Clinical characteristics of pulmonary non-tuberculous mycobacterial disease with CFTR variants in the Japanese population

Abstract

Background

Pulmonary non-tuberculous mycobacterial (NTM) disease is a respiratory infection with an increasing incidence worldwide, including Japan. Host factors may also be involved in the establishment of pulmonary NTM disease. Cystic fibrosis transmembrane conductance regulator (CFTR) variants are associated with pulmonary NTM disease and bronchiectasis. However, data on CFTR variants in the Japanese population remain limited.

Objectives

We aimed to determine the frequency of CFTR variants and the impact on the clinical features of pulmonary NTM disease and bronchiectasis in the Japanese population.

Methods

We analysed 458 patients with either pulmonary NTM disease, non-cystic fibrosis bronchiectasis or both at Keio University Hospital from February 2016 to March 2019. CFTR variants were identified using exome sequencing, Sanger sequencing and multiplex ligation-dependent probe amplification (MLPA). These variants were determined to be deleterious using CFTR2 and in silico tools. Clinical characteristics of patients with and without CFTR variants were compared in a 1:8 age-matched and sex-matched ratio. Additionally, exome sequencing was performed for the family of a patient with a family history of pulmonary NTM disease.

Results

Deleterious CFTR variants were identified in 16 patients (3.5%). One variant was identified by MLPA, and 15 by Sanger sequencing. All patients harboured a CFTR variant in one allele. Compared with matched controls, these patients had lower sputum culture conversion rates and higher rates of macrolide resistance. In one family cluster, members with pulmonary NTM disease were found to carry the same CFTR variant.

Conclusions

We defined the frequency and clinical characteristics of CFTR variants among the Japanese population with either pulmonary NTM disease, non-cystic fibrosis bronchiectasis or both and found that patients with CFTR variants may be refractory to pulmonary Mycobacterium avium complex disease. Further comprehensive research is needed to assess the impact of CFTR variants on pulmonary NTM disease and bronchiectasis in non-European populations.

WHAT IS ALREADY KNOWN ON THIS TOPIC

• In European populations, cystic fibrosis transmembrane conductance regulator variants have been associated with pulmonary non-tuberculous mycobacterial disease and bronchiectasis.

WHAT THIS STUDY ADDS

• This study describes the frequency of these variants in Japanese patients and highlights their link to poor treatment outcomes in pulmonary Mycobacterium avium complex disease.

HOW THIS STUDY MIGHT AFFECT RESEARCH, PRACTICE OR POLICY

• These findings may help guide management strategies for affected people in Japan.

Introduction

Pulmonary non-tuberculous mycobacterial (NTM) disease is a respiratory infection with an increasing incidence worldwide, including in Japan.1,3 Non-tuberculous mycobacteria are environmental mycobacteria found ubiquitously in water and soil. Pulmonary Mycobacterium avium complex (MAC) disease is more common in thin, middle-aged women without pre-existing lung disease.⁴ In addition, a Hawaiian cohort study revealed a higher incidence of pulmonary NTM disease in an Asian population.⁵ Thus, host factors may be involved in the establishment of pulmonary NTM disease.

Cystic fibrosis (CF) is an autosomal recessive disease caused by mutations in the CF transmembrane conductance regulator (CFTR) gene.⁶ CF is characterised by loss of CFTR function, which results in impaired water transport into the lumen, abnormal viscous mucus secretion and postnatal ileus, causing meconium ileus, pancreatic insufficiency and recurrent lower respiratory tract infections.⁷ Clinically, European guidelines recommend that patients with clinical features of CF, such as young onset, upper lobe-predominant bronchiectasis, sinusitis, nasal polyps, recurrent pancreatitis and male infertility, should be tested for CF using methods such as a sweat chloride test and genetic analysis.⁸ Pulmonary disease is the main determinant of prognosis in people with CF. Bronchiectasis progresses because of repeated lower respiratory tract infections, resulting in respiratory failure and death.⁹

