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ABSTRACT Different genes related to alveolar stability have been associated with familial interstitial pneumonia (FIP). Here, we report a novel, rare _SFTPA1_ variant in a family with

idiopathic interstitial pneumonia (IIP). We performed whole-exome sequencing on germline DNA samples from four members of one family; three of them showed signs of pulmonary fibrosis

(idiopathic interstitial pneumonia) with autosomal-dominant inheritance. A heterozygous single nucleotide variant c.532 G > A in the _SFTPA1_ gene has been identified. This variant

encodes the substitution p.(Val178Met), localized within the carbohydrate recognition domain of surfactant protein A and segregates with the genes causing idiopathic interstitial pneumonia.

This rare variant has not been previously reported. We also analyzed the detected sequence variant in the protein structure _in silico_. The replacement of valine by the larger methionine

inside the protein may cause a disruption in the protein structure. The c.532 G > A variant was further validated using Sanger sequencing of the amplicons, confirming the diagnosis in all

symptomatic family members. Moreover, this variant was also found by Sanger sequencing in one other symptomatic family member and one young asymptomatic family member. The

autosomal-dominant inheritance, the family history of IIP, and the evidence of a mutation occurring in part of the _SFTPA1_ gene all suggest a novel variant that causes FIP. SIMILAR CONTENT

BEING VIEWED BY OTHERS WHOLE-EXOME SEQUENCING IDENTIFIES SUSCEPTIBILITY GENES AND PATHWAYS FOR IDIOPATHIC PULMONARY FIBROSIS IN THE CHINESE POPULATION Article Open access 14 January 2021

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PULMONARY FIBROSIS Article 28 June 2023 INTRODUCTION Familial interstitial pneumonia (FIP) is defined as idiopathic interstitial pneumonia (IIP) and affects two or more first-degree

relatives who have been diagnosed with characteristics of IIP1. IIP belongs to a group of interstitial lung diseases (ILDs). ILDs are a heterogeneous group of predominantly chronic diseases

characterized by various degrees of inflammation and pulmonary fibrosis at the level of the interstitium, alveolar ducts, alveoli, pulmonary capillaries and respiratory bronchioles2. There

is evidence that the development of pulmonary fibrosis is genetically determined, and genetic testing is considered for IIP cases with (1) familial clustering of IIP; (2) IIP associated with

other inherited disorders; or (3) when there is significant variability in the development of pulmonary fibrosis among individuals exposed to a dusty environment3. Moreover, several

phenotypes of fibrotic interstitial lung processes may be present in members of one family. Idiopathic pulmonary fibrosis/usual idiopathic pneumonia (IPF/UIP) is the most frequently observed

phenotype in the familial occurrence of IIP4. There are no differences in clinical, radiologic, or histological features between familial IPF and nonfamilial IPF cases5. In FIP, many genes

may be pathogenic. FIP risk factor genes include telomerase catalytic activity genes (_TERT –_ telomerase reverse transcriptase; _TERC –_ telomerase RNA component), genes affecting

telomerase biogenesis (_DKC1_ – dyskerin; PARN _–_ polyadenylation-specific ribonuclease deadenylation nuclease; _NAF1 –_ nuclear assembly factor 1 ribonucleoprotein) and genes that alter

telomeres (_TINF2 –_ telomere-interacting factor 2; _RTEL1 –_ regulator of telomere-elongation helicase-1). Mutations associated with adult-onset FIP are also rarely found in genes that

encode surfactant proteins, such as the heterozygous mutations of surfactant proteins A and C (_SFTPA1, SFTPA2_, and _SFTPC_). Rare biallelic variants in the genes encoding surfactant

protein B (_SFTPB_) and branched-chain amino acid aminotransferase (_BCA3_) have also been described. Altogether, mutations in _SFTPC, SFTPA2, TERT_, and _TERC_ clarify a maximum of 20% of

all FIP cases6,7,8,9,10,11,12,13,14,15,16,17. However, common variants (an allele frequency in the population above 5%) in genes associated with a risk of FIP and IPF have been observed.

