Biomedical subjects

B McInnes

Publications and source records attributed to B McInnes.

17 recordsLinked to original sources

PubMed1992

Characterization of two HEXB gene mutations in Argentinean patients with Sandhoff disease.

Beta-hexosaminidase A (beta-N-acetyl-D-hexosaminidase, EC 3.2.1.5.2) is a lysosomal hydrolase composed of an alpha- and a beta-subunit. It is responsible for the degradation of GM2 ganglioside. Mutations in the HEXB gene encoded beta-subunit cause a form of GM2 gangliosidosis known as Sandhoff disease. Although this is a rare disease in the general population, several geographically isolated groups have a high carrier frequency. Most notably, a 1 in 16-29 carrier frequency has been reported for an Argentinean population living in an area contained within a 375-km radius from Córdoba. Analysis of the genomic DNA of two patients from this region revealed that one was homozygous for a G to A substitution at the 5' donor splice site of intron 2. This mutation completely abolishes normal mRNA splicing. The other patient was a compared of the intron 2 G-->A substitution and a second allele due to a 4-bp deletion in exon 7. The beta-subunit mRNA of this allele is unstable, presumably as a result of an early stop codon introduced by the deletion. Two novel PCR-based assays were developed to detect these mutations. We suggest that one of these assays could be modified and used as a rapid screening procedure for 5' donor splice site defects in other genes. These results provide a further example of the genetic heterogeneity that can exist even in a small geographically isolated population.

Argentina↗

PubMed1992

Two small deletion mutations of the HEXB gene are present in DNA from a patient with infantile Sandhoff disease.

Lysosomal beta-hexosaminidase (EC 3.2.1.52) occurs as two major isozymes hexosaminidase A (alpha beta) and B (beta beta). The alpha subunit is encoded by the HEXA gene and the beta subunit by HEXB gene. Defects in the alpha or beta subunits lead to Tay-Sachs or Sandhoff disease, respectively. While many HEXA gene mutations have been reported only three HEXB gene mutations are known. We report the characterization of two rare HEXB mutations present in genomic DNA from a single fibroblast cell line, GM203, taken from a patient with the infantile form of Sandhoff disease. The first is a single base pair deletion in exon 7 changing the codon for Gly-258, GGA, to GA and the second, a two base pair deletion in exon 11 changes the codons for Arg-435/Val-436, AGA/GTC, to AGTC. Each mutation produces a frame shift in the affected allele that results in a premature stop codon 17 or 20 codons downstream, respectively. These mutations also result in the inability to detect beta-mRNA by Northern blot analysis of total mRNA. These data are consistent with the idea that the severe infantile form of Tay-Sachs or Sandhoff disease is associated with a total lack of residual hexosaminidase A activity.

Amino Acid Sequence↗

PubMed1992

Identification of a processed pseudogene related to the functional gene encoding the GM2 activator protein: localization of the pseudogene to human chromosome 3 and the functional gene to human chromosome 5.

The GM2 activator protein is an essential substrate cofactor for the hydrolysis of GM2 ganglioside by lysosomal beta-hexosaminidase A (EC 3.2.1.52). There have been conflicting reports as to the chromosomal localization of the gene encoding the activator. We demonstrate here that these conflicts were caused by the presence of a previously unidentified processed activator-pseudogene on chromosome 3, and we confirm a previous ELISA-based localization of the functional activator gene to chromosome 5. Our data indicate that the functional activator locus can still be considered a candidate site for defects causing some forms of spinal muscular atrophy.

Animals↗

PubMed1992

An unusual splicing mutation in the HEXB gene is associated with dramatically different phenotypes in patients from different racial backgrounds.

Sandhoff disease is caused by mutations affecting the beta subunit of lysosomal beta-hexosaminidase (EC 3.2.1.52) and displays a wide spectrum of clinical phenotypes. We report a 57-year-old patient with a very mild phenotype, although residual hexosaminidase A activity in his cultured fibroblasts was less than 3% of normal activity, a level observed in juvenile onset patients. Northern and Western blot analyses confirmed a similar low level of beta subunit-mRNA and mature beta-protein, respectively. Two mutations of the HEXB gene were identified in this patient, a partial 5' gene deletion (a null allele), and a C----T transition 8 nucleotides downstream from the intron 10/exon 11 junction affecting the splicing of the beta subunit-mRNA. In their homozygous forms, the 5' deletion has been previously shown to result in a severe infantile phenotype, and the C----T transition in a juvenile phenotype. The genotype and the low level of residual hexosaminidase A activity would be expected to produce a juvenile Sandhoff phenotype in this patient, as well as in four of his six clinically normal siblings. The biochemical basis of his mild phenotype is uncertain, but may result from genetic variations in the RNA splicing machinery.

