**4. Telomeres and TAPs in human diseases: Telomeropathies**

Telomeres shorten with each cell division. When telomeres become excessively short, they lose their protective role and activate a DNA damaging signal response resulting in genomic instability, cell cycle arrest, and senescence. TAPs play an essential role in maintaining telomere length, and genetic mutations affecting their activity can result in telomere dysfunc‐ tion. This manifests into a wide variety of diseases collectively named as "telomeropathies" or "telomere syndromes", which exhibit impaired telomere maintenance.

**Figure 2.** Schematic representation showing Telomere associated proteins interacting with several cell-signaling path‐ ways.

#### **4.1. Telomere-shortening syndromes**

Inherited mutations, which hamper telomerase or telomere maintenance genes, result in progressive shortening of telomeres. Telomere shortening has major impact on highly proliferating tissues, such as bone marrow, where stem cells reach senescence stage and organ failure might ensue. Clinical conditions associated with shortened telomeres may be very different. This may be partly due to genetic anticipation since telomere length is inherited [63].

#### *4.1.1. Dyskeratosis congenita*

Dyskeratosis congenita (DC) arises primarily due to bone marrow failure and is associated with a diagnostic triad of oral leukoplakia, skin hyperpigmentation, nail dystrophy, and other manifestations. Dyskerin (encoded by *DKC1*), which is an essential component of telomerase enzyme in vivo, was the first gene identified as a cause of DC, and was thus named after this syndrome. DC is a heterogeneous disease showing all modes of inheritance. To date, 11 genes have been associated with DC. These include genes encoding products involved in telomere elongating enzyme, telomerase components (TERT and *TR*), telomerase stability (dyskerin, NOP10, NHP2), telomerase recruitment (TIN2 and TPP1), telomerase trafficking (TCAB1), telomerase docking (CTC1), and telomere replication (RTEL1) [130].

Figure 3 shows schematic representation of telomere-interacting proteins with domains and positions of reported germ-line mutations, which result in various forms of DC.

**4.1. Telomere-shortening syndromes**

MITOCHONDRIA

Tert

p65 p50

Inhibition of gene expression

CYTOPLASM

ways.

Regulates Apoptosis

Oxidative Stress

RAP1 IKK2 IKK1

NF-κB pathway

56 Telomere - A Complex End of a Chromosome

Cytokine Ligand

*4.1.1. Dyskeratosis congenita*

Inherited mutations, which hamper telomerase or telomere maintenance genes, result in progressive shortening of telomeres. Telomere shortening has major impact on highly proliferating tissues, such as bone marrow, where stem cells reach senescence stage and organ failure might ensue. Clinical conditions associated with shortened telomeres may be very different. This may be partly due to genetic anticipation since telomere length is inherited [63].

**Figure 2.** Schematic representation showing Telomere associated proteins interacting with several cell-signaling path‐

WNT target genes

NUCLEOPLASM

Genomic DNA RNA polymerase I TERT

▲ transcription of MYC target genes

▲ Increase

Phosphorylation

▲MYC stability

Genomic DNA

CELL

MYC Tert

MYC

▲ribosomal RNA and thus biogenesis

Tert Brg1 β-catenin

Inhibits DNA damage signaling

TRF2 TRF1

TIN2

TPP1 POT1

Rap1

Telomeres

Tert ▲ transcription of

Tert

Cytokine Ligand

β-catenin

TIN2

Hyperproliferative signal

Regulates mitochondrial structure

β-catenin pathway

▲ transcription of NF-κB target genes

p65 p50 p65 p50

Dyskeratosis congenita (DC) arises primarily due to bone marrow failure and is associated with a diagnostic triad of oral leukoplakia, skin hyperpigmentation, nail dystrophy, and other manifestations. Dyskerin (encoded by *DKC1*), which is an essential component of telomerase enzyme in vivo, was the first gene identified as a cause of DC, and was thus named after this syndrome. DC is a heterogeneous disease showing all modes of inheritance. To date, 11 genes have been associated with DC. These include genes encoding products involved in telomere elongating enzyme, telomerase components (TERT and *TR*), telomerase stability (dyskerin,

**Figure 3.** Schematic representation showing protein structure and localization of reported mutations in telomere asso‐ ciated proteins. The information is adapted from Espinoza et al [144].

