AD DC and Mutations in TERC and TERT 421 Mutations in TERC

The Autosomal-dominant form of DC is caused by mutations in the telomerase RNA component gene, TERC. Since the first report establishing the link between TERC mutations and DC (Vulliamy et al. 2001a), 26 mutations have been identified

(Table 5.3; Fig. 5.3), including two polymorphisms G58A and G228A that do not reduce telomerase activity. The secondary structure of TERC is highly conserved among different species, but the primary sequence is highly variable (Chen et al. 2000). The highly conserved domains include the pseudoknot domain containing the template region, the CR4/CR5 domain, the highly variable paired region, and the box H/ACA and CR7 domain (Chen and Greider 2004). The pseudoknot and CR4/CR5 domain, which mediate the interaction with TERT protein at bases 33-147 and 163-330, seem to be important for telomerase activity (Autexier et al. 1996, Tesmer et al. 1999, Mitchell and Collins 2000, Martin-Rivera and Blasco 2001, Chen and Greider 2003, Ly et al. 2003). The H/ACA and CR7 domains are essential for localization, maturation, dimerization, and stability of TERC molecule. It has been demonstrated that most mutations affect the telomerase activity via disrupting the structural integrity of TERC (Comolli et al. 2002, Fu and Collins 2003, Ly et al. 2003, Theimer et al. 2003, Marrone et al. 2004, Ly et al. 2005a).

The conventional method of investigating the effect of mutations has been to use the rabbit reticulocyte lysate assay or a WI-38 VA13 cell line devoid of telomerase activity to reconstitute the activity with wild-type or mutant TERC and TERT. By using this analysis, mutations located in the P2a, P2b, and P3 of the pseudoknot domain (C72G, 96-97ACT, GC107-108AG, 110-113AGACT, A79C, C116T, A117C) have been shown to abolish telomerase activity, thereby highlighting the importance of this structure in maintaining telomerase activity. The GC107-108AG mutation was proposed to affect the conformation state of the P2b and P3 pseudo-knot stem structure. In normal conditions, there is an equilibrium between two conformational states: formation of P2b and P3 stems (conformation 1), and disassociation of stem P3 and extension of stem P2b (conformation 2) (Theimer et al. 2003). The mutation GC107-108AG was observed to hyperstablize the conformation 2 state and repress the switch between the two states, thereby leading to a decrease in telomerase activity (Antal et al. 2002, Comolli et al. 2000, Ly et al. 2003, Theimer et al. 2003).

There are two mutations found in the P1a stem: C204G on the upper strand and A28-34 on the lower strand. While A28-34 was not shown to affect the telomerase activity both in vitro and in vivo by TRAP assay (Xin et al. 2006), abolishment of activity was observed in the C204G mutation. It was speculated that sequences near the 5'end of human TERC are not functionally important for telomerase activity, which is in accordance with the observation that this portion of the sequence is absent from the TERC of mouse (Chen et al. 2000, Ly et al. 2003). The two nucleotide changes A48G and A52-55 occurring in the template region both exhibited strong inhibition of telomerase activity (Xin et al. 2006), which suggests that the correct sequence in the template region is essential for the function of telomerase. Mutation analysis has shown that the P1b stem close to the template is important for the definition of the template boundary and precise copying of the telomeric sequence (Chen and Greider 2003, Moriarty et al. 2005). Disruption of this paired structure led to abrogation of telomerase activity (Ly et al. 2003).

