Background C-type natriuretic peptide (CNP), its endogenous receptor, natriuretic peptide receptor-B (NPR-B), as well as its downstream mediator, cyclic guanosine monophosphate (cGMP) dependent protein kinase II (cGKII), have been shown to play a pivotal role in chondrogenic differentiation and endochondral bone growth. In humans, biallelic variants in NPR2, encoding NPR-B, cause acromesomelic dysplasia, type Maroteaux, while heterozygous variants in NPR2 (natriuretic peptide receptor 2) and NPPC (natriuretic peptide precursor C), encoding CNP, cause milder phenotypes. In contrast, no variants in cGKII, encoded by the protein kinase cGMP-dependent type II gene (PRKG2), have been reported in humans to date, although its role in longitudinal growth has been clearly demonstrated in several animal models.
Methods Exome sequencing was performed in two girls with severe short stature due to acromesomelic limb shortening, brachydactyly, mild to moderate platyspondyly and progressively increasing metaphyseal alterations of the long bones. Functional characterisation was undertaken for the identified variants.
Results Two homozygous PRKG2 variants, a nonsense and a frameshift, were identified. The mutant transcripts are exposed to nonsense-mediated decay and the truncated mutant cGKII proteins, partially or completely lacking the kinase domain, alter the downstream mitogen activation protein kinase signalling pathway by failing to phosphorylate c-Raf 1 at Ser43 and subsequently reduce ERK1/2 activation in response to fibroblast growth factor 2. They also downregulate COL10A1 and upregulate COL2A1 expression through SOX9.
Conclusion In conclusion, we have clinically and molecularly characterised a new acromesomelic dysplasia, acromesomelic dysplasia, PRKG2 type (AMDP).
- bone diseases
- gene expression regulation
- human genetics
- molecular medicine
Data availability statement
Data are available on reasonable request. All data relevant to the study are included in the article or uploaded as supplementary information.
Statistics from Altmetric.com
If you wish to reuse any or all of this article please use the link below which will take you to the Copyright Clearance Center’s RightsLink service. You will be able to get a quick price and instant permission to reuse the content in many different ways.
Longitudinal bone growth is achieved by endochondral ossification in the cartilaginous growth plate. Here, the chondrocytes undergo biochemical and morphological transformation from the resting states, during proliferation and hypertrophy and terminating in their replacement by bone. This process is tightly regulated, requiring a precise temporal and spatial interaction between hormonal and growth factors.
C-type natriuretic peptide (CNP), its endogenous receptor, natriuretic peptide receptor-B (NPR-B), as well as its downstream mediator, cyclic guanosine monophosphate (cGMP) dependent protein kinase II (cGKII), have been shown to play a pivotal role in chondrogenic differentiation and endochondral bone growth.1–3 The binding of CNP to NPR-B leads to synthesis and accumulation of intracellular cGMP that subsequently activates cGKII. In humans, NPR-B and CNP are encoded by the natriuretic peptide receptor 2 (NPR2) and natriuretic peptide precursor C (NPPC), respectively. Homozygous or compound heterozygous variants in NPR2 cause acromesomelic dysplasia, type Maroteaux (AMDM (MIM 602875)), a rare autosomal recessive skeletal dysplasia characterised by severe disproportionate short stature, occurring soon after birth with adult height standard deviation score (SDS) <−5, acromesomelic shortening of the extremities, platyspondyly with kyphosis or scoliosis and other dysmorphic features.4 Heterozygous NPR2 variants are associated with a milder phenotype, with short stature, mild mesomelic shortening of the limbs and brachydactyly (MIM 616255) and have also been identified in individuals with idiopathic short stature (ISS).5–9 Although the relevance of CNP to skeletal growth has been clearly demonstrated in various animal models,10–12 it was only recently that we reported the first heterozygous variants in NPPC in humans.13 The two loss-of-function CNP variants showed a diminished ability to activate NPR-B and subsequently reduced cGMP synthesis, leading to short stature and small hands.13
In contrast, no variants in cGKII, encoded by the protein kinase cGMP dependent type II gene (PRKG2), have been reported in humans to date, although its role in longitudinal growth has been demonstrated in several animal models. In 1996, Pfeiffer et al 1 were the first to describe the link between cGKII and endochondral ossification, when postnatal dwarfism, cranial alterations and shortened limbs were unexpectedly observed in cGKII−/− mice, later shown to be a consequence of elongated growth plates and impaired chondrocyte hypertrophy.1 14 Another model is the Komeda miniature rat Ishikawa (KMI), a naturally occurring homozygous prkg2 mutant (deletion), which exhibits longitudinal growth retardation starting at 4 weeks after birth and resulting in their limbs being 20%–30% shorter than wild-type (WT) littermates.15 Dwarfism in American Angus cattle was also found to be due to a homozygous nonsense PRKG2 mutation, where once again, the mutant cattle were smaller with shorter limbs and metatarsals, fused ulnae and radii and vertebral abnormalities.16
Here, we report the first homozygous variants in PRKG2 identified in two unrelated individuals, both with a skeletal dysplasia associated with severe disproportionate short stature due to acromesomelia. We describe the clinical and radiological features observed in these individuals and demonstrate the pathogenic mechanism of the identified variants.
