NPR2 Research Articles

Unveiling the pathogenic mechanisms of NPR2 missense variants: insights into the genotype-associated severity in acromesomelic dysplasia and short stature

Introduction: Natriuretic peptide receptor 2 (NPR2 or NPR-B) plays a central role in growth development and bone morphogenesis and therefore loss-of-function variations in NPR2 gene have been reported to cause Acromesomelic Dysplasia, Maroteaux type 1 and short stature. While several hypotheses have been proposed to underlie the pathogenic mechanisms responsible for these conditions, the exact mechanisms, and functional characteristics of many of those variants and their correlations with the clinical manifestations have not been fully established.Methods: In this study, we examined eight NPR2 genetic missense variants (p.Leu51Pro, p.Gly123Val, p.Leu314Arg, p.Arg318Gly, p.Arg388Gln, p.Arg495Cys, p.Arg557His, and p.Arg932Cys) Acromesomelic Dysplasia, Maroteaux type 1 and short stature located on diverse domains and broadly classified as variants of uncertain significance. The evaluated variants are either reported in patients with acromesomelic dysplasia in the homozygous state or short stature in the heterozygous state. Our investigation included the evaluation of their expression, subcellular trafficking and localization, N-glycosylation profiles, and cyclic guanosine monophosphate (cGMP) production activity.Results and Discussion: Our results indicate that variants p.Leu51Pro, p.Gly123Val, p.Leu314Arg, p.Arg388Gln have defective cellular trafficking, being sequestered within the endoplasmic reticulum (ER), and consequently impaired cGMP production ability. Conversely, variants p.Arg318Gly, p.Arg495Cys, and p.Arg557His seem to display a non-statistically significant behavior that is slightly comparable to WT-NPR2. On the other hand, p.Arg932Cys which is located within the guanylyl cyclase active site displayed normal cellular trafficking profile albeit with defective cGMP. Collectively, our data highlights the genotype-phenotype relationship that might be responsible for the milder symptoms observed in short stature compared to acromesomelic dysplasia. This study enhances our understanding of the functional consequences of several NPR2 variants, shedding light on their mechanisms and roles in related genetic disorders which might also help in their pathogenicity re-classification.

Clinical-genetic analysis of selected genes involved in the development of the human skeleton in 128 Czech patients with suspected congenital skeletal abnormalities

BackgroundCongenital skeletal abnormalities are a heterogeneous group of diseases most commonly associated with small or disproportionate growth, cranial and facial dysmorphisms, delayed bone maturation, etc. Nonetheless, no detailed genotype-phenotype correlation in patients with specific genetic variants is readily available. Ergo, this study focuses on the analysis of patient phenotypes with candidate variants in genes involved in bone growth as detected by molecular genetic analysis. MethodsIn this study we used molecular genetic methods to analyse the ACAN, COL2A1, FGFR3, IGFALS, IGF1, IGF1R, GHR, NPR2, STAT5B and SHOX genes in 128 Czech children with suspected congenital skeletal abnormalities. Pathogenic variants and variants of unclear clinical significance were identified and we compared their frequency in this study cohort to the European non-Finnish population. Furthermore, a prediction tool was utilised to determine their possible impact on the final protein. All clinical patient data was obtained during pre-test genetic counselling. ResultsPathogenic variants were identified in the FGFR3, GHR, COL2A1 and SHOX genes in a total of six patients. Furthermore, we identified 23 variants with unclear clinical significance and high allelic frequency in this cohort of patients with skeletal abnormalities. Five of them have not yet been reported in the scientific literature. ConclusionCongenital skeletal abnormalities may lead to a number of musculoskeletal, neurological, cardiovascular problems. Knowledge of specific pathogenic variants may help us in therapeutic procedures.

