Haemolytic anaemia in Basenji dogs. 2. Partial deficiency of erythrocyte pyruvate kinase (PK; EC 2.7.1.40) in heterozygous carriers

Congenital nonspherocytic haemolytic anaemia (HA) in dogs of the Basenji breed is inherited as a simple, autosomal recessive trait. Previous results of pyruvate kinase (PK) assays suggest a causal relationship between the anaemia and PK deficiency in erythrocytes. In the present investigation assays of this enzyme have been performed on haemolysates from 45 Basenji dogs, comprising 3 anaemic and 42 non-anaemic individuals of which 13 were known heterozygotes. The PK activity in haemolysates from the 42 non-anaemic dogs exhibited a bimodal distribution corresponding to the genotypic classes: heterozygotes and normal homozygotes. The results indicate that heterozygotes have a partial, detectable enzyme deficiency, not reflected in clinical disease, and thus give evidence of a gene dosage effect in agreement with observations in man. The proposed genotypes PK PK, PK pk and pk pk refer to normal homozygotes, heterozygotes, and anaemic individuals, respectively. The findings strengthen the genetic hypothesis of recessiveness of the anaemia by direct detection of heterozygosity of parents of affected individuals. Moreover, the results are of value in comparative studies and they have practical application in connection with eradication programmes.     

Canine erythrocyte pyruvate kinase. II. Properties of the abnormal enzyme associated with hemolytic anemia in the basenji dog

We have compared the solubility, kinetic, immunological, and electrophoretic properties of erythrocyte pyruvate kinase from normal dogs and Basenji dogs with congenital hemolytic anemia due to pyruvate kinase deficiency. Differences can be detected between the two enzymes by all methods. The enzyme from the affected animals has a greater solubility in ammonium sulfate. It has a lower K _m for phosphoenolpyruvate, while the K _m for ADP is increased. This enzyme is not inhibited by ATP or activated by fructose 1,6-diphosphate. The enzyme from the affected animals has none of the allosteric properties characteristic of the normal canine enzyme. No difference can be detected by enzyme inactivation with rabbit antiserum against the human erythrocyte enzyme, but a slight spur is observed on comparison of the two enzymes by Ouchterlony immunodiffusion. The enzymes also differ in their electrophoretic mobilities on starch gel electrophoresis.     

Establishment and characterization of B cell lines from individuals with various types of adenine phosphoribosyltransferase deficiencies

Patients with 2,8-dihydroxyadenine urolithiasis are either completely or partially deficient in adenine phosphoribosyltransferase activities. Patients with partial enzyme deficiencies, all of whom have been found among Japanese, are homozygotes having a unique mutant adenine phosphoribosyltransferase gene (APRT*J) in double dose (Japanese type deficiency). We have established B-cell lines from heterozygotes and homozygotes of complete and Japanese type adenine phosphoribosyltransferase deficiencies as well as normal individuals. Characterization of the cell lines indicated that all homozygous cells were deficient in adenine phosphoribosyltransferase function while all heterozygous and normal cells had functional adenine phosphoribosyltransferase.     

HETEROZYGOSITY AND DEVELOPMENTAL RATE IN A STRAIN OF RAINBOW TROUT (SALMO GAIRDNERI)

Rainbow trout (Salmo gairdneri) with greater heterozygosity at enzyme loci also have greater developmental stability, as measured by bilateral symmetry of five meristic traits. Fish with increased amounts of liver phosphoglucomutase activity have greater developmental stability and develop faster than fish with normal activity. These observations suggest that the differences in developmental stability between homozygotes and heterozygotes may be the result of differences in developmental rate. Faster developmental rates are expected to decrease the probability of accidents during critical periods of development, resulting in a more stable or uniform phenotype. As indicated by differences in hatching time, heterozygotes tend to develop more rapidly than homozygotes. This association is not strongly expressed within families at any locus except Pgm1-t. However, heterozygotes for Mdh3,4 and Hex hatched significantly sooner than homozygotes in a population sample. These results suggest that differences in developmental rate between homozygotes and heterozygotes may account for the positive association between developmental stability and heterozygosity in rainbow trout.     

