Short Stature is Progressive in Patients with Heterozygous NPR2 Mutations

Abstract

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.

The molecular basis for growth failure in many children with idiopathic short stature (ISS) has been clarified through application of next-generation sequencing and high-throughput genomic analysis. These studies have revealed mutations in a growing number of genes that encode proteins that are directly involved in growth plate function (1-5), including heterozygous mutations in the NPR2 gene encoding the transmembrane receptor atrial natriuretic peptide receptor 2 (ANPRB, previously NPR-B or NPR2) (6-8). ANPRB is a homodimeric plasma membrane guanylate cyclase that is highly expressed in chondrocytes (9), and when bound to its ligand, C-type natriuretic peptide (CNP), produces the second messenger cyclic GMP (cGMP) (10-12). The ANPRB signaling pathway represents a critical regulator of longitudinal growth and skeletal development (13), and genome-wide association studies and microarray expression analyses have strongly implicated genes in the CNP-ANPRB signaling pathway with regulation of the growth plate and height variation (14). Moreover, mutations in the CNP or NPR2 genes have been shown to cause monogenic growth disorders (7, 13). For example, mutations in NPR2 that lead to a gain-of-function are a cause of tall stature (15-18). By contrast, loss-of-function mutations in NPPC encoding CNP are a cause of autosomal dominant short stature (19), and mice deficient in CNP (20) or ANPRB (21) show impaired skeletal development and decreased length (22-28).

Biallelic loss-of-function mutations in NPR2 cause acromesomelic dysplasia Maroteaux type (AMDM; OMIM 602875), a distinctive skeletal dysplasia that is characterized by short limbs and dwarfism (29). Length is normal at birth, but skeletal growth quickly decelerates and results in severe short stature (30). Recent literature describes short stature in heterozygous carriers of NPR2 mutations, many of whom also manifest nonspecific skeletal anomalies (6, 8, 29, 31-35). Although most of these missense mutations show dominant negative effects when co-expressed with wild-type ANPRB protein in heterologous mammalian cells, studies in knockout mice that are heterozygous for deletion of Npr2 indicate that haploinsufficiency can also produce a growth-restricted phenotype (36).

Here we report a comprehensive analysis of the growth patterns and auxological characteristics of an extended, multigenerational family in which 2 sisters with AMDM are compound heterozygotes for NPR2 missense mutations, 6 subjects have short stature due to heterozygous NPR2 mutations, and 6 relatives with wild-type NRP2 sequences have taller stature than their heterozygous NPR2 siblings.

Patients and Methods

Subjects

This study was approved by the Institutional Review Board of the Children’s Hospital of Philadelphia (CHOP). Written informed consent or assent was obtained from all participants or their parents when applicable. Clinical information was extracted from medical records. One NPR2 heterozygote (III-6) and 1 wild-type subject (III-18) were excluded from most analyses because they were less than 1 month of age at the time of evaluation.

Molecular genetics and protein studies

Genomic DNA was isolated from peripheral blood mononuclear cells or saliva from members of an extended Ashkenazi Jewish family in which there was no history of consanguinity (Fig. 1B). DNA samples from the parents of subfamily A (II-1 and II-2) and their daughters with severe short stature and brachydactyly (III-1 and III-4) were subjected to commercial whole exome sequencing by conventional techniques (GeneDx, Gaithersburg, MD). NPR2 mutations were confirmed by Sanger sequencing, which was used to genotype all other subjects. The effect of sequence variants was analyzed using in silico prediction tools (Supplemental Table 1 (37),) that included CADD (Complete Annotation Dependent Depletion, http://cadd.gs.washington.edu/), SIFT (http://sift.jcvi.org), PolyPhen-2 (Polymorphism Phenotyping v2, http://genetics.bwh.harvard.edu/pph2/) and MutationTaster (http://www.mutationtaster.org). Protein function was summarized using information found in the UniProt database (www.uniprot.org).

Figure 1.

Family pedigree and electropherograms of NPR2 mutations. Panel A shows the sequence electropherograms for the 2 novel mutations found in NPR2, p.P93S and p.R989L. Electropherograms of portions of exon 1 and exon 20 with the specific mutations notated by arrows, c.277 C>T (p.P93S) and c.2966 G>T (p.R989L). Panel B shows the pedigree of the studied family, subfamily A and B denoted. Squares represent male family members and circles female family members. Asterisk symbols = unknown genetics. Open white symbols = WT/WT. Black symbols = compound heterozygotes p.P93S/p.R989L. Symbols that are half white and black on the left side of the symbol = p.P93S/WT. Symbols that are half white and black on the right side of the symbol = p.R989L/WT. Numbers next to subjects in row III are used to identify subjects in family A or B when described in manuscript.