ВАРИАБЕЛЬНОСТЬ ГЕНОВ НАТРИЙУРЕТИЧЕСКИХ ПЕПТИДОВ И АНТИОКСИДАНТНОЙ ЗАЩИТЫ У ПАЦИЕНТОВ С ИНФАРКТОМ МИОКАРДА

Introduction. Cardiovascular diseases (CVD) continue to be the subject of scientific research in relation to molecular genetic predictors of their development. It has been proven that diseases of the cardiovascular continuum have a multifactorial nature with a significant genetic component, including the hereditary risk of myocardial infarction (MI). CVDs are characterized by a complex genetic structure with diverse combinations of single nucleotide polymorphic variants (SNVs). A promising task is to study the relationship of SNV adaptogenesis genes: inflammatory response, myocardial and endothelial dysfunction with developed MI in the Siberian population. Purpose: to reveal the relationship between SNV genes of natriuretic peptides and the antioxidant defense system with the development of myocardial infarction. Materials and methods. The material for the study was genomic DNA isolated from the peripheral blood of patients (n=146, 38 women and 108 men) admitted for treatment at the Kuzbass Clinical Cardiological Dispensary, Kemerovo, with a diagnosis of MI. The control group is represented by a population sample of residents of the city of Kemerovo (n=300, 190 women and 110 men). 20 polymorphic variants of 7 natriuritic peptide genes (NPPA, NPPA-AS1, NPPB, NPPC, NPR1, NPR2, NPR3) and 5 genes of the antioxidant defense system (SOD2, NCF4, CBR1, CBR3, CAT) were selected for the study. Genotyping of selected polymorphic variants was performed by real-time PCR using TaqMan technology. Statistical data analysis was carried out using GraphPad Prism 8 and SNPstats software. Results. Protective associations of allelic variants of the CBR1 rs9024 genes (OR=0.21 (95% CI 0.13-0.34); p=0.0001) and CBR3 (OR=0.44 (95% CI 0.28-0) .69); p=0.001) regardless of gender according to the dominant model of inheritance. When separating patients by gender, it was found that in men, the rs9024 CBR1 allele A (OR=0.20 (95% CI 0.11-0.36); p<0.0001) and the rs1056892 CBR3 allele A (OR=0, 51 (95% CI 0.28-0.91); p=0.022) have a protective effect in the development of MI according to the dominant model of inheritance. Allele T rs2236289 (OR=1.93 (95% CI 1.04-3.58); p=0.035) and allele A rs7034957 (OR=1.88 (95% CI 1.03-3.45); p =0.038) of the NPR2 gene are associated with a risk effect on the development of myocardial infarction in men according to the dominant model of inheritance. In women, rare alleles of the polymorphic loci rs13288085 (OR=0.25 (95% CI 0.08-0.73); p=0.0034) and rs7034957 (OR=0.30 (95% CI 0.11-0, 79); p=0.007) of the NPR2 gene, rs9024 (OR=0.21 (95% CI 0.09-0.47); p=0.00001) of CBR1, as well as rs1056892 (OR=0.31 (95% CI 0.15-0.64); p=0.0014) CBR3 are characterized by a protective effect on the development of MI according to the dominant model of inheritance. Conclusion. It has been shown that polymorphism of genes for natriuretic peptides and antioxidant protection may have a risky and protective effect in relation to predisposition to the development of MI. The obtained preliminary results indicate the need for further studies of the identified SNV genes of adaptogenesis in relation to the severity of MI and the risk of recurrent cardiovascular events in the long-term period after MI.

Pharmacological and Genetic Disruption of C-Type Natriuretic Peptide (nppcl) Expression in Zebrafish (Danio rerio) Causes Stunted Growth during Development.

Human patients with mutations within NPPC or NPR2 genes (encoding C-type natriuretic peptide (CNP) and guanylyl cyclase-B (GC-B), respectively) display clinical signs associated with skeletal abnormalities, such as overgrowth or short stature. Mice with induced models of Nppc or Npr2 deletion display profound achondroplasia, dwarfism and early death. Recent pharmacological therapies to treat short stature are utilizing long-acting CNP analogues, but the effects of manipulating CNP expression during development remain unknown. Here, we use Danio rerio (zebrafish) as a model for vertebrate development, employing both pharmacological and reverse genetics approaches to alter expression of genes encoding CNP in zebrafish. Four orthologues of CNP were identified in zebrafish, and spatiotemporal expression profiling confirmed their presence during development. Bioinformatic analyses suggested that nppcl is the most likely the orthologue of mammalian CNP. Exogenous CNP treatment of developing zebrafish embryos resulted in impaired growth characteristics, such as body length, head width and eye diameter. This reduced growth was potentially caused by increased apoptosis following CNP treatment. Expression of endogenous nppcl was downregulated in these CNP-treated embryos, suggesting that negative feedback of the CNP system might influence growth during development. CRISPR knock-down of endogenous nppcl in developing zebrafish embryos also resulted in impaired growth characteristics. Collectively, these data suggest that CNP in zebrafish is crucial for normal embryonic development, specifically with regard to growth.