Recently poly(A)-specific ribonuclease (*PARN*) gene mutations have been reported in a subgroup of patients with DC wherein *PARN* deficiency results in reduced stability of several key TAPs (dyskerin, TRF1, RTEL1, and *TR*) and specifically leads to telomere attrition [131]. Almost all modes of inheritance have been reported in DC, which include autosomal recessive, autosomal dominant, and X-linked. Based on functional relevance of mutated proteins and their penetrance, clinically diverse variant manifestations of DC are reported.

Calado et al reported a study of five families who were suffering from liver disease (familial liver cirrhosis) in combination with hematologic and autoimmune disorders [132]. They analyzed the mutations associated with the disease and found rare loss of function mutations in *TERT* or *TR* (3.7% vs 0.8%). Hoyeraal–Hreidarsson syndrome (HHS) is associated with intrauterine growth retardation (IUGR), microcephaly, cerebellar hypoplasia, and thrombo‐ cytopenia along with various nonspecific enteropathies. HHS patients are also found to harbor DC mucocutaneous triad in adulthood. Detailed analysis revealed mutations in *DKC1*, *TIN2* along with some cases showing autosomal recessive mutations in *TERT*, *NHP2*, *NOP10*, *TPP1,* and *RTEL1* genes [133, 134]. Revesz syndrome (RS) is associated with various disease mani‐ festations mainly bilateral exudative retinopathy. Other symptoms reported include IUGR, intracranial calcifications, developmental delay, and nail dystrophy in different cases, which were highly overlapping with DC symptoms. It was discovered that RS patients have short telomeres and harbor germ-line mutations in *TINF2* gene [135]. Coats plus syndrome (CCS) is a rare recessive disorder that is characterized by intracranial calcifications, hematological abnormalities, and retinal vascular defects. CCS patients display shortened telomeres indicat‐ ing telomere dysfunction as a major cause. Missense mutations in *CTC1* gene whose protein is a part of CST complex has been reported to occur in CCS patients [136]. HHS, RS, and CCS represent severe forms of DC.

About 10% of DC patients develop cancer at a very young age. Various DC families display an increased incidence of acute myeloid leukemia and myelodysplastic syndrome [137]. Spontaneous reversion to the functional *TR* allele in hematopoietic stem cells of haploinsuffi‐ cient DC patients has been observed predisposing them to hematological disorders. The mechanism behind high cancer incidence, in spite of short telomeres that should have cancerprotecting effect, remains largely unexplained. The only proposed mechanism is genomic instability due to fusion of chromosome ends by NHEJ as has been observed in mutation carriers and in *TR-*knockout mice [138].

#### *4.1.2. Pulmonary fibrosis*

TCAB1

H376Y

NHP2

R398W  G435R 

F164L 

CTC1

RTEL1

G503R  R840W 

V871M 

H945Sfs 

R975G  R987W 

del985C 

E251K

Helicase DEAD-like Helicase Rad3-type

TRF2 and TPP1 binding

TRFH

M492I

E591D ★

A621T ✖

I699M

L710R

G739V

★ V745M

★

★

★

★

★

Q7X

Q241X 

V259M  R287X 

58 Telomere - A Complex End of a Chromosome

K242Lfs 

TINF2

P236S 

ciated proteins. The information is adapted from Espinoza et al [144].

S245Y

Q269X Q271X 

K280E K280X ★ E281K R282C 

R282H ★ P283A P283H 

CBX3 and SIAH2-bining

their penetrance, clinically diverse variant manifestations of DC are reported.