CF occurs in 1 in 3500 individuals in Europe and the USA.¹⁰ However, it is estimated to occur in 1 in 590 000 people in Japan.¹¹ More than 2000 variants have been identified in the CFTR gene, of which approximately 800 are defined as CF-causing variants in the CFTR2 database.¹² CF-causing variants vary by region and population. Different profiles of CFTR variants have been reported in Asian and European populations. The p.Phe508del mutation accounts for 85% of CF-causing variants in the Europe and the USA.¹³ In Japan, exon 16a-17 is the most common CF-causing variant in East Asian/Japanese-origin patients with CF, whereas p.Phe508del is detected in less than 25% of CF cases.¹⁴ As exon deletions can be missed by exome or Sanger sequencing, tests to detect exon copy number abnormalities (eg, multiplex ligation-dependent probe amplification (MLPA)) might be useful for detecting CFTR variants in Japanese patients.¹⁵

The CFTR-related disorder (CFTR-RD) caused by one CFTR variant is defined as a condition that does not meet the diagnostic criteria for CF but displays single-organ symptoms due to CFTR dysfunction.¹⁶ From the perspective of pulmonary NTM disease, two studies from America reported that CFTR variants were found in half and 36%, respectively.^{4 17} Thus, CFTR-RD is assumed to be a risk factor for pulmonary NTM disease.

The CFTR variant discovered in a Korean population, p.Gln1352His, has been reported to be associated with susceptibility to pulmonary NTM disease.¹⁸ A previous study showed an association between the T5 allele in intron 8 of CFTR variants and pulmonary MAC disease in a Japanese population with pulmonary NTM disease¹⁹; however, there has been a lack of comprehensive exploration of the frequency and types of CFTR variants among Japanese patients with pulmonary NTM disease/bronchiectasis. Therefore, this study aimed to determine the frequency of CFTR variants in a Japanese population with either pulmonary NTM disease, non-CF bronchiectasis (NCFB) or both as well as to evaluate their clinical characteristics.

Methods

Study population

This study included 458 patients who were enrolled at the Keio University Hospital (Tokyo, Japan) from February 2016 to March 2019, all of whom had a diagnosis of either pulmonary NTM disease, NCFB or both. Patients were recruited from our previous genome-wide association study of pulmonary NTM disease (UMIN000021692),²⁰ including patients attending our hospital because their clinical information was easily accessible. Pulmonary NTM disease and bronchiectasis were diagnosed according to the diagnostic criteria of the American Thoracic Society/Infectious Disease Society of America for NTM lung disease²¹ and recommendations for clinical trials on bronchiectasis,²² respectively. NTM isolates were identified as previously described.²³ Patients and the public were not involved in the design, conduct, reporting or dissemination of this research.

Whole-exome and Sanger sequencing

Genomic DNA was extracted from the peripheral blood of patients. Exon regions in genomic DNA with variants were detected via exome analysis as previously described.²⁴ CFTR variants were confirmed using Sanger sequencing. Regions of interest for exons were amplified via PCR with gene-specific primers (online supplemental table 1) and the AmpliTaq Gold 360 Master Mix (Thermo Fisher Scientific, Waltham, Massachusetts) using an Applied Biosystems GeneAmp PCR System 9700 (Thermo Fisher Scientific). The PCR products were processed using ExoSAP-IT (Thermo Fisher Scientific), and then sequenced with sequencing primers. Sanger sequencing was performed by Azenta Life Sciences (Tokyo, Japan), and sequencing data were assessed using ApE and UGENE software.

Quantitative fragment analysis via MLPA

MLPA analysis is a molecular genetic technique that can be used to detect copy number variations. A SALSA P091-C1 CFTR MLPA kit (MRC Holland, Amsterdam, Netherlands) was used in accordance with the manufacturer’s protocol. Briefly, PCR amplification was performed for 35 cycles, and 15 µL unlabelled PCR product was separated on a 2% agarose gel. Fragments were separated on a 3500 Genetic Analyzer (Applied Biosystems, Waltham, Massachusetts) using GeneScan 500 LIZ dye Size Standard (4322682; Applied Biosystems), then analysed using Coffalyzer Net Software (MRC Holland).

