Novel Pathogenic NPR2 Variants in Short Stature Patients and the Therapeutic Response to rhGH

doi:10.21203/rs.3.rs-1991300/v1

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Research Article

Novel Pathogenic NPR2 Variants in Short Stature Patients and the Therapeutic Response to rhGH

https://doi.org/10.21203/rs.3.rs-1991300/v1

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Journal Publication

published 27 Jul, 2023

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Objective

A heterozygous loss-of-function mutation in the NPR2 gene causes short stature with non-specific skeletal abnormalities, accounting for approximately 2 ~ 6% of all idiopathic short stature cases. The aim of this study was to analyze and identify pathogenic variants in the NPR2 gene, and to examine the therapeutic response to recombinant growth hormone (rhGH).

Methods

NPR2 was sequenced in three Chinese Han patients with short stature via whole-exome sequencing. In vitro functional experiments, homology modeling, and molecular docking analysis of variants were conducted to investigate putative protein changes and pathogenicity. Three patients received rhGH therapy for two years.

Result

Two NPR2 heterozygous variants were identified: c.1579C > T,p.Leu527Phe in patient 1 and c.2842dupC,p.His948Profs*5 in patient 2. A small gene model was constructed, and transcriptional analysis of the synonymous mutation (c.2643G > A) was performed in patient 3, which revealed the deletion of exon 17 and the premature formation of a stop codon (p.His840Gln*). Functional studies showed that both NPR2 variants, His948Profs*5 and His840Gln*, failed to produce cGMP in the homozygous state. Furthermore, the Leu527Phe variant of NPR2 was almost unresponsive to the stimulatory effect of ATP on CNP-dependent guanylyl cyclase activity. This loss of response to ATP has not been previously reported. The average age of patients at the start of treatment was about 6.5 ± 1.8 years old, and their height increased by 1.59 ± 0.1 SDS after 2 years of treatment.

Conclusion

we report 2 cases of novel pathogenic mutations in the NPR2 gene. We broadened the genotypic spectrum of NPR2 variants in short stature patients and provided insights into rhGH's efficacy.

NPR2 variants

short stature

loss-of-function mutation

rhGH treatment

Short stature is defined as a body height below the two standard deviations (SD) of the population (of the same gender and chronological age), and it is one of the most common causes of referral to pediatric endocrinologists[1]. During long bone development, chondrocytes of the cartilaginous growth center, called the epiphyseal growth plate, are responsible for driving linear growth. The regulatory mechanisms of growth plate are extraordinarily complicated and involve multiple factors, such as extracellular matrix molecules, intracellular signaling pathways, paracrine factors, endocrine factors and epigenetic regulation[2]. In recent years, genes involved in paracrine functions such as C-type natriuretic peptide (CNP) and its receptor Atrial natriuretic peptide receptor 2 (NPR2), Indian Hedgehog signaling pathway, parathyroid hormone-related protein, and bone morphogenetic protein/TGF-β superfamily signaling have attracted more attention, especially NPR2[3, 4].

In the growth plate, CNP and NPR2 regulate endochondral ossification through the hypertrophic region [5, 6]. CNP-NPR2 stimulates the production of chondrocytes and cartilage matrix, which promotes the formation of growth plates[7, 8]. Moreover, CNP-NPR2 has been shown to stimulate bone growth in growth plate chondrocytes through autonomic Ca2 + entry[9]. The NPR2 gene heterozygous loss-of-function variant causes short stature with non-specific skeletal abnormalities (MIM#616255), accounting for about 2 ~ 6% of idiopathic short stature[10]. The recombinant growth hormone (rhGH) treatment appears to be beneficial in short stature patients with NPR2 heterozygous variants[4]. Despite this, there is relatively little information on the cause of short stature arising from NPR2 gene variants. Furthermore, related genotype-phenotype and rhGH therapy efficacy correlations require functional and clinical analysis, which include parameters such as age, treatment dose, and duration.

