Genetic study of eight cases of spondylo-epiphyseal dysplasia tarda and its autosomal recessive transmission

Spondylo-epiphyseal dysplasia tarda is an hereditary disease of bone first described by Maroteaux in 1957. The disease affects only boys, then is transmitted on X-linked recessive pattern. The authors describe two tunisians families with 8 patients presenting clinical and radiologic symptoms resembling spondylo-epiphyseal dysplasia tarda but affecting both sexes. Autosomal recessive inheritance is probable in these families. Conclusion is that genetic heterogeneity of the disease must be precise.     

A novel nonsense mutation of the sedlin gene in a family with spondyloepiphyseal dysplasia tarda

We report a new nonsense mutation in the human sedlin (SEDL) gene in a family with X-linked spondyloepiphyseal dysplasia tarda. A substitution of cytosine for adenine at nucleotide position 329 causing a nonsense mutation (S110X) in exon 6 was identified in the affected patient in the family.     

TRPV4-associated skeletal dysplasias

Dominant mutations in the TRPV4 gene result in a bone dysplasia family and form a continuous phenotypic spectrum that includes, in decreasing severity, lethal, and nonlethal metatropic dysplasia (MD), spondylometaphyseal dysplasia Kozlowski type (SMDK), and autosomal dominant brachyolmia. Several rare variant phenotypes that have some overlap but deviate in some ways from the general pattern have also been described. The known variant phenotypes are spondyloepiphyseal dysplasia Maroteaux type (Pseudo-Morquio type 2), parastremmatic dysplasia, and familial digital arthropathy with brachydactyly. Interestingly, different TRPV4 mutations have been associated with dominantly inherited neurologic disorders such as congenital spinal muscular atrophy and hereditary motor and sensory neuropathy. Finally, a small number of patients have been identified in whom a TRPV4 mutation results in a phenotype combining skeletal dysplasia with peripheral neuropathy. The TRPV4 gene encodes a regulated calcium channel implicated in multiple and diverse cellular processes. Over 50 different TRPV4 mutations have been reported, with two codons appearing to be mutational hot spots: P799 in exon 15, mostly associated with MD, and R594 in exon 11, associated with SMDK. While most pathogenic mutations tested so far result in activation of the calcium channel in vitro, the mechanisms through which TRPV4 activation results in skeletal dysplasia and/or peripheral neuropathy remain unclear and the genotype-phenotype correlations in this group of disorders remains somewhat mysterious. Since the phenotypic expression of most mutations seems to be relatively constant, careful clinical and radiographic assessment is useful in directing molecular analysis.     

No skeletal dysplasia in the nariokotome boy KNM‐WT 15000 (homo erectus)—A reassessment of congenital pathologies of the vertebral column

The Nariokotome boy skeleton KNM‐WT 15000 is the most complete Homo erectus fossil and therefore is key for understanding human evolution. Nevertheless, since Latimer and Ohman (2001) reported on severe congenital pathology in KNM‐WT 15000, it is questionable whether this skeleton can still be used as reference for Homo erectus skeletal biology. The asserted pathologies include platyspondylic and diminutive vertebrae implying a disproportionately short stature; spina bifida; condylus tertius; spinal stenosis; and scoliosis. Based on this symptom complex, the differential diagnosis of spondyloepiphyseal dysplasia tarda, an extremely rare form of skeletal dysplasia, has been proposed. Yet, our reanalysis of these pathologies shows that the shape of the KNM‐WT 15000 vertebrae matches that of normal modern human adolescents. The vertebrae are not abnormally flat, show no endplate irregularities, and thus are not platyspondylic. As this is the hallmark of spondyloepiphyseal dysplasia tarda and related forms of skeletal dysplasia, the absence of platyspondyly refutes axial dysplasia and disproportionate dwarfism. Furthermore, we neither found evidence for spina bifida occulta nor manifesta, whereas the condylus tertius, a developmental anomaly of the cranial base, is not related to skeletal dysplasias. Other fossils indicate that the relatively small size of the vertebrae and the narrow spinal canal are characteristics of early hominins rather than congenital pathologies. Except for the recently described signs of traumatic lumbar disc herniation, the Nariokotome boy fossil therefore seems to belong to a normal Homo erectus youth without pathologies of the axial skeleton. Am J Phys Anthropol, 2013. © 2013 Wiley Periodicals, Inc.     

