Review of clinical presentation and diagnosis of mucopolysaccharidosis IVA.

Mucopolysaccharidosis type IVA (MPS IVA) was described in 1929 by Luis Morquio from Uruguay and James Brailsford from England, and was later found as an autosomal recessive lysosomal storage disease. MPS IVA is caused by mutations in the gene encoding the enzyme, N-acetylgalactosamine-6-sulfate sulfatase (GALNS). Reduced GALNS activity results in impaired catabolism of two glycosaminoglycans (GAGs), chondroitin-6-sulfate (C6S) and keratan sulfate (KS). Clinical presentations of MPS IVA reflect a spectrum of progression from a severe "classical" phenotype to a mild "attenuated" phenotype. More than 180 different mutations have been identified in the GALNS gene, which likely explains the phenotypic heterogeneity of the disorder. Accumulation of C6S and KS manifests predominantly as short stature and skeletal dysplasia (dysostosis multiplex), including atlantoaxial instability and cervical cord compression. However, abnormalities in the visual, auditory, cardiovascular, and respiratory systems can also affect individuals with MPS IVA. Diagnosis is typically based on clinical examination, skeletal radiographs, urinary GAG, and enzymatic activity of GALNS in blood cells or fibroblasts. Deficiency of GALNS activity is a common assessment for the laboratory diagnosis of MPS IVA; however, with recently increased availability, gene sequencing for MPS IVA is often used to confirm enzyme results. As multiple clinical presentations are observed, diagnosis of MPS IVA may require multi-system considerations. This review provides a history of defining MPS IVA and how the understanding of the disease manifestations has changed over time. A summary of the accumulated knowledge is presented, including information from the International Morquio Registry. The classical phenotype is contrasted with attenuated cases, which are now being recognized and diagnosed more frequently. Laboratory based diagnoses of MPS IVA are also discussed.

Peroxisome assembly factor-2, a putative ATPase cloned by functional complementation on a peroxisome-deficient mammalian cell mutant.

Rat peroxisome assembly factor-2 (PAF-2) cDNA was isolated by functional complementation of peroxisome deficiency of a mutant CHO cell line, ZP92, using transient transfection assay. This cDNA encodes a 978-amino acid protein with two putative ATP-binding sites. PAF-2 is a member of a putative ATPase family, including two yeast gene products essential for peroxisome assembly. A stable transformant of ZP92 with the cDNA was morphologically and biochemically restored for peroxisome biogenesis. Fibroblasts derived from patients deficient in peroxisome biogenesis (complementation group C) were also complemented with PAF-2 cDNA, indicating that PAF-2 is a strong candidate for the pathogenic gene of group C peroxisome deficiency.

A human gene responsible for Zellweger syndrome that affects peroxisome assembly.

The primary defect arising from Zellweger syndrome appears to be linked to impaired assembly of peroxisomes. A human complementary DNA has been cloned that complements the disease's symptoms (including defective peroxisome assembly) in fibroblasts from a patient with Zellweger syndrome. The cause of the syndrome in this patient was a point mutation that resulted in the premature termination of peroxisome assembly factor-1. The homozygous patient apparently inherited the mutation from her parents, each of whom was heterozygous for that mutation.

Animal cell mutants represent two complementation groups of peroxisome-defective Zellweger syndrome.

Generalized peroxisome-deficient disorders including cerebro-hepato-renal Zellweger syndrome, neonatal adrenoleukodystrophy, and infantile Refsum disease are autosomal recessive diseases, where catalase-containing particles (peroxisomes) are morphologically absent. We previously isolated two Chinese hamster ovary (CHO) cell mutants (Z24 and Z65) that resemble the fibroblasts from patients with such diseases, in their defective peroxisome assembly (Tsukamoto, T., S. Yokota, and Y. Fujiki. 1990. J. Cell Biol. 110:651-660). Here we report isolation by the P9OH/UV method of a peroxisome-deficient CHO mutant, ZP92, of the third complementation group distinct from those of Z24 and Z65. Peroxisomal membrane ghosts were noted by immunochemical staining in all of the CHO mutants. Complementation analysis by cell fusion of the CHO mutants with cultured fibroblasts from patients with generalized peroxisomal disorders revealed that two CHO mutants (Z24 and ZP92) represent the human complementation groups, E (the same as group 1 in the U.S.) and C (the same as group 4), respectively. These CHO cell mutants are an apparently relevant animal cell model for studies on the molecular bases and primary defects of human peroxisome-deficient diseases.