In this study, the clinical features, laboratory results, genetic information, treatment, and follow-up data of three patients with short stature were analyzed. In addition, to elucidate pathogenic mechanisms associated with NPR2 variants, a series of functional assays and molecular dynamics simulations were performed.

2.1 Subjects

This study was approved by the Ethics Committee of Fuzhou Children’s Hospital of Fujian Medical University (approval number 201921) and was conducted in agreement with the Declaration of Helsinki. Written informed consent was obtained from their parents.

2.2 Clinical Evaluations

Medical history and clinical manifestations were assessed in all patients, as well as physical examinations. Thereafter, hormonal analysis, including thyroid hormone, growth hormone, insulin-like growth factor-1 (IGF-1), and insulin-like growth factor binding protein-3 (IGFBP3), and imaging examination (X-ray of the spine and left hand) were performed.

2.3 Genetic Examination

A standard procedure was used for obtaining genomic DNA from the probands and their parents via peripheral blood leukocytes. As described previously, whole-exon sequencing (WES) was performed [11]. NCBI entry NG_009249 (NM_003995.4) describes the NPR2 gene. Nomenclature for sequence variants was adopted from the Human Genome Variation Society (HGVS) [12].

2.4 Conservation and Pathogenicity Analysis of the Variant

Conservation analysis of amino acids was completed as previously described[13]. The PREDICTSNP webserver (https://loschmidt.chemi.muni.cz/predictsnp1/) predicted changes in NPR2 function caused by missense variants. Several predictors are employed in this tool (PredictSNP, PhD-SNP, PolyPhen-2, SIFT, and SNAP) along with a confidence score[14].

2.5 Minigene Construction

The wild-type (WT) human NPR2 cDNA was obtained from NCBI. To confirm the possible consequences of the splice site mutation, and in vitro analysis was performed using a minigene splicing assay based on the pSPL3 exon trapping vector (Invitrogen). The minigene included the 169-bp exon 15, the 147-bp exon 16, the 124-bp exon 17, the 69-bp exon 18, and the intron of 15, 16, 17 and 18. The minigene sequence was inserted into the multiple cloning site of the pSPL3 vector (restriction sites: EcoRI and BamHI). The pSPL3-WT plasmid contained the gene of interest. The c.2643G > A mutation was introduced into the sequence using PCR with mutagenic primers (5’-CCCATGCAAGTGAGAGCCAT-3’), and then the PCR products were ligated using the In-Fusion® Snap Assembly Master Mix (TaKara, Beijing, China).

2.6 Homology Modeling and Molecular docking of Human NPR2

The ATP-binding domain of Human NPR2 was modeled as follows. The wild-type of NPR2 (amino acids:512–783) was modeled on the crystal structure (PDB entry 6JUT). Bioinformatic tools from HOPE (https://www3.cmbi.umcn.nl/hope/) were used to model and predict the effects of the p.Leu527Phe variant on NPR2. We downloaded the 3D structures of ATP from the PubChem database (CID: 5957), and then used Open Babel (http://openbabel.org/) to minimize the energy of the structures[15]. AutoDock Vina 1.1.2 software was used for molecular docking[16]. Prior to docking, the receptor protein was treated with PyMol 2.5, resulting in the removal of water molecules, salt ions, and small molecules[17]. The docking box was then set up to contain the active site. Subsequently, all the processed small molecules and receptor proteins were converted to PDBQT format using ADFRsuite 1.0[18]. From the docking results, the highest scoring docked model was chosen. Finally, PyMOL 2.5 was used for the visualization of the docking results. The interactions between amino acid residues were analyzed using DynaMut (http://biosig.unimelb.edu.au/dynamut/)[19].

2.7 Wild-Type and Mutant NPR2 Gene Expression Plasmid Construction

Chemically synthesized full-length human NPR2 complementary DNA was cloned into a 3×FLAG-CMV vector. The c.1579C > T, c.2842dupC, and c.2643G > A variants were introduced into the NPR2 sequence using homologous recombination (TaKara, Beijing, China). The ligation mixture was transformed into Escherichia coli DH5α. Subsequently, positive clones were PCR amplified with the universal primers CMV-F (forward) and hGH (reverse) and Sanger sequenced. The mutagenesis primer sequences are indicated in Supplemental Table 1.

