NPR2 Mutations Research Articles

OR18-5 A Prospective Clinical Trial of Vosoritide in Selected Genetic Causes of Short Stature

Abstract Objectives Vosoritide is a C-type natriuretic peptide analog which binds its receptor on chondrocytes, leading to increased chondrocyte proliferation and differentiation via its inhibition of the ERK1/2-MAPK pathway. It was recently approved for increasing linear growth in children with achondroplasia. Our study aims to assess the safety and efficacy of vosoritide in children with genetic mutations in 6 categories: hypochondroplasia, Rasopathies (including Noonan syndrome), aggrecan deficiency, carriers of heterozygous NPR2 mutations, CNP deficiency, and SHOX deficiency. Methods This is a prospective, Phase II study with a goal N of 35 subjects. All subjects must have a documented genetic mutation in one of the 6 categories, be prepubertal between the ages of 3 and 11 for boys and 3 and 10 for girls, and have a height < -2.25 SD. Subjects are followed for a 6-month observation period to establish a baseline annualized growth velocity (AGV) and then receive daily subcutaneous vosoritide (15 mcg/kg/day) for 12 months. The primary outcomes are rate of AEs and change in AGV from baseline. Pharmacokinetic studies and pharmacodynamic studies, using cGMP production as a pharmacodynamic marker, are completed at Day 1 and Months 6 and 12. Results To date, 27 subjects have enrolled in the trial (20 hypochondroplasia, 3 Noonan, 2 NPR2, 2 Aggrecan). Median baseline height is -3.1 SD (IQR -3.6, -2.6). 14 subjects have initiated on vosoritide, and 10 completed 6 months of therapy. Median increase in AGV was 3.9 cm/yr (IQR 2.2, 4.6). The two subjects with NPR2 mutations had increased AGVs of 4.4 and 9.2 cm/yr. The two subjects with Noonan syndrome had an increase in AGV of 7.5 and 3.3 cm/yr. One subject with Noonan syndrome, a 4-year-old male, had previously been treated with growth hormone (GH) for 2 years. His AGV was 6.7 cm/yr while on GH, 4.1 cm/yr during the 6-month observation period, and 11.6 cm/year during the first 6 months of vosoritide treatment. There were no serious adverse events related to vosoritide treatment. One subject had a brief episode of syncope with her first injection which self-resolved, and she continued on therapy with no further incidents. PK parameters were similar to previously published parameters for vosoritide in children with achondroplasia. In preliminary data, vosoritide Cmax and AUC correlated strongly with peak increase in cGMP (r=0.70, p=0.01;r=0.84, p<0.01, respectively) but not with increase in AGV (r=0.35, p=0.32). Conclusions This is the first clinical trial of vosoritide for children with genetic short stature who do not have achondroplasia. Vosoritide treatment may work as a precision therapy to improve growth in multiple genetic conditions which interact with the ERK1/2-MAPK pathway. Presentation: Monday, June 13, 2022 12:00 p.m. - 12:15 p.m.

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.

Clinical characteristics of short-stature patients with an NPR2 mutation and the therapeutic response to rhGH

CONTEXT The natriuretic peptide receptor 2 gene (NPR2) is a causative gene of idiopathic short stature (ISS) with an incidence rate of 2% to 6%. The clinical characteristics of patients with NPR2 heterozygous mutations are atypical, and data on the efficacy of recombinant human growth hormone (rhGH) treatment in patients with NPR2 mutations are limited. OBJECTIVES This work reports 6 cases with NPR2 mutation and explores the characteristics of patients with an NPR2 mutation and their therapeutic response to rhGH. DESIGN, SETTINGS, AND PATIENTS Six Chinese short-stature patients in our hospital with NPR2 mutations by whole-exome sequencing were included. We also searched all previously published NPR2 mutation cases as of August 10, 2020, and information about their medical history, mutations, and rhGH treatment were recorded and summarized. RESULTS The clinical characteristics of patients with an NPR2 heterozygous mutation mainly included short stature, facial anomalies, and skeletal dysplasia. Skeletal dysplasia mainly included brachydactyly (56.2%), shortened metacarpals or metatarsals (particularly fourth to fifth; 26.1%), and clinodactyly (21.7%). rhGH treatment significantly improved the height SD score (SDS) of patients with NPR2 heterozygous mutations (median, -2.1 vs -2.9, P < .001), especially in girls. The height SDS change correlated negatively with initial age of treatment (r = -0.477; P = .034), and height SDS change of patients with NPR2 heterozygous mutations in the carboxyl-terminal guanylyl cyclase catalytic domain was significantly higher than that of the extracellular ligand-binding region domain (median, 1.9 vs 0.6, P = .019). CONCLUSIONS ISS patients with skeletal deformities should be tested for an NPR2 mutation. rhGH treatment is beneficial for short-stature patients with NPR2 heterozygous mutations and needs further study.