To determine the functional and structural consequences of NPR2 mutations, we used site-directed mutagenesis to insert each nucleotide substitution into a human wild-type ANPRB cDNA (BC166642) that was cloned into a mammalian expression vector that generates an ANPRB fusion protein containing an epitope tag at the C-terminus consisting of 3 copies of either FLAG or HA. For immunoblot analysis, we transfected HEK293T cells using Lipofectamine 3000 per the manufacturer’s protocol (Thermo Fisher, USA) in 24-well dishes. Cells were cultured in Dulbecco’s Modified Eagle Medium with high glucose supplemented with 10% FBS and 1% penicillin-streptomycin 10 000 U/mL (Thermo Fisher Scientific) at 37 °C with 5% CO₂. After 48 hours of culture we extracted total protein using either Laemmli buffer or “killer buffer” (8% sucrose, 2M urea, 4% SDS). Protein samples (50 μg) were subjected to electrophoresis through 4% to 12% SDS-polyacrylamide gels (ExpressPlus™ PAGE Gel, Catalog no- M41212, GenScript; Piscataway, NJ) and proteins were transferred to PVDF for immunoblot analysis. Membranes were incubated sequentially with antisera against β Actin (1:5000, ab8227, Abcam), HA Tag (1:1000, 11583816001, Roche), and FLAG Tag (1:1000, F3165, Sigma Aldrich).

To perform subcellular localization studies, we transfected HEK293T cells that had been plated onto 4-well Nunc Labtek II Chamber Slides (Millipore Sigma) with ANPRB plasmids expressing HA-tagged mutant proteins and FLAG-tagged wild-type fusion proteins. After 24 hours, the cells were washed twice with ice cold phosphate-buffered saline (PBS). The cells were then fixed with ice cold 10% formalin on ice for 10 min. After washing 4 times with PBS, the cells were then simultaneously blocked and permeabilized by incubation for 30 minutes on ice in PBS containing 0.1% Tween 20 and 5% bovine serum albumin. After 30 minutes the solution was aspirated, and the cells were washed 4 times with PBS. The fixed cells were then incubated at room temperature for 1 hour with labeled primary antibodies (DyLight 650-coupled mouse anti-HA IgG1 (1:25; 26183-D650, Thermofisher) and/or an Alexa 488-coupled rat anti-FLAG IgG2a (1:25; MA1-142-A488, Thermofisher). We treated cells with ProLong Gold Antifade mounting medium with DAPI (P36935 Thermo Fisher) and imaged them with a Leica STED 3× Super-resolution confocal microscope.

To determine the guanylate cyclase activity of the ANPRB mutants, we transfected HEK293T cells with empty vector or combinations of wild-type and mutant ANPRB cDNAs in 24-well dishes, and after 24 hours we replaced the regular media with serum-starved medium. Cells were cultured for an additional 24 hours and then medium was replaced with PBS supplemented with 20 mM HEPES (pH 7.5) and 0.5 mM IBMX plus the indicated concentration of CNP. Cells were incubated for 10 minutes at 37 °C and then we extracted total cGMP using 0.1 N HCl, and processed extracts as described previously (38). We assayed cGMP by ELISA (Enzo Life Sciences, Catalog no- 89141–242).

We performed co-immunoprecipitation experiments to analyze interactions between wild-type and mutant epitope-tagged recombinant ANPRB fusion proteins. HEK293T cells were transfected in 6-well dishes and after culture for 48 hours cell lysates were prepared by addition of lysis buffer (50 mM Tris HCl, pH 7.4, with 150 mM NaCl, 1 mM EDTA, and 1% TRITON X-100). Fusions proteins were captured using anti-FLAG M2 Magnetic Beads (Catalog Number M8823, Sigma-Aldrich, St. Louis, MO) according to the manufacturer’s protocol. FLAG-tagged proteins and interacting partners were eluted from the M2 beads using 1X SDS sample preparation buffer without reducing agents. Aliquots of whole cell lysates and eluates were subjected to SDS-polyacrylamide gel electrophoresis and immunoblot analysis with antibodies against FLAG, HA, and β-actin (above) and protein binding was detected by chemiluminescence using Amersham ECL Prime Western Blotting Detection Reagent (Cytiva Life Sciences, Marlborough, MA). Blots were imaged using the Alpha Innotech Fluor Chem FC2 Imaging System, and quantification of the optical density of protein bands was performed using Image Studio Lite Ver 5.2 (LI-COR Biotechnology, Lincoln NE). To analyze interactions of wild-type FLAG-tagged ANPRB with each specific HA-tagged ANPRB protein, we calculated the ratio of HA-tagged ANPRB fusion protein in the eluate to that present in the whole cell lysate.

Auxology

We plotted the height and weight for all subjects on the Centers for Disease Control and Prevention growth charts and z-scores for height and weight were calculated using the National Health and Nutrition Examination Survey (NHANES) III growth data set (39). We classified pubertal stage according to the methods described by Tanner et al (40, 41), and assessed bone age (BA) according to the method of Greulich and Pyle (42) using radiographs of the left hand and wrist. All bone age radiographs were examined and interpreted by the investigators (M.A.L. and P.C.H.) and in many cases by a radiologist as well. The bone ages were assigned after discussion to achieve consensus. In figures, an asterisk indicates that the BA reading was performed by a non-CHOP radiologist and confirmed by the referring pediatric endocrinologist. We used data from the Brush Foundation study of child growth and development (43) to generate BA z-scores for our patients. The arm span to height (AS:H), and seated height to height (SH:H) ratios used to define disproportionate shortening were according to phenotyping scoring data determined by Rappold and colleagues based on 1608 unrelated normal individuals (44) and data from Hawkes et al were used to generate z-scores for SH:H ratios (45). The longitudinal height measurements were obtained from either measurements taken in the endocrine clinic at CHOP, or from pediatrician growth charts.

