A new variant in the ZCCHC8 gene: diverse clinical phenotypes and expression in the lung

Abstract

Introduction: Pulmonary fibrosis is a severe disease which can be familial. A genetic cause can only be found in ∼40% of families. Searching for shared novel genetic variants may aid the discovery of new genetic causes of disease.

Methods: Whole-exome sequencing was performed in 152 unrelated patients with a suspected genetic cause of pulmonary fibrosis from the St Antonius interstitial lung disease biobank. Variants of interest were selected by filtering for novel, potentially deleterious variants that were present in at least three unrelated pulmonary fibrosis patients.

Results: The novel c.586G>A p.(E196K) variant in the ZCCHC8 gene was observed in three unrelated patients: two familial patients and one sporadic patient, who was later genealogically linked to one of the families. The variant was identified in nine additional relatives with pulmonary fibrosis and other telomere-related phenotypes, such as pulmonary arterial venous malformations, emphysema, myelodysplastic syndrome, acute myeloid leukaemia and dyskeratosis congenita. One family showed incomplete segregation, with absence of the variant in one pulmonary fibrosis patient who carried a PARN variant. The majority of ZCCHC8 variant carriers showed short telomeres in blood. ZCCHC8 protein was located in different lung cell types, including alveolar type 2 (AT2) pneumocytes, the culprit cells in pulmonary fibrosis. AT2 cells showed telomere shortening and increased DNA damage, which was comparable to patients with sporadic pulmonary fibrosis and those with pulmonary fibrosis carrying a telomere-related gene variant, respectively.

Discussion: The ZCCHC8 c.586G>A variant confirms the involvement of ZCCHC8 in pulmonary fibrosis and short-telomere syndromes and underlines the importance of including the ZCCHC8 gene in diagnostic gene panels for these diseases.

FIGURE 1

a) Condensed pedigrees of family 1; a sporadic patient, later named family 2; and family 3. Arrows indicate probands. Roman numerals indicate the generation. Genealogical research linked family 1 to the sporadic patient (dashed lines) through a common ancestor (generation I). Age at time of death is indicated at the upper right of each symbol. +: heterozygosity for ZCCHC8 c.586G>A; −: wild type for this variant; dx: age at time of diagnosis; IPF: idiopathic pulmonary fibrosis; TL: telomere length in blood; <1: <1st percentile for age; <10: <10th percentile for age; >10: >10th percentile for age; ND: not determined; MDS: myelodysplastic syndrome; AML: acute myeloid leukaemia. In family 3, subject III.11 does not carry the ZCCHC8 c.586G>A variant, but does carry PARN c.1045C>T. Smoking status “never”: Fam1.IV.1, Fam1.IV.2, Fam2.IV.1, Fam2.V.1, Fam3.III.4, Fam3.III.10; “former”: Fam1.IV.3, Fam3.III.1, Fam3.III.5, Fam3.III.7, Fam3.III.8, Fam3.III.9, Fam3.III.11. ^#: autopsy tissue available; ^¶: lung biopsy tissue available. b) ZCCHC8 E196K conservation across nine species. Darker shading indicates a more conserved residue (Homo sapiens used as reference). c) Telomere length in blood plotted against age from patients carrying the novel ZCCHC8 c.586G>A or PARN c.1045C>T or the ZCCHC8 c.557C>T variant. Symbol coloration indicates the initial telomere-related presentation, being haematological disease, pulmonary fibrosis or emphysema. Lines indicate percentiles of healthy control subjects. T/S ratio: relative telomere to single copy gene ratio.

FIGURE 2

Example of ZCCHC8 protein expression in a) bronchioles and b) alveoli of non-fibrotic and c) fibrotic tissue of an idiopathic pulmonary fibrosis patient carrying a disease-associated TERT variant. Green: ZCCHC8 immunofluorescence staining; blue: 4′,6-diamidino-2-phenylindole (DAPI) DNA staining; A3 red: CC10 staining as a marker for club cells. B3,C3 red: pro-surfactant protein (SP)-C as a marker for alveolar type 2 cells.

FIGURE 3

ZCCHC8 protein and RNA expression. a) Fluorescence in situ hybridisation quantification of ZCCHC8 protein expression levels in alveolar type 2 (AT2) cells as measured by relative fluorescence compared to 4′,6-diamidino-2-phenylindole (DAPI) staining of lung tissue of controls (Ctrls) and patients with sporadic IPF (sIPF), or pulmonary fibrosis with a variant in TERT (PF-TERT) or ZCCHC8 c.586G>A (PF-ZCCHC8). Lines indicate the median. Each dot represents one AT2 cell. b) Western blot results of ZCCHC8 protein expression in primary skin fibroblast homogenates from Ctrls, non-carrier (non-c) relatives, symptomatic ZCCHC8 c.586G>A and both asymptomatic (upward facing triangle) and symptomatic (downward facing triangle) ZCCHC8 c.557C>T variant. Kruskal–Wallis test p=0.20. c) Quantitative PCR results for relative ZCCHC8 RNA expression in whole-blood-derived samples. ZCCHC8 expression was calculated compared to the household gene ARF3 and expressed relatively to the unrelated healthy controls. Kruskal–Wallis test p=0.08. b,c) Levels are relative to the mean of controls; each symbol represents one individual.

FIGURE 4

Cell-specific quantification of telomere length in lung tissue in control (n=5), sporadic IPF (sIPF; n=10) and pulmonary fibrosis with a variant in TERT (PF-TERT; n=6) or ZCCHC8 c.586G>A (PF-ZCCHC8; n=2). Telomere length is measured using fluorescence in situ hybridisation in a) alveolar type 2 (AT2) cells, b) club cells and c) myofibroblasts. No telomere length is depicted for the control samples in (c), as myofibroblasts were not observed in control tissue. The two individuals included in the PF-ZCCHC8 group are depicted with different shades of blue (light for Fam1.IV.2 and dark for Fam3.III.6). *: p<0.05; **: p<0.01; ***: p<0.001; ****: p<0.0001, calculated using Kruskal–Wallis with Dunn's multiple comparison tests.