Base Sequence↗

PubMed1991

Isolation and expression of a full-length cDNA encoding the human GM2 activator protein.

We report the construction of a cDNA clone encoding a functional GM2-activator protein. The sequence of the complete 5' end of the coding region was determined by direct nucleotide sequencing of a fragment generated by multiple RACE PCR procedures from Hela cell cDNA. Specific oligonucleotides were synthesized from these data which allowed us to produce a PCR fragment that contained the complete coding sequence of the protein. This was then cloned into a mammalian expression vector. The ability of purified hexosaminidase A (beta-N-acetylhexosaminidase, EC 3.2.1.52) to hydrolyse labeled GM2 ganglioside was enhanced 10-fold more by the addition in the assay mix of lysate from transfected COS-1 cells than by the addition of identical amounts of lysate from mock transfected cells. Direct sequencing of PCR fragments from two sources also identified three polymorphisms.

Amino Acid Sequence↗

PubMed1991

Structure-function studies of human interferons-alpha: enhanced activity on human and murine cells.

To identify functionally important regions of the human interferon (IFN)-alpha molecule, mutagenesis in vitro of human IFN-a genes was used to create analogs with deletions or specific amino acid replacements. These analogs were expressed in vitro using SP6 RNA polymerase and a rabbit reticulocyte lysate protein synthesis system. Deletion of 7 highly conserved hydrophilic amino acids from the C-terminus of human IFN-alpha 4 reduced, but did not abolish, antiviral activity on human cells. However, analogs with deletions of 15 or 25 amino acids from the C-terminus, or 28 amino acids from the N-terminus, had no measurable antiviral activity. The antiviral activity of human IFN-alpha 4 was increased by substitution of cysteine for serine at position 86, and lysine for arginine at position 121. However, other amino acid substitutions at positions 121, 122 or 123 reduced antiviral activity. The size of the side chain of the amino acid residue at position 130 was shown to be important. Replacement of the absolutely conserved leucine residue at position 131 with glutamine had little effect on antiviral activity. However, the introduction of a proline residue at this position abolished antiviral activity, probably due to the formation of a beta turn in the polypeptide chain. The antiviral activity of human IFN-alpha 4 on murine cells was increased by substitutions at positions 86, 121 and 133. This study illustrates the utility of the in vitro mutagenesis and rabbit reticulocyte lysate systems for the investigation of structure-function relationships, and extends our knowledge of the biologically active regions and species specificity of the human IFN-alpha molecule.

Animals↗

PubMed1990

Structure-function studies of interferon-alpha based on random mutagenesis and expression in vitro.

An efficient procedure for random chemical mutagenesis was used to create analogs of human interferon (IFN)-alpha 4. Unique restriction enzyme sites were introduced into the human IFN-alpha 4 gene to enable cassetting of the gene for localized random mutagenesis. Single-stranded IFN-alpha 4 DNA was treated with nitrous acid, followed by second-strand synthesis using reverse transcriptase. A 72 base pair cassette spanning the coding region for amino acid residues 120 to 136 (120-136 region) was isolated and cloned into a phagemid vector adjacent to a GC-rich sequence. A DNA segment comprising the IFN-alpha 4 cassette sequence and the GC clamp was excised and electrophoresed on a denaturing gradient gel, which allowed the separation from unmutated DNA of DNA fragments with single base pair changes. DNA fragments with mobility different from that of the unmutated fragment were pooled and cloned into an expression vector. Using this procedure, mutations were found in the DNA of 48% of the clones analyzed. However, mutations at two "hot spots" accounted for 89% of these clones. Four of the IFN-alpha 4 analogs with mutations in the 120-136 region were expressed in vitro. The antiproliferative activities on human Daudi cells of most of the analogs were less than 0.2% of the activity of unmodified IFN-alpha 4, suggesting that the integrity of the carboxy terminus is important for the antiproliferative activity of human IFN-alpha 4.

