(C) PLOS One [1]. This unaltered content originally appeared in journals.plosone.org.
Licensed under Creative Commons Attribution (CC BY) license.
url:https://journals.plos.org/plosone/s/licenses-and-copyright

------------



A homozygous stop-gain variant in ARHGAP42 is associated with childhood interstitial lung disease, systemic hypertension, and immunological findings

['Qifei Li', 'Division Of Newborn Medicine', 'Boston Children S Hospital', 'Harvard Medical School', 'Boston', 'Massachusetts', 'United States Of America', 'Division Of Genetics', 'Genomics', 'The Manton Center For Orphan Disease Research']

Date: 2021-09

ARHGAP42 encodes Rho GTPase activating protein 42 that belongs to a member of the GTPase Regulator Associated with Focal Adhesion Kinase (GRAF) family. ARHGAP42 is involved in blood pressure control by regulating vascular tone. Despite these findings, disorders of human variants in the coding part of ARHGAP42 have not been reported. Here, we describe an 8-year-old girl with childhood interstitial lung disease (chILD), systemic hypertension, and immunological findings who carries a homozygous stop-gain variant (c.469G>T, p.(Glu157Ter)) in the ARHGAP42 gene. The family history is notable for both parents with hypertension. Histopathological examination of the proband lung biopsy showed increased mural smooth muscle in small airways and alveolar septa, and concentric medial hypertrophy in pulmonary arteries. ARHGAP42 stop-gain variant in the proband leads to exon 5 skipping, and reduced ARHGAP42 levels, which was associated with enhanced RhoA and Cdc42 expression. This is the first report linking a homozygous stop-gain variant in ARHGAP42 with a chILD disorder, systemic hypertension, and immunological findings in human patient. Evidence of smooth muscle hypertrophy on lung biopsy and an increase in RhoA/ROCK signaling in patient cells suggests the potential mechanistic link between ARHGAP42 deficiency and the development of chILD disorder.

Childhood interstitial lung disease (chILD) is a heterogeneous group of rare disorders characterized by diffuse pulmonary infiltrates, respiratory signs and symptoms, and impaired gas exchange. These disorders are complex to diagnose and are associated with substantial morbidity and mortality. Although pathogenic variants in a number of genes have been described to cause chILD, these known genetic etiologies explain only a minority of the cases and there are additional genes yet to be identified. We have identified an 8-year-old girl with chILD, systemic hypertension, and immune abnormalities who carries a homozygous stop-gain variant in the ARHGAP42 gene. Functional studies demonstrate that this stop-gain variant leads to exon 5 skipping and reduced levels of ARHGAP42 protein. We also show enhanced RhoA expression and its activity in the patient’s lymphoblastoid cell lines. ARHGAP42 is involved in regulation of blood pressure and its deficiency causes hypertension in murine models and human adults. This is the first report to link a homozygous stop-gain variant in ARHGAP42 with a chILD disorder, systemic hypertension, and immunological findings in a pediatric patient. Identification of additional chILD patients carrying ARHGAP42 mutations will better define its role in chILD.

Funding: PBA was supported by R01 AR068429 from the National Institute of Arthritis and Musculoskeletal and Skin Diseases of National Institute of Health (NIH). DR was supported by GAUK n°740120 and project “Center for Tumor Ecology - Research of the Cancer Microenvironment Supporting Cancer Growth and Spread” (reg. No. CZ.02.1.01/0.0/0.0/16_019/0000785), Operational Programme Research, Development and Education. Exome sequencing was performed at the Yale Center for Genome Analysis supported by National Institutes of Health (NIH) (grant no. U54 HG006504). Sanger sequencing was performed by the Boston Children’s Hospital IDDRC Molecular Genetics Core Facility supported by NIH award U54HD090255 from the National Institute of Child Health and Human Development. The ARHGAP42 expression results in S2 Fig A and B are based upon data generated by the LungMAP Consortium [U01HL122642] and downloaded from ( www.lungmap.net ), on August 9, 2020. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Copyright: © 2021 Li et al. This is an open access article distributed under the terms of the Creative Commons Attribution License , which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

Although much effort has been made to understand the mechanism of ARHGAP42 in hypertension, its effect on childhood interstitial lung disease (chILD) and immune abnormalities has not been described. Here, we report an 8-year-old girl with chILD and persistent lymphocytosis who carries a homozygous stop-gain variant in ARHGAP42 (NM_152432.2:c.469G>T, p.(Glu157Ter)). To determine the pathogenicity of this variant, we performed anatomic and functional studies to link it with the patient’s phenotype.

