The genetics of oncology.
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Breast Neoplasms↗
Biomedical subjects
J A Lowden
Publications and source records attributed to J A Lowden.
Genetic discrimination and insurance underwriting.
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Genetic Diseases, Inborn↗
Molecular heterogeneity in the infantile and juvenile forms of Sandhoff disease (O-variant GM2 gangliosidosis).
There are two major beta-hexosaminidase, EC 3.2.1.52, isozymes in normal human tissues. They exist as active dimers of alpha- and/or beta-subunits. A defect of their beta-subunit results in Sandhoff disease (O-variant GM2 gangliosidosis), an inherited, clinically heterogeneous, lysosomal storage disease. The status of the HEXB gene, pre beta-polypeptide chain mRNA, and residual beta-hexosaminidase activities were examined in a clinically and ethnically diverse collection of 16 fibroblast cell lines from patients with Sandhoff disease. Differentiation of the two major clinical types, infantile and juvenile onset, could be made by the determination of the activity of the residual beta-hexosaminidase eluting in the same pH range as hexosaminidase A. All the juvenile lines were found to have normal or reduced levels of pre beta-chain mRNA and no gross abnormalities in the HEXB gene. Of the 11 infantile type cell lines examined, four were found to contain no detectable pre beta-chain mRNA. Two cell lines in this group contained partial gene deletions localized to the 5' end of the HEXB gene. One of these cell lines has previously been assigned to the single complementation group in Sandhoff disease, conclusively demonstrating that the primary gene defect in the majority of Sandhoff cases is in the HEXB gene itself. These data suggest that each clinical group is made up of a collection of different HEXB mutations.
Base Sequence↗
Isolation of cDNA clones coding for the alpha-subunit of human beta-hexosaminidase. Extensive homology between the alpha- and beta-subunits and studies on Tay-Sachs disease.
The lysosomal beta-hexosaminidases (N-acetyl-beta-glucosaminidase, EC 3.2.1.30) occur as two major isozymes, hexosaminidase A (alpha beta a beta b) and hexosaminidase B (2(beta a beta b)). To facilitate the investigations of the biosynthesis and structure of the enzymes and the nature of mutation in Tay-Sachs disease, we have isolated cDNA clones coding for the alpha-subunit. The polypeptide chains of hexosaminidase A (30 mg) were digested with trypsin, and peptides were isolated by reverse phase high pressure liquid chromatography and their amino acid sequences determined. One of alpha-chain peptides contained a string of seven amino acids from which two sets of oligonucleotides were specified. They were used to screen the SV40-transformed human fibroblast cDNA library of Okayama and Berg. Three cDNA clones, designated pHexA, identified from among 5 X 10(5) clones screened, contained the deduced amino-acid sequences of five alpha-chain peptides. Genomic DNA homologous to pHexA cDNA mapped to human chromosome 15 in somatic cell hybrids, as expected for the pre-alpha-polypeptide. Two of the clones contained identical polyadenylation sites, while the third was polyadenylated about 450 base pairs downstream. The two types of clones were found to correspond to a major 2.0-kilobase pair and a minor 2.3-kilobase pair mRNA species. Blot hybridizations of mRNA and DNA from Tay-Sachs variant fibroblasts revealed absence or reduction of levels of both mRNA species among infantile and juvenile variants, but no observable DNA alterations. Alignment of the pre-alpha- and pre-beta-polypeptides revealed 55% nucleotide and 57% amino acid homology. These data suggest a common origin of the HEXA and HEXB genes and account for the similar substrate specificities of the alpha-dimer subunit, hexosaminidase S, and hexosaminidase B.
Amino Acid Sequence↗
Fatal metabolic disease and family breakdown.
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Adjustment Disorders↗
HPLC analysis of urinary sulfatide: an aid in the diagnosis of metachromatic leukodystrophy.
Metachromatic leukodystrophy (MLD) presents as six separate variant forms, four allelic and two non-allelic. It is diagnosed in the laboratory by a decrease in the fibroblast or leukocyte arylsulfatase A activity, generally against an artificial substrate. Since residual enzyme activity is not always an indicator of presence or absence of disease, it may be helpful to supplement this information with that of the presence or absence of sulfatide storage in the body. We have improved the HPLC analysis of sulfatide by the use of a sulfated internal standard, sulfatoxymonoalkylmonoacylgalactosylglycerol. Normal urines contain approximately 0 to 0.2 nmol sulfatide/mg creatinine, whereas MLD urines may contain 5 to 7.5 nmol/mg. There is no increase in plasma sulfatide compared to controls in the age group of MLD patients which we studied (up to 4 years).