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.

Novel Mutations and Genes That Impact on Growth in Short Stature of Undefined Aetiology: The EPIGROW Study.

BackgroundChildren with short stature of undefined aetiology (SS-UA) may have undiagnosed genetic conditions.PurposeTo identify mutations causing short stature (SS) and genes related to SS, using candidate gene sequence data from the European EPIGROW study.MethodsFirst, we selected exonic single nucleotide polymorphisms (SNPs), in cases and not controls, with minor allele frequency (MAF) < 2%, whose carriage fitted the mode of inheritance. Known mutations were identified using Ensembl and gene-specific databases. Variants were classified as pathogenic, likely pathogenic, or variant of uncertain significance using criteria from the American College of Medical Genetics and Genomics and the Association for Molecular Pathology. If predicted by ≥ 5/10 algorithms (eg, Polyphen2) to be deleterious, this was considered supporting evidence of pathogenicity. Second, gene-based burden testing determined the difference in SNP frequencies between cases and controls across all and then rare SNPs. For genotype/phenotype relationships, we used PLINK, based on haplotype, MAF > 2%, genotype present in > 75%, and Hardy Weinberg equilibrium P > 10–4.ResultsFirst, a diagnostic yield of 10% (27/263) was generated by 2 pathogenic (nonsense in ACAN) and a further 25 likely pathogenic mutations, including previously known missense mutations in FANCB, IGFIR, MMP13, NPR2, OBSL1, and PTPN11. Second, genes related to SS: all methods identified PEX2. Another 7 genes (BUB1B, FANCM, CUL7, FANCA, PTCH1, TEAD3, BCAS3) were identified by both gene-based approaches and 6 (A2M, EFEMP1, PRKCH, SOS2, RNF135, ZBTB38) were identified by gene-based testing for all SNPs and PLINK. ConclusionsSuch panels improve diagnosis in SS-UA, extending known disease phenotypes. Fourteen genes related to SS included some known to cause growth disorders as well as novel targets.

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.

Role of NPR2 mutation in idiopathic short stature: Identification of two novel mutations.

BackgroundC‐type natriuretic peptide (CNP, NPPC) and its receptor, natriuretic peptide receptor‐B (NPR‐B, NPR2), are critical for endochondral ossification. A monoallelic NPR2 mutation has been suggested to mildly impair long bone growth. This study was performed to identify the NPR2 mutations in Korean patients with idiopathic short stature (ISS).MethodsOne hundred and sixteen subjects with nonsyndromic ISS were enrolled in this study, and the NPPC and NPR2 were sequenced. In silico prediction and in vitro functional analysis, using a cell‐based assay, were performed to confirm their protein derangement.ResultsMean age at diagnosis of ISS was 8.0 years, and the height z‐score was −2.65. Three pathogenic variants (R921Q, R495C, and Y598N) and one benign variant (R787W) of the NPR2 were identified, while no novel sequence variant of the NPPC was found in all subjects. Two novel pathogenic mutants (R495C and Y598N) were predicted as highly pathogenic by several computational methods. In vitro study involving stimulation with CNP, R495C‐, and Y598N‐transfected cells showed decreased cGMP production compared to wild type‐transfected cells.ConclusionHeterozygous NPR2 mutations were found in 2.6% of ISS Korean subjects. This prevalence and the dominant‐negative effect of mutant NPR‐B on growth signals imply that it is one of genetic causes of ISS.

A novel NPR2 mutation (p.Arg388Gln) in a patient with acromesomelic dysplasia, type Maroteaux.