As mentioned earlier, the CR4-CR5 domain provides one of the binding sites for TERT. In addition, recent studies have identified another important region in the

Clinical

Short

Telomerase

Sequence variant

Domain

Diagnosis

Family History

telomere

activity (%)

References

A48G

template

DC

Normal

+++

-

(Vulliamy et al. 2006)

A52-55CTAA

template

DC

Normal

+++

-

(Vulliamy et al. 2006)

A79C

Pseudoknot P2a

AA

AA

+++

-

(Vulliamy et al. 2006)

A28-34

Pseudoknot Pla

AA

Thrombocytopenic purpura

+++

100

(Xin et al. 2006)

G58A

Near template

AA or healthy

Normal(4%)

100

(Vulliamy et al. 2002; Ly et al. 2003; Wilson et al. 2003)

A378-747

H/ACA

DC

DC

+++

0

(Vulliamy et al. 2001a)

A-2664-316

5TJTR and 5'TERC

AA

MDS/AML

+++

0

(Vulliamy et al. 2004)

A96.97CT

Pseudoknot P2b

DC

DC

+++

0

(Vulliamy et al. 2004)

A110-113GACT

Pseudoknot P3

AA

Normal

+++

0

(Vulliamy et al. 2002)

A389.390CC

CR7 P7b

ET

AA

NA

0

(Ly et al. 2005a)

C-99G

5'UTR

PNH

NA

NA

NA

(Keith et al. 2004)

C-21T

5'UTR

Normal

NA

NA

NA

(Yamaguchi et al. 2003)

C72G

Pseudoknot P2a. 1

AA

Normal

+++

5

(Vulliamy et al. 2002)

107-108GC->AG

Pseudoknot P3

DC

DC

+++

5

(Vulliamy et al. 2001a)

C116T

Pseudoknot P2b

AA

AA

+++

0

(Fogarty et al. 2003), Ortmann

A117C

Pseudoknot P2b

AA

NA

+++

0

(Ly et al. 2005a)

G143A

Pseudoknot P2a. 1

AA/MDS

AA/MDS

+++

NA

(Vulliamy et al. 2004)

C204G

Pseudoknot Pla

AA

AA

+++

0

(Fogarty et al. 2003)

G228A

CR4-CR5 P4.1

AA

Normal (2%)

-

100

(Bryan et al. 1997; Wilson et al. 2003)

G305A

CR4-CR5 P6.1

AA

AA

+++

5

(Yamaguchi et al. 2003)

G322A

CR4-CR5 P5

MDS

NA

NA

5

(Yamaguchi et al. 2003)

C408G

CR7 P8b

DC

DC

+++

25

(Vulliamy et al. 2001a)

G450A

H/ACA

AA

Normal

-

100

(Fogarty et al. 2003)

T+16C

3'-UTR

AA

Normal

NA

NA

(Yamaguchi et al. 2003)

G+63A

3'-UTR

Normal

Normal (50%)

-

100

(Bryan et al. 1997)

A37G and A216-229

Pseudoknof Plb and

DC

Normal (30%)

+++

0

(Ly et al. 2005b)

hyperviriable region hyperviriable region

The reconstituted telomerase activities for the TERC mutants were from Cornolli et al, 2002; Fu and Collins 2003; Ly et al. 2003; Theimer et al. 2003; Marrone et al 2004; Ly et al. 2005a; Xin et al. 2006. +++, short telomere; -, normal telomere.

Fig. 5.3 Secondary structure of human TERC and nucleotide alterations. The mature human TERC consists of 451 nucleotides. The highly conserved secondary structure that is composed of four domains is shown: the pseudoknot domain, the CR4-CR5 domain, the hypervariable region, and the CR7 and H/ACA domain. Twenty-two nucleotide alterations within this molecule are

Fig. 5.3 Secondary structure of human TERC and nucleotide alterations. The mature human TERC consists of 451 nucleotides. The highly conserved secondary structure that is composed of four domains is shown: the pseudoknot domain, the CR4-CR5 domain, the hypervariable region, and the CR7 and H/ACA domain. Twenty-two nucleotide alterations within this molecule are

CR4-CR5 domain, the junction P6.1 between P5 and P6 that is important for RNA-RNA interaction within the telomerase complex at the template region in vitro (Ueda and Roberts 2004). Among the three mutations identified in the CR4-CR5 domain, G228A was shown to fully reconstitute telomerase activity, and appears to be a polymorphism (Bryan et al. 1997, Wilson et al. 2003). The effects of two other mutations (G305A, G322A) were not clear.

