The frequency of 844ins68 mutation in the cystathionine beta-synthase gene is not increased in patients with venous thrombosis

Background and Objectives. A frequent mutation in the cystathionine beta-synthase (CBS) gene (844ins68, a 68-bp insertion in the coding region of exon 8) was recently discovered. In the present study we Investigated this mutation as a candidate risk factor for venous thrombosis.Design and Methods. The prevalence of the 844ins68 CBS mutation was determined in 101 patients with objectively diagnosed deep venous thrombosis and in 101 healthy controls matched for age, sex and race. PCR amplification of a DNA fragment containing exon 8 of the CBS gene was employed to determine the genotypes, Additionally, Bsrl restriction enzyme digestion of the PCR products was performed in all samples from carriers of the insertion, to test for concurrent presence of a second mutation (T833C) in the CBS gene.Results. The insertion was found in 21 out of 101 patients (20.8%; allele frequency 0.109) and in 20 out of 101 controls (19.8%; allele frequency 0.114), yielding a relative risk for venous thrombosis related to the 844ins68 CBS mutation close to 1.0. In addition, the T833C GBS mutation was detected in all alleles carrying the 844ins68 CBS insertion, confirming the co-inheritance of the two mutations.Interpretation and Conclusions. Our findings do not support the hypothesis that the 844ins68 mutation in the CBS gene is a genetic risk factor for venous thrombosis. (C)1998, Ferrata Storti Foundation.

The frequency of 844ins68 mutation in the cystathionine β-synthase gene is not increased in patients with venous thrombosis

Background and Objectives. A frequent mutation in the cystathionine β- synthase (CBS) gene (844ins68, a 68-bp insertion in the coding region of exon 8) was recently discovered. In the present study we investigated this mutation as a candidate risk factor for venous thrombosis. Design and Methods. The prevalence of the 844ins68 CBS mutation was determined in 101 patients with objectively diagnosed deep venous thrombosis and in 101 healthy controls matched for age, sex and race. PCR amplification of a DNA fragment containing exon 8 of the CBS gene was employed to determine the genotypes. Additionally, Bsrl restriction enzyme digestion of the PCR products was performed in all samples from carriers of the insertion, to test for concurrent presence of a second mutation (T833C) in the CBS gene. Results. The insertion was found in 21 out of 101 patients (20.8%; allele frequency 0.109) and in 20 out of 101 controls (19.8%; allele frequency 0.114), yielding a relative risk for venous thrombosis related to the 844ins68 CBS mutation close to 1.0. In addition, the T833C CBS mutation was detected in all alleles carrying the 844ins68 CBS insertion, confirming the co- inheritance of the two mutations. Interpretation and Conclusions. Our findings do not support the hypothesis that the 844ins68 mutation in the CBS gene is a genetic risk factor for venous thrombosis.

Linker insertion mutagenesis based on IS21 transposition: isolation of an AMP-insensitive variant of catabolic ornithine carbamoyltransferase from Pseudomonas aeruginosa.

The bacterial insertion sequence IS21 when repeated in tandem efficiently promotes non-replicative cointegrate formation in Escherichia coli. An IS21-IS21 junction region which had been engineered to contain unique SalI and BglII sites close to the IS21 termini was not affected in the ability to form cointegrates with target plasmids. Based on this finding, a novel procedure of random linker insertion mutagenesis was devised. Suicide plasmids containing the engineered junction region (pME5 and pME6) formed cointegrates with target plasmids in an E.coli host strain expressing the IS21 transposition proteins in trans. Cointegrates were resolved in vitro by restriction with SalI or BglII and ligation; thus, insertions of four or 11 codons, respectively, were created in the target DNA, practically at random. The cloned Pseudomonas aeruginosa arcB gene encoding catabolic ornithine carbamoyltransferase was used as a target. Of 20 different four-codon insertions in arcB, 11 inactivated the enzyme. Among the remaining nine insertion mutants which retained enzyme activity, three enzyme variants had reduced affinity for the substrate ornithine and one had lost recognition of the allosteric activator AMP. The linker insertions obtained illustrate the usefulness of the method in the analysis of structure-function relationships of proteins.

A first case of protease codon 35 amino acid insertion in a HIV-1 subtype B sequence detected in the Bauru region, state of São Paulo, Brazil: case report

Amino acid insertions in the protease have rarely been described in HIVinfected patients. One of these insertions has recently been described in codon 35, although its impact on resistance remains unknown. This study presents a case of an HIV variant with an insertion in codon 35 of the protease, described for the first time in Bauru, State of Sao Paulo, Brazil, circulating in a 38-year-old caucasian male with asymptomatic HIV infection since 1997. The variant isolated showed a codon 35 insertion of two amino acids in the protease: a threonine and an aspartic acid, resulting in the amino acid sequence E35E_TD.