Cerebroside-Sulfatase↗
Isolation of cDNA clones coding for the beta subunit of human beta-hexosaminidase.
The major forms of beta-hexosaminidase (2-acetamido-2-deoxy-beta-D-glucoside acetamidodeoxyglucohydrolase, EC 3.2.1.30) occur as multimers of alpha and beta chains--hexosaminidase A (alpha beta a beta b) and hexosaminidase B 2(beta a beta b). To facilitate the investigation of beta-chain biosynthesis and the nature of mutation in Sandhoff disease, a human hexosaminidase beta-chain cDNA clone was isolated. Hexosaminidase B (10 mg) was treated with CNBr, five peptide fragments were isolated by reverse-phase HPLC, and their amino acid sequences were determined. One of these contained a string of six amino acids from which an oligonucleotide probe was defined. The simian virus 40-transformed human fibroblast cDNA library of Okayama and Berg was screened by colony hybridization with the radiolabeled probe. Thirteen probe-binding clones were selected out of 50,000 clones screened. Four of these designated pHex were shown to be identical at their 3' ends by restriction enzyme mapping, differing only in their 5' extensions (1.4-1.7 kilobases). The nucleotide sequence of a 174-base-pair segment contained the deduced amino acid sequence of two of the five CNBr peptides, indicating that the pHex clones encode the beta subunit of hexosaminidase. In addition, pHex cDNA was found homologous to multiple bands in digests of genomic human DNA totaling 43 kilobases (kb), all of which were mapped to chromosome 5 in somatic cell hybrids, as expected of the HEXB gene. The pHex cDNA also hybridized to a 2.2-kilobase RNA that apparently codes for the pre-beta-polypeptide of hexosaminidase. This RNA species was absent in the fibroblasts of one of three patients with Sandhoff disease examined. We anticipate that these clones will be of value to diagnosis and carrier detection of Sandhoff disease in affected families.
Amino Acid Sequence↗
Discrimination of the isozymes of human placental hexosaminidase by kinetic parameter estimation.
Human placental beta-N-acetylhexosaminidase (EC 3.2.1.52) (HEX) is a lysosomal glycosyl hydrolase with an acidic pH optimum. Four isozymes (HEX B, HEX I1, HEX I2, and HEX A) have been isolated from human placenta. HEX BA derived from the subunit rearrangement of HEX A was also prepared. To determine if the isozymes of hexosaminidase differ in their kinetic parameters, the conditions for 4-methylumbelliferyl-beta-D-N-acetylglucosaminide hydrolysis were optimized for each isozyme. The Km values were essentially similar and varied from 0.64 +/- 0.06 for HEX BA to 0.85 +/- 0.13 for HEX I1. The Vmax values were similar only for HEX I1 (3.90 +/- 0.28 kat kg-1) and HEX I2 (4.40 +/- 0.17). Vmax values varied significantly between HEX A (9.68 +/- 0.52), HEX B (8.00 +/- 0.75), HEX BA (4.81 +/- 0.17), and the HEX I values.
Electrophoresis, Polyacrylamide Gel↗
A statistical comparison of parameter estimation for the Michaelis-Menten kinetics of human placental hexosaminidase.
To enable the most effective method of kinetic discrimination between a group of isozymes such as those of human placental hexosaminidases (HEX), three methods estimating the parameters of the Michaelis-Menten equation were evaluated. Computer-simulated experiments were performed under various conditions. They indicated that, in the presence of constant absolute or relative errors, the method of unweighted nonlinear least squares yielded slightly more precise and accurate parameters than the method of the direct linear plot. Parameters calculated from the Lineweaver-Burk plot were very imprecise and inaccurate. The direct linear plot was comparatively resistant to outlier observations; however, only when outliers were substantial did the method become superior to nonlinear least squares. The calculation of a confidence limit is necessary for the evaluation of any resulting differences in the kinetic parameters for a set of isozymes. This can easily be calculated from either the Lineweaver-Burk plot or the nonlinear least-squares method. However, those calculated from the Lineweaver-Burk plot are biased, especially at higher levels of experimental errors. Therefore, the nonlinear least-squares method is the one most suited for the discrimination of a group of enzymes based on their kinetic parameters.