The H/ACA and CR7 domain has been predicted to be involved in localization, 3'-end processing and stability of TERC. The mutation C408G and a large deletion that removes 74 bases at the 3' end of TERC were both shown to result in reduced telomerase activity in vivo, though they were able to reconstitute activity comparable to WT in vitro (Fu and Collins 2003, Marrone et al. 2004). The impairment of telomerase activity was speculated to be induced by diminished accumulation of TERC rather than a direct influence on the catalytic activity of telomerase (Theimer et al. 2003). The CR7 domain also contains a region called J7b-8a loop (the junction between P7b and 8a), which may play a role in RNA-RNA interaction (Ren et al. 2003). Along with what has been mentioned above, three regions-the template region in pseudoknot domain, the P6.1 stem-loop structure in CR4-CR5 domain, and the J7b-8a loop in the CR7 domain-are important sites for RNA binding in the telomerase complex. This has brought about a new model for the telomerase complex, which may contain at least two molecules of TERC and two molecules of TERT (Keppler and Jarstfer 2004).

While most of the mutations in DC are heterozygous (i.e., only one allele is affected), a rare case was also reported where a patient had a A37G mutation in one of the alleles and A216-229 in the other (Ly et al. 2005b). The nucleotide substitution A37G is located in the lower strand of P1b stem, and the deletion from 216 to 229 occurs at the highly variable region which removes part of the P4.1 stem and the junction between P4.1 and P4. The A37G mutation was not seen to result in a detectable reduction of telomerase activity, although it has been inferred as previously mentioned that disruption of the P1b region can markedly affect telomerase function. A possible explanation might be that the base change is not sufficient to disrupt the P1b helix. The 14-base deletion in the highly variable region, on the other hand, led to a pronouncedly diminished enzymatic activity, suggesting that the highly variable region may also be important for telomerase function.

Two mechanisms, a dominant negative effect, or haploinsufficiency, could explain the phenotype of a disease with dominant inheritance. In the dominant negative effect, the mutated gene product is thought to interact with the normal gene product in a multiprotein complex, thereby inhibiting its function. The reduction in activity is thus expected to be more than 50% of normal. Haploinsufficiency, on the other hand implies that 50% of gene product is not enough to maintain normal function.

Fig. 5.3 (continued) labeled. The two polymorphisms G58A and G228A are marked in blue, and other nucleotide alterations are in red. The majority of the nucleotide alterations are located in the pseudoknot domain. While most alterations lead to a reduction of telomerase activity, G450A and A28-34 showed normal telomerase activity in both in vitro and in vivo assay (See Color Plate)

Dominant negative effects have been observed to be induced by TERC mutations in experimental models (Martin-Rivera and Blasco 2001). However, except for two mutations (A52-55, A48G) located in the template region (Xin et al. 2006), the majority of mutations identified in DC patients affect telomerase activity via haploin-sufficiency. Therefore, it is plausible that 50% or more of the normal expression level of TERC is not sufficient for its normal function, i.e., telomere maintenance. Indeed, with the exception of the one patient who carries mutations in both alleles (above) all the patients are heterozygous for the TERC mutation. A phenotype mediated by a dominant negative effect may be presented earlier and be more severe than that induced by haploinsufficiency (Xin et al. 2006)