Patients and methods
The proband, a Moroccan girl aged 12 years old, was referred for molecular studies due to severe disproportionate short stature (−4.01 SDS, according to Spanish growth charts, http://www.aeped.es/noticias/estudios-espanoles-crecimiento-2010), brachydactyly and mild dysmorphic features. She was the second of three children, born to consanguineous parents (third cousins). Although no birth data were available, at 3 weeks old she had reduced body length (48 cm, −1.93 SDS) and head circumference (35 cm, −0.42 SDS) according to http://www.aeped.es/noticias/estudios-espanoles-crecimiento-2010. She had a congenital complete atrioventricular block (Mobitz IIa) and a pacemaker was implanted at the age of 1 year.
At the age of 10, she presented with a height of 119 cm (−3.21 SDS, https://www.aeped.es) and body mass index SDS of 1. She was at Tanner stage 2–3. She had a normal oval-shaped head and no dysmorphic facial features. Physical examination revealed mesomelic shortening of the limbs and short, stubby fingers. Metabolic and hormonal work-up was normal. Both her parents exhibited normal height (mother: 164 cm, −0.01 SDS; father: 165 cm, −1.75 SDS) as did her two brothers. No skeletal abnormalities were observed in any of them. At the age of 12, she had a height of 126 cm (−4.01 SDS). Her sitting height to height ratio was 0.579 (1.21 SDS)17 and her arm span to height ratio was 0.933. She underwent both femoral and humeral limb lengthening; thus, no further growth data are available. She obtained a final postoperative height of 146 cm (−2.92 SDS) and limb proportionality. She continued to have short, stubby fingers (figure 1F). Her growth curve is shown in online supplemental figure S1.
Radiological analysis of proband 1 at the age of 12 (figure 1) revealed mildly flattening of the thoracic vertebral bodies, mild thoracic scoliosis, lumbar hyperlordosis and short pedicles of the lumbar spine (figure 1A,B). Her lower limbs exhibited mild mesomelic shortening with very mild flaring of the metaphyses and mild genu valgum. The growth plate at the knee was prematurely fused (figure 1C). Her radius and ulna were mildly bowed and mild mesomelic shortening was present (figure 1D). She had short broad phalanges and metacarpals (especially the third, fourth and fifth), all of which presented premature fusion (figure 1E). She had an advanced bone age (+2.6 SDS). The clinical and radiological features were similar to AMDM but with a milder radiological phenotype, no cone-shaped epiphyses in the hands and relatively mild mesomelia.
An Indian girl aged 7 years was first seen in our genetic clinic with a history of short stature and skeletal deformities. She had been referred as a follow-up case of short stature with complaints of gaining little weight since the age of 4 years (height 79 cm, −5.51 SDS, weight 10 kg, −3.61 SDS) according to Indian Academy of Pediatrics (IAP) growth charts (https://iapindia.org/iap-growth-charts/). Her upper to lower segment ratio was 1.45 suggestive of disproportionate short stature. She was the second child, born at full term by vaginal delivery, to parents with known consanguinity (third cousins). There was no history of chronic illness in the past or any other relevant medical history. Both parents had short stature according to Centers for Disease Control and Prevention 2000 growth charts (https://www.cdc.gov/growthcharts/), mother (147 cm; −2.50 SDS) and father’s (158 cm; −2.59 SDS), while her 1-year old brother, at the age of 11.75 years had normal stature (136.7 cm, −1.54 SDS) according to IAP growth charts. No dysmorphic features, brachydactyly or skeletal abnormalities were observed in any of them. The proband, now age 11 years has severe short stature with a height of 102.4 cm (−5.06 SDS), upper to lower segment ratio 1.41 and arm span of 92.7 cm.
Facial dysmorphic features included triangular face with a broad nasal bridge and pointed chin, synophrys, hypertelorism, low set ears. Physical examination revealed generalised hirsutism, prominent costochondral cartilages, sternal prominence, widening of wrists, short stubby fingers, brachydactyly, genu varum and sandal gap. There was no other member in the family with similar clinical features. Routine biochemical evaluation was not significant.