Statistics

We analyzed height z-scores and immunoprecipitation efficiency using Kruskal-Wallis test by ranks with posttest analysis of group differences determined by Dunn’s multiple comparison test. We analyzed specific sample pairs for stochastic dominance in post hoc testing using Dunn’s test. To assess differences in activity between ANPRB mutant and wild-type recombinant proteins we first conducted ANOVA and then performed post hoc testing of paired means using the Student-Newman-Keuls Multiple Comparisons test. Statistical analyses were performed using GraphPad InStat version 3.1 and GraphPad Prism (GraphPad Software, San Diego, California).

Results

Clinical studies in patients with biallelic NPR2 mutations

The proband (III-1) and her sister (III-4), aged 11 years 9 months and 5 years, respectively at initial evaluation, were referred for evaluation of severe short stature and brachydactyly type E of the hands and feet (x-ray images in Supplemental Figure 1 (37). Whole exome sequence analysis showed they were compound heterozygous for NPR2 mutations, confirming the diagnosis of AMDM. The sisters had normal weight and length at birth and no obvious skeletal deformity, but subsequently showed declining statural growth (Supplemental Figure 2, panel D and E (37) and developed disproportionate short stature (Table 1) with reduced AS:H ratios, but neither had an abnormal SH:H ratio (44).

Table 1.

Demographics and Anthropomorphic Measurements in Compound Heterozygotes, Heterozygotes, and Wild-type NPR2 Subjects

	Sex	Age	NPR2 genotype	Height (cm)	Height centile	Height z-score	Height z-score soon after birth	SH:H ratio	SH:H ratio z-score	AS:H ratio	AS+leg length (cm)	BMI	BMI centile	Rappold Score	Brachydactyly D
Biallelic Subjects
III-1	F	13 yr, 9 mo	p.P93S/p. R989L	137.1	0.03	-3.42	NA	0.54	1.23	0.93	190.5	17.61	26.81	NA	Yes
III-4	F	7 yr, 1 mo	p.P93S/p. R989L	110.1	1.15	-2.27	NA	0.55	1.53	0.95	153.6	17.16	80.21	NA	Yes
Mean ± SD					0.59 ± 0.79	-2.85 ± 0.81		0.55 ± 0.01	1.38 ± 0.21	0.94 ± 0.01		17.39 ± 0.32	53.51 ± 37.76
Heterozygotes
II-1	F	39 yr	WT/p.P93S	154.94	15.7	-1.00	NA	NA	NA	NA	NA	NA	NA	NA	No
II-2	M	40 yr	WT/p.R989L	167.64	13.2	-1.10	NA	NA	NA	NA	NA	NA	NA	NA	No
II-4	F	43 yr	WT/p.R989L	151.1	6.1	-1.50	NA	0.50	NA	1.02	229.1	NA	NA	NA	No
III-2	M	12 yr, 4 mo	WT/p.R989L	141.16	9.19	-1.33	NA	0.53	1.00	1.02	210.26	19.95	75.07	4	No
III-5	F	4 yr, 1 mo	WT/p.P93S	99.3	32.14	-0.46	NA	0.54	-0.81	0.95	139.9	14.6	26.68	2	No
III-6a	M	0 mo	WT/p.P93S	49.53	43	-0.17	-0.173	NA	NA	NA	NA	NA	NA	NA	NA
III-8	F	17 yr, 10 mo	WT/p.R989L	144.9	0.25	-2.80	NA	0.56	2.13	0.95	201.2	25.05	82.63	8	Yes
III-9	F	16 yr, 4 mo	WT/p.R989L	143.8	0.17	-2.93	-0.426	0.57	3.44	0.97	201	27.66	92.92	4	Yes
III-16	F	5 yr, 0 mo	WT/p.R989L	96.5	0.6	-2.50	-0.923	0.55	0.67	0.93	130.7	NA	NA	NA	Yes
III-17	F	2 yr, 11 mo	WT/p.R989L	85.6	1.02	-2.32	0.098	NA	NA	NA	NA	17.06	83.9	NA	No
Mean ± SD					7.23 ± 12.69	-2.06 ± 0.97	-0.36 ± 0.43	0.55 ± 0.02	1.29 ± 1.6	0.96 ± 0.03		20.86 ± 5.44	72.24 ± 26.25
Wild-type
II-3	M	46 yr	WT/WT	161.6	2.7	-1.9	NA	0.51	NA	0.98	237.8	NA	NA	NA	No
II-3	M	8 yr, 7 mo	WT/WT	130.4	42.99	-0.18	0.207	NA	NA	1.03	NA	16.29	56.57	NA	No
III-7	M	19 yr, 4 mo	WT/WT	165.7	6.3	-1.50	NA	0.51	NA	0.96	240	NA	NA	NA	Yes
III-10	F	15 yr, 4 mo	WT/WT	151.8	5.5	-1.60	NA	0.54	0.66	0.98	219.7	NA	NA	NA	No
III-11	M	13 yr, 8 mo	WT/WT	138.4	0.27	-2.78	-0.173	0.54	1.87	0.97	198	20.2	67.75	4	Yes
III-12	M	12 yr, 5 mo	WT/WT	140.6	6.3	-1.53	-3.109	0.51	-0.26	0.99	207.9	NA	NA	NA	No
III-13	F	10 yr, 1 mo	WT/WT	126.8	3.42	-1.82	-0.923	0.55	2.04	0.99	183	18.6	73.15	4	No
III-14	F	8 yr, 7 mo	WT/WT	121.9	5.94	-1.56	-0.371	0.54	1.42	0.91	166.4	NA	NA	NA	Yes
III-18a	F	1 mo	WT/WT	NA	NA	NA	NA	NA	NA	NA	NA	NA	NA	NA	No
Mean ± SD					8.92 ± 13.99	-1.69 ± 0.78	-0.87 ± 1.13	0.53 ± 0.01	1.15 ± 0.95	0.97 ± 0.04		18.36 ± 1.97	65.82 ± 8.46

