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. 2023;1(1):100811.
doi: 10.1016/j.gimo.2023.100811.

Biallelic variants in the calpain regulatory subunit CAPNS1 cause pulmonary arterial hypertension

Alex V Postma  1   2 , Christina K Rapp  3 , Katrin Knoflach  3 , Alexander E Volk  4 , Johannes R Lemke  5   6 , Maximilian Ackermann  7 , Nicolas Regamey  8 , Philipp Latzin  9 , Lucas Celant  10 , Samara M A Jansen  10 , Harm J Bogaard  10 , Aho Ilgun  2 , Mariëlle Alders  2 , Karin Y van Spaendonck-Zwarts  2 , Danny Jonigk  11 , Christoph Klein  3 , Stefan Gräf  12   13 , Christian Kubisch  4 , Arjan C Houweling  2 , Matthias Griese  3
Affiliations

Biallelic variants in the calpain regulatory subunit CAPNS1 cause pulmonary arterial hypertension

Alex V Postma et al. Genet Med Open. 2023.

Abstract

Purpose: The aim of this study was to identify the monogenic cause of pulmonary arterial hypertension (PAH), a multifactorial and often fatal disease, in 2 unrelated consanguine families.

Methods: We performed exome sequencing and validated variant pathogenicity by whole-blood RNA and protein expression analysis in both families. Further RNA sequencing of preserved lung tissue was performed to investigate the consequences on selected genes that are involved in angiogenesis, proliferation, and apoptosis.

Results: We identified 2 rare biallelic variants in CAPNS1, encoding the regulatory subunit of calpain. The variants cosegregated with PAH in the families. Both variants lead to loss of function (LoF), which is demonstrated by aberrant splicing resulting in the complete absence of the CAPNS1 protein in affected patients. No other LoF CAPNS1 variant was identified in the genome data of more than 1000 patients with unresolved PAH.

Conclusion: The calpain holoenzyme was previously linked to pulmonary vascular development and progression of PAH in patients. We demonstrated that biallelic LoF variants in CAPNS1 can cause idiopathic PAH by the complete absence of CAPNS1 protein. Screening of this gene in patients who are affected by PAH, especially with suspected autosomal recessive inheritance, should be considered.

Keywords: Angiogenesis; CAPNS1; Calpain; Pulmonary arterial hypertension.

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Conflict of interest statement

The authors declare no conflicts of interest.

Figures

Figure 1
Figure 1
Detailed representation of the biallelicCAPNS1variants identified in the probands. A. Schematic drawing of the pedigrees. Clinically affected individuals with confirmed diagnosis of pulmonary alveolar hypertension are depicted in solid black, and individuals with suspected pulmonary hypertension diagnosis are depicted in grey. Healthy individuals are depicted in solid white. Abortion is illustrated as a triangle. Deceased individuals are denoted by a slash, with age of death indicated. Consanguinity is indicated by double lines. +/+ variant was identified in homozygote, +/− variant was identified in heterozygote, na/na no genetic information. B. Consequences on splicing of the identified CAPNS1 variants. The variant identified in family A resulted in skipping of coding exon 8. The variant identified in family B resulted in 2 aberrant transcripts, 1 with skipping of coding exon 2 and 1 using a cryptic splice site within coding exon 2 (between c.213 and c.214), indicating by an asterisk. C. Complementary DNA analysis in patient P3 confirmed the skipping of coding exon 8, without a shift in the reading frame. D. Schematic protein structure. Calpain consists of the catalytic subunit (CAPN1/2: blue) and the regulatory subunit (CAPNS1: orange). Both harbor the penta EF-hand motif, essential for Ca2+ binding and heterodimerization. The in-frame deletion of coding exon 8 in family A will potentially result in a CAPNS1 protein lacking the fourth and fifth EF-hand domain (shaded in red). EF, Helix-Loop-Helix; GERP, Genomic Evolutionary Rate Profiling.
Figure 2
Figure 2
Further analyses in the probands' blood and lung tissue samples. A. CAPNS1 western blot analysis. In all members of family A with identified heterozygote CAPNS1 variant (c.721+1G>A), no aberrant CAPNS1 protein was detectable compared with HEK cells that overexpressed this CAPNS1 variant (lane 1). All samples demonstrated only 1 band, at the correct height, similar to healthy controls (CAPNS1 = 28 kDA). Of note, GAPDH was used as a loading control (separate blot). B. The aberrant CAPNS1 protein (of the c.721+1G>A variant) was increasingly detectable by adding increasing amounts of the proteasome blocker MG-132. In addition, GAPDH was used as a loading control (separate blot). C. In patient P5, family B, CAPNS1 western blot demonstrates total absence of CAPNS1, in comparison with healthy controls. Furthermore, GAPDH was used as a loading control (separate blot). D. Scanning electron microscopy analysis of lung biopsies. Vascular corrosion casts from patient P2 and a healthy control show a cross-sectional view of the preserved lung parenchyma with (a/b) proliferation around the bronchovascular bundles and (c/d) dilated capillary plexuses within the alveolar septae. (e/f) For comparison, less and small alveolar capillaries in a healthy human lung. Bars: a = 500 μm, b/f = 200 μm, c/e = 100 μm, d = 50 μm. cDNA, complementary DNA; GAPDH, glyceraldehyde 3-phosphate dehydrogenase; HEK, human embryonic kidney.
Figure 3
Figure 3
Downstream signaling of calpain in theory and RNA analysis in lung tissue sample of proband P2. A. The calpain holoenzyme is inactive in bound form because of the inhibition via calpastatin. After ubiquitination of CAPNS1 subunit, calpain is active. B. The loss of calpain subunit is assumed to cause a loss of inhibition via calpastatin. Calpain plays a key role in TGF-β pathway by degradation of phosphorylated SMAD2. The TGF-β and BMP pathways build 2 arms of an angiogenesis balance. The loss of calpain regulation is suspected to lead to an unbalanced angiogenesis. C. Multiple t test analysis using GraphPad Prism 9.3.1 is illustrated in volcano plot, by plotting the significance (false discovery rate) vs the differences in the counts of RNA molecules. Volcano plot marks the genes of interests of RNA NanoString analysis in patient P2. D. The counts of RNA molecules were normalized, and the gene expression ratio between patient P2 compared with the mean of controls was calculated using nSolver Analysis Software v.4.0. The normalized log2 counts of most informative changes in the gene expression are displayed.
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