A novel dyskerin (DKC1) mutation is associated with familial interstitial pneumonia

Abstract

Short telomeres are frequently identified in patients with idiopathic pulmonary fibrosis (IPF) and its inherited form, familial interstitial pneumonia (FIP). We identified a kindred with FIP with short telomeres who did not carry a mutation in known FIP genes TERT or hTR . We performed targeted sequencing of other telomere-related genes to identify the genetic basis of FIP in this kindred. The proband was a 69 year-old man with dyspnea, restrictive pulmonary function test results, and reticular changes on high-resolution CT scan. An older male sibling had died from IPF. The proband had markedly shortened telomeres in peripheral blood and undetectably short telomeres in alveolar epithelial cells. Polymerase chain reaction-based sequencing of NOP10 , TINF2 , NHP2 , and DKC1 revealed that both affected siblings shared a novel A to G 1213 transition in DKC1 near the hTR binding domain that is predicted to encode a Thr405Ala amino acid substitution. hTR levels were decreased out of proportion to DKC1 expression in the T405A DKC1 proband, suggesting this mutation destabilizes hTR and impairs telomerase function. This DKC1 variant represents the third telomere-related gene identified as a genetic cause of FIP. Further investigation into the mechanism by which dyskerin contributes to the development of lung fibrosis is warranted.

Figure 1.

Clinical features of affected subjects. A, Affected subjects manifested a hyperpigmented rash on the hands with dyskeratotic nail changes. B, Reticular changes and mild bronchiectasis on high-resolution CT scan. C, Hematoxylin and eosin (H&E)-stained transbronchial biopsy specimen from the proband demonstrates interstitial fibrosis (original magnification × 200). D, H&E-stained surgical lung biopsy specimen from an affected relative demonstrates temporal heterogeneity and extensive interstitial fibrosis consistent with a usual interstitial pneumonia pattern (original magnification × 100).

Figure 2.

Four-generation pedigree of the affected kindred. Numbers below individual symbols are age at death or study. DKC1 mutation status is also depicted below age in subjects for whom DNA was available for sequencing. Subject II-2 is an obligate carrier; however, no DNA was available for analysis. Insufficient historical information was available to clinically phenotype generation I.

Figure 3.

Affected subjects have marked telomere shortening. A, TRF analysis of the proband, his daughters (obligate carriers), and his maternal aunt (age 87 y), in addition to a healthy control subject. Lane markers correspond to generation and number, as depicted in Figure 2. B, TRF percentile for age plot. Affected proband’s TRF at age 69 y is shown in red, confirmed carrier daughters’ TRFs in pink, and unaffected maternal aunt’s in blue. C, Fluorescence in situ hydridization (FISH) showing relative telomere length in type 2 alveolar epithelial cells (AECs) from lungs of the affected proband. D, FISH showing relative telomere length in type 2 AECs from a normal control lung. Red = telomeres; green = prosurfactant protein C; blue = 4’,6-diamidino-2-phenylindole (nuclear staining, original magnification × 640). E, Quantification of AEC telomere length by FISH in normal lung (n = 8), sporadic idiopathic pulmonary fibrosis (n = 7), and affected siblings (n = 2). Telomere length is quantified by ratio of red to blue fluorescence. Data are presented as mean ± SD. ∗P = .003. IPF = idiopathic pulmonary fibrosis; TRF = telomere restriction fragment.

Figure 4.

Sanger sequencing confirms DKC1 mutation in affected sibling and obligate carriers. An A-to-G transition was identified at position 1213 that is predicted to encode a Thr405Ala amino acid substitution in the mature peptide.

Figure 5.

The Thr405Ala DKC1 substitution is associated with disproportionate reduction of hTR levels compared with DKC1 expression. A, Lymphoblastoid DKC1 mRNA quantified by quantitative polymerase chain reaction (qPCR). B, hTR RNA expression quantified by qPCR. Results are expressed a copies relative to β-actin normalized to healthy control subjects (n = 4). C, hTR levels were plotted vs DKC1 expression for the proband and control subjects. Sample size precluded formal statistical analysis between the groups.