Data are presented as number of subjects or percentiles. When applicable for each group, calculated data are represented as mean percentages ± standard deviation. All length measurements represented in centimeters. ^aSubjects III-11 was not included in table because of GHD, and subject III-18 not included in analysis because no measurements available.

Abbreviations: AS, arm span; BMI, body mass index calculated as weight in kilograms divided by the square of height in meters; CM, centimeters; E, extremity; F, female; GHD, growth hormone deficiency; H, height; HC, head circumference; M, male; NA, not available; SH, seated height.

The BA and chronological age (CA) for subjects III-1 and III-4 are shown in Supplemental Table 2 and supplemental Figure 5 (37). The earliest BAs for when both subjects were young were markedly delayed, but BAs progressively advanced for both subjects as they matured, consistent with a progressive loss of adult height.

Statural growth was poor for both III-1 and III-4 (Supplemental Figure 2, panels D and E (37), and serum concentrations of insulin-like growth factor 1 (IGF-1) were normal in both subjects (Supplemental Table 3 (37). The proband (III-1) showed a minimal and nonsustained increase in growth velocity when treated with progressively increasing doses of growth hormone (GH) (up to 0.52 mg/kg/week) (Supplemental Figure 2D (37). She had normal pubertal development with breast development by age 10 years, but failed to undergo menarche first due to treatment with leuprolide acetate for depot suspension for 1 year between the ages of 11 and 12 years, and then due to low body weight caused by an eating disorder. She completed her linear growth between age 13 and 14 years.

Her younger sister (III-4) showed a modest improvement in height z-score while taking GH (0.32 to 0.36 mg/kg/week) for 12 months beginning at age 6 years, which then declined after discontinuation of GH therapy (Supplemental Figure 2E (37). Her serum IGF-1 level increased appropriately in response to GH treatment (Supplemental Table 3 (37). Although her linear velocity increased in response to GH therapy, it should be noted her BA advanced during this time, possibly offsetting any benefits on final adult height. Subject III-4 underwent an arginine and clonidine GH stimulation test approximately 2 years after discontinuation of GH therapy and had a normal peak GH response (13.8 ng/mL).

Clinical studies in subjects with heterozygous NPR2 mutations

We studied 7 children and 3 adults who were heterozygous for either the NPR2 p.P93S or p.R989L mutation (Fig. 1). During the study, a child with a single NPR2 mutation (III-6) and a wild-type sibling (III-18) were born, and both were excluded for most comparison analysis due to young age and lack of comprehensive measurements. Another wild-type sibling (subject III-11), was excluded because he was diagnosed with GH deficiency due to poor linear growth velocity and a deficient peak GH response (3.7 ng/mL). All NPR2 heterozygotes were born full term, and at birth (or shortly thereafter) their length z-scores and percentiles were within the normal range (Table 1). As shown in Figs. 2A and 2B, during childhood and into adolescence and adulthood, the height z-scores of the patients with heterozygous NPR2 mutations gradually worsened, leading to more pronounced short stature. Three of the 7 children (subjects III-2, III-8, and III-9) with heterozygous NPR2 mutations had either completed or started puberty at the time of evaluation, while the other 4 heterozygous children were 5 years of age or younger at the time of evaluation. Two heterozygote females (subjects III-8 and III-9) had menarche at ages 14 and 14.5 years, respectively, and 1 heterozygote male (subject III-2) had Tanner 2 testicular enlargement at age 12 years. Subjects III-8 and III-9 who had completed menarche, had normal IGF-1 levels for age and Tanner stage when evaluated at 17 years 10 months (subject III-8, IGF-1 256 ng/mL), and at 16 years 4 months (subject III-9, IGF-1 295 ng/mL). The effect of NPR2 mutations on height z-scores is illustrated most clearly when analyzing subfamily B (Fig. 2B and 2C). The height z-scores of the wild-type siblings remained relatively stable and continued to track consistently in their respective growth channels (Fig. 2C), but the NPR2 heterozygote siblings had declining height z-scores leading to progressively more severe short stature and increasing loss of adult height potential (Fig. 2B). Kruskal-Wallis rank analysis with post hoc testing using Dunn’s test showed no differences in height z-scores between heterozygotes and wild-type children at ages 0 to 24 months and 25 to 96 months. By contrast, height z-scores for NPR2 heterozygotes were significantly less than wild-type children at both ages 97 to 168 months (P < 0.05) and 169 to 240 months (P < 0.0003). Of the 6 NPR2 heterozygotes and 6 wild-type siblings included in the data analysis, 2 in each group reached their final adult height. All 4 subjects who reached their final adult heights were from subfamily B (subjects III7-III10 in Fig. 2). The height z-scores for final adult height were −2.8 and −2.93 respectively in the NPR2 heterozygotes, and −1.5 and −1.6 in the wild-type siblings.