2.8 Cell Culture and Transfection

At 37°C with 5% CO2, HEK293T cells (ATCC® CRL-11268) were cultured in high-glucose DMEM (Hyclone®), containing 10% fetal bovine serum and 1% penicillin-streptomycin. HEK293T cells were cultured in 24-well plates. For transient transfections, 70% confluent HEK293T cells were transfected with 500ng plasmid DNA per well by Hieff Trans™ Liposomal Transfection Reagent (Cat#40802, Yeasen, China).

2.9 cGMP Synthesis and Enzyme-linked Immunosorbent Assay (ELISA)

Seven groups were tested, including a negative control with only plasmid 3×FLAG-CMV, WT with 3×FLAG-CMV-WT-NPR2, and experimental groups with 3xFLAG-CMV-MUT-NPR2 of c.1579C > T, c.2842dupC, c.2643G > A, WT/c.1579C > T, WT/c. 2842dupC or WT/c.2643G > A. After 24 hours, the cells were starved with opti-MEM for 4 hours. The cells were pretreated with 0.1mM 3-isobutyl-1-methylxanthine (GLPBIO, Cat#GC11730, USA) for 15 minutes and then exposed to 100nM CNP (GLPBIO, Cat#GC43328, USA) and different concentrations of ATP (0 ~ 1000nM) (GLPBIO, Cat#GC35420, USA) for another 15 minutes. Subsequently, 0.1M HCl containing 0.4% TritonX-100 was added to stop endogenous phosphodiesterase activity, stabilize the released cGMP, and cause cell lysis. A centrifuge at 1200×g for 3 minutes isolated the supernatants from cell lysates. cGMP levels were detected using the cGMP ELISA detection Kit (GenScript, Cat# L00461, USA). At least three independent experiments were conducted. A t-test was used for analysis of the statistical data, which was expressed as mean ± SD.

2.10 Western Blot Analysis

As described previously, Western blotting was performed [13]. Antibodies against FLAG-tag (Cat#14793) were purchased from Cell Signaling Technology (Beverley, MA, USA). Antibodies against α-Tublin (Cat#ab52866) were obtained from Abcam (Shanghai, China). The grayscale was measured by ImageJ software. In addition, all experiments were carried out in triplicate. The data were expressed as a ratio of relative optical density over β-actin levels.

2.11 Quantitative Real-Time Polymerase Chain Reaction

Total RNA was prepared using Trizol reagent (Invitrogen), and reverse transcription into cDNA was completed with HiScript III RT SuperMix (Vazyme Biotech, Cat#R323) for qPCR. Previously described[13], we carried out quantitative real-time PCR. β-actin (ACTB gene) was used as a reference gene for normalization.

2.12 Statistical Analysis

Western blotting images were processed using Fiji/Image J software (https://imagej.net/Fiji). The Graph Pad Prism 8 was used for statistical analysis. A t-test was used to analyze statistical significance. At least three independent experiments were conducted to verify the results. Data are represented as mean ± SD (n ≥ 3). * indicates P < 0.05, ** indicates P < 0.01 and *** indicates P < 0.001.

3.1 Clinical evaluations

3.1.1 Patient 1

The proband, a 7.6-year-old girl, was admitted to hospital due to short stature since early childhood. She was delivered by cesarean section at gestational week 40. Her birth weight was 2650g (-1.0SDS), and her birth length was 48cm (-1.0SDS). Her height and weight were 110.2cm (-3.06SDS) and 17.5kg (-0.33SDS), respectively. Her sitting height was 62.2cm, with a sitting height/height ratio of 0.56 (2.35SDS). A spinal X-ray revealed slight lumbar scoliosis (Supplemental Fig. 1A). The bone age (BA) was 5.3 years using the Tanner-Whitehouse 3 (TW3) method (Supplemental Fig. 1B). The GH stimulation test (levodopa and clonidine) resulted in GH levels of 7.9 ng/mL (normal: ≥10ng/mL), and IGF1 levels of 116ng/mL (Table 1). Her IGFBP3, thyroid function, plasma calcium, phosphate and parathyroid hormone were all within normal limits. Pituitary magnetic resonance imaging showed a normal pituitary gland. The heights of the father, mother, and grandmother were 167cm (-0.93SDS), 146.5cm (− 2.6SDS), and 150 cm (− 1.96SDS), respectively. Her parents had proportionate bodies.