Hereditary hepatic and systemic amyloidosis caused by a new deletion/insertion mutation in the apolipoprotein AI gene.

We report a Spanish family with autosomal-dominant non-neuropathic hereditary amyloidosis with a unique hepatic presentation and death from liver failure, usually by the sixth decade. The disease is caused by a previously unreported deletion/insertion mutation in exon 4 of the apolipoprotein AI (apoAI) gene encoding loss of residues 60-71 of normal mature apoAI and insertion at that position of two new residues, ValThr. Affected individuals are heterozygous for this mutation and have both normal apoAI and variant molecules bearing one extra positive charge, as predicted from the DNA sequence. The amyloid fibrils are composed exclusively of NH2-terminal fragments of the variant, ending mainly at positions corresponding to residues 83 and 92 in the mature wild-type sequence. Amyloid fibrils derived from the other three known amyloidogenic apoAI variants are also composed of similar NH2-terminal fragments. All known amyloidogenic apoAI variants carry one extra positive charge in this region, suggesting that it may be responsible for their enhanced amyloidogenicity. In addition to causing a new phenotype, this is the first deletion mutation to be described in association with hereditary amyloidosis and it significantly extends the value of the apoAI model for investigation of molecular mechanisms of amyloid fibrillogenesis.

A natural structural variant of the mouse TCR beta-chain displays intrinsic receptor function and antigen specificity.

The Cbeta0 alternate cassette exon is located between the Jbeta1 and Cbeta1 genes in the mouse TCR beta-locus. In T cells with a VDJbeta1 rearrangement, the Cbeta0 exon may be included in TCRbeta transcripts (herein called TCRbeta-Cbeta0 transcripts), potentially inserting an additional 24 aa between the V and C domains of the TCR beta-chain. These TCRbeta splice isoforms may be differentially regulated after Ag activation, because we detected TCRbeta-Cbeta0 transcripts in a high proportion (>60%) of immature and mature T cells having VDJbeta1 rearrangements but found a substantially reduced frequency (<35%) of TCRbeta-Cbeta0 expression among CD8 T cells selected by Ag in vivo. To study the potential activity of the TCRbeta-Cbeta0 splice variant, we cloned full-length TCR cDNAs by single-cell RT-PCR into retroviral expression vectors. We found that the TCRbeta-Cbeta0 splice isoform can function during an early stage of T cell development normally dependent on TCR beta-chain expression. We also demonstrate that T hybridoma-derived cells expressing a TCRbeta-Cbeta0 isoform together with the clonally associated TCR alpha-chain recognize the same cognate peptide-MHC ligand as the corresponding normal alphabetaTCR. This maintenance of receptor function and specificity upon insertion of the Cbeta0 peptide cassette signifies a remarkable adaptability for the TCR beta-chain, and our findings open the possibility that this splice isoform may function in vivo.

Hereditary hepatic and systemic amyloidosis caused by a new deletion/insertion mutation in the apolipoprotein AI gene.

We report a Spanish family with autosomal-dominant non-neuropathic hereditary amyloidosis with a unique hepatic presentation and death from liver failure, usually by the sixth decade. The disease is caused by a previously unreported deletion/insertion mutation in exon 4 of the apolipoprotein AI (apoAI) gene encoding loss of residues 60-71 of normal mature apoAI and insertion at that position of two new residues, ValThr. Affected individuals are heterozygous for this mutation and have both normal apoAI and variant molecules bearing one extra positive charge, as predicted from the DNA sequence. The amyloid fibrils are composed exclusively of NH2-terminal fragments of the variant, ending mainly at positions corresponding to residues 83 and 92 in the mature wild-type sequence. Amyloid fibrils derived from the other three known amyloidogenic apoAI variants are also composed of similar NH2-terminal fragments. All known amyloidogenic apoAI variants carry one extra positive charge in this region, suggesting that it may be responsible for their enhanced amyloidogenicity. In addition to causing a new phenotype, this is the first deletion mutation to be described in association with hereditary amyloidosis and it significantly extends the value of the apoAI model for investigation of molecular mechanisms of amyloid fibrillogenesis.

