August F. Van Elsen

ASSIGNMENT OF X-LINKED HYDROCEPHALUS TO XQ28 BY LINKAGE ANALYSIS

X-linked recessive hydrocephalus (HSAS) occurs at a frequency of approximately 1 per 30,000 male births and consists of hydrocephalus, stenosis of the aqueduct of Sylvius, mental retardation, spastic paraparesis, and clasped thumbs. Prenatal diagnosis of affected males by ultrasonographic detection of hydrocephalus is unreliable because hydrocephalus may be absent antenatally. Furthermore, carrier detection in females is not possible because they are asymptomatic. Using four families segregating HSAS, we performed linkage analysis with a panel of X-linked probes that detect restriction fragment length polymorphisms. We report here that HSAS, in all tested families, is closely linked to marker loci mapping in Xq28 (DXS52, lod = 6.52 at theta of 0.03; F8, lod = 4.32 at theta of 0.00; DXS15, lod = 3.40 at theta of 0.00). These data assign HSAS to the gene-dense chromosomal band Xq28 and allow for both prenatal diagnosis and carrier detection by linkage analysis.

Infantile metachromatic leukodystrophy. Confirmation of a prenatal diagnosis.

Abstract Infantile metachromatic leukodystrophy was diagnosed in utero by the absence of arylsulfatase A in cultured amniotic-fluid cells. The diagnosis was confirmed since enzyme activity was not demonstrable in cultured fetal skin fibroblasts, liver, or brain, and since characteristic histologic metachromatic deposits were present in kidney. Electron microscopy showed abnormal cytoplasmic membrane-bound inclusions, composed in part of stacked lamellar structures, in renal tubular cells and in Schwann and endoneurial cells of the sciatic nerve. Intrauterine diagnosis of metachromatic leukodystrophy is thus a potentially valuable method. (N Engl J Med 288:1365–1369, 1973)

Arylsulphatases A and B in human diploid fibroblasts: Differential assay with 4-methylumbelliferylsulphate and AgNO3

A new technique is introduced for the differential assay of arylsulphatases A and B in centrifuged homogenates of cultured human skin fibroblasts, using 4-methylumbelliferyl-sulphate as a substrate and AgNO3 as a selective inhibitor of arylsulphatase A. The method can be applied in the diagnosis of metachromatic leucodystrophy, mucopolysaccharidosis type VI and mucosulphatidosis. Normal arylsulphatase activities were found in fibroblasts derived from patients with mucopolysaccharidoses types II, III-A and IV, known to be caused by deficiencies of various other sulphatases.

pH-Dependent association-dissociation of high and low activity plasma a-l-fucosidase

SummaryPopulation and family studies have confirmed the existence of a plasma a-l-fucosidase polymorphism in humans and the autosomal recessive inheritance of the low activity trait. The frequency of the latter is estimated at 11%. The low activity individuals or variants can also be distinguished by the fact that their plasma a-l-fucosidase is heat-inactivated at acidic pH. Sucrose gradient centrifugation results indicate the transition of non-variant plasma a-l-fucosidase with a molecular weight of 66,000 at pH 8.4 to an enzyme form with a molecular weight of 193,000 at pH 3.0. The former is thermolabile, the latter thermostable. Interconversion is pH-dependent. It is hypothesized that the non-variant enzyme, a monomer at alkaline pH, changes upon acidification into a trimeric conformation via dimerization. The thermolabile variant a-l-fucosidase monomer is not converted into a trimer, but only partially into a dimer.

Human elongation factor 1α: a polymorphic and conserved multigene family with multiple chromosomal localizations

SummaryOne of the genes activated in human melanoma cells by the tumor-promoting phorbol ester is that of the elongation factor 1α. A cDNA clone containing the complete 3′-end untranslated region and the nucleotide sequences coding for 227 carboxyterminal amino acids was isolated. Computer-assisted comparison with known sequences of elongation factors from other species revealed homologies up to 73% and 63% on amino acid and nucleotide sequences, respectively. Northern blot analysis of mRNA from unstimulated and phorbol ester-treated cells showed a 3- to 5-fold increase in cytoplasmic elongation factor 1α mRNA after phorbol ester induction. When compared with the phorbol ester-inducible single-copy gene transcripts coding for the tissue-type plasminogen activator, the cellular mRNA content of elongation factor 1α is 30 times higher. By Southern blot analysis experiments on human genomic DNA, a multi-gene family was found showing polymorphisms in restriction endonuclease fragment lengths (RFLP). Several polymorphisms were studied more extensively in the population on more than 100 DNA samples from normal individuals and in three-generation families. In situ hybridization of the cDNA probe to normal human metaphase chromosomes showed multiple chromosomal localizations of the elongation factor gene(s), with peak hybridization on the chromosomes 1, 2, 4, 5, 6, 7, and 15. The estimate of the gene copy number in humans is more than ten copies per (haploid) genome.