Figure 2.

Height z-score versus age over time. Panel A shows the height z-score versus age in all heterozygous NPR2 subjects. In Panel on the left shows the 7 heterozygotes: at 0 to 24 months (subjects III-5, 6, 9, 16, 17); at 25 to 96 months (subjects III-5, 9, 16, 17); at 97 to 168 months subjects III-2, 8, 9); at 169 to 240 months (subjects III-8, 9). The Panel on the right shows the height z-score versus age in all wild-type subjects (n = 7). Linear regression equations for the 2 lines are: Wild-type: Y = 0.001788*X − 1.741 and NPR2 Het Y = −0.009009*X − 0.7978.

There was a trend towards increasing SH:H ratio z-scores based on the number of defective NPR2 alleles that reflected increasing loss of height due to progressive impairment of growth in the long bones (normal, 0.96 ± 0.99, mean ± SD; heterozygotes, 1.29 ± 1.60; biallelic, 1.38 ± 0.21) (Table 1). Although these differences did not meet statistical significance, it is conceivable that greater variances will emerge as the subjects reach final adult height and/or more subjects are assessed.

There was a similar progressive advancement in BA for CA in subjects with 1 or 2 NPR2 mutant alleles compared with wild-type siblings (Fig. 3). Three of the 6 NPR2 heterozygotes had a BA that was above CA age, but all BA were within 2 SDs for age. The youngest 3 patients who were NPR2 heterozygotes and available BAs all initially had a BA less than their CA. Interestingly, 1 of the 3 had an initial BA that was 6 months behind her CA age (CA 42 months, BA 36 months) and then later had a repeat BA that was advanced 4 months beyond her CA (CA 78 months, BA 82 months). The other 2 subjects were less than 6 years old and had BA of 2 months and 4 months less than CA respectively. Overall, BAs were delayed in the younger children and progressively advanced in the older children.

Figure 3.

Bone age z-score over time in AMDM and heterozygous NPR2 subjects. Dot plots of calculated BA z-scores over time in AMDM and NPR2 heterozygote subjects. Circle symbols = AMDM subjects. Square symbols = NPR2 heterozygotes. Data points generated as described in methods section of manuscript using data from the Brush Foundation study of child growth and development to generate BA z-scores for our patients, using the formula z-score = (X - µ)/σ, where X is the BA-CA for the subject, µ is the BA-CA of the corresponding normal group for sex, and σ is the corresponding SD for the µ group. Abbreviations: AMDM, acromesomelic dysplasia Maroteaux type; BA, bone age; CA, chronological age. Linear regression line, Y = 0.02450*X − 2.156 (AMDM) and Y = 0.009519*X − 1.149 (heterozygotes).

In subfamily A, the heterozygous siblings had reduced stature regardless of the NPR2 mutations, with heights in the ninth percentile for the p.R989L variant (subject III-2), and the 32nd percentile for the p.P93S variant (subject III-5), respectively. By contrast, the height for the normal wild-type sibling was at the 43rd percentile(subject III-3), which is well above his predicted mid-parental height (10th percentile). Growth charts for the siblings in subfamily A are shown in Supplemental Figure 2 (37), clearly showing the allelic dose effect of NPR2 mutations on height.

Other skeletal features

Two siblings in subfamily A with AMDM had brachydactyly type E. By contrast, 7 of the 12 children in subfamily B, but neither parent, had brachydactyly type D (BDD; OMIM 113200), which did not segregate with the NPR2 genotype or height (Table 1). Gene sequencing of HOXD13 was negative in subjects, III-7, III-15, and III-16, and whole exome sequencing of subjects III-7 and III-15 did not disclose a genetic basis for brachydactyly type D in this family.

Molecular genetic analyses

We identified 2 NPR2 mutations, c.277 C>T; p.P93S in exon 1 and c.2966 G>T; p.R989L in exon 20 (Fig. 1A); in both cases, the reference amino acid is highly conserved across multiple species, and the replacement is predicted by in silico analyses (Supplemental Table 1 (37) to be pathogenic. Both variants are predicted to be damaging (see Supplemental data (37). The p.P93S variant (NM_003995.4:c.277C>T (p.Pro93Ser) ID rs773934765) originated from the mother (II-2) and the p.R989L variant originated from the father (II-1) of the proband (III-1) as seen in pedigree of Subfamily A (Fig. 1B).

The p.R989L NPR2 mutation (NM_003995.3 (NPR2):c.2966G>T (p.Arg989Leu) ID rs771373457) is present in the gnomAD database (http://gnomad.broadinstitute.org/) with a frequency of 0.00001193, but is not present in the EXAC database (http://exac.broadinstitute.org/) or the 1000 Genomes Project. The R989 residue is highly conserved in all species through Caenorhabditis elegans (nematode). This amino acid substation occurs in the guanylate cyclase (GC) domain of ANPRB.

