Mutations in GRK2 cause Jeune syndrome by impairing Hedgehog and canonical Wnt signaling
2020; Springer Nature; Volume: 12; Issue: 11 Linguagem: Inglês
10.15252/emmm.201911739
ISSN1757-4684
AutoresMichaela Kunova Bosakova, Sara P. Abraham, Alexandru Niţă, Eva Hrubá, Marcela Buchtová, S. Paige Taylor, Iván Durán, Jorge Martı́n, Katerina Svozilova, Tomáš Bárta, Miroslav Vařecha, Lukáš Bálek, Jiří Kohoutek, Tomasz Radaszkiewicz, Ganesh V. Pusapati, Vı́tězslav Bryja, Eric T. Rush, Isabelle Thiffault, Deborah A. Nickerson, Michael J. Bamshad, Rajat Rohatgi, Daniel H. Cohn, Deborah Krakow, Pavel Krejčı́,
Tópico(s)Ocular Disorders and Treatments
ResumoArticle14 October 2020Open Access Source Data Mutations in GRK2 cause Jeune syndrome by impairing Hedgehog and canonical Wnt signaling Michaela Bosakova Michaela Bosakova orcid.org/0000-0002-7627-0344 Department of Biology, Faculty of Medicine, Masaryk University, Brno, Czech Republic International Clinical Research Center, St. Anne's University Hospital, Brno, Czech Republic Institute of Animal Physiology and Genetics of the CAS, Brno, Czech Republic Search for more papers by this author Sara P Abraham Sara P Abraham Department of Biology, Faculty of Medicine, Masaryk University, Brno, Czech Republic Search for more papers by this author Alexandru Nita Alexandru Nita Department of Biology, Faculty of Medicine, Masaryk University, Brno, Czech Republic Search for more papers by this author Eva Hruba Eva Hruba Institute of Animal Physiology and Genetics of the CAS, Brno, Czech Republic Search for more papers by this author Marcela Buchtova Marcela Buchtova Institute of Animal Physiology and Genetics of the CAS, Brno, Czech Republic Search for more papers by this author S Paige Taylor S Paige Taylor Department of Orthopaedic Surgery, David Geffen School of Medicine at UCLA, Los Angeles, CA, USA Search for more papers by this author Ivan Duran Ivan Duran Department of Orthopaedic Surgery, David Geffen School of Medicine at UCLA, Los Angeles, CA, USA Search for more papers by this author Jorge Martin Jorge Martin Department of Orthopaedic Surgery, David Geffen School of Medicine at UCLA, Los Angeles, CA, USA Search for more papers by this author Katerina Svozilova Katerina Svozilova Department of Biology, Faculty of Medicine, Masaryk University, Brno, Czech Republic Institute of Animal Physiology and Genetics of the CAS, Brno, Czech Republic Search for more papers by this author Tomas Barta Tomas Barta Department of Histology and Embryology, Faculty of Medicine, Masaryk University, Brno, Czech Republic Search for more papers by this author Miroslav Varecha Miroslav Varecha Department of Biology, Faculty of Medicine, Masaryk University, Brno, Czech Republic Search for more papers by this author Lukas Balek Lukas Balek Department of Biology, Faculty of Medicine, Masaryk University, Brno, Czech Republic Search for more papers by this author Jiri Kohoutek Jiri Kohoutek Veterinary Research Institute, Brno, Czech Republic Search for more papers by this author Tomasz Radaszkiewicz Tomasz Radaszkiewicz orcid.org/0000-0003-4850-9933 Institute of Experimental Biology, Faculty of Science, Masaryk University, Brno, Czech Republic Search for more papers by this author Ganesh V Pusapati Ganesh V Pusapati Department of Biochemistry, Stanford University, Palo Alto, CA, USA Department of Medicine, Stanford University, Palo Alto, CA, USA Search for more papers by this author Vitezslav Bryja Vitezslav Bryja orcid.org/0000-0002-9136-5085 Institute of Experimental Biology, Faculty of Science, Masaryk University, Brno, Czech Republic Search for more papers by this author Eric T Rush Eric T Rush Children's Mercy Kansas City, Center for Pediatric Genomic Medicine, Kansas City, MO, USA Department of