A Novel Missense Variant of ZC3H12A in Pulmonary Arterial Hypertension

Abstract

Background: Because Regnase-1, encoded by ZC3H12A, suppresses the development of pulmonary arterial hypertension (PAH) by controlling pro-inflammatory cytokines, we aimed to identify ZC3H12A variants in patients with PAH.

Methods and Results: We analyzed whole-genome sequence data of patients with PAH to search for disease-associated ZC3H12A variants. The Regnase-1 p.D426G variant was identified in 2 patients, 1 of whom presented with prominent infiltration of inflammatory cells in the lung. The protein level of the variant was decreased in vitro.

Conclusions: We identified a novel missense variant of ZC3H12A that is directly involved in regulating inflammation in patients with PAH.

Pulmonary arterial hypertension (PAH) is a progressive disease characterized by pulmonary arterial remodeling, leading to right ventricular failure.^1–3 Both genetic and environmental factors are thought to be involved in the onset of PAH.⁴ Numerous gene variants, including in genes of the bone morphogenic protein pathway, are associated with PAH through endothelial dysfunction. Although accumulating evidence has shown the importance of inflammation in the pathogenesis of PAH, there have been no reports of variants in inflammation-related genes in PAH.⁵ Among the pro-inflammatory cytokines, interleukin-6 (IL-6) is important for the pathophysiology of pulmonary hypertension.^6–8 Regnase-1, encoded by ZC3H12A, regulates the levels of various pro-inflammatory cytokines, including IL-6 and IL-1β, by degrading their mRNAs.⁹ We recently reported that ZC3H12A expression in peripheral blood mononuclear cells was decreased in patients with PAH, and that its expression inversely correlated with the severity of the disease, especially in connective tissue disease (CTD)-PAH.¹⁰ Furthermore, mice lacking Regnase-1 in alveolar macrophages spontaneously develop severe PAH.¹⁰ These findings strongly indicate a significant role for Regnase-1 in the pathophysiology of PAH, and on this basis we analyzed whole-genome sequence data of patients with PAH registered in the National Cerebral and Cardiovascular Center (NCVC) Biobank to search for disease-associated variants in ZC3H12A.

Methods

We retrospectively analyzed data from 202 consecutive patients with PAH. DNA was isolated from peripheral blood samples collected at between January 2001 and May 2021. Whole-genome sequencing was performed using the NovaSeq 6000 platform (Illumina, San Diego, CA, USA) with 150-bp paired-end reads. Variant calling was conducted by the Genome Analysis Toolkit (GATK) pipeline. Sequence reads were aligned to the human reference genome (GRCh38). Single-nucleotide variants and small insertions/deletions were called by the GATK. Allele frequency was determined according to the Tohoku Medical Megabank Organization database (ToMMo 38KJPN) and the Genome Aggregation Database (GnomAD). The study is being conducted in accordance with the Declaration of Helsinki. All participants provided written informed consent, and the Research Ethics Committee of the NCVC approved the study (M30-135).

HeLa cells (JCRB) were cultured at 37℃ in a 5% CO₂ humidified environment in Dulbecco’s modified Eagle’s medium supplemented with 10% fetal bovine serum and 1% penicillin/streptomycin. HeLa cells were transiently transfected using Lipofectamine 3000 reagent (Thermo Fisher Scientific, Waltham, MA, USA) with either N-terminal flag-tagged wild-type ZC3H12A or ZC3H12A variants (I140V, D426G, I440L) cDNAs (GenScript, Piscataway, NJ, USA), inserted into the pIRES2-EGFP vector for western blotting or the pIRES2-ZsGreen1 vector for immunocytochemistry. Total cell lysates were analyzed by western blotting using an anti-FLAG M2 antibody (F1804, Sigma Aldrich, St. Louis, MO, USA) and an anti-GFP antibody (ab6556, Abcam, Cambridge, UK). Immunocytochemistry was performed with the anti-FLAG M2 antibody and an Alexa 594-conjugated secondary antibody. Imaging was performed using a BZ-X810 fluorescence microscope (Keyence Corporation, Osaka, Japan). ZC3H12A expression was quantified using a hybrid cell count application (BZ-H4C, Keyence Corporation, Osaka, Japan) in BZ-X800 Analyzer.

Results

Among the 202 patients with PAH registered in the NCVC Biobank, we identified 3 missense variants in six patients (Figure 1A). All missense variants were heterozygous. No nonsense or frame shift variants were found. Although the most frequent missense variant identified in the examined cohort was p.I140V, the allele frequency of this variant in the general Japanese population was 0.0072, which was considerably higher than the global allele frequency. To estimate the significance of these variants, we calculated the Combined Annotation-Dependent Depletion (CADD) phred scores.¹¹ For all variants, the CADD phred scores were >15, the threshold for significance stated in the original study.¹¹ The p.D426G variant is located near the DSGxxS motif of Regnase-1, and was identified in 2 patients with PAH. This variant has not been registered in gnomAD, and the allele frequency was as low as 0.00075, even in the general Japanese population (Figure 1B). The ratio of the frequency of this variant in PAH to that in the general Japanese population was 6.61. No variants of genes previously reported to be associated with PAH, such as BMPR2, ACVRL1, and EIF2AK4, were found in either patient. Only the 2 patients harboring the ZC3H12A p.D426G variant presented with a phenotype similar to that of refractory PAH (Table), as described below.