.Acromesomelic dysplasia, type Maroteaux (AMDM) is a congenital bone dysplasia characterized by disproportionate, acromesomelic shortening of the limbs and mild spondylar dysplasia. AMDM is caused by biallelic loss-of-function mutations in NPR2 encoding natriuretic peptide receptor-B. We report on a 25-yr-old Japanese woman with AMDM. Her height was 119.0 cm (–7.4 SD) and weight 35 kg (–2.3 SD). She had acromesomelic shortening of limbs and severe brachydactyly. Radiological examination showed that her metacarpals and phalanges were short and wide, and her vertebral bodies were mildly flattened. Molecular analysis revealed a novel homozygous NPR2 mutation (c.1163G>A, p.Arg388Gln). We performed in vitro functional studies using HA-tagged wild-type (WT) and Arg388Gln vectors (HA-WT-NPRB and HA-R388Q-NPRB). Cells expressing HA-R388Q-NPRB showed negligible cGMP responses to C-type natriuretic peptide (CNP) stimulation, indicating that the mutation led to severe loss-of-function. By immunofluorescence experiments under permeabilized conditions, HA-WT-NPRB was expressed on plasma membrane, while HA-R388Q-NPRB co-localized with an Endoplasmic Reticulum marker. Cells co-expressing R388Q and the WT exhibited lower responses under CNP treatment than cells co-expressing the WT and empty vectors. Thus, it was thought that R388Q caused a dominant-negative effect with a defect in cellular trafficking to the plasma membrane.

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.

Novel variants in natriuretic peptide receptor 2 in unrelated patients with acromesomelic dysplasia type Maroteaux

Acromesomelic dysplasia are a heterogeneous group of disorders with variable spectrum and severity of skeletal anomalies in the affected individuals. Acromesomelic dysplasia type Maroteaux (AMDM) is characterized by extreme shortening of the forelimbs and disproportionate short stature. Several homozygous inactivating mutations in NPR2 have been identified in different AMDM patients. We report five novel variants in affected individuals in four different families. These include two nonsense and three missense variants. This study broadens the genotypic spectrum of NPR2 mutations in individuals with AMDM and also describes the intra- and inter-familial phenotypic variability due to NPR2 variants.

Molecular and in silico analyses validates pathogenicity of homozygous mutations in the NPR2 gene underlying variable phenotypes of Acromesomelic dysplasia, type Maroteaux.

Homozygous and/or heterozygous loss of function mutations in the natriuretic peptide receptor B (NPR2) have been reported in causing acromesomelic dysplasia, type Maroteaux with variable clinical features and idiopathic short stature with nonspecific skeletal deformities. On the other hand, gain of function mutations in the same gene result in overgrowth disorder suggesting that NPR2 and its ligand, natriuretic peptide precursor C (CNP), are the key players of endochondral bone growth. However, the precise mechanism behind phenotypic variability of the NPR2 mutations is not fully understood so far. In the present study, three consanguineous families of Pakistani origin (A, B, C) with variable phenotypes of acromesomelic dysplasia, type Maroteaux were evaluated at clinical and molecular levels. Linkage analysis followed by Sanger sequencing of the NPR2 gene revealed three homozygous mutations including p.(Leu314 Arg), p.(Arg371*), and p.(Arg1032*) in family A, B and C, respectively. In silico structural and functional analyses substantiated that a novel missense mutation [p.(Leu314 Arg)] in family A allosterically affects binding of NPR2 homodimer to its ligand (CNP) which ultimately results in defective guanylate cyclase activity. A nonsense mutation [p.(Arg371*)] in family B entirely removed the transmembrane domain, protein kinase domain and guanylate cyclase domains of the NPR2 resulting in abolishing its guanylate cyclase activity. Another novel mutation [p.(Arg1032*)], found in family C, deteriorated the guanylate cyclase domain of the protein and probably plundered its guanylate cyclase activity. These results suggest that guanylate cyclase activity is the most critical function of the NPR2 and phenotypic severity of the NPR2 mutations is proportional to the reduction in its guanylate cyclase activity.

