Detection of somatic mutants in man: HPRT mutations in lymphocytes and hemoglobin mutations in erythrocytes.
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Adult↗
Biomedical subjects
L F Bernini
Publications and source records attributed to L F Bernini.
Hemoglobin Tilburg: alpha 2-beta 2 73 (E 17) Asp----Gly. A new hemoglobin with reduced oxygen affinity.
A new hemoglobin variant has been found in a Dutch Caucasian girl and detected also in members of three generations of her family. This variant is characterized by the substitution of an aspartic acid at position 73 (E 17) of the beta-chain with a glycine residue. Hemoglobin Tilburg makes up to 42% of the total hemoglobin in the blood of the proposita, it is stable at the isopropanol test, and not associated with significant hematological abnormalities in heterozygous carriers. The oxygen dissociation curve of the purified variant, carried out at different pH values, shows a definite reduction of the affinity for oxygen and a normal alkaline Bohr effect. Three more hemoglobins with a single amino acid substitution at the same site have been previously described: Hb Korle-Bu (Asp----Asn), Hb Mobile (Asp----Val) and Hb Vancouver (Asp----Tyr). In all these proteins the affinity for oxygen is lowered to an extent which is variable and characteristic of each mutant. In this paper we discuss the possible mechanism responsible for the abnormal behaviour of hemoglobins substituted at beta 73.
Amino Acids↗
[Allogeneic bone marrow transplantation in beta thalassemia].
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Bone Marrow Transplantation↗
Fetal blood group determination in first-trimester pregnancy for the management of severe immunization.
The presence or absence of Rho (D) and Kell antigens on fetal red blood cells was determined in the first trimester of pregnancy on erythrocytes obtained by chorionic villi sampling with the use of mixed agglutination and solid phase microimmunofluorescence techniques. Pregnancies in one Kell-sensitized woman and seven severely RH-sensitized women with a poor obstetric history and a partner heterozygous for the offending antigen were examined. A conclusive diagnosis could be made in seven of the eight cases studied.
Blood Grouping and Crossmatching↗
[Hemoglobin G Georgia in a Turkish family in the Netherlands].
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Adult↗
ABO and Rhesus phenotyping of fetal erythrocytes in the first trimester of pregnancy.
Determination of fetal red blood cell antigens in early pregnancy can be important in cases with a history of severe haemolytic disease of the newborn. From chorionic villus biopsies (CVB) between the 8th and 12th week of gestation a small number of fetal red blood cells was obtained, inevitably highly contaminated with maternal blood cells. Two techniques were used to demonstrate the minor (fetal) cell population with a blood group antigen differing from the major (maternal) cell population: a solid-phase microfluorescence technique (introduced in this paper) which was compared with the mixed agglutination technique. In series of artificial mixtures of erythrocytes it was shown that with the microfluorescence technique the ABO and Rhesus phenotypes of minor cell populations could be determined at a ratio of 1 in 4000 erythrocytes of the major population, making this technique 4 times as sensitive as the mixed agglutination technique. We further investigated the reliability of the microfluorescence technique to demonstrate antagonistic fetal blood groups in the first trimester of pregnancy. Of 18 women undergoing CVB prior to therapeutic abortion, blood group antagonism (ABO and Rhesus systems) was demonstrate in all 11 cases in which it was present. Therefore, it seems that CVB can be reliably used for the prenatal diagnosis of (recurrent) blood group antagonism.
ABO Blood-Group System↗
Molecular evidence of triplication in the haptoglobin Johnson variant gene.
The protein and gene structure of the Hp Johnson variant (Hp3) were analyzed in two related heterozygous individuals. The molecular weight (23 kd) and amino acid composition of Hp3 alpha chain were in agreement with the triplicated structure first suggested by Smithies in 1964. Direct gene analysis by Southern blotting showed a three-fold tandem repeat of the same 1.7 kb DNA segment implicated in the Hp2 gene duplication. On the basis of these data a nine exon model for the Hp3 gene is proposed.
Amino Acid Sequence↗
Chorionic biopsy in management of severe rhesus isoimmunisation.
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Biopsy↗
Genetic linkage between erythrokeratodermia variabilis and Rh locus.
A genetic linkage study was performed in a large Dutch kindred with erythrokeratodermia variabilis (EKV, McKusick no. 13320). The autosomal-dominant trait appeared to segregate rather consistently with the cde (r) gene complex of the Rh system. Only one recombinant was found amongst 27 informative individuals. Lod score calculations gave strong evidence for close linkage between the loci for EKV and Rh (with a maximum lod score of 5.55 at a recombination fraction of 0.044).