Amino Acid Sequence↗

PubMed1990

Structure and distribution of an Alu-type deletion mutation in Sandhoff disease.

Sandhoff disease is a recessively inherited lysosomal storage disease resulting from a deficiency of beta-hexosaminidase activity. The enzyme occurs in two major forms, beta-hexosaminidase A, composed of an alpha- and beta-subunit and beta-hexosaminidase B, composed of two beta-subunits. Both isozyme activities are deficient in Sandhoff disease, owing to mutations of the HEXB gene encoding the common beta-subunit. We have cloned and fully characterized a deletion at the HEXB gene from fibroblasts of a patient with the infantile form of Sandhoff disease. The deletion removes approximately 16 kb of DNA including the HEXB promoter, exons 1-5 and part of intron 5. It most likely arose from recombination between two Alu sequences, with the breakpoints occurring at the midpoint between the left and right arms in each case and regenerating an intact Alu element in the deletion sequence. The deletion allele accounts for 27% of the Sandhoff mutant alleles we analyzed. Two cell lines were shown to be homozygous for the deletion and both had the infantile form of the disease. Four additional patients were compound heterozygotes with other mutations, all of whom displayed a different clinical phenotype. Finally, the mutant allele was present in different ethnic backgrounds, suggesting that it may have been subject to genetic drift.

Base Sequence↗

PubMed1989

Functional significance of amino acid residues within conserved hydrophilic regions in human interferons-alpha.

Site-directed in vitro mutagenesis was used to create analogs of human interferons (IFNs)-alpha 1 and -alpha 4. Analogs were expressed in vitro using SP6 RNA polymerase and a rabbit reticulocyte lysate cell-free protein synthesis system. Amino acid substitutions for the highly conserved residues at positions 33, 121, 122 and 123 greatly reduced the antiviral and antiproliferative activities on human cells of IFNs-alpha 1 and -alpha 4. In general, the amino acid substitutions had much less effect on the antiviral activities on bovine, compared with human, cells. Substitutions at positions 31, 41, 42, 124, 134, 135 and 136 had little or no effect on the biological activities of the IFN analogs. The abrogation of antiviral activity resulting from amino acid substitutions for the arginine residue at position 33 suggests that this arginine residue is required for binding to the IFN-alpha receptor on the cell surface.

Amino Acid Sequence↗

PubMed1989

Structure-function studies of interferons-alpha: amino acid substitutions at the conserved residue tyrosine 123 in human interferon-alpha 1.

Analogs of human interferon-alpha 1 (IFN-alpha 1) were created in vitro by site-directed mutagenesis to investigate the structural requirements at amino acid position 123 for binding to the IFN receptor, antiviral activity, and antiproliferative activity. The tyrosine residue 123, which is conserved in all known mammalian IFNs-alpha and -beta, was replaced by each of 6 amino acids or was deleted from the protein. Several of the substitutions at position 123 partly or completely abrogated antiviral and antiproliferative activities of human IFN-alpha 1 when human or murine cells were used but not when bovine cells were used. However, with analogs in which amino acids structurally related to tyrosine, phenylalanine, or tryptophan were substituted at position 123, there was retention of antiviral and antiproliferative activities using homologous cells. Thus, although there is not an absolute requirement for tyrosine at position 123, conformational changes associated with alterations of this residue are prejudicial to the biological functions of the IFN-alpha molecule.

Amino Acids↗

PubMed1988

Amino acid substitutions which alter the antiviral activity of human interferon-alpha 1 on mouse cells.

Human interferon-alpha 1 (IFN-alpha 1) is one of only three human (Hu) IFN-alphas having significant antiviral activity on mouse cells. Specific amino acid substitutions in HuIFN-alpha 1 in the region from amino acids 121 to 136 indicate that this region is critical to the determination of mouse and human cell antiviral activities. Bovine cell activities are relatively unaffected by changes in this region. In particular we have identified the arginine residue at position 125 of human IFN-alpha 1 as a major mediator of the molecules antiviral activity on mouse cells. Various substitutions in the carboxy-terminal region of human IFN-alpha 1 are also evaluated and discussed in the context of recently published data.

Amino Acids↗

PubMed1979