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1.  A novel mutation in DDR2 causing spondylo-meta-epiphyseal dysplasia with short limbs and abnormal calcifications (SMED-SL) results in defective intra-cellular trafficking 
BMC Medical Genetics  2014;15:42.
Background
The rare autosomal genetic disorder, Spondylo-meta-epiphyseal dysplasia with short limbs and abnormal calcifications (SMED-SL), is reported to be caused by missense or splice site mutations in the human discoidin domain receptor 2 (DDR2) gene. Previously our group has established that trafficking defects and loss of ligand binding are the underlying cellular mechanisms of several SMED-SL causing mutations. Here we report the clinical characteristics of two siblings of consanguineous marriage with suspected SMED-SL and identification of a novel disease-causing mutation in the DDR2 gene.
Methods
Clinical evaluation and radiography were performed to evaluate the patients. All the coding exons and splice sites of the DDR2 gene were sequenced by Sanger sequencing. Subcellular localization of the mutated DDR2 protein was determined by confocal microscopy, deglycosylation assay and Western blotting. DDR2 activity was measured by collagen activation and Western analysis.
Results
In addition to the typical features of SMED-SL, one of the patients has an eye phenotype including visual impairment due to optic atrophy. DNA sequencing revealed a novel homozygous dinucleotide deletion mutation (c.2468_2469delCT) on exon 18 of the DDR2 gene in both patients. The mutation resulted in a frameshift leading to an amino acid change at position S823 and a predicted premature termination of translation (p.S823Cfs*2). Subcellular localization of the mutant protein was analyzed in mammalian cell lines, and it was found to be largely retained in the endoplasmic reticulum (ER), which was further supported by its N-glycosylation profile. In keeping with its cellular mis-localization, the mutant protein was found to be deficient in collagen-induced receptor activation, suggesting protein trafficking defects as the major cellular mechanism underlying the loss of DDR2 function in our patients.
Conclusions
Our results indicate that the novel mutation results in defective trafficking of the DDR2 protein leading to loss of function and disease. This confirms our previous findings that DDR2 missense mutations occurring at the kinase domain result in retention of the mutant protein in the ER.
doi:10.1186/1471-2350-15-42
PMCID: PMC4001364  PMID: 24725993
DDR2; Spondylo-meta-epiphyseal dysplasia; Trafficking defect; SMED-SL; ERAD; Optic atrophy
2.  Reversible Daptomycin Tolerance of Adherent Staphylococci in an Implant Infection Model ▿ 
Daptomycin (DAP) is bactericidal against methicillin-resistant Staphylococcus aureus (MRSA) in vitro, but it failed to eradicate MRSA in an experimental model of implant-associated infection. We therefore investigated various factors which could explain treatment failure by evaluating DAP activity, including the role of different cell wall components, adherence, biofilm, and calcium ions (Ca2+) in vitro and in vivo. In the tissue cage infection model, DAP was active only prophylactically and against low inocula. To identify the mechanisms of treatment failure, the in vitro activity of DAP against planktonic and adherent growing S. aureus and S. epidermidis mutants, differing in their capacity of biofilm formation and adherence, was determined. For planktonic staphylococci, the MIC was 0.625 μg/ml. For adherent staphylococci, DAP reduced biofilms at 30 μg/ml. However, it did not kill adherent bacteria up to 500 μg/ml, independent of biofilm biosynthesis (the ica mutant strain), nuclease (the nuc1/nuc2 mutant strain), LPXTG-anchored adhesin (the srtA mutant strain), autolysin (the atl mutant strain), or alanyl-LTA (the dltA mutant strain). Resistance of adherent staphylococci was not due to mutations of adherent bacteria, since staphylococci became DAP susceptible after detachment. Phenotypic tolerance was not explained by inactivation of DAP or inability of initial Ca2+-DAP complex formation. However, the addition of up to 100 mg/liter (2.5 mmol/liter) Ca2+ gradually improved bactericidal activity toward adherent staphylococci in vitro and increased the prevention rate in the cage model from 40% to 60%. In summary, adherent staphylococci are resistant to DAP killing unless Ca2+ is supplemented to physiologic concentrations.
doi:10.1128/AAC.00172-11
PMCID: PMC3122382  PMID: 21576433
3.  Endoplasmic Reticulum Quality Control Is Involved in the Mechanism of Endoglin-Mediated Hereditary Haemorrhagic Telangiectasia 
PLoS ONE  2011;6(10):e26206.
