ORMDL3 but not neighboring 17q21 gene LRRC3C is expressed in human lungs and lung cells of asthmatics

To the Editor:

The chromosome 17q21 region has strong linkage to asthma in genetic association studies.¹ This region contains a cluster of nine genes, including ORMDL3, GSDMB, LRRC3C, GSDMA, ZPBP2, IKZF3, GRB7, ERBB2, and PGAP3 of which the role of ORMDL3 and GSDMB have been most studied in relation to the pathogenesis of asthma¹. Although ORMDL3 expression in human CD4 cells^E1,E2 and epithelial cells² is well documented there are currently no studies examining its expression in human lungs from asthmatics which is the focus of this study. As ORMDL3 is linked in genetic association studies to asthma^1,E3, but not to allergic rhinitis³, ORMDL3 expression in the lung rather than its ability to regulate Th2 responses^E1 (which are present both in asthma and allergic rhinitis) may potentially be more important to its role in the pathogenesis of asthma. The pathway(s) mediating the linkage of ORMDL3 to asthma are currently unknown, but there is in vitro and in vivo evidence in mouse models to support effects of ORMDL3 on airway smooth muscle and AHR⁴, sphingolipids⁵, ceramide⁵, ATF6α², SERCA2b², cytokines/chemokines², and adhesion molecules^E4 as potential candidates. In contrast to studies investigating the biology of ORMDL3 in relation to the pathogenesis of asthma, there are currently no published studies examining whether LRRC3C, a neighboring gene located immediately next to ORMDL3 on chromosome 17q21 (Figure 1A-C), is expressed in asthmatic lung or lung cells which is a second focus of this study.

Figure 1. ORMDL3 and LRRC3C mRNA expression in human asthma and control lungs.

(A) Chromosome 17q21 localized genes with ORMDL3 and LRRC3C in red. SNP rs2872507, rs8076131 and rs4794820 (small black arrows). (B) ORMDL3 gene structure (coding exons in black boxes) and SNP rs8076131 (black arrow). (C) LRRC3C gene structure (two coding exons in black boxes) and SNP rs4794820 (black arrow). (D) ORMDL3 and LRRC3C mRNA expression in human asthma and control lungs. (E) ORMDL3 mRNA expression in human lung by SNP rs8076131 genotype. (F) LRRC3C mRNA expression in human lung by SNP rs2872507 genotype. (G) Cycle threshold (Ct) levels of LRRC3C mRNA at different human LRRC3C cDNA clone concentrations.

The chromosome 17q21 locus has not only been implicated in the pathogenesis of asthma but also in the pathogenesis of inflammatory bowel disease (IBD)^E5,E6 and auto-immune disease^E7. Studies of IBD and chromosome 17q21 have demonstrated that LRRC3C is linked to IBD and that levels of LRRC3C expression in colon are linked to chromosome 17q21 SNPs⁶. In addition, genetic epidemiologic studies of asthma have demonstrated that a SNP rs4794820 located in chromosome 17q21 within LRRC3C (Figure 1C) was significantly associated with severe asthma compared to mild to moderate asthma⁷, as well as with atopic asthma and lower PC₂₀⁸ while a second SNP rs2872507 (Figure 1A) linked to LRRC3C expression in IBD⁶ was associated with poor ICS response in asthma⁹. LRRC3C has two exons (Figure 1C) and encodes a 275 amino acid predicted membrane protein with one predicted transmembrane region^E8. In addition to LRRC3C expression in colon², a RNAseq data set in normal humans demonstrates highest levels of LRRC3C expression in testis^E9. Interestingly another chromosome 17q21 gene ZPBP2 is not only highly expressed in testis^E10,E11, but is also expressed in bronchial epithelial cells and linked to asthma in mouse model studies^E10,E11. As there are no studies investigating whether ORMDL3 and its neighboring gene LRRC3C are expressed in human lungs from asthmatics in this study we investigated this possibility.

To determine whether ORMDL3 and LRRC3C mRNA were expressed in the lungs of asthmatics we extracted total RNA from postmortem human lungs of asthmatics (n=12) and control non-asthmatics (n=14)(clinical characteristics described in Supplement Table 1) which was processed for RT-qPCR (all methods described in the Supplementary Online Repository)^E1-E21. ORMDL3 mRNA was readily detected in the lung samples from both asthmatics (10/12 or 83% of asthmatics had detectable ORMDL3 mRNA) and normal controls (11/14 or 78% of controls had detectable ORMDL3 mRNA) with levels trending but not statistically significantly higher in the asthma lungs compared to control lungs (Figure 1D). As prior studies have demonstrated that the 17q21 association with asthma is mainly driven by haplotype H1 SNPs rs8076131 and rs4065275^E1 located in close proximity and present in approximately 61% of childhood allergic asthmatics and 49% of controls^E1, we genotyped the asthma and control lungs to determine the proportion of lungs expressing the ORMDL3 asthma risk allele haplotype H1 SNP AA rs8076131^E1. The presence of the AA SNP rs8076131 was associated with detectable lung ORMDL3 mRNA in 7/7 asthmatics (100%) and 7/9 controls (78%) who had this SNP (Fig 1E).