Skeletal surveys were performed at ages 4, 7 and her current age of 11 (figure 1 and online supplemental figure S2). At age 4, moderate platyspondyly with anterior beaking of vertebral borders of dorsolumbar spine was observed (figure 1G,H). Her long bones showed relatively large epiphyses and widening and some irregularity of the metaphyses (online supplemental figure S2). Prominent deltoid tuberosities of the humeri were seen. She had short and broad phalanges and metaphyseal irregularity of metacarpals (online supplemental figure S2) and metatarsals. Her bone age was within normal limits (−1.5 SDS). Her ilia were short with flaring of the iliac wings (online supplemental figure S2). Skeletal survey at 7 years showed increased metaphyseal irregularity of her long bones (figure 1I and online supplemental figure S2). Vertebral alterations were less prominent and restricted to the thoracic region with mild shortening of the pedicles of her lumbar spine (online supplemental figure 1). Her pelvic radiograph showed minor irregularity of the acetabula (Fig S2). The skeletal anomalies were persistent at 11 years old (figure 1J–M and online supplemental figure S2).
Thus, in general, the radiographic hallmarks in both patients include: (1) mild to moderate platyspondyly, (2) moderate brachydactyly, (3) iliac flaring and (4) progressively increasing metaphyseal alterations of the long bones. The overall pattern resembles that of AMDM with a milder phenotype including an absence of cone-shaped epiphyses in the hands.
All participants provided informed consent for the performed genetic studies and ethical approval was obtained from the respective institutions. Genomic DNA and RNA was isolated from peripheral blood lymphocytes.
The proband was initially assessed for variants in known skeletal dysplasia genes by using a custom-designed NGS skeletal dysplasia panel (SkeletalSeq.V4, n=327 genes, SeqCap, Roche Nimblegen, Wisconsin, USA). As no causative gene defect was identified, the proband and parents (trio) were subsequently subjected to exome sequencing using Sure Select Human All exon V6 targeted capture (Agilent Technologies, Santa Clara, California, USA), paired-end 150 bp sequencing on a NovaSeq 6000 (Illumina, San Diego, California, USA). Reads were aligned to the hg19 assembly. Variant filtering was performed with the help of VarSeq (Golden Helix, Bozeman, Montana, USA), primarily focusing on mode of inheritance, variants absent in controls (gnomAD), highly conserved nucleotide (GERP++, http://mendel.stanford.edu/SidowLab/downloads/gerp) and amino acid (Alamut 2.14, Interactive Biosoftware, France), predicted to be pathogenic by multiple in silico programs: CADD V1.4 (https://cadd.gs.washington.edu/), SIFT (https://sift.bii.a-star.edu.sg/), Polyphen2 (http://genetics.bwh.harvard.edu/pph2/) and Mutation Assessor (http://mutationassessor.org/r3). Splice variants were assessed with the aid of Alamut which included MaxEntScan, NNSplice, GeneSplicer and Human Splicing Finder. Non-synonymous or splice site variants were then selected based on first recessive inheritance and then other modes of inheritance and genes associated with Human Phenotype Ontology (HPO) codes. Variants of interest were validated by Sanger sequencing.
Exome sequencing was performed for proband 2 using the Nextera Expanded exome kit (Illumina) and paired-end 100 bp sequencing on a HiSeq2000 (Illumina). The obtained sequencing reads were processed for variant calling using standard pipelines, as previously described.18 Variant assessment was similar to that previously mentioned. Variants of interest were validated in proband and family by Sanger sequencing.
Genetic screening of PRKG2
After the identification of a homozygous variant in PRKG2 in one proband, PRKG2 variant screening (NM_006259.2 - exons and intron:exon boundaries) was performed in a cohort of 298 patients with proportionate/disproportionate short stature with or without other skeletal abnormalities by Sanger sequencing. Oligonucleotide sequences for coding regions and intron : exon boundaries are available on request.
Nonsense-mediated decay (NMD) assay
Peripheral blood samples were obtained in ACD (Anticoagulant Citrate Dextrose) buffer vials from probands 1 and 2. Peripheral blood mononuclear cells (PBMCs) were isolated by Ficoll (GE Healthcare, Upsala, Sweden) gradient method. The buffy coat (PBMC-lymphocyte) layer was transferred to a new falcon tube and cells were washed with 1× phosphate-buffered saline (Gibco, Thermo Fisher Scientific, Waltham, Massachusetts, USA) until clearance of the red blood cell ring. Cell viability was verified using trypan blue.
Cells were then maintained in RPMI-1640 supplemented with 15% fetal bovine serum and 1% antibiotic–antimycotic solution (Gibco, Thermo Fisher Scientific). One hour later, 25 μg/mL cycloheximide (CHX) (Millipore, Darmstadt, Germany) was added to the appropriate wells (treated) and incubated at 37°C, 5% CO2 and 95% humidity. After 6 hours, treated and untreated cells were harvested and total RNA was isolated using the RNeasy Mini kit (Qiagen, Hilden, Germany) following the manufacturer’s recommendations or by the Trizol method. cDNA was generated using the high-capacity cDNA reverse transcription kit (Thermo Fisher Scientific). TaqMan probes for human PRKG2 (Hs_00922440_m1, Applied Biosystems, Thermo Fisher Scientific) and the endogenous control glyceraldehyde 3-phosphate dehydrogenase (GAPDH) (4 326 317E, Applied Biosystems, Thermo Fisher Scientific) and the TaqMan Universal PCR Master Mix (Thermo Fisher Scientific) were employed for quantitative real-time PCR, according to manufacturer’s instructions.