Identification of three mutant alleles of the gene for mitochondrial acetoacetyl-coenzyme A thiolase. A complete analysis of two generations of a family with 3-ketothiolase deficiency.

3-Ketothiolase deficiency (3KTD) stems from a deficiency of mitochondrial acetoacetyl-coenzyme A thiolase (T2). We analyzed the molecular basis of 3KTD in two generations of a family. A boy (patient 2, GK04), his father (patient 1, GK05), his mother, and his brother were studied; three mutant alleles of T2 gene were identified. Patient 1 is a compound heterozygote: one allele has a point mutation of G to A at position 547 on his T2 cDNA, causing Gly150 to Arg substitution of the mature T2 subunit, and the other allele has GT to TT transition at the 5' splice site of intron 8, causing exon 8's skipping of the T2 cDNA. Patient 2 is also a compound heterozygote: one allele inherited from his mother has AG to CG transition at the 3' splice site of intron 10, causing exon 11's skipping of the T2 cDNA, and the other allele derived from patient 1 has the G to A mutation (Gly to Arg). The brother of patient 2 is an obligatory carrier with the mutant allele causing the exon 8 skipping. This report seems to be the first complete molecular definition of 3KTD at the gene level.

Defect in biosynthesis of mitochondrial acetoacetyl-coenzyme A thiolase in cultured fibroblasts from a boy with 3-ketothiolase deficiency.

The etiology of 3-ketothiolase deficiency has been attributed to a defect of mitochondrial acetoacetyl-CoA thiolase because the acetoacetyl-CoA thiolase activity in related materials is not activated by K+, a property characteristic for this enzyme. We studied the enzyme protein and the biosynthesis of mitochondrial acetoacetyl-CoA thiolase, using cultured skin fibroblasts from a 5-yr-old boy with 3-ketothiolase deficiency. The following results were obtained. (a) Activation of acetoacetyl-CoA thiolase activity by K+ was nil; (b) The enzyme activity was not affected by treatment with the antibody against mitochondrial acetoacetyl-CoA thiolase; (c) A signal for mitochondrial acetoacetyl-CoA thiolase protein was not detected in the immunoblot analysis; and (d) Pulse-chase experiments of skin fibroblasts, using [35S]methionine, revealed no incorporation of radioactivity into this enzyme. Therefore, fibroblasts from this patient lacked mitochondrial acetoacetyl-CoA thiolase protein due to a defect in its biosynthesis.

Identification of a novel exonic mutation at -13 from 5' splice site causing exon skipping in a girl with mitochondrial acetoacetyl-coenzyme A thiolase deficiency.

We identified a novel exonic mutation which causes exon skipping in the mitochondrial acetoacetyl-CoA thiolase (T2) gene from a girl with T2 deficiency (GK07). GK07 is a compound heterozygote; the maternal allele has a novel G to T transversion at position 1136 causing Gly379 to Val substitution (G379V) of the T2 precursor. In case of in vivo expression analysis, cells transfected with this mutant cDNA showed no evidence of restored T2 activity. The paternal allele was associated with exon 8 skipping at the cDNA level. At the gene level, a C to T transition causing Gln272 to termination codon (Q272STOP) was identified within exon 8, 13 bp from the 5' splice site of intron 8 in the paternal allele. The mRNA with Q272STOP could not be detected in GK07 fibroblasts, presumably because pre-mRNA with Q272STOP was unstable because of the premature termination. In vivo splicing experiments revealed that the exonic mutation caused partial skipping of exon 8. This substitution was thought to alter the secondary structure of T2 pre-mRNA around exon 8 and thus impede normal splicing. The role of exon sequences in the splicing mechanism is indicated by the exon skipping which occurred with an exonic mutation.