Pediatrics, University of Missouri, Kansas City, MO, USA Search for more papers by this author Isabelle Thiffault Isabelle Thiffault Children's Mercy Kansas City, Center for Pediatric Genomic Medicine, Kansas City, MO, USA Department of Pediatrics, University of Missouri, Kansas City, MO, USA Search for more papers by this author Deborah A Nickerson Deborah A Nickerson Department of Genome Sciences, University of Washington, Seattle, WA, USA Search for more papers by this author Michael J Bamshad Michael J Bamshad Department of Genome Sciences, University of Washington, Seattle, WA, USA Department of Pediatrics, University of Washington, Seattle, WA, USA Division of Genetic Medicine, Seattle Children's Hospital, Seattle, WA, USA Search for more papers by this author University of Washington Center for Mendelian Genomics University of Washington Center for Mendelian Genomics Search for more papers by this author Rajat Rohatgi Rajat Rohatgi Department of Biochemistry, Stanford University, Palo Alto, CA, USA Department of Medicine, Stanford University, Palo Alto, CA, USA Search for more papers by this author Daniel H Cohn Daniel H Cohn Department of Orthopaedic Surgery, David Geffen School of Medicine at UCLA, Los Angeles, CA, USA Department of Molecular Cell and Developmental Biology, University of California at Los Angeles, Los Angeles, CA, USA Search for more papers by this author Deborah Krakow Corresponding Author Deborah Krakow [email protected] orcid.org/0000-0001-9906-4968 Department of Orthopaedic Surgery, David Geffen School of Medicine at UCLA, Los Angeles, CA, USA Department of Human Genetics, David Geffen School of Medicine at UCLA, Los Angeles, CA, USA Department of Obstetrics and Gynecology, David Geffen School of Medicine at UCLA, Los Angeles, CA, USA Search for more papers by this author Pavel Krejci Corresponding Author Pavel Krejci [email protected] orcid.org/0000-0003-0618-9134 Department of Biology, Faculty of Medicine, Masaryk University, Brno, Czech Republic International Clinical Research Center, St. Anne's University Hospital, Brno, Czech Republic Institute of Animal Physiology and Genetics of the CAS, Brno, Czech Republic Search for more papers by this author Michaela Bosakova Michaela Bosakova orcid.org/0000-0002-7627-0344 Department of Biology, Faculty of Medicine, Masaryk University, Brno, Czech Republic International Clinical Research Center, St. Anne's University Hospital, Brno, Czech Republic Institute of Animal Physiology and Genetics of the CAS, Brno, Czech Republic Search for more papers by this author Sara P Abraham Sara P Abraham Department of Biology, Faculty of Medicine, Masaryk University, Brno, Czech Republic Search for more papers by this author Alexandru Nita Alexandru Nita Department of Biology, Faculty of Medicine, Masaryk University, Brno, Czech Republic Search for more papers by this author Eva Hruba Eva Hruba Institute of Animal Physiology and Genetics of the CAS, Brno, Czech Republic Search for more papers by this author Marcela Buchtova Marcela Buchtova Institute of Animal Physiology and Genetics of the CAS, Brno, Czech Republic Search for more papers by this author S Paige Taylor S Paige Taylor Department of Orthopaedic Surgery, David Geffen School of Medicine at UCLA, Los Angeles, CA, USA Search for more papers by this author Ivan Duran Ivan Duran Department of Orthopaedic Surgery, David Geffen School of Medicine at UCLA, Los Angeles, CA, USA Search for more papers by this author Jorge Martin Jorge Martin Department of Orthopaedic Surgery, David Geffen School of Medicine at UCLA, Los Angeles, CA, USA Search for more papers by this author Katerina Svozilova Katerina Svozilova Department of Biology, Faculty of Medicine, Masaryk University, Brno, Czech Republic Institute of Animal Physiology