Mutations in C16orf57 and normal-length telomeres unify a subset of patients with dyskeratosis congenita, poikiloderma with neutropenia and Rothmund-Thomson syndrome

Abstract

Dyskeratosis congenita (DC) is an inherited poikiloderma which in addition to the skin abnormalities is typically associated with nail dystrophy, leucoplakia, bone marrow failure, cancer predisposition and other features. Approximately 50% of DC patients remain genetically uncharacterized. All the DC genes identified to date are important in telomere maintenance. To determine the genetic basis of the remaining cases of DC, we undertook linkage analysis in 20 families and identified a common candidate gene region on chromosome 16 in a subset of these. This region included the C16orf57 gene recently identified to be mutated in poikiloderma with neutropenia (PN), an inherited poikiloderma displaying significant clinical overlap with DC. Analysis of the C16orf57 gene in our uncharacterized DC patients revealed homozygous mutations in 6 of 132 families. In addition, three of six families previously classified as Rothmund-Thomson syndrome (RTS-a poikiloderma that is sometimes confused with PN) were also found to have homozygous C16orf57 mutations. Given the role of the previous DC genes in telomere maintenance, telomere length was analysed in these patients and found to be comparable to age-matched controls. These findings suggest that mutations in C16orf57 unify a distinct set of families which clinically can be categorized as DC, PN or RTS. This study also highlights the multi-system nature (wider than just poikiloderma and neutropenia) of the clinical features of affected individuals (and therefore house-keeping function of C16orf57), a possible role for C16orf57 in apoptosis, as well as a distinct difference from previously characterized DC patients because telomere length was normal.

Figure 1.

Identification of a common region of homozygosity in four families with DC. (A) Homozygosity of all SNPs on chromosome 16. Black boxes indicate a homozygous call, white spaces heterozygous calls. The red bar indicates the overlapping region. (B) LOD score plot of families DCR279, DCR070, DCR224 and DCR107 showing the maximum LOD score of 8.2 on chromosome 16.

Figure 2.

Pedigrees of families with mutations in C16orf57. Sequence panel on the left shows the normal trace while the right shows the observed mutation. The arrow highlights the point of mutation. c., coding nucleotide number; nd, sample unavailable for analysis; open shape, unaffected individual; filled shape, affected individual.

Figure 3.

Schematic structure of C16orf57. (A) Relative position of the mutations to the exons (grey boxes). (B) Relative protein lengths predicted to remain after mutation. Text colour relates to disease type: blue, DC; red, RTS; black, PN/DC: bar, correct amino acid sequence; line, mutant amino acid sequence before stop codon. The effect of the splice-site mutation is not shown because of the lack of appropriate material in which to determine the sequence of the aberrant transcript.

Figure 4.

Telomere lengths are not short in patients with mutations in C16orf57 compared with healthy controls. (A) Sample of a Southern blot using the subtelomeric probe pTelBam8. This shows the variety of lengths obtained from different individuals both with and without disease. (B) Age-adjusted ▵ tel values for controls compared with patients with C16orf57 mutations, uncharacterized patients with DC and patients with known DKC1 mutations. (C) T/S ratio as a measure of telomere length comparing controls with patients with C16orf57 mutations and uncharacterized DC patients. AA, patient with aplastic anaemia; Unch, genetically uncharacterized DC patients; Tinf2, DC patient with TINF2 mutation; solid line, median length obtained for the sample group, P-values from the Mann–Whitney U test relative to controls.

Figure 5.

Effects of knockdown of C16orf57 on apoptosis and cell cycle. (A) Levels of knockdown achieved in 7 days after treatment with lentiviral-shRNA. (B) Corresponding apoptosis levels in HT1080 cells. (C) Corresponding cell cycle analysis. Vertical stripes, unmanipulated cells; horizontal stripes, cells transduced with a negative shRNA lentiviral construct; solid black bars, cells transduced with a C16orf57 shRNA lentiviral construct. Numbers on graphs relate to the data point for each experiment.