Table 1

Clinical data of patients.
Items	Patient 1	Patient 2
Gender	Female	Female
Birth weight (kg/SDS)	2.65 /-1.0	2.9/+1.9
Birth height (cm/SDS)	48/-1.0SDS	46/-2.18
Age at diagnosis	7.6	4.5
Height (cm)	110.2	96.3
HSDS	-3.06	-2.6
Weight (kg/SDS)	17.5/-0.33	14/-0.33
Sitting height	62.2	52.2
Sitting height/height (ratio/SDS)	0.56/2.35	0.54/2.36
Bone age (years)	5.3	4.5
Blood hormonal characteristics			Normal value
Peak growth hormone	7.9	25.6	≥ 10ng/mL
IGF1	116	158	4.5 year-old: 31.0 ~ 222.5 pg/mL 7.6 year-old: 47.0 ~ 890.5 pg/mL
IGFBP3	3.05	4.55	0.7 ~ 10mg/L
SDS: standard deviation score; HSDS: height standard deviation score; IGF1: insulin-like growth factor-1; IGFBP3: insulin-like growth factor binding protein-3.

3.1.2 Patient 2

The proband, a 4.5-year-old girl, was admitted to hospital due to short stature since early childhood. Her height and weight were 96.3cm (-2.6SDS) and 14.0kg (-0.33SDS), respectively. She was delivered by cesarean section at gestational week 40. Her birth weight was 2900g (+ 1.9SDS), and her birth length was 46cm (-2.18SDS). Her sitting height was 52.2cm, with a sitting height/height ratio of 0.54 (2.36SDS). The bone age (BA) was 4.5 years (Supplemental Fig. 1C). A spinal X-ray revealed slight lumbar scoliosis (Supplemental Fig. 1D). Blood hormone levels are listed in Table 1. The heights of the father and mother were 168cm (-0.77SDS) and 148cm (− 2.25SDS), respectively.

3.1.3 Patient 3

The patient 3 characteristics, including diagnoses and detailed clinical features, have been described in our previous paper [20]. Here, the treatment and follow-up are further described to provide an explanation of the synonymous mutation (NPR2 c.2643 G > A) carried by the patient and verify its pathogenicity (see below).

3.1.4 Treatment and follow-up

Three patients received rhGH therapy (0.13 ~ 0.19IU/kg/d) for two years (Table 2). Average age at initiation of treatment was 6.5 ± 1.8 years. In the first year of treatment, the average height increased by 1.10 ± 0.15SDS. In the second year of treatment, the average height further increased by 0.48 ± 0.11SDS (Fig. 1A-1D). The three patients showed a good response to rhGH treatment. No significant side effects were noted during the course of treatment. Details of follow-up, treatment and outcome treatment are provided in Table 2.