Chromosomes, Human, 1-3↗
Familial benign hypercalcaemia (FBH; McK. No. 14598, 1983): linkage studies in a large Dutch family.
By screening 27 hypercalcaemic and 21 normocalcaemic subjects in a large Dutch pedigree with familial benign hypercalcaemia (FBH; McK. No. 14598) (McKusick 1983) for more than 35 genetic markers, it was found that linkage of FBH can be excluded at about 25 centimorgans (cM) from GM, 20 cM from ABO, 15 cM from MNS and HLA, 10 cM from JK and PI, and 5 cM each from ACP1, AK1, ADA, GPT1, and PGP.
Female↗
Inheritance of rDNA spacer length variants in man.
We studied the rDNA spacer length polymorphism in a sample of 121 individuals belonging to families of 2-3 generations. Our data, obtained by restriction pattern analysis of genomic DNA, confirmed the limited and discrete nature of this polymorphism. Using the pattern as a genetic marker, we analyzed the segregation of length variants in the different families and we investigated the possible occurrence of unequal crossing-over events among homologous and nonhomologous rDNA clusters. No direct evidence of recombination in the spacer region that we analyzed emerged from our study. All the differences in the restriction patterns observed among individuals from the same family could be explained as resulting from meiotic segregation. Family data showed a multichromosomal distribution of NTS length variants and demonstrated a direct correspondence between the frequency of a variant in the population and its degree of spreading on the different rDNA clusters.
Base Sequence↗
Development of a screening system for detection of somatic mutations. II. The use of peptides and insoluble protein fragments in a non-competitive solid-phase enzyme immunoassay.
A system proposed for measurement of mutational risk consists in detection of hemoglobin mutations expressed in erythrocytes. For this detection the production of antibodies specific for Hb variants is essential. Recently we reported a sensitive solid-phase EIA for the production and selection of polyclonal and monoclonal antibodies specific for hemoglobin determinants. An important characteristic of this EIA was the coating of water-insoluble proteins to polystyrene microtiter plates. Here we report that with this system, insoluble protein fragments and small peptides may also be covalently coated to a polystyrene surface. Coating is independent of the length of the peptides. This allows direct, non-competitive titration of the antibody response to small peptides and avoids the drawbacks of competitive assay.
Animals↗
Different quantitative expression of the hemoglobin alpha-chain genes in sheep.
We previously reported that the alpha-chain locus of sheep hemoglobin is duplicated and that the II alpha gene is less efficient than the I alpha gene. The II alpha locus controls two allelic alpha chains, one containing leucine (identical to that directed by the I alpha locus) and the other one having histidine in position 113 or 114 (called II alpha 113Leu and II alpha 113His chains, respectively). In sheep homozygous at the II alpha locus for the II alpha 113His allele, the I alpha 113Leu/II alpha 113His-chain ratio was about 1.8:1; in those heterozygous for the II alpha 113Leu and the II alpha 113His alleles, the ratio between the chains was 4-5:1. We report here the detection of a new alpha 113Leu/II alpha 113His-chain ratio of 12-15:1, observed in 16 sheep belonging to four domestic breeds, during a survey of 245 animals. This phenotype was found associated in five sheep with the alpha D-chain variant. The occurrence of a quantitative polymorphism of the II alpha 113His gene is considered.
Alleles↗
Duplication of the hemoglobin alpha-chain gene in sheep: characterization of a new alpha-chain variant present in animals possessing the alpha Leu and the II alpha His chains.
The structural characterization of a third type of alpha chain, detected in the hemoglobin of sheep also possessing the alpha Leu and the II alpha His chains, is reported. The new alpha-chain variant is an allele of the common alpha Leu chain controlled by the 1 alpha locus and differs from it in the replacement of the serine residue at position 8 with an alanine (I alpha 8 Ser leads to Ala). The alanine variant was observed in 38 of 206 sheep, whose hemolysates were analyzed by isoelectric focusing, and was found exclusively among the 57 animals also possessing the II alpha His chain.
Alleles↗
Absence of close linkage between Hereditary spherocytosis (SPH) and 24 genetic marker systems including HLA and GM.
