Glycogen storage disease type Ia: recent experience with mutation analysis, a summary of mutations reported in the literature and a newly developed diagnostic flow chart.

UNLABELLED: We studied the glucose-6-phosphatase (G6Pase) gene of 30 unrelated glycogen storage disease type Ia (GSD Ia) patients using single strand conformational polymorphism (SSCP) prior to automated sequencing of exons revealing an aberrant SSCP pattern. In all patients we could identify mutations on both alleles of the G6Pase gene, indicating that this method is a reliable procedure. A total of 14 different mutations were identified. R83C (16/60), 158delC (12/60), Q347X (7/60), R170X (6/60) and deltaF327 (4/60) were found most frequently. Nine other mutations accounted for the other 15 mutant alleles. Two DNA-based prenatal diagnoses were performed successfully. At present, 56 mutations in the G6Pase gene have been reported in 300 unrelated GSD Ia patients and an overview of these mutations is presented. Evidence for a clear genotype-phenotype correlation could be established neither from our data nor from those in the literature. With increased knowledge about the genetic basis of GSD Ia and GSD Ib and the high detection rate of mutations, it is our opinion that the diagnoses GSD Ia and GSD Ib can usually be based on clinical and biochemical abnormalities combined with mutation analysis instead of enzyme assays in liver tissue obtained by biopsy. A newly developed flowchart for the diagnosis of GSD I is presented. CONCLUSION: Increased knowledge of the genetic basis of glycogen storage disease type I provides a DNA-based diagnosis, prenatal DNA-based diagnosis in chorionic villus samples and carrier detection.

Identification of a novel mutation (867delA) in the glucose-6-phosphatase gene in two siblings with glycogen storage disease type Ia with different phenotypes.

We identified a novel mutation (867delA) in the glucose-6-phosphatase gene of two siblings with glycogen storage disease type Ia. Although both siblings share the same mutations, their phenotype regarding adult height and hepatomegaly differs. In glycogen storage disease type Ia, substantial heterogeneity in phenotype is observed. So far, no evidence for a clear genotype-phenotype correlation has been found. Hum Mutat 15:381, 2000.

Glycogen storage disease type Ia: four novel mutations (175delGG, R170X, G266V and V338F) identified. Mutations in brief no. 220. Online.

Deficient activity of glucose-6-phosphatase (G6Pase) causes glycogen storage disease type Ia (GSD Ia). We analysed the G6Pase gene of 16 GSD Ia patients using single strand conformation polymorphism (SSCP) analysis prior to automated sequencing of exon(s) revealing an aberrant SSCP pattern. In all GSD Ia patients we were able to identify mutations on both alleles of the G6Pase gene, indicating that this method is a reliable procedure to identify mutations. Four novel mutations (175delGG, R170X, G266V and V338F) were identified.

A general system for generating unlabelled gene replacements in bacterial chromosomes.

A general system is described that facilitates gene replacements such that the recombinant strains are not labelled with antibiotic resistance genes. The method is based on the conditional replication of derivatives of the lactococcal plasmid pWV01, which lacks the repA gene encoding the replication initiation protein. Replacement vectors can be constructed in and isolated from gram-positive and gram-negative helper strains that provide RepA in trans. Cointegrate formation of the integration vectors with the chromosome of the target strain is selected by antibiotic resistance. Resolution of the cointegrate structure is identified in the second step of the procedure by the loss of the lacZ reporter gene present in the delivery vector. The second recombination event results either in gene replacement or in restoration of the original copy of the gene. As no antibiotic resistance marker is present in the genome of the mutant the system can be used to introduce multiple mutations in one strain. A feasibility study was performed using Lactococcus lactis and Bacillus subtilis as model organisms. The results indicate that the method should be applicable to any non-essential gene in numerous bacterial species.

A general method for the consecutive integration of single copies of a heterologous gene at multiple locations in the Bacillus subtilis chromosome by replacement recombination.

We have devised a two-step procedure by which multiple copies of a heterologous gene can be consecutively integrated into the Bacillus subtilis 168 chromosome without the simultaneous integration of markers (antibiotic resistance). The procedure employs the high level of transformability of B. subtilis 168 strains and makes use of the observation that thymine-auxotrophic mutants of B. subtilis are resistant to the folic acid antagonist trimethoprim (Tmpr), whereas thymine prototrophs are sensitive. First, a thymine-auxotrophic B. subtilis mutant is transformed to prototrophy by integration of a thymidylate synthetase-encoding gene at the desired chromosomal locus. In a second step, the mutant strain is transformed with a DNA fragment carrying the heterologous gene and Tmpr colonies are selected. Approximately 5% of these appear to be thymine auxotrophic and contain a single copy of the heterologous gene at the chromosomal locus previously carrying the thymidylate synthetase-encoding gene. Repetition of the procedure at different locations on the bacterial chromosome allows the isolation of strains carrying multiple copies of the heterologous gene. The method was used to construct B. subtilis strains carrying one, two, and three copies of the Bacillus stearothermophilus branching enzyme gene (glgB) in their genomes.