Regulation of mucoidy in the biological control agent Pseudomonas fluorescens CHA0 and construction of an exopolysaccharide-overproducing variant

Pseudomonas fluorescens CHA0, an effective biological control agent of soilborne plant diseases, is naturally non-mucoid. We have isolated a highly mucoid Tn5 insertion mutant of strain CHA0. The mucoid phenotype was found to be due to the overproduction of exopolysaccharide (EPS), as a result of a mutation in the mucA gene. The wild-type mucA gene was cloned by a two-step, Tn5-dependent cloning procedure previously described and the deduced amino acid sequence showed 71% identity with MucA of P. aeruginosa, a negative regulator of the alternative sigma factor AlgU (=s22, sE). As in P. aeruginosa, mucA is preceded by the algU gene encoding s22 (91% identity at the amino acid sequence level). A mucA in-frame deletion mutant of CHA0 overproduced EPS and formed mucoid colonies, whereas an algU in-frame deletion mutant showed a non-mucoid phenotype. Pyoluteorin, an antibiotic produced by P. fluorescens, was found to be entrapped in EPS of a mucoid mutant. In natural soil, mucoidy negatively affected survival of the bacteria, suggesting that under these conditions the potential to produce abundant EPS does not confer a selective advantage on the bacteria.

A first case of protease codon 35 amino acid insertion in a HIV-1 subtype B sequence detected in the Bauru region, state of São Paulo, Brazil: case report

Inserções de aminoácidos na protease têm sido raramente descritas em pacientes infectados pelo HIV. Uma destas inserções foi, recentemente, descrita no codon 35, embora seu impacto na resistência mantém-se pouco conhecido. Este trabalho apresenta um caso de uma variante viral com inserção no codon 35 da protease, descrita pela primeira vez em Bauru, São Paulo, Brasil, circulante em um homem, caucasiano, com 38 anos, o qual apresenta infecção assintomática pelo HIV desde 1997. A variante isolada mostrou uma inserção no codon 35 da protease de dois aminoácidos: uma treonina e um ácido aspártico, resultando na sequência de aminoácidos E35E_TD.

A RAB3GAP1 SINE Insertion in Alaskan Huskies with Polyneuropathy, Ocular Abnormalities and Neuronal Vacuolation (POANV) Resembling Human Warburg Micro Syndrome 1 (WARBM1)

We observed a hereditary phenotype in Alaskan Huskies, which was characterized by polyneuropathy with ocular abnormalities and neuronal vacuolation (POANV). The affected dogs developed a progressive severe ataxia, which led to euthanasia between 8 and 16 months of age. The pedigrees were consistent with a monogenic autosomal recessive inheritance. We localized the causative genetic defect to a 4 Mb interval on chromosome 19 by a combined linkage and homozygosity mapping approach. Whole genome sequencing of one affected dog, an obligate carrier and an unrelated control revealed a 218 bp SINE insertion into exon 7 of the RAB3GAP1 gene. The SINE insertion was perfectly associated with the disease phenotype in a cohort of 43 Alaskan Huskies and it was absent from 541 control dogs of diverse other breeds. The SINE insertion induced aberrant splicing and led to a transcript with a greatly altered exon 7. RAB3GAP1 loss-of-function variants in humans cause Warburg Micro Syndrome 1 (WARBM1), which is characterized by additional developmental defects compared to canine POANV, whereas Rab3gap1 deficient mice have a much milder phenotype than either humans or dogs. Thus the RAB3GAP1 mutant Alaskan Huskies provide an interesting intermediate phenotype that may help to better understand the function of RAB3GAP1 in development. Furthermore, the identification of the presumed causative genetic variant will enable genetic testing to avoid the non-intentional breeding of affected dogs.

A transposable element insertion in APOB causes cholesterol deficiency in Holstein cattle

Cholesterol deficiency, a new autosomal recessive inherited genetic defect in Holstein cattle, has been recently reported to have an influence on the rearing success of calves. The affected animals show unresponsive diarrhea accompanied by hypocholesterolemia and usually die within the first weeks or months of life. Here, we show that whole genome sequencing combined with the knowledge about the pedigree and inbreeding status of a livestock population facilitates the identification of the causative mutation. We resequenced the entire genomes of an affected calf and a healthy partially inbred male carrying one copy of the critical 2.24-Mb chromosome 11 segment in its ancestral state and one copy of the same segment with the cholesterol deficiency mutation. We detected a single structural variant, homozygous in the affected case and heterozygous in the non-affected carrier male. The genetic makeup of this key animal provides extremely strong support for the causality of this mutation. The mutation represents a 1.3kb insertion of a transposable LTR element (ERV2-1) in the coding sequence of the APOB gene, which leads to truncated transcripts and aberrant splicing. This finding was further supported by RNA sequencing of the liver transcriptome of an affected calf. The encoded apolipoprotein B is an essential apolipoprotein on chylomicrons and low-density lipoproteins, and therefore, the mutation represents a loss of function mutation similar to autosomal recessive inherited familial hypobetalipoproteinemia-1 (FHBL1) in humans. Our findings provide a direct gene test to improve selection against this deleterious mutation in Holstein cattle.