In contrast to detecting ORMDL3 expression in asthma lungs, LRRC3C mRNA was not detected in any of the lungs from either asthmatics (0/12 asthmatics detectable LRRC3C mRNA) or controls (0/14 controls detectable LRRC3C mRNA)(Figure 1F). As the chromosome 17q21 SNP rs2872507 has been linked to expression of LRRC3C in colon tissue as assessed by gene expression assay², we also genotyped the asthma and control lungs to determine whether the SNP rs2872507 would influence our ability to detect LRRC3C mRNA in human lungs. The lung genotyping demonstrated that the rs2872507 GG (risk allele for asthma)⁹ was detected in 3/10 (30%) of the asthma lungs, and 8/13 (61%) of the control lungs (Figure 1F), none of whose lungs expressed LRRC3C mRNA. All of the lung samples were genotyped for a second SNP rs4794820 and had the AG allele (data not shown) making comparisons between the risk allele (GG) and non-risk allele (AA) not possible. As a LRRC3C positive control for our RT-qPCR, we measured LRRC3C mRNA in a human LRRC3C cDNA clone, and identified high Cycle threshold (Ct) values at seven different clone concentrations of LRRC3C cDNA (0.01 to 10⁵ ng/μL) (Figure 1G).

In addition, to investigating whether ORMDL3 and LRRC3C mRNA were expressed in human lung we also examined whether they were expressed in primary human lung cells pertinent to the pathogenesis of asthma including primary bronchial epithelial cells, BAL macrophages, and PBMCs from both normal and asthmatics as previously described^E12,E13. We readily detected expression of ORMDL3 mRNA in asthmatics in all cell types studied (bronchial epithelial cells, BAL macrophages, PBMC) (Figure 2A-C). In contrast, no LRRC3C mRNA was detected in either primary bronchial epithelial cells (Figure 2D), BAL macrophages (Figure 2E), or PBMCs (Figure 2F) in both asthmatics and normals.

Figure 2. ORMDL3 and LRRC3C mRNA expression in human asthma and control cells.

(A) ORMDL3 mRNA expression in primary human bronchial epithelial cells. (B) ORMDL3 mRNA expression in primary human BAL macrophages. (C) ORMDL3 mRNA expression in human PBMCs. (D) LRRC3C mRNA expression in primary human bronchial epithelial cells. (E) LRRC3C mRNA expression in primary human BAL macrophages. (F) LRRC3C mRNA expression in human PBMCs.

In summary, this study demonstrates that ORMDL3 mRNA can readily be detected in the lungs as well as lung cells of asthmatics. In contrast, expression of LRRC3C mRNA, the gene neighboring ORMDL3 on chromosome 17q21, cannot be detected in the same lung samples, or in lung cells such as epithelial cells, macrophages, or PBMCs. These studies suggest that in unselected subjects, ORMDL3 is expressed at significant levels in the human lung and in selected lung cells pertinent to the pathogenesis of asthma. In contrast, LRRC3C was not detected in human lung suggesting that LRCC3C may be more important to intestinal diseases such as IBD⁶ than asthma. Further study is needed to determine whether there are SNPs that regulate LRRC3C expression and whether in such a preselected smaller subset of asthma or control lungs LRRC3C expression could be detected or not. Overall, this study demonstrates that ORMDL3 is significantly expressed in the lungs of asthmatics underscoring the potential importance of lung cell expression of ORMDL3 to the pathogenesis of asthma.

Abbreviations:

AHR

Airway hyperreactivity

ASM

Airway smooth muscle

ATF6α

Activating transcription factor 6α

BAL

Bronchoalveolar lavage

GSDMA

Gasdermin A

GSDMB

Gasdermin B

IBD

Inflammatory bowel disease

LRRC3C

Leucine rich repeat containing 3C

ORMDL3

ORM1-like-3

SERCA2b

Sarco/endoplasmic reticulum Ca²⁺ ATPase 2b

SNP

Single nucleotide polymorphism

PBMC

Peripheral blood mononuclear cell

ZPBP2

Zona Pellucida Binding Protein 2