Mutations in prickle orthologs cause seizures in flies, mice, and humans

Epilepsy is heritable, yet few causative gene mutations have been identified, and thus far no human epilepsy gene mutations have been found to produce seizures in invertebrates. Here we show that mutations in prickle genes are associated with seizures in humans, mice, and flies. We identified human epilepsy patients with heterozygous mutations in either PRICKLE1 or PRICKLE2. In overexpression assays in zebrafish, prickle mutations resulted in aberrant prickle function. A seizure phenotype was present in the Prickle1-null mutant mouse, two Prickle1 point mutant (missense and nonsense) mice, and a Prickle2-null mutant mouse. Drosophila with prickle mutations displayed seizures that were responsive to anti-epileptic medication, and homozygous mutant embryos showed neuronal defects. These results suggest that prickle mutations have caused seizures throughout evolution.     

Mutations in spalt cause a severe but reversible neurodegenerative phenotype in the embryonic central nervous system of Drosophila melanogaster

The gene spalt is expressed in the embryonic central nervous system of Drosophila melanogaster but its function in this tissue is still unknown. To investigate this question, we used a combination of techniques to analyse spalt mutant embryos. Electron microscopy showed that in the absence of spalt, the central nervous system cells are separated by enlarged extracellular spaces populated by membranous material at 60% of embryonic development. Surprisingly, the central nervous system from slightly older embryos (80% of development) exhibited almost wild-type morphology. An extensive survey by laser confocal microscopy revealed that the spalt mutant central nervous system has abnormal levels of particular cell adhesion and cytoskeletal proteins. Time-lapse analysis of neuronal differentiation in vitro, lineage analysis and transplantation experiments confirmed that the mutation causes cytoskeletal and adhesion defects. The data indicate that in the central nervous system, spalt operates within a regulatory pathway which influences the expression of the beta-catenin Armadillo, its ligand N-Cadherin, Notch, and the cell adhesion molecules Neuroglian, Fasciclin 2 and Fasciclin 3. Effects on the expression of these genes are persistent but many morphological aspects of the phenotype are transient, leading to the concept of sequential redundancy for stable organisation of the central nervous system.     

Cloning and characterization of mitochondrial methionyl-tRNA synthetase from a pathogenic fungi Candida albicans

A genomic sequence encoding mitochondrial methionyl-tRNA synthetase (MetRS) was determined from a pathogenic fungi Candida albicans. The gene is distinct from that encoding the cytoplasmic MetRS. The encoded protein consists of 577 amino acids (aa) and contains the class I defining sequences in the N-terminal domain and the conserved anticodon-binding amino acid, Trp, in the C-terminal domain. This protein showed the highest similarity with the mitochondrial MetRSs of Saccharomyces cerevisiae and Shizosaccharomyces pombe. The mitochondrial MetRSs of these fungi were distinguished from their cytoplasmic forms. The protein lacks the zinc binding motif in the N-terminal domain and the C-terminal dimerization appendix that are present in MetRSs of several other species. Escherichia coli tRNA^Met was a substrate for the encoded protein as determined by genetic complementation and in vitro aminoacylation reaction. This cross-species aminoacylation activity suggests the conservation of interaction mode between tRNA^Met and MetRS.     

Purification of the yeast mitochondrial methionyl-tRNA synthetase. Common and distinctive features of the cytoplasmic and mitochondrial isoenzymes

Yeast-mitochondrial methionyl-tRNA synthetase was purified 1060-fold from mitochondrial matrix proteins of Saccharomyces cerevisiae using a four-step procedure based on affinity chromatography (heparin-Ultrogel, tRNA(Met)-Sepharose, Agarose-hexyl-AMP) to yield to a single polypeptide of high specific activity (1800 U/mg). Like the cytoplasmic methionyl-tRNA synthetase (Mr 85,000), the mitochondrial isoenzyme is a monomer, but of significantly smaller polypeptide size (Mr 65,000). In contrast, the corresponding enzyme of Escherichia coli is a dimer (Mr 152,000) made up of identical subunits. The measured affinity constants of the purified mitochondrial enzyme for methionine and tRNA(Met) are similar to those of the cytoplasmic isoenzyme. However, the two yeast enzymes exhibit clearly different patterns of aminoacylation of heterologous yeast and E. coli tRNA(Met). Furthermore, polyclonal antibodies raised against the two proteins did not show any cross-reactivity by inhibition of enzymatic activity and by the highly sensitive immunoblotting technique, indicating that the two enzymes share little, if any, common antigenic determinants. Taken together, our results further support the belief that the yeast mitochondrial and cytoplasmic methionyl-tRNA synthetases are different proteins coded for by two distinct nuclear genes. Like the yeast cytoplasmic aminoacyl-tRNA synthetases, the mitochondrial enzymes displayed affinity for immobilized heparin. This distinguishes them from the corresponding enzymes of E. coli. Such an unexpected property of the mitochondrial enzymes suggests that they have acquired during evolution a domain for binding to negatively charged cellular components.     

