Novel Robinow syndrome causing mutations in the proximal region of the frizzled-like domain of ROR2 are retained in the endoplasmic reticulum
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- Ali, B.R., Jeffery, S., Patel, N. et al. Hum Genet (2007) 122: 389. doi:10.1007/s00439-007-0409-0
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ROR2 is a member of the cell surface receptor tyrosine kinase (RTKs) family of proteins and is involved in the developmental morphogenesis of the skeletal, cardiovascular and genital systems. Mutations in ROR2 have been shown to cause two distinct human disorders, autosomal recessive Robinow syndrome and dominantly inherited Brachydactyly type B. The recessive form of Robinow syndrome is a disorder caused by loss-of-function mutations whereas Brachydactyly type B is a dominant disease and is presumably caused by gain-of-function mutations in the same gene. We have previously established that all the missense mutations causing Robinow syndrome in ROR2 are retained in the endoplasmic reticulum and therefore concluded that their loss of function is due to a defect in their intracellular trafficking. These mutations were in the distal portion of the frizzled-like cysteine rich domain and kringle domain. Here we report the identification of two novel mutations in the frizzled-like cysteine-rich domain of ROR2 causing Robinow syndrome. We establish the retention of the mutated proteins in the endoplasmic reticulum of HeLa cells and therefore failure to reach the plasma membrane. The clustering of Robinow-causing mutations in the extracellular frizzled-like cysteine-rich domain of ROR2 suggests a stringent requirement for the correct folding of this domain prior to export of ROR2 from the endoplasmic reticulum to the plasma membrane.
Robinow syndrome is a skeletal dysplasia that was initially described by Robinow et al. (1969) as a new form of dwarfism. This syndrome is characterised by short stature, mesomelic limb shortening, brachydactyly, spinal segmental abnormalities, genital hypoplasia and dysmorphic facial appearance including hypertelorism, prominent forehead and broad nasal base (Soliman et al. 1998). Robinow syndrome can be inherited as an autosomal dominant (DRS; MIM 180700) or autosomal recessive (RRS; MIM 268310) disorder with the latter exhibiting a more severe phenotype (Wadia 1978; Teebi 1990; Robinow 1993; Patton and Afzal 2002; Afzal and Jeffery 2003). The molecular basis of DRS is still largely unknown whereas loss-of-function mutations in the orphan receptor tyrosine kinase ROR2 have been shown to cause RRS (Afzal et al. 2000; van Bokhoven et al. 2000). RRS is genetically allelic to autosomal dominantly inherited Brachydactyly type B (BDB1; MIM 113000), a distinctive skeletal dysplasia with hypoplasia of distal phalanges. Brachydactyly type B is caused by gain-of-function mutations in ROR2 presumably resulting from a constitutively active ROR2-mediated signaling pathway (Oldridge et al. 2000; Schwabe et al. 2000).
RRS causing mutations in ROR2 can be missense, frameshift or nonsense and are located within the various domains of the protein including the frizzled-like, kringle and tyrosine kinase domains (Afzal et al. 2000; van Bokhoven et al. 2000; Schwabe et al. 2000). From the recessive mode of inheritance of Robinow syndrome and the striking similarities in the phenotype of the complete knockout mouse and human patients, it was predicted that mutations causing RRS would leads to loss-of-function of ROR2. We have recently shown by cellular studies that missense mutations in the distal region of the frizzeled-like domain produce this loss of function by retention of the mutated mRor2 proteins in the endoplasmic reticulum (ER) of the cell (Chen et al. 2005).
The retention of malfolded proteins in the ER is part of a stringent quality control system operating in this organelle and have been shown to be responsible for the mechanisms of dozens of human genetic disorders including the loss of function of CFTR and α1-antitrypsin in cystic fibrosis and emphysema patients, respectively (Aridor and Hannan 2000, 2002; Welch 2004; Chen et al. 2005). Almost a third of all cellular proteins are targeted to the ER where they enter into their folding pathways and assemble into complexes assisted by the large quantities of molecular chaperones present in the ER lumen. Misfolded proteins and unassembled subunits of protein complexes are rejected by the ER quality control system, and therefore exported to the cytosol where they get ubiquitinated and degraded by the ubiquitin/proteasome systems. This process of misfolded protein recognition, their retranslocation to the cytosol and degradation has been called ER-associated protein degradation or ERAD (McCracken and Brodsky 2003). In the present study we report that novel mutations causing Robinow syndrome in the proximal portion of the frizzled-like cysteine rich domain of ROR2 also cause ERAD. This strengthens the data supporting a trafficking defect as the mechanism for the loss of function of this protein in RRS patients, and underscores the essential role of all of this domain for proper folding of the protein before its passage through the secretory pathway.