Table 2

Genetic information and response to growth hormone therapy in patients
	Age (years)	Duration of GH treatment (years)	Dose of rhGH (IU/kg/d)	Hight (cm)	HSDS	Bone age (years)	IGF1 (pg/mL)	IGFBP3 (mg/L)	Genetic information
Patient 1	7.6	0	0.13	110.2	-3.06	5.8	116	3.05	NPR2 c.1579C > T,p.Leu527Phe De novo
	8.1	0.5	0.14	115.5	-2.4	ND	176	3.03
	8.6	1	0.15	121	-1.78	8.0	324	3.45
	9.1	1.5	0.16	125	-1.56	8.6	181	3.74
	9.6	2	0.17	128.6	-1.4	9.5	153	3.26
Patient 2	4.5	0	0.15	96.3	-2.6	4.5	158	4.55	NPR2 c.2842dupC,p.His948Profs*5 Inherited from her mother
	5	0.5	0.15	101.8	-2.0	ND	383	6.58
	5.5	1	0.14	106.7	-1.58	5.6	318	5.99
	6	1.5	0.14	110.7	-1.28	ND	236	7.41
	6.5	2	0.14	114.9	-0.96	6.6	292	6.39
Patient 3	7.5	0	0.15	110.8	-3.11	4.5	185	3.90	NPR2 c.2643 G > A, p.His840Gln* Inherited from his father
	8	0.5	0.15	115.8	-2.77	ND	325	5.80
	8.5	1	0.166	119.7	-2.1	6.6	369	5.94
	9	1.5	0.17	123.8	-1.87	7.3	334	4.97
	9.5	2	0.19	128.2	-1.63	8.0	387	5.98
GH: growth hormone; HSDS: height standard deviation score; rhGH: recombinant human growth hormone; IGF1: insulin-like growth factor-1; IGFBP3: insulin-like growth factor binding protein-3; ND: not done.

3.2 Genetic diagnosis

WES and Sanger sequencing revealed a heterozygous NPR2 variant c.1579C > T,p.Leu527Phe in patient 1 (Fig. 2A). Neither parent carried this variant, suggesting a de novo occurrence. A frameshift variant of the NPR2 gene (c.2842dupC,p.His948Profs*5) was identified in patient 2, which was inherited from the mother (Fig. 2B). Additionally, neither of the variants was discovered in the publicly accessible gnomAD (http://gnomad), Chinese Millionome (https://db.cngb.org), Clinvar (https://www.ncbi.nlm.nih.gov/clinvar/), or Human Gene Mutation databases (http://www.hgmd.org/). According to the ACMG/AMP guidelines, the frameshift variant (c.2842dupC) was classified as pathogenic (ACMG criteria: PVS1, PM2 _Supporting, PP1, and PP3), whereas the missense variants appeared as variants of unknown significance (VUS) (c.1579C > T, ACMG criteria: PM2 _Supporting, PP2, PP3, and PP4). Conservative analysis of the protein sequence showed the Leu527 was highly conserved among various species (Fig. 2D). In addition, the missense variant Leu527Phe was predicted to be deleterious on NPR2 from four out of five predictors (Table 3).

Table 3. Predictions and confidence scores for the NPR2 variants were obtained from the PREDICT-SNP server.

Variants Predictors	Leu527Phe
PredictSNP (Accuracy)	Deleterious （51%）
PhD-SNP (Confidence score)	Deleterious（61%）
PolyPhen-2 (Confidence score)	Deleterious（40%）
SIFT (Confidence score)	Deleterious（53%）
SNAP (Confidence score)	Neutral（83%）

A detailed description of the clinical manifestations and genetic testing results of patient 3 has been reported in our previous study[20]. Briefly, a synonymous mutation (NPR2 c.2643G > A) was identified in patient 3 that appeared to affect mRNA splicing. To assess the effect of the NPR2 c.2643G > A variant on splicing, a minigene construct model was established and was transiently transfected into HEK293T cells for expression. The empty pSPL3 plasmid vector was used as a negative control, and WT was used as a positive control. Expectedly, a significant difference in the cDNA fragment size between WT and c.2643G > A was observed by agarose gel electrophoresis. WT produced a PCR product of 509 bp, whereas c.2643G > A only produced a 385bp band, which corresponded to the exons 15, 16, and 18 (Fig. 3A, 3B). Thus, the c.2643G > A variant caused a complete deletion of exon 17 and a truncation of the NPR2 protein (His840Gln*) (Fig. 3C).

3.3 Wild-type or mutant NPR2 gene expression in HEK293T cell lines

To assess the effect of each NPR2 variant, wild-type and mutant NPR2 genes were expressed in HEK293T cells. The mRNA levels of NPR2 showed no difference among all of the groups (Supplemental Fig. 2). His948Profs*5 and His840Gln* exhibited a smaller molecular weight than the wild type, consistent with truncated variants. The protein level of His840Gln* was slightly decreased, while the protein expression of other variants did not seem to change significantly. Furthermore, the protein level after co-transfection of His840Gln* and wild-type was 1.4 times higher than that of wild-type in HEK293T cells (Fig. 4A, 4B).