Filtering of CFTR variants

We assessed the pathogenicity of CFTR variants using the following steps. Initially, CFTR variants identified through Sanger sequencing were checked for pathogenicity using the CFTR2 database. Next, CFTR variants annotated with uncertain significance in CFTR2 or those not listed in the database were evaluated for their functional effects. Synonymous variants were excluded, and missense variants were evaluated by using in silico tools (CADD,²⁵ AlphaMissense²⁶ and REVEL²⁷) to predict variant function. Splice region and intron variants were evaluated by using CADD and SpliceAI.²⁸ All variants were referred to in ClinVar²⁹ for consistency with previous reports. We applied the following threshold for each in silico prediction tool: CADD score >15, REVEL score >0.5, AlphaMissense score >0.564 and SpliceAI score >0.2. Variants were included for further analysis if missense variants exceeded the thresholds in at least two out of three prediction tools, and if splice region and intronic variants exceeded the thresholds for both CADD and SpliceAI. The frequency of variants in the global population was obtained from the Genome Aggregation Database (gnomAD; https://gnomad.broadinstitute.org),³⁰ and the frequency in the Japanese population was obtained from TogoVar (https://togovar.org).³¹

Clinical characteristics of patients with CFTR variants

The clinical information of patients with deleterious variants was collected from their medical records. The following data were collected at the time of diagnosis of pulmonary NTM disease or bronchiectasis: age at onset, sex, body mass index, smoking history, comorbidities, familial history of pulmonary NTM disease, CT findings, pulmonary function tests, acid-fast bacilli smears and mycobacterial/bacterial cultures. For bronchiectasis without a diagnosis of pulmonary NTM disease, data were collected at the onset of bronchiectasis.

Comparison of the clinical characteristics of patients with and without CFTR variants

To compare clinical characteristics between patients with and without CFTR variants, we included only those with a confirmed diagnosis of pulmonary NTM disease in the case group. These patients were compared at a 1:8 ratio with age-matched and sex-matched (within 5 years) patients without CFTR variants, who were selected only from those diagnosed with pulmonary MAC disease, the most common causative pathogen of pulmonary NTM disease in Japan. Findings from physical, CT imagings and pulmonary function tests were used at or near the time of diagnosis, whereas bacterial findings and treatment details were used within the observation period. Next, we compared only the patients who had received treatment between the case and control groups. Coinfection was defined as repeated isolation of the same pathogen from sputum samples within 1 year before or after the initiation of treatment. Culture conversion, treatment failure and cure were defined according to the NTM-NET consensus statement.³² For patients who could not expectorate sputum, culture conversion was deemed positive if there was an improvement in imaging and clinical symptoms.³³ The history of exacerbation was defined as requiring a change in bronchiectasis treatment.³⁴ Clinical information was retrospectively collected from electronic medical records for the period available up to February 2023.

Statistical analysis

The R statistical software (V.4.3.2) was used for all analyses. Quantitative values are presented as medians and categorical variables are presented as numbers and percentages. Comparison of culture conversion, treatment failure and macrolide resistance rate between patients with and without CFTR variants was analysed using Fisher’s exact test. Values of p<0.05 were considered statistically significant.

Results

Genetic CFTR variants in the Japanese population

Figure 1 shows the result of the identification of CFTR gene variants. We identified 30 variants of the CFTR gene via exome sequencing. Three samples were excluded because Sanger sequencing could not be performed on them, owing to insufficient specimens available. One sample was excluded because the Sanger sequencing results revealed no variants. Twenty-six variants were evaluated using in silico analysis tools. Through screening with CFTR2, we found that one case (p.Arg764Ter) was listed as a pathogenic variant, and another (p.Ser912Leu) listed as having uncertain significance in the database; the rest were not listed. We then used in silico functional prediction tools (CADD, AlphaMissense and REVEL) and included variants of uncertain significance. We also performed evaluations in ClinVar and referred to previously published data. Using our algorithm, we excluded 11 variants: five synonymous, two missense, three splice region and one intron variant. Finally, 15 CFTR variants were passed through our filtering criteria (table 1). There were no cases of patients who had CFTR variants in both alleles.

Figure 1. Diagram illustrating the steps used for extracting CFTR variants in this study. The MLPA flow is illustrated in the middle and that of exome and Sanger sequencing illustrated on the right. CFTR, cystic fibrosis transmembrane conductance regulator; MAC, Mycobacterium avium complex; MLPA, multiplex ligation-dependent probe amplification; NTM, non-tuberculous mycobacteria.

