Xlinked DC and Mutations in DKC1

The DKC1 gene located on Xq28 that encodes dyskerin is responsible for the X-linked recessive form of the disorder (Heiss et al. 1998). Dyskerin is a component of a small nucleolar ribonucleoprotein (snoRNP) that is highly conserved throughout evolution. The orthologs in yeast, fly, and rat are Cbf5p (Cadwell et al. 1997), N60B/minifly (Phillips et al. 1998), and NAP57 (Meier and Blobel 1994), respectively. The ubiquitous expression of dyskerin in all tissues of the body suggests a vital housekeeping function (Meier 2005), which correlates with the multisystem phenotype of the disease. Dyskerin, together with three other proteins, GAR1, NOPIO, and NHP2 (homologous to Garlp, Nhp2p, and Nop10p, respectively, in yeast), bind to small nucleolar RNAs (snoRNAs) containing the box H and box ACA motif (Henras et al. 1998, Lafontaine et al. 1998, Henras et al. 2004, Wang and Meier 2004) (Fig. 5.1A). The snoRNAs guide the snoRNP complex to newly synthesized ribosomal RNA (rRNA) via specific residue base pairing. Subsequently, the rRNA is modified by pseudouridylation, a process that transforms uridine to pseudouridine to stabilize RNA helices and aid in the formation of protein binding sites (Newby and Greenbaum 2002). Pseudouridylation is essential for biogenesis, maturation, and assembly of ribosomes (Tollervey and Kiss 1997, Filipowicz and Pogacic 2002). It is likely that dyskerin is the active human pseudouridine synthase that catalyzes the isomerization of uridine to pseu-douridine, based on the structural similarity with pseudouridine synthases in yeast and bacteria (Cadwell et al. 1997).

In addition to its involvement in RNA processing, dyskerin has been shown to be associated with TERC, which also contains H/ACA consensus sequence (Mitchell et al. 1999a, Mitchell et al. 1999b) (Fig. 5.1B). The interaction between dyskerin and the telomerase complex was demonstrated by coimmunoprecipitation of dyskerin with TERC in a telomerase-positive cell line (Pogacic et al. 2000). SnoRNA was also immunoprecipitated with dyskerin, suggesting the structural association among dyskerin, telomerase, and snoRNAs.

Fig. 5.1 Dyskerin is an important component of both the H/ACA small nucleolar (snoRNA) ribonucleoprotein (RNP) particles and telomerase complex. (A) Dyskerin, together with GAR1, NHP2, NOPIO, and H/ACA snoRNA, comprises snoRNP particles. The H/ACA RNA-protein complex catalyzes pseudouridylation and processing of ribosomal (r) RNA. (B) TERC (telomerase RNA component) and TERT (telomerase reverse transcriptase) are the core components of telomerase complex. As an H/ACA snoRNA, TERC associates with dyskerin, GAR1, NHP2, and NOPIO, which are also present in the telomerase complex (See Color Plate)

Fig. 5.1 Dyskerin is an important component of both the H/ACA small nucleolar (snoRNA) ribonucleoprotein (RNP) particles and telomerase complex. (A) Dyskerin, together with GAR1, NHP2, NOPIO, and H/ACA snoRNA, comprises snoRNP particles. The H/ACA RNA-protein complex catalyzes pseudouridylation and processing of ribosomal (r) RNA. (B) TERC (telomerase RNA component) and TERT (telomerase reverse transcriptase) are the core components of telomerase complex. As an H/ACA snoRNA, TERC associates with dyskerin, GAR1, NHP2, and NOPIO, which are also present in the telomerase complex (See Color Plate)

DC was hypothesized to be a disease resulting from defective telomere maintenance based on the discovery of interaction between dyskerin protein and TERC, and clinical observation of abnormally short telomeres and reduced level of TERC in DC individuals (Mitchell et al. 1999b, Vulliamy et al. 2001a). On the other hand, significant defects in pseudouridylation or processing of 18S or 28S rRNA were not detected (Mitchell et al. 1999b). Telomerase activity in fibroblasts from DC-affected male cells is lower than in cells from DC female carriers when transfected with TERT; and the diminished telomerase activity in DC patients could be recovered by expressing additional TERC (Wong and Collins 2006). A recent study showed that co-expression of TERT and TERC in dermal fibroblasts from DC patients carrying AL37 dyskerin mutation or A386T mutation could gain and stabilize telomere length, whereas telomere shortening was not inhibited by expressing TERT alone (Wong and Collins 2006). These findings indicate that dyskerin plays a role in telomere maintenance by stabilizing TERC.