3.4 Functional characterization of the NPR2 variants

We assessed the CNP-dependent guanylate cyclase activity of the NPR2 variants to confirm their predicted pathogenicity. Both NPR2 variants, His948Profs*5 and His840Gln*, failed to produce cGMP in the homozygous state. When co-transfected with the wild-type construct (ratio 1:1), cGMP synthesis showed a significant decrease of 49% and 31% compared to the wild-type, respectively. Compared with the wild type, the Leu527Phe variant demonstrated 38.2% cGMP production capacity while the heterozygous variant WT/Leu527Phe retained 50.2% of the activity of the wild-type (Fig. 5).

3.5 Homology Modeling and Molecular Docking

The Protein kinase domain of NPR2 was modeled on the HOPE online server. Molecular docking is an application used for predicting the interactions between small molecules (ligands or substrates) and proteins (enzymes) using molecular modeling techniques[21]. Herein, the Vina 1.1.2 software was used to perform a docking study of the compound ATP with the protein kinase domain of NPR2. The docked conformers were evaluated using the Glide (G) Score (kcal/mol). The negative number of ΔG represents the positive binding affinity between the ligand and receptor. Generally, a ΔG value less than − 6 kcal/mol is considered to be more likely to bind. The ΔG of this complex was − 9.1 kcal/mol, implying a reasonable binding mode of ATP to the protein kinase domain of NPR2.

The molecular docking results are displayed in Figs. 6A and 6C. The data showed that the Leu527Phe variant decreased the flexibility of the ATP-binding pocket (ΔΔS_VibENCoM: -0.911 kcal/mol/K) (Fig. 6B). Here, molecular docking results revealed adequate binding between ATP and the active pocket of NPR2. The adenine moiety of ATP formed two hydrogen bonds with the hinge backbone (Glu597 and Cys599) of the kinases (Fig. 6D), and the ribose part of ATP formed hydrogen bonds with Ser603 and Asp606. The phosphate part of ATP also formed hydrogen bonds with Ser648, Arg520, Leu527 and Ser526. In addition, ATP also has salt bridge interactions with Arg520 and Lys551 (Fig. 6D).

Last but not least, the HOPE and DynaMut online servers were used to predict the local interactions between wild-type amino acid residues and variant amino acid residues. As a result of the variant, leucine (Leu) changed to phenylalanine (Phe) and the sizes of the individuals were significantly different. The mutated residue is located in the protein kinase domain, which is important for the binding of ATP. Moreover, the variant introduced a more hydrophobic residue that could disrupt the folding of proteins and/or cause hydrogen bond loss (Fig. 6E, 6F). Thus, the Leu527Phe variant might disturb the interaction between ATP and the domain and affect the function of the protein.

3.6 Leu527Phe exchange decreases the ATP-dependent activation of NPR2

To investigate the influence of the Leu527Phe variant on the modulatory effect of ATP on NPR2 activity, CNP-induced guanylyl cyclase activity (100nM CNP) assays were performed in the presence of different concentrations of ATP. HEK293T cells were transfected with wild-type or mutant NPR2 genes (500 ng/well) or cotransfected with the wild-type and mutant NPR2 genes (ratio 1:1). In wild-type NPR2, cGMP levels increased significantly with the increase of ATP concentration: 1.3-fold (in response to 10 µM ATP), 2.0-fold (100 µM ATP), and 3.2-fold (1000 µM ATP) increase compared to baseline activity. However, the Leu527Phe variant was unresponsive to ATP. When the ATP concentration was increased to 100 and 1000 µM, the cGMP levels of the Leu527Phe variant were approximately 22.3% and 14.4% that of the wild type, respectively. In contrast, the cGMP levels of co-expressed NPR2 were approximately 46.6% (100 µM ATP) and 35.7% (1000 µM ATP) of the wild type, respectively (Fig. 7A, 7B).

