The second most commonly mutated gene in DC is TINF2
, which bears mutations in approximately 15% of all probands [13
]. All reported mutations are heterozygous, resulting in autosomal dominant disease. Although a family with autosomal inheritance of a TINF2
mutation has been reported [27
], these mutations are most often de novo, and result in a dramatic telomere shortening within the first generation [27
]. Moreover, compared to DC patients with other mutations, those with TINF2
mutations have shorter telomere lengths, earlier onset of disease, and are more likely to present with bone marrow failure prior to manifesting any signs of the classic triad [27
]. Not surprisingly, TINF2
mutations have been identified in some patients with the severe DC variants Revesz and Hoyeraal Hreidarsson syndromes and, thus far, are only gene mutations associated with Revesz syndrome.
Unlike the other factors implicated in DC, TIN2 is a component of the shelterin complex, where it occupies a central position, binding the double-stranded telomeric DNA binding factors, TRF1 and TRF2, and, thereby recruiting another binding partner, TPP1 (and secondarily its binding partner POT1), to regions of double-stranded telomeric DNA () [67
]. Knockdown of TIN2 in human cell lines results in telomere elongation indicating it functions as a negative regulator of telomere length [68
]. TIN2’s positive modulation of the levels of TRF1, another known negative telomere length regulator, has been proposed to underlie this regulation. Conversely, TIN2 depletion has also been shown to impair the recruitment of telomerase to telomeres, likely via its crucial role in anchoring TPP1 at telomeres [69
]. Thus, TIN2 may have both positive and negative roles in telomere length regulation. However, because TIN2 depletion is accompanied by reduction in both TRF1 and TPP1 levels [69
], TIN2-independent contributions to length regulation have not been established. TIN2 also functions in sister telomere cohesion, and secondarily in telomere break repair, via its interaction with the cohesin subunit SA1 [70
]. Lastly, two isoforms of TIN2 are expressed in human cells, including a longer isoform, known as TIN2L, which is formed by the inclusion of three C-terminal exons through alternative splicing [72
]. While both isoforms appear to bind similarly to TRF1, TRF2, and TPP1, TIN2L has been shown to a have greater association with the nuclear matrix and telomeres. Therefore, it is unlikely to perform functions identical to those of the shorter isoform, analyzed in the prior functional studies.
Understanding the mechanism(s) responsible for the rapid and severe shortening observed in patients with germline TINF2
mutations has been of great interest, but to date remains unclear. In contrast to the reduction in hTR levels that is observed with certain telomerase associated mutations (e.g., in DKC1
), hTR levels are unaffected in TIN2 mutant cells [66
]. Additionally, ectopically overexpressed TIN2 bearing the most common DC-associated mutations does not differentially impact telomerase activity levels (as measured in a semiquantitative assay); hTR, TRF1, TPP1, or TRF2 levels; the ability of TIN2 to interact with TRF1, TRF2 or TPP; or the subcellular and telomere localization of TIN2 [73
]. Instead, the DC mutations lead to a reduction in TIN2’s association with telomerase, consistent with a role for TIN2 in modulating telomerase recruitment. The extent to which this accounts for the mechanism by which TIN2 mutations result in telomere shortening in patients, however, is questioned given only a modest effect is observed in the in vitro
telomerase-association assay (at most a 40% reduction), whereas dramatic shortening is observed in patient-derived cells, which still also carry a wild type allele.
Strikingly, nearly all of the pathogenic TIN2 mutations reported to date cluster within a highly conserved 18 amino acid region from K280 to Q298 () [27
]. The function of this segment of TIN2 remains to be elucidated, but prior interaction mapping indicates it lies C-terminal to its TRF1, TRF2, TPP1, and SA1 binding domains and, therefore, is unlikely to impact significantly on TIN2’s interaction with any of these proteins [70
]. Recently, two additional nonsense mutations at amino acids 269 and 271 have been reported in patients with DC, extending the mutated region of TIN2 to include amino acids directly adjacent to the TRF1 binding domain [77
]. These truncation proteins are expressed, and the most N terminal truncation protein, encompassing the first 269 amino acids of TIN2, was found to have markedly reduced interactions with TRF1, while the most common missense mutation, TIN2p.R282H, had little effect on this interaction. This suggests that while some mutations may affect TRF1 binding to TIN2, it is unlikely that this is the underlying mechanism driving pathogenesis in all DC patients with TINF2
Figure 3 Structural organization of TIN2 with localization of disease-associated mutations. See the Telomerase Database for a periodically updated compilation of disease-associated TINF2 mutations [60, 105]. Abbreviations: AA, aplastic anemia; BD, binding domain; (more ...)
The identification of TINF2
mutations in patients with DC raises the question as to whether mutations in genes encoding other shelterin components might be present in among the 40 to 50 percent genetically uncharacterized cases. To address this question, Savage et al, analyzed the sequences of the other five shelterin genes (ACD, POT1, TERF1, TERF2
, which encode TPP1, POT1, TRF1, TRF2 and hRAP1, respectively) in a cohort of 16 patients with DC and 7 patients with very short telomeres in some or all leukocyte subsets, bone marrow failure, and features suggestive of DC, but who did meet diagnostic criteria [78
]. Although rare sequence variants were identified, none were predicted to alter coding sequence or affect splicing. Therefore, mutations in the other shelterin components, at the very least, do not appear to be a common cause of DC.