Mutations affecting the Rossman fold of isoleucyl-tRNA synthetase are correlated with low-level mupirocin resistance in Staphylococcus aureus.

The isoleucyl-tRNA synthetase (ileS) gene was sequenced in toto from 9 and in part from 31 Staphylococcus aureus strains with various degrees of susceptibility to mupirocin. All strains for which the mupirocin MIC was greater than 8 µg/ml contained point mutations affecting the Rossman fold via Val-to-Phe changes at either residue 588 (V588F) or residue 631 (V631F). The importance of the V588F mutation was confirmed by an allele-specific PCR survey of 32 additional strains. Additional mutations of uncertain significance were found in residues clustered on the surface of the IleS protein.

JAK2 exon 12 mutations in polycythemia vera and idiopathic erythrocytosis

Background The V617F mutation, which causes the substitution of phenylalanine for valine at position 617 of the Janus kinase (JAK) 2 gene (JAK2), is often present in patients with polycythemia vera, essential thrombocythemia, and idiopathic myelofibrosis. However, the molecular basis of these myeloproliferative disorders in patients without the V617F mutation is unclear. Methods We searched for new mutations in members of the JAK and signal transducer and activator of transcription (STAT) gene families in patients with V617F-negative polycythemia vera or idiopathic erythrocytosis. The mutations were characterized biochemically and in a murine model of bone marrow transplantation. Results We identified four somatic gain-of-function mutations affecting JAK2 exon 12 in 10 V617F-negative patients. Those with a JAK2 exon 12 mutation presented with an isolated erythrocytosis and distinctive bone marrow morphology, and several also had reduced serum erythropoietin levels. Erythroid colonies could be grown from their blood samples in the absence of exogenous erythropoietin. All such erythroid colonies were heterozygous for the mutation, whereas colonies homozygous for the mutation occur in most patients with V617F-positive polycythemia vera. BaF3 cells expressing the murine erythropoietin receptor and also carrying exon 12 mutations could proliferate without added interleukin-3. They also exhibited increased phosphorylation of JAK2 and extracellular regulated kinase 1 and 2, as compared with cells transduced by wild-type JAK2 or V617F JAK2. Three of the exon 12 mutations included a substitution of leucine for lysine at position 539 of JAK2. This mutation resulted in a myeloproliferative phenotype, including erythrocytosis, in a murine model of retroviral bone marrow transplantation. Conclusions JAK2 exon 12 mutations define a distinctive myeloproliferative syndrome that affects patients who currently receive a diagnosis of polycythemia vera or idiopathic erythrocytosis.

Mice lacking alpha-tocopherol transfer protein gene have severe alpha-tocopherol deficiency in multiple regions of the central nervous system

Ataxia with vitamin E deficiency is caused by mutations in a-tocopherol transfer protein (a-TTP) gene and it can be experimentally generated in mice by a-TTP gene inactivation (a-TTP-KO). This study compared a-tocopherol (a-T) concentrations of five brain regions and of four peripheral organs from 5 months old, male and female, wild-type (WT) and a-TTP-KO mice. All brain regions of female WT mice contained significantly higher a-T than those from WT males. a-T concentration in the cerebellum was significantly lower than that in other brain regions of WT mice. These sex and regional differences in brain a-T concentrations do not appear to be determined by a-TTP expression which was undetectable in all brain regions. All the brain regions of a-TTP-KO mice were severely depleted in a-T. The concentration of another endogenous antioxidant, total glutathione, was unaffected by gender but was decreased slightly but significantly in most brain regions of a-TTP-KO mice. The results show that both gender and the hepatic a-TTP, but not brain a-TTP gene expression are important in determining a-T concentrations within the brain. Interestingly, functional abnormality (ataxia) develops only very late in a-TTP-KO mice in spite of the severe a-tocopherol deficiency in the brain starting at an early age.

Erythrocytosis-associated HIF-2alpha mutations demonstrate a critical role for residues C-terminal to the hydroxylacceptor proline.