Novel pathogenic NPR2 variants in short stature patients and the therapeutic response to rhGH

ObjectiveHeterozygous loss-of-function variants in the NPR2 gene cause short stature with nonspecific skeletal abnormalities and account for about 2 ~ 6% of idiopathic short stature. This study aimed to analyze and identify pathogenic variants in the NPR2 gene and explore the therapeutic response to recombinant growth hormone (rhGH).MethodsNPR2 was sequenced in three Chinese Han patients with short stature via exome sequencing. In vitro functional experiments, homology modeling and molecular docking analysis of variants were performed to examine putative protein changes and the pathogenicity of the variants.ResultThree patients received rhGH therapy for two years, and two NPR2 heterozygous variants were identified in three unrelated cases: c.1579 C > T,p.Leu527Phe in patient 1 and c.2842dupC,p.His948Profs*5 in patient 2. Subsequently, a small gene model was constructed, and transcriptional analysis of the synonymous variant (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 6.5 ± 1.8 years old, and their height increased by 1.59 ± 0.1 standard deviation score after 2 years of treatment.ConclusionIn this report, two novel variants in NPR2 gene were described. Our findings broaden the genotypic spectrum of NPR2 variants in individuals with short stature and provid insights into the efficacy of rhGH in these patients.

Роль генов систем натрийуретических пептидов и антиоксидантной защиты в формировании риска развития инфаркта миокарда (предварительные результаты пилотного исследования)

Aim: to reveal a relationship of single-nucleotide polymorphic variants of the genes of the natriuretic peptides, antioxidant defense systems and the endothelial function with the development of myocardial infarction. Patients and Methods: the study included 146 patients with myocardial infarction (MI) and maintained or moderately decreased left ventricular ejection fraction: 108 males and 38 females with a mean age of 57 (51; 64) years. The control group was composed of Kemerovo city residents without acute MI (n=300, 190 females and 110 males). To make the evaluation, genomic DNA was isolated from the peripheral blood by phenol- chloroform extraction according to the standard protocol. Real-time PCR method was used for genotyping. To conduct the study, 24 polymorphic variants of 14 genes (those of the natriuretic peptides, antioxidant defense systems and the endothelial function) were selected. Results: significant associations with the allelic variants of CBR1 and CBR3 genes (carbonyl reductase 1 and 3 genes) of the antioxidant defense system were found in the sample of studied patients with MI (not divided by gender or age). When the patients were divided by gender, the following associations were found: in males the single-nucleotide polymorphic variant (SNPV) genotypes CBR1 rs9024 and CBR3 rs1056892 had the protective effect against susceptibility to myocardial infarction (dominant inheritance pattern). It was demonstrated that allelic variants in the NPR2 gene (the gene of atrial natriuretic peptide receptor 2) comprised a risk factor for MI development. In females, a relationship between the assessed genetic factors and the MI development (dominant inheritance pattern) could be associated with SNPV genotypes NPR2 rs13288085 and rs7034957, CBR1 rs9024, and CBR3 rs1056892 which are characterized by the protective effect. Conclusion: some polymorphic variants of the genes of the natriuretic peptide and antioxidant defense systems are associated with the risk and the protective effect against MI susceptibility. The preliminary findings indicate that it is necessary to continue investigations of the identified single-nucleotide polymorphic variants and to assess their impact on the MI severity and the risk of recurrent cardiovascular events in the long-term perspective. KEYWORDS: myocardial infarction, gene, natriuretic peptides, antioxidant defense, polymorphic variants. FOR CITATION: Khutornaya M.V., Khryachkova O.N., Sinitskaya A.V. et al. Role of the genes of the natriuretic peptide and antioxidant defense systems in creating the risk of myocardial infarction development (the preliminary results of a pilot study). Russian Medical Inquiry. 2023;7(1):5–12 (in Russ.). DOI: 10.32364/2587-6821-2023-7-1-5-12.

Multifunctional tunable ZnFe2O4@MnFe2O4 nanoparticles for dual-mode MRI and combined magnetic hyperthermia with radiotherapy treatment.

With the increase in non-communicable diseases, cancer is becoming one of the most lethal ailments of the coming decades. Significant progress has been made in the development of NPs that combine diagnostic and therapeutic properties in a single system. Multimodal NPs that sequentially perform MRI diagnostics with increased contrast and then act as synergistic agents for magnetic hyperthermia and radiotherapy can be considered as next-generation anticancer drugs. Thus, we propose a systematic study of composite theranostic ZnFe2O4@MnFe2O4 NPs for the first time. Two types of magnetic NPs with MnFe2O4 shell thicknesses of 0.5 (ZM0.5) and 1.7 nm (ZM3) were prepared via hydrothermal synthesis. Tuning the shell thickness was shown to influence the NP r2 and r1 relaxivities and allow T1-T2 dual-mode contrast agents to be obtained. A radiotherapy study demonstrated a significant dose factor enhancement (about 40%) for both NP types. The specific absorption rate of ZM3 in a 100 Oe alternating magnetic field with a frequency of 75 kHz was found to be 8 W g-1, which results in heating up to 42 °C within a few seconds. This work presents high-performance multifunctional NPs capable of combining different diagnostic and therapeutic methods for a full course of treatment using only one type of NP.