The empty Myc-DDK-tagged expression pCMV6-Entry plasmid and the human PRKG2 (NM_006259.2; pCMV6-PRKG2-WT) WT plasmid were obtained from Origene Technologies (Rockville, Maryland, USA). PRKG2 mutants were generated by site-directed mutagenesis of this WT vector according to the manufacturer’s instructions (QuikChange Site-Directed Mutagenesis Kit, Agilent Technologies). Mutagenesis primers (mutated base underlined) were the following: 5′-GCTCATTACAGATGCCCTTAA A TAAAAATCAGTTTCTGAAAAG-3′ for the c.491dup; p.Asn164Lysfs*2 (N164Kfs*2) variant, 5′-AGAAGCATTTGATTACCTGCAT T GACTAGGTATTATCTACAGAG-3′ for the c.1705C>T; p.Arg569* (R569*) variant and 5′-CCCAGGAAGATAACACGA T GACCTGAGGATTTGATTC-3′ for the positive control and the American Angus cattle variant, c.2032C>T;p.Arg678* (R678*). All constructs were verified by Sanger sequencing.
Cell culture and transient transfection
HEK293T cells were maintained in 1× Dulbecco’s Modified Eagle’s Medium (DMEM) supplementedwith 10% fetal bovine serum and 1% penicillin/streptomycin (Gibco, Thermofisher) at 37ºC, 5% CO2 and 95% humidity. Cells were seeded in 6-well plates,and a total of 2 µg of DNA was transfected using jetPRIME (Polyplus-transfection, Illkirch, France) at a 1:2 ratio DNA to jetPRIME reagent following themanufacturer’s instructions. In co-transfection studies, equal amount of thetwo plasmids (1 µg) was added, thus maintaining the 1:2 ratio.
Western blot analysis
Initially we performed dose-dependent experiments using WT PRKG2 to determine the minimal concentration of fibroblast growth factor 2 (FGF2) and cGMP required to observe reductions in the downstream mitogen activation protein kinase (MAPK) signalling pathway by analysing the expression of phosphorylated extracellular-signal-regulated kinase (ERK) 1/2 by western blot. HEK293T cells were transiently transfected with the empty vector (EV) WT PRKG2 (pCMV6-PRKG2-WT). Twenty-4 hours post-transfection, cells were treated with 100 or 250 µM of 8-pCPT-cGMP (Sigma Aldrich, Merck, Darmstadt, Germany) for 30 min, followed by the addition of 1 or 2 nM of FGF2 (PeproTech, London, UK) to the appropriate wells for another 30 min to stimulate the MAPK pathway. The cells were then harvested and whole cell protein extract was extracted with the Nuclear Extract Kit (Active Motif, Belgium) according to the manufacturer’s instructions. Total protein concentration was determined by Bradford method (Bio-Rad Laboratories, California, USA). Proteins (20 µg) were resolved under denaturing conditions on a 10% or 12% (for cGKII protein detection) sodium dodecyl sulfate-acrylamide gels and transferred onto nitrocellulose membranes.
Subsequently, to analyse the effect of the two PRKG2 variants, N164Kfs*2 and R569*, on the MAPK signalling pathway, HEK293T cells were transiently transfected with the EV (pCMV6-Myc-DDK), WT PRKG2 (pCMV6-PRKG2-WT), one of the two identified mutants (pCMV6-PRKG2-N164Kfs*2, pCMV6-PRKG2-R569*) or the bovine positive control mutant (pCMV6-PRKG2-R678*). Twenty-four hours post-transfection, cells were treated with 100 µM of 8-pCPT-cGMP (Sigma-Aldrich, Merck, Darmstadt, Germany) for 30 min, followed by addition of 1 nM of FGF2 (PeproTech, London, UK) to the appropriate wells for another 30 min to stimulate the MAPK pathway. The proteins were extracted as previously described and western blots were undertaken.
The following antibodies were employed: anti-cGKII (1:1000, ab196960; Abcam, Cambridge, UK), phospho-ERK1/2 (1:1000, catalogue no: 9101; Cell Signalling, Leiden, The Netherlands), ERK1/2 (1:1000, catalogue no: 9102; Cell Signalling), phospho-Raf1-Ser43 (1:1000, AP3332a; Abgent, San Diego, California, USA), C-Raf-1 (1:2000, catalogue no: 610152; BD Biosciences, San Jose, California, USA). The proteins were detected using Western Lightning Plus- ECL kit (PerkinElmer, USA). All blots were rehybridised with either GAPDH (1:2000, ab9485; Abcam) or the corresponding non-phosphorylated form of the protein to check for loading variability. Three biological experiments were performed.