Activity of the respiratory inhibitor RF in human red cells in anaemia

Stroma-free haemolysates of red cells from patients with acute or latent anaemia inhibit the NADH oxidase activity of submitochondrial particles from beef heart. The inhibitory activities appeared significantly more frequently in anaemic patients than in normal persons and are likely due to the action of a factor which is identical or similar to the inhibitory protein RF present in rabbit reticulocytes. The possible use of the inhibitor assay for clinical purpose is under discussion.     

On the nature of sickle-cell disease in the Arabian Peninsula

Summary The sickle-cell gene contributes substantially to the presentation of anaemia in certain areas of the Arabian Peninsula. However, the clinical presentation of the homozygous state of Hb S is less severe than that observed in other ethnic groups, such as American negroes. In the present paper, biosynthesis studies performed on reticulocytes from heterozygotes and homozygotes for the Hb S give further indications of the mild nature of sickle-cell disease in Arabia. Comparison of two affected families, from Saudi Arabia and Jordan, showed that clinical manifestation of the disease is mirrored by the biochemical and haematological findings in affected individuals. The results are discussed in terms of the effect of co-existing thalassaemia and/or iron deficiency with Hb S. It is suggested that both genetic and acquired conditions play a role in the clinical features of the disease. The mechanisms responsible for regulation of -chain synthesis by iron (haem) deficiency are discussed.     

Neonatal screening for sickle haemoglobinopathies in Birmingham.

During 1978-81 there were about 43,500 births in Birmingham, of which 10.3% were to Negroes and 22.6% to Asians. Cellulose acetate electrophoresis of red cell haemolysates from capillary specimens collected for phenylketonuria screening was performed for these babies to assess the feasibility, cost, and benefits of detecting sickle haemoglobinopathies early. Eight babies had important haemoglobinopathies; four were homozygotes for haemoglobin S (HbS), three were mixed heterozygotes for HbS and haemoglobin C (HbC), and one had haemoglobin E (HbE) and beta-thalassemia. Also, 534 (1.19%) were heterozygotes for HbS or haemoglobin D (HbD) and 205 (0.46%) for HbC or HbE, 453 (1.01%) were heterozygotes with a fast-moving band, one was a heterozygote for haemoglobin Norfolk, and one a heterozygote for both HbS and haemoglobin G Philadelphia. The cost of neonatal screening for haemoglobinopathies was 12.5 p per baby (705 pounds for each serious abnormality).     

APOB‐associated cholesterol deficiency in Holstein cattle is not a simple recessive disease

In 2015, cholesterol deficiency (CD) was reported for the first time as a new recessive defect in Holstein cattle. After GWAS mapping and identification of a disease‐associated haplotype, a causative loss‐of‐function variant in APOB was identified. CD‐clinically affected APOB homozygotes showed poor development, intermittent diarrhea and hypocholesterolemia and, consequently, a limited life expectation. Herein, we present a collection of 18 cases clinically diagnosed as CD‐affected APOB heterozygotes. CD‐clinically affected heterozygotes show reduced cholesterol and triglyceride blood concentrations. The differences in total blood cholesterol and triglycerides between nine CD‐clinically affected and 36 non‐affected heterozygotes were significant. As only some APOB heterozygotes show the clinical CD phenotype, we assume that the penetrance is reduced in heterozygotes compared to the fully penetrant effect observed in homozygotes. We conclude that APOB‐associated CD represents most likely an incomplete dominant inherited metabolic disease with incomplete penetrance in heterozygotes.     

