The presence of at least two different H-blood-group-related beta-D-gal alpha-2-L-fucosyltransferases in human serum and the genetics of blood group H substances.

Sera from H normal, secretors and nonsecretors (H/-, Se/- and H/-, se/se), as well as from H-deficient secretors (h/h, Se/- or Bombay secretors) contain enzyme(s) for the transfer of L-fucose in the alpha-configuration to the 2-position of suitable beta-D-galactopyranosyl units. Sera from H-deficient nonsecretors (h/h, se/se; i.e., Bombay nonsecretors) are devoid of such beta-D-Gal alpha-2-L-fucosyltransferase(s). In order to study these enzymes, a comparison was made of the kinetic properties of the enzymes present in the sera of H-normal nonsecretors (H/-, se/se) with those of H-deficient secretors (h/h, Se/se) with those of H-deficient secretors (h/h, Se/-). These studies revealed a clear difference between the two sources of enzyme: (1) the apparent Km for GDP-fucose was four times lower with the H-normal nonsecretor serum (0.008 mM) than with the H-deficient secretor serum (0.028 mM); (2) acceptors with a type 1 or type 3 chain proved to be better than acceptors with a type 2 chain or than phenyl-beta-D-galactopyranoside for the enzyme present in the serum of H-deficient secretor individuals. Indeed, the synthetic type 2 compound, betaDGal (1-->4)-3-deoxy-beta-DGlcNAc-1-OCH3, which cannot act as an acceptor of beta DGlcNAc alpha-3/4-L-fucosyltransferases, remained unchanged in the serum of an H-deficient secretor but was a good acceptor in the serum of an H-normal nonsecretor, and (3) the alpha-2-L fucosyltransferease activity of the H-deficient secretor serum was more sensitive to heat inactivation than that of the H-normal nonsecretor serum (t1/2 at 46 degrees C were 10 min and 75 min, respectively). These results show that at least two distinct alpha-2-L-fucosyltransferases are present in human serum. It is concluded that the enzymatic activity found in the H-deficient secretor serum (h/h, Se/-) could be the product of the Se gene and the enzymatic activity found in the H-normal nonsecretor serum (H/-, se/se) could be the product of the H gene. This conclusion correlates well with the finding that H and Se genes are closely linked and might have derived by gene duplication in the course of evolution.     

H-deficient blood groups of Reunion island. II. Differences between Indians (Bombay Phenotype) and whites (Reunion phenotype).

Two variants of recessive, H-deficient nonsecretor individuals (h/h, se/se) were identified on Reunion Island: (1) H-negative individuals corresponding to the classical Bombay phenotypes (OhO, OhA, OhB, OhAB) who lack completely the H antigen on their red cells; all of them were Indian and had strong anti-H antibodies reacting with normal O and Oh red cells from whites; and (2) H-weak individuals (Oh, Ah, Bh, ABh). This phenotype represented the majority (85%) of the H-deficient phenotypes on Reunion Island, and all of them were white. They had only a weak expression of the H antigen and showed small but detectable amounts of ABH antigens on their red cells. Their anti-H antibodies reacted with normal O erythrocytes, but failed to react with Oh red cells, regardless of the ethnic origin of the donor. They were all from the same geographical area on the Island (Cilaos) and showed homogeneous titers of anti-H antibodies in sera. We propose to call this particular variant of weak H phenotype, belonging to the so-called para-Bombay series, Reunion.     

H-deficient blood groups of Reunion Island. III. alpha-2-L-fucosyltransferase activity in sera of homozygous and heterozygous individuals

Small amounts of H-enzyme activity (from 500 to 1,280 counts per minute [cpm]) were detected in sera of white H-deficient individuals (h/h; Reunion phenotype), while no significant H activity (less than 500 cpm) could be detected in sera of Indian H-deficient individuals (h/h; Bombay phenotype). The H-enzyme levels of the unaffected members of H-deficient pedigrees showed an H-related dosage effect. Heterozygous H/h individuals had only half as much H activity in sera (17,000 +/- 3,400 cpm) compared with homozygous H/H individuals (31,600 +/- 3,700 cpm). Data suggesting interactions between the H enzyme and the other gene specified glycosyltransferases working on the same oligosaccharide chains carrying combined ABH and Lewis antigens are analyzed in terms of their possible genetic or biochemical origin.     

A new genetic model proposing that the Se gene is a structural gene closely linked to the H gene.