Molecular cloning and sequence of comK, a gene required for genetic competence in Bacillus subtilis.

The transformation-deficient strain E26, isolated as a pHV60 insertion mutant, was used to isolate comK, a novel transcription unit required for genetic competence in Bacillus subtilis. Mutational analysis and sequence determination showed that comK contained one open reading frame (ORF), which could encode a protein of 192 amino acid residues with a predicted molecular weight of 22,500. An integrated copy of comK not only complemented the competence deficiency of a comK deletion mutant, but also that of strains E26 and FB93. Expression of comK occurred exclusively in glucose-based minimal medium during the transition to stationary growth phase. Furthermore, the expression of late competence genes appeared to be dependent on the gene product of comK, the expression of which in turn depended on the presence of a functional comL (or srfA) transcription unit. These epistatic interactions indicate that comK is a competence locus occupying an intermediate position in the competence signal transduction network. Primer extension analysis showed that comK has one major transcription start site, preceded by a sequence resembling the consensus promoter used by the sigma A form of RNA polymerase.

Two putative insertion sequences flank a truncated glycogen branching enzyme gene in the thermophile Bacillus stearothermophilus CU21.

We have isolated a region from the Bacillus stearothermophilus CU21 chromosome hybridizing strongly to a fragment of the B. caldolyticus glycogen operon. Sequence analysis of this region revealed the presence of a truncated glgB gene encoding the N-terminus of branching enzyme. A region highly similar to an internal fragment of B. caldolyticus glgC encoding ADP-glucose pyrophosphorylase was located approximately 1kb downstream from the incomplete glgB gene. The two truncated genes appeared to flank a sequence with characteristics of bacterial Insertion Sequences, which was designated RSBst-alpha. The presence of RSBst-alpha at this position indicates that integration of (an) IS-like element(s) may have been involved in deletion formation in the putative glycogen operon. Upstream of glgB an additional incomplete ORF was found with significant similarity to putative transposases from bacterial Insertion Sequences. This region was designated RSBst-beta. Both RSBst-alpha and RSBst-beta appeared to be present in multiple copies in the B. stearothermophilus CU21 chromosome.

Nucleotide sequence of the Synechococcus sp. PCC7942 hemE gene encoding the homologue of mammalian uroporphyrinogen decarboxylase.

We have determined the complete nucleotide sequence of a Synechococcus sp. PCC7942 gene encoding the homologue of mammalian uroporphyrinogen decarboxylase (UROD). The gene, designated hemE, encoded a polypeptide of 354 amino acids with a molecular weight of 39,283. The primary sequences of the polypeptide encoded by hemE and human and rat UROD had 32.5% identical amino acid residues. No invariant cysteine residues were found, despite the fact that UROD isolated from different sources has been shown to be inhibited by sulfhydryl reagents. The knowledge of the primary structure of this cyanobacterial protein may be helpful in better understanding the structural alterations and functional abnormalities of UROD in patients suffering from Porphyria Cutanea Tarda (PCT).

Isolation of a catabolite repression mutant of yeast as a revertant of a strain that is maltose negative in the respiratory-deficient state.

A character originating from Saccharomyces cerevisiae 1403-7A is described which interferes with maltose growth in the respiratory-deficient state. This character is inherited in an apparently non-Mendelian way, but at present no statement can be made concerning the localization of this character on a plasmid or the involvement of multiple genes. As a revertant of this character, a flaky mutant was isolated, showing a heavy flocculation during growth on liquid medium and resistance to catabolite repression for maltase, alpha-methyl-glucosidase, invertase, and succinate dehydrogenase. In wild-type cells, repression (caused by growth on 2% glucose) and derepression (caused by growth on 2% galactose) can be correlated with a lower and a higher level of cyclic 3',5'-adenosine monophosphate (cAMP), respectively. In cells of flaky mutant, growth on these carbon sources results in the same levels of cAMP as observed for the wild type. Consequently, in this mutant derepression in the presence of 2% glucose is not reflected in a higher level of cAMP.