Quantitative real-time PCR (qPCR)
HEK293T cells were transiently transfected (equal ratio) with combinations of SOX9 and WT or mutant PRKG2 plasmids. EV and the cattle mutant, R678* were employed as negative and positive controls, respectively. Twenty-four hours post-transfection, 250 µM of 8-pCPT-cGMP (Sigma-Aldrich, Merck) was added to the cells as previously described.19 Total RNA was isolated 72 hours after treatment using the RNeasy Mini Kit (Qiagen, Hilden, Germany) according to the manufacturer’s recommendations. Reverse transcription was performed using the high-capacity cDNA reverse transcription kit (Applied Biosystems, Thermo Fisher Scientific). Expression of collagen type II (COL2A1) and type X (COL10A1), markers of chondrogenic proliferation and hypertrophic differentiation, respectively, was evaluated using TaqMan probes (Hs_00264051 and Hs_00166657, respectively; Applied Biosystems, Thermo Fisher Scientific) and TaqMan Universal PCR Master Mix (Applied Biosystems, Thermo Fisher Scientific). Each plasmid combination was transfected in duplicate, qPCRs were run in triplicate and four biological replicates were performed. The ∆∆CT method was used to determine relative expression levels using RPL13A (Hs_04194366; Applied Biosystems, Thermo Fisher Scientific) as the endogenous control. All data are expressed as percentages compared with the EV.
Two homozygous PRKG2 variants, NM_006259.2: c.1705C>T: p.(Arg569*) and c.491dup: p.(Asn164Lysfs*2), were identified in exons 14 and 3, in probands 1 and 2, respectively (figure 3). The variants were confirmed to be present in heterozygosity in their respective parents (figure 3). The frameshift variant is absent from the gnomAD population databases, while the nonsense variant is present in the heterozygous state in one South Asian individual (MAF 0.0033%). The nonsense variant p.(Arg569*) is predicted to produce a truncated protein partially lacking the catalytic domain affecting the substrate-binding region but maintains the regulatory domain, the cyclic nucleotide binding domains, (figure 3) while the frameshift variant p.(Asn164Lysfs*2) is predicted to ablate both the regulatory and catalytic domains (figure 3).
A summary of the exome sequencing data is provided in online supplemental data. A list of all homozygous variants detected in probands 1 and 2 are presented in online supplemental tables S1 and S2, respectively.
PRKG2 variants lead to reduced mRNA expression in proband PBMCs
As both variants were predicted to result in prematurely truncated proteins, we assessed whether the mutant mRNA was subjected to NMD using PBMCs from the patients in the presence or absence of NMD inhibitor, CHX. PRKG2 expression was significantly increased in the treated compared with untreated cells for both variants (c.491dup; p.(Asn164Lysfs*2), p<0.01 and c.1705C>T; p.(Arg569*), p<0.05), thus confirming that NMD occurs for both variants (figure 4A).
PRKG2 mutants are unable to downregulate MAPK signalling
First, we investigated whether the variants affected the synthesis of cGKII by performing western blot analysis of HEK293T cell lysates transfected with WT or mutant PRKG2. All truncated mutant proteins were detected, although their expression levels were reduced compared with WT (figure 2A).
Second, we determined whether the cGKII mutants were able to inhibit the FGF-induced MAPK pathway by transiently transfecting HEK293T cells with WT or mutant PRKG2 and treated with a cGMP analogue (8 pCPT-cGMP) and FGF2 to stimulate the MAPK pathway. We analysed their ability to phosphorylate Raf-1 at Ser-43 and ERK1/2. FGF2 was unable to induce phosphorylation of Raf-1 at Ser-43 per se, but phosphorylated ERK1/2 in the absence of 8 pCPT-cGMP, as previously described.20 WT PRKG2/cGKII phosphorylated Raf-1 and reduced FGF2-induced ERK1/2 phosphorylation in a cGMP-dependent manner. However, all mutants significantly failed to phosphorylate Raf-1 at Ser-43 and thus did not reduce ERK1/2 phosphorylation induced by FGF2 (figure 2B,C).
