Genetic heterogeneity in autosomal recessive dyskeratosis congenita with one subtype due to mutations in the telomerase-associated protein NOP10

Abstract

Dyskeratosis congenita (DC) is characterized by multiple features including mucocutaneous abnormalities, bone marrow failure and an increased predisposition to cancer. It exhibits marked clinical and genetic heterogeneity. DKC1 encoding dyskerin, a component of H/ACA small nucleolar ribonucleoprotein (snoRNP) particles is mutated in X-linked recessive DC. Telomerase RNA component (TERC), the RNA component and TERT the enzymatic component of telomerase, are mutated in autosomal dominant DC, suggesting that DC is primarily a disease of defective telomere maintenance. The gene(s) involved in autosomal recessive DC remains elusive. This paper describes studies aimed at defining the genetic basis of AR-DC. Homozygosity mapping in 16 consanguineous families with 25 affected individuals demonstrates that there is no single genetic locus for AR-DC. However, we show that NOP10, a component of H/ACA snoRNP complexes including telomerase is mutated in a large consanguineous family with classical DC. Affected homozygous individuals have significant telomere shortening and reduced TERC levels. While a reduction of TERC levels is not a universal feature of DC, it can be brought about through a reduction of NOP10 transcripts, as demonstrated by siRNA interference studies. A similar reduction in TERC levels is also seen when the mutant NOP10 is expressed in HeLa cells. These findings identify the genetic basis of one subtype of AR-DC being due to the first documented mutations in NOP10. This further strengthens the model that defective telomere maintenance is the primary pathology in DC and substantiates the evidence in humans for the involvement of NOP10 in the telomerase complex and telomere maintenance.

Figure 1

Homozygosity mapping used to identify a locus for autosomal recessive DC. (A) Pedigrees of consanguineous families studied. DNA samples were not available on all individuals shown. Open circles and squares represent normal females and males, respectively. Black circles and squares represent affected females and males, respectively. Affected individuals marked with asterisk were not available for analysis. (B) The five most homozygous markers demonstrate the degree of genetic heterogeneity that exists in AR-DC. Where there is more than one affected individual in a family a consensus for the family is shown. Filled boxes indicate homozygosity for the same alleles within a family. For comparison the homozygosity profile of D15S1007 is also shown.

Figure 2

Localization of NOP10 as a candidate gene in a family with AR-DC. (A) Pedigree from family DCR 148 showing partial chromosome 15 genotype data. Open circles and squares represent normal females and males, respectively. Black circles and squares represent affected females and males, respectively. The black and white bars highlight the parental disease carrying haplotypes (black, maternal; white, paternal). (B) LOD score graph for chromosome 15 identifying a potential candidate locus. This also shows the approximate location of NOP10 relative to the microsatellite markers.

Figure 3

Identification of a homozygous mutation in NOP10. (A) Examples of sequence traces showing the C to T change at nucleotide 100 which causes an amino acid change at codon 34 from arginine to tryptophan. Both parents and the three unaffected children (I-1, I-2, II-1, II-4, II-6) are heterozygous for this mutation (C/T, left panel trace shown from II-1), whereas all three patients (II-2, II-3 and II-5) are homozygous for the mutation (T/T, right panel trace shown from II-5). (B) NOP10 is highly conserved. Human NOP10 was aligned with Nop10 of the mouse, Danio rerio, Caenorhabditis elegans, Arabidopsis thaliana and Saccharomyces cerevisiae. The p.R34W mutation is in a highly conserved region of the molecule. Highly conserved residues are shown in red and moderately conserved residues in blue. Exclamation mark equals I or V; hash symbol equals D, E, N or Q; bullets equal no significant homology between codons. The alignment was obtained with the MultAlin program (43).

Figure 4

Delta Tel measurements in dyskeratosis congenita (DC) patients. Age-adjusted telomere length measurements from whole blood are shown for different subtypes of DC emphasizing how short telomeres are in the homozygous p.R34W individuals relative to both the heterozygous individuals, healthy controls and other DC types. P-values are expressed relative to the healthy individual group; NS, P-value not significant.

Figure 5

Effect of p.R34W on TERC levels. TERC was quantitated and expressed relative to ABL in all individuals with NOP10 p.R34W. TERC/ABL ratios were also determined for DC patients with other mutations from whole blood. P-values are expressed relative to the healthy individual group. Unknown, the genetic basis of the disease remains unidentified. NS, P-value not significant.

Figure 6

Effects of siRNAs against NOP10 in HeLa cells in a transient system. (A) Levels of NOP10 expression are reduced by siRNA I and II at all time points studied when compared with mock-transfected and negative siRNA transfected cells (24 h, solid black bar; 48 h, grey bar; 72 h, solid white bar). (B) TERC levels are reduced from 48 h in the siRNA-treated cells when compared with controls.

Figure 7

Stable expression of mutant and wild-type NOP10. (A) Endogenous NOP10 expression is reduced with siRNA treatment but modest plasmid-derived NOP10 is detected in cell lines containing either wild-type or mutant plasmid. NOP10 expression in stable cell lines is shown relative to time = 0 in the cell line with the empty vector (empty vector, dotted bar; wild-type plasmid, vertical lines; mutant plasmid, horizontal lines). (B) TERC levels are partially rescued in the cell line expressing wild-type NOP10 but not in the cell line expressing the mutant NOP10. TERC expression is shown relative to time = 0 for each cell line.