Materials and methods
Clinical features of the new RRS patients
The first affected case was Egyptian in origin and the result of a consanguineous marriage, with two other normal siblings and no other affected family members. The index case was a boy with short stature (−3.0 SD) and abnormal features. His dysmorphic features included; fetal facies with macrocephaly and bulging forehead, hypertelorism, wide palpebral fissures, short nose with upturned nasal tip and wide mouth. He had crowded teeth with gingival hyperplasia. Skeletal deformities were mesomelic shortening of all limbs with short arm span and brachydactyly. He also had severe micropenis. Moreover, he had a ventricular septal defect of the heart identified by echocardiography.
The second case was a female from the UK and was examined upon referral to a clinical geneticist due to abnormal features and mild feeding difficulties at the age of 2 months. She was the product of an uneventful pregnancy and was born at 39 weeks and weighed 3.25 kg. Her head circumference was >97 centile, and her height was between the 10th and 25th centile. Her features were compatible with typical Robinow syndrome. Dysmorphic features included; midface hypoplasia, hypertelorism, a depressed nasal bridge and nasal tip, and gingival hyperplasia. On skeletal examination she had pectus carinatum, broad thumbs and fingers. At 17 months of age she had a delay in walking. At the age of 3 years, she was again reviewed in the clinic. She had crowded teeth. Skeletal X-rays showed increased bone density. It also confirmed shortening of first metacarpal, brachydactyly with shortening of middle phalanges. There was a degree of camptodactyly of ring and index fingers and broad thumbs and fingers. She had no apparent spinal abnormalities. Hypopigmentation on the left shoulder was noticed. Her Karyotype was normal (46XX). Skin biopsy of abnormally pigmented area showed no sign of chromosomal mosaicism. She was last seen by the paediatrician at the age of 13 years and no educational difficulties were reported. Her mother was examined and did not have any abnormal features or brachydactyly. The father was not available for examination but was said to have some similarity in facial features. The parents were not related. Three other children were normal.
DNA analysis and identification of the new Robinow syndrome-causing mutations from patients
The DNA from peripheral blood was extracted using standard methodology (Afzal et al. 2000). PCR reactions contained Red Hot (5 U/μl) Taq polymerase enzyme (0.5 U), genomic DNA (250 ng), primers (20 pmol each) and Mg2+ (1.5 mM) in a total volume of 25 μl. Primer sets were used as previously described (Afzal et al. 2000). These products were subsequently sequenced on both forward and reverse strands using cycle sequencing on an ABI 3100 automated sequencer (Applied Biosystems) to identify the site and the nature of the mutation in each case. We repeated this on a separate PCR product to confirm the validity of the result. All exons of ROR2 were sequenced. Numbering of the bases assumes the first base of the initiation codon as 1.
Construction of mouse Ror2 mutants
The plasmid pcDNA3-ROR2WT-HA (gift of Drs Y. Minami Kobe University, Japan) served as a template to generate Ror2 mutants (pcDNA3-Ror2Y192D-HA, pcDNA3-Ror2R244W-HA) whereas pcDNA3-Ror2A245T-HA was used as the template for the double mutant pcDNA3-Ror2R244W/A245T-HA. QuickChange™ Mutagenesis (Stratagene) was performed according to the manufacturer’s instructions using the following primers (mutagenic bases are underlined and in bold): Y192D; Ror2-Y192D-F 5′-GGGAACCGGACTATCGATGTGGACTCCCTCCAG-3′ and Ror2-Y192D-R 5′-CTGGAGGGAGTCCACATCGATAGTCCGGTTCCC-3′, a novel Cla I site was introduced for diagnostic digestion. R244W; Ror2-R244W-F 5′-GCGACGCATGCTCCTGGGCGCCCAAGCCTCGC-3′ and Ror2-R244W-R 5′-GCGAGGCTTGGGCGCCCAGGAGCATGCGTCGC-3′; a Sma I site was abolished for diagnostic digestion. R244W/A245T; Ror2-R244W + A245T-F 5′-GCGACGCATGCTCCTGGACGCCCAAGCCTCGC-3′ and Ror2-R244W + A245T-R 5′-GCGAGGCTTGGGCGTCCAGGAGCATGCGTCGC-3′; a Sma I site was abolished for diagnostic digestion.