Table 1. CFTR variants identified via Sanger sequencing.

Patient number	cDNA	Amino acid*	Variant function	SNP ID*	SNP position†	Exon	Frequency		in silico tool
							gnomAD*	ToMMo*	CADD	AlphaMissense	REVEL	SpliceAI
1	c.113A>G	p.Tyr38Cys	missense	rs758826243	7:117504312	2	0.0000319	0.0009909	26.4	0.299	0.858	0
2	c.727A>C	p.Met243Leu	missense		7:117535395	6			23.1	0.0997	0.548	0
3	c.971C>T	p.Pro324Leu	missense	rs397508822	7:117540201	8		0.0000354	25.8	0.645	0.779	0.02
4	c.1306C>G	p.Leu436Val	missense	rs2115903382	7:117548737	10			19.57	0.0914	0.233	0
5	c.1558G>A	p.Val520Ile	missense	rs77646904	7:117559629	11	0.0000659	0.0001522	20.5	0.103	0.585	0
6	c.2042A>T	p.Glu681Val	missense	rs201295415	7:117592209	14	0.0017694	0.0001355	24.2	0.146	0.621	0.36
7	c.2290C>T	p.Arg764Ter	stop gained	rs121908810	7:117592457	14		0.0000078	34		NA	0.02
8	c.2294G>A	p.Arg765Lys	missense	rs557228597	7:117592461	14	0.0000045	0.0002123	26.3	0.204	0.717	0
9	c.2626G>C	p.Ala876Pro	missense	rs1328356747	7:117602832	16	0.0000066	0.0000708	8.919	0.765	0.648	0
10	c.2735C>T	p.Ser912Leu	missense	rs121909034	7:117603609	17	0.0009937	0.0001769	15.77	0.125	0.543	0
11	c.3140–3C>G		splice region		7:117611578	20			26.9		NA	0.91
12	c.3406G>A	p.Ala1136Thr	missense	rs755968404	7:117614651	21	0.0000389	0.0000708	27.1	0.408	0.845	0.01
13	c.3468G>T	p.Leu1156Phe	missense	rs139729994	7:117614713	21	0.0001316	0.0259042	33	0.391	0.604	0.71
14	c.4110T>G	p.Asp1370Glu	missense	rs1793345654	7:117664834	25		0.0000659	23	0.956	0.941	0
15	c.4225G>A	p.Glu1409Lys	missense	rs397508699	7:117665547	26	0.000004	0.0003185	28.3	0.511	0.75	0

^*Amino acid, SNP ID, gnomAD, ToMMo: empty fields are not named or listed in the database.

^†SNP position: GRCh38.

CFTR, cystic fibrosis transmembrane conductance regulator.

MLPA was performed for all 458 cases, where one case was found to have a numerical exon abnormality (online supplemental figure 1). The variant was exon 16-17b del (new numbering: exon 18–20 del), and the other allele had no CFTR variant. This variant was listed as a pathogenic variant of CFTR2.

In our study, a total of 16 cases (3.5%), 15 identified via exome and Sanger sequencing and one via MLPA, showed CFTR variants that may be damaging variants. The CFTR variant, which is recommended for newborn screening tests, was not detected in this study.³⁵ Figure 2 visualises the position on the CFTR gene of variants found in our study and those recommended for screening.

Figure 2. Positions of variants in the CFTR gene. Exon numbers represent new numbering. Upper row. CFTR variants identified in our study. Lower row. CFTR variants recommended by the ACMG in screening for cystic fibrosis. ACMG, American College of Medical Genetics; CFTR, cystic fibrosis transmembrane conductance regulator.

Clinical characteristics of cases with CFTR variants

Clinical characteristics of the 16 patients with CFTR variants are shown in online supplemental table 2. The median age of onset was 60.5 years old. Fourteen patients (87.5%) had a bacteriological diagnosis of pulmonary NTM disease; 13 of them were infected with MAC and one with M. lentiflavum. The imaging patterns were identified as an NB type in all cases; three patients (18.8%) had a family history of pulmonary NTM disease and five (31.2%) had been treated for pulmonary MAC disease. There were no cases of pancreatitis, and five patients (31.2%) had findings of chronic sinusitis.