Molecular cloning and sequence of the complementary DNA encoding human mitochondrial acetoacetyl-coenzyme A thiolase and study of the variant enzymes in cultured fibroblasts from patients with 3-ketothiolase deficiency.

Complementary DNAs encoding the precursor of human hepatic mitochondrial acetoacetyl-CoA thiolase (T2) (EC 2.3.1.9) were cloned and sequenced. The cDNA inserts in these clones were 1,518 bases in length when overlapped, and encoded the 427-amino acid precursor of this enzyme (45,199 mol wt). This amino acid sequence included a 33-residue leader peptide moiety and a 394-amino acid subunit of the mature enzyme (41,385 mol wt). The T2 gene expression in fibroblasts from four patients with 3-ketothiolase deficiency was analyzed by Northern blotting. The T2 mRNA in all four cell lines had the same 1.7 kb as that of the control. However, the amounts of T2 mRNA differed: the content was reduced in two cell lines (cases 1 and 3), whereas it was within a normal range in others (cases 2 and 4). Pulse labeling followed by subcellular fractionation revealed that the T2 proteins in the fibroblasts from these patients are present in the mitochondria. These results suggest that different mechanisms are involved in the enzyme defects in the four patients.

International Morquio A Registry: clinical manifestation and natural course of Morquio A disease.

Mucopolysaccharidosis IVA (MPS IVA; Morquio A disease) is a lysosomal storage disorder caused by deficiency of N-acetylgalactosamine-6-sulfate sulfatase. The natural history of this disease is incompletely understood. To study which variables influence the clinical outcome, we conducted a study in which MPS IVA patients were asked to fill out a questionnaire with inquiries regarding family history, diagnosis, signs and symptoms, height, weight, surgical history, physical activity, and general complaints. A total of 326 patients (172 male, 154 female) from 42 countries enrolled in the Morquio A Registry programme. The mean age of patients enrolled was 14.9 years for males and 19.1 years for females, with a wide range of 1-73 years. Sixty-four per cent of the patients were under 18 years. Initial symptoms were recognized between 1 and 3 years of age (mean age 2.1 years) and mean age at diagnosis for the patients was 4.7 years. A progressive skeletal dysplasia was commonly observed among the MPS IVA patients. Fifty per cent of patients underwent surgical operations to improve their quality of life. The most frequent surgical sites include neck (51%), ear (33%), leg (26%) and hip (25%). The birth length for affected males and females was 52.2 +/- 4.7 cm and 52.2 +/- 4.5 cm, respectively. The final adult height for affected males and females was 122.5 +/- 22.5 cm and 116.5 +/- 20.5 cm, respectively. The results of this study provide a reference for assessment of efficacy for studies of novel therapies.

Effect of 'attenuated' mutations in mucopolysaccharidosis IVA on molecular phenotypes of N-acetylgalactosamine-6-sulfate sulfatase.