Mutations in the gene encoding the Wnt-signaling component R-spondin 4 (RSPO4) cause autosomal recessive anonychia

Anonychia is an autosomal recessive disorder characterized by the congenital absence of finger- and toenails. In a large German nonconsanguineous family with four affected and five unaffected siblings with isolated total congenital anonychia, we performed genomewide mapping and showed linkage to 20p13. Analysis of the RSPO4 gene within this interval revealed a frameshift and a nonconservative missense mutation in exon 2 affecting the highly conserved first furin-like cysteine-rich domain. Both mutations were not present among controls and were shown to segregate with the disease phenotype. RSPO4 is a member of the recently described R-spondin family of secreted proteins that play a major role in activating the Wnt/ beta -catenin signaling pathway. Wnt signaling is evolutionarily conserved and plays a pivotal role in embryonic development, growth regulation of multiple tissues, and cancer development. Our findings add to the increasing body of evidence indicating that mesenchymal-epithelial interactions are crucial in nail development and put anonychia on the growing list of congenital malformation syndromes caused by Wnt-signaling-pathway defects. To the best of our knowledge, this is the first gene known to be responsible for an isolated, nonsyndromic nail disorder.     

Mutations in the ABCA4 (ABCR) gene are the major cause of autosomal recessive cone-rod dystrophy

The photoreceptor cell-specific ATP-binding cassette transporter gene (ABCA4; previously denoted "ABCR") is mutated, in most patients, with autosomal recessive (AR) Stargardt disease (STGD1) or fundus flavimaculatus (FFM). In addition, a few cases with AR retinitis pigmentosa (RP) and AR cone-rod dystrophy (CRD) have been found to have ABCA4 mutations. To evaluate the importance of the ABCA4 gene as a cause of AR CRD, we selected 5 patients with AR CRD and 15 patients from Germany and The Netherlands with isolated CRD. Single-strand conformation-polymorphism analysis and sequencing revealed 19 ABCA4 mutations in 13 (65%) of 20 patients. In six patients, mutations were identified in both ABCA4 alleles; in seven patients, mutations were detected in one allele. One complex ABCA4 allele (L541P;A1038V) was found exclusively in German patients with CRD; one patient carried this complex allele homozygously, and five others were compound heterozygous. These findings suggest that mutations in the ABCA4 gene are the major cause of AR CRD. A primary role of the ABCA4 gene in STGD1/FFM and AR CRD, together with the gene's involvement in an as-yet-unknown proportion of cases with AR RP, strengthens the idea that mutations in the ABCA4 gene could be the most frequent cause of inherited retinal dystrophy in humans.     

Binding of the anticodon domain of tRNA(fMet) to Escherichia coli methionyl-tRNA synthetase

A stem and loop RNA domain carrying the methionine anticodon (CAU) was designed from the tRNA(fMet) sequence and produced in vitro. This domain makes a complex with methionyl-tRNA synthetase (Kd = 38(+/- 5) microM; 25 degrees C, pH 7.6, 7 mM-MgCl2). The formation of this complex is dependent on the presence of the cognate CAU anticodon sequence. Recognition of this RNA domain is abolished by a methionyl-tRNA synthetase mutation known to alter the binding of tRNA(Met).     

Mutations in mitochondrial tRNA genes: a frequent cause of neuromuscular diseases.

We have sequenced the tRNA genes of mtDNA from patients with chronic progressive external ophthalmoplegia (CPEO) without detectable mtDNA deletions. Four point mutations were identified, located within highly conserved regions of mitochondrial tRNA genes, namely tRNA(Leu)(UAG), tRNA(Ser)(GCU), tRNA(Gly) and tRNA(Lys). One of these mutations (tRNA(Leu)(UAG)) was found in four patients with different forms of mitochondrial myopathy. An accumulation of three different tRNA point mutations (tRNA(Leu)(UAG)), tRNA(Ser)(GCU) and tRNA(Gly) was observed in a single patient, suggesting that mitochondrial tRNA genes represent hotspots for point mutations causing neuromuscular diseases.     

Mutations in the gene encoding tight junction claudin-14 cause autosomal recessive deafness DFNB29

Tight junctions in the cochlear duct are thought to compartmentalize endolymph and provide structural support for the auditory neuroepithelium. The claudin family of genes is known to express protein components of tight junctions in other tissues. The essential function of one of these claudins in the inner ear was established by identifying mutations in CLDN14 that cause nonsyndromic recessive deafness DFNB29 in two large consanguineous Pakistani families. In situ hybridization and immunofluorescence studies demonstrated mouse claudin-14 expression in the sensory epithelium of the organ of Corti.     