Detection of heterozygotes for galactokinase deficiency in a human population

A simple method for assaying galactokinase in red blood cells has been described. The assay is proportional with respect to time and enzyme concentration within the limits studied. The pH optimum of the enzyme is between pH 7.8 and 8.2. In whole blood kept at 4 C, the enzyme is stable for at least 8 days, but activity is rapidly lost when the cells are washed and frozen. Employing this method, blood samples from 642 individuals were assayed for galactokinase activity. Six individuals had activity about one-half the normal value and were considered to be heterozygotes for galactokinase deficiency. Family studies on four of the individuals further substantiated this conclusion. The calculated gene frequency indicates that approximately 1 in 46,000 individuals would be expected to have galactokinase deficiency. This work was supported by a grant from the Children's Bureau.     

Q192R polymorphism of <Emphasis Type="Italic">PON-1</Emphasis> gene in type 2 diabetes patients

The Q192R polymorphism of PON-1 gene was genotyped in 96 individuals with diabetes mellitus type 2 (T2DM) and 123 nondiabetic control individuals from Kharkiv. Allele frequencies do not differ significantly between T2DM (p _Q = 0.65 and p _R = 0.35) and healthy individuals (p _Q = 0.70 and p _R = 0.30). Genotype distribution for healthy people complies with the Hardy-Weinberg equilibrium, and the T2DM patients have excess of both homozygotes and deficiency of heterozygotes. The risk of T2DM for QQ homozygotes is 1.47 times higher and for QR heterozygote is twice lower than the population average (2%). The RR homozygote individuals have statistically insignificant, 1.86 times, increase in T2DM risk.     

Polymorphism of the kinase domain of the S-locus receptor kinase gene (SRK) in Brassica oleracea L.

 DNA polymorphism of the S-locus receptor kinase gene (SRK) participating in self-incompatibility in Brassica was analyzed by PCR-RFLP and nucleotide sequencing. In the screening of primers for specific amplification of polymorphic DNA fragments of SRK, the best combination was that of a forward primer (PK1) having the nucleotide sequence of the second exon of S6 SRK and a reverse primer (PK4) having the complementary nucleotide sequence of the fifth exon of S6 SRK. PCR using this primer pair amplified DNA fragments of 0.9–1.0 kb from 36 S haplotypes out of 42 tested. These DNA fragments showed high polymorphism in polyacrylamide-gel electrophoresis after digestion with restriction endonuclease(s): 25 types were found in a double digestion with MboI and AfaI. Nucleotide sequencing of the DNA fragments amplified from five S haplotypes showed that the third, fourth, and fifth exons of SRK are highly conserved, and that there are high variations of the second and third introns of SRK, which produced polymorphism of the band pattern in PCR-RFLPs. Another forward primer (PK5) having the nucleotide sequence of the second exon, which is derived from S2 SRK, amplified DNA fragments of almost the same region of SRK from 27 S haplotypes in combination with PK4. Although SRK alleles of the class-II S haplotypes were not amplified, all of the class-I S-haplotypes were amplified with a primer mixture of PK1, PK4 and PK5. The DNA fragments of both SRK alleles in S heterozygotes, or a 1 : 1 mixture of the genomic DNA of different S homozygotes, were amplified without exception, suggesting the usefulness of these primers for the identification of S heterozygotes. The DNA fragment sizes obtained by digestion with restriction endonucleases served as markers for the identification of S haplotypes. Received: 15 December 1996 / Accepted: 14 February 1997     

Short alleles revealed by PCR demonstrate no heterozygote deficiency at minisatellite loci D1S7, D7S21, and D12S11.

Short VNTR alleles that go undetected after conventional Southern blot hybridization may constitute an alternative explanation for the heterozygosity deficiency observed at some minisatellite loci. To examine this hypothesis, we have employed a screening procedure based on PCR amplification of those individuals classified as homozygotes in our databases for the loci D1S7, D7S21, and D12S11. The results obtained indicate that the frequency of these short alleles is related to the heterozygosity deficiency observed. For the most polymorphic locus, D1S7, approximately 60% of those individuals previously classified as homozygotes were in fact heterozygotes for a short allele. After the inclusion of these new alleles, the agreement between observed and expected heterozygosity, along with other statistical tests employed, provide additional evidence for lack of population substructuring. Comparisons of allele frequency distributions reveal greater differences between racial groups than between closely related populations.     