Mucopolysaccharidosis IVA is an autosomal recessive disease caused by the deficiency of N-acetylgalactosamine-6-sulfate sulfatase (GALNS). Mutation screening of the GALNS gene was performed for seven MPS IVA patients with attenuated phenotypes from three unrelated families. Four of 5 missense mutations identified in this study (p.F167V, p.R253W, p.R380S, p.P484S) and two reported (p.F97V, p.N204K), associated with attenuated phenotypes, were characterized using in vitro stable expression experiments, enzyme kinetic study, protein processing and structural analysis. The stably expressed mutant enzymes defining the attenuated phenotype exhibited a considerable residual activity (1.2-36.7% of the wild-type GALNS activity) except for p.R380S. Enzyme kinetic studies showed that p.F97V, p.F167V and p.N204K have lower affinity to the substrate compared with other mutants. The p.F97V enzyme was the most thermolabile at 55 degrees C. Immunoblot analyses indicated a rapid degradation and/or an insufficiency in processing in the mutant proteins. Tertiary structure analysis revealed that although there was a tendency for 'attenuated' mutant residues to be located on the surface of GALNS, they have a different effect on the protein including modification of the hydrophobic core and salt-bridge formation and different potential energy. This study demonstrates that 'attenuated' mutant enzymes are heterogeneous in molecular phenotypes, including biochemical properties and tertiary structure.

Effect of Hunter disease (mucopolysaccharidosis type II) mutations on molecular phenotypes of iduronate-2-sulfatase: enzymatic activity, protein processing and structural analysis.

Mucopolysaccharidosis II (Hunter disease), a lysosomal storage disorder caused by a deficiency of iduronate-2-sulfatase (IDS), has variable clinical phenotypes. Nearly 300 different mutations have been identified in the IDS gene from patients with Hunter disease, but the correlation between the genotype and phenotype has remained unclear. We studied the characteristics of 11 missense mutations, which were detected in the patients or artificially introduced, using stable expression experiments and structural analysis. The mutants found in the attenuated phenotype showed considerable residual activity (0.2-2.4% of the wild-type IDS activity) and those in the severe phenotype had no activity. In immunoblot analysis, both the 73-75 kDa precursor and processed forms were detected in the expression of 'attenuated' mutants (R48P, A85T and W337R) and the artificial active site mutants (C84S, C84T). The 73-75 kDa initial precursor was detected in the 'severe' mutants (P86L, S333L, S349I, R468Q, R468L). The truncated 68 kDa precursor form was synthesized in the Q531X mutant. The results of immunoblotting indicated rapid degradation and/or insufficiency in processing as a result of structural alteration of the IDS protein. A combination of analyses of genotype and molecular phenotypes, including enzyme activity, protein processing and structural analysis with an engineered reference protein, could provide an avenue to understanding the molecular mechanism of the disease and could give a useful tool for the evaluation of possible therapeutic chemical compounds.

Dimorphism of sister chromatid exchange in Bloom's syndrome B- and T-cell lines transformed with Epstein-Barr and adult T-cell leukemia viruses.

The present study describes the establishment of both B- and T-cell lines from the peripheral blood cells of two Bloom's syndrome (BS) patients and one healthy female by using Epstein-Barr (EBV) and adult T-cell leukemia viruses (ATLV). The cell lines from normal and BS subjects exhibited cell surface markers compatible with B- and T-cell origin; in addition, the BS B- and T-cell lines retained the original cytogenetic characteristics of the syndrome. Even though phytohemagglutinin-stimulated BS lymphocytes from the two BS patients studied all showed high levels of sister chromatid exchange (SCE), the established BS B-lines with EBV yielded two separate lines each, i.e., one with increased SCE and another with normal levels of SCE; also, one of the BS T-lines retained high SCE levels in 100% of the cells, whereas the other BS T-line contained two populations, one with high SCE (70%) and the other with normal SCE levels (30%), at a relatively constant frequency over a period of 6 months. Neither EBV nor ATLV caused a significant increase in chromosome instability in the established lines compared to fresh lymphocytes. Reinfection of the BS B- and T-cell lines with EBV or ATLV did not alter the SCE or karyotypes. These results strongly suggest that BS patients have two populations in vivo, one with high and another with normal levels of SCE, at least in the lymphoid cell system.

Molecular basis of 3-ketothiolase deficiency: identification of an AG to AC substitution at the splice acceptor site of intron 10 causing exon 11 skipping.

