1. The truncating mutations R155X and Q7TfsX27 were unable to translate to protein with abrogative localization in Cajal bodies, while the R155X mutation affected RNA stability through nonsense-mediated decay. The Y345C mutant was translated, but it was severely diminished in the nucleus and demonstrated defective localization in the Cajal bodies. The decline of TCAB1 signal in Cajal bodies was also observed in patients 1&3 with biallelic mutations.

Next, to describe the effect of mutations on cellular processes we performed RNA-sequencing (RNAseq) and mass spectrometric-based quantitative proteomics using SILAC on LCLs from patients/carriers and WT controls. The principal component analysis (PCA) of RNAseq data clusters the transcriptomes of patients with missense mutations (Y345C/G435R, Y345C) and truncating mutations (R155X, Q7TfsX27) into separate groups, while index 1 (R155X/Y345C) demonstrated a very distinct transcriptomic profile (Fig 1A). The telomere maintenance processes were highly downregulated for index 1 and clinically healthy carrier parents (Fig 1B). This might suggest that heterozygous mutations also affect telomere biology; however the compensatory effect from the WT allele allows maintaining normal homeostasis. Based on disease severity and established pathogenic effect of mutations we then focused on index patients 1 and 3 carrying biallelic mutations. The differential gene expression analysis suggested RPSA, LILRB1 (downregulated) and SH3BP5, TSPYL5 (upregulated) as top candidates. Strikingly, the identification of downregulated RPSA points at dysregulated ribosomal function as a possible novel mechanism in TCAB1-mutated cells. Finally, positive correlation was observed for differential gene regulation on transcriptome and protein level. A gene set variation analysis indicated down-regulation of telomere maintenance, protein processing and ubiquitination. Surprisingly the members of the telomerase holoenzyme complex were enriched on the protein level however they were detected transcriptionally down-regulated in patients (Fig 1C). This might hint at defective protein transport or slower protein turnover due to decreased ubiquitination.

In summary, we expand the clinical spectrum of TCAB1-associated DC ranging from BMF without mucocutaneous symptoms to severe HHS. We provide additional insights into the underlying cellular effects of TCAB1 mutations. The preliminary insight into the gene networks affected by TCAB1 mutations allows for first comprehension into the pleiotropic effects observed in patients.

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