PRKG2 mutants uncouple the switch from proliferative to hypertrophic differentiation through impaired SOX9 function
To investigate the effect of PRKG2 variants in the regulation of proliferation and hypertrophic differentiation of chondrocytes by SOX9, expression of COL2A1 and COL10A1, markers of chondrocyte differentiation and hypertrophy, respectively, was determined by qPCR. Cells transfected with SOX9 showed a twofold increase in COL2A1 expression compared with the EV control (237±18.4, p<0.001; figure 4B). Cotransfection with WT PRKG2 significantly reduced SOX9-induced COL2A1 levels (p<0.01) in HEK293T cells. In contrast, neither of the PRKG2 mutants were able to suppress COL2A1 expression (N164Kfs*2, p=0.058; R569*, p<0.05; R678*, p<0.01; figure 4B). Cotransfection with WT PRKG2 revealed a twofold increase in COL10A1 expression compared with SOX9-transfected cells (p<0.01) (figure 4C). Both human PRKG2 variants, N164Kfs*2 and R569*, failed to restore COL10A1 expression to WT PRKG2 (N164Kfs*2, p<0.0001; R569*, p<0.01). However, the bovine mutant R678* did not reduce COL10A1 expression, unlike the two human mutants, as previously described (figure 4C).16
In this study, two homozygous loss-of-function variants in PRKG2 were identified in two individuals with an acromesomelic skeletal dysplasia. No other PRKG2 variants were found in the screening of 298 individuals with proportionate/disproportionate short stature with or without other skeletal abnormalities.
The two girls have severe disproportionate short stature with acromesomelic shortening of the limbs and mild to moderate platyspondyly. Metaphyseal dysplasia of the long bones was present in both probands, although in proband 2, this was not evident in early childhood but was most noted at the age of 7. The skeletal anomalies are similar to those observed in AMDM, although the platyspondyly and brachydactyly appears to be milder, as is the mesomelic shortening, and to a greater degree in proband 1 compared with proband 2. The identification of further cases will enable us to determine if indeed this is a milder phenotype or whether there is a broad phenotypic spectrum. These clinical and radiological similarities are logical since AMDM is caused by biallelic variants in the gene encoding NPR-B, the main receptor for CNP and is located just upstream from cGKII.
The importance of CNP/NPR-B signalling in the skeletal human growth has been widely demonstrated as variants in NPR-B and CNP have been associated with various skeletal dysplasias and growth disorders. Common phenotypic features such as short stature and brachydactyly are observed but the severity is variable, principally depending on the mode of inheritance but also due to other factors such as polymorphisms in modifying genes, the majority of which remain unknown, epigenetics, diet and so on. The most severe phenotype is observed in AMDM individuals with homozygous NRP2 variants, whereas the phenotype is milder in some heterozygous affected individuals with only isolated short stature, and even individuals may be asymptomatic.5 21
While no pathogenic variants in human PRKG2 have been reported until this study, its role in skeletal growth has been widely supported by animal models such as cGKII null mice, KMI rat with a Prkg2 deletion and American Angus cattle with the nonsense mutation (p.Arg678*), all of which developed growth retardation with shortened limbs (>30%) and trunk, soon after birth.1 14 16 19 The shortened limbs and radiological features are similar to those observed in the two probands.
As both PRKG2 variants, c.491dup; p.(Asn164Lysfs*2) in exon 3 and c.1705C>T: p.(Arg569*) in exon 14, are predicted to cause premature truncations of cGKII, we investigated if the transcripts were exposed to NMD. Using lymphocyte cells from the patients, both mutant transcripts were degraded or partially degraded by NMD. This is in contrast to that observed for the bovine mutant located in exon 16, c.2032C>T; p.Arg678* which was not found to be degraded by NMD.16 However, using in silico predictions, both the human and bovine nonsense variants may be exposed to NMD while the frameshift variant was not thus, the location of the mutants along the 19 exons of PRKG2 does not appear to explain these results. In addition, due to the lack of tissue availability, we cannot demonstrate that our findings in lymphocytes can be representative of what is occurring in the chondrocytes. Tissue-specific NMD has been shown in various disorders including in Schmid metaphyseal dysplasia (MIM 156500), where NMD was shown to be specific to cartilage and did not occur in the non-cartilage lymphoblast or bone cells.22
We were also able to observe the truncated mutant proteins by western analysis. Moreover, the mutant proteins showed a reduced expression in vitro compared with WT (figure 2A). Taken together, these results provide evidence that NMD reduces the amount of mRNA transcribed from the alleles carrying the p.Asn164Lysfs*2 and p.Arg569* variants, hence preventing or reducing the synthesis of abnormal cGKII protein.