3-Ketothiolase deficiency (3KTD) is the result of a deficiency in mitochondrial acetoacetyl-CoA thiolase (T2). The molecular basis of 3KTD was analyzed in a patient (GK10) and his family at the protein, cDNA and gene levels. Protein analyses showed that GK10's T2 protein was undetectable in fibroblasts even with the pulse-protein labeling method and that his parents were carriers of 3KTD. Complementary DNA analyses with PCR showed that T2 cDNA in the patient lacked the normal exon 11 sequence and that his parents were obligatory carriers of the DNA sequence which canceled exon 11. When the PCR-amplified genomic fragments around exon 11 were sequenced, an AG to AC mutation at the 3' splice site of intron 10 was detected. This mutation is presumed to be responsible for exon 11 skipping.

Succinyl-CoA:3-ketoacid coenzyme A transferase (SCOT): development of an antibody to human SCOT and diagnostic use in hereditary SCOT deficiency.

Succinyl-CoA:3-ketoacid CoA transferase (SCOT) is a key enzyme for ketone body utilization. Hereditary SCOT deficiency in humans (McKusick catalogue number 245050) is characterized by intermittent ketoacidotic attacks and permanent hyperketonemia. Since previously-available antibody to rat SCOT did not crossreact with human SCOT, we developed an antibody against recombinant human SCOT expressed in a bacterial system. The recombinant SCOT was insoluble except under denaturing conditions. Antibody raised to this polypeptide recognized denatured SCOT and proved useful for immunoblot analysis. On immunoblots, SCOT was easily detectable in control fibroblasts and lymphocytes but was detected neither in fibroblast extracts from four SCOT-deficient patients, nor in lymphocytes from two SCOT-deficient patients. These data indicate that immunoblot analysis is useful for diagnosis of SCOT deficiency in combination with enzyme assay.

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.

A nonsense mutation (TGG [Trp116]-->TAG [Stop]) in CYP11B1 causes steroid 11 beta-hydroxylase deficiency.

Steroid 11 beta-hydroxylase deficiency (11 beta OHD), an autosomal recessive hereditary disease, accounts for 5-8% of cases of congenital adrenal hyperplasia. In this study, we carried out a molecular genetic analysis of CYP11B1 encoding steroid 11 beta-hydroxylase (P450c11) from a Japanese patient affected with this disease. Nucleotide sequence analysis of polymerase chain reaction-amplified products of the patient's genome revealed the occurrence of a stop codon in exon 2 due to a point mutation, TGG-->TAG (Trp116-->Stop). To further analyze the role of CYP11B2 encoding steroid 18-hydroxylase (P450c18) in the 11 beta OHD patient, CYP11B2 of the patient was also amplified and sequenced. In contrast to CYP11B1, there was no mutation in CYP11B2. Restriction fragment length polymorphism analysis indicated that the 11 beta OHD patient is homozygous and his unaffected parents are heterozygous for the mutation. When a cDNA corresponding to CYP11B1 of the 11 beta OHD patient was transfected into COS-7 cells, steroid 11 beta-hydroxylase activity was not detectable in mitochondria of the cells. These results demonstrate that intact P450c11 was not produced at all due to the nonsense mutation in CYP11B1 of the patient without any mutation in CYP11B2.

The organization and the complete nucleotide sequence of the human NADH-cytochrome b5 reductase gene.