Most often, the single nucleotide polymorphism (SNP) rs35705950 of the _MUC5B_ gene has been strongly associated with both FIP and IPF across multiple different cohorts18,19. Here, we

present the FIP case in which we have revealed a novel germline pathogenic variant in the _SFTPA1_ gene. MATERIALS AND METHODS SUBJECTS THE PROBAND Figure 1 (_II-1_) was a male nonsmoker who

was referred to the Department of Pneumology and Phtiseology, University Hospital, Brno, Czech Republic because of IIP at the age of 46 years. The diagnosis of IIP was based on standard

criteria2. The proband presented with dyspnea and a dry cough. Lung function testing showed a restrictive syndrome with a mild reduction in diffuse lung capacity. High-resolution computed

tomography (HRCT) of the patient’s chest showed interlobular septal thickening and ground glass opacities, especially in the lower lung areas (Fig. 2a). Video-assisted thoracoscopic (VATS)

lung biopsy revealed pulmonary fibrosis with a nonspecific interstitial pneumonia pattern. He died of respiratory failure due to acute exacerbation of pulmonary fibrosis 11 years after

diagnosis. A detailed family history revealed that several of the patient’s family members had pulmonary fibrosis or interstitial lung involvement. The proband´s mother (_I-2_), a nonsmoker,

died of respiratory failure at the age of 73 years. She was diagnosed with IIP seven years ago. HRCT of the thorax initially showed predominant ground glass opacities, inter- and

intralobular septal thickening and bronchiectasis that did not correspond with the possible, typical interstitial pneumonia (Fig. 2b). However, a VATS lung biopsy and histological

examination revealed a pattern of typical interstitial pneumonia. The clinical course gradually deteriorated over time despite therapy. THE PROBAND´S SISTER (II-2), a nonsmoker with digital

clubbing, developed dyspnea at the age of 44 years. An HRCT of her thorax showed ventral and dorsobasal subpleural interlobular septal thickening. THE PROBAND´S SON (III-1), a nonsmoker, had

suffered from shortness of breath during exercise since the age of 25. He also developed digital clubbing. An HRCT of his thorax revealed discrete nonspecific ground glass opacities in the

upper and lower right lobes. THE PROBAND´S DAUGHTER (_III-2_), a nonsmoker, was asymptomatic. All results of the clinical and radiology examinations, functional tests and treatment of family

members with pulmonary symptoms are summarized in Supplemental Table S1. The pedigree analysis (Fig. 1) indicates an autosomal-dominant mode of inheritance. FIP was therefore suspected, and

diagnostics were extended by genetic testing of the family members, who gave written informed consent according to the Declaration of Helsinki. MUTATIONAL SCREENING Blood samples from nine

family members were collected and processed for genomic DNA isolation using the MagCore® Genomic DNA Whole Blood Kit (RBC Bioscience, USA). We performed whole-exome sequencing (WES) on

samples from four family members (I-2, I-3, II-1, and III-1). Whole-exome libraries were prepared using the Kapa Hyper Prep Kit (Roche, USA) according to the protocol for NimbleGen SeqCap EZ

Exome v3 (Roche, USA). Paired-end 2 × 75 bp sequencing was performed on an Illumina NextSeq 500 sequencer (Illumina Inc., USA). The raw sequencing reads were aligned to the GRCh37 human

reference genome using the BWA mem algorithm, version 0.7.15. PCR duplicates were identified with the MarkDuplicates tool from Picard version 2.9.2. GATK HaplotypeCaller, version 3.7, was

used to detect germline single nucleotide variants (SNV) and indels. Obtained variants/indels have been annotated using Annovar program version (2018Apr16). On the basis of the current

knowledge, we have chosen 30 candidate genes previously associated with _IPF_: _TERC_, _TERT_, _SFTPC_, _SFTPA1_, _SFTPA2_, _MUC5B_, _MUC5C_, _RTEL1_, _PARN_, _ABCA3_, _DKC1_, _TINF2_,

_IL1RN_, _IL8_, _FAM13A_, _TLR3_, _HLA- DRB1_, _HLA- DQB1_, _DSP_, _OBFC1_, _MUC2_, _TOLLIP_, _ATP11A_, _MDGA2_, _MAPT_, _SPPL2C_, _DPP9_, _TGFB1_, _NAF1_, and

_OBFC1_7,8,9,10,11,12,13,14,15,16,17. We then looked more deeply into the exonic variants of these genes. RESULTS The analysis revealed a novel variation c.532 G > A in exon 6 of the

_SFPTA1_ gene (reference sequence: NM_005411.4), located within the carbohydrate recognition domain of surfactant protein A. The variant was found to be heterozygous in three affected family

members (I-2, II-1, III-1) with IIP but was absent in the healthy individual I-3 (Fig. 1). The coverage range of c.532 G > A was 66–126 in all affected samples, and the variant allele

frequency range was 41.41%-59.09%. This identified variant (c.532 G > A) has not been described previously and is absent in the ExAC, 1000 genomes, ESP (exome sequencing projects) 6500,