ARHGAP42 (Rho GTPase Activating Protein 42), also known as GRAF3, is a member of the GRAF (GTPase-activating protein for Rho associated with focal adhesion kinase) family of Rho-specific GAP (GTPase-activating protein). It is highly expressed in the smooth muscle cell (SMC) layers of blood vessels, stomach, intestine, and lung [ 1 ]. Previous reports have shown that several single nucleotide polymorphisms (SNPs: rs604723, rs633185, rs607562, and rs667575) of ARHGAP42 are blood pressure (BP)-associated loci [ 1 – 4 ]. Arhgap42-deficient mice, homozygous for a gene-trap-mediated reduction in Arhgap42 mRNA levels, exhibit significant hypertension with no other manifestations [ 1 ]. The Arhgap42 heterozygous mice also display significant hypertension [ 1 , 5 ]. RhoA kinase inhibitor can abrogate this response in these Arhgap42-deficient mice [ 1 , 5 ]. Functional studies indicated that ARHGAP42 acts preferentially as a GAP for RhoA and that it plays a key role in maintaining normal BP homeostasis by reducing RhoA-dependent phosphorylation of the myosin light chain (MLC) and Ca2 + -mediated SMC contractility in resistance vessels [ 1 , 6 ]. Recently, a SNP (rs633185) of ARHGAP42 has been reported to be associated with chronic obstructive pulmonary disease [ 7 ], although the relationship between ARHGAP42 and lung diseases has not yet been elucidated.

Results

Clinical history The proband, a female child, with a birth weight of 2,780 g (16th percentile), was born at 39 weeks gestation following an uncomplicated pregnancy, via vaginal delivery through meconium.

Childhood interstitial lung disease (chILD) The infant had tachypnea with normal oxygen saturation soon after birth and was discharged home. Poor feeding, failure to thrive, and labored breathing developed soon after. Evaluation at 2 months of age showed retractions, tachypnea, and rales needing hospitalization. Oxygen saturations were in the low 80’s on room air and required 0.5 L/min supplemental oxygen by nasal cannula to normalize. A chest X-ray (Fig 1A) demonstrated bilateral diffuse opacities and low lung volumes, concerning for ILD. A chest computed tomography (CT) scan (Fig 1B) at age 3 months showed diffuse ground glass opacities with subpleural sparing and patchy subpleural opacities in the dependent portions of both lower lobes; air trapping was not observed. Serial echocardiograms revealed no evidence of pulmonary hypertension or structural heart disease. The patient was treated with oxygen and required bi-level positive airway pressure (BiPAP) at night to maintain normocarbia. She also had aspiration during swallow and was treated with thickened feeds. PPT PowerPoint slide

PNG larger image

TIFF original image Download: Fig 1. Clinical findings of the proband with homozygous ARHGAP42 stop-gain variant. (A) Chest X-ray at age 3 months showed bilateral diffuse opacities and low lung volumes; (B) Initial chest CT scan at age 3 months displayed diffuse ground glass opacities with subpleural sparing and patchy subpleural opacities; (C) Patient growth chart for weight through two years of age. https://doi.org/10.1371/journal.pgen.1009639.g001 The child subsequently demonstrated a gradual, yet sustained, improvement and near-complete resolution of her lung disease over the subsequent 8 years of follow-up. She had no history of recurrent pneumonia or frequent lower respiratory tract infections. Her weight recovered to the 10th percentile by age 18 months with nutritional supplementation (Fig 1C). She was weaned off of daytime oxygen at two years of age, and she continued nighttime oxygen therapy until she was 4 years and 9 months old. Repeat chest CT showed radiographic improvement in her lung disease. At her most recent follow-up at age 8 years, she showed continued clinical stability, with normal body mass index, excellent interval linear growth, regular participation in multiple athletic interests, and rare use of nighttime supplemental oxygen with only occasional upper respiratory tract infections.