The organization and the complete nucleotide (nt) sequence of the b5R gene encoding human NADH-cytochrome b5 reductase (b5R; EC 1.6.2.2) have been determined by a combination of restriction mapping and nt sequence analysis of overlapping genomic DNA clones. The entire gene is about 31 kb in length and contains nine exons and eight introns. Exon 2 contains the junction of the membrane-binding domain and the catalytic domain of b5R, indicating that two forms of b5R, a soluble and a membrane-bound form, are generated by post-translational processing. The 5' portion of the b5R gene lacks the canonical 5' transcriptional regulatory elements, but contains five copies of the GC box sequence G-G-G-C-G-G. While the average G + C content of the b5R gene is 55%, that of the 5' portion of the gene is extraordinarily high (86%). The CpG dinucleotide sequence was found at a very high frequency in this G + C-rich region. These structural features are very similar to those of the regulatory regions of constitutively expressed 'housekeeping' genes. Several transcription start points were identified by the primer extension experiment. Seventeen complete and twelve incomplete Alu family sequences were found in introns. An uncanonical polyadenylation signal was detected in the 3'-untranslated region of the gene as A-G-T-A-A-A instead of A-A-T-A-A-A.

Structure and expression of the human mitochondrial acetoacetyl-CoA thiolase-encoding gene.

To examine 3-ketothiolase deficiency at the gene level, we analyzed the structure of the human mitochondrial acetoacetyl-CoA thiolase (MAT; EC 2.3.1.9)-encoding gene (MAT). From the genomic library of a normal subject in lambda EMBL3, we isolated seven overlapping clones covering the entire length of MAT and the structural organization was determined. The gene spans approx. 27 kb and contains twelve exons interrupted by eleven introns. The 5'-flanking region of the gene lacks a conventional TATA box, but is G + C-rich and contains two CAAT boxes. Included are a putative binding site for the transcription factor, Sp1, and sequences resembling the binding sites of several other transcription factors, all features characteristic of housekeeping genes. A CAT assay revealed that a 101-bp DNA fragment immediately upstream from the cap site has promoter activity, and suggested that a DNA fragment from bp -888 to -102 probably contains a negative regulatory element(s).

Molecular basis of mucopolysaccharidosis type VII: replacement of Ala619 in beta-glucuronidase with Val.

We have identified a mutation causing beta-glucuronidase (beta Gl) deficiency in a 6-year-old girl with mucopolysaccharidosis type VII. Enzyme assay of lysates of a girl's lymphocytes or cultured fibroblasts showed little residual activity and a normal beta Gl-specific mRNA level, as revealed by Northern-blot analysis. Sequencing of the full-length mutated cDNA revealed a C----T transition, an event causing a single Ala619----Val change (we designated this variant beta GGifu). This change is detected by loss of the cleavage site for the enzyme Fnu4HI in the mutated cDNA. On the basis of the loss of Fnu4HI restriction site, the patient was shown to be a homozygote with the beta GGifu mutation and her parents and brother were heterozygotes. This mutation disrupts a functional domain consisting of a region of sequence highly conserved among human, rat and bacterial beta Gls, and it reduces the enzyme activity, as tested by transfection of COS cells with expression vectors harboring the mutated cDNA.

Assessment of liver graft function after cold preservation using 31P and 23Na magnetic resonance spectroscopy.

We investigated the functional damages of the cytoplasm and the cell membrane of liver grafts in male Wister Kyoto rats after 24-hr and 48-hr cold preservations using the University of Wisconsin solution in vitro. Fructose (10 mM) or NH4Cl (50 mM) was added to the perfusate, and synthesis of fructose-1-phosphate (F-1-P) and Na- and H-ion transports through the cell membrane were evaluated by magnetic resonance spectroscopy (MRS), 31P-MRS and 23Na-MRS. After 30 min of reperfusion, beta-ATP/(inorganic phosphate: Pi) of the 48-hr preserved group was significantly lower than the control group and the 24-hr preserved group. The changes of F-1-P in the control group and the 24-hr preserved group were almost the same, but F-1-P synthesis was lower in the 48-hr preserved liver than those of the other groups. Intracellular pH began to drop after the cessation of NH4Cl loading, and then it recovered to the preloading level. At the same time Nain+ was increased in the control group. However, in the other two groups, the increasing rates of Nain+ were lower, and the recoveries of Nain+ were less. In conclusion, the function of cell membrane was more fragile than that of mitochondria and cytoplasmic sugar metabolism in the liver graft.