Identification of Diagnostic Variants in FGFR2 and NPR2 Genes in a Chinese Family Affected by Crouzon Syndrome and Acromesomelic Dysplasia, Type Maroteaux.

This study aims to conduct a comprehensive clinical and genetic investigation on a large family with members having various phenotypes, including acromesomelic dysplasia, type Maroteaux (AMDM), idiopathic short stature (ISS), Crouzon syndrome (CS). Prenatal diagnosis was performed on the high-risk fetus. We performed the whole-exome sequencing on three members with AMDM, ISS, or CS. Detailed genotypes and phenotypes were investigated on members of this 4-generation family. Genetic analysis identified three variants, which were designated as p.Val548del, p.Arg989Gln in natriuretic peptide receptor B/guanylate cyclase B (NPR2), and p.Cys342Tyr in fibroblast growth factor receptor-2 (FGFR2). Compound heterozygous variation consisting of p.Val548del and p.Arg989Gln caused AMDM. NPR2 heterozygous variant carriers exhibited normal height or ISS. The p.Cys342Tyr mutation of FGFR2 causes the typical clinical phenotype of CS. The fetus carried the heterozygous p.Val548del and p.Cys342Tyr mutations, with ultrasound results showing exophthalmos, parrot-beaked nose, low and flat frontal skull, and intrauterine growth retardation at the second and third trimesters of gestation. We are reporting those two novel mutations (p.Val548del and p.Arg989Gln) in NPR2 and a p.Cys342Tyr mutation in FGFR2 in an extended Chinese family. This finding extended the genotype-phenotype spectra of ISS, AMDM, and CS related to pathogenic variants.

Heterozygous NPR2 Variants in Idiopathic Short Stature.

Heterozygous variants in the NPR2 gene, which encodes the B-type natriuretic peptide receptor (NPR-B), a regulator of skeletal growth, were reported in 2–6% cases of idiopathic short stature (ISS). Using next-generation sequencing (NGS), we aimed to assess the frequency of NPR2 variants in our study cohort consisting of 150 children and adolescents with ISS, describe the NPR2 phenotypic spectrum with a growth pattern including birth data, and study the response to growth hormone (GH) treatment. A total of ten heterozygous pathogenic/likely pathogenic NPR2 variants and two heterozygous NPR2 variants of uncertain significance were detected in twelve participants (frequency of causal variants: 10/150, 6.7%). During follow-up, the NPR2 individuals presented with a growth pattern varying from low–normal to significant short stature. A clinically relevant increase in BMI (a mean gain in the BMI SDS of +1.41), a characteristic previously not reported in NPR2 individuals, was observed. In total, 8.8% participants born small for their gestational age (SGA) carried the NPR2 causal variant. The response to GH treatment was variable (SDS height gain ranging from −0.01 to +0.74). According to the results, NPR2 variants present a frequent cause of ISS and familial short stature. Phenotyping variability in growth patterns and variable responses to GH treatment should be considered.

EP115: Two novel NPR2 variants in a patient with acromesomelic dysplasia - Maroteaux type

