Background
Interstitial lung disease (ILD) in children represents a heterogeneous group of respiratory disorders that are characterized by impaired gas exchange and diffuse infiltrates [
1]. In children, ILD is most frequently diagnosed in the first year of life with a predominance of genetic entities [
2]. In the past decade, significant advances have been made in understanding the underlying causes for childhood ILD (chILD) such as genetic disorders of surfactant dysfunction which result from mutations in genes critical for the function and metabolism of pulmonary surfactant [
3]. Among these genes is the surfactant protein C gene (
SFTPC) located on chromosome 8p21. Its encoding protein, surfactant protein C (SP-C), is a hydrophobic 35-amino-acid polypeptide secreted into the alveolar space by alveolar type II epithelial cells to help reduce surface tension [
4]. Since Nogee et al. first reported a case caused by an
SFTPC mutation in 2001 [
5], more than 60 mutations in
SFTPC have been identified in pediatric ILD patients to date.
Lung disease caused by different
SFTPC mutations varies greatly, from respiratory distress syndrome (RDS) in neonates to ILD in adults [
6,
7]. However, up to now a large proportion of the reported cases are of Caucasian or African descent. Only a few cases with Asian origin were reported [
8‐
10]. Whether patients with different geographic and ethnic origins differ in clinical and genetic spectrum remains unclear. With the increase of awareness of this disease and advances in diagnostic technique, although rare, we discover that
SFTPC mutations account for a substantial proportion of unexplained ILD with early onset in our Chinese population. Recently, Chen J et al. [
11] reported 18 Chinese cases with surfactant dysfunction. Among the 15 patients who had
SFTPC mutations, 5 different mutations were identified. However, the information regarding the genotype as well as the choice of treatments and therapeutic response of Chinese patients is still limited. Here, we report the clinical features and genetic findings in 6 Chinese subjects heterozygous for
SFTPC mutations to expand the genetic and clinical spectrum.
Methods
Patients
In this study the subjects were identified among symptomatic infants and children who were clinically diagnosed as chILD and suspected of having genetic surfactant dysfunction and then referred for candidate gene sequencing in the laboratory at Children’s Hospital of Fudan University between 2013 and 2018. According to an American Thoracic Society guideline [
12], a child is regarded as having chILD if at least three of the following four criteria are present: (1) respiratory symptoms (cough, rapid and/or difficult breathing, or exercise intolerance); (2) respiratory signs (tachypnea, adventitious sounds, retractions, digital clubbing, failure to thrive, or respiratory failure); (3) hypoxemia; and (4) diffuse abnormalities on a chest radiograph or CT scan. Meanwhile, common diseases that can cause ILD were excluded as primary diagnosis by echocardiography and the screening of pathogens, autoimmune antibodies and immune deficiency. Clinical data were collected during the study. This study was approved by the ethics committees of Children’s Hospital of Fudan University. Written informed consent was obtained from all parents or guardians of the patients.
Genetic analysis
Genomic DNA was isolated from blood of the patients and their parents using the QIAamp DNA Blood Mini kit (Qiagen, Hilden, Germany). Molecular analysis of the disease-causing genes SFTPB, SFTPC, ABCA3, NKX2–1, CSF2RA and CSF2RB were performed through a self-designed gene panel using Ion Torrent PGM (Life Technologies). Targeted genomic regions covered exons and their flanking sequences of these six genes responsible for surfactant dysfunction. Library preparation was conducted by multiplex amplification using the Ion AmpliSeq Library Kit 2.0 (Life Technologies). Sequencing was performed using 316 v2 chips (Life Technologies) on the Ion Torrent PGM platform. We use Torrent Suite software (Life Technologies) to compare base calls. Then we use NextGENe software (SoftGenetics) to read alignments and to call variants with the human reference genome hg19 (NCBI). The variants were then compared with dbSNP. Novel variants were analyzed with in silico tools MutationTaster, SIFT and PolyPhen2.
The validation of the variants was performed by PCR followed by direct Sanger sequencing using 3500XL Genetic Analyzer (Applied Biosystems).
Functional analysis of SFTPC D105G mutation
The methods used to characterize
SFTPC D105G mutation such as
SFTPC cDNA expression constructs, A549 cell line transfection, Western blotting and immunofluorescence were described previously [
13].
For construction of mutant Flag/SP-CD105G, mutagenesis was performed by inverse PCR using KOD Plus Mutagenesis Kit (Toyobo, Japan) with pCDH-EGFP-Flag/SP-CWT serving as a template. The 5′ (forward) primer used for mutagenesis: GCTACCAGCAGCTGCTGATC. The 3′ (reverse) primer: CATACACCACGAGGCCAGTG. All constructs were confirmed by Sanger sequencing.