The clinical manifestation of patients with the autosomal-dominant form of DC is generally less severe than the X-linked form. Some TERC mutation carriers only present mild signs of bone marrow failure and lack mucocutaneous features. In the absence of the diagnostic triad, diagnosis of DC is difficult. Therefore, it is worthwhile to analyze the TERC gene in patients with either familial or acquired aplastic anemia. Based on this hypothesis, TERC gene mutations have been identified in patients diagnosed as AA (Vulliamy et al. 2002, Fogarty et al. 2003, Yamaguchi et al. 2000, Ly et al. 2005b, Ortmann et al. 2006). In addition, mutations in TERC have also been found in patients with an initial diagnosis of myelodysplastic syndrome (MDS), acute myelogenous leukemia arising from MDS (Yamaguchi et al. 2003, Vulliamy et al. 2004), PNH (Keith et al. 2004), and thrombocythemia (Ly et al. 2005a). The identification of TERC mutations in these patients suggests that the defects in telomere maintenance should be responsible for their symptoms and they should be classified as DC. DC, on the other hand, may have a wider clinical spectrum than has been realized so far.

4.2.2 Anticipation

Clinical observation of families with the autosomal-dominant form of DC showed that patients exhibited a phenomenon called disease anticipation, which means disease was presented with early onset and more severity in successive generations (Vulliamy et al. 2004, Armanios et al. 2005). A possible explanation for disease anticipation might be that both a TERC gene mutation and short telomeres are required to be inherited from the parents for the development of clinical manifestations (Marrone and Mason 2003). There have been several pedigrees reported in which the parents of autosomal DC patients did not display clinical features although they carried a TERC mutation (Vulliamy et al. 2004, Vulliamy et al. 2006), and siblings who did not inherit the mutation did not develop the disease either, although they inherited short telomeres (Shay and Wright 2004). A study on 32 members of a three-generation family in which a partial deletion of TERC is segregating demonstrated that in gene deletion carriers, telomeres of paternal and maternal origin are similarly short despite the unaffected parent having normal telomere length. The children of affected parents who had a normal TERC gene again showed similar telomere length between paternal and maternal chromosomes.

Short telomeres could be restored after several generations in offspring that have normal genotype (Vulliamy et al. 2001a). These observations are in accordance with the hypothesis that telomerase preferentially acts on the shortest telomeres (Hemann et al. 2001). Progressive telomere shortening in successive generations anticipation has also been seen in mice carrying a null mutation of telomerase (Rudolph et al. 1999, Herrera et al. 1999). When mice with long and short telomeres were crossed, it was shown that the offspring inherited both long and short telomeres, and telomerase preferentially elongated the short telomeres. Therefore, several generations may be required for the development of the disease caused by haploinsufficiency for telomerase activity (Hemann et al. 2001). Still, it remains to be determined whether asymptomatic individuals with short telomeres are prone to present clinical manifestations under situations of replicative stress.

4.2.3 Mutations in TERT

As the catalytic component of telomerase, TERT has also been implicated in DC or other bone marrow failure syndromes (Table 5.4; Fig. 5.4). Some of the missense mutations were demonstrated to induce short telomeres and decreased telomerase activity. However, due to the much larger size of the TERT gene that is composed of 16 exons, more mutations resulting in defective telomere maintenance should be observed if TERC and TERT have comparable effect. The fact that some mutations in TERT did not produce a phenotype raises the question whether haploinsufficiency of TERT has the same effect on telomere length as haploinsufficiency for TERC.

Human TERT protein (reviewed by (Autexier and Lue 2006) contains a universally conserved central RT-like domain composed of seven RT motifs termed 1, 2, A, B', C, D, E, a large N-terminal extension (NTE) of about 400 amino acid, and a small C terminal extension (CTE) of about 200 amino acid (Cristofari and Lingner 2003, Lue et al. 2003) (Fig. 5.4B). Distinct from viral RTs, the central RT- like domain of the TERT protein has an insertion (insertion in fingers domain, IFD) between motif A and B'. The central RT-like domain provides the essential catalytic function of reverse transcription, consistent with the conserved sequence with RTs. The major difference between TERT and other RTs is its property of specific binding with telomerase RNA and repetitive addition of telomeric sequence onto the same DNA substrate (repeat addition processivity). The extra RT domains contribute to the catalytic activity as well as the assembly of the telomerase complex. A linker domain separates the N-terminal end named as GQ or RNA interaction domain 1 (RID1) from the rest of NTE. The RID1 domain is the most variable region of the protein among different species in terms of sequence and size. Extensive muta-genesis studies have suggested an important role of this domain in DNA interaction. The CTE domain was observed to play a role in localization and processivity of telomerase.