The NPR2 gene has been implicated as a cause of short stature by heterozygous loss-of-function variants [22]. As a homodimeric receptor, NPR2 regulates skeletal growth by activating guanylate cyclase upon binding its ligand [22, 23]. Four domains make up the NPR2 protein, including the extracellular ligand-binding domain, the transmembrane domain, the kinase homology domain (KHD), and the guanylyl cyclase domain [24]. NPR2 gene is located on chromosome 9p13.3 and contains 22 exons. Two variants of the NPR2 gene were identified in patient 1 and patient 2, both of which are novel. Patient 3 carried a synonymous mutation (NPR2 c.2643 G > A)[20] at a conserved site of the intron-exon boundary region. A minigene model was constructed with the exon capture plasmid pSPL3 for transcript analysis. The variant resulted in aberrant splicing, in which exon 17 was skipped, leading to the deletion of exon 17 and the premature formation of a stop codon (NPR2 gene c.2643G > A,p.His840Gln*). Therefore, this synonymous variant is pathogenic, possibly affecting splicing. Functional studies showed that cells co-transfected with His840Gln* variant and wild-type NPR2 exhibited a significant decrease in cGMP levels after CNP stimulation in comparison with wild-type, suggesting a dominant-negative effect.

Most short stature associated NPR2 variants result in abnormal protein trafficking to the plasma membrane, reduced CNP binding affinity for ligands, or inhibition of NPR2 activity[25]. These mechanisms lead to a significant reduction in cGMP production. Studies have shown that ATP binding in KHD is required for sensitization of NPRs, which is followed by ligand-induced conformational change and enzyme activation[26, 27]. Hannema et al. have reported an activating variant in KHD that significantly increases the stimulatory effect of ATP on CNP-dependent guanylyl cyclase activity, resulting in increased function[24]. The c.1579C > T mutation described in patient 1 resulted in the replacement of a leucine with phenylalanine at residue 527 in KHD. As shown in Fig. 6, the modeling of KHD indicated that Leu527 was located in the ATP binding pocket and directly bound to ATP through hydrogen bonding. The prediction results of the DynaMut online server showed a significant change in the local structure of the Leu527Phe variant, and the rigidification of the local protein increased with unique structural disturbance of Leu527Phe, including changes in local hydrogen bond and hydrophobic bond patterns for NPR2. Known protein-substrate binding requires protein-local flexibility for the protein function and activity[28]. These results suggest that the Leu527Phe variant may impair the interaction with ATP. The almost ineffective stimulation of guanylyl cyclase activity by ATP in the Leu527Phe variant of NPR2 supports this hypothesis.

Heterozygous mutations in the NPR2 gene are associated with short stature, and the clinical manifestations were nonspecific[4, 29, 30]. Patient 1 and patient 2 presented with disproportionate short stature and scoliosis. Recent studies have shown significant differences in the efficacy of rhGH in the treatment of short stature caused by variants of the NPR2 gene[4, 29, 31, 32]. Vasque et al. found that GH did not improve height in older children (13 years old)[29]. A recent study showed that the initiation of rhGH treatment is inversely related to age in patients with heterozygous NPR2 mutations and that the treatment significantly improves height[4]. In addition, the effect of growth hormone treatment was also related to the location of the mutation. Patients with mutations in the extracellular ligand-binding domain appear to respond poorly to growth hormones [4]. The children in our study started treatment at a younger age, on average, about 6.5 ± 1.8 years old. Height improved by 1.59 ± 0.1SDS over 2 years of treatment, with a height improvement of 1.10 ± 0.15SDS in the first year. All three patients showed a good response to growth hormone treatment. Due to the progressive development of short stature in patients with NPR2 variants[33], it is worth considering rhGH treatment to prevent short stature. Despite this, a larger population with long-term follow-up is still needed to determine the efficacy of rhGH treatment for patients with NPR2 heterozygous variants.