P283S ★ T284A 

T284K  L287P F288L  P289S P290Lfs 

**Figure 3.** Schematic representation showing protein structure and localization of reported mutations in telomere asso‐

Recently poly(A)-specific ribonuclease (*PARN*) gene mutations have been reported in a subgroup of patients with DC wherein *PARN* deficiency results in reduced stability of several key TAPs (dyskerin, TRF1, RTEL1, and *TR*) and specifically leads to telomere attrition [131]. Almost all modes of inheritance have been reported in DC, which include autosomal recessive, autosomal dominant, and X-linked. Based on functional relevance of mutated proteins and

Calado et al reported a study of five families who were suffering from liver disease (familial liver cirrhosis) in combination with hematologic and autoimmune disorders [132]. They analyzed the mutations associated with the disease and found rare loss of function mutations in *TERT* or *TR* (3.7% vs 0.8%). Hoyeraal–Hreidarsson syndrome (HHS) is associated with

R291G Q298Rfs 

T284Hfs 

⏏ R282S ⏏

⏏

K280Rfs ⏏

K897E

R957W

R964L

RING/U-BOX

R974X ★

R986X ★

C1244R

R1264H

★

del1196L 1202R 

OB folds P-rich WD40 G-rich

★

★

★

★

POT1

S270N

NOP10

OB folds

R34W 

H/ACA RNP

R273L

A532P

Q623H

⌘

⌘

V126M  Y139H

⌘

⌘

H/ACA RNP Ribosomal L7A

D224N

⌘

Y89C

Q94E

R137H

⌘

⌘

⌘

Idiopathic pulmonary fibrosis (IPF) disease is characterized by progressive lung scarring and fibrotic changes. The disease is associated with abnormal telomere maintenance and is an attenuated form where fibrosis develops with cumulative age-related changes. This disease arises from mutations in genes encoding *TERT* and *TR* leading to reduced telomerase activity and subsequently shorter telomeres, resulting in impaired growth of lung stem cells [139]. Surprisingly, short telomeres have been detected in IPF patients with intact telomerase genes, indicating that IPF may develop in people who have short telomere lengths [140]. This study also showed the development of liver cirrhosis in 3% of sporadic IPF patients, demonstrating a complication of telomere-mediated disease outside the lung even in the absence of telomerase mutations. Also, increased incidence of insulin-dependent diabetes is detected in IPF patients [141]. Short telomeres have been shown to cause insulin secretion defects and glucose intol‐ erance in telomerase-deficient mice [142].

#### *4.1.3. Bone marrow failure*

Many bone marrow failure disease cases have been linked to telomere biology. Mutations in telomeric proteins can lead to accelerated telomere attrition in hematopoietic compartment leading to bone marrow failure. The most common gene associated with bone marrow failure is *TERT*, which generally harbors point mutations in its gene [143, 144].

#### **4.2. Role of TAPs in cancer**

The role of TAPs in cancer development is well known. People with long telomeres are at a greater genetic risk of developing cancers [145]. Thus, examining the role of telomere proteins in cancer holds immense prognostic, diagnostic, and therapeutic value.

#### *4.2.1. Shelterin proteins and cancer*

The shelterin complex member POT1 was found to be somatically inactivated in chronic lymphocytic leukemia where it led to telomere deprotection and length extension [146]. Recently, two studies reported occurrence of rare, germ-line variants in *POT1*, making them susceptible to the development of familial melanoma [145]. In these cohorts, carrier individ‐ uals displayed significantly longer and more fragile telomeres than controls, and in some cases developed cancer in other tissues along with melanoma. Molecular and functional analysis showed that some of the variants abrogate the binding of POT1 to ssDNA, thus raising the possibility that carriers are predisposed to malignancy via telomere uncapping and a more permissive extension of chromosome ends. However, the exact biological mechanism needs further investigation. Mutation in *RAP1*, another shelterin protein member was reported in a melanoma cohort. RAP1 is involved in negative regulation of telomere length and functions by repressing homology-directed repair [147]. Mutations were report‐ ed to occur in TRF1-interacting region of RAP1. This loss of interaction with shelterin may increase the risk of cancer development.