Comparison of clinical characteristics of cases with and without CFTR variants

We compared the clinical information of 14 patients with pulmonary NTM disease with CFTR variants, excluding two patients who did not have a diagnosis of pulmonary NTM disease from the above 16 patients, and 112 patients with pulmonary MAC disease without CFTR variants matched for age and sex. Sex was matched with the same ratio (male:female=3:11), and age of onset showed no difference (case 59.0 years (53.5–63.8) vs control 59.0 years (52.8–64.0), median (IQR)). All patients with CFTR variants had nodular bronchiectasis (NB) on imaging. While patients with CFTR variants exhibited higher modified Reiff scores than those without (p=0.02), the median score was 2 (table 2).

Table 2. Comparison of clinical characteristics between pulmonary NTM patients with and without CFTR variants.

	CFTR variant (+) (n=14)	CFTR variant (−) (n=112)
Age at diagnosis (years)	59.0 (53.5–63.8)	59.0 (52.8–64.0)
Sex male/female	3/11 (21.4/78.6)	24/88 (21.4/78.6)
Duration of follow-up (years)	8.5 (6.1–13.1)	11.2 (8.0–14.7)
Duration of disease (years)	12.7 (9.7–17.9)	11.4 (8.4–14.9)
Never/ex or current smoker	10/4 (71.4/28.6)	87/25 (77.7/22.3)
Body mass index (kg/m2)	19.3 (18.7–20.9)	19.1 (18.0–21.5)
Family history of pulmonary NTM disease	2 (14.3)	6 (5.4)
Comorbidity
Cancer	1 (7.1)	16 (14.3)
Connective tissue disease	1 (7.1)	3 (2.7)
Underlying pulmonary disease	0 (0.0)	3 (2.7)
Sinusitis	4 (28.6)	28 (25.0)
Radiological findings
Modified Reiff score	2 (1.0–3.0)*	1 (0.0–2.0)
Radiographic pattern
FC type	0 (0.0)	4 (3.6)
NB type	14 (100.0)	102 (91.1)
Unclassified	0 (0.0)	6 (5.4)
Presence of cavitary lesion	1 (7.1)	17 (15.2)
Pulmonary functional test
FVC (% predicted)	107.2 (85.9–111.7)	97.0 (91.5–109.0)
FEV1 (% predicted)	93.1 (80.0–103.9)	92.1 (81.8–100.8)
FEV1/FVC (%)	75.9 (73.5–77.2)	75.1 (71.2–80.7)
Microbiology
AFB smear positive at diagnosis	1 (7.1)	33 (29.5)
Mycobacterium species
Mycobacterium avium complex	13 (92.9)	112 (100)
Mycobacterium lentiflavum	1 (7.1)	0 (0.0)
Pseudomonas aeruginosa	2 (14.3)	7 (6.2)

Data shown median (IQR) or number (%) of patients.

^*P<0.05: significant compared with patients without CFTR variant.

AFB, acid fast bacilli; CFTR, cystic fibrosis transmembrane conductance regulator; FC, fibrocavitary; FEV₁, forced expiratory volume in 1 s; FVC, forced vital capacity; NB, nodular bronchiectasis; NTM, non-tuberculous mycobacterial.

During the follow-up, 5 patients with CFTR variants (35.7%) and 76 patients without CFTR variants (67.9%) received treatment for pulmonary NTM disease. All patients received treatment with multiple drug regimens including macrolides, except for one patient without CFTR variants who could not use macrolides due to side effects. There were no differences between patients with and without CFTR variants in age at the initiation of treatment, nutritional status (body mass index and serum albumin), modified Reiff score or extent of radiological lesions and the presence of coinfections. Among treated patients, those with CFTR variants had lower 12-month culture conversion rates (20.0% vs 69.7%, p=0.04) than those without CFTR variants (table 3). No patients were confirmed of macrolide resistance at the start of treatment. During the treatment, patients with CFTR variants developed macrolide resistance more than those without variants (60.0% vs 11.8%, p=0.02). The median time from treatment initiation to the detection of macrolide resistance was 5.0 years (3.3–6.1). The results of sputum cultures for patients with CFTR variants over a period of 2 years after the initiation of treatment are shown in online supplemental figure 2, and all of them developed macrolide resistance 2 years after the initiation of antibacterial treatment.