However, the observation of the function of dyskerin in mouse models suggested that the involvement in ribosome biosynthesis cannot be ruled out. When two human mutations in the DKC1 gene were introduced into murine embryonic stem cells (A353V and G402E), defects in pseudouridylation and pre-rRNA processing were observed in cell lines with each mutation. On the other hand, only mutation A353V induced destabilization of TERC and reduced telomerase activity (Mochizuki et al. 2004). Furthermore, mice carrying a hypomorphic Dkc1 mutation display some features of DC before the onset of significant telomere shortening (Ruggero et al. 2003). The Dkc1 mutant was shown to lead to a defect in IRES (internal ribosome entry site)-dependent translation, which resulted in impaired translation of mRNA containing IRES elements (Yoon et al. 2006). Therefore the relative contribution of ribosome biogenesis and telomere maintenance to the phe-notype of DC is still controversial, at least in the mouse model. The importance of the two mechanisms might be dependent on the site of mutation within dyskerin.

Forty-eight mutations, including five polymorphisms, have been identified in the DKC1 gene to date (Marrone and Dokal 2004, Kanegane et al. 2005, Mason et al. 2005) (Table 5.2; Fig. 5.2). The majority of the mutations (38) (Fig. 5.2A) are missense mutations resulting in single amino acid substitutions. One large deletion removes the last exon of the DKC1 gene transcript; one mutation is located in the promoter region, and three in noncoding regions. The absence of frameshift mutations or nonsense mutations indicates that null mutations might be lethal to organisms as demonstrated by the observation that disruption of Dkc1 caused embryonic lethality in mice (He et al. 2002), which could be due to defects in ribosomal RNA processing. So far, the functional consequences of the mutations are far from clear.

The recent elucidation of the crystal structure of the dyskerin-N0P10-GAR1 complex facilitates the identification of the domains in dyskerin where most mutations cluster (Li and Ye 2006; Rashid et al. 2006). Based on information from orthologs in other species, it was inferred that dyskerin has several functional domains: nuclear localization signals, a pseudouridine synthase domain (TruB/ PUS), a pseudouridine synthase and archaeosine-spicific transglycosylase domain (PUA), and lysine-rich carboxy domains (Meier and Blobel 1994, Ni et al. 1997,

Table 5.2 Mutations in DKC1

Amino Acid

Clinical

Mutation

Exon

Change

Manifestation

Refs

-141C->G

Promotor

NA

DC

(Knight et al. 2001)

C5T

1

A2V

DC

(Knight et al. 1999b; Safa et al. 2001)

IVS1 592C->G

IVS1

-

DC

(Knight et al. 2001)

C29T

2

P10L

DC/HH

(Vulliamy et al. 2006)

IVS2 473C->G

IVS2

-

DC

(Knight et al. 1999b)

C91G

3

Q31E

DC

(Wong et al. 2004)

C91A

3

Q31K

DC

(Kanegane et al. 2005)

T106G

3

F36V

DC

(Heiss et al. 1998)

109-111 DCTT

3

D37L

DC

(Heiss et al. 1998; Mitchell et al. 1999b)

T113C

3

I38T

HH

(Cossu et al. 2002)

A115G

3

K39E

DC

(Knight et al. 1999b)

C119G

3

P40R

DC

(Heiss et al. 1998)

G121A

3

E41K

HH

(Knight et al. 1999b)

A127G

3

K43E

DC

(Heiss et al. 2001)

C146T

3

T49M

HH

(Knight et al. 1999b; Heiss et al. 2001; Sznajer et al. 2003)

G194C

4

R65T

DC

(Knight et al. 1999b)

A196G

4

T66A

DC

(Hassock et al. 1999; Knight et al. 1999b; Mitchell et al. 1999b)

C200T

4

T67I

DC/HH

(Vulliamy et al. 2006)

C204A

4

H68Q

DC/HH

(Vulliamy et al. 2006)

CT214-215TA

4

L72Y

DC

(Heiss et al. 1998)

A361G

5

S121G

HH

(Knight et al. 1999b)

C472T

6

R158W

DC

(Knight et al. 2001)

IVS 6 T40G

IVS6

-

polymorphism

(Marrone and Dokal 2004)

A838C

9

S280R

DC

(Knight et al. 2001)

A941G

10

K314R

DC/HH

(Vulliamy et al. 2006)

C949T

10

L317F

DC

(Rostamiani et al. 1999)

C961G

10

L321V

DC

(Knight et al. 1999b)

G965A

10

R322Q

DC

(Rostamiani et al. 1999)

T1049C

11

M350T

DC

(Knight et al. 1999b)

G1050A/C

11

M350I

DC

(Knight et al. 1999b)

C1058T

11

A353V

DC/HH

(Knight et al. 1999b; Yaghmai et al. 2000; Yoshimoto et al. 2000; Treister et al. 2004)

A1069G

11

T357A

DC

(Kanegane et al. 2005)

G1075A

11

D359N

DC

(Vulliamy et al. 2006)

C1150T

11

P384S

DC

(Rostamiani et al. 1999)

C1151T

11

P384L

DC

(Knight et al. 2001)

G1156A

12

A386T

DC/HH

(Vulliamy et al. 2006)