Abstract A classic physiologic response to hypoxia in humans is the up-regulation of the ERYTHROPOIETIN (EPO) gene, which is the central regulator of red blood cell mass. The EPO gene, in turn, is activated by hypoxia inducible factor (HIF). HIF is a transcription factor consisting of an alpha subunit (HIF-alpha) and a beta subunit (HIF-beta). Under normoxic conditions, prolyl hydroxylase domain protein (PHD, also known as HIF prolyl hydroxylase and egg laying-defective nine protein) site specifically hydroxylates HIF-alpha in a conserved LXXLAP motif (where underlining indicates the hydroxylacceptor proline). This provides a recognition motif for the von Hippel Lindau protein, a component of an E3 ubiquitin ligase complex that targets hydroxylated HIF-alpha for degradation. Under hypoxic conditions, this inherently oxygen-dependent modification is arrested, thereby stabilizing HIF-alpha and allowing it to activate the EPO gene. We previously identified and characterized an erythrocytosis-associated HIF2A mutation, G537W. More recently, we reported two additional erythrocytosis-associated HIF2A mutations, G537R and M535V. Here, we describe the functional characterization of these two mutants as well as a third novel erythrocytosis-associated mutation, P534L. These mutations affect residues C-terminal to the LXXLAP motif. We find that all result in impaired degradation and thus aberrant stabilization of HIF-2alpha. However, each exhibits a distinct profile with respect to their effects on PHD2 binding and von Hippel Lindau interaction. These findings reinforce the importance of HIF-2alpha in human EPO regulation, demonstrate heterogeneity of functional defects arising from these mutations, and point to a critical role for residues C-terminal to the LXXLAP motif in HIF-alpha.

Identification of gene networks associated with acute myeloid leukemia by comparative molecular methylation and expression profiling

Around 80% of acute myeloid leukemia (AML) patients achieve a complete remission, however many will relapse and ultimately die of their disease. The association between karyotype and prognosis has been studied extensively and identified patient cohorts as having favourable [e.g. t(8; 21), inv (16)/t(16; 16), t(15; 17)], intermediate [e.g. cytogenetically normal (NK-AML)] or adverse risk [e.g. complex karyotypes]. Previous studies have shown that gene expression profiling signatures can classify the sub-types of AML, although few reports have shown a similar feature by using methylation markers. The global methylation patterns in 19 diagnostic AML samples were investigated using the Methylated CpG Island Amplification Microarray (MCAM) method and CpG island microarrays containing 12,000 CpG sites. The first analysis, comparing favourable and intermediate cytogenetic risk groups, revealed significantly differentially methylated CpG sites (594 CpG islands) between the two subgroups. Mutations in the NPM1 gene occur at a high frequency (40%) within the NK-AML subgroup and are associated with a more favourable prognosis in these patients. A second analysis comparing the NPM1 mutant and wild-type research study subjects again identified distinct methylation profiles between these two subgroups. Network and pathway analysis revealed possible molecular mechanisms associated with the different risk and/or mutation sub-groups. This may result in a better classification of the risk groups, improved monitoring targets, or the identification of novel molecular therapies.

Murine model of autosomal dominant retinitis pigmentosa generated by targeted deletion at codon 307 of the rds-peripherin gene

We introduced a targeted single base deletion at codon 307 of the rds-peripherin gene in mice, similar mutations being known to cause autosomal dominant retinitis pigmentosa (RP) in man. Histopathological and electroretinographic analysis indicate that the retinopathy in mice homozygous for the codon 307 mutation appears more rapid than that in the naturally occurring null mutant, the rds(-/-) mouse, suggesting that the rds-307 mutation displays a dominant negative phenotype in combination with that due to haplosufficiency. RP is the most prevalent cause of registered visual handicap in those of working age in developed countries, the 50 or so mutations so far identified within the RDS-peripherin gene accounting for up to 10% of dominant cases of the disease. Given the sequence homologies that exist between the murine rds-peripherin and the human RDS-peripherin gene, this disease model, the first to be generated for peripherin-based RP using gene targeting techniques, should in principle be of value in the work-up in mice of therapeutics capable of targeting transcripts derived from the human gene.

Mutations in sodium-borate cotransporter SLC4A11 cause recessive congenital hereditary endothelial dystrophy (CHED2)

Congenital hereditary endothelial dystrophy ( CHED) is a heritable, bilateral corneal dystrophy characterized by corneal opacification and nystagmus. We describe seven different mutations in the SLC4A11 gene in ten families with autosomal recessive CHED. Mutations in SLC4A11, which encodes a membrane-bound sodium-borate cotransporter, cause loss of function of the protein either by blocking its membrane targeting or nonsense-mediated decay.