and Genetics of the CAS, Brno, Czech Republic Search for more papers by this author Tomas Barta Tomas Barta Department of Histology and Embryology, Faculty of Medicine, Masaryk University, Brno, Czech Republic Search for more papers by this author Miroslav Varecha Miroslav Varecha Department of Biology, Faculty of Medicine, Masaryk University, Brno, Czech Republic Search for more papers by this author Lukas Balek Lukas Balek Department of Biology, Faculty of Medicine, Masaryk University, Brno, Czech Republic Search for more papers by this author Jiri Kohoutek Jiri Kohoutek Veterinary Research Institute, Brno, Czech Republic Search for more papers by this author Tomasz Radaszkiewicz Tomasz Radaszkiewicz orcid.org/0000-0003-4850-9933 Institute of Experimental Biology, Faculty of Science, Masaryk University, Brno, Czech Republic Search for more papers by this author Ganesh V Pusapati Ganesh V Pusapati Department of Biochemistry, Stanford University, Palo Alto, CA, USA Department of Medicine, Stanford University, Palo Alto, CA, USA Search for more papers by this author Vitezslav Bryja Vitezslav Bryja orcid.org/0000-0002-9136-5085 Institute of Experimental Biology, Faculty of Science, Masaryk University, Brno, Czech Republic Search for more papers by this author Eric T Rush Eric T Rush Children's Mercy Kansas City, Center for Pediatric Genomic Medicine, Kansas City, MO, USA Department of Pediatrics, University of Missouri, Kansas City, MO, USA Search for more papers by this author Isabelle Thiffault Isabelle Thiffault Children's Mercy Kansas City, Center for Pediatric Genomic Medicine, Kansas City, MO, USA Department of Pediatrics, University of Missouri, Kansas City, MO, USA Search for more papers by this author Deborah A Nickerson Deborah A Nickerson Department of Genome Sciences, University of Washington, Seattle, WA, USA Search for more papers by this author Michael J Bamshad Michael J Bamshad Department of Genome Sciences, University of Washington, Seattle, WA, USA Department of Pediatrics, University of Washington, Seattle, WA, USA Division of Genetic Medicine, Seattle Children's Hospital, Seattle, WA, USA Search for more papers by this author University of Washington Center for Mendelian Genomics University of Washington Center for Mendelian Genomics Search for more papers by this author Rajat Rohatgi Rajat Rohatgi Department of Biochemistry, Stanford University, Palo Alto, CA, USA Department of Medicine, Stanford University, Palo Alto, CA, USA Search for more papers by this author Daniel H Cohn Daniel H Cohn Department of Orthopaedic Surgery, David Geffen School of Medicine at UCLA, Los Angeles, CA, USA Department of Molecular Cell and Developmental Biology, University of California at Los Angeles, Los Angeles, CA, USA Search for more papers by this author Deborah Krakow Corresponding Author Deborah Krakow [email protected] orcid.org/0000-0001-9906-4968 Department of Orthopaedic Surgery, David Geffen School of Medicine at UCLA, Los Angeles, CA, USA Department of Human Genetics, David Geffen School of Medicine at UCLA, Los Angeles, CA, USA Department of Obstetrics and Gynecology, David Geffen School of Medicine at UCLA, Los Angeles, CA, USA Search for more papers by this author Pavel Krejci Corresponding Author Pavel Krejci [email protected] orcid.org/0000-0003-0618-9134 Department of Biology, Faculty of Medicine, Masaryk University, Brno, Czech Republic International Clinical Research Center, St. Anne's University Hospital, Brno, Czech Republic Institute of Animal Physiology and Genetics of the CAS, Brno, Czech Republic Search for more papers by this author Author Information Michaela Bosakova1,2,3, Sara P Abraham1, Alexandru Nita1, Eva Hruba3, Marcela Buchtova3, S Paige Taylor4, Ivan Duran4, Jorge Martin4, Katerina