KAVIAR, gnomAD, and HGMD databases20,21. The c.532 G > A variant was further validated using PCR and Sanger sequencing of the amplicons, confirming the diagnosis in all affected family

members (I-2, II-1, and III-1). Due to the similarity of the _SFTPA1_ and _SFTPA2_ genes, we designed a specific primer set corresponding to the differences in the nucleotide sequence

(forward primer: 5´-TGGTCAGTGGCCTGACCC-3´ and reverse primer: 5´-AGAGTCAGGGCCCATCAGA-3´). PCR was performed with an annealing temperature of 60 °C using Q5 High-Fidelity DNA Polymerase (New

England Biolabs Inc., England) according to the manufacturer´s protocol. PCR products were purified using a Qiaquick PCR purification kit (QIAGEN, Germany). Capillary sequencing was

performed using BigDye-terminator chemistry on a 3500 Genetic Analyzer (Applied Biosystems, USA). The analysis showed that the variant c.532 G > A was heterozygous in the I-2, II-1, II-2

and III-1 family members (Fig. 3). Subsequently, PCR and Sanger sequencing of c.532 G > A was performed in the other family members (I-1, II-2, II-3, II-4, and III-2). Moreover, the c.532

 G > A heterozygous variant was found in two additional family members: II-2 with disease symptoms and III-2, the young but still asymptomatic family member. IN SILICO ANALYSIS Currently,

there is no evidence for any known functional impacts of the c.532 G > A; p.Val178Met variant in the _SFPTA1_ gene, which encodes the missense substitution valine to methionine in the

α-helix (the carbohydrate recognition domain of surfactant protein A1). Valine178 is a highly conserved amino acid and is found in up to 11 frog species. We performed an assessment of the

severity of the identified variant using _in silico_ prediction analysis: the Sorting Intolerant from Tolerant (SIFT), Polymorphism Phenotyping v2 (Poly-Phen-2), MutationTaster, Align GVGD

and CADD_phred programs (http://sift.jcvi.org/www/SIFT_seq_submit2.html; http://genetics.bwh.harvard.edu/pph/; http://www.mutationtaster.org/; http://agvgd.hci.utah.edu/agvgd_input.php)22.

According to SIFT, this variant with a score of 0.01 was “deleterious” (the score of predicted damage was < 0.05). The Poly-Phen-2 program evaluated p.Val178Met as “probably damaging”

with a score of 1,0 (a maximum score). On the other hand, MutationTaster predicted this variant as a “polymorphism” with a p-value of 1 (score of predicted damage is < 0.09), and the

Align GVGD (Grantham Variation and Grantham Deviation) defined “the lowest risk” for the prediction of missense substitutions with the grade C0 (C0 is the first of a total of seven grades).

However, the scaled CADD_phred score was calculated as 16.72. This result of scaled transformation was assigned to the top 1% of single nucleotide variants and may predict a potentially

pathogenic variant. We therefore analyzed the c.532 G > A; p.Val178Met variant effects via protein structure modeling. Currently, there is no available crystal structure of this human

protein, so we used the homolog protein structure of porcine surfactant protein D neck and carbohydrate recognition domains complexed with mannose (PDB code 4DN8), which shows 43% sequence

homology with the human protein. In the homology model, valine178 is positioned in the α-helix, which is buried inside the protein. The replacement of this valine (its volume is 140.0 Å3)23

by larger methionine (its volume is 162.9 Å3) will probably cause a disruption of the protein structure (supplemental Figure S1). DISCUSSION Unfortunately, we do not have accurate FIP

prevalence and incidence data. However, some studies estimate that the incidence of FIP is approximately 2-10% of all IIP cases1,3,8,12. The inheritance of FIP is most commonly

autosomal-dominant with incomplete penetrance4,5,19,24,25,26. Autosomal-recessive types of inheritance have rarely been described27. Our case shows signs of an autosomal-dominant

inheritance. Both familial and sporadic cases of pulmonary fibrosis are not clinically or histologically recognizable, although familial cases can be diagnosed in younger individuals3. Rosas

et al.28 determined the clinical, radiographic, and histological features of asymptomatic ILDs in the relatives of patients with FIP. Histopathological findings in patients with FIP are

heterogeneous29. Steele et al.4 described several histopathological subtypes of IIP found in members of one family. Our case also shows different histological, clinical and radiological

findings in affected individuals. FIP is caused mainly by rare pathogenic gene variants (with an allele frequency in the population of less than 0.1%)30. Familial studies have found