HIF pathway mutations and erythrocytosis

Erythrocytosis is present when there is an increase in the red cell mass, usually accompanied by an elevated hemoglobin and hematocrit. This occurs when there is an intrinsic defect in the erythroid component of the bone marrow or for secondary reasons when an increase in erythropoietin production drives red cell production. In normoxic conditions, HIF-alpha interacts with the other proteins in the HIF pathway and is destroyed, but in hypoxic conditions, HIF-alpha binds to HIF-beta. and alters the expression of downstream genes, including the erythropoietin gene. The end result is an increase in erythropoietin production. Mutations in any of the genes in the HIF pathway could lead to changed proteins, abnormalities in the degradation of HIF-alpha and, ultimately, result in increased erythropoietin levels. A number of mutations in the VHL, PHD2, and HIF2A genes have been identified in individuals. These mutations lead to erythrocytosis. The clinical results of these mutations may include some major thromboembolic events in young patients.

Erythrocytosis due to a mutation in the erythropoietin receptor gene

Familial erythrocytosis, associated with high haemoglobin levels and low serum erythropoietin (Epo), has been shown to co-segregate with a sequence repeat polymorphism at the 5' region of the erythropoietin receptor (EpoR) in a large Finnish family. We have investigated the cause of erythrocytosis in an English boy. Sequencing of the cytoplasmic region of the EpoR detected a de novo transition mutation of G to A at nucleotide 6002. This mutation resulted in the formation of a stop codon at amino acid 439 with the loss of 70 amino acids from the carboxy terminus. The mutation (G6002A) has arisen independently in a Finnish family and de novo in this English boy. Patients with unexplained erythrocytosis and low serum Epo levels should be investigated for EpoR mutations.

Exome sequencing of gastric adenocarcinoma identifies recurrent somatic mutations in cell adhesion and chromatin remodelling genes

Gastric cancer is a major cause of global cancer mortality. We surveyed the spectrum of somatic alterations in gastric cancer by sequencing the exomes of 15 gastric adenocarcinomas and their matched normal DNAs. Frequently mutated genes in the adenocarcinomas included TP53 (11/15 tumors), PIK3CA (3/15) and ARID1A (3/15). Cell adhesion was the most enriched biological pathway among the frequently mutated genes. A prevalence screening confirmed mutations in FAT4, a cadherin family gene, in 5% of gastric cancers (6/110) and FAT4 genomic deletions in 4% (3/83) of gastric tumors. Frequent mutations in chromatin remodeling genes (ARID1A, MLL3 and MLL) also occurred in 47% of the gastric cancers. We detected ARID1A mutations in 8% of tumors (9/110), which were associated with concurrent PIK3CA mutations and microsatellite instability. In functional assays, we observed both FAT4 and ARID1A to exert tumor-suppressor activity. Somatic inactivation of FAT4 and ARID1A may thus be key tumorigenic events in a subset of gastric cancers.

Downregulation of the motA gene delays the escape of the obligate predator Bdellovibrio bacteriovorus 109J from bdelloplasts of bacterial prey cells

Bdellovibrio bacteriovorus is a Gram-negative bacterium that preys on other Gram-negative bacteria. The lifecycle of B. bacteriovorus alternates between an extracellular flagellated and highly motile non-replicative attack-phase cell and a periplasmic non-flagellated growth-phase cell. The prey bacterium containing periplasmic bdellovibrios becomes spherical but osmotically stable, forming a structure known as the bdelloplast. After completing the growth phase, newly formed bdellovibrios regain their flagellum and escape the bdelloplast into the environment, where they encounter more prey bacteria. The obligate predatory nature of B. bacteriovorus imposes a major difficulty to introducing mutations in genes directly involved in predation, since these mutants could be non-viable. This work reports the cloning of the B. bacteriovorus 109J motAB operon, encoding proteins from the flagellar motor complex, and a genetic approach based on the expression of a motA antisense RNA fragment to downregulate motility. Periplasmic bdellovibrios carrying the plasmid expressing antisense RNA displayed a marked delay in escaping from bdelloplasts, while the released attack-phase cells showed altered motility. These observations suggest that a functionally intact flagellar motor is required for the predatory lifecycle of B. bacteriovorus. Also, the use of antisense RNA expression may be a useful genetic tool to study the Bdellovibrio developmental cycle.

