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

Division of Endocrinology (R.C.O.), Nemours Children’s Clinic, Jacksonville, Florida 32207; Departments of Epidemiology and Biostatistics (H.B., M.L.W.), Genetics (C.F.B.), and Pediatrics (M.L.W.), Case Western Reserve University School of Medicine, Cleveland, Ohio 44106; Department of Medicine (T.C.R.P., E.A.E.), Christchurch School of Medicine and Health Sciences, Christchurch, New Zealand; and Department of Biochemistry, Molecular Biology, and Biophysics (L.R.P.), University of Minnesota-Twin Cities, Minneapolis, Minnesota 55455

Address all correspondence and requests for reprints to: Robert C. Olney, M.D., Division of Endocrinology, Nemours Children’s Clinic, Jacksonville, Florida 32207. E-mail: rolney{at}nemours.org.

Abstract

Context: 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.

Objective: The objective of the study was to determine the phenotypic features of heterozygous carriers of NPR2 mutations.

Design and Setting: This was a case-control study from the general community.

Subjects: Thirty-nine members of a family in which one member has AMDM were studied.

Intervention: This was an observational study.

Main Outcome Measure: The primary measure was stature, with the hypothesis that carriers have reduced stature compared with noncarriers.

Results: 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.

Conclusions: 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.

C-TYPE NATRIURETIC peptide (CNP) is a small, secreted peptide and a member of the natriuretic peptide family. The natriuretic peptides bind to three membrane receptors, natriuretic peptide receptor (NPR)-A, -B, and -C (1). Of these, NPR-B binds CNP with the highest affinity and, in physiological conditions, mediates the known effects of CNP. CNP binding to NPR-B activates a guanylyl cyclase domain, generating cGMP. Further intracellular signaling involves a number of cGMP-dependent proteins, including ion channels, protein kinases, and phosphodiesterases.

CNP is an important regulator of skeletal growth. The knockout mice for CNP (2) and NPR-B (3) are dwarfed, and CNP-overexpressing mice are overgrown (4). Organ culture experiments with fetal rat metatarsals (5) and fetal mouse tibias (6, 7) revealed that CNP increases intracellular cGMP levels in the growth plate and increases the bone growth rate. Both CNP and NPR-B are expressed in the growth plate (2, 3), and blood levels of CNP are very low (8), suggesting that CNP acts through an autocrine/paracrine mechanism in the growth plate.

In humans, loss-of-function mutations in the NPR-B gene (NPR2) cause acromesomelic dysplasia, Maroteaux type (AMDM; OMIM no. 602875) (9). This autosomal recessive skeletal dysplasia has characteristic radiological changes and results in extreme, disproportionate short stature. Patients with AMDM are near normal-sized at birth with the growth abnormalities becoming apparent postnatally. AMDM is not associated with impaired intelligence or other phenotypic features. Interestingly, parents of patients with AMDM (obligate heterozygotes) were noted to be shorter than expected for their population of origin (9).

We report here studies of a large kindred in which multiple members carry a loss-of-function mutation in NPR2. This enables more accurate comparisons to be made between carriers and noncarriers.

Subjects and Methods

Experimental subjects

The participants of this study were all members of a single kindred, identified through a proband with AMDM. Forty subjects were studied. Unrelated spouses were not included. Table 1 shows the characteristics of the participants. This study was approved by the institutional review boards of University Hospitals of Cleveland and Nemours Children’s Clinic. Signed consent was obtained for all participants.

Height and sitting height were determined using a portable stadiometer/digital scale, and head circumference (occipital-frontal circumference) was measured by fiberglass tape. Measurements were made by a single observer. Coefficients of variation for these measurements were 0.17% (height), 0.24% (sitting height/height ratio), and 0.42% (head circumference). Weight was determined using a digital scale (Detecto Model 6439; Cardinal Scale Manufacturing Co., Webb City, MO), and heart rate and blood pressure were determined using a DinaMap Pro 100 (GE Healthcare Technologies, Waukesha, WI).