Computers↗
Characterization by nuclear magnetic resonance of the concanavalin A binding oligosaccharide on the beta b chain of placental beta-hexosaminidase B: lectin binding to the separated polypeptide chains of hexosaminidases A and B.
The type and distribution of the oligosaccharides on each polypeptide of human placental beta-hexosaminidases A (alpha (beta a beta b)) and B (2(beta a beta b)) were examined. The denatured polypeptides were separated by isoelectric focussing in a polyacrylamide slab gel and each gel was then overlaid with 125I-labelled lectins. The study indicated that the beta a chain contains negligible carbohydrate, the beta b chain contains both the high-mannose and a complex type oligosaccharide, and the alpha chain contains predominantly high-mannose or hybrid type moieties. Two asparagine-linked high-mannose type oligosaccharides present on the beta b polypeptide of beta-hexosaminidase B were isolated by concanavalin A chromatography and by reverse-phase high pressure liquid chromatography. Proton nuclear magnetic resonance characterization of the oligosaccharides revealed an equimolar glycan mixture of the high-mannose type structure Man5 and Man6.
Autoradiography↗
The lysosomal hexosaminidase isozymes.
In the 15 years since the demonstration that HEX A is the defective enzyme in patients with TSD, intensive efforts in many laboratories have revealed much about the HEX group of enzymes. In contradistinction to the two isozymes described by Robinson and Stirling [1968], we now know that there are several different species. They include the products of at least three genes which code for the alpha and beta polypeptides as well as for an enzyme that we have called HEX D. The different species of HEX found in human tissues and fluids include significant amounts of larger, unprocessed polypeptides as well as mature enzyme. Thus the HEX A of serum (HEX AS) is a more primitive form of the enzyme than that found in lysosomes. The role of biosynthesis in the formation of multiple species of HEX is not confined to the polypeptide chains of the enzyme. All lysosomal enzymes are glycosylated and HEX is not an exception. The carbohydrate side-chains are essential to the packaging process that places HEX in the lysosome. Carbohydrates on lysosomal HEX species clearly differ from those on HEX in serum. Characterization of HEX oligosaccharides is still in the preliminary stages. Many minor species of HEX have been described. The more important ones are the intermediate isozymes (HEX Is). In tissues the HEX Is appear to contain mixtures of processed and unprocessed alpha and beta polypeptides. In serum, on the other hand, they contain unprocessed beta chains and differ in the carbohydrate side-chains. Most species of HEX show microheterogeneity. Native, mature HEX B separates into several bands on isoelectric focusing. The nature of this microheterogeneity has not been defined. Clear differences have been described, however, between the two chains in the beta subunit. The chains are always united in non-random fashion and are probably derived by cleavage of a single gene product. Studies of hydrolytic activity have been interesting. Like other lysosomal enzymes, HEX A requires a specific protein activator for optimum activity. This small polypeptide has been partially characterized but its mode of action is as yet unclear. Defects in activator synthesis lead to a form of GM2 ganglioside storage disease. Clinically many different phenotypes have been identified which appear to result from defects in each of the HEX genes. The differences between the defects have not been characterized in molecular terms.(ABSTRACT TRUNCATED AT 400 WORDS)
Animals↗
The beta 2 subunit of human placenta hexosaminidase (Hex) A and B is a non-random association of the two polypeptides beta a and beta b.
The subunit structures of placental Hex A and B have previously been assigned as alpha beta 2 and 2 beta 2, respectively. The beta 2 subunit is composed of two non-identical polypeptide chains, beta a and beta b. Purified Hex A and B were fractionated on a chromatofocusing column, and the fractions were reduced and then alkylated with iodo-1-14C-acetamide. The polypeptide chains were separated by polyacrylamide-gel isoelectric focusing. From the radioactivity measurements of the polypeptides a constant value for beta a/beta b was obtained in all the chromatofocusing fractions, demonstrating a non-random structure of (beta a beta b) in each beta 2 subunit.