Thirteen missense mutations have been found in the TERT gene. Four amino acid changes (A202T, A279T, H412Y, and K570N) were located in the NTE domain.

Table 5.4 Mutations in TERT

Amino

Acid

Clinical

Telomere

Telomerase

Mutation

Exon

Change

Presentation

Length

Activity

Refs

G604A

2

A202T

Moderate-severe AA

4.7(6.7)

Yamaguchi et al. 2005; Vulliamy et al. 2005

G835A

2

A279T

AA or healthy

NA

+++

(Vulliamy et al. 2005; Yamaguchi et al. 2005; Vulliamy et al. 2006)

C1234T

2

H412Y

Moderate-severe AA

5.8(6.6)

+++

Yamaguchi et al. 2005

G1710C/T

3

K570N

Severe AA

3.1(7.5)

+/-

(Xin et al. 2006)

G1945A

5

G682D

AA

NA

+/-

(Liang et al. 2006)

G2080A

5

V694M

Moderate AA

-4.7(7)

-

Yamaguchi et al. 2005

C2162G

6

P721R

DC

NA

+++

(Vulliamy et al. 2006)

C2177T

6

T726M

Severe or no symptom

-4(14), -13 (12.5)

+++

(Liang et al. 2006)

A2315G

7

Y772C

Moderate AA

-3.2(6.2)

-

Yamaguchi et al. 2005

G2706C/T

11

K902N

DC

135 (506)*

+/-

Armanios et al. 2005

C2935T

12

R979W

DC

-15.5(16.5)

+++

(Vulliamy et al. 2005)

G3268A

15

V1090M

Severe AA

-5.7(6)

-

Yamaguchi et al. 2005

T3379C

16

F1127L

DC

-14(16)

+++

(Vulliamy et al. 2005; Vulliamy et al. 2006)

Numbers in parenthesis denote the telomere length of controls. -, undetectable; +/-, < 1% telom-erase activity of WT; +, 1-2%; ++, 2-20%; +++, 20-100%. \*, The reconstitutions of telomerase activity with mutant TERT are from Yamaguchi et al. 2005; Xin et al. 2006. Telomere lengths and telomerase activity were measured in peripheral blood cells.

Numbers in parenthesis denote the telomere length of controls. -, undetectable; +/-, < 1% telom-erase activity of WT; +, 1-2%; ++, 2-20%; +++, 20-100%. \*, The reconstitutions of telomerase activity with mutant TERT are from Yamaguchi et al. 2005; Xin et al. 2006. Telomere lengths and telomerase activity were measured in peripheral blood cells.

A202T and K570 N were shown to result in a dramatic reduction of telomerase activity to <1% of the wild type in the in vivo telomerase activity reconstitution assay (Yamaguchi et al. 2005, Xin et al. 2006), whereas H412Y induced a decrease of telomerase activity of only 50% (Yamaguchi et al. 2005). A279T did not appear to affect the telomerase activity (Xin et al. 2006). Both A202T and A279T are located in the linker domain within the NTE, which is not essential for the enzymatic activity (Armbruster et al. 2001). The abrogation of activity incurred by the K570N mutation in the T (telomerase-specific) domain is likely to be induced by disrupted protein-RNA binding. Mutations in the T, CP, or CP2 motifs in Tetrahymena thermophila, all of which belong to the RNA binding domain, have also been shown to eliminate the interaction between TERT and TERC (Bryan et al. 2000, Lai et al. 2002). There are six nonsynonymous mutations identified in the RT domain, with three in the highly conserved motifs and three in the nonconserved linker motifs. The residue changes G682D, V694M,

