Mutations in the telomerase component NHP2 cause the premature ageing syndrome dyskeratosis congenita - PubMed (original) (raw)

Mutations in the telomerase component NHP2 cause the premature ageing syndrome dyskeratosis congenita

Tom Vulliamy et al. Proc Natl Acad Sci U S A. 2008.

Abstract

Dyskeratosis congenita is a premature aging syndrome characterized by muco-cutaneous features and a range of other abnormalities, including early greying, dental loss, osteoporosis, and malignancy. Dyskeratosis congenita cells age prematurely and have very short telomeres. Patients have mutations in genes that encode components of the telomerase complex (dyskerin, TERC, TERT, and NOP10), important in the maintenance of telomeres. Many dyskeratosis congenita patients remain uncharacterized. Here, we describe the analysis of two other proteins, NHP2 and GAR1, that together with dyskerin and NOP10 are key components of telomerase and small nucleolar ribonucleoprotein (snoRNP) complexes. We have identified previously uncharacterized NHP2 mutations that can cause autosomal recessive dyskeratosis congenita but have not found any GAR1 mutations. Patients with NHP2 mutations, in common with patients bearing dyskerin and NOP10 mutations had short telomeres and low TERC levels. SiRNA-mediated knockdown of NHP2 in human cells led to low TERC levels, but this reduction was not observed after GAR1 knockdown. These findings suggest that, in human cells, GAR1 has a different impact on the accumulation of TERC compared with dyskerin, NOP10, and NHP2. Most of the mutations so far identified in patients with classical dyskeratosis congenita impact either directly or indirectly on the stability of RNAs. In keeping with this effect, patients with dyskerin, NOP10, and now NHP2 mutations have all been shown to have low levels of telomerase RNA in their peripheral blood, providing direct evidence of their role in telomere maintenance in humans.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.

Fig. 1.

NHP2 mutations cause autosomal recessive dyskeratosis congenita. Sequencing traces from within the NHP2 gene are shown next to each family member. Mutations are indicated by arrows and specified below each panel. The age in years and abnormalities observed are given below each individual, with nail dystrophy illustrated in one case. (A) Family DCR096. (B) Family DCR177.

Fig. 2.

Fig. 2.

Location of amino acid substitutions in NHP2. Mutations of NHP2 in dyskeratosis congenita patients (along with mutations of dyskerin and NOP10) are mapped onto the crystal structure of Pyrococcus furiosus H/ACA RNP. Mutated residues are shown as orange Cα spheres and side chains of the P. furiosus structure. Dots represent the C-terminal extension caused by the stop codon mutation. The three NHP2 mutations are labeled. L7Ae (equivalent to NHP2) is colored gray, NOP10 is purple, Cbf5 (equivalent to dyskerin) is green, GAR1 is blue, and the H/ACA RNA is yellow. The figure was prepared by using PyMOL (DeLano Scientific LLC) by Keqiong Ye.

Fig. 3.

Fig. 3.

NHP2 mutations result in short telomeres and reduced TERC levels. (A) Telomere length measurements in 112 healthy control subjects (open circles) are plotted against age with a line of best fit. Members of families in which NHP2 mutations are segregating are shown as gray triangles (Y139H heterozygotes), a black triangle (Y139H homozygote), gray square (V126M heterozygote), gray diamond (X154R heterozygote), black diamond (V126M/X154R, compound heterozygote), and open diamond (normal sibling). (B) Age-adjusted telomere lengths (delta tel, the difference between the observed telomere length and the length expected from the line of best fit drawn in panel a) for healthy control subjects (open circles, n = 112), NHP2 family members (as in A), and patients with dyskerin mutations (dashes, n = 67). (C) TERC levels, expressed as a TERC/ABL ratio, in healthy control individuals (open circles, n = 24), NHP2 family members (as in A), and patients with dyskerin mutations (dashes, n = 27).

Fig. 4.

Fig. 4.

Depletion of NHP2 but not GAR1 results in reduced TERC levels. The amount of NHP2, GAR1, and TERC, measured by QRT-PCR and normalized to ABL, is expressed relative to the level seen in the untransfected cells at the 24 h time point. The different manipulations are indicated below each panel. Each bar shows the mean of two experiments: blue bars, 24 h; green bars, 48 h; yellow bars, 72 h. (A) NHP2 expression. (B) TERC levels under manipulation with NHP2 siRNAs. (C) GAR1 expression. (D) TERC levels under manipulation with GAR1 siRNAs.

Fig. 5.

Fig. 5.

Wild-type, but not mutant, NHP2 can increase TERC accumulation. Expression levels of endogenous NHP2 (A), total NHP2 (B), and accumulation of TERC (C) are shown in HeLa cell lines into which either an empty vector (black bars), wild-type NHP2 (gray bars), or c.415T>C mutant NHP2 (dotted bars) have been stably transfected. Endogenous and total NHP2 are distinguished by using different TaqMan probes specific to either the 3′ UTR (endogenous only) or to the coding sequence (total) of NHP2. The effect of NHP2 siRNA, which is specific to the endogenous transcript, is shown relative to the untransfected cells as indicated beneath each panel.

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