Mutations in alpha-tubulin cause abnormal neuronal migration in mice and lissencephaly in humans

The development of the mammalian brain is dependent on extensive neuronal migration. Mutations in mice and humans that affect neuronal migration result in abnormal lamination of brain structures with associated behavioral deficits. Here, we report the identification of a hyperactive N-ethyl-N-nitrosourea (ENU)-induced mouse mutant with abnormalities in the laminar architecture of the hippocampus and cortex, accompanied by impaired neuronal migration. We show that the causative mutation lies in the guanosine triphosphate (GTP) binding pocket of alpha-1 tubulin (Tuba1) and affects tubulin heterodimer formation. Phenotypic similarity with existing mouse models of lissencephaly led us to screen a cohort of patients with developmental brain anomalies. We identified two patients with de novo mutations in TUBA3, the human homolog of Tuba1. This study demonstrates the utility of ENU mutagenesis in the mouse as a means to discover the basis of human neurodevelopmental disorders.     

A missense mutation in the nuclear gene coding for the mitochondrial aspartyl-tRNA synthetase suppresses a mitochondrial tRNA(Asp) mutation

The nuclear suppressor allele NSM3 in strain FF1210-6C/170-E22 (E22), which suppresses a mutation of the yeast mitochondrial tRNA^Asp gene in Saccharomyces cerevisiae, was cloned and identified. To isolate the NSM3 allele, a genomic DNA library using the vector YEp13 was constructed from strain E22. Nine YEp13 recombinant plasmids were isolated and shown to suppress the mutation in the mitochondrial tRNA^Asp gene. These nine plasmids carry a common 4.5-kb chromosomal DNA fragment which contains an open reading frame coding for yeast mitochondrial aspartyl-tRNA synthetase (AspRS) on the basis of its sequence identity to the MSD1 gene. The comparison of NSM3 DNA sequences between the suppressor and the wild-type version, cloned from the parental strain FF1210-6C/170, revealed a G to A transition that causes the replacement of amino acid serine (AGU) by an asparagine (AAU) at position 388. In experiments switching restriction fragments between the wild type and suppressor versions of the NSM3 gene, the rescue of respiratory deficiency was demonstrated only when the substitution was present in the construct. We conclude that the base substitution causes the respiratory rescue and discuss the possible mechanism as one which enhances interaction between the mutated tRNA^Asp and the suppressor version of AspRS.     

Mutations in the novel mitochondrial protein REEP1 cause hereditary spastic paraplegia type 31

Hereditary spastic paraplegia (HSP) comprises a group of clinically and genetically heterogeneous diseases that affect the upper motor neurons and their axonal projections. For the novel SPG31 locus on chromosome 2p12, we identified six different mutations in the receptor expression-enhancing protein 1 gene (REEP1). REEP1 mutations occurred in 6.5% of the patients with HSP in our sample, making it the third-most common HSP gene. We show that REEP1 is widely expressed and localizes to mitochondria, which underlines the importance of mitochondrial function in neurodegenerative disease.     

Importance of the anticodon sequence in the aminoacylation of tRNAs by methionyl-tRNA synthetase and by valyl-tRNA synthetase in an Archaebacterium

The mode of recognition of tRNAs by aminoacyl-tRNA synthetases and translation factors is largely unknown in archaebacteria. To study this process, we have cloned the wild type initiator tRNA gene from the moderate halophilic archaebacterium Haloferax volcanii and mutants derived from it into a plasmid capable of expressing the tRNA in these cells. Analysis of tRNAs in vivo show that the initiator tRNA is aminoacylated but is not formylated in H. volcanii. This result provides direct support for the notion that protein synthesis in archaebacteria is initiated with methionine and not with formylmethionine. We have analyzed the effect of two different mutations (CAU-->CUA and CAU-->GAC) in the anticodon sequence of the initiator tRNA on its recognition by the aminoacyl-tRNA synthetases in vivo. The CAU-->CUA mutant was not aminoacylated to any significant extent in vivo, suggesting the importance of the anticodon in aminoacylation of tRNA by methionyl-tRNA synthetase. This mutant initiator tRNA can, however, be aminoacylated in vitro by the Escherichia coli glutaminyl-tRNA synthetase, suggesting that the lack of aminoacylation is due to the absence in H. volcanii of a synthetase, which recognizes the mutant tRNA. Archaebacteria lack glutaminyl-tRNA synthetase and utilize a two-step pathway involving glutamyl-tRNA synthetase and glutamine amidotransferase to generate glutaminyl-tRNA. The lack of aminoacylation of the mutant tRNA indicates that this mutant tRNA is not a substrate for the H. volcanii glutamyl-tRNA synthetase. The CAU-->GAC anticodon mutant is most likely aminoacylated with valine in vivo. Thus, the anticodon plays an important role in the recognition of tRNA by at least two of the halobacterial aminoacyl-tRNA synthetases.