In conclusion, we report 2 cases of novel pathogenic mutations in the NPR2 gene. The Leu527Phe variant in KHD significantly decreases the stimulatory effect of ATP on CNP-dependent guanylyl cyclase activity, resulting in loss of function. This study enriches our knowledge of the NPR2 gene mutation spectrum. GH therapy may be an effective approach to improving height in patients with heterozygous mutations in the NPR2 gene, particularly those located in the cytosolic compartment.

BA: bone age; CNP: C-type natriuretic peptide; IGF-1:insulin-like growth factor-1; IGFBP3: insulin-like growth factor binding protein-3; KHD：kinase homology domain; NPR2: natriuretic peptide receptor 2; rhGH: recombinant growth hormone; WES: whole-exome sequencing; WT: wild-type.

Acknowledgements

We thank the patients and their families for participating in this study. This work was sponsored by grants from the Clinical medical center (Grant No. 201808310), and the Basic and Clinical Study of Rare Disease (No. ZD-2019-01).

Authors’ contributions

HC, SZ, YS, and RC conceived and designed the research. RC, JC, KY, YZ and XY provided details of clinical data of patients. HC, SZ, and YS performed the experiments and analyzed the data. HC, SZ, and RC wrote the manuscript.

Conflict of interest

The authors declare no conflict of interest.

Funding

This work was sponsored by the grants from the Key Clinical Special Discipline Construction Program of Fuzhou, Fujian, P.R.C (No:201610191) and The Basic and Clinical Research of Rare Disease (No: ZD-2019-01).

Availability of data and materials

The dataset supporting the conclusions of this article is included within the article. Novel variants have been submitted to ClinVar (http://www.ncbi.nlm.nih.gov/clinvar/) [Accession numbers: SCV002540213 (NPR2 c.1579C>T,p.Leu527Phe) and SCV002498790 (NPR2, c.2842dupC,p.His948Profs*5)].

Ethics approval and consent to participate

This study was approved by the Ethics Committee of Fuzhou Children’s Hospital of Fujian, and written informed consents were obtained from the participants’ legal guardians.

Consent for publication

Not applicable.

Competing interests

The authors declare that they have no competing interests.

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FigureS1.png
Supplemental Figure 1. X-ray imaging of patient 1 and patient 2. (A) Radiographs of the hands and wrists of patient 1. (B) X-ray revealing the presence of scoliosis in the spine plain film. (C) Radiographs of the hands and wrists of patient 2. (D) The spine X-ray of patient 2 shows scoliosis.
FigureS2.tif
Supplemental Figure 2. Relative levels of NPR2 overexpression were measured by a qPCR analysis.
TableS1.docx

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published 27 Jul, 2023

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Novel Pathogenic NPR2 Variants in Short Stature Patients and the Therapeutic Response to rhGH

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Journal Publication

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Abstract

Objective

Methods

Result

Conclusion

Figures

1. Introduction

2. Patients And Methods

2.1 Subjects

2.2 Clinical Evaluations

2.3 Genetic Examination

2.4 Conservation and Pathogenicity Analysis of the Variant

2.5 Minigene Construction

2.6 Homology Modeling and Molecular docking of Human NPR2

2.7 Wild-Type and Mutant NPR2 Gene Expression Plasmid Construction

2.8 Cell Culture and Transfection

2.9 cGMP Synthesis and Enzyme-linked Immunosorbent Assay (ELISA)

2.10 Western Blot Analysis

2.11 Quantitative Real-Time Polymerase Chain Reaction

2.12 Statistical Analysis

3. Results

3.1 Clinical evaluations

3.1.1 Patient 1

3.1.2 Patient 2

3.1.3 Patient 3

3.1.4 Treatment and follow-up

3.2 Genetic diagnosis

3.3 Wild-type or mutant NPR2 gene expression in HEK293T cell lines

3.4 Functional characterization of the NPR2 variants

3.5 Homology Modeling and Molecular Docking

3.6 Leu527Phe exchange decreases the ATP-dependent activation of NPR2

4. Discussion

Abbreviations

Declarations

References

Supplementary Files

Status:

Journal Publication

Version 1