Standing height z-scores for subjects less than 20 yr old were calculated using National Health and Nutrition Examination Survey (NHANES) general population data (10). For subjects over 20 yr old, NHANES data for non-Hispanic white adults were used (11). Weight and BMI z-scores were also calculated using NHANES data (10), sitting height to height ratio z-scores using data were reported by Frediks et al. (12), head circumference z-scores were from data reported by Roche et al. (13), and heart rate z-scores were from data reported by Davignon et al. (14). Except for standing height, z-scores for subjects age 18 yr or over were calculated using the 18-yr endpoints of the reference data.

Genotyping

DNA was obtained by buccal swab or from blood. Sequencing of NPR2 (GenBank accession no. NM_000907) was done as described (9). The disease-causing mutation in the proband (c.1092delT) results in the loss of a Tsp509I cleavage site. Genotyping for this mutation was done by restriction fragment length analysis. Exon 4 of NPR2 was amplified by PCR from genomic DNA with primers targeted to flanking introns: forward primer, 5'-CCCTTCCTGAGAGTCAGGTCAAG; and reverse primer, 5'-GTGGGCAACTCTGCCATATT. The amplification reaction volume was 50 µl and included 50–100 ng genomic DNA. The thermal profile was 94 C for 5 min, 35 cycles of 94 C for 1 min, 60 C for 50 sec, and 72 C for 50 sec, followed by a single incubation at 72 C for 10 min. Five microliters of product was examined on an agarose gel, and the remainder was purified using QiaQuick columns (QIAGEN, Valencia, CA) and digested with Tsp509I (New England Biolabs, Ipswich, MA) at 65 C for 1 h. Digests were analyzed by electrophoresis in 2% standard agarose. Tsp509I cut the amplified wild-type allele into fragments of 379 and 150 bp. Amplimer containing the 1092delT mutation remained uncut and had a length of 529 bp. For quality control purposes, the genotypes of seven heterozygous subjects were confirmed by sequencing.

Immunoassays

Assays for serum IGF-I (immunochemiluminometric assay) and IGFBP-3 (RIA) were run at Quest Diagnostics’ Nichols Institute. Values were transformed to z-scores using data reported by Brabant et al. (15) (IGF-I) or Nichols’ internal data (IGFBP-3). RIAs for plasma CNP and amino-terminal CNP propeptide (NT-proCNP) were run as described (8) and transformed into z-scores using data reported by Prickett et al. (8).

Statistical methods

Because subjects were of both sexes and from a wide range of ages, all values were transformed into z-scores, using the stated norms. Data are presented as mean ± SD. For each measurement, ANOVA was used to compare carriers, noncarriers, and the general population. Where ANOVA showed significance, pair-wise comparisons were made using Holm’s t tests. Statistical significance was assumed for P < 0.05.

Results

The proband

The proband was diagnosed at 1 yr of age with AMDM, based on characteristic physical and x-ray findings. Table 2 lists her anthropometric and biochemical measurements and highlights the short-limbed nature of this skeletal dysplasia. Repeated measurements have shown she has low IGF-I levels. An arginine/insulin-induced hypoglycemia GH stimulation test done at 1.5 yr of age showed a baseline GH level of 2.9 ng/ml and an exaggerated peak level of 29 ng/ml. IGFBP-3 and GH binding protein levels were within the normal range. A random GH level of 15 ng/ml was measured at 8 yr of age. An IGF-I generation test (0.05 mg/kg GH daily for 4 d) resulted in a 1.7-fold increase in IGF-I level. During 4 yr of treatment with GH (0.05 mg/kg GH daily, 1.5–5.5 yr of age), her height z-score decreased from –6.3 to –7.2.

Sequencing of NPR2 revealed the proband is homozygous for a deletion of a single thymidine nucleotide in exon 4 of the gene (c.1092delT, where nucleotide 1 corresponds to the adenosine nucleotide in the translation initiation codon). The mutation is predicted to cause a reading frame shift, creating a premature termination codon in the NPR2 mRNA (p.I364fsX376). Loss of NPR-B function would result either from non-sense-mediated mRNA decay or from production of a severely truncated receptor that lacks the transmembrane and guanylyl cyclase domains.