FIP-associated genes influencing alveolar stability: _SFTPA1_15, _SFTPA2_14, and _SFTPC_;30,31,32 _ABCA3_ (ATP-binding cassette - type 3);32 genes related to telomerases, including _TERT_29,

and _TERC_;6,29 _DKC1_ (dyskeratin);8,29 _TINF2_;7,9,29 or _RTEL1_10,11,29,33. On the other hand, common variants are also observed in FIP; most often, the variant rs35705950 is located in

the promoter region of the _MUC5B_ gene29,34. Surfactant proteins (SPs) are divided into hydrophilic (SP-A and SP-D) and hydrophobic (SP-B and SP-C) categories. SP-A, SP-B, SP-C and SP-D are

synthesized by type II alveolar epithelial cells. SP-A and SP-D play roles in lung defense, and SP-B and SP-C ensure proper surfactant function35,36. The main protein of pulmonary

surfactant is SP-A37. The two closely related genes, _SFTPA1_ and _SFTPA2_, are located near _SFTPD_ on chromosome 10q22.3 and encode SP-A. The nucleotide sequence of the _SFTPA1_ gene is

70% identical to that of _SFTPA2_. The pathogenic variants of _SFTPA1_ and _SFTPA2_ can cause FIP and pulmonary adenocarcinoma. All pathogenic heterozygous mutations in the _SFTPA1_ and

_SFTPA2_ genes are missense and lead to a decreased secretion of mature protein by alveolar cells15,38,39,40. In our case, the novel single nucleotide variant c.532 G > A was detected in

the _SFTPA1_ gene and encodes a p.Val178Met substitution. To date, 5 missense/nonsense _SFTPA1_ variants have been identified (https://portal.biobase-international.com/hgmd/pro/gene.php?gene

 = SFTPA1; 09 AUG 2018)15,20,41. Nathan et al. described in detail a molecular defect in _SFTPA1_ in FIP15. Unfortunately, there is no consensus to determine which IPF patient could benefit

from genetic testing. The international guidelines for IPF did not recommend genetic testing42, and genetic testing is not even mentioned in the guidelines43. On the other hand, the

international guidelines for IIP published in 2013 propose searching for genetic abnormalities in patients with FIP44. As proposed by Kropski et al.45, genetic testing is not recommended for

every ILD patient. Genetic counseling should be offered to patients with FIP, to patients with IIP in the context of rare inherited disorders, to individuals with disease onset before the

age of 18 years, or to patients with significant variability in the development of pulmonary fibrosis after exposure to a dusty environment3,45. For asymptomatic patients (similar to our

patient III-2), a chest HRCT is recommended at age 40 or 10 years before the age of disease onset in the proband. If signs of ILD are not present, an HRCT scan should be repeated every 5

years35. In conclusion, we have described a novel _SFTPA1_ heterozygous variant in family members with IIP. Such findings are of the utmost importance, and an early FIP diagnosis for the

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Care. Med._ 195, 1423–1428 (2017). Article  CAS  PubMed  PubMed Central  Google Scholar  Download references ACKNOWLEDGEMENTS This work was supported by Research Grant AZV 16–29447 A of

Ministry of Health, Czech Republic and CEITEC 2020 (LQ1601) of Ministry of Education, Youth and Sports. We also acknowledge the CF Genomics CEITEC MU supported by the NCMG research

infrastructure (LM2015091 funded by MEYS CR) for their support in obtaining the scientific data presented in this paper. AUTHOR CONTRIBUTIONS All authors were involved in the data analysis

and interpretation. Moreover, MD, JT, ZV and MD2 were involved in clinical data acquisition and manuscript preparation. All authors reviewed the manuscript and approved the final version for

submission. AUTHOR INFORMATION Author notes * These authors contributed equally: Martina Doubková and Kateřina Staňo Kozubík. AUTHORS AND AFFILIATIONS * Department of Pneumology and

Phtiseology, University Hospital and Faculty of Medicine, Brno, Czech Republic Martina Doubková * Central European Institute of Technology, Masaryk University, Brno, Czech Republic Kateřina

Staňo Kozubík, Lenka Radová, Michaela Pešová, Jakub Trizuljak, Karol Pál, Klára Svobodová, Kamila Réblová, Hana Svozilová, Zuzana Vrzalová, Šárka Pospíšilová & Michael Doubek *

Department of Internal Medicine, Hematology and Oncology, University Hospital and Faculty of Medicine, Brno, Czech Republic Kateřina Staňo Kozubík, Jakub Trizuljak, Hana Svozilová, Zuzana

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