Molecular basis and PCR-DNA typing of the Fya/fyb blood group polymorphism

Spondyloepiphyseal dysplasia tarda (SEDL) is an X-linked recessive disorder characterized in affected males by short stature resulting from a growth defect of the vertebral bodies. We have extended our earlier studies by analyzing 15 families with newly identified microsatellite DNA markers; analysis of recombination events with these markers indicates that the gene responsible for SEDL is located in Xp22 between DXS 16 and DXS 987 on an interval spanning approximately 2 Mb.     

Vaccine efficacy of recombinant GAPDH of Edwardsiella tarda against Edwardsiellosis

Thirty-seven kilodalton glyceraldehyde-3-phosphate dehydrogenase (GAPDH) of Edwardsiella tarda was suggested to be an effective vaccine candidate against E. tarda infection in previous research. For developing a vaccine, obtaining GAPDH in large quantities is necessary. In this study, we determined the complete nucleotide sequence of the gene that encodes GAPDH of E. tarda, and overexpressed the GAPDH of E. tarda by using the Escherichia coli expression system. We immunized Japanese flounder with recombinant GAPDH (rGAPDH) and evaluated its vaccine efficacy. Our results showed that rGAPDH effectively protected Japanese flounder from experimental E. tarda infection, and will contribute to the development of a vaccine against E. tarda.     

Glyceraldehyde-3-phosphate dehydrogenase of Edwardsiella tarda has protective antigenicity against Vibrio anguillarum in Japanese flounder

Edwardsiella tarda glyceraldehyde-3-phosphate dehydrogenase (GAPDH) may be an effective vaccine candidate against infection by E. tarda in Japanese flounder Paralichthys olivaceus. The GAPDH of E. tarda is highly homologous to that of Vibrio cholerae (91%), and therefore E. tarda GAPDH may have protective antigenicity against Vibrio species. In this study, we immunized Japanese flounder with GAPDH of E. tarda and infected the fish with V anguillarum. The result showed that GAPDH prepared from E. tarda protected Japanese flounder effectively in a challenge of V anguillarum. Therefore, E. tarda GAPDH should be considered as a multi-purpose vaccine candidate against several kinds of pathogenic bacteria.     

Isolation of an unusual strain of Edwardsiella tarda from turbot and establish a PCR detection technique with the gyrB gene

Aims:  The aim of this study was to report an unusual Edwardsiella tarda and develop an effective method to identify this bacterium.
Methods and Results:  During the spring and summer of 2006, an epizootic occurred among cultured turbot ( Scophthalmus maximus ) in Qingdao, China. A gram-negative, rod-shaped bacterium (designated as LTB-4) was isolated from the infected fish, and was proved to be virulent to turbot. Based on the 16S rDNA sequencing and phenotypic tests, the bacterial pathogen was identified as E. tarda. Unlike those commonly described E. tarda strains, no flagellate was observed. Partial gyrB genes were amplified from E. tarda using the universal primers of gyrB genes and sequenced. The polymerase chain reaction (PCR) primers for the gyrB gene were designed specific to E. tarda . It revealed positive amplification of the gyrB fragment in E. tarda , whereas other bacterial species were negative. In addition, the technique enabled the recognition of E. tarda from diseased fish.
Conclusions:  The isolate was identified as E. tarda without flagellate and an effective method was developed to identify E. tarda based on using the gyrB gene as a taxonomic marker.
Significance and Impact of the Study:  The unusual E. tarda was first reported in China and the PCR allowed the rapid and sensitive detection of E. tarda.     

Edwardsiella comparative phylogenomics reveal the new intra/inter-species taxonomic relationships, virulence evolution and niche adaptation mechanisms