Immunological findings The proband was noted to have persistent leukocytosis and lymphocytosis since the age of 3 months (S1 Fig). Absolute lymphocyte count (ALC) and total white blood cells (WBC) were persistently high or above normal for age, with normal numbers of circulating neutrophils, monocytes, and eosinophils. By 5 years of age, the proband had developed extensive verrucae vulgares on her hands and lip. Limited immunologic phenotyping at age 7 showed that the patient’s lymphocytosis was comprised of proportionately elevated numbers of T cells, B cells and NK cells, which were all present at ~2x the upper normal range of absolute cell counts for age. The CD4+ T cell population showed a slightly high percentage of recent thymic emigrant (RTE) cells (66.3% CD4+ CD31+ CD45RA+, normal range: 45.3%-63.6%), but this did not correlate with an increase in the proportion of naïve CD4+ T cells. Percentages of naïve vs. memory/effector CD4+ and CD8+ T cells were normal, except for a slightly low proportion of TEMRA CD8+ T cells (8.7% CD45RA+ CCR7+, normal range: 9.1%-49.1%). Functional T cell testing showed normal lymphocyte proliferation to stimulation with mitogens (PHA, ConA and anti-CD3) and antigens (Candida and Tetanus). Neutrophil oxidative function (DHR) was normal. Analysis of circulating B cell populations showed slightly high proportions of naïve B cells (78.1% CD19+CD27-IgD+, normal range: 47.3%-77%), with slightly low proportions of transitional B cells (6.7% CD24hi CD38hi, normal range: 7.2%-23.8%) and plasmablasts (0.1% CD24low CD38high, normal range: 0.4%-5.2%). IgG, IgE and IgA levels were normal for age at 5 years. Although she had received all recommended vaccinations, at 7 years, she had non-protective specific IgG antibody responses for hepatitis B surface antibody and 9/12 Streptococcus pneumoniae serotypes. She did have a protective tetanus IgG titer at the same time point. Vaccine challenges were not performed.

Systemic hypertension The proband was noted to have intermittent systemic hypertension. An ambulatory blood pressure monitoring was performed at 8 years of age, which showed both mean 24-hour systolic (119 mmHg, 95%ile) and diastolic (75 mmHg, 95%ile) ambulatory blood pressure to be at or above the 95%ile for sex-age (S1 Table). The family history is notable for father developing hypertension in his 20s, and the mother with a recent diagnosis of hypertension. Both are under no treatment so far although they are being monitored closely. Both parents have a history of exercise-induced asthma during childhood. In addition, the proband’s father had recurrent streptococcal pharyngitis during childhood, multiple antibiotic reactions and gastroesophageal reflux. The mother is recently diagnosed with autoimmune sclerosis of her left eye and treated with adalimumab (Humira). The paternal grandfather has a history of recurrent bronchitis and pneumonia and asthma since childhood, as well as uncontrollable hypertension beginning in his mid-50s. He is currently on losartan and amlodipine. The maternal grandfather also has hypertension and is on medication, but no further information is available.

A homozygous stop-gain variant in ARHGAP42 identified by exome sequencing (ES) The pedigree of the family is shown in Fig 3A. Clinical testing for common genetic causes of chILD, including genes encoding the ATP-binding cassette transporter A3 (ABCA3) and the surfactant proteins (SFTPB and SFTPC), was negative. Trio ES was performed and a novel homozygous variant in ARHGAP42 (hg19, chr11:100784267, NM_152432.2:c.469G>T, p.(Glu157Ter)) was identified in the proband (III:1) and confirmed by Sanger sequencing (Fig 3B). While the family is not known to be consanguineous, the parents shared 0.7% of the genome of which the 19.72 MB region containing ARHGAP42 was the largest (hg19, Chr11: 82924358–102649954). There are two other smaller shared regions of 4.8 Mb and 3.2 Mb on chromosomes 2 and 15 respectively. Additional homozygous variants were evaluated and no additional candidate variant was identified. Both parents (II:1 and II:2) and the paternal grandfather (I:1) were heterozygous for this variant. PPT PowerPoint slide