Hereditary haemorrhagic telangiectasia (HHT) is an autosomal dominant genetic condition affecting the vascular system and is characterised by epistaxis, arteriovenous malformations and mucocutaneous and gastrointestinal telangiectases. This disorder affects approximately 1 in 8,000 people worldwide. Significant morbidity is associated with this condition in affected individuals, and anaemia can be a consequence of repeated haemorrhages from telangiectasia in the gut and nose. In the majority of the cases reported, the condition is caused by mutations in either ACVRL1 or endoglin genes, which encode components of the TGF-beta signalling pathway. Numerous missense mutations in endoglin have been reported as causative defects for HHT but the exact underlying cellular mechanisms caused by these mutations have not been fully established despite data supporting a role for the endoplasmic reticulum (ER) quality control machinery. For this reason, we examined the subcellular trafficking of twenty-five endoglin disease-causing missense mutations. The mutant proteins were expressed in HeLa and HEK293 cell lines, and their subcellular localizations were established by confocal fluorescence microscopy alongside the analysis of their N-glycosylation profiles. ER quality control was found to be responsible in eight (L32R, V49F, C53R, V125D, A160D, P165L, I271N and A308D) out of eleven mutants located on the orphan extracellular domain in addition to two (C363Y and C382W) out of thirteen mutants in the Zona Pellucida (ZP) domain. In addition, a single intracellular domain missense mutant was examined and found to traffic predominantly to the plasma membrane. These findings support the notion of the involvement of the ER's quality control in the mechanism of a significant number, but not all, missense endoglin mutants found in HHT type 1 patients. Other mechanisms including loss of interactions with signalling partners as well as adverse effects on functional residues are likely to be the cause of the mutant proteins' loss of function.
doi:10.1371/journal.pone.0026206
PMCID: PMC3194820  PMID: 22022569
4.  Trafficking defects and loss of ligand binding are the underlying causes of all reported DDR2 missense mutations found in SMED-SL patients 
Human Molecular Genetics  2010;19(11):2239-2250.
Spondylo-meta-epiphyseal dysplasia (SMED) with short limbs and abnormal calcifications (SMED-SL) is a rare, autosomal recessive human growth disorder, characterized by disproportionate short stature, short limbs, short broad fingers, abnormal metaphyses and epiphyses, platyspondyly and premature calcifications. Recently, three missense mutations and one splice-site mutation in the DDR2 gene were identified as causative genetic defects for SMED-SL, but the underlying cellular and biochemical mechanisms were not explored. Here we report a novel DDR2 missense mutation, c.337G>A (p.E113K), that causes SMED-SL in two siblings in the United Arab Emirates. Another DDR2 missense mutation, c.2254C>T (p.R752C), matching one of the previously reported SMED-SL mutations, was found in a second affected family. DDR2 is a plasma membrane receptor tyrosine kinase that functions as a collagen receptor. We expressed DDR2 constructs with the identified point mutations in human cell lines and evaluated their localization and functional properties. We found that all SMED-SL missense mutants were defective in collagen-induced receptor activation and that the three previously reported mutants (p.T713I, p.I726R and p.R752C) were retained in the endoplasmic reticulum. The novel mutant (p.E113K), in contrast, trafficked normally, like wild-type DDR2, but failed to bind collagen. This finding is in agreement with our recent structural data identifying Glu113 as an important amino acid in the DDR2 ligand-binding site. Our data thus demonstrate that SMED-SL can result from at least two different loss-of-function mechanisms: namely defects in DDR2 targeting to the plasma membrane or the loss of its ligand-binding activity.
doi:10.1093/hmg/ddq103
PMCID: PMC2865377  PMID: 20223752
5.  Defective cellular trafficking of missense NPR-B mutants is the major mechanism underlying acromesomelic dysplasia-type Maroteaux 
Human Molecular Genetics  2008;18(2):267-277.