Figure 1.

ZC3H12A missense variants identified in patients with pulmonary arterial hypertension. (A) Distribution of the ZC3H12A variants in patients with PAH (red triangles). (B) Characteristics of each variant. AF, allele frequency; CADD, Combined Annotation-Dependent Depletion

Table.

Characteristics of ZC3H12A p.D426G Variant Carriers at Diagnosis

	Case 1	Case 2
Age at diagnosis, years	63	16
Sex	Female	Female
Comorbidity	SSc	ASD
Family history	Sudden cardiac death	SLE
ANA titer	1 : 40	1 : 1,280
PAWP (mmHg)	8	1
Mean PAP (mmHg)	39	62
PVR (WU)	16	45
Cardiac index (L/min/m2)	1.56	2.1
Medication for PAH	Tadalafil 10 mg Beraprost 120 μg	Tadalafil 40 mg Bosentan 187.5 mg

ASD, atrial septal defect; PAH, pulmonary arterial hypertension; PAP, pulmonary artery pressure; PAWP, pulmonary artery wedged pressure; PVR, pulmonary vascular resistance; SLE, systemic lupus erythematosus; SSc, systemic sclerosis.

Case 1

A 68-year-old woman presented with a diagnosis of CTD-PAH. She exhibited progressive dyspnea upon exercise, classified as New York Heart Association (NYHA) functional class III. She had previously been diagnosed with systemic sclerosis at 38 years of age. Although details were unavailable, her mother had a sudden cardiac death and her sister died from heart failure. At the age of 67, her dyspnea worsened and she was diagnosed with PAH at another hospital. Upon presentation at the NCVC, right heart catheterization revealed elevated pulmonary artery pressure (69/28 [39] mmHg). She was treated with tadalafil and beraprost, although she required careful administration of pulmonary vasodilators because of complications of left heart disease and lung disease associated with systemic sclerosis. She also received home oxygen therapy but, despite these treatments, her B-type natriuretic peptide level continued to increase and her 6-min walking distance continued to decrease (Figure 2A). She died aged 71.

Figure 2.

Clinical features of 2 cases of refractory pulmonary arterial hypertension with the ZC3H12A p.D426G variant. (A) Temporal trends of plasma B-type natriuretic peptide (BNP) levels and 6-min walking distance (6MWD) of the 2 patients with the ZC3H12A p.D426G variant. (B) Histopathological images of lung specimens from a patient with the ZC3H12A p.D426G variant (Case 2). Bars=100 μm. HE, hematoxylin-eosin; EVG, elastica van Gieson; IL, interleukin.

Case 2

A 14-year-old girl was diagnosed with atrial septal defect (ASD) due to the presence of a heart murmur, and was referred to the NCVC. She had a maternal family history of systemic lupus erythematosus. Upon presentation, she also had pressure overload of the right ventricle and was diagnosed with PAH. She underwent a lung biopsy in 1984 to determine indications for radical surgery for ASD. The Heath–Edwards classification was grade III, and radical surgery was not indicated.¹² This pathology showed marked infiltration of inflammatory cells, including IL-6-expressing CD68⁺ macrophages and aggregation of CD20⁺ B cells in the lung, which was an atypical inflammatory response for ASD-associated PAH (Figure 2B). She was subsequently treated with diuretics and other pharmacological treatments. At age 25, she started home oxygen therapy. At age 37, her dyspnea worsened, progressing to NYHA classification III. Right heart catheterization revealed elevated pulmonary artery pressure (102/42 [62] mmHg) with bidirectional atrial shunt. Although she was treated with bosentan and tadalafil, her B-type natriuretic peptide level continued to increase and her 6-min walking distance continued to decrease, resulting in a current NYHA classification of IV (Figure 2A).

In Vitro Study

In both cases described, the patients were refractory to treatment in their inflammatory pathology. The ZC3H12A p.D426G variant shared by them might therefore be associated with this pathology. The amino acid sequence surrounding D426 of human ZC3H12A is conserved across species (Figure 3A). Post-translational protein stability of Regnase-1 contributes to prolonged decay time of mRNAs involved in immune responses.^13–15 Therefore, we examined the protein levels of Regnase-1 variants in a reconstituted system using HeLa cells, which are commonly used cells that express a battery of immune signaling molecules. Regnase-1 variants were exogenously expressed using bicistronic vectors. Protein levels of GFP, the transfection control, were similar among variants (Figure 3B). In contrast, compared with the wild-type, only the protein level of the p.D426G variant of Regnase-1 was significantly decreased, as determined by western blotting (Figure 3B,C). The decreased level of the p.D426G variant was also confirmed by immunocytochemistry (Figure 3D,E).