Svozilova1,3, Tomas Barta5, Miroslav Varecha1, Lukas Balek1, Jiri Kohoutek6, Tomasz Radaszkiewicz7, Ganesh V Pusapati8,9, Vitezslav Bryja7, Eric T Rush10,11, Isabelle Thiffault10,11, Deborah A Nickerson12, Michael J Bamshad12,13,14, , Rajat Rohatgi8,9, Daniel H Cohn4,15, Deborah Krakow *,4,16,17 and Pavel Krejci *,1,2,3 1Department of Biology, Faculty of Medicine, Masaryk University, Brno, Czech Republic 2International Clinical Research Center, St. Anne's University Hospital, Brno, Czech Republic 3Institute of Animal Physiology and Genetics of the CAS, Brno, Czech Republic 4Department of Orthopaedic Surgery, David Geffen School of Medicine at UCLA, Los Angeles, CA, USA 5Department of Histology and Embryology, Faculty of Medicine, Masaryk University, Brno, Czech Republic 6Veterinary Research Institute, Brno, Czech Republic 7Institute of Experimental Biology, Faculty of Science, Masaryk University, Brno, Czech Republic 8Department of Biochemistry, Stanford University, Palo Alto, CA, USA 9Department of Medicine, Stanford University, Palo Alto, CA, USA 10Children's Mercy Kansas City, Center for Pediatric Genomic Medicine, Kansas City, MO, USA 11Department of Pediatrics, University of Missouri, Kansas City, MO, USA 12Department of Genome Sciences, University of Washington, Seattle, WA, USA 13Department of Pediatrics, University of Washington, Seattle, WA, USA 14Division of Genetic Medicine, Seattle Children's Hospital, Seattle, WA, USA 15Department of Molecular Cell and Developmental Biology, University of California at Los Angeles, Los Angeles, CA, USA 16Department of Human Genetics, David Geffen School of Medicine at UCLA, Los Angeles, CA, USA 17Department of Obstetrics and Gynecology, David Geffen School of Medicine at UCLA, Los Angeles, CA, USA *Corresponding author. Tel: +13109831252; E-mail: [email protected] *Corresponding author. Tel: +420549495395; E-mail: [email protected] EMBO Mol Med (2020)12:e11739https://doi.org/10.15252/emmm.201911739 PDFDownload PDF of article text and main figures. Peer ReviewDownload a summary of the editorial decision process including editorial decision letters, reviewer comments and author responses to feedback. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Abstract Mutations in genes affecting primary cilia cause ciliopathies, a diverse group of disorders often affecting skeletal development. This includes Jeune syndrome or asphyxiating thoracic dystrophy (ATD), an autosomal recessive skeletal disorder. Unraveling the responsible molecular pathology helps illuminate mechanisms responsible for functional primary cilia. We identified two families with ATD caused by loss-of-function mutations in the gene encoding adrenergic receptor kinase 1 (ADRBK1 or GRK2). GRK2 cells from an affected individual homozygous for the p.R158* mutation resulted in loss of GRK2, and disrupted chondrocyte growth and differentiation in the cartilage growth plate. GRK2 null cells displayed normal cilia morphology, yet loss of GRK2 compromised cilia-based signaling of Hedgehog (Hh) pathway. Canonical Wnt signaling was also impaired, manifested as a failure to respond to Wnt ligand due to impaired phosphorylation of the Wnt co-receptor LRP6. We have identified GRK2 as an essential regulator of skeletogenesis and demonstrate how both Hh and Wnt signaling mechanistically contribute to skeletal ciliopathies. Synopsis This study identifies GRK2 as a regulator of human skeletogenesis. Loss of GRK2 deregulates the function of two major morphogens in the bone - Hedgehog and canonical Wnt signaling, and manifests in autosomal recessive skeletal ciliopathy syndrome, asphyxiating thoracic dystrophy or Jeune syndrome. GRK2 loss leads to bone defects involving the proliferation and hypertrophic differentiation of