Acromesomelic dysplasia, Maroteaux type (AMDM) is a rare autosomal recessive skeletal dysplasia characterized by severe disproportionate short stature (adult height < 120 cm), dolichocephaly, acro- and mesomelic shortening of the limbs, short, broad fingers and flat feet. Radiographic findings include bowed radius with a short ulna, dolichocephaly, platyspondyly and narrowing of the lumbar interpedicular distances. Intelligence is unaffected. The condition is typically diagnosed at birth or in infancy, but features become more apparent over time because of the loss of linear growth. AMDM is caused by biallelic loss of function variants in the NPR2 gene which encodes the natriuretic peptide receptor-B (NPR-B). The NPR2 gene encodes a transmembrane receptor that is critical in endochondral bone formation. Forty four percent of variants in NPR2 occur in the ligand binding domain, 8% in the transmembrane domain, 26% in the protein kinase domain and 21% in the nucleotide cyclase domain. A seven-month-old was seen with clinical findings suggestive of a skeletal dysplasia. She was born at 36 weeks following a pregnancy complicated by maternal polysubstance use and poor prenatal care. Clinical findings were shortening of all limb segments, trident hands, weak grasp, and overlapping second and third toes bilaterally. Radiographic findings were shortening of all the long bones with metaphyseal flaring, short and broad metatarsals, metacarpals and phalanges also with mild metaphyseal widening, platyspondyly with mild anterior vertebral body beaking in the upper lumbar spine. Cervical CT examination revealed platyspondyly of the suboccipital vertebrae and decreased AP diameter of the spinal canal at C1 secondary to anterior bowing of the posterior arch. A multi-gene skeletal dysplasia panel identified twelve variants, including two variants in NPR2. NPR2 c.1699G>T was classified as pathogenic, causing a premature termination at amino acid 567 in the kinase homology domain of the transmembrane protein. While classified as a VUS, the c.1123G>A missense variant was predicted to cause a substitution at amino acid 375 and falls at the last nucleotide of exon 4, which is part of the consensus splice site for this exon. The patient’s mother tested positive for the pathogenic variant and negative for the VUS in NPR2. Acromesomelic dysplasia, Maroteaux type is a rare skeletal dysplasia with limited published phenotypic and molecular information available. The described patient presented a diagnostic conundrum because only one of the variants had been characterized as pathogenic. The variant of uncertain significance is located in the ligand binding domain of the NPR2 gene, in which the majority of reported variants in this gene are found. In silico algorithms predict this variant to be deleterious to the protein structure and function but these predictions have not been confirmed by functional studies. Furthermore, missense variants within the consensus splice sites commonly cause abnormal splicing. This variant is not found in population databases. While paternal samples are not available to confirm that the variants are in trans, the patient was given the diagnosis of AMDM based on her clinical presentation. Molecular analysis and clinical description of other patients with AMDM will continue to characterize disease causing variants and natural history of this rare disease.

Association Between Natriuretic Peptide Receptor 2 (NPR2) RS208158047 Polymorphism and Fattening Performance of Young Bulls

Abstract The objective of this study was to determine fattening performance data for Charolais, Limousin and Blonde d’Aquitaine beef cattle and associate these data with NPR2 gene 8:g.59961937 T&gt;C (rs208158047) mutation. Experiments were conducted with 176 beef cattle (77 Charolais, 66 Limousin and 33 Blonde d’Aquitaine) at nine months of age. Experiments lasted for 9 months and animals were slaughtered at the age of 18 months. Cattle body weights were determined at four different periods: beginning of fattening (d0), 60th day of fattening (d60), 120th day of fattening (d120) and at the end of fattening (sw). In terms of rs208158047 mutation of Charolais, Limousin and Blonde d’Aquitaine breeds, TT and CT genotypes were identified, and CC genotype was not encountered. The association of average daily gain (ADG) in d0-d60, d0-d120 and d0-sw periods with the genotypes of rs208158047 mutation was found to be significant (P&lt;0.05). Greater ADGs were observed in rs208158047-CT genotypes compared to rs208158047-TT genotypes. These results indicate that the selection of bovine NPR2 gene could be used to ensure the breeding direction for growth related traits of the beef cattle.

Short stature is progressive in patients with heterozygous NPR2 mutations

Background NPR2 encodes atrial natriuretic peptide receptor B (ANPRB), a regulator of skeletal growth. Biallelic loss-of-function mutations in NPR2 result in acromesomelic dysplasia Maroteaux type (AMDM; OMIM 602875), while heterozygous mutations may account for 2% to 6% of idiopathic short stature (ISS). Objective Describe the physical proportions and growth characteristics of an extended family with novel NPR2 mutations including members with AMDM, ISS, or normal stature. Design and participants We performed whole exome sequencing in 2 healthy parents and 2 children with AMDM. Detailed genotyping and phenotyping were performed on members of a multigenerational family in an academic medical center. We expressed mutant proteins in mammalian cells and characterized expression and function. Results The sisters with AMDM were compound heterozygotes for missense mutations in the NPR2 gene, a novel p.P93S (maternal) and the previously reported p.R989L (paternal). Both mutant ANPRB proteins were normally expressed in HEK293T cells and exhibited dominant negative effects on wild-type ANPRB catalytic activity. Heterozygous relatives had proportionate short stature (height z-scores -2.06 ± 0.97, median ± SD) compared with their wild-type siblings (-1.37 ± 0.59). Height z-scores progressively and significantly decreased as NPR2-heterozygous children matured, while remaining constant in their wild-type siblings. Conclusions Biallelic NPR2 mutations cause severe skeletal dysplasia (AMDM), whereas heterozygous mutations lead to a subtler phenotype characterized by progressive short stature with by increasing loss of height potential with age.