The p.P93S NPR2 mutation was previously reported in the EXAC database with a frequency of 0.00000824, but it is absent in the gnomAD database and 1000 Genomes Project. The P93 residue is also highly conserved in all species through Danio rerio (zebrafish). This mutation was previously identified in the mother (height 160 cm, z-score −0.5) of 2 daughters with severe AMDM who were compound heterozygotes (p.Arg989Leu and p.Arg989ter) (35). This amino acid replacement occurs in the ligand binding domain of ANPRB.

ANPRB expression studies

Immunoblot analysis (Fig. 4) showed that both mutant ANPRB proteins are of normal size and were expressed in amounts that are similar to the wild-type ANPRB protein. Fig. 7 shows more clearly that both P93S and R989L, as well as the previously described NPR2 p.R110C mutant used here as a control (32), proteins migrate through reduced SDS polyacrylamide gels as a doublet band consisting of a slower migrating species (upper band) of ANPRB protein that has been completely processed by glycosylation and phosphorylation and a faster migrating species (lower band) that corresponds to ANPRB protein that has not been completely processed (46). We analyzed the subcellular localization of the mutant ANPRB proteins that contained epitope tags in the c-terminal intracellular domain after permeabilization of transfected cells. Both the P93S and R989L proteins co-localized with wild-type ANPRB, with predominant expression on the cell surface (merged images, Fig. 5; other data not shown).

Figure 4.

Immunochemical characterization of ANPRB proteins. HEK293T cells were transiently transfected with empty vector (EV) or cDNA encoding HA-tagged wild-type (WT) or the indicated mutant ANPRB fusion proteins. Other cells were untransfected (Un) or mock-transfected (M). Blots were sequentially incubated with anti-HA antibody (upper panel) and anti-β actin antibody (lower panel).

Figure 7.

Interaction analyses of mutant ANPRB proteins. Coimmunoprecipitation analysis of WT and the mutants is shown. Panel on left shows a representative immunoblot of (upper) proteins eluted from anti-FLAG-beads after incubation with anti-HA antibody or (lower) proteins present in whole cell lysates (WCL) from HEK293 cells that had been co-transfected with cDNAs encoding wild-type ANPRB-FLAG plus either HA-tagged wild-type, P93S, or R989L ANPRB fusion proteins. The panel on the right shows the relative ratio of scanned bands corresponding to eluted/WCL proteins. Data are N = 3 and are expressed as mean ± SEM. The symbol * denotes P < 0.05 versus wild-type.

Figure 5.

Confocal microscopy of ANPRB proteins. Expression of ANPRB fusion proteins as disclosed by confocal immunofluorescence microscopy. HEK293T cells were transfected with a total of 200 ng of DNA consisting of either 200 ng of 1 cDNA or 100 ng each of 2 cDNAs, with the following conditions: S1, empty vector (EV); S2, EV plus wild-type (WT); S3, WT; S4, P93S; S5, R989L; S6, EV plus MP93S; S7, EV plus R989L; S8, WT plus P93S; S9, WT plus R989L; S10, mock transfection (no DNA). Cells were permeabilized, and incubated with anti-FLAG to detect ANPRB wild-type fusion proteins or anti-HA to detect ANPRB mutant fusion proteins, and antibody binding was detected with fluorescent secondary antibodies (see Methods) such that WT ANPRB was green and mutant ANPRB proteins were red. Nuclei were stained with DAPI. Only merged images are shown, at 40×. There was co-localization of WT and mutant ANPRB protein on the cell surface.

Functional studies

Both variant ANPRB proteins showed low basal activity, similar to the wild-type protein. By contrast, cells expressing the 2 mutant proteins showed little or no generation of cGMP after incubation with CNP at 2 different concentrations, 1 µM and 0.1 µM, whereas cells expressing the wild-type ANPRB showed robust, concentration-dependent cGMP responses to CNP (Fig. 6). In addition, co-expression of each mutant ANPRB with an equivalent amount of wild-type ANPRB showed significant reductions in cGMP after stimulation with CNP compared with incubation of cells that had been transfected with the wild-type ANPRB and an empty vector, indicating that both mutant ANPRB proteins exerted dominant negative effects (Fig. 6). To determine the basis for the dominant negative effect of the mutant ANPRB proteins, we evaluated the interaction between wild-type ANPRB-FLAG and HA-tagged ANPRB proteins corresponding to either wild-type, mutants P93S and R989L and a control mutant ANPRB protein, R110C (32), by co-immunoprecipitation (Fig. 7). After precipitation of total cell lysates with an anti-FLAG antibody, immunoblotting of the precipitates with an anti-HA antibody showed that the interactions between FLAG-ANPRB with both HA-P93S-ANPRB and HA-R89L-ANPRB were similar to HA-ANPRB, consistent with the ability of these 2 proteins to compete with wild-type protein to generate nonfunctional ANPRB heterodimers (Fig. 6). By contrast, HA-R110C-NPRB showed significantly greater interaction with FLAG-ANPRB than HA-ANPRB, suggesting that increased binding of this mutant to wild-type ANPRB protein might explain its extreme dominant negative effects (32).

Figure 6.