chondrocytes in the growth plate cartilage, and sulfation of the cartilage extracellular matrix. GRK2 loss causes under-phosphorylation of Smoothened and its exclusion from the cilia, and inhibits Hedgehog pathway. GRK2 loss inhibits canonical Wnt signaling through reduced LRP6 phosphorylation and Frizzled-βArrestin2 interaction. The paper explained Problem Herein, we identified patients with Jeune syndrome or asphyxiating thoracic dystrophy (ATD), a genetically heterogeneous disorder that is both multisystemic disorder and often associated with lethality. In two independent families, we identified homozygosity for a loss-of-function variant and biallelic variants predicting loss of functional protein. Because of this novel finding that links GRK2 to the skeletal development, we focused on identification of the molecular mechanisms underlying this disorder since loss of Grk2 in mice is an early embryonic lethal. Results We found that the loss of GRK2 leads to specific changes in the bone that indicated impaired function of two major regulators of bone development, both Hedgehog and Wnt signaling. We indeed found that loss of GRK2 in patient's cells and model cell lines led to deregulation of these two pathways, suggesting in part the molecular mechanisms underlying this phenotype. Impact Development skeletal disorders, including ATD, are often severe, lethal syndromes with no cure or treatment options. Identification of the molecular pathogenesis of the disease therefore expands our understanding of the genetic heterogeneity associated with this disorder, provides families with reproductive options, and uncovers the role of GRK2 in skeletogenesis. Introduction A single primary cilium protrudes from nearly every post-mitotic vertebrate cell, and cilia sense and transduce a vast array of extracellular cues. Cilia utilize intraflagellar transport (IFT), a bidirectional system that builds and maintains the cilium while also facilitating protein entry, exit and trafficking through the organelle. IFT is governed by a large multimeric protein complex with two main subcomplexes, IFT-A and IFT-B. The anterograde IFT is driven by the kinesin motor KIF3 and mediates transport from the base to the tip of cilia, while retrograde IFT is driven by the dynein-2 motor and transports cargo from the tip to the base of the cilium (Kozminski et al, 1993). Vertebrate primary cilia act as signaling centers for the Hedgehog (Hh) family of morphogens and also orchestrate a variety of other cell signaling cascades (Gerhardt et al, 2016). Mutations in genes affecting primary cilia structure or function cause ciliopathies, a group of pleiotropic disorders. Among these disorders is a subset with profound abnormalities in the skeleton, termed skeletal ciliopathies. Asphyxiating thoracic dystrophy (ATD) or Jeune syndrome is considered an autosomal recessively inherited skeletal ciliopathy characterized by a long narrow chest, shortened long bones, and occasional polydactyly. Other affected organs can include the brain, retina, lungs, liver, pancreas, and kidneys. Characteristic radiographic findings include handlebar shaped clavicles, short horizontal ribs, shortened appendicular bones with irregular metaphyseal ends, a small pelvis with a trident shaped acetabular roof, and brachydactyly with cone-shaped epiphyses. Other skeletal ciliopathies with overlapping features include short rib polydactyly syndrome (SRPS), Ellis-van Creveld dysplasia (EVC), and cranioectodermal dysplasia (CED). ATD is clinically and radiographically most similar to the perinatal-lethal SRPS. However, while ATD is not uniformly lethal, long-term survivors often have multi-organ system complications. Mutations in genes affecting ciliogenesis or the IFT process produce a broad phenotypic