Short Stature is Progressive in Patients with Heterozygous NPR2 Mutations.

NPR2 encodes atrial natriuretic peptide receptor B (ANPRB), a regulator of skeletal growth. Biallelic loss-of-function mutations in NPR2 result in acromesomelic dysplasia Maroteaux type (AMDM; OMIM 602875), while heterozygous mutations may account for 2% to 6% of idiopathic short stature (ISS). Describe the physical proportions and growth characteristics of an extended family with novel NPR2 mutations including members with AMDM, ISS, or normal stature. We performed whole exome sequencing in 2 healthy parents and 2 children with AMDM. Detailed genotyping and phenotyping were performed on members of a multigenerational family in an academic medical center. We expressed mutant proteins in mammalian cells and characterized expression and function. The sisters with AMDM were compound heterozygotes for missense mutations in the NPR2 gene, a novel p.P93S (maternal) and the previously reported p.R989L (paternal). Both mutant ANPRB proteins were normally expressed in HEK293T cells and exhibited dominant negative effects on wild-type ANPRB catalytic activity. Heterozygous relatives had proportionate short stature (height z-scores -2.06 ± 0.97, median ± SD) compared with their wild-type siblings (-1.37 ± 0.59). Height z-scores progressively and significantly decreased as NPR2-heterozygous children matured, while remaining constant in their wild-type siblings. Biallelic NPR2 mutations cause severe skeletal dysplasia (AMDM), whereas heterozygous mutations lead to a subtler phenotype characterized by progressive short stature with by increasing loss of height potential with age.

Сlinical and Radiological Characteristics of Two Patients with Acromesomelic Dysplasia Maroteaux Type with New Mutation in the &lt;i&gt;NRP2&lt;/i&gt; Gene

Relevance. Acromezomelic dysplasia Maroteaux type (AMDM) is a rare variant of autosomal recessive skeletal disorder. The disease is caused by mutations in the NPR2 gene, coding the protein product which is one of the main regulators of endochondral ossification. To date, 49 mutations in this gene have been identified, more than half of which are missense substitutions. The presence of polymorphism of phenotypic manifestations makes it necessary to describe the features of clinical and radiological characteristics of the disease in patients with newly identified mutations in the gene, which will help to optimize its diagnosis. Case presentation. The clinical and radiological characteristics of two siblings with newly identified mutations c.125_126insTGGCG (p.Trp42CysfsTer12) and (p.Arg767Ter) in the NPR2 gene are described. Intra-family polymorphism of clinical manifestations is shown. Discussion. Clinical manifestations and radiological data in two siblings with AMDM caused by new mutations in the NPR2 gene and analysis of the literature data allowed us to conclude that there is no correlation of the severity of clinical signs and the type of mutations in the gene. Patients are born with normal growth and weight, and clinical manifestations (disproportionate dwarfism) appeared during the first year of life. The main radiological signs are shortening of tubular bones, most pronounced in the upper limbs and wedge-shaped formation of the vertebral bodies. Genotype-phenotype correlations confirmed the hypothesis that the majority of mutations leading to the disease is localized within the ligand-binding and guanylate cyclase domains. Conclusion. The obvious genetic heterogeneity, the similarity of the clinical manifestations of individual nosological groups of skeletal dysplasias, as well as the presence of intrafamily and interfamily polymorphism of clinical manifestations allows us to consider sequencing of a clinical exome or whole exome as the optimal method for diagnosing this group of diseases.

A novel nonsense mutation in NPR2 gene causing Acromesomelic dysplasia, type Maroteaux in a consanguineous family in Southern Punjab (Pakistan)