Female↗
Congenital ascites as a presenting sign of lysosomal storage disease.
Neonatal ascites is usually attributed to hematologic, genitourinary, gastrointestinal tract, or congenital heart disease. When these lesions have been excluded, metabolic storage disorders should be considered in the differential diagnosis. We report eight cases of neonatal ascites associated with different types of lysosomal storage disease: infantile sialidosis, Salla disease, GM1 gangliosidosis, and Gaucher disease. In each case there was a history of sibling of perinatal death resulting from the disease. In three cases the diagnosis of ascites was made in utero by ultrasound examination. These diseases are characterized by excretion in the fetal urine of abnormal catabolic products or by measurement of decreased activity of specific lysosomal hydrolases in cultured amniocytes. Thin-layer chromatography of the oligosaccharides in amniotic fluid may be indicated when a diagnosis of persistent fetal ascites has been established.
Adult↗
Human placental beta-galactosidase: structural and immunological observations.
beta-Galactosidase purified to apparent homogeneity from human placenta occurred in two separable fractions. A low molecular mass form (relative mass (Mr) 170 000) is composed of a single polypeptide chain (Mr 70 000). This was derived from a larger form by molecular sieve chromatography at both low (10 mM) and high (500 mM) NaCl concentration. The larger form of beta-galactosidase also contains small amounts of two polypeptides with apparent Mr values of 23 000 and 35 000 daltons. Both forms of the enzyme hydrolyze synthetic aryl galactosides and natural glycolipid substrates at comparable rates. Antibodies raised in rabbits against the low Mr beta-galactosidase also cross-reacts with the high Mr enzyme. The antibody preparation also cross-reacted with beta-hexosaminidase even though the latter was found at very low levels in the antigen, as judged by lack of detection of representative protein bands on sodium dodecyl sulfate - polyacrylamide gel electrophoresis and enzyme activity measurements. A portion of this cross-reactivity (35%) against beta-hexosaminidase could not be absorbed from the preparation without the simultaneous loss of beta-galactosidase activity, suggesting that the two enzymes show a degree of antigenic identity.
Amino Acids↗
HPLC analysis of neutral glycolipids: an aid in the diagnosis of lysosomal storage disease.
Concentrations of GL-la (glucocerebroside) (8.36 nmol/ml), GL-2a (lactosylceramide) (4.03 nmol/ml), GL-3a (globotriosylceramide) (2.25 nmol/ml) and GL-4a (globotetraosylceramide) (2.87 nmol/ml) have been determined in normal plasma and compared to concentrations in the plasma from patients with Gaucher, Krabbe, Fabry, Sandhoff and Tay-Sachs diseases as well as with hypercholesterolemia. HPLC analysis of perbenzoylated glycolipid derivatives (isolated and purified by modification of an existing procedure) was performed on samples equivalent to 50 to 100 microliter of plasma. The sensitivity could be readily increased ten-fold. We have employed a novel internal standard-monogalactosyl diglyceride, a plant glycolipid, commercially available in pure form. Analysis was performed on a 5 micron ultrasphere silica column, using a gradient of isopropanol in hexane rather than the more usual dioxane in hexane. Our gradient exhibited an essentially flat baseline precluding the necessity of a reference cell. Recoveries of glycolipids added to plasma (95%), experimental yields (60%) and standard curves are presented and discussed. A method is also presented for the separation of GL-la and monogalactosyl diglyceride derivatives for rapid (8 minute) isocratic analysis of multiple samples from Gaucher patients. The benefits of such a simple, reproducible HPLC technique are discussed.
Chromatography, High Pressure Liquid↗
Rapid molecular weight determination for native glucocerebrosidase from human placenta using high-performance liquid chromatography.
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Chromatography, High Pressure Liquid↗
Biliary disease in metachromatic leukodystrophy.
This paper illustrates the previously unreported sonographic changes in the biliary tract in metachromatic leukodystrophy (MLD). Gallbladder wall thickening due to sulfatide deposition, intraluminal globules of sulfatide or papillomatosis may cause symptoms referrable to the biliary tract in these patients. A series of patients whom we have studied is briefly alluded to in order to bring to the attention of the radiologist MLD as a rare cause of biliary disease in children.
Adolescent↗