Molecular cloning of the Haemophilus influenzae gmhA (lpcA) gene encoding a phosphoheptose isomerase required for lipooligosaccharide biosynthesis

We have determined that gene HI#1181 of Haemophilus influenzae is a homolog of Escherichia coli gmhA (previously designated lpcA) (J. S. Brooke and M. A. Valvano, J. Biol. Chem. 271:3608-3614, 1996), which encodes a phosphoheptose isomerase catalyzing the first step of the biosynthesis of ADP-L-glycero-D-manno heptose. Mutations in this gene are associated with a heptoseless core lipopolysaccharide which determines an increased outer membrane permeability to hydrophobic compounds. The cloned H. influenzae gmhA restored the synthesis of a complete core in the gmhA-deleted E. coli strain chi711. Amino acid sequence comparisons of the GmhA proteins of E. coli and H. influenzae with other proteins in the databases revealed the existence of a novel family of phosphosugar a1do-keto isomerases.

Structure-Phenotype Correlations of Human CYP21A2 Mutations in Congenital Adrenal Hyperplasia

Congenital Adrenal Hyperplasia (CAH) is a family of autosomal recessive disorders involving impaired synthesis of cortisol from cholesterol by adrenal cortex. The predominant causes of the disorder are mutations in the CYP21A2 gene that encodes a Cytochrome P450 21-hydroxylase enzyme, which is central to steroidogenesis. The severity of the disease depends upon the extent of impaired enzymatic activity and can be classified under severe Classical form or the mild Non-Classical form, Molecular characterisation of CYP21A2 mutations can be used to predict clinical phenotype and disease severity based upon changes it brings in 21-hydroxylase enzyme structure. A humanized model of CYP21A2 has been used to map and investigate the structural role of all known disease-causing mutations. A structural explanation of clinical manifestation allows us to put forward criteria that might allow the prediction of clinical severity of the disease.

An analysis of ABCR mutations in British patients with recessive retinal dystrophies

PURPOSE. Several reports have shown that mutations in the ABCR gene can lead to Stargardt disease (STGD)/fundus flavimaculatus (FFM), autosomal recessive retinitis pigmentosa (arRP), and autosomal recessive cone-rod dystrophy (arCRD). To assess the involvement of ABCR in these retinal dystrophies, the gene was screened in a panel of 70 patients of British origin. METHODS. Fifty-six patients exhibiting the STGD/FFM phenotype, 6 with arRP, and 8 with arCRD, were screened for mutations in the 50 exons of the ABCR gene by heteroduplex analysis and direct sequencing. Microsatellite marker haplotyping was used to determine ancestry. RESULTS. In the 70 patients analyzed, 31 sequence changes were identified, of which 20 were considered to be novel mutations, in a variety of phenotypes. An identical haplotype was associated with the same pair of in-cis alterations in 5 seemingly unrelated patients and their affected siblings with STGD/FFM. Four of the aforementioned patients were found to carry three alterations in the coding sequence of the ABCR gene, with two of them being in-cis. CONCLUSIONS. These results suggest that ABCR is a relatively polymorphic gene. Because putative mutations have been identified thus far only in 25 of 70 patients, of whom only 8 are compound heterozygotes, a large number of mutations have yet to be ascertained. The disease haplotype seen in the 5 patients carrying the same 'complex' allele is consistent with the presence of a common ancestor.

Mutational spectrum of the ZEB1 gene in corneal dystrophies supports a genotype-phenotype correlation

Mutations in ZEB1 have been reported in posterior polymorphous corneal dystrophy (PPCD3; MIM #609141) and Fuchs' endothelial corneal dystrophy (FECD6; MIM #613270). Although PPCD and keratoconus are clinically and pathologically distinct, PPCD has been associated with keratoconus, suggesting a common genetic basis. The purpose of our study was to perform mutational screening of the ZEB1 gene in patients affected with keratoconus or PPCD.