Next generation sequencing-based mutation screening of 86 patients with idiopathic short stature

Although mutations in ACAN, FGFR3, NPR2, and SHOX typically lead to skeletal dysplasia, and mutations in GHRHR, GH1, GHR, STAT5B, IGF1, IGFALS, and IGF1R usually underlie hormonal defects of the growth hormone (GH)-insulin-like growth factor 1 (IGF1) axis, such mutations have also been identified in patients with idiopathic short stature (ISS). Of these, SHOX abnormalities are known to account for a certain percentage of ISS cases, whereas the frequency of mutations in the other 10 genes in ISS cohorts remains unknown. Here, we performed next-generation sequencing-based mutation screening of the 10 genes in 86 unrelated Japanese ISS patients without SHOX abnormalities. We searched for rare protein-altering variants. The functional significance of the identified variants was assessed by in silico analyses. Consequently, we identified 18 heterozygous rare variants in 19 patients, including four probable damaging variants in ACAN, six pathogenicity-unknown variants in FGFR3, GHRHR, GHR, and IGFALS, and eight possible benign variants. Pathogenic variants in NPR2, GH1, and IGF1 were absent from our cohort. Unlike previously reported patients with ACAN mutations, our four patients with ACAN variants manifested non-specific short stature with age-appropriate or mildly delayed bone ages, and had parents of normal stature. These results indicate that ACAN mutations can underlie ISS without characteristic skeletal features, and that such mutations are possibly associated with de novo occurrence or low penetrance. In addition, our data imply that mutations in FGFR3, NPR2, and GH-IGF1 axis genes play only limited roles in the etiology of ISS.

Novel mutations in the transmembrane natriuretic peptide receptor NPR-B gene in four Indian families with acromesomelic dysplasia, type Maroteaux.

Acromesomelic dysplasia, type Maroteaux is a disorder characterized by disproportionate short stature predominantly affecting the middle and distal segments of the upper and lower limbs. It is an autosomal recessive disorder due to mutation in NPR2 gene which impairs skeletal growth. To screen the mutations in the gene NPR2, all of its coding exons and splice junction sites were PCR amplified from genomic DNA of affected individuals of four families and sequenced. Four homozygous mutations in four different families were identified. These include three novel mutations including a deletion frameshift mutation (p.Cys586Ter), one nonsense mutation (p.Arg479Ter), one missense mutation (p.Val187Asp) and one reported missense mutation (p.Tyr338Cys). The study describes phenotypes of Indian patients and expands the mutation spectrum of the disorder.

Heterozygous NPR2 Mutations Cause Disproportionate Short Stature, Similar to Léri-Weill Dyschondrosteosis.

SHOX mutations have been detected in approximately 70% of Léri-Weill dyschondrosteosis (LWD) and approximately 2.5% of idiopathic short stature (ISS) cases, suggesting the implication of other genes or loci. The recent identification of NPR2 mutations in ISS suggested that NPR2 mutations may also be involved in disproportionate short stature. The objective of the study was to investigate whether NPR2 mutations can account for a proportion of the cases referred for LWD and ISS in whom no SHOX mutation was detected. We undertook NPR2 mutation screening in 173 individuals referred for suspected LWD and 95 for ISS, with no known defect in SHOX or its enhancers. Intracellular localization and natriuretic peptide precursor C-dependent guanylate cyclase activity were determined for the identified NPR2 variants. Eight NPR2 variants were identified in nine individuals, seven referred for suspected LWD and two for ISS. Six were demonstrated to affect NPR-B cell trafficking and/or its ability to synthesize cyclic GMP (cGMP) under response to natriuretic peptide precursor C/brain natriuretic peptide stimulation. All pathogenic mutations were detected in the suspected LWD referral group (∼3%). Interestingly, one of these patients is currently being treated with recombinant human GH and in contrast to previous reports is showing a positive response to the treatment. NPR2 mutations account for approximately 3% of patients with disproportionate short stature and/or clinical or radiographic indicators of SHOX deficiency and in whom no SHOX defect has been identified. However, no patient has yet presented with Madelung deformity. Thus, NPR2 should be screened in the SHOX-negative LWD referrals.