BackgroundAcromesomelic dysplasia, type Maroteaux (AMDM) is a rare skeletal dysplasia following autosomal recessive mode of inheritance and characterized by abnormal growth plates, short and abnormal bones in the extremities and spine.ObjectivePresent study was designed to report the molecular basis of AMDM in enrolled consanguineous family from Pakistan.MethodsA consanguineous family from Vehari District in Pakistan having multiple siblings suffering from AMDM was enrolled in present study. Whole exome sequencing (WES) approach was adopted to identify causative agent of AMDM. Human full length NPR2 gene and sequence with nonsense mutation was amplified by using Myc-tagged pXN vector and transformed in E. coli DH5α cells to confirm mutation. SDS-PAGE and Western blotting were done to confirm the production of truncated protein. Computational three dimensional structure generation through homology modeling approach was done to compare protein structure between patients and controls.ResultsWES reveled a nonsense mutation (c.613 C>T, p.R205X) in exon 1 of NPR2 gene leading to premature termination codon in mRNA of NPR2 gene resulting in a truncated protein with 204 amino acid residues that was confirmed by SDS-PAGE and Western blotting. Sanger sequencing confirmed that mutation in all subjects and mutation followed Mendalian pattern of inheritance. Multiple sequence alignment by ClustalW revealed that mutated domain of NPR2 is conserved region. Proetin structure comparison revealed a significant structural part of NPR2 was missing in truncated protein as compared to control.ConclusionWe are reporting that a novel nonsense mutation (c.613 C>T, p.R205X) in exon 1 of NPR2 gene is causing AMDM in a consanguineous Pakistani family.

NPR2 Variants Are Frequent among Children with Familiar Short Stature and Respond Well to Growth Hormone Therapy.

The C-type natriuretic peptide receptor encoded by the NPR2 gene is a paracrine regulator of the growth plate; heterozygous NPR2 variants cause short stature with possible presence of different signs of bone dysplasia. To date, the effect of growth hormone (GH) treatment has been described in a few individuals with NPR2 gene variants with inconsistent results. To identify NPR2 gene variants among children with familial short stature (FSS) and to describe their phenotype, including GH treatment response. Out of 747 patients with short stature treated with GH in a single center, 87 with FSS met the inclusion criteria (pretreatment height ≤ -2 standard deviation in both the patient and the shorter parent, unknown genetic etiology). Next-generation sequencing methods were performed to search for NPR2 gene variants. The results were evaluated using the American College of Medical Genetics and Genomics guidelines. The GH treatment response (growth velocity improvement and height standard deviation score development over the first 5 years of treatment) was evaluated. In 5/87 children (5.7%), a (likely) pathogenic variant in the NPR2 gene was identified (p.Ile558Thr [in 2], p.Arg205*, p.Arg557His, p.Ser603Thr). Two children had disproportionate short-limbed short stature, 1 a dysplastic 5th finger phalanx. The growth velocity in the first year of GH treatment accelerated by 3.6 to 4.2 cm/year; the height improved by 1.2 to 1.8 SD over 5 years of treatment. NPR2 gene variants cause FSS in a significant proportion of children. Their GH treatment response is promising. Studies including final height data are necessary to assess the long-term efficacy of this therapy.

Stabilization of Perivascular Mast Cells by Endothelial CNP (C-Type Natriuretic Peptide).

Activated perivascular mast cells (MCs) participate in different cardiovascular diseases. Many factors provoking MC degranulation have been described, while physiological counterregulators are barely known. Endothelial CNP (C-type natriuretic peptide) participates in the maintenance of vascular barrier integrity, but the target cells and mechanisms are unclear. Here, we studied whether MCs are regulated by CNP. Approach and Results: In cultured human and murine MCs, CNP activated its specific GC (guanylyl cyclase)-B receptor and cyclic GMP signaling. This enhanced cyclic GMP-dependent phosphorylation of the cytoskeleton-associated VASP (vasodilator-stimulated phosphoprotein) and inhibited ATP-evoked degranulation. To elucidate the relevance in vivo, mice with a floxed GC-B (Npr2) gene were interbred with a Mcpt5-CreTG line to generate mice lacking GC-B in connective tissue MCs (MC GC-B knockout). In anesthetized mice, acute ischemia-reperfusion of the cremaster muscle microcirculation provoked extensive MC degranulation and macromolecule extravasation. Superfusion of CNP markedly prevented MC activation and endothelial barrier disruption in control but not in MC GC-B knockout mice. Notably, already under resting conditions, such knockout mice had increased numbers of degranulated MCs in different tissues, together with elevated plasma chymase levels. After transient coronary occlusion, their myocardial areas at risk and with infarction were enlarged. Moreover, MC GC-B knockout mice showed augmented perivascular neutrophil infiltration and deep vein thrombosis in a model of inferior vena cava ligation. CNP, via GC-B/cyclic GMP signaling, stabilizes resident perivascular MCs at baseline and prevents their excessive activation under pathological conditions. Thereby CNP contributes to the maintenance of vascular integrity in physiology and disease.