T1193C

12

L398P

DC

(Hiramatsu et al. 2002)

G1204A

12

G402R

DC

(Knight et al. 1999b)

G1205A

12

G402E

DC

(Heiss et al. 1998)

(continued)

(continued)

Mutation

Exon

Amino Acid Change

Clinical Manifestation

Refs

C1223T 12

C1226T 12

AG1258-1259TA 12

C1461T 14

IVS 14 G403T IVS 14

IVS 14 A473G IVS14

AExon15 15

1494insAAG G1551A

15 15

T408I P409L S420Y

AD493-514

498insK

DC DC DC

polymorphism polymorphism DC DC

polymorphism polymorphism

(Hassock et al. 1999) (Marrone and Dokal 2004) (Vulliamy et al. 2006) (Heiss et al. 1998; Vulliamy et al. 1999) (Dokal 2000) (Hassock et al. 1999)

Fig. 5.2 Nonsynonymous amino acid substitutions in dyskerin affected in dyskeratosis congen-ita. (A) Linear representation of dyskerin protein that contains 514 amino acids. The functional domains: nuclear localization signals (NL), pseudouridine synthase domain (TruB/PUS), pseu-douridine synthase, and archaeosine-spicific transglycosylase domain (PUA) are shown. Locations of nonsynonymous amino acid substitutions are labeled. (B) 3D structure of dyskerin with amino acid changes identified in dyskeratosis congenita. Mutations in the regions 1-35 and 359-513 are not shown due to the unavailability of modeling in these regions (Courtesy of Dr. Hong Li, Department of Chemistry and Biochemistry, Florida State University) (See Color Plate)

Fig. 5.2 Nonsynonymous amino acid substitutions in dyskerin affected in dyskeratosis congen-ita. (A) Linear representation of dyskerin protein that contains 514 amino acids. The functional domains: nuclear localization signals (NL), pseudouridine synthase domain (TruB/PUS), pseu-douridine synthase, and archaeosine-spicific transglycosylase domain (PUA) are shown. Locations of nonsynonymous amino acid substitutions are labeled. (B) 3D structure of dyskerin with amino acid changes identified in dyskeratosis congenita. Mutations in the regions 1-35 and 359-513 are not shown due to the unavailability of modeling in these regions (Courtesy of Dr. Hong Li, Department of Chemistry and Biochemistry, Florida State University) (See Color Plate)

Youssoufian et al. 1999). Recently, the three-dimensional structure of dyskerin was modeled on the proposed crystal structure of Cbf5 from the archaebacterium Pyrococcus furiosus and sequence homology between dyskerin and Cbf5 (Li and Ye 2006, Rashid et al. 2006). In the primary sequence of dyskerin, most mutations found in DC are clustered in the N-terminal 100 residues and PUA domain (Fig. 5.2B). In the crystal three-dimension structure, the PUA domain is encircled by the N terminus. Therefore, the regions where the mutations are concentrated fall within or near PUA domain, which is necessary for recognition and binding of H/ACA RNA and telomerase RNA (Li and Ye 2006, Rashid et al. 2006).

Amino acid substitutions in the PUA domain were predicted, though the important residues were not those that specifically affect the interaction between dyskerin and target RNA (see Li 2006). It has been observed that certain mutations lead to decreased accumulation of H/ACA RNA or telomerase RNA (Mitchell et al. 1999b, Mochizuki et al. 2004), thereby substantiating the importance of functional interaction of dyskerin and telomerase in the assembly, processing, and stability of telomerase RNA.

The A353V mutation located in the PUA domain has been found in more than 40% of individuals with DC. Only two mutations have been found in the TruB domain, one of which causes amino acid substitution S121G. This locus is only three residues from the highly conserved essential aspartic acid residue D125 required for pseudouridylation (Ramamurthy et al. 1999). A large deletion removing the last exon was identified that induces a truncated protein devoid of 22 amino acids at the C-terminus (Vulliamy et al. 1999). A C-G mutation in the promoter region at -141 is at a potential Sp1 binding site, inducing decreased transcription in an in vitro promoter assay. (Knight et al. 2001, Salowsky et al. 2002). The intronic mutation C592G has been shown to affect splicing with generation of aberrantly spliced mRNA, and G473C and IVS 14 G403T change the splice site acceptor motif (Knight et al. 2001, Vulliamy et al. 2006).

In the most severe form of DC-HH syndrome-nine mutations have been identified which are distributed in the two clusters, the N-terminal 100 residues and PUA domain, as well as in the TruB domain. The amino acid substitutions I38T and T49M have, to date, only been seen in the HH patients (Knight et al. 1999a, Sznajer et al. 2003). On the other hand, the most frequent mutation A353V leads to DC with different extents of severity, including HH (Vulliamy et al. 2006). Therefore, although there appears to be some correlation between severity and mutation, the phenotype can be affected by other genetic and environmental factors.

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