Analysis of any point mutation in DNA. The amplification refractory mutation system (ARMS).

We have improved the "polymerase chain reaction" (PCR) to permit rapid analysis of any known mutation in genomic DNA. We demonstrate a system, ARMS (Amplification Refractory Mutation System), that allows genotyping solely by inspection of reaction mixtures after agarose gel electrophoresis. The system is simple, reliable and non-isotopic. It will clearly distinguish heterozygotes at a locus from homozygotes for either allele. The system requires neither restriction enzyme digestion, allele-specific oligonucleotides as conventionally applied, nor the sequence analysis of PCR products. The basis of the invention is that unexpectedly, oligonucleotides with a mismatched 3'-residue will not function as primers in the PCR under appropriate conditions. We have analysed DNA from patients with alpha 1-antitrypsin (AAT) deficiency, from carriers of the disease and from normal individuals. Our findings are in complete agreement with allele assignments derived by direct sequencing of PCR products.     

Genetic variability in Muscari comosum L. (Liliaceae) IV Geographical distribution and adaptive role of the polymorphic variants of chromosome 2

Muscari comosum L. (Liliaceae) has a chromosomal polymorphism for a pericentric inversion and a supernumerary chromosome segment probably due to an unequal interchange or insertional translocation. Both arrangements are widely distributed throughout the species range and the mean genetic distance among populations is D=0.131±0.075. There are no correlations between genetic distance and geographic distance or latitude. Only appreciable decreases in the frequencies of the inversion are detected in populations with ecologically marginal characteristics. There is a permanent and extended association between chromosomal inversion and an enzymatic locus (ADH). An excess of individuals heterozygous for the inversion was found and female productivity of heterozygotes is higher than that of corresponding homozygotes. A low rate of inversion heterozygosity in populations with ecologically marginal characteristics could be explained by natural selection. With respect to the adaptive role of the segment, although no homozygotes are found and may be selected against, heterozygotes could have heterotic effects.     

The frequency of the C854 mutation in the aspartoacylase gene in Ashkenazi Jews in Israel.

Canavan disease (CD) is an infantile neurodegenerative disease that is transmitted in an autosomal recessive manner and has mainly been reported in Ashkenazi Jewish families. The primary enzymatic defect is aspartoacylase deficiency, and an A-to-C transition at nucleotide 854 of the cDNA has recently been reported. We screened 18 patients with CD and 879 healthy individuals, all Israeli Ashkenazi Jews, for the mutation. All 18 patients were homozygotes for the mutation, and 15 heterozygotes were found among the healthy individuals. The results disclose a carrier rate of 1:59 and suggest that a screening for the mutation is warranted among Ashkenazi Jewish couples.     

Disparity between dietary iron intake and iron status of children aged 10-12 years

Iron status was assessed in a representative sample of 188 adolescents living in a medium-sized city in Poland. Dietary intakes were evaluated using records of diet over a period of seven consecutive days. Subjects were considered to be iron deficient when two or more of the following parameters were abnormal: serum ferritin, transferrin saturation or mean corpuscular haemoglobin concentration. Based on this definition, the prevalence of iron deficiency in the investigated sample of children aged from ten to twelve years was 12.7%. Iron deficiency anaemia was defined using the following criteria: haemoglobin values less than 12.0 g. dl (-1) in girls or less than 12.2 g. dl(-1) in boys, combined with an iron deficiency. With such a definition, the prevalence of iron deficiency anaemia in all subjects was 6.3%. Four boys (3.9%) and six girls (6.8%) were diagnosed as anaemic. The values for Hb in the anaemic boys ranged from 10.9 to 12.2 g. dl (-1) and in anaemic girls from 8.7 to 12.0 g. (-1). It was found that the majority of the individuals studied had a dietary haem-iron intake lower than that recommended. No relationship was found between the level of serum ferritin and total iron and vitamin C dietary intake, but there was positive correlation between serum ferritin and intake of haem iron. A seven-day dietary history questionnaire correctly identified children at risk of iron deficiency anaemia.     