Table 3. Comparison of outcome between treated pulmonary MAC patients with and without CFTR variants.

	CFTR variant (+) (n=5)	CFTR variant (−) (n=76)
Age at diagnosis (years)	60.0 (57.0–62.0)	57.5 (48.8–64.0)
Sex male/female	2/3 (40.0/60.0)	12/64 (15.8/84.2)
Duration of follow-up (years)	8.5 (7.0–8.6)	11.6 (7.9–14.6)
Status at treatment initiation
Age (years)	64.0 (61.0–65.0)	63.0 (54.0–69.3)
Body mass index (kg/m2)	19.7 (18.7–21.9)	18.9 (17.3–21.1)
Serum albumin (g/dL)	4.1 (4.1–4.2)	4.1 (4.0–4.4)
Modified Reiff score	1 (1.0–1.0)	1 (0.0–3.0)
The number of lobes involved	2 (2.0–3.0)	3 (2.0–4.0)
Coinfection	1 (20.0)	24 (31.6)
Pseudomonas aeruginosa	1 (20.0)	7 (9.2)
Staphylococcus aureus	0 (0.0)	6 (7.9)
Klebsiella species	0 (0.0)	7 (9.2)
Aspergillus species	0 (0.0)	1 (1.3)
History of exacerbations	0 (0.0)	19 (25.0)
Duration of treatment (years)	8.6 (6.8–15.1)	5.2 (3.3–7.6)
Culture conversion*	1 (20.0)†	53 (69.7)
Treatment failure	2 (40.0)	13 (17.1)
Cure	2 (40.0)	37 (48.7)
Resistance of macrolide	3 (60.0)†	9 (11.8)

Data shown median (IQR) or number (%) of patients.

^*Culture conversion 12 months after starting treatment.

^†P <0.05: significant compared with patients without CFTR variant.

CFTR, cystic fibrosis transmembrane conductance regulator; MAC, Mycobacterium avium complex.

CFTR variant of familial clusters

Three patients with CFTR variants had a family history of pulmonary NTM disease. We performed exome sequencing for three relatives of one of these patients, patient number 13 (table 1) who harboured a p.Leu1156Phe (c.3468G>T) variant. The mother and aunt with pulmonary MAC disease harboured one allele of p.Leu1156Phe. In contrast, the aunt, who did not have pulmonary MAC disease, did not have the CFTR variant at either allele (online supplemental figure 3).

Discussion

We revealed that CFTR variants were found in 3.5% (16/458 cases) of patients with either pulmonary NTM disease, NCFB or both. This frequency is lower than that in a previous report showing that CFTR variants were found in 50% of patients with pulmonary NTM disease in the USA.¹⁷ It is considered to reflect the difference in prevalence of CF between Japan (1:590 000 live birth)¹¹ and the USA (1:3,500),¹⁰ indicating differences in the frequency of causing variants.

CFTR modulators have recently been shown to improve lung function in people with CF. In particular, combination therapy with elexacaftor plus tezacaftor plus ivacaftor has demonstrated significant improvement in the percentage predicted forced expiratory volume in 1 s in people with CF who harbour p.Phe508del.³⁶ Moreover, the use of CFTR modulators (ivacaftor monotherapy or combination therapy with lumacaftor or tezacaftor) has been reported to reduce the risk of NTM positivity in people with CF.³⁷ These therapeutic benefits are attributed to the modulator’s ability to improve CFTR function. In the study with a limited number of patients, CFTR modulators were reported to be effective in NCFB patients with a single pathogenic CFTR variant,³⁸ suggesting that they may also be effective for CFTR-RD. Currently available CFTR modulators are primarily used to treat more frequently detected variants (ex. p.Phe508del) in Europeans and Americans.³⁹ Recent studies have highlighted that the spectrum of CFTR variants differs across populations, such as Asian and African.^{40 41} In particular, the profile of CFTR variants in Japanese people with CF differs from that in European people with CF .¹⁴ In our present study, we identified a subset of Japanese patients with pulmonary NTM disease who carried heterozygous CFTR variants. Notably, the profile of CFTR variants observed in these patients also differed from that in Europe. Clinical trials have been conducted on minor CFTR variants, and people with non p.Phe508del variants in France and Israel have responded to CFTR modulators.^{42 43} These findings raise the possibility that CFTR modulators may have therapeutic potential in patients with pulmonary NTM disease who carry certain CFTR variants, even in the heterozygous state, and could contribute to improved clinical outcomes. In addition to CFTR modulators, supportive and systemic management approaches used in people with CF, such as nutrition education and mucus clearance therapies including inhaled dornase alfa and inhaled hypertonic saline,⁴⁴ may be beneficial for patients with CFTR-RD. Further evaluation of the profile and function of CFTR variants in the Japanese population is needed to develop therapeutic strategies for pulmonary NTM disease that encompass both antimicrobial treatment and comprehensive management approaches.

