The mouse N-acetylgalactosamine-6-sulfate sulfatase (Galns) gene: cDNA isolation, genomic characterization, chromosomal assignment and analysis of the 5'-flanking region.

Deficiency of lysosomal enzyme N-acetylgalactosamine-6-sulfate sulfatase (GALNS) leads to mucopolysaccharidosis IV A (MPS IV A), for which there is no definitive treatment so far. Although a number of mutations of the GALNS gene of MPS IV A patients have been described, pathogenesis of the disorder still remains elusive. In order to facilitate in vivo studies using model animals for MPS IV A, we isolated and performed molecular characterization of the mouse homolog of human GALNS. The 2.3-kb cDNA contains a 1560-bp open reading frame encoding 520 amino acid residues. The coding region has 84% similarity to the human GALNS cDNA at amino acid level. The mouse Galns gene was mapped by interspecific backcross analysis to the distal region of chromosome 8 where it co-segregates with Aprt. Northern blot analysis showed a wide expression of a single-copy gene, being higher especially in liver and kidney. The Galns gene was isolated from S129vJ genomic library and its genomic organization was characterized. The mouse Galns gene was about 50-kb long and organized into 14 exons and 13 introns. All intron-exon splice junctions conformed to the GT/AG consensus sequence except exon 8/intron 8 junction. Primer extension shows multiple transcription initiation sites between -44 and -75 although major transcription initiation site was observed at -90 bp from the ATG codon. The 5'-flanking region lacks canonical TATA and CAAT box sequences, but is G+C rich with 10 GC boxes (potential Sp1 binding sites), characteristic of a housekeeping gene promoter.

Mitochondrial trifunctional protein deficiency associated with recurrent myoglobinuria in adolescence.

A 23-year-old man with recurrent myoglobinuria had low muscle-free carnitine levels and deficient fasting ketogenesis. Urinary organic acid analysis showed large amounts of C6-C14 3-hydroxydicarboxylic acids. Mitochondrial trifunctional protein (TP), harboring long-chain enoyl-coenzyme A (CoA) hydratase, long-chain 3-hydroxyacyl-CoA dehydrogenase, and long-chain 3-ketoacyl-CoA thiolase showed markedly decreased activity in fibroblasts. On immunoblot analysis, the TP content of his fibroblasts was less than 2% that of the control cells. TP deficiency can be a life-threatening disorder with early infantile onset, but it can also present in adolescence with recurrent myoglobinuria.

Long-term duration of reduced serum complement level following burn injury.

We report the case of a boy whose serum CH50 was below the detection limit following burn injury and skin transplantation. The APCH50 level was slightly decreased, although C3, C4, and the other complements were within the normal range. Cold activation was not detected in his plasma. His peripheral blood monocyte ratio slightly elevated to 19% and then decreased to 5.9%. In this case, burn injury caused the depletion of the complement, particularly in the alternative pathway, and resulted in the reduced CH50 level, although C3 did not show the typical pattern of alternative pathway depletion. In previously reported cases of burn injury, the CH50 level returned to the normal range within 2 weeks. In this patient the reduced level of CH50 continued for 4 months. We should consider burn injury one of the causes of complement deficiency even in cases with a duration of more than 1 month.

Fluorescence in situ hybridization mapping of the alpha and beta subunits (HADHA and HADHB) of human mitochondrial fatty acid beta-oxidation multienzyme complex to 2p23 and their evolution.

Mitochondrial fatty acid beta-oxidation multienzyme complex/trifunctional protein has an alpha4beta4 structure and catalyzes the second through fourth reactions of the fatty acid beta-oxidation cycle. The alpha and beta subunits (HADHA and HADHB) are members of the enoyl-CoA hydratase/3-hydroxyacyl-CoA dehydrogenase and 3-ketoacyl-CoA thiolase families, respectively. We analyzed the localization of each of these two genes (HADHA and HADHB) by in situ hybridization and found that both can be assigned to human chromosome band 2p23. Since the distance between the two loci is quite short, the two genes seem to exist side by side, as do the two (A and B subunit) genes of the bacterial fatty acid beta-oxidation multienzyme complex. This is an important and interesting finding in that two entirely different genes, encoding two independent proteins forming a multienzyme complex, are adjacent on chromosome band 2p23.

Peroxisome proliferator-induced long chain acyl-CoA thioesterases comprise a highly conserved novel multi-gene family involved in lipid metabolism.