A total of 250 individuals belonging to 19 different families, identified through established propositi were simultaneously screened for hereditary spherocytosis (SPH), using stringent criteria, and 27 well-known polymorphic genetic marker systems. The segregation analysis indicated that the pattern of inheritance of SPH in these families, being autosomal and dominant, had a 100% penetrance. A statistical analysis, using the LIPED computer program of Ott (1974), revealed the absence of close linkage between SPH and ABO, ACP1, ADA, AK1, C3, DIA2, ESD, FY, GC, GLO1, GM, GPT1, HLA, HPA, JK, K, KM, MNS, P, PGD, PGM1, PGP, PI, and RH. Since an earlier study by other investigators had convincingly suggested a linkage between SPH and GM, we subjected the data to further analysis and found no significant heterogeneity in our recombination values of linkage between SPH and GM, or any of the other informative loci.
Chromosome Mapping↗
Development of a screening system for detection of somatic mutations. I. Enzyme immunoassay for detection of antibodies against specific hemoglobin determinants.
A solid-phase enzyme immunoassay for the detection of antibodies, specific for hemoglobin (Hb) is described. The application of glutaraldehyde resulted in a sensitive assay and allowed the use of urea, which is an important advantage if polypeptides not soluble in aqueous buffers are to be used. Mutation-carrying Hb chains can be purified, solubilized in urea and used in the immunoassay to monitor the purification and selection of antibodies specific for these variants. Specific antibodies are the main tools for the development of a hemoglobin-locus mutation system for detection of potentially mutagenic environmental agents. With erythrocytes as target cells, this system permits in vivo monitoring of subjects under exposure. Conventional antibody production, however, frequently turns out to be unsuccessful. The production of monoclonal antibodies has several advantages over conventional antibody production, but a sensitive antibody screening system is essential. Because of the sensitivity and the ease with which a large panel of antibody fractions against a vast panel of Hb antigens can be examined, the described immunoassay has potential value for the screening of hybridoma cultures.
Adult↗
Delta beta (F)-thalassaemia in Sardinia.
A population survey carried out in southern sardinia on more than 5000 people has shown that delta beta (F)-thalassaemia, with a gene frequency of 0-00088, is a rare trait in this population. We examined the members of three families segregating for both delta beta- and beta(0)-thalassemia and a number of delta beta carriers identified during the screening. The doubly heterozygous children suffer from a mild form of Cooley's disease with non-alpha/alpha biosynthetic ratios within the range of values observed in beta (0)-thalassaemia homozygotes. Three of them have been transfusion dependent for some time. The delta beta carriers, although in many respects showing the usual picture of delta beta-thalassaemia, such as abnormal red cell indices, normal Hb A2, Hb F heterogeneously distributed in the erythrocytes, and low beta/alpha synthetic ratios, have unusually high levels of Hb F (range 10 to 20%) and particularly low glycine content (range 0.02 to 0.14 residues) in the isolated gamma CB3 peptide. These results have led us to the conclusion that the delta beta-thalassaemia found in Sardinia is different from the similar kind of delta beta defect found in Negroes and in other Mediterranean populations, including continental Italians.
Adolescent↗
The molybdoenzyme system of Drosophila melanogaster. I. Sulfite oxidase: identification and properties. Expression of the enzyme in maroon-like (mal), low-xanthine dehydrogenase (lxd), and cinnamon (cin) flies.
Sulfite oxidase (sulfite: ferricytochrome c oxidoreductase; EC 1.8.2.1) has been detected in Drosophila melanogaster and some of its properties have been studied. In most respects this enzyme resembles the mammalian sulfite oxidases except for its molecular weight (148,000), which is somewhat higher than that of rat sulfite oxidase (116,000). Cytochrome c, potassium-ferricyanide, and oxygen can serve as electron acceptors in the oxidation of sulfite by the enzyme. Although definite evidence can be obtained only through the analysis of the pure enzyme, experiments involving tungstate feeding suggest that Drosophila sulfite oxidase is most probably a molybdoenzyme. Extracts of mal flies show normal levels of sulfite oxidase, whereas lxd flies have only 5-10% of the activity of wild type, and in cin flies the enzyme is apparently absent. While it is possible that the lxd and cin mutations are at some level responsible for the defective synthesis of a molybdenum-containing cofactor (supposed to be present in most molybdoenzymes), the evidence accumulated so far by several authors and the results of the present investigation argue against the involvement of a Mo cofactor in the multiple enzyme deficiencies observed in mal flies.
Animals↗