All constructs were confirmed by the diagnostic restriction digests and by direct DNA sequencing (Advanced Biotechnology Centre, Imperial College London).
Cell culture and transfection
HeLa cells were cultured in Dulbecco’s modified Eagle’s medium (Invitrogen) supplemented with 10% foetal calf serum, 2 mM l-glutamine, and 100 U/ml penicillin/streptomycin at 37°C with 10% CO2. For immunofluorescence, HeLa cells were grown on coverslips in a 24-well plate for 24 h and transiently transfected using the liposomal transfection reagent FuGENE 6 (Roche Biochemicals) according to the manufacturer’s instructions. In co-transfection, a mixture of 0.5 μg of EGFP-hRas, 1 μg of mRor2 wildtype or mutant DNA and 5 μl of FuGENE 6 in 94 μl of OPTIMEM I medium (Invitrogen) was applied to each well of the HeLa cells at about 60% confluence. In single transfection, only a mixture of 1 μg of mRor2 wildtype or mutant DNA and 4 μl of FuGENE 6 in 94 μl of OPTIMEM I medium was applied to each well of the HeLa cells. The cells were then fixed and processed for microscopy 24 h later.
Antibodies were purchased from the following sources: mouse anti-HA-Tag monoclonal antibody (dilution 1:200 for immunofluorescence 1:1,000 for Western blotting; Cell Signaling Technology), rabbit anti-calnexin polyclonal antibody (dilution 1:500; StressGen Biotechnologies), Alexa Fluor 568-goat anti-mouse IgG (dilution 1:200; Molecular Probes), Alexa Fluor 488-goat anti-rabbit IgG (dilution 1:200; Molecular Probes).
For immunofluorescence, coverslip-grown HeLa cells were washed with phosphate-buffered saline (PBS), fixed in cold methanol at −20°C for 4 min, washed in PBS three times and incubated in 1× PBS containing 0.5% BSA for 15 min. The fixed cells were then incubated at room temperature for 1 h with either mouse monoclonal anti-HA antibody alone, or co-stained with both mouse monoclonal anti-HA antibody and rabbit polyclonal anti-calnexin antibody. After washing with PBS, the cells were incubated with the appropriate secondary antibodies for 1 h at room temperature, washed several times with PBS and mounted in Immuno Fluor medium (ICN Biomedicals), and visualised under a Leica DM-IRBE confocal microscope. Images were acquired using Leica TCS-NT software associated with the microscope and processed with Adobe Photoshop® (Adobe Inc.).
Novel homozygous mutations in the frizzled-like cysteine-rich domain of ROR2 are responsible for the RRS phenotype
Sequencing of ROR2 in the Egyptian subject revealed a homozygous mutation in exon 5 of the gene causing a nucleotide change 574T > G and resulting in a missense change of tyrosine to aspartic acid in position 192 (Y192D) in the frizzled domain (Fig. 1). Parents were both heterozygous for the above change. Sequencing of ROR2 in the UK subject revealed a homozygous mutation in exon 6 of the gene causing a nucleotide change 730C > T and thus resulting in a missense change of arginine to tryptophan in position 244 (R244W); again in the frizzled-like domain. Additionally the subject is homozygous for the 733G > A variant causing an A245T change. The mother was heterozygous for both molecular changes (R244W and A245T). DNA sample from the father was not available for analysis. The above mutations were not observed in any of the 50 ethnically matched control subjects (100 chromosomes). Moreover they have not been reported in the previous studies by Afzal et al. (2000) Schwabe et al. (2004) Oldridge et al. (2000) and van Bokhoven et al. (2000) in Caucasian background.