Identification and Functional Characterization of Two Novel NPR2 Mutations in Japanese Patients With Short Stature

C-type natriuretic peptide-natriuretic peptide receptor B (NPR-B) signaling is critical for endochondral ossification, which is responsible for longitudinal growth in limbs and vertebrae. Biallelic NPR2 mutations cause acromesomelic dysplasia, type Maroteaux, which is bone dysplasia characterized by severe short stature and short limbs. A monoallelic NPR2 mutation has been suggested to mildly impair long bone growth. The goal of this study was to identify and characterize NPR2 mutations among Japanese patients with short stature. We enrolled 101 unrelated Japanese patients with short stature. NPR2 and NPPC were sequenced, and the identified variants were characterized in vitro. In two subjects, we identified two novel heterozygous NPR2 mutations (R110C and Q417E) causing a loss of C-type natriuretic peptide-dependent cGMP generation capacities and having dominant-negative effects. R110C was defective in trafficking from the endoplasmic reticulum to the Golgi apparatus. In contrast, Q417E showed clear cell surface expression. We identified heterozygous NPR2 mutations in 2% of Japanese patients with short stature. Our in vitro findings indicate that NPR2 mutations have a dominant negative effect, and their dominant-negative mechanisms vary corresponding to the molecular pathogenesis of the mutations.

Heterozygous Mutations in Natriuretic Peptide Receptor-B (NPR2) Gene as a Cause of Short Stature in Patients Initially Classified as Idiopathic Short Stature

Based on the stature observed in relatives of patients with acromesomelic dysplasia, type Maroteaux, homozygous for mutations in natriuretic peptide receptor B gene (NPR2), it has been suggested that heterozygous mutations in this gene could be responsible for the growth impairment observed in some children with idiopathic short stature (ISS). The objective of the study was to investigate the presence of NPR2 mutations in a group of patients with ISS. The NPR2 coding region was directly sequenced in 47 independent patients with ISS. The functional consequences of NPR2 nonsynonymous variations were established using in vitro cell-based assays. Three novel heterozygous NPR2 mutations were identified: c.226T>C (p.Ser76Pro), c.788G>C (p.Arg263Pro), and c.2455C>T (p.Arg819Cys). These allelic variants were not found in our controls or in the 1000 Genomes database. In silico analysis suggested that the three missense mutations are probably damaging. All of them were selected for in vitro functional evaluation. Cells transfected with the three mutants failed to produce cyclic GMP after treatment with C-type natriuretic peptide. Cells cotransfected with mutant and wild-type-NPR-B (1:1) showed a significant decrease in cGMP levels after C-type natriuretic peptide stimulation in comparison with cells cotrasnfected with empty vector and wild type, suggesting a dominant-negative effect. These three mutations segregated with short stature phenotype in an autosomal dominant pattern (height SD score ranged from -4.5 to -1.7). One of these patients and two relatives have disproportionate short stature, whereas in another patient a nonspecific skeletal abnormality was observed. All three of these patients were treated with recombinant human GH (33-50 μg/kg · d) without significant height SD score change during therapy. We identified heterozygous NPR2 mutations in 6% of patients initially classified as ISS. Affected patients have mild and variable degrees of short stature without a distinct phenotype. Heterozygous mutations in NPR2 could be an important cause of nonsyndromic familial short stature.

Novel mutations in natriuretic peptide receptor-2 gene underlie acromesomelic dysplasia, type maroteaux

BackgroundNatriuretic peptides (NPs) are peptide hormones that exert their biological actions by binding to three types of cell surface natriuretic peptide receptors (NPRs). The receptor NPR-B binding C-type natriuretic peptide (CNP) acts locally as a paracrine and/or autocrine regulator in a wide variety of tissues. Mutations in the gene NPR2 have been shown to cause acromesomelic dysplasia-type Maroteaux (AMDM), an autosomal recessive skeletal disproportionate dwarfism disorder in humans.MethodsIn the study, presented here, genotyping of six consanguineous families of Pakistani origin with AMDM was carried out using polymorphic microsatellite markers, which are closely linked to the gene NPR2 on chromosome 9p21-p12. To screen for mutations in the gene NPR2, all of its coding exons and splice junction sites were PCR amplified from genomic DNA of affected and unaffected individuals of the families and sequenced.ResultsSequence analysis of the gene NPR2 identified a novel missence mutation (p.T907M) in five families, and a splice donor site mutation c.2986 + 2 T > G in the other family.ConclusionWe have described two novel mutations in the gene NPR2. The presence of the same mutation (p.T907M) and haplotype in five families (A, B, C, D, E) is suggestive of a founder effect.