The First Turkish Family with Skeletal Abnormality and Novel NPR2 Gene Mutation

The First Turkish Family with Skeletal Abnormality and Novel NPR2 Gene Mutation

The Protective Role of Natriuretic Peptide Receptor 2 against High Salt Injury in the Renal Papilla

Mutations in natriuretic peptide receptor 2 (Npr2) gene cause a rare form of short-limbed dwarfism, but its physiological effects have not been well studied. Human and mouse genetic data suggest that Npr2 in the kidney plays a role in salt homeostasis. Herein, we described anatomic changes within renal papilla of Npr2 knockout (Npr2-/-) mice. Dramatic reduction was found in diuresis, and albuminuria was evident after administration of 1% NaCl in drinking water in Npr2-/- and heterozygous (Npr2+/-) mice compared with their wild-type (Npr2+/+) littermates. There was indication of renal epithelial damage accompanied by high numbers of red blood cells and inflammatory cells (macrophage surface glycoproteins binding to galectin-3) and an increase of renal epithelial damage marker (T-cell Ig and mucin domain 1) in Npr2-/- mice. Addition of 1% NaCl tended to increase apoptotic cells (cleaved caspase 3) in the renal papilla of Npr2-/- mice. Invitro, genetic silencing of the Npr2 abolished protective effects of C-type natriuretic peptide, a ligand for Npr2, against death of M-1 kidney epithelial cells exposed to 360 mmol/L NaCl. Finally, significantly lower levels of expression of the NPR2 protein were detected in renal samples of hypertensive compared with normotensive human subjects. Taken together, these findings suggest that Npr2 is essential to protect renal epithelial cells from high concentrations of salt and prevent kidney injury.

SUN-252 Comparison of Sitting Height/Height Ratio for Age in Children with Short Stature Caused by Defects in Growth Plate Genes

Introduction: Abnormal body proportion disclosed by sitting height/height ratio for age and sex (SH/H SDS) > 2 indicates an impairment of appendicular growth. This is frequently observed in patients with skeletal dysplasia and can be the only clinical sign in nonsyndromic short stature children that harbor heterozygous defects in genes expressed in growth plate. The aim of the present study is to compare the degree of body disproportion among children with different genetic cause of growth impairment. Methods: We evaluated 75 children with short stature caused by defects in known growth plate genes, including FGFR3 gene (16 mutations in 7 patients with achondroplasia - ACH and in 9 patients with hypochondroplasia - HCH), SHOX gene (35 defects in 15 patients with Leri-weill dyschondrosteosis - DLW and in 20 patients with isolated short stature, defined as absence of Madelung deformity clinically and radiologically), NPR2 gene (8 heterozygous mutations) and IHH gene (16 heterozygous mutations). Variants in NPR2 and IHH genes were identified in children with isolated short stature or with nonspecific skeletal abnormalities. Results: At first evaluation, patients had a mean age of 8.7±4.9 years. Children with ACH were significantly shorter than those of the other groups (mean height SDS: -4.4 vs -2.5, p<0.05). All patients with clinically recognizable skeletal dysplasia (ACH, HCH and DLW) had abnormal body proportion (SH/H SDS >2). The frequency of abnormal body proportion observed in children initially classified as idiopathic short stature (ISS) was 85% for SHOX defects and 56% for IHH mutations (p = 0.073). None children with heterozygous mutations in NPR2 presented abnormal body proportion, though 62.5% had SH/H SDS >1. Children with ACH had SH/H SDS higher than all other groups (mean: 9.9±2.8; p<0.001). The degree of abnormal body proportion observed in children with HCH was similar to that of DLW (mean: 5.7±1.1 and 4.6±2.3, respectively; p = 0.14) and was higher than in children with isolated short stature, independently of the genetic defect (p=0.003 for SHOX and p<0.001 for IHH and NPR2). The mean SH/H SDS observed in children initially classified as ISS was similar between SHOX and IHH groups (mean: 2.9 ± 0.9 and 2.3 ± 1.4, respectively; p = 0.26), which was significantly higher in comparison to NPR2 group (mean: 0.7 ± 1.3; p = 0.004 for SHOX and p=0.024 for IHH). Conclusion: All patients with ACH, HCH and DLW had abnormal body proportion and their SH/H SDS were significantly higher than that observed in ISS children with SHOX, IHH or NPR2 defects. Furthermore, among nonsyndromic children with short stature, abnormal body proportion is associated with SHOX and IHH defects, but not with NPR2 defects. The authors have nothing to disclose.