RRS-causing mutations fail to reach the plasma membrane
RRS-causing mutations are retained in the ER
Autosomal recessive Robinow syndrome is caused by different homozygous missense (see Fig. 1), nonsense and frameshift mutations in the ROR2 gene (Afzal and Jeffery 2003). Moreover, two cases of compound heterozygosity has been reported by Afzal et al. (2000) and Tufan et al. (2005). The reported mutations are in exons 3, 5–6, 7, 8, and 9 of the gene corresponding to the Ig-like domain, frizzled-like, the kringle domain, the region between the KD and TK domains, and the tyrosine kinase domain, respectively (Afzal et al. 2000; van Bokhoven et al. 2000; Schwabe et al. 2000; Afzal and Jeffery 2003; Tufan et al. 2005). The extracellular portion of the ROR2 protein contains three distinct motifs (Fig. 1), all of which are known to have properties for protein–protein interactions possibly ligand binding functions. We report here two new cases of RRS in two different families. Both cases are caused by missense mutations located in the proximal region of the extracellular frizzled-like domain, highlighting a considerable clustering of missense mutations causing RRS in this cysteine-rich area. All these mutations point to structurally/functionally important residues of this domain in the ROR2 protein. The frizzled cysteine-rich domain is a protein sub-structure that was first identified in G-protein-coupled receptors (Chan et al. 1992). This domain usually contains ten highly conserved cysteine residues participating in disulfide bridges, characteristic of a family of proteins that includes the extracellular ligand-binding region of frizzled receptors (the receptors for Wnt) and smoothened (the co-receptor for sonic hedgehog) (Masiakowski and Yancopoulos 1998; Xu and Nusse 1998; Billiard et al. 2005). Recently Roszmusz et al. (2001) have defined the structure of this important module using the frizzled domain of rat Ror1 receptor tyrosine kinase, by proteolytic digestion and amino-acid sequencing. They identified this region to be a single, compact, and remarkably stable folding domain, possessing both alpha-helices and beta-strands (Roszmusz et al. 2001). The cysteine-rich domain in frizzled proteins consists of 120–125 amino acids and is necessary and sufficient for Wnt binding. The crystal structure of the cysteine-rich domain of mFz8 and sFRP-3 revealed a predominantly alpha helical structure forming a unique protein fold (Dann et al. 2001). However, our attempts to model the structure of cysteine-rich domain of ROR2 using the above structures as templates was unsuccessful due low sequence homology (data not shown).
The Frizzled genes encode membrane proteins that are involved in multiple cellular signalling pathways regulating embryonic development, tissue and cell polarity, formation of neural synapses and proliferation (Huang and Klein 2004). The cysteine-rich domain of Frizzled proteins is involved in Wnt binding and hence involved in the various signalling pathways. Abnormal Wnt-mediated signalling pathways have been implicated in various human diseases including developmental diseases and certain cancers (Reya and Clevers 2005). Disruption of the cysteine-rich domain in frizzled has been implicated in human disease or animal models (Kameya et al. 2002; Toomes et al. 2004). Missense mutations in the FZD4 cysteine-rich domain have been shown to cause familial exudative vitreoretinopathy (Toomes et al. 2004). However, the possible involvement of ER retention in this case has not been examined. In another case, disruption of the cysteine-rich domain of FZD4 by truncation has been shown to cause retention of both wild type and the truncated form of the protein leading to dominant negative effect and causing the same disease (Kaykas et al. 2004). Therefore, it is reasonable to consider variations in the cysteine-rich domain of frizzled protein as disease susceptibility or risk factors.
Recently we have shown that ER-associated protein degradation is the most common mechanism underpinning numerous monogenic diseases (Chen et al. 2005). Retention of proteins in the ER and their degradation by ERAD components may result from mild changes to the native protein structure including malfolding due to amino acid substitution mutations or lack of post-translational modifications or partners for assembly into complexes. It is clear from our studies that loss of ROR2 function due to missenses mutations results from trafficking defects rather than affecting an active site in the protein. It is also apparent that mutations throughout the frizzled-like cystein rich domain can produce this results, highlighting the essential nature of this part of the protein for correct folding and transport. Establishing the mechanisms of genetic diseases will inevitably have impact on therapeutic approaches. Overcoming ER retention as a potential target for therapy of genetic diseases has been the subject for active research over the last few years (Romisch 2004).
We are grateful to Prof. Y. Minami and Department of Biomedical Regulation, Kobe University School of Medicine, Kobe, Japan for providing the mouse pcDNA3-Ror2WT-HA plasmid. Thanks to Vanda Lopes for assistance and Prof. Miguel Seabra (supported by the Wellcome Trust) for reagents and facilities. ARA was supported by the Wellcome Trust.