Natriuretic peptides (NPs) comprise a family of structurally related but genetically distinct hormones that regulate a variety of physiological processes such as cardiac growth, blood pressure, axonal pathfinding and endochondral ossification leading to the formation of vertebrae and long bones. The biological actions of NPs are mediated by natriuretic peptide receptors (NPRs) A, B and C that are located on the cell surface. Mutations in NPR-B have been shown to cause acromesomelic dysplasia-type Maroteaux (AMDM), a growth disorder in humans and severe dwarfism in mice. We hypothesized that missense mutations of NPR-B associated with AMDM primarily affect NPR-B function by the arrest of receptor trafficking at the endoplasmic reticulum (ER), due to conformational change, rather than an impairment of ligand binding, transmission of signal through the membrane or catalytic activity. Twelve missense mutations found in AMDM patients and cn/cn mice were generated by site-directed mutagenesis and transiently overexpressed in HeLa cells. Confocal microscopy revealed that 11 out of 12 mutants were retained in the ER. Determination of the ligand-dependent cGMP response confirmed that ER-retained NPR-B mutants are non-functional. Meanwhile, the only cell surface-targeted NPR-B missense mutant (D176E) displayed greatly reduced enzymatic activity due to impaired ligand binding. Thus, in the majority of cases of AMDM associated with missense NPR-B mutation, disease appears to result from defects in the targeting of the ER receptor to the plasma membrane.
doi:10.1093/hmg/ddn354
PMCID: PMC2638773  PMID: 18945719
6.  Efficacy of Daptomycin in Implant-Associated Infection Due to Methicillin-Resistant Staphylococcus aureus: Importance of Combination with Rifampin▿  
Limited treatment options are available for implant-associated infections caused by methicillin (meticillin)-resistant Staphylococcus aureus (MRSA). We compared the activity of daptomycin (alone and with rifampin [rifampicin]) with the activities of other antimicrobial regimens against MRSA ATCC 43300 in the guinea pig foreign-body infection model. The daptomycin MIC and the minimum bactericidal concentration in logarithmic phase and stationary growth phase of MRSA were 0.625, 0.625, and 20 μg/ml, respectively. In time-kill studies, daptomycin showed rapid and concentration-dependent killing of MRSA in stationary growth phase. At concentrations above 20 μg/ml, daptomycin reduced the counts by >3 log10 CFU/ml in 2 to 4 h. In sterile cage fluid, daptomycin peak concentrations of 23.1, 46.3, and 53.7 μg/ml were reached 4 to 6 h after the administration of single intraperitoneal doses of 20, 30, and 40 mg/kg of body weight, respectively. In treatment studies, daptomycin alone reduced the planktonic MRSA counts by 0.3 log10 CFU/ml, whereas in combination with rifampin, a reduction in the counts of >6 log10 CFU/ml was observed. Vancomycin and daptomycin (at both doses) were unable to cure any cage-associated infection when they were given as monotherapy, whereas rifampin alone cured the infections in 33% of the cages. In combination with rifampin, daptomycin showed cure rates of 25% (at 20 mg/kg) and 67% (at 30 mg/kg), vancomycin showed a cure rate of 8%, linezolid showed a cure rate of 0%, and levofloxacin showed a cure rate of 58%. In addition, daptomycin at a high dose (30 mg/kg) completely prevented the emergence of rifampin resistance in planktonic and adherent MRSA cells. Daptomycin at a high dose, corresponding to 6 mg/kg in humans, in combination with rifampin showed the highest activity against planktonic and adherent MRSA. Daptomycin plus rifampin is a promising treatment option for implant-associated MRSA infections.
doi:10.1128/AAC.00047-09
PMCID: PMC2704655  PMID: 19364845
7.  Unique Acquisition of Cytotoxic T-Lymphocyte Escape Mutants in Infant Human Immunodeficiency Virus Type 1 Infection†  
Journal of Virology  2005;79(18):12100-12105.
The role of cytotoxic T-lymphocyte (CTL) escape in rapidly progressive infant human immunodeficiency virus type 1 (HIV-1) infection is undefined. The data presented here demonstrate that infant HIV-1-specific CTL can select for viral escape variants very early in life. These variants, furthermore, may be selected specifically in the infant, despite the same CTL specificity being present in the mother. Additionally, pediatric CTL activity may be compromised both by the transmission of maternal escape variants and by mother-to-child transmission of escape variants that originally arose in the father. The unique acquisition of these CTL escape forms may help to explain the severe nature of some pediatric HIV infections.
doi:10.1128/JVI.79.18.12100-12105.2005
PMCID: PMC1212591  PMID: 16140787

Results 1-7 (7)