PNG larger image

TIFF original image Download: Fig 3. Genetic findings in the family with ARHGAP42 stop-gain variant. (A) Pedigree of the family carrying ARHGAP42 variant. Half-filled symbol: heterozygous; Filled symbol: homozygous; (B) Sanger sequencing chromatogram for the family of the ARHGAP42 variant; (C) Amino acid 157 (arrowhead) is evolutionally conserved in vertebrates; (D) Agarose gel electrophoresis result of ARHGAP42 pre-variant, in-variant, and post-variant amplification by PCR in cDNA samples extracted from blood. The smaller band (arrow) corresponds with exon 5 deletion confirmed by Sanger sequencing. Pt: patient; Mo: mother; Fa: father; C1: control; gDNA: genomic DNA. Pre-variant: Exon 1 to first 7 bp of Exon 5, 279 bp for ARHGAP42 cDNA. A 373 bp band seen with gDNA is from non-specific amplification of another locus; In-variant: Exon 4 to Exon 7, 298 bp for ARHGAP42 cDNA. A 618 bp band was seen with gDNA from non-specific amplification from another locus; Post-variant: Exon 7~8, 204 bp for ARHGAP42 cDNA. (E) Schematic of ARHGAP42 functional domains. The amino acid change is depicted by an arrow, and the skipped exon 5 is depicted as a shaded box in the BAR domain. https://doi.org/10.1371/journal.pgen.1009639.g003 This variant is absent from both the publicly available Exome Aggregation Consortium (ExAC) and genome aggregation database (gnomAD) databases, and is classified as damaging by multiple in silico prediction software packages, including MutationTaster and Combined Annotation-Dependent Depletion (CADD). The probability of being Loss-Of-Function (LOF) Intolerant (pLI) score for ARHGAP42 in gnomAD is 1, indicating that the ARHGAP42 cannot tolerate protein truncating variation. In addition, no homozygous LOF variant is present in the ExAC or gnomAD databases. The glutamate 157 residue is highly conserved in vertebrates (Fig 3C). No additional de novo or biallelic variants associated with her phenotype were identified by ES.

ARHGAP42 is highly expressed in the lung of human neonates and mice at postnatal (P) day 7–10 Publicly available RNA-seq data (LungMAP database, https://lungmap.net/) indicate that ARHGAP42 expression is high in the lung of human neonates and mice at P7-10 (S2A and S2B Fig). In addition, single-cell RNA-seq data from fetal lung tissues [8] (https://descartes.brotmanbaty.org/) show ARHGAP42 expression in stromal, vascular endothelial, bronchiolar and alveolar epithelial cells, myeloid, and lymphoid cells (S2C Fig).

ARHGAP42 stop-gain variant leads to exon 5 skipping To evaluate the effect of ARHGAP42 stop-gain variant, total RNA was extracted from Epstein-Barr virus-transformed lymphoblastoid cell lines (EBV-LCLs) of the proband, both parents, and an age-matched control. ARHGAP42 cDNA sequences including the pre-variant, in-variant, and post-variant regions were amplified by PCR (primers shown in S2 Table). Agarose gel electrophoresis (AGE) results (Fig 3D) displayed a clear band in both the pre-variant and post-variant regions in the family and control, indicating that their transcription was not affected by the variant. However, all three family members exhibited two bands (196 and 298 bp) in the in-variant region, whereas the control showed only one (298 bp). The smaller 196 bp band was due to the skipping of exon 5 (ΔExon5), confirmed by Sanger sequencing. This band was much stronger in the proband compared to parents, suggesting a predominance of ARHGAP42 ΔExon5 transcript. Quantitative real-time PCR (qRT-PCR) analysis of the ARHGAP42 mRNA expression from EBV-LCLs of the proband, both parents, and an age-matched control confirmed the AGE findings (S3 Fig). A schematic of ARHGAP42 functional domains carrying this variant was shown in Fig 3E. To verify the importance of ARHGAP42 exon 5 in lung tissue, RNA was extracted from mouse lung tissues of different ages. The AGE of ARHGAP42 RT-PCR products after cDNA synthesis from mouse lung tissue (10 days to eight months old) is shown in S4A Fig, and the ARHGAP42 exon 5 was present in all the samples. In addition, the exon 5 was also present in the skeletal muscle tissues extracted from 10 days to one month old mice (S4B Fig). Sanger sequencing (S4C Fig) confirmed the exon 5 sequence of ARHGAP42, and demonstrated a high degree of sequence homology of exon 5 between human and mouse.

