A large kindred of pulmonary fibrosis associated with a novel ABCA3 gene variant

Abstract

Background: Interstitial lung disease occurring in children is a condition characterized by high frequency of cases due to genetic aberrations of pulmonary surfactant homeostasis, that are also believed to be responsible of a fraction of familial pulmonary fibrosis. To our knowledge, ABCA3 gene was not previously reported as causative agent of fibrosis affecting both children and adults in the same kindred.

Methods: We investigated a large kindred in which two members, a girl whose interstitial lung disease was first recognized at age of 13, and an adult, showed a diffuse pulmonary fibrosis with marked differences in terms of morphology and imaging. An additional, asymptomatic family member was detected by genetic analysis. Surfactant abnormalities were investigated at biochemical, and genetic level, as well as by cell transfection experiments.

Results: Bronchoalveolar lavage fluid analysis of the patients revealed absence of surfactant protein C, whereas the gene sequence was normal. By contrast, sequence of the ABCA3 gene showed a novel homozygous G > A transition at nucleotide 2891, localized within exon 21, resulting in a glycine to aspartic acid change at codon 964. Interestingly, the lung specimens from the girl displayed a morphologic usual interstitial pneumonitis-like pattern, whereas the specimens from one of the two adult patients showed rather a non specific interstitial pneumonitis-like pattern.

Conclusions: We have detected a large kindred with a novel ABCA3 mutation likely causing interstitial lung fibrosis affecting either young and adult family members. We suggest that ABCA3 gene should be considered in genetic testing in the occurrence of familial pulmonary fibrosis.

Figure 1

Patient A - chest CT at diagnosis. High-resolution CT scan demonstrates fibrotic architectural distortion with intra and inter-lobular thickening, associated with apical honey-combing. No ground glass opacity is present.

Figure 2

Patient A Hematoxylin and Eosin staining. Two surgical samples were taken from the upper and the lower left lobes. The sample from the upper lobe (a, b) shows limited architectural changes, with centrilobular inflammation and fibrosis. Fibroblastic foci and small aggregates of multinucleated giant cells engulfing cholesterol clefts are visible in centrilobular areas. Granular pink material is visible in some alveolar spaces (b). The sample from the lower lobe (c, d) shows diffuse microcysts, fibrosis and smooth muscle proliferation, without residual normal tissue. Fibroblastic foci and bronchiolar metaplasia are prominent (d). (a) magnification 2x, scale bar 0,5 cm; (b) magnification 10x, scale bar 200 micron; (c) magnification 2x, scale bar 0,5 cm; (d) magnification 20x, scale bar 30 micron.

Figure 3

Analysis of pro and mature SP-B and SP-C in BALF. Patient A (Pt A). Molecular weights (MW, kDa) are indicated on the left side. 5 μg of total protein was added to each lane from lavage of a patient. All bands were analyzed under non-reducing conditions. A small amount of pro-SP-B was detected at 21 kDa in Pt A (second lane from left). As positive control pro-SP-B of a lavage from a healthy subject (Con, nl) is given (first lane from the left). SP-B was detected in fair amounts (third lane compared to the standard (STD) of which 20 ng was applied). Aberrant proforms of SP-C were not detected (third lane from the right). As positive control pro-SP-C of a lavage from a subject with SFTPC mutation I73T, which usually have aberrant pro-SP-C at about 14 kDa (Con, I73T) is given (fourth lane from the right). No SP-C was detected (second lane from the right; compared to the standard STD of 25 ng applied to the first lane from the right).

Figure 4

ABCA3 gene sequence analysis. a) Sequence analysis of ABCA3 G > A transition at nucleotide 2891 in Patient A, her parents and sisters. b) Partial amino acid alignment of ABCA3 sequences (codons 949–986), showing that Gly964 (indicated by an arrow) is extremely conserved.

Figure 5

Family tree. The family genetic screening by the Taqman assay resulted in the identification of two other paternal members (black arrows), who were homozygous for the same ABCA3 Gly964Asp mutation. The proband is indicated by the dashed arrow.

Figure 6

Patient B Hematoxylin and Eosin staining. A single sample of lung tissue was obtained. The site of the biopsy could not be documented. a) Low power evaluation showed a patchy fibrotic process, characterized by areas of interstitial scarring (upper left) in an otherwise uninvolved alveolar background; b) fibrotic area characterized by an NSIP pattern; c) isolated centrilobular fibrosis and inflammation; d) areas with normal lung parenchyma.

Figure 7

Thorax HRTC Scan of the two brothers with the homozygous ABCA3 mutation. a, b) Patient B. Diffuse areas of reticular ground glass opacities, prevalent in the upper right lobe. Initial honeycombing in the dorsal segment. Diffuse pleural thickening. c and d) Patient C. The involvement of the upper lobes is very similar to that of the brother, patient B.

Figure 8

Cellular effects of 48 h transient ABCA3 expression in A549 cells. a. ABCA3 mRNA expression levels analyzed by quantitative real time PCR (P < 0.001 for WT/G964D vs. A549/Mock). b. Western immunoblot analysis of HA-tagged ABCA3 in total cell lysates. β-actin was used as a loading control. c. Ratio of the upper/lower ABCA3 processing form. d. Co-immunostaining of HA-tagged ABCA3 with the lysosomal (lamellar body) marker LAMP3. e. Co-staining of HA-tagged ABCA3 with the ER marker calnexin. f. ER (calnexin, BiP) and early endosome markers (EEA1) in cells transiently transfected with ABCA3. β-actin was used as a loading control. Scale bars 7.5 μm, *P < 0.05.

Figure 9

Levels of phospholipids and free cholesterol in HEK cells. Shown are cellular contents of phosphatidylcholine, phosphatidylglycerol, phosphatidylinositol, and free cholesterol in HEK cells expressing wild type ABCA3 and ABCA3 G964D, respectively. Values are given in nmol/mg protein. *P < 0.05, **P < 0.01, ***P < 0.001.

Figure 10

Transmission electron microscopy of stable transfected HEK293-cells. Top: overview of one cell showing cytoplasm, containing mitochondria (light grey) and organelles resembling lamellar bodies (dark grey). Bottom: single ‘lamellar bodies resembling organelles’ in higher magnification. The boxed area is shown in the top image as inset in higher magnification. Note the concentric arrangement of the lamellae in the ABCA3-WT strain. Dark appearing lamellae are mostly wound around a centre of less electron density and appear as parallel lines in the high magnification. In the strain G964D these organelles appear less organized: lamellae are often not arranged perfectly parallel (see inset), and the concentric organisation is not as distinct as in the WT.