Germ-line mutations affecting other proteins that interact with shelterin complex members and increase cancer risk have also been reported. For example, ku80, which interacts with RAP1 and PARP1, which in turn interacts with TRF2, has been found to be associated with diffuse large B-cell lymphomas.

#### *4.2.2. Telomerase and cancer*

Telomerase activity is essential for immortalization. Thus, targeting telomerase activity represents an attractive approach for both cancer diagnosis and treatment [148, 149]. As described previously, TERT is the limiting factor for telomerase activity. Therefore, its reactivation mechanisms hold great significance in understanding the development of cancer and thus designing targeted therapies.

Two hot-spot mutations in the *TERT* promoter, -228 C>T and -250 C>T, were recently reported to occur at high frequency in several solid tumors, for example: melanoma, gliomas, carcinoma of bladder, urothelial cancer, thyroid and squamous cell carcinoma of the tongue, as well as in liposarcomas and hepatocellular carcinomas, which have relatively low rates of self-renewal [85, 150–153]. It was recently shown that *TERT* promoter mutations create novel binding sites for GABP, which belongs to Ets family of transcription factors [154]. These mutations have strong clinical implications with worse prognosis and poor survival, and thus may represent a novel therapeutic target [153].

#### *TERT* promoter mutation in skin cancers

**4.2. Role of TAPs in cancer**

60 Telomere - A Complex End of a Chromosome

*4.2.1. Shelterin proteins and cancer*

increase the risk of cancer development.

diffuse large B-cell lymphomas.

and thus designing targeted therapies.

may represent a novel therapeutic target [153].

*4.2.2. Telomerase and cancer*

The role of TAPs in cancer development is well known. People with long telomeres are at a greater genetic risk of developing cancers [145]. Thus, examining the role of telomere proteins

The shelterin complex member POT1 was found to be somatically inactivated in chronic lymphocytic leukemia where it led to telomere deprotection and length extension [146]. Recently, two studies reported occurrence of rare, germ-line variants in *POT1*, making them susceptible to the development of familial melanoma [145]. In these cohorts, carrier individ‐ uals displayed significantly longer and more fragile telomeres than controls, and in some cases developed cancer in other tissues along with melanoma. Molecular and functional analysis showed that some of the variants abrogate the binding of POT1 to ssDNA, thus raising the possibility that carriers are predisposed to malignancy via telomere uncapping and a more permissive extension of chromosome ends. However, the exact biological mechanism needs further investigation. Mutation in *RAP1*, another shelterin protein member was reported in a melanoma cohort. RAP1 is involved in negative regulation of telomere length and functions by repressing homology-directed repair [147]. Mutations were report‐ ed to occur in TRF1-interacting region of RAP1. This loss of interaction with shelterin may

Germ-line mutations affecting other proteins that interact with shelterin complex members and increase cancer risk have also been reported. For example, ku80, which interacts with RAP1 and PARP1, which in turn interacts with TRF2, has been found to be associated with

Telomerase activity is essential for immortalization. Thus, targeting telomerase activity represents an attractive approach for both cancer diagnosis and treatment [148, 149]. As described previously, TERT is the limiting factor for telomerase activity. Therefore, its reactivation mechanisms hold great significance in understanding the development of cancer

Two hot-spot mutations in the *TERT* promoter, -228 C>T and -250 C>T, were recently reported to occur at high frequency in several solid tumors, for example: melanoma, gliomas, carcinoma of bladder, urothelial cancer, thyroid and squamous cell carcinoma of the tongue, as well as in liposarcomas and hepatocellular carcinomas, which have relatively low rates of self-renewal [85, 150–153]. It was recently shown that *TERT* promoter mutations create novel binding sites for GABP, which belongs to Ets family of transcription factors [154]. These mutations have strong clinical implications with worse prognosis and poor survival, and thus

in cancer holds immense prognostic, diagnostic, and therapeutic value.