Figure 3.

Amino acid sequences of ZC3H12A either side of human D426 and post-translational stability of Regnase-1 variant proteins in HeLa cells. (A) Alignment of amino acid sequences of ZC3H12A surrounding human D426. Sequences identical in all species are highlighted in gray. (B–E) HeLa cells were transiently transfected with IRES-based bicistronic expression vectors encoding N-terminus FLAG-tagged Regnase-1 variants and fluorescent protein (GFP for western blotting in B and C, ZsGreen1 for immunocytochemistry in D and E). (B) Representative images of western blots of FLAG (Regnase-1) and GFP. (C) Quantification of FLAG (Regnase-1) normalized to GFP, a transfection control. (D) Representative merged images of ZsGreen1 (Green) and immunofluorescence staining of FLAG (Regnase-1, Red), and Hoechst staining of nuclei (Blue). (E) Quantification of the percentages of FLAG (Regnase-1)-positive cells among ZsGreen1-positive cells. Statistical analysis was performed using one-way analysis of variance with Tukey’s post hoc test. *P<0.05, **P<0.01. WT, wild-type.

Discussion

We identified a novel missense variant of ZC3H12A that may be associated with PAH. We recently reported that Regnase-1 suppresses the development of PAH by controlling IL-6 and platelet-derived growth factor mRNA in alveolar macrophages.¹⁰ This is the first report of a gene variant being directly involved in the regulation of inflammation in patients with PAH.

We previously identified somatic mutations in ZC3H12A in colon epithelial cells in patients with ulcerative colitis.¹⁶ Given the importance of Regnase-1 in the regulation of inflammatory cytokines, variations in ZC3H12A could lead to systemic autoimmunity. The p.D426G variant, which we identified in 2 PAH patients, is located near the DSGxxS motif of Regnase-1 and is very rare in the general population. In Case 1 the patient had systemic sclerosis with interstitial lung disease, which had a clear association with IL-6 because anti-IL-6 antibody treatment has been approved worldwide for the disease.¹⁷ Whereas in Case 2, the patient had prominent infiltration of inflammatory cells with high expression of IL-6 in lung biopsy samples, which were atypical pathological changes in ASD-associated PAH. These findings demonstrate that ZC3H12A p.D426G may contribute to the development of PAH. Moreover, our in vitro study revealed that the level of the Regnase-1 p.D426G variant protein was lower than that of the wild-type, which indicated that post-translational protein stability of Regnase-1 was reduced in the ZC3H12A p.D426G variant. In response to inflammatory stimuli, such as Toll-like receptor ligands and IL-1β, Regnase-1 is phosphorylated at S438 and S442, and is subsequently recognized by a ubiquitin ligase, βTRCP, and degraded by the proteasome.¹⁵ D426 is close to the phosphorylation sites, S438 and S442; therefore, the p.D426G variant might be phosphorylated at the serine residues and recruit βTRCP without inflammatory stimuli.

Study Limitations

First, it was a Japanese single-center study that included a relatively small number of PAH patients. The frequency of the ZC3H12A p.D426G variant should be confirmed in a larger prospective multicenter study. Second, because of a lack of blood samples, we could not examine the expression of ZC3H12A in peripheral blood mononuclear cells or serum, or the pro-inflammatory cytokine levels. Further studies will reveal the significance of Regnase-1 variants in the development of PAH.

In summary, we identified a novel missense variant of ZC3H12A in 2 patients with PAH. Given the importance of Regnase-1 in the pathophysiology of PAH, ZC3H12A variants may contribute to the development of CTD-PAH via dysregulation of pro-inflammatory cytokines.

Acknowledgments

This research was performed using samples and data acquired from the National Center Biobank Network/NCVC Biobank resource. For further details see http://www.ncbiobank.org/ and http://www.ncvc.go.jp/biobank/. We thank Jeremy Allen, PhD, and Sarah Williams, PhD, from Liwen Bianji (Edanz) (www.liwenbianji.cn) for editing a draft of this manuscript.

Conflicts of Interest

This study was supported by a grand from a Janssen Pharmaceutical K.K. Contracted Research Grant.

Source of Funding

This study was funded by the Intramural Research Fund for Cardiovascular Diseases of the NCVC (21-1-5 to Y.N.), a Research Grant from the Japan Agency for Medical Research and Development (AMED) (JP22ek0109592 to Y.N.), JSPS KAKENHI Grant Number JP23H02914 (to Y.N.), and a Janssen Pharmaceutical K.K. Contracted Research Grant (to Y.N.).

Disclosures

T.O. is a member of Circulation Reports’ Editorial Team.

IRB Information

The ethical approval was obtained from the Research Ethics Committee of the NCVC (M30-135).

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