Edwardsiella bacteria are leading fish pathogens causing huge losses to aquaculture industries worldwide. E. tarda is a broad-host range pathogen that infects more than 20 species of fish and other animals including humans while E. ictaluri is host-adapted to channel catfish causing enteric septicemia of catfish (ESC). Thus, these two species consist of a useful comparative system for studying the intricacies of pathogen evolution. Here we present for the first time the phylogenomic comparisons of 8 genomes of E. tarda and E. ictaluri isolates. Genome-based phylogenetic analysis revealed that E. tarda could be separate into two kinds of genotypes (genotype I, EdwGI and genotype II, EdwGII) based on the sequence similarity. E. tarda strains of EdwGI were clustered together with the E. ictaluri lineage and showed low sequence conservation to E. tarda strains of EdwGII. Multilocus sequence analysis (MLSA) of 48 distinct Edwardsiella strains also supports the new taxonomic relationship of the lineages. We identified the type III and VI secretion systems (T3SS and T6SS) as well as iron scavenging related genes that fulfilled the criteria of a key evolutionary factor likely facilitating the virulence evolution and adaptation to a broad range of hosts in EdwGI E. tarda. The surface structure-related genes may underlie the adaptive evolution of E. ictaluri in the host specification processes. Virulence and competition assays of the null mutants of the representative genes experimentally confirmed their contributive roles in the evolution/niche adaptive processes. We also reconstructed the hypothetical evolutionary pathway to highlight the virulence evolution and niche adaptation mechanisms of Edwardsiella. This study may facilitate the development of diagnostics, vaccines, and therapeutics for this under-studied pathogen.     

Edwardsiella tarda endotoxin as an immunopotentiator in Singhi,Heteropneustes fossilis fingerlings

Endotoxin of E. tarda grown in brain heart infusion broth at 30 degrees C for 18 hr was extracted by differential centrifugation. Fingerlings of H. fossilis (weighing 1-2 g) were allowed for hyperosmotic infiltration in the endotoxin at the rate of 0,2,4,8,16 and 20 mg/l. Mortality varied from 20-50% at 2 to 20 mg/ml. Toxin treated fishes were challenged 21 days post treatment with the same E. tarda strain containing 2.1 x 10(9) CFU/ml. There was 80% mortality in the control group whereas only 20% mortality in toxin treated group at 2 mg/l concentration after challenge with homologous E. tarda. Subsequently a second challenge of E. tarda was given to the survivors of fish one month after first challenge using same concentration where no mortality could be observed. It was concluded that the endotoxin could enhance percentage of survival against E. tarda infection in Singhi.     

Defective human erythrocyte uroporphyrinogen decarboxylase in familial porphyria cutanea tarda: the metabolic lesion or the result of endogenous porphyrinemia?

We have demonstrated that oral charcoal therapy is as effective as therapeutic phlebotomy in reducing porphyrinemia in porphyria cutanea tarda. The effects of immediate and sustained reduction of porphyrinemia on the catalytic properties of partially purified (approximately 200-fold) preparations of red cell uroporphyrinogen decarboxylase of a patient with familial porphyria cutanea tarda were studied. All populations of the patient's red cells exhibited defective enzyme activity, and the apparent Michaelis constants (Km) determined with penta-, hepta-, and octa-carboxylic I porphyrinogen substrates were approximately 3-4 times higher as compared to the normal controls. Mixing experiments (normal and defective enzyme), and preincubation of the normal enzyme with porphyric plasma prior to purification, yielded data supporting the concept that the catalytic defects of red cell uroporphyrinogen decarboxylase in familial porphyria cutanea tarda are independent of interactions between circulating endogenous porphyrins and the enzyme.     

Porphyria cutanea tarda: clinical and laboratory features.

Eleven patients with porphyria cutanea tarda were studied. Biochemical confirmation of the clinical diagnosis required only determination of the total urine porphyrin concentration in a sample of urine voided on rising in the morning. The patients were divided for convenience of discussion into four groups differing in age, sex and etiologic factors. Of the six patients in whom a liver biopsy was done one was shown to have micronodular cirrhosis. Except for a modest elevation in the serum glutamic oxaloacetic transaminase values when the patients were first seen, no evidence was found for liver disease apart from the presence of porphyria cutanea tarda. One patient recovered solely by abstaining from alcohol consumption. Five patients underwent phlebotomy; their iron stores had been found to be between 2 and 3 g. Decreasing urine porphyrin values correlated well with decreasing serum ferritin values during the course of phlebotomy. Porphyria cutanea tarda, which is due to a deficiency of uroporphyrinogen decarboxylase, is manifested in association with alcohol abuse, estrogen therapy, exposure to chlorinated hydrocarbons or increased tissue iron stores, or a combination of these factors. Although relatively uncommon, this condition raises important and unresolved issues regarding the hepatotoxicity of alcohol, estrogens, chlorinated hydrocarbons and iron.     