Functional characterization of ANPRB proteins. CNP-induced production of intracellular cGMP by ANPRB proteins in transfected HEK293T cells. Data are presented as means ± SEM for 3 separate experiments. cGMP production increased in a dose-dependent manner for wt-ANPRB compared to negligible production by the 2 mutant ANPRB proteins. Accumulation of cGMP by HEK293T cells that had been transfected with 1:1 ratios (total 400 ng DNA per well) of wild-type (WT) ANPRB cDNA plus either empty vector (EV), P93S mutant, or R989L mutant showed that each mutant exerted a dominant negative effect. The symbol * indicates P < 0.05 compared to cells transfected with the wild-type protein and stimulated with 10^–7 M CNP, while ** indicates P < 0.05 versus cells transfected with wild-type protein and stimulated with 10 × ⁻⁶ M CNP.

Discussion

Heterozygous mutations in NPR2 may be present in up to 6% of patients who are classified as having ISS (8, 31, 32), and are associated with an average reduction in adult height of 9 cm for women and 10 cm for men (ie, height z-scores from −0.4 to −1.8) (33). Our work confirms the association of biallelic NPR2 mutations with AMDM, characterized by severe skeletal dysplasia (29, 30) and short stature (6, 31, 32), and refines previous studies that indicated that heterozygous NPR2 mutations cause a form of short stature that resembles ISS (6, 8, 31, 32, 35) or Leri-Weil dyschondrosteosis (34). Here we demonstrate for the first time that patients who carry heterozygous NPR2 mutations manifest a progressive form of disproportionate short stature that is distinct from typical ISS.

Similar to the functional studies presented here, most NPR2 missense mutations encode ANPRB proteins that have dominant negative properties. Because ANPRB exists as a dimer, only 25% of the dimers in plasma membranes of target cells of heterozygous subjects are predicted to be wild-type; 50% will be hybrid molecules consisting of 1 wild-type and 1 mutant protein, and 25% will consist of only abnormal proteins. By contrast, subjects with mutations that lead to little or no mutant ANPRB protein (ie, haploinsufficiency) will be predicted to have 50% of the normal numbers of dimers. Therefore, AMDM patients with biallelic NPR2 mutations are expected to have little or no functional ANPRB activity, while 25% to 50% of normal levels cause only short stature.

Because we studied subjects from a single, extended family, the auxological data we collected allows for a more comprehensive analysis of the patterns of growth in children with heterozygous NPR2 mutations than has been previously reported. Height is strongly influenced by both environmental and genetic factors (43, 47), and the relative genetic contribution to height increases with age and is greatest in adolescence (up to 0.83 in boys and 0.76 in girls) (48). In addition, height is influenced by conditions of assortative mating, such that shorter men tend to marry shorter women. Studies of genetic loci that influence height are further challenged by genetic variance, which is greatest in North America and Australia and lowest in East Asia. Therefore, it is advantageous to analyze height within family groups rather than across families or in the general population. This was clear in our study, in which the subjects carrying wild-type NPR2 alleles had mean height z-scores of between −1.5 and −1.6.

In the extreme, the 2 sisters with AMDM and severe short stature were disproportionate in their AS:H ratios, and had elevated SH:H ratio z-scores; this pattern reflects the important role that ANPRB signaling plays in endochondral bone development, particularly in long bone growth. These features can be compared to the phenotypes of patients with defects in SHOX and FGFR3, and emphasize the interrelationships between the proteins encoded by these genes. SHOX induces expression of the NPPB gene encoding brain natriuretic peptide (BNP), which can directly influence ANPRB signaling or indirectly increase local CNP levels (49). Moreover, cGMP produced by activated ANPRB can inhibit the MAPK pathway and thereby antagonize fibroblast growth factor receptor (FGFR) signaling (50). These effects occur primarily in the mesomelic skeletal region where SHOX is strongly expressed. This gives a plausible explanation for the phenotypes and limb findings seen in patients with mutations in FGFR3 (such as achondroplasia) or SHOX defects (such as Léri-Weill dyschondrosteosis). Although previous studies have reported several skeletal features in subjects with heterozygous NPR2 mutations that are more typical of SHOX defects, these subjects were recruited from a cohort of patients suspected to have Léri-Weill dyschondrosteosis (34).

Our study also provides several important insights into the effect(s) of ANPRB on growth. First, previous studies have suggested that patients with heterozygous NPR2 mutations have greater defects in arm span (AS) than height (H) based on SD (31). Although the smaller size of our cohort limits our ability to detect statistically significant differences in auxology, the trend of the SH:H ratio z-scores in NPR2 heterozygotes was toward the abnormal value of >55.5% for the Rappold cohort, and the AS:H ratio was decreased toward the abnormal value of < 96.5% (Table 1) (44). Specifically, we found that the short stature of young NPR2 heterozygotes is proportionate, as patients had normal Rappold scores (Table 1). By contrast, with advancing age, short stature became more disproportionate, suggesting that the progressive loss of statural potential in NPR2 heterozygotes is the result of a mechanism that preferentially affects endochondral long bone growth. Therefore, younger but not older children who have NPR2 haploinsufficiency will be more distinct from ISS patients who have SHOX haploinsufficiency (44, 51).