Growth induction in cystic fibrosis fibroblasts with low dexamethasone concentrations. Experience with application to genotyping

Summary Dexamethasone (DM) resistance was evaluated in fibroblasts from a pool of five patients with cystic fibrosis (CF) homozygotes, ten of their parental obligate heterozygotes, and seventeen age-matched controls of both sexes. The CF heterozygotes showed a mean DM resistance greater than homozygotes and both groups exhibited a higher mean DM resistance at every DM concentration than controls. However, substantial interassay variability rendered these differences in the total pooled data to non-significance. One control showed a consistently increased resistance and was possibly a covert heterozygote. It was concluded that the phenomenon of DM resistance was exhibited by CF heterozygotes and homozygotes but was not discrete enough for genotyping in the prenatal diagnosis of CF.     

Mouse mammary tumor virus promoter directs high‐level expression of bovine αS1 casein in the milk of transgenic heterozygous and homozygous mice

Abstract

To test the mouse mammary tumor virus (MMTV) promoter as an inducible mammary‐specific promoter, we have produced 3 lines of transgenic mice carrying bovine αS1 casein cDNA under the control of MMTV promoter. The RNA analysis showed that in line 27, heterozygotes expressed 25%, and homozygotes 37% of the endogenous αSI casein mRNA of a mid‐lactation cow. Dexamethasone increased expression by about 3 fold in both heterozygotes and homozygotes. A similar increase in the level of mRNA was observed in both heterozygotes and homozygotes from line 42 with/without induction by dexamethasone. The transgenes were expressed predominantly in the mammary gland with low levels in the salivary gland, spleen, lung, and kidney. Their expression in mammary glands was lactation‐specific. The offspring from lines 27 and 42 expressed a high‐level bovine αS1 casein in their milk. Their expression in milk were 0.21 and 0.11 g/L for heterozygotes, 0.36 and 0.19 g/L for homozygotes, respectively. Dexamethasone further increased the levels of expression by approximately two fold for both heterozygotes and homozygotes.     

Genetic study of psoriasis in the Kharkov population

By means of genetic analysis of 400 individuals suffering from psoriasis the mendelian inheritance model have been rejected: the segregation frequencies are SFNxN = 0.083 and SFNxA = 0.1474. The heritability of psoriasis in the polygenic model is about 100%. The main gene model with incomplete penetration have been proposed (p = 0.044, [symbol: see text]1 = 6.1%, [symbol: see text]2 = 82.2%). 0.189% residents of Kharkov population are homozygotes and 8.32% heterozygotes on psoriatic gene, 0.155% residents are suffering from psoriasis heterozygotes and 0.508% heterozygotes. Among individuals suffering from psoriasis 23% are homozygotes and 77% heterozygotes.     

G6PD Genotype and Its Associated Enzymatic Activity in a Chinese Population

Knowledge of the G6PD genotype and its associated enzyme activity is significant for population genetics, diagnosis of disease, and management of patients. We tested 2,872 unrelated subjects from a Hakka population in China for G6PD activity by the WHO standard method and for genotype by DHPLC and DNA sequencing. Among female heterozygotes, 78.5% had relatively normal enzyme activity. The phenotype frequency of G6PD deficiency is 0.028, and the causal allele frequency is 0.060 in females. The accuracy, sensitivity, and specificity of DHPLC are more than 98% for detecting G6PD-deficient hemizygotes, heterozygotes, and homozygotes. Measuring enzyme activity alone is not sufficient for the diagnosis of heterozygotes. A combination of enzyme activity and DNA analysis should be used.