The kindred

The proband’s parents (who share a common ancestor) are heterozygous carriers of the NPR2 1092delT mutation. The proband’s father’s and mother’s height z-scores were –2.0 and –3.6, respectively.

The proband is a member of a large Caucasian kindred, primarily of Irish ancestry. Data were obtained on 39 family members from four generations (see supplemental Fig. 1, published on The Endocrine Society’s Journals Online web site at http://jcem.endojournals.org). Sixteen are heterozygous carriers of the 1092delT mutation, and 23 are not. The mean height z-score for the carriers was –1.8 ± 1.1 (mean ± SD) vs. –0.4 ± 0.8 for the noncarriers (P < 0.0005) (Fig. 1). The height z-score of the carriers was also less than the general population (P < 0.0005). There was no difference in the sitting height to height ratio z-scores between the carriers and noncarriers (Fig. 2). Body mass index was significantly higher in the carriers (P < 0.05 vs. noncarriers; P < 0.005 vs. the general population). The only other parameter that differed between the groups was heart rate z-score, which was –0.9 ± 0.8 for the carriers and 0.0 ± 1.1 for the noncarriers (P < 0.05 vs. noncarriers, P < 0.05 vs. the general population). Neither blood pressure nor history of cardiovascular disease were different between the groups. In none of the parameters measured were the noncarriers significantly different from the general population.

View larger version (12K): [in this window] [in a new window]   FIG. 1. Stature of carriers of the NPR2 1092delT mutation. The heights of carriers and noncarrier family members were measured, and values were transformed into z-scores. The points show the individual results (circles, female subjects; squares, male subjects). Bars, Mean for each group; error bars, SD. Horizontal line, Population mean (z-score of 0). For the carriers, n = 16; and for the noncarriers, n = 23. *, P < 0.0005 vs. noncarrier family members. , P < 0.0005 vs. general population (ANOVA with Holm’s t test post hoc comparisons).

View larger version (18K): [in this window] [in a new window]   FIG. 2. Other characteristics of carriers of the NPR2 1092delT mutation. The values for BMI, sitting height to height ratio (SH/Ht), head circumference (HC), heart rate, CNP, NT-proCNP, IGF-I, and IGFBP-3 of carriers and noncarrier family members were determined and transformed into z-scores. White points, Mean of the carriers; black points, mean of the noncarriers. Error bars, SD. Horizontal line, Population mean (z-score of 0). For the carriers, n = 14 for BMI, HC, and heart rate, and n = 8 for the other measures. For the noncarriers, n = 23 for BMI, HC, and heart rate, and n = 13 for the other measures. *, P < 0.05 vs. noncarrier family members. , P < 0.05 vs. the general population.

The heights of confirmed carriers of NPR2 mutations were previously reported (9). When these heights were transformed to z-scores using population-specific reference standards (see supplemental Table 1, published on The Endocrine Society’s Journals Online web site at http://jcem.endojournals.org), the mean height z-score was –1.1 ± 0.8 (n = 35) and was significantly different from the general population (P < 0.0005).

Discussion

In the initial description of NPR2 mutations in patients with AMDM (9), data from adult mutation carriers showed they were on average 5.7 cm shorter than population-matched controls. Further analysis showed these carriers had a mean height 1.1 SD values below the mean for their population of origin. However, these individuals came from a wide range of geographic and ethnic backgrounds, and measurements were done be a number of observers, which complicated the comparison. Here, we present data from a single kindred that allowed for a simpler comparison between carriers and noncarriers. Because height is subject to assortive mating (i.e. short men tend to marry short women), it is preferable to make comparisons within family groups, rather than relying on population norms. This was demonstrated in our kindred, where the noncarriers had a mean height 0.4 SD values below the general population. Our results show that the NPR2 mutation carriers have a mean height that is 1.4 SD values less than their noncarrier family members. This translates into an average reduction in adult height of 9 cm for woman and 10 cm for men. Although causality cannot yet be determined, these data demonstrate a strong association of heterozygous NPR2 mutations with reduced stature. Future studies of other families will be important to confirm this association.