Intramolecular rearrangements as a consequence of the dephosphorylation of phosphoaspartate residues in proteins

Sedlin is an evolutionarily conserved protein encoded by the causative gene SEDL for spondyloepiphyseal dysplasia tarda. Nevertheless, how Sedlin mutations cause the disease remains unknown. Here, the intracellular chloride channel protein CLIC1 was shown to associate with Sedlin by yeast two-hybrid screening. Green fluorescence protein-CLIC1 readily co-immunoprecipitated with FLAG-Sedlin. In addition, both proteins colocalized extensively in cytoplasmic vesicular/reticular structures in COS-7 cells, suggesting their interaction at intracellular membranous organelles. Sedlin also associated with CLIC2 in yeast two-hybrid assays. The link between Sedlin and the intracellular chloride channels is the first step to understand their functional interplays.     

Identification of novel putative virulence factors, adhesin AIDA and type VI secretion system, in atypical strains of fish pathogenic Edwardsiella tarda by genomic subtractive hybridization

Edwardsiella tarda, which is known to be the causative agent of edwardsiellosis in freshwater and marine fish, has two motility phenotypes. Typical strains exhibiting motility are isolated mainly from freshwater fish and Japanese flounder. Atypical strains exhibiting non-motility are isolated mainly from marine fish, with the exception of Japanese flounder. Subtractive hybridization was performed to identify genomic differences between these two phenotypes. Two fragments which showed homology to potential virulence factors were isolated from atypical strains: the autotransporter adhesin AIDA and a component of T6SS. We analysed DNA sequences of about 5 kbp containing these fragments and identified two partial ORF, and ORF encoding for other components of T6SS. The predicted amino acid sequences showed remarkably low homology to components of T6SS reported in the typical E. tarda strain PPD130/91. Furthermore, the organization of these ORF was different from the gene cluster of the typical E. tarda strain. AIDA and T6SS may therefore be associated with different pathogenicity in typical and atypical E. tarda hosts.     

Generation of two auxotrophic genes knock-out Edwardsiella tarda and assessment of its potential as a combined vaccine in olive flounder (Paralichthys olivaceus)

Two auxotrophic genes that play essential roles in bacterial cell wall biosynthesis--alanine racemase (alr) gene and aspartate semialdehyde dehydrogenase (asd) gene--knock-out Edwardsiella tarda (Δalr Δasd E. tarda) was generated by the allelic exchange method to develop a combined vaccine system. Green fluorescent protein (GFP) was used as a model foreign protein, and was expressed by transformation of the mutant E. tarda with antibiotic resistant gene-free plasmids harboring cassettes for GFP and asd expression (pG02-ASD-EtPR-GFP). In vitro growth of the mutant E. tarda was similar to wild-type E. tarda when D-alanine and diaminopimelic acid (DAP) were supplemented to growth medium. However, without d-alanine and/or DAP supplementation, the mutant showed very limited growth. The Δalr Δasd E. tarda transformed with pG02-ASD-EtPR-GFP showed a similar growth pattern of wild-type E. tarda when D-alanine was supplemented in the medium, and the expression of GFP could be observed even with naked eyes. The virulence of the auxotrophic mutant E. tarda was decreased, which was demonstrated by approximately 10? fold increase of LD?? dose compared to wild-type E. tarda. To assess vaccine potential of the present combined vaccine system, olive flounder (Paralichthys olivaceus) were immunized with the GFP expressing mutant E. tarda, and analyzed protection efficacy against E. tarda challenge and antibody titers against E. tarda and GFP. Groups of fish immunized with 10? CFU of the Δalr Δasd E. tarda harboring pG02-ASD-EtPR-GFP showed no mortality, which was irrespective to boost immunization. The cumulative mortality rates of fish immunized with 10? or 10? CFU of the mutant bacteria were lowered by a boost immunization. Fish immunized with the mutant E. tarda at doses of 10?-10? CFU/fish showed significantly higher serum agglutination activities against formalin-killed E. tarda than PBS-injected control fish. Furthermore, fish immunized with 10?-10? CFU/fish of the mutant E. tarda showed significantly higher ELISA titer against GFP antigen than fish in other groups. These results indicate that the present double auxotrophic genes knock-out E. tarda coupled with a heterologous antigen expression has a great strategic potential to be used as combined vaccines against various fish diseases.     