Long chain acyl-CoA esters are important intermediates in degradation and synthesis of fatty acids, as well as having important functions in regulation of intermediary metabolism and gene expression. Although the physiological functions for most acyl-CoA thioesterases have not yet been elucidated, previous data suggest that these enzymes may be involved in lipid metabolism by modulation of cellular concentrations of acyl-CoAs and fatty acids. In line with this, we have cloned four highly homologous acyl-CoA thioesterase genes from mouse, showing multiple compartmental localizations. The nomenclature for these genes has tentatively been assigned as CTE-I (cytosolic), MTE-I (mitochondrial), and PTE-Ia and Ib (peroxisomal), based on the identification of putative targeting signals. Although the various isoenzymes show between 67% and 94% identity at amino acid level, each individual enzyme shows a specific tissue expression. Our data suggest that all four genes are located within a very narrow cluster on chromosome 12 in mouse, similar to a sequence cluster on human chromosome 14, which identified four genes homologous to the mouse thioesterase genes. Four related genes were also identified in Caenorhabditis elegans, all containing putative PTS1 targeting signals, suggesting that the ancestral type I thioesterase gene(s) is/are of peroxisomal origin. All four thioesterases are differentially expressed in tissues examined, but all are inducible at mRNA level by treatment with the peroxisome proliferator clofibrate, or during the physiological condition of fasting, both of which conditions cause a perturbation in overall lipid homeostasis. These results strongly support the existence of a novel multi-gene family cluster of mouse acyl-CoA thioesterases, each with a distinct function in lipid metabolism.

Genes for the human mitochondrial trifunctional protein alpha- and beta-subunits are divergently transcribed from a common promoter region.

Human HADHA and HADHB genes encode the subunits of an enzyme complex, the trifunctional protein, involved in mitochondrial beta-oxidation of fatty acids. Both genes are located in the same region of chromosome 2p23. We isolated genomic clones, including 5' flanking regions, for HADHA and HADHB. Sequencing revealed that both of these genes are linked in a head-to-head arrangement on opposite strands and have in common a 350-bp 5' flanking region. The 5' flanking region has bidirectional promoter activity within this region; two cis elements proved critical for the activity. Transcription factor Sp1 functions as an activator for the bidirectional promoter by binding to both elements. Therefore, expression of trifunctional protein subunits are probably coordinately regulated by a common promoter and by Sp1.

Genomic DNA organization of human mitochondrial very-long-chain acyl-CoA dehydrogenase and mutation analysis.

Very-long-chain acyl-CoA dehydrogenase (VLCAD) is a major enzyme catalyzing long-chain fatty acids in the first step of mitochondrial beta-oxidation system. Inborn error of this enzyme can cause sudden infant death syndrome and hypertrophic cardiomyopathy is present at a significantly high frequency. To investigate VLCAD deficiency at the genomic DNA level, we cloned the VLCAD gene and analyzed the structure. The gene is about 5.4 kb long and contains 20 exons. We performed mutation analysis in two patients, both having a 105 bp deletion encompassing bases 1078-1182 in cDNA. A point mutation (GT-->AT) at 5' splice site of intron 11 was identified in both patients. This mutation seems to cause skipping of exon 11 corresponding to the 105 bp deletion. This is the first documentation of aberrant splicing in the VLCAD gene.

Molecular basis of very long chain acyl-CoA dehydrogenase deficiency in three Israeli patients: identification of a complex mutant allele with P65L and K247Q mutations, the former being an exonic mutation causing exon 3 skipping.

Very long chain acyl-CoA dehydrogenase (VLCAD) deficiency is a life-threatening disorder of mitochondrial fatty acid beta-oxidation. We identified four novel mutations in three unrelated patients. All patients had the severe childhood form of VLCAD deficiency with early onset and high mortality. Immunoblot analysis revealed that VLCAD protein was undetectable in patients 2 and 3, whereas normal-size VLCAD protein and an aberrant form of VLCAD (4kDa smaller) were detected in patient 1. As expected, null mutations were found in patients 2 and 3: patient 2 is homozygous for a frameshift mutation, del 4 bp at 798-801, and patient 3 is homozygous for a nonsense mutation 65C>A(S22X). Patient 1 was homozygous for a complex mutant allele containing two alterations, including a 194C>T transition (P65L) and 739A>C transversion (K247Q); in the case of P65L, the amino acid change does not reduce enzyme activity. However, the nucleotide change resulted in exon 3 skipping, whereas the latter K247Q mutation had a drastic effect on enzyme activity. We verified these events by in vivo splicing experiments and transient expression analysis of mutant cDNAs. The P65L mutation locates 11 bases upstream of a splice donor site of intron 3. This is an example of an exonic mutation which affects exon-splicing.