Discussion
The identification of SFTPC mutations has led to significant advances in the diagnosis of interstitial lung disease in infancy and childhood. Due to the lack of diagnostic techniques, patients with SP-C dysfunction were frequently misdiagnosed in the past decades in China. In this study, we described 6 Chinese ILD patients with detailed clinical and genetic information which may help to provide a recognisable pattern for identifying such rare cases in clinical practice.
Most of the patients reported in our study had symptoms within the first year of life and then gradually developed dependence of oxygen with a finding of ground-glass pattern on chest CT. This was consistent with other studies reported by pediatric centers in Western countries [
15,
16]. Our study showed an earlier age of onset and a more prevalent failure to thrive in group with
SFTPC mutations. Referral for genetic analysis should be preferred in ILD patients with these features.
The severity of individuals with
SFTPC mutations vary greatly, from severe RDS in neonates to mild interstitial lung disease in adults [
6,
17,
18]. Clinical outcome at follow-up in our report varied from healthy (age 3 years) to deceased (age 8 and 22 months). Hydroxychloroquine has been reported to improve the clinical status of cases with
SFTPC mutations. In some case series, 50 to 100% patients responded well to hydroxychloroquine treatment [
16,
19,
20]. The exact mechanism of action of hydroxychloroquine is unknown. In addition to having anti-inflammatory properties, hydroxychloroquine has been shown to cause inhibition of the intracellular processing of the precursor of SP-C [
21], which may explain its therapeutic effects. 50% (2/4) of our patients responded well to hydroxychloroquine (initiated from 11 months and 13 months of life respectively) and one responded partially (initiated from 24 months of life). However, it should be noted that in the only case not responsive to hydroxychloroquine (initiated from 7 months of life), treatment had just begun for 1 month until he died of pulmonary exacerbation. Nowadays there are still few centers choosing hydroxychloroquine to treat ILD patients resulting from
SFTPC mutations in China. In the future, more cases and long-term follow-up will be needed to determine the efficacy.
In terms of the genetic findings, we identified 3 different mutations in 6 patients, including two known and a novel mutations. I73T was the most common mutation accounting for 66.7% (4/6) of our patients which was consistent with other literature (28–68%) [
15,
20,
22]. Mutations in half of the cases were inherited from parents and only one had family history suggesting imcomplete penetrance. The mechanism of imcomplete penetrance in this disease was still elusive. It was reported that heterozygosity for
ABCA3 (another gene responsible for surfactant dysfunction) mutations modifies the severity of lung disease in individuals with the same
SFTPC mutation suggesting modifier genes may be involved [
23]. In addition, Kaltenborn et al. [
24] discovered infection with respiratory syncytial virus potentiated the mutational effects on loss of lung epithelial cell differentiation induced by
ABCA3 mutation. This study indicated that environmental factors such as viral infections may also have a key role in modulating the disease course thus contributing to the phenomenon of imcomplete penetrance.
The mutation D105G identified in patient 5 was once reported [
14]. However, the father and sister of the patient who also carried the mutation showed no signs of any lung diseases leading us to questioning the pathogenicity of the mutation. Moreover, no functional data of this mutation is currently available. So together with the novel mutation Y113H, in vitro functional study was performed. According to previous research, many
SFTPC mutations such as exon 4 deletion cause chronic accumulation of misfolded proSP-C leading to endoplasmic reticulum (ER) stress and alveolar type II cell apoptosis [
25,
26]. In our Western bloting analysis, multiple bands of proSP-C
Y113H and proSP-C
D105G were missing or significantly reduced when compared with the wild-type proprotein suggesting aberrant protein processing of both mutant proteins. However, an accumulated proprotein at 20 kDa was observed for proSP-C
D105G while not for proSP-C
Y113H indicating distinct proprotein processing. Immunofluorescence assay of transfected A549 cells showed proSP-C
Y113H and proSP-C
D105G both predominantly colocalized with EEA1 but not with lamellar body marker CD63. So we speculate, unlike proSP-C
WT secreted via lamellar body fusion with the plasma membrane and then catabolized mainly by alveolar macrophages, misfolded proSP-C
Y113H and proSP-C
D105G were endocytosed into endosome. ProSP-C
D105G may trigger the unfolded protein response and result in ER stress while proSP-C
Y113H may be degraded by the ubiquitin–proteasome system as previously described [
13]. The difference in molecular pathogenesis between D105G and Y113H may partially explain why imcomplete penetrance occured in family of patient 5 but not patient 3.
A limitation of the study is that no lung samples of these patients were available. Therefore in vivo data regarding mutant SP-C expression and ultrastructure of alveolar type II cells were unclear. Besides, due to the rareness of this disease, our study is limited by small sample size and further multicenter study will be needed in the future. Also, a longer follow-up period is needed to determine the long-term outcome of the patients and the efficacy of the treatment.
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