The protein cGKII is a 762-amino acid enzyme that belongs to the serine/threonine protein kinase family. It consists of three distinctive domains: an amino-terminal (N), a regulatory (R) and a carboxy-terminal catalytic domain (C) (figure 3C). The N-terminus has been shown to be involved in dimerisation, autoinhibition of the catalytic activity and cellular localisation of the protein to the cell membrane.23 24 The regulatory domain has two cyclic nucleotide-binding sites in tandem (CNB-A and CNB-B) that exhibit different affinities for cGMP binding (CNB-B > CNB-A).25 Finally, the C-terminal domain or active domain contains ATP and substrate-binding sites necessary for phosphorylation of consensus sequences on target proteins.26 Both PRKG2 variants lack the catalytic domain and thus have no kinase activity. Moreover, the p.(Asn164Lysfs*2) mutant also lacks the regulatory domain, which would mean that it is also unable to bind cGMP, necessary for the activation of the enzyme. Therefore, the p.(Arg569*) and p.(Asn164Lysfs*2) variants are likely to be abnormal and catalytically inactive proteins.
To demonstrate this, we examined their ability to downregulate the MAPK pathway in vitro. CNP promotes chondrocyte proliferation by antagonising FGF-induced MAPK signalling in the growth plate.27 Activation of fibroblast growth factor receptor-3 (FGFR3)-MAPK pathway (FGFR3/Ras/Raf/MEK/ERK) by FGFs is the underlying mechanism of the chondrocyte growth arrest observed in various skeletal dysplasias and growth disorder such as achondroplasia (MIM 100800).27 28 Conversely, activation of cGKII, by CNP-mediated intracellular elevation of cGMP, has been reported to inhibit FGF-induced MEK and ERK1/2 phosphorylation in fibroblasts and tibia explants thus restoring and promoting chondrocyte proliferation in the growth plate.29 30 Moreover, chemical inhibition of FGFR3 and MEK1/2 rescued FGF2-induced growth arrest in vitro25 27 and overexpression of CNP in the cartilage growth plate prevented dwarfism in a mouse model of achondroplasia by attenuating ERK1/2 activation.30 Both PRKG2 human mutants, R569* and N164Kfs*2, as well as the bovine R678* mutant, failed to inhibit FGF2-induced ERK1/2 phosphorylation in the presence of the cGMP analogue, 8-pCPT-cGMP, previously shown to downregulate ERK1/2 activation.20 Moreover, we also investigated whether the PRKG2 mutants were able to phosphorylate Raf-1 at Serine 43, necessary for the cGKII inhibitory effect on FGF-induced MAP kinase activation. Neither of the two mutants were able to phosphorylate and thus inactivate Raf-1 at Ser-43 and consequently reduce ERK activation. Therefore, these data confirm that both variants are unable to abolish MAPK cascade activation in response to FGF2, thus leading to growth retardation.
Based on previous studies in the KMI rat with a Prkg2 deletion, cGKII was shown to act as a molecular switch by impeding the translocation of SOX9 to the nucleus and thus attenuating its ability to induce chondrocyte differentiation, impeding both chondrocyte proliferation and conversion of proliferating chondrocytes into hypertrophic chondrocytes.19Both R569* and N164Kfs*2 PRKG2 mutants showed increased expression of COL2A1, a marker of chondrocyte proliferation, and decreased expression of COL10A1, a marker of hypertrophy, compared with WT in HEK293T cells. These results are consistent with those observed in chondrocyte primary cultures from KMI rats tibia and in ATDC5 and HuH-7 cell lines transfected with cGKII mutant lacking kinase domain.19 However, the bovine R678* mutant increased COL2A1 expression but did not significantly decrease COL10A1 expression, duplicating the result observed by Koltes et al 16 who postulated that these differences may be due to tissue collection or sample source as they evaluated expression levels in bovine growth plate cartilage. However, in this current study, we analysed all three mutants using the in vitro cell culture method described by Chikuda et al.19 Despite discrepant results of COL10A1 expression in the investigations of PRKG2 mutants and no proof of what is occurring in the growth plate, it is tempting to speculate that aberration of type X collagen expression would interfere with terminal maturation of chondrocytes and thus the metaphyseal abnormalities observed in the probands.
Interestingly, although Prkg2-/- animal models showed similar growth restraint phenotypes to those observed in Npr2 and Nppc mice models,10 31 the histology of the growth plate is quite different. Conversely to Npr2-/- and Nppc-/- mice that exhibited shorter growth plates but regular organisation of chondrocyte zones, the elongated growth plate of Prkg2-/- mice and rats maintained an organised pattern but revealed an intermediate layer between the proliferative and hypertrophic zones, where chondrocytes had ceased proliferation but had failed to start hypertrophic differentiation. However, the Angus cattle dwarf growth plate showed a general loss of organisation but once again, mixing of proliferative and hypertrophic chondrocytes.16
GKII is not only implicated in growth but has also been shown to play a role in other pathways including gastrointestinal secretion of chloride and water via phosphorylation of CFTR (cystic fibrosis transmembrane conductance regulator protein)1 32 and might also regulate aldosterone secretion and blood pressure33 although the latter appears to be due to overexpression of cGKII rather than loss.34 In addition, these systems may be regulated by GKII due to NPR-A activation in response to CNP rather than via NPR-B. The clinical features observed in both probands seem to be at present restricted to the skeletal system.