The finding of reduced stature in heterozygotes was also reported in the NPR-B knockout mouse, where mice heterozygous for the mutation were shorter (nasoanal length) than their wild-type litter mates (3).

Our proband appeared to have an abnormality in the GH/IGF-I system, characterized by low IGF-I levels, high GH levels, and lack of a growth response to GH treatment. This is a similar to findings in the NPR-B knockout mouse, where IGF-I levels were 62% of those from wild-type litter mates at 70–120 d of age (3). These data imply an interaction between the CNP/NPR-B and the GH/IGF-I pathways during postnatal growth and that the growth failure in AMDM may be due (at least in part) to low IGF-I levels. Studies in other children with AMDM will test this hypothesis.

Plasma CNP and NT-proCNP levels were very high in our patient with AMDM. Because AMDM results from absence of the primary receptor for this ligand, this suggests the presence of a feedback loop that regulates CNP production. For the heterozygous carriers of the mutation, these levels were not different from the noncarriers. Similarly, although IGF-I levels were low in our proband, they were not in the carriers. This finding is also similar to the NPR-B knockout mouse, where heterozygotes have normal IGF-I levels (3). In the normal population, blood levels of CNP, NT-proCNP (8), and IGF-I are all lower in adults than in children. It is possible that NPR2 mutation carriers have abnormal levels of these peptides during childhood but normal levels in adulthood. This would not have been detected in our study because most of our participants were adults.

Individuals with no functional NPR2 alleles have AMDM, a skeletal dysplasia with extreme short stature and body disproportion. Although we found no evidence of skeletal dysplasia or body disproportion in individuals with only one functional NPR2 allele, we did find that haploinsufficiency reduces linear growth, resulting in the phenotype of idiopathic short stature. Therefore, it is reasonable to consider whether NPR2 mutations may be a common cause of idiopathic short stature in the general population. The fraction of NPR2 carriers in the general population with short stature can be calculated using Hardy-Weinberg equilibrium to estimate the NPR2 mutation carrier frequency. We first estimated the prevalence of AMDM in the United States by comparing the number of individuals with AMDM with the number of individuals with achondroplasia in the Little People of America (LPA) membership database. This is a conservative estimate because achondroplasia is likely overrepresented in LPA because of its autosomal dominant inheritance pattern and hence the enrollment of multiple affected family members. There are about 100-fold fewer members of LPA with AMDM than with achondroplasia. Because achondroplasia has a prevalence of one in 20,000, we estimate the prevalence of AMDM to be one in 2,000,000 and frequency of heterozygous carriers of AMDM mutations to be approximately one in 700. We observed that these carriers have a mean height z-score of –1.8. Using the definition of idiopathic short stature as having a height z-score of less than –2.25, we calculate that one person in 30 with idiopathic short stature will be a carrier of an NPR2 mutation. Consequently, heterozygosity for a loss-of-function mutation in NPR2 should be considered in the differential diagnosis of idiopathic short stature.

Acknowledgments

We thank the family for participating in this study.

Footnotes

This work was supported in part by a grant from the National Heart Foundation of New Zealand. M.L.W. is an investigator with the Howard Hughes Medical Institute and a recipient of a Clinical Scientist Award in Translational Research from the Burroughs Wellcome Fund.

First Published Online December 29, 2005

Abbreviations: AMDM, Acromesomelic dysplasia, Maroteaux type; CNP, C-type natriuretic peptide; LPA, Little People of America; NHANES, National Health and Nutrition Examination Survey; NPR, natriuretic peptide receptor.

Received August 31, 2005.

Accepted December 20, 2005.

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This Article Abstract Full Text (PDF) Submit a related Letter to the Editor Alert me when this article is cited Alert me when eLetters are posted Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Request Copyright Permission Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Olney, R. C. Articles by Warman, M. L. Search for Related Content PubMed PubMed Citation Articles by Olney, R. C. Articles by Warman, M. L. Related Collections Pediatric Endocrinology