spectrum of skeletal ciliopathies, and there is significant allelic and locus heterogeneity. Many of the genes mutated in this spectrum of disorders encode IFT-A proteins and their motors: the cytoplasmic dynein-2 motor heavy chain, DYNC2H1 (MIM 603297), WDR34 (MIM 615633), WDR60 (MIM 615462), WDR19 (also known as IFT144) (MIM 614376), IFT140 (MIM 266920), WDR35 (also known as IFT121) (MIM 614091), TTC21B (also known as IFT139) (MIM 612014), IFT43 (MIM 614068), TCTEX1D2 (MIM 617353), IFT122 (NIM 606045), IFT140 (MIM 614620), and DYNC2LI1 (MIM 617083) (Gilissen et al, 2010; Walczak-Sztulpa et al, 2010; Arts et al, 2011; Bredrup et al, 2011; Davis et al, 2011; Perrault et al, 2012; Huber et al, 2013; McInerney-Leo et al, 2013; Schmidts et al, 2013, 2015; Taylor et al, 2015). Mutations also have been reported in the genes encoding IFT-B members: IFT80 (MIM 611263), IFT52 (MIM 617094), IFT81 (MIM 605489), and IFT172 (MIM 615630) (Beales et al, 2007; Halbritter et al, 2013; Duran et al, 2016; Zhang et al, 2016). Additional locus heterogeneity in these disorders results from mutations in the genes that encode the centrosomal kinase NEK1 (MIM 604588), MAP kinase family member ICK (MIM 612325), CEP290 (MIM 6101142), KIAA05866 (MIM 610178), and C21ORF2 (Thiel et al, 2011; Alby et al, 2015; Paige Taylor et al, 2016; McInerney-Leo et al, 2017), as well as two proteins involved in planar cell polarity, INTU (MIM 610621) and FUZ (MIM 610622) (Toriyama et al, 2016; Zhang et al, 2018); three centrosomal proteins, EVC (NIM 604831), EVC2 (NIM 607261) and CEP120 (NIM 613446) (Ruiz-Perez et al, 2000; Galdzicka et al, 2002; Shaheen et al, 2015); and the nuclear membrane protein LBR (NIM 600024) (Zhang et al, 2018). In an effort to identify additional ATD genes and increase our understanding of ciliary function in skeletal development, exome sequence analysis was carried out in a cohort of patients within the skeletal ciliopathy spectrum, and two families were identified with pathogenic variants in the adrenergic receptor kinase beta 1 gene (ADRBK1 or GRK2). GRK2 is one of seven G protein-coupled receptor kinases (GRKs), which bind to and rapidly desensitize activated G protein-coupled receptors (GPCRs). GPCRs localize to the cell membrane where they sense extracellular cues and, through coupling with G proteins, relay signals to the cell. The wide repertoire of GPCR ligands includes photons, peptides, hormones, lipids, and sugars. With over 800 known members, the GPCR family represents the largest and most diverse class of eukaryotic membrane receptors (Fredriksson et al, 2003). In its canonical signaling cascade, GRK2 is activated by protein kinase A and phosphorylates the beta-adrenergic receptor, desensitizing it, and thus preventing the cells from overstimulation by catecholamines (Hausdorff et al, 1990). In addition to GPCR signaling, the ubiquitously expressed GRK2 participates in many other processes, including regulation of phospholipase C, PI3 kinase, and RAF kinase activity, modulation of cytoskeletal proteins and activation of Smad and NFκB signaling through direct phosphorylation (Evron et al, 2012). GRK2 also functions in the Hh pathway. Hh signaling is initiated by Hh ligand binding to the Patched 1 (PTCH1) receptor, followed by phosphorylation-dependent accumulation of Smoothened (SMO) GPCR at the cell membrane (Drosophila) or in the primary cilia (vertebrates). This results in activation of the Hh gene expression through degradation of the GLI2/GLI3 transcriptional repressors and production of GLI activators (Forbes et al, 1993; Van den Heuvel & Ingham, 1996; Dai et al, 1999; Sasaki et al, 1999; Aza-Blanc et al, 2000; Wang & Holmgren, 2000; Haycraft et al, 2005; Rohatgi et al, 2007; Tukachinsky et al, 2010; Su et al, 2011). The