Our finding suggests that patients with CFTR variants might have the characteristic of poor outcome. It is reported that the presence of cavitary (fibrocavitary form or cavitary NB form) and smear positive is associated with poor outcome of pulmonary NTM disease.⁴⁵ In our study, all patients with CFTR variants had NB type, and few people had cavitary or smear positive at diagnosis. In our cohort, the culture conversion rate was lower in patients with pulmonary NTM disease harbouring CFTR variants (20.0%) than in those without CFTR variants and in previous Japanese reports, which consistently reported rates around 70%.^{46 47} Additionally, patients with CFTR variants developed macrolide resistance more frequently than those without CFTR variants. Macrolide resistance is a known risk factor for poor clinical outcomes in pulmonary NTM disease.⁴⁸ Previous studies have reported that macrolide monotherapy is associated with an increased risk of developing macrolide resistance.⁴⁹ In our study, all patients who developed macrolide resistance were initially macrolide-susceptible and received appropriate combination antimicrobial therapy. Notably, patients with pulmonary NTM disease who harboured CFTR variants exhibited macrolide resistance more frequently, which may contribute to poorer clinical outcomes in this subgroup.

Variants in genes involved in innate immunity, such as MPEG1,⁵⁰ MST1R⁵¹ and NRAMP1,⁵² have been reported to be associated with susceptibility to pulmonary NTM disease. In contrast, CFTR, a gene associated with epithelial function, has also been implicated in NTM disease pathogenesis. In six families with familial cases of pulmonary NTM disease, it was reported that 5 of 12 relatives had CFTR variants (5’UTR-680T>6, p.Glu681Val and p.Val754Met), all of which were heterozygous.⁵³ In our study, exome analysis of CFTR variants in one family revealed that relatives who developed pulmonary MAC disease harboured the same CFTR variant (p.Leu1156Phe). The missense variant of p.Leu1156Phe has been reported as a risk factor for idiopathic pancreatitis in children⁵⁴ and alcoholic chronic pancreatitis⁵⁵ in Japan, suggesting that it is associated with reduced CFTR function. As a host genetic factor, natural resistance-associated macrophage protein 1 (NRAMP1) gene was reported in Japanese familial pulmonary MAC disease.⁵² In our study, CFTR is also suggested to be a host genetic factor for familial onset of Japanese pulmonary NTM disease.

This study had some limitations. We filtered CFTR variants using only in silico tools. Due to the lack of analysis of CFTR function, such as the evaluation of sweat chloride ion concentrations or CFTR transcript levels in vitro, it is unclear whether the identified CFTR variants caused pulmonary NTM disease and bronchiectasis. In addition, this study involved a cohort from a single hospital with specialised care for pulmonary NTM disease, which resulted in selection bias. Further analysis of CFTR variants in the Japanese population is required, where the number of subjects should be increased and CFTR function evaluated.

In conclusion, we clarified the clinical characteristics of either pulmonary NTM disease, NCFB or both with CFTR variants in a non-European Japanese population. CFTR variants have been found to be host factors for pulmonary NTM disease in the Japanese population and might be associated with treatment failure.

Data availability statement

Data are available upon reasonable request.