AsaGEI2b: a new variant of a genomic island identified in the Aeromonas salmonicida subsp. salmonicida JF3224 strain isolated from a wild fish in Switzerland.

Aeromonas salmonicida subsp. salmonicida is the causal agent of furunculosis in salmonids. We recently identified a group of genomic islands (AsaGEI) in this bacterium. AsaGEI2a, one of these genomic islands, has almost exclusively been identified in isolates from North America. To date, Aeromonas salmonicida subsp. salmonicida JF3224, a strain isolated from a wild brown trout (Salmo trutta) caught in Switzerland, was the only European isolate that appeared to bear AsaGEI2a. We analyzed the genome of JF3224 and showed that the genomic island in JF3224 is a new variant of AsaGEI, which we have called AsaGEI2b. While AsaGEI2b shares the same integrase gene and insertion site as AsaGEI2a, it is very different in terms of many other features. Additional genomic investigations combined with PCR genotyping revealed that JF3224 is sensitive to growth at 25°C, leading to insertion sequence-dependent rearrangement of the locus on the pAsa5 plasmid that encodes a type three secretion system, which is essential for the virulence of the bacterium. The analysis of the JF3224 genome confirmed that AsaGEIs are accurate indicators of the geographic origins of A. salmonicida subsp. salmonicida isolates and is another example of the susceptibility of the pAsa5 plasmid to DNA rearrangements.

Structure and function of the Bacillus hybrid enzyme GluXyn-1: Native-like jellyroll fold preserved after insertion of autonomous globular domain

The 1,3–1,4-β-glucanase from Bacillus macerans (wtGLU) and the 1,4-β-xylanase from Bacillus subtilis (wtXYN) are both single-domain jellyroll proteins catalyzing similar enzymatic reactions. In the fusion protein GluXyn-1, the two proteins are joined by insertion of the entire XYN domain into a surface loop of cpMAC-57, a circularly permuted variant of wtGLU. GluXyn-1 was generated by protein engineering methods, produced in Escherichia coli and shown to fold spontaneously and have both enzymatic activities at wild-type level. The crystal structure of GluXyn-1 was determined at 2.1 Å resolution and refined to R = 17.7% and R(free) = 22.4%. It shows nearly ideal, native-like folding of both protein domains and a small, but significant hinge bending between the domains. The active sites are independent and accessible explaining the observed enzymatic activity. Because in GluXyn-1 the complete XYN domain is inserted into the compact folding unit of GLU, the wild-type-like activity and tertiary structure of the latter proves that the folding process of GLU does not depend on intramolecular interactions that are short-ranged in the sequence. Insertion fusions of the GluXyn-1 type may prove to be an easy route toward more stable bifunctional proteins in which the two parts are more closely associated than in linear end-to-end protein fusions.

Inactive conformation of the serpin α1-antichymotrypsin indicates two-stage insertion of the reactive loop: Implications for inhibitory function and conformational disease

The serpins are a family of proteinase inhibitors that play a central role in the control of proteolytic cascades. Their inhibitory mechanism depends on the intramolecular insertion of the reactive loop into β-sheet A after cleavage by the target proteinase. Point mutations within the protein can allow aberrant conformational transitions characterized by β-strand exchange between the reactive loop of one molecule and β-sheet A of another. These loop-sheet polymers result in diseases as varied as cirrhosis, emphysema, angio-oedema, and thrombosis, and we recently have shown that they underlie an early-onset dementia. We report here the biochemical characteristics and crystal structure of a naturally occurring variant (Leu-55–Pro) of the plasma serpin α1-antichymotrypsin trapped as an inactive intermediate. The structure demonstrates a serpin configuration with partial insertion of the reactive loop into β-sheet A. The lower part of the sheet is filled by the last turn of F-helix and the loop that links it to s3A. This conformation matches that of proposed intermediates on the pathway to complex and polymer formation in the serpins. In particular, this intermediate, along with the latent and polymerized conformations, explains the loss of activity of plasma α1-antichymotrypsin associated with chronic obstructive pulmonary disease in patients with the Leu-55–Pro mutation.

Comment On 'practical Advice For Avoidance Of Pain Associated With Insertion Of Intrauterine Contraceptives': Authors' Response.

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