ciliary cAMP levels appear to have a central role in this process, regulating activity of protein kinase A (PKA) that is critical for formation of GLI repressors (Humke et al, 2010; Tuson et al, 2011; Mukhopadhyay et al, 2013; Niewiadomski et al, 2014). In this context, GRK2 acts as a positive regulator of Hh signaling, necessary for maximal Hh response in both Drosophila and vertebrates (Jia et al, 2004; Maier et al, 2014; Li et al, 2016); yet, the molecular mechanisms are not fully understood. GRK2 was shown to phosphorylate SMO C-tail, leading to ciliary accumulation of SMO and Hh pathway activity (Chen et al, 2011); the former has been disputed and appears cell context dependent (Zhao et al, 2016; Pusapati et al, 2018). GRK2-mediated SMO phosphorylation was shown to induce β-arrestin (ARRB) recruitment, leading to KIF3A-dependent cilia accumulation of SMO (Chen et al, 2004; Kovacs et al, 2008); however, later studies showed normal cilia accumulation of SMO in ARRB knock-out cells (Pal et al, 2016; Desai et al, 2020). Ligand-dependent removal of the Hh pathway inhibitor GPR161 was shown to depend on GRK2 activity (Pal et al, 2016; Pusapati et al, 2018). More recently, the GRK2 activity was shown necessary for SMO to sequester the catalytic subunit of PKA which in turn can no longer phosphorylate GLI3 in order to produce the GLI3 repressor (Niewiadomski et al, 2014; preprint: Happ et al, 2020). Despite the limited understanding of the complex role of GRK2 in Hh signaling, its depletion by RNA interference in cell cultures clearly inhibits Hh activity (Meloni et al, 2006; Chen et al, 2010, 2011; Maier et al, 2014), and the Grk2−/− NIH3T3 do not respond to Hh stimulation as they fail to degrade GLI3 repressor and to activate Hh gene expression (Zhao et al, 2016; Pusapati et al, 2018). This effect is recapitulated by morpholino-mediated knock-down of grk2 and in the maternal-zygotic grk2 mutant zebrafish embryos (Philipp et al, 2008; Evron et al, 2011; Zhao et al, 2016). Loss of grk2 in zebrafish results in a curved body axis, U-shaped body somites and severe cyclopia (Zhao et al, 2016), phenocopying the smo mutant (Chen et al, 2001). In contrast, the Grk2 mouse knock-out shows milder phenotypes, at least in the neural tube patterning, yet is lethal around midgestation (Jaber et al, 1996; Philipp et al, 2008). This lethality was associated with developmental heart defects (Jaber et al, 1996; Matkovich et al, 2006), yet the timing did not allow for studying the effect of Grk2 loss on later developing tissues and organs such as skeleton. Herein, we demonstrate that pathogenic variants in GRK2 produce ATD and modulate both Hh and Wnt signaling, demonstrating that GRK2 is an essential regulator of skeletogenesis. Results Loss of GRK2 results in ATD The first proband (R05-365A) was born at 38 weeks to second-cousin parents. Prenatal ultrasound showed shortened limbs with a lag of approximately 8–9 weeks from the estimated due date. The pregnancy was complicated by ascites and hydrops fetalis that arose in the third trimester. The proband was delivered at term and had a very small chest with underlying pulmonary insufficiency. Additionally, she had low muscle tone, an atrial septal defect, hypoplastic nails, but no polydactyly. Radiographic findings included long narrow clavicles, short horizontal bent ribs with lack of normal distal flare, short humeri, mesomelia with bending of the radii, short femora and tibiae with broad metaphyses, diminished mineralization, and no endochondral ossification delay (Fig 1A and C). She expired 5 days after birth. The findings compared to characteristic ATD are delineated in Table 1. Figure 1. Asphyxiating thoracic dystrophy (ATD) probands R05-365A and Cmh001543-01 A. AP radiograph demonstrates