A second important finding of our study is that heterozygous carriers of NPR2 mutations have a progressive loss of statural potential as they age that leads to increasing severity of short stature (Figs. 2A, 2B and Fig. 3). In AMDM, skeletal growth quickly decelerates after birth due to abnormal endochondral ossification, resulting in severe short stature (30). Our data (Fig. 3) show that with age, patients with heterozygous NPR2 mutations experience a similar but less severe decline in height z-score, and therefore worsening height potential, as patients with AMDM. Moreover, this progressive decline in height z-score is associated with an unusual pattern of skeletal maturation that is characterized by delayed BA in very young children, with a subsequent acceleration in BA with age (Fig. 3). Previous studies had described delayed BA in younger NPR2 heterozygote children (31, 32, 34), but later BA measurements for these children were not provided. The basis for this apparent acceleration in skeletal maturation is presently unknown, but it would appear that the consequences of NRP2 inactivation on the physiological process of endochondral bone development are dependent on spatial and temporal considerations. Moreover, we would note that this pattern of skeletal maturation is also distinct from the BA advancement that has been described in many but not all subjects with heterozygous ACAN mutations, who also have disproportionate short stature due to disordered long bone growth (2, 52). The progressive short stature and advancing BA in patients with NPR2 haploinsufficiency distinguish this condition from typical ISS, SHOX deficiency, or constitutional delay of growth and maturation.

Third, although the present study was not designed to assess the genotype-phenotype association of NPR2 mutations, and we identified only 2 allelic variants, we nevertheless note that our study suggests a subtle genotype-phenotype association between the 2 variants we identified, which previously was not observed among the various mutations in NPR2 (6, 31-33). In the literature, heterozygous patients’ height z-scores range from −0.98 to −2.75 (6, 7), and height z-scores range from −1.5 to −4.15 (31-34). In subfamily A, siblings III-2 and III-5 each inherited different parental NPR2 mutations, and had heights in the 9th percentile (p.R989L variant) and 32nd percentile (p.P93S variant) respectively, highlighting the possibility of differential effects based on specific mutation and initial genetic potential affecting analysis among cohorts. An alternative but equally important consideration is that genetic background can have a significant influence on the height-attenuating effect of an NPR2 variant, which can even account for differences even between siblings (53).

Finally, GH treatment has not been shown to improve final adult height in patients who are homozygous for NPR2 mutations (this work). Both AMDM subjects had appropriate therapeutic IGF-1 levels while receiving GH therapy (Supplemental Table 3 (37), but in subject III-1 an initial increase in height velocity was not sustained, and in subject III-4 there was BA advanced on GH treatment, so it is unclear whether an initial increase in linear velocity would have improved final adult height. By contrast, GH appears to be more effective for NPR2 heterozygotes (8, 31, 32). Several reports have described sustained increases in height velocity with GH treatment of NPR2 heterozygotes (8, 54), but in many other cases the initial increase was not sustained (31, 32). Nevertheless, most published studies of GH treatment indicate that final adult height was either not improved or not yet achieved (8, 33, 34, 54). Although it is not yet clear whether GH will improve final adult height in patients with heterozygous NPR2 mutations, the ANPRB signaling pathway suggests some alternative approaches. Because ANPRB activation inhibits FGFR3 signaling, CNP analogs have been proposed as therapeutic agents to counteract the growth-impairing effects of activating mutations in FGFR3 that cause achondroplasia or the milder phenotype of hypochondroplasia (22-25), and recent clinical studies of these agents have shown great promise (26-28). Accordingly, CNP therapy might be used to activate remaining ANPRB receptors, while other therapeutics that act downstream of the receptor and increase cGMP (eg, type 5 phosphodiesterase inhibitors), or inhibit FGRFR3 signaling, may hold future promise (26, 28, 55-57).

The strengths of our study include our ability to perform targeted analysis of the effect of NPR2 mutations due to the utilization of a single, extended family that includes subjects with 2, 1 or 0 mutant NPR2 alleles. Another strength is the extensive auxological measurements on the subject cohort we studied, which permitted the most comprehensive and longitudinal description of the growth phenotype of subjects with NPR2 mutations to date.

One limitation of our study is that we did not have complete auxological measurements or BA on all children included, with several having reached or been close to final adult height before being evaluated. In addition, although we have a large number of subjects in our cohort with many growth points, in some subjects we do not have earlier growth points during the first year of life. Lastly, our observation that there is a progressive advancement of bone age in NPR2 carriers is based upon a relatively small cohort. This important distinction from typical ISS will need confirmation in larger numbers of subjects who are heterozygous for NPR2 mutations.

In conclusion, we report 2 missense NPR2 mutations that encode dominant negative ANPRB proteins and describe the novel findings that height velocity decreases and BA advancement accelerates with age in NPR2 heterozygotes, leading to progressive loss of adult statural height and disproportionate short stature. These observations may provide useful clinical tools that help identify short patients who have heterozygous NPR2 mutations.

Glossary

Abbreviations

AMDM

acromesomelic dysplasia Maroteaux type

ANPRB

atrial natriuretic peptide B

AS

arm span

AS:H

arm span to height ratio

BA

bone age

CA

chronological age

cGMP

cyclic guanosine monophosphate

CHOP

The Children’s Hospital of Philadelphia

CNP

C-type natriuretic peptide

FGFR

fibroblast growth factor receptor

GH

growth hormone

IGF-1

insulin-like growth factor 1

ISS

idiopathic short stature

PBS

phosphate-buffered saline

SD

standard deviation

SH:H

seated height to height ratio

Additional Information

Disclosure Summary: The authors have declared that no conflict of interest exists.

Data Availability

All data generated or analyzed during this study are included in this published article or in the data repositories listed in References.