Hypomorphic pathogenic variant in SFTPB leads to adult pulmonary fibrosis

Abstract

Biallelic pathogenic variants in the surfactant protein (SP)-B gene (SFTPB) have been associated with fatal forms of interstitial lung diseases (ILD) in newborns and exceptional survival in young children. We herein report the cases of two related adults with pulmonary fibrosis due to a new homozygous SFTPB pathogenic variant, c.582G>A p.(Gln194=). In vitro transcript studies showed that this SFTPB synonymous pathogenic variant induces aberrant splicing leading to three abnormal transcripts with the preservation of the expression of a small proportion of normal SFTPB transcripts. Immunostainings on lung biopsies of the proband showed an almost complete loss of SP-B expression. This hypomorphic splice variant has thus probably allowed the patients' survival to adulthood while inducing an epithelial cell dysfunction leading to ILD. Altogether, this report shows that SFTPB pathogenic variants should be considered in atypical presentations and/or early-onset forms of ILD particularly when a family history is identified.

Fig. 1. Family tree, CT scan and histological features.

A Genealogical tree of the described family. Consanguineous unions are indicated with double lines. Black symbols show subjects with pulmonary fibrosis. The age at the time of the study is mentioned within symbols. The arrow indicates the proband. The genotypes are provided with the reference nucleotide in green and the pathogenic variant in red. The CT scan of the proband (III.2) at 51 years (before lung transplantation) shows parenchymal distortion, ground glass opacities, centrilobular nodules and mosaic attenuation with area of decreased attenuation and pulmonary fibrosis. The CT scan of his son (IV.7) at 16 years shows mild ground glass opacities, large areas of attenuation especially in the lingula and subtle distortion. B The native lung explant examination of the proband (III.2) using Haematoxylin and eosin (HE) staining shows fibroblastic foci alternating with less affected areas consistent with a usual interstitial pneumonia pattern. Pulmonary hemosiderosis was not observed nor pulmonary alveolar proteinosis. The SP-B-deficient neonate showed thickened septa with moderate pulmonary alveolar proteinosis and the adult and infant controls showed a normal lung parenchyma. SP-B antibody targets pro-SP-B as it has been developed against amino acids 121–197. SP-B immunostaining (magnification ×20 and ×40) showed an almost complete loss of SP-B expression in the patient’s type 2 alveolar epithelial cells (AEC)2, a complete loss of SP-B expression in SP-B-deficient neonate and a normal intracytoplasmic granular AEC2 SP-B expression in both adult and infant controls. Note that the anti-SP-B antibody does not target the peptide corresponding to the in-phase deleted transcript (p.(Asp132_Gln194del)) which lacks most of the epitope (Fig. 2). SP-C antibody has been developed against full-length SP-C (amino acids 1-197) and can thus detect both pro-SP-C and mature SP-C. SP-C immunostaining (magnification ×20 and ×40) showed a higher degree of expression in the patient’s and the SP-B-deficient neonate’s type 2 alveolar epithelial cells than in adult and infant controls. HE haematoxylin and eosin, AEC alveolar epithelial cell.

Fig. 2. In vitro assessment of SFTPB splicing defects associated with c.582G>A p.(Gln194=).

SFTPB exons 4–6 pre-mRNA corresponding to plasmids pSFTPB-WT, pSFTPB_mut and pSFTPB_ctr are represented. The reference nucleotide is in green and the pathogenic variant is in red. The dotted line highlights a cryptic acceptor splice site in exon 6 (nucleotide 636). After transfection of A549 cells with the above-mentioned plasmids, the migration on a 1.5% agarose-BET gel of the RT-PCR products obtained from extracted RNAs is shown. The main amplicons characterized by Sanger sequencing and obtained from the normal and mutant constructs are represented on the right of the gel image. Two amplicons were observed for the pSFTPB_WT: a 363 bp (canonical splicing) and a 310 bp (using exon 6 cryptic splice-acceptor site corresponding to putative p.(Asp195Glyfs*39)). Both the c.582G>A (pSFTPB_mut) and the c.582+1G>T (pSFTPB_ctr) pathogenic variants resulted in three specific and aberrant amplicons, i.e., from top to bottom: one 464 bp transcript related to the retention of the first 101 bp of intron 5 and predicting a frameshift with a premature Stop codon in exon 6 (p.(Asp195Valfs*54)), and two 174 and 121 bp amplicons lacking exon 5 and corresponding to the use of intron 5 canonical splice-acceptor site (p.(Asp132_Gln194del) in-phase deletion), or exon 6 cryptic splice-acceptor site (p.(Asp132Glyfs*39)) respectively. The data are representative from 3 independent experiments. WT wild type.