characteristic findings of ATD in the R05-365A proband. Note the shortened humeri (closed arrowhead) and elongated clavicles (arrow). B. Radiographs of the Cmh001543-01 proband showing similar findings. C. Family R05-365A pedigree; * indicates common ancestors. CHD, congenital heart disease, SAB, spontaneous abortion. Abn, abnormalities. Download figure Download PowerPoint Table 1. Clinical and radiographic phenotype of ATD and the R05-365A and Cmh001543-01 and -02 cases Clinical and Radiographic Phenotype ATD/Jeune syndrome R05-365A Cmh001543-01 and -02 Autosomal recessive + + + Retinal insufficiency (age dependent) + − − Pulmonary insufficiency/hypoplasia + + + Polycystic liver disease/hepatic fibrosis (age dependent) + − − Cystic kidneys/chronic renal failure (age dependent) + − − Congenital heart defect + + − Ascites + + − Lethality due to pulmonary hypoplasia + + + Long narrow thorax + + + Short horizontal ribs + + + Handlebar clavicles + − + Small pelvis + + + Hypoplastic iliac wings (infancy) + + + Short long bones + + + Irregular metaphyses + + + Short fibulae (relative) + + Unknown Short ulnae (relative) + + (bowed) Unknown Brachydactyly + Unknown Unknown Cone-shaped epiphyses (childhood) + Unknown Unknown Occasional polydactyly + − − R05-365A, International Skeletal Dysplasia Registry Number, +, present, −, absent. In the second family, the elder of the two affected female siblings (proband Cmh001543-01) was delivered at 39 weeks' gestational age by cesarean section. The pregnancy was complicated by exposure to cannabis and the antiseizure medication, levetiracetam. Prenatal ultrasound at 20 weeks showed shortened long bones and a small chest and evaluation of the chest to abdominal circumference was predictive of lethality. At delivery, the proband was cyanotic with Apgar scores were 31, 75, and 810 and the head circumference was 32.5 cm, birth length 46 cm, and weight 2.82 kg. She was intubated at 12 min of life and initially required high levels of respiratory support and was extubated on day 14 of life, but continued to have chronic respiratory issues. Radiographic findings showed similar findings to the first proband (R05-365A) and included handlebar clavicles, short horizontal bent ribs with lack of normal distal flare, short humeri, broad irregular metaphyses, diminished mineralization, axial clefts, and odontoid hypoplasia (Fig 1B). She died at 5.5 months of age related to respiratory sequelae due to chronic lung disease. The second affected sibling had similar radiographic findings, but did not require prolonged respiratory support. She had questionable seizure events, but otherwise exhibited a much less severe clinical course and was alive at 12 months of age. Using exome sequence analyses of the R05-365A proband, homozygosity for a nonsense variant in exon 6 of GRK2, c. 469 C>T predicting the amino acid change p.R158*, was identified. The pathogenic variant localizes to the G protein signaling (RGS) domain of GRK2 (Fig 2A and C). The pathogenic variant occurred within a 13 Mb block of homozygosity on chromosome 11 and has not been seen in population databases. Detection of GRK2 expression, by RT–PCR of cDNA and Western blot analysis of protein, respectively, demonstrated loss of both GRK2 transcript and protein in cultured patient fibroblasts (Fig 2D and E). The data thus demonstrate that the p.R158* pathogenic variant results in a GRK2 null (GRK2−/−) genotype. Figure 2. Homozygosity and compound heterozygosity for null mutations in GRK2 in ATD A. Sanger sequence trace showing homozygosity for the c.469C>T mutation, predicting a stop codon in exon 6 of GRK2 (arrow). B. Exome analysis showing the biallelic changes c. 1348_1349del and c.555 +1G
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