Protection of tilapia (Oreochromis mosambicus) from edwardsiellosis by vaccination with Edwardsiella tarda ghosts

The vaccine potential of Edwardsiella tarda ghosts produced by gene E mediated lysis was investigated using tilapia (Oreochromis mosambicus). Tilapia immunized with E. tarda ghosts (ETG) and formalin killed E. tarda (FKC) vaccines showed significantly higher serum agglutination titers than control fish. Fish immunized with ETG showed no significant differences with fish immunized with FKC in serum agglutination titers, but showed significantly higher bactericidal activity than fish immunized with FKC. Furthermore, fish immunized with ETG showed higher protection than fish immunized with FKC. As this promising type of a non-living whole cell envelope preparation seems to be favorable over conventional vaccines, we suggest E. tarda ghosts as a new vaccine candidate.     

Isolation of Edwardsiella tarda from Swine

Edwardsiella tarda was isolated from the intestinal tract of a 2-month-old pig. This is the first reported isolation of Edwardsiella tarda from swine in the United States. Swine have been reported as potential carriers of Edwardsiella tarda, but pathogenicity of this organism for swine has not been determined. Although the pig had access to several farm ponds, the exact source of infection was not determined.     

Mutations in the gyrB, parC, and parE genes of quinolone-resistant isolates and mutants of Edwardsiella tarda

The full length genes gyrB (2,415 bp), parC (2,277 bp), and parE (1,896 bp) in Edwardsiella tarda were cloned by PCR with degenerate primers based on the sequence of the respective quinolone resistance-determining region (QRDR), followed by elongation of 5' and 3' ends using cassette ligation-mediated PCR (CLMP). Analysis of the cloned genes revealed open reading frames (ORFs) encoding proteins of 804 (GyrB), 758 (ParC), and 631 (ParE) amino acids with conserved gyrase/topoisomerase features and motifs important for enzymatic function. The ORFs were preceded by putative promoters, ribosome binding sites, and inverted repeats with the potential to form cruciform structures for binding of DNA-binding proteins. When comparing the deduced amino acid sequences of E. tarda GyrB, ParC, and ParE with those of the corresponding proteins in other bacteria, they were found to be most closely related to Escherichia coli GyrB (87.6% identity), Klebsiella pneumoniae ParC (78.8% identity) and Salmonella typhimurium ParE (89.5% identity), respectively. The two topoisomerase genes, parC and parE, were found to be contiguous on the E. tarda chromosome. All 18 quinoloneresistant isolates obtained from Korea thus far did not contain subunit alternations apart from a substitution in GyrA (Ser83→Arg). However, an alteration in the QRDR of ParC (Ser84→Ile) following an amino acid substitution in GyrA (Asp87→Gly) was detected in E. tarda mutants selected in vitro at 8 microng/ml ciprofloxacin (CIP). A mutant with a GyrB (Ser464→Leu) and GyrA (Asp87→Gly) substitution did not show a significant increase in the minimum inhibitory concentration (MIC) of CIP. None of the in vitro mutants exhibited mutations in parE. Thus, gyrA and parC should be considered to be the primary and secondary targets, respectively, of quinolones in E. tarda.     

Retention of virulence in a viable but nonculturable Edwardsiella tarda isolate

Edwardsiella tarda is pathogen of fish and other animals. The aim of this study was to investigate the viable but nonculturable (VBNC) state and virulence retention of this bacterium. Edwardsiella tarda CW7 was cultured in sterilized aged seawater at 4 degrees C. Total cell counts remained constant throughout the 28-day period by acridine orange direct counting, while plate counts declined to undetectable levels (<0.1 CFU/ml) within 28 days by plate counting. The direct viable counts, on the other hand, declined to ca. 10(9) CFU/ml active cells and remained fairly constant at this level by direct viable counting. These results indicated that a large population of cells existed in a viable but nonculturable state. VBNC E. tarda CW7 could resuscitate in experimental chick embryos and in the presence of nutrition with a temperature upshift. The resuscitative times were 6 days and 8 days, respectively. The morphological changes of VBNC, normal, and resuscitative E. tarda CW7 cells were studied with a scanning electron microscope. The results showed that when the cells entered into the VBNC state, they gradually changed in shape from short rods to coccoid and decreased in size, but the resuscitative cells did not show any obvious differences from the normal cells. The VBNC and the resuscitative E. tarda CW7 cells were intraperitoneally inoculated into turbot separately, and the fish inoculated with the resuscitative cells died within 7 days, which suggested that VBNC E. tarda CW7 might retain pathogenicity.