Genomic and mutational analysis of the mitochondrial trifunctional protein beta-subunit (HADHB) gene in patients with trifunctional protein deficiency.

Mitochondrial trifunctional protein (TP), an enzyme of beta-oxidation, is a multienzyme complex composed of four molecules of the alpha-subunit (HADHA) containing the enoyl-CoA hydratase and 3-hydroxyacyl-CoA dehydrogenase domains and four molecules of the beta-subunit (HADHB) containing the 3-ketoacyl-CoA thiolase domain. An inborn error of this enzyme complex can cause sudden infant death syndrome, acute hepatic encephalopathy or liver failure, skeletal myopathy, or hypertrophic cardiomyopathy. TP deficiency is classified into two different biochemical phenotypes: one represents the existence of both subunits and the lack of only the 3-hydroxyacyl-CoA dehydrogenase activity and the other represents the absence of both subunits and the lack of all three TP activities, although their clinical features are similar. We have identified two Japanese patients with this disorder. Three enzyme activities of TP were undetectable in fibroblasts from these two patients. We detected two mutations in the HADHB gene from two Japanese patients, an exonic single T insertion which created a new cryptic 5' splice site and a G1331A transition (R411 K). Patient 1 was a compound heterozygote, while patient 2 was a homozygote of a G1331A transition.

Formation of the enzyme complex in mitochondria is required for function of trifunctional beta-oxidation protein.

The first Japanese patient with mitochondrial trifunctional protein deficiency has been identified. The patient's alpha and beta-subunits were synthesized, transported into the mitochondria, and converted to the mature size, but rapidly disappeared. The newly synthesized mature alpha and beta-subunits in the control cells were incorporated into the enzyme complex, alpha4beta4, whereas those in the patient's cells were present as monomers. We propose that formation of the enzyme complex is required for stabilization of trifunctional protein.

Characterization of N93S, I312T, and A333P missense mutations in two Japanese families with mitochondrial acetoacetyl-CoA thiolase deficiency.

Mitochondrial acetoacetyl-CoA thiolase (T2) deficiency is an inborn error of ketone body and isoleucine catabolisms. Japanese patients, GK01 and GK19, were found to be compound heterozygotes of 149delC and A333P, and N93S and I312T, respectively. The latter three missense mutations were individually characterized by analyses of transient expression of the cDNAs and heat stability. A333P and I312T subunits showed aberrant electrophoretic mobility on SDS-PAGE. T2 protein was destabilized by A333P and existed as an insoluble form in the mitochondria. I312T mutation also destabilized T2 protein; however, some T2 protein was retained in soluble form and reduced residual activity was apparent. N93S mutation did not change the heat stability of T2 activity and the reduced residual activity was retained, however a considerable amount was observed in an insoluble form. The effects of mutations were interpreted based on a tertiary structural model of a subunit of the human T2. This model was constructed from the X-ray crystal structure of the homologous peroxisomal 3-ketoacyl-CoA thiolase of Saccharomyces cerevisiae. On the basis of this model, the positions of Ala333 and Ile312 were far from the active site and the mutations would be expected to destabilize the tertiary structure of T2 subunit. By contrast, Asn93 is located near the active site and may function to maintain a local loop structure. The mutation of Asn93 could directly disrupt disposition of the active site.

Succinyl-CoA:3-ketoacid CoA transferase (SCOT): cloning of the human SCOT gene, tertiary structural modeling of the human SCOT monomer, and characterization of three pathogenic mutations.

The activity of succinyl-CoA:3-ketoacid CoA transferase (SCOT; locus symbol OXCT; EC 2.8.3.5) is the main determinant of the ketolytic capacity of tissues. Hereditary SCOT deficiency causes episodic ketoacidosis. Here we describe the human SCOT gene, which spans more than 100 kb and contains 17 exons, on chromosome 5p13. We report pathogenic missense mutations in three SCOT-deficient patients designated GS04, 05, and 06. GS04 is a G219E/G324E compound; GS05 is a V221M homozygote, and GS06 is a G324E homozygote. We constructed a tertiary structural model of human SCOT by homology modeling based on the known structure of Acidaminococcus fermentans glutaconate CoA transferase. The model predicts that V221 and G219 are on the dimerizing surface, whereas G324 is near the active site. SCOT activity was reduced to a comparable degree in all three patients, but in a transient expression assay in SCOT-deficient fibroblasts, cDNAs containing G219E and G324E produced no detectable activity, whereas V221M constructs yielded approximately 10% of the control peptide level and detectable specific activity. Interestingly, GS05 had the mildest clinical course reported to date and detectable levels of SCOT protein in fibroblasts.