Arginase isoenzymes in human diploid fibroblasts

Arginase activity has been demonstrated in cultured diploid fibroblasts and compared with that of the known arginases in other human tissues. In liver, kidney, erythrocytes and fibroblasts the ratios of specific activities are 750/45/45/1 respectively. Enzyme kinetics, pH-optimum, Km-values and Mn++ effect are similar in all tissues. The known DEAE isoenzymes A1, A3 and A4 also exist in fibroblast strains, which consistently show either A1 and A3 (liver pattern), or A1 and A4 (kidney pattern) or the mixed pattern A1, A3 and A4.

Isoenzymes of serum N-acetyl-beta-D-glucosaminidase in the I cell disease heterozygote

SummarySerum N-acetyl-beta-D-hexosaminidase is compared quantitatively and qualitatively in 14 obligate heterozygotes for the mutant gene causing I cell disease (ICD) or mucolipidosis II and in 31 normal controls. The average specific activity in either group is significantly different but reliable heterozygote detection cannot be achieved because of some overlapping of the ranges of individual results. Fractionation of the enzyme either by DEAE cellulose column chromatography, or by heat inactivation, yields a typical average result for each genotype. Also, mere expression of the various components as percentages of the total activity is not useful for certain identification of the ICD heterozygote. There is considerable overlapping of the percents hexosaminidase I1 and A in both groups of sera. If enzymatic hydrolysis by any component is expressed as a partial activity, a much between though not absolute distinction between the ICD heterozygote and the normal control becomes possible. Only the latter way of expression of hexosaminidase results makes distinction between the ICD heterozygote and the Tay-Sachs heterozygote very probable.

α-l-Fucosidase in human fibroblasts. I. The enzyme activity polymorphism

Low plasma α-l-fucosidase activity is a recessive polymorphic trait observed in 8% of the normal population. The molecular basis of this polymorphism remains unclear and its expression is tissue specific. As the low-activity (variant) phenotype is expressedin vitro in cultured human fibroblasts, this cell type was chosen to study the enzyme activity polymorphism. Fibroblast cell lines derived from individuals with low plasma fucosidase activity (variants) have less than 30% of the fucosidase activity of fibroblast cell lines established from individuals with high plasma fucosidase activity (nonvariants). No qualitative differences in the synthesis, processing, and extracellular release of newly made α-l-fucosidase could be demonstrated among variant and nonvariant cell strains. Cells pulsed with3H-leucine for 10 min produce a 51-kDa protein which is rapidly processed to a 55-kDa intermediate. The latter is converted to a mature 59-kDa intracellular and a 61-kDa extracellular end product, in both variant and nonvariant fibroblast cell lines. Variant and nonvariant fibroblast cell lines also release relatively equal amounts of fucosidase into the extracellular medium. Therefore, differences in processing or extracellular release of fucosidase between variants and nonvariants are not the basic mechanism of this tissue-specific activity polymorphism.

Frequency of the phenylalanine deletion (ΔF508) in the CF gene of Belgian cystic fibrosis patients

Cloning and sequencing of the CF gene has identified a three‐base‐pair deletion (ΔF508) responsible for CF in the majority of CF patients (Kerem et al. 1989). We have used the polymerase chain reaction with oligonucleotide primers bridging the ΔF308 deletion to analyze the presence or absence of this mutation in the Belgian CF population. The ΔF306 mutation was present in 80% (57 on 71) of CF chromosomes from 36 unrelated Belgian CF families from the region of Antwerp. This mutation was associated with haplotype B for the KM.19–XV‐2c RFLPs as 93% (53 on 57) of the CF chromosomes with the Δ306 mutation carried haplotype B.