Fig. 5.4 Nonsynonymous amino acid substitutions identified in TERT. (A) Linear representation of the TERT transcript composed of 16 exons. Each box represents an exon. The location of nonsynonymous amino acid substitutions are indicated. (B) Structure of the TERT Protein represented with functional domains. The TERT protein includes the RT motif (1,2, A, B', C,D,E); the telomerase-specific motif (T); and N- and C- terminal extension (NTE, CTE). A linker domain separates the NTE; and IFD (insertion in fingers domain) separates A and B'. The functionally defined domains comprise I-A, DAT (disassociated activities of telomerase), I-B, II, III, RID (RNA interaction domain), NRS (nuclear export signal) (See Color Plate)

Fig. 5.4 Nonsynonymous amino acid substitutions identified in TERT. (A) Linear representation of the TERT transcript composed of 16 exons. Each box represents an exon. The location of nonsynonymous amino acid substitutions are indicated. (B) Structure of the TERT Protein represented with functional domains. The TERT protein includes the RT motif (1,2, A, B', C,D,E); the telomerase-specific motif (T); and N- and C- terminal extension (NTE, CTE). A linker domain separates the NTE; and IFD (insertion in fingers domain) separates A and B'. The functionally defined domains comprise I-A, DAT (disassociated activities of telomerase), I-B, II, III, RID (RNA interaction domain), NRS (nuclear export signal) (See Color Plate)

and Y772C in the linker region, and K902N in the conserved D motif, exhibited strong inhibition of telomerase activity. However, two variants in the motif A of the RT domain presented telomerase activity comparable to the WT level (Armanios et al. 2005, Xin et al. 2006, Xin 2006). Extensive studies on viral RT, yeast, and Tetrahymena have revealed that mutations in three conserved motifs (1, 2, and E) led to impairment of nucleotide addition processivity by affecting substrate binding (Miller et al. 2000, Bosoy and Lue 2001, Peng et al. 2001). There is no evidence on the precise role of other motifs of the RT domain, including the linker region in the enzymatic function or structure of the telomerase complex. As mentioned before, CTE contributes to promoting nucleotide addition processivity. This has been demonstrated by multiple mutation analysis in both yeast and human (Hossain et al. 2002, Huard et al. 2003). The CTE domain was also postulated to form a "thumb domain" of telomerase, thereby functioning in the repeat addition processivity. An additional role of this domain is implicated in the regulation of localization of TERT to telomeres. The N-terminus and C-terminus of telomerase both contain a DAT domain (dissociates activities of telomerase) that is important for recruiting regulatory proteins or associating with telomeres. Mutations in this region resulted in disassociation of TERT from telomeres, but did not influence telomerase activity (Counter et al. 1998, Friedman and Cech 1999, Xia et al. 2000, Armbruster et al. 2001, Banik et al. 2002). In accordance with these findings, the two mutations found in the CTE domain among patients with DC or AA, R979W, and F1127L were not shown to impact on telomerase activity (Xin et al. 2006). Another missense mutation V1090M, however, drastically reduced telomerase activity to less than 1% (Xin et al. 2006).

Investigations of TERT gene mutations in DC or AA patients suggest that mutations in TERT may be different in penetrance and in the range of phenotypes produced compared with mutations in TERC. Among the mutations identified, only one family, with members heterozygous for the K902N mutation, displays autosomal-dominant inheritance, telomere shortening, and disease anticipation (Armanios et al. 2005). In a family with an A202T mutation, although all the family members with one mutated allele showed shortened telomere length in leukocytes, only the proband presented with clinical manifestation of a hematological disorder (Yamaguchi et al. 2005). There are also mutations (e.g., F1127L) that do not segregate with disease. Interestingly, in several pedigrees inherited idiopathic pulmonary fibrosis has been found to be associated with TERT mutations. (Armanios et al. 2005, Armanios et al, 2007, Tsakiri et al, 2007).

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