Stem cells differentiate into normal somatic cells and as a consequence repress TERT tran‐ scription. Upon subsequent cell division, progressive telomere shortening occurs due to lack of telomerase activity. This acts as a barrier for tumor development and progression. Skin epidermal cells are highly differentiated cells, possess short telomeres, and are thus capable of undergoing limited proliferation [155]. However, in melanoma, increased telomerase activity is reported and this has been associated with high proliferation rate and early meta‐ stasis [156, 157].

High frequency of *TERT* promoter mutations has been reported in familial and sporadic melanoma (about 29–73%) [150, 151]. In primary cutaneous melanoma, *TERT* promoter mutations were found to be associated with BRAFV600E mutations, worse prognostic features, and shorter disease-free and overall survival [158, 159]. *TERT* promoter mutations have also been reported to be common in nonmelanoma skin cancer ranging from 39 to 74% in sporadic basal cell carcinoma and up to 50% mutation frequency in squamous cell carcinoma [158, 160, 161]. Various studies have assessed the association between telomere length and risk of developing skin cancer [162]. Some reports suggest no association between telomere length in peripheral blood leukocytes (PBL) and risk of nonmelanoma skin cancer [163]. On the contrary, other authors have reported that longer telomeres in PBL are protective for certain skin cancer types [162].

#### *TERT* promoter mutations in central nervous system (CNS) tumors

Within CNS tumors, gliomas have been shown to possess the highest frequency of *TERT* promoter mutations, while medulloblastoma and meningioma show lower frequencies [164]. Within gliomas, the percentage of cases with *TERT* promoter mutations varies depending on the histopathological type of tumor. *TERT* promoter mutations are reported in a large number of cases of glioblastoma multiforme (GBM), which is the most frequent and aggressive form of glioma, and in oligodendrogliomas, in contrast to astrocytoma and ependymoma, where only a small percentage of the tumors possess such mutations [159, 164]. Furthermore, the frequency of *TERT* promoter mutations in oligoastrocytomas, gliomas with a mixed origin, is in between that of oligodendrogliomas and astrocytomas [152]. These findings are consistent with the reported data on telomerase activity in gliomas, which is significantly higher in GBM (50–89%) and oligodendrogliomas (75–100%) than in astrocytomas (0–45%) [165–167].

Some studies also reported an association between single-nucleotide polymorphisms (SNPs) in the *TERT* gene and an increased risk of glioma development [168, 169].

#### *TERT* promoter mutations in other cancers

Telomerase role in bladder carcinoma (BC) has been reported. In majority of BC tumor samples, telomerase activity was detected, while it was absent in the respective normal parallel samples [170, 171]. In some reports, telomerase activity was associated with lower grade and lower stage BC [170, 172]. Other studies showed that both telomerase activity and telomerase expression are associated with more advanced and higher grade of cancers [171, 173]. Pre‐ liminary evidence obtained in cell lines suggests that BC might have *TERT* promoter mutations [150]. *TERT* promoter mutations are also frequently detected in BC cell lines, with a prevalence ranging from 47 to 85%. These results have clearly shown that *TERT* promoter mutations represent one of the most common genetic events, perhaps the most frequent, in BC [85].

*TERT* promoter mutations also occur at high frequency in other cancer types, for example: hepatocellular carcinoma (56%), several soft tissue tumors histotypes (e.g., 93% in atypical fibroxanthoma, 79% in myxoid liposarcoma, and 76% in pleomorphic dermal sarcoma) and carcinoma of the renal pelvis (64%). Tumor histotypes with intermediate frequencies of *TERT* promoter mutations comprise laryngeal carcinoma (27%) and clear cell carcinoma of the ovary (16%) [174]. *TERT* promoter mutations are not frequently found in leukemias and colorectal cancers [174].

The high prevalence of *TERT* promoter mutations suggests the significance of telomere maintenance in cancers. Clinically, *TERT* promoter mutations represent a potential biomarker in cancer prognosis. Furthermore, *TERT* promoter mutations also serve as an attractive therapeutic target since they occur specifically in cancer cells and are absent in surrounding healthy tissues.