In the current nosology of skeletal dysplasias, there are four acromesomelic dysplasias (Group 1635): the three autosomal recessively inherited dysplasias, AMDM caused by variants in NPR2 (MIM 6002875); Grebe dysplasia (MIM 200700, 609441) encompassing also Hunter-Thompson dysplasia (MIM 201250) and acromesomelic dysplasia with genital anomalies (Demirhan type (MIM 603248)) caused by variants in GDF5 and BMPR1B; fibular hypoplasia and complex brachdactyly (Du pan dysplasia (MIM 228900)) also caused by variants in GDF5 and BMPR1B and lastly acromesomelic dysplasia Osebold-Remondini type (MIM 112910) in which the molecular defect has not been identified and has only been reported in one multigenerational family.36 The clinical and radiological similarities and differences of the various forms in comparison with the new entity, acromesomelic dysplasia PRKG2 type, are summarised in table 1.
We investigated whether heterozygous carriers of PRKG2 variants are unaffected or have a milder phenotype, similar to that observed in some heterozygotes of NPR2, GDF5 and BMPR1B variants (MIM 616255, 615072, 112600, 113100, 610017, 615298, 616849), genes implicated in other acromesomelic dysplasias. The parents of proband 1 have normal stature, although the father’s height is at the lower end (−1.75 SDS), while the parents of proband 2 both have short stature (mother −2.50 SDS, father −2.59 SDS). All four do not have any skeletal or dysmorphic features. Thus, at present we cannot conclude if heterozygous PRKG2 variants have an influence on height. The identification of further cases will be necessary to determine this.
At present, no therapies are available for children with PRKG2 variants. Currently, there are various clinical trials being undertaken or completed in children with achondroplasia including: CNP analogues (Vosoritide (BMN111), BioMarin, California, USA37; TransCon CNP, Ascendis Pharma, Denmark,38 soluble FGFR3 (TA-46; Pfizer, California, USA)39 and FGFR3 selective tyrosine kinase inhibitors (infigratinib, compound BGJ398; QED Therapeutics, California, USA).40 The CNP analogues would not be able to repress the activation of the MAPK pathway by the PRKG2 mutants but soluble FGFR3 or FGFR selective tyrosine kinase inhibitors may be able to counteract this activation.
In conclusion, we describe a new skeletal dysplasia caused by biallelic variants in PRKG2. The inability to downregulate MAPK signalling as well as modulate SOX9 actions confirms the pathogenicity of the two identified variants. We suggest the naming of this new acromesomelic dysplasia as acromesomelic dysplasia, PRKG2 type (AMDP).
Data availability statement
Data are available on reasonable request. All data relevant to the study are included in the article or uploaded as supplementary information.
Patient consent for publication
Ethical approval was obtained from Hospital Universitario La Paz, Madrid, Spain, and CSIR—Institute of Genomics and Integrative Biology, New Delhi, India. All participants provided informed consent for the performed genetic studies. All the procedures were performed under the Declaration of Helsinki and relevant policies in Spain and India.
We wish to thank the patients and families for their help during this study.
MF and KEH are joint senior authors.
Correction notice The article has been corrected since it was published online first. The caption of figures 2, 3 and 4 were incorrectly ordered; they have been amended.
Contributors KEH and MF established and obtained financing of the project. SKu, LS-M, MA-B, MBR-M, HS, SKo clinically diagnosed and followed up the patients. GN and ACO examined and reported the radiological data. SW, MR-Z, MA-C, IA, SZ, DK, NK, US, MF and KEH analysed genetic data. FD-G, SW, IH, MR-Z, MA-C performed functional characterisation. KEH and MF analysed the data and supervised the project. FD-G and KEH wrote the first draft of the manuscript, tables and figures. All the authors revised the manuscript and approved the final version.
Funding This work was supported in part by the following grants: SAF2017-84646-R from MINECO (to KEH), Council of Scientific & Industrial Research (CSIR), India (to MF) and Indian Council of Medical Research (to MF). FG-D was supported by an FPU studentship from the Spanish Ministry of Education.
Competing interests None declared.
Provenance and peer review Not commissioned; externally peer reviewed.
Supplemental material This content has been supplied by the author(s). It has not been vetted by BMJ Publishing Group Limited (BMJ) and may not have been peer-reviewed. Any opinions or recommendations discussed are solely those of the author(s) and are not endorsed by BMJ. BMJ disclaims all liability and responsibility arising from any reliance placed on the content. Where the content includes any translated material, BMJ does not warrant the accuracy and reliability of the translations (including but not limited to local regulations, clinical guidelines, terminology, drug names and drug dosages), and is not responsible for any error and/or omissions arising from translation and adaptation or otherwise.