Gastrointestinal Tract Disorder in Natriuretic Peptide Receptor B Gene Mutant Mice

Natriuretic peptide receptor B (NPR-B), which has high affinity for C-type natriuretic peptide (CNP) and synthesizes intracellular cGMP, may be involved in gastrointestinal tract (GIT) regulation. A mutant allele of the NPR-B-encoding gene (Npr2) is responsible for the phenotype of the short-limb dwarfism (SLW) mouse. Homozygosity for this autosomal-recessive gene (slw/slw) leads to dwarfism and death before weaning because of milk retention in the stomach and intestinal distention. To elucidate the relationship between CNP/NPR-B signaling and GIT function, we investigated the association between Npr2 mutation and the GIT phenotype in slw/slw mice. The pylorus and large intestine of the mutants did not respond to CNP stimulation; further, they showed pyloric lumen narrowing with randomly aligned circular muscle cells. Comparison of the cGMP and neuronal marker distribution in GIT tissues confirmed cGMP expression in neuronal tissues. An Auerbach's plexus and submucosal tissues of the mutants didn't express cGMP and expressed Ca(2+). In contrast, those of normal mice (controls) expressed both cGMP and Ca(2+). Sequencing revealed that the causative Npr2 mutation was a 7-base deletion in exon 8, resulting in a frameshift and premature termination codon appearance. Therefore, the GIT phenotype of slw/slw mice is because of a CNP/NPR-B-signaling defect caused by an Npr2 mutation. These results facilitate better understanding of the role of CNP/NPR-B signaling in GIT motility.

Heterozygous Mutations in Natriuretic Peptide Receptor-B (NPR2) Are Associated with Short Stature

C-type natriuretic peptide (CNP) is an important regulator of skeletal growth. Loss-of-function mutations affecting the CNP receptor natriuretic peptide receptor-B (gene NPR2) cause the autosomal recessive skeletal dysplasia, acromesomelic dysplasia, Maroteaux type (AMDM). The phenotype of heterozygous carriers of NPR2 mutations is less clear. The objective of the study was to determine the phenotypic features of heterozygous carriers of NPR2 mutations. This was a case-control study from the general community. Thirty-nine members of a family in which one member has AMDM were studied. This was an observational study. The primary measure was stature, with the hypothesis that carriers have reduced stature compared with noncarriers. Sixteen family members were NPR2 mutation carriers. Height z-scores of these carriers were -1.8 +/- 1.1 (mean +/- sd), which was significantly less than the 23 noncarrier family members (-0.4 +/- 0.8, P < 0.0005) and the general population (P < 0.0005). However, there was no difference in body proportion between carriers and noncarriers. The proband with AMDM had low IGF-I levels and evidence of GH resistance, as well as very high plasma levels of CNP and its amino-terminal propeptide. Levels of these peptides were normal in the heterozygous carriers. We have shown that heterozygous mutations in NPR2 are associated with short stature. Assuming one in 700 people unknowingly carry an NPR2 mutation, our data suggest that approximately one in 30 individuals with idiopathic short stature are carriers of NPR2 mutations.

Mutations in the Transmembrane Natriuretic Peptide Receptor NPR-B Impair Skeletal Growth and Cause Acromesomelic Dysplasia, Type Maroteaux

The homodimeric transmembrane receptor natriuretic peptide receptor B (NPR-B [also known as guanylate cyclase B, GC-B, and GUC2B]; gene name NPR2) produces cytoplasmic cyclic GMP from GTP on binding its extracellular ligand, C-type natriuretic peptide (CNP). CNP has previously been implicated in the regulation of skeletal growth in transgenic and knockout mice. The autosomal recessive skeletal dysplasia known as "acromesomelic dysplasia, type Maroteaux" (AMDM) maps to an interval that contains NPR2. We sequenced DNA from 21 families affected by AMDM and found 4 nonsense mutations, 4 frameshift mutations, 2 splice-site mutations, and 11 missense mutations. Molecular modeling was used to examine the putative protein change brought about by each missense mutation. Three missense mutations were tested in a functional assay and were found to have markedly deficient guanylyl cyclase activity. We also found that obligate carriers of NPR2 mutations have heights that are below the mean for matched controls. We conclude that, although NPR-B is expressed in a number of tissues, its major role is in the regulation of skeletal growth.