ARHGAP42 stop-gain variant leads to reduced ARHGAP42 expression To assess the effect of ARHGAP42 stop-gain variant on protein expression, we performed immunostaining against ARHGAP42 in the patient EBV-LCLs. Immunofluorescence analyses demonstrated a lower expression of ARHGAP42 in the patient than that in her parents (Fig 4A). Immunoblotting was not applicable due to a lack of good-quality antibodies. PPT PowerPoint slide

PNG larger image

TIFF original image Download: Fig 4. ARHGAP42 stop-gain variant causes reduced ARHGAP42 expression. (A) Immunofluorescence of ARHGAP42 in the patient and her parents lymphoblastoid cells. Lymphoblastoid cells were fixed, permeabilized, and stained with DAPI (blue), phalloidin (green), and ARHGAP42 (red) (scale bar 10 μm); (B) Expression of control, GFP-ARHGAP42 WT and GFP-ARHGAP42 ΔExon5 in transfected HT1080 and U2OS cells by immunoblotting (black arrow indicates a lower level of ARHGAP42 ΔExon5 protein). https://doi.org/10.1371/journal.pgen.1009639.g004 We have shown that this stop-gain variant leads to exon 5 skipping in the patient. To evaluate the effects of ARHGAP42 ΔExon5 at the protein level, immunoblotting in HT1080 and U2OS cells transfected with ARHGAP42 ΔExon5 construct were undertaken. Both U2OS and HT1080 cells were transfected with GFP-ARHGAP42 WT or GFP-ARHGAP42 ΔExon5 and successfully transfected cells were selected by fluorescence-activated cell sorter (S5 Fig). Both cell lines showed less ARHGAP42-ΔExon5 expression than ARHGAP42-WT (Fig 4B).

ARHGAP42 stop-gain variant contributes to enhanced RhoA activity ARHGAP42 is a member of the RhoGAP family, and it acts as a GAP for RhoA and Cdc42. First, we evaluated the levels of RhoA and Cdc42 protein in the patient’s EBV-LCLs. Both were higher in the patient than that in the parents and healthy controls by immunoblotting (Fig 5A). To further test the effects of ARHGAP42 stop-gain variant on the RhoA activity, a GST-Rhotekin-RBD (Rho binding domain) pull-down assay was performed on the patient’s EBV-LCLs (Fig 5B). The RhoA-GTP levels were significantly increased in the patient relative to both parents (Fig 5C). PPT PowerPoint slide

PNG larger image

TIFF original image Download: Fig 5. ARHGAP42 stop-gain variant contributes to enhanced RhoA activity and Cdc42 expression. (A) Expression of RhoA and Cdc42 in the patient’s EBV-LCLs compared with her parents and healthy controls by immunoblotting; (B) Measurement of RhoA activity by GST-Rhotekin-RBD (Rho binding domain) pull-down assay in the patient and her parents. The level of RhoA-GTP was determined by western blot with anti-RhoA antibody following a pull-down assay; (C) The RhoA-GTP data are expressed as the mean ± SD of three independent experiments. *p<0.05; **p<0.01. https://doi.org/10.1371/journal.pgen.1009639.g005

[END]

[1] Url: https://journals.plos.org/plosgenetics/article?id=10.1371/journal.pgen.1009639

(C) Plos One. "Accelerating the publication of peer-reviewed science."
Licensed under Creative Commons Attribution (CC BY 4.0)
URL: https://creativecommons.org/licenses/by/4.0/


via Magical.Fish Gopher News Feeds:
gopher://magical.fish/1/feeds/news/plosone/

You are viewing proxied material from magical.fish. The copyright of proxied material belongs to its original authors. Any comments or complaints in relation to proxied material should be directed to the original authors of the content concerned. Please see the disclaimer for more details.