The Se gene is classically considered as a regulatory gene controlling the expression of the structural gene H in external secretions. Under this hypothesis, Bombay (h/h) individuals should not be able to express the Se gene. Statistical analysis of the 44 published Bombay pedigrees suggests on the contrary that there is no suppression of Se in Bombay individuals, and that both Se and H loci can be fully expressed at the phenotypic level. Based on a lod score of 12.9 at 1% recombination units and the existence of two different acceptors for the biosynthesis of the H antigen, a new genetic model is proposed in which H and Se would be two closely linked structural genes coding for two different 2-alpha-L-fucosyltransferases.     

Action of glycosyl transferases upon "Bombay" (Oh) erythrocytes. Conversion to cells showing blood-group H and A specificities.

Individuals of the rare "Bombay" (Oh) blood-group phenotype lacking, due to a genetic defect, the alpha(1-2)fucosyl transferase, which is responsible for converting blood-group H precursor substances to H-specific structures. Treatment with GDP-fucose and alpha(1-2)fucosyl transferase prepared from gastric mucosa of O individuals to transform native or ficin-treated "Bombay" erythrocytes into cells phenotypically resembling O cells. The transformation was achieved, however, after prior incubation of the "Bombay" erythrocytes with neuraminidase, indicating that blood-group H precursor molecules on the surface of these cells are masked by sialyl residues. Blood-group A specificity was conferred upon neuraminidase-treated "Bombay" cells by enzymatic transfer of alpha-N-acetylgalactosamine residues, in addition to alpha-fucose residues.     

A test for linkage and association in general pedigrees: the pedigree disequilibrium test

Family-based tests of linkage disequilibrium typically are based on nuclear-family data including affected individuals and their parents or their unaffected siblings. A limitation of such tests is that they generally are not valid tests of association when data from related nuclear families from larger pedigrees are used. Standard methods require selection of a single nuclear family from any extended pedigrees when testing for linkage disequilibrium. Often data are available for larger pedigrees, and it would be desirable to have a valid test of linkage disequilibrium that can use all potentially informative data. In this study, we present the pedigree disequilibrium test (PDT) for analysis of linkage disequilibrium in general pedigrees. The PDT can use data from related nuclear families from extended pedigrees and is valid even when there is population substructure. Using computer simulations, we demonstrated validity of the test when the asymptotic distribution is used to assess the significance, and examined statistical power. Power simulations demonstrate that, when extended pedigree data are available, substantial gains in power can be attained by use of the PDT rather than existing methods that use only a subset of the data. Furthermore, the PDT remains more powerful even when there is misclassification of unaffected individuals. Our simulations suggest that there may be advantages to using the PDT even if the data consist of independent families without extended family information. Thus, the PDT provides a general test of linkage disequilibrium that can be widely applied to different data structures.     

Incidence of ABO(H) blood group variants among the Bulgarian population.

The result of a screening of ABO (Hh) variants after investigating the blood group of 106,980 persons are presented. The Ax variant is registered most frequently among the Bulgarian population. As a whole the frequencies of ABO blood group variants Ax, Ael, A3, Aend and Am among the Bulgarian population are similar to that established by other authors among the French population and nearly twice as high as among the population of Bombay. The group of H-deficient phenotypes includes AA1Xh, AHm, OHm variants. Their frequency is significantly lower when compared with the frequency of AHm and OHm variants among the population of Thailand. Variants A1 and Aint with unusually high H-content are classified as A1H, A1Hint, Aint H and integrated as a category of H-excess phenotypes. Their incidence among the Bulgarian population is significantly lower than that registered among Maharastrian, South African Bantu and Indian.     

Further evidence for a locus for cutaneous malignant melanoma-dysplastic nevus (CMM/DN) on chromosome 1p, and evidence for genetic heterogeneity.

Assignment of a susceptibility locus for cutaneous malignant melanoma-dysplastic nevus (CMM/DN) to chromosome 1p remains controversial. We examined the relationship between CMM/DN and markers D1S47, PND, and D1S160 on seven new families (set B) plus updated versions of six previously reported families (set A). Three linkage analyses were performed: (1) CMM alone--all individuals without confirmed melanoma or borderline lesions were considered unaffected (model I); (2) CMM/DN with variable age at onset and sporadics (model II); and (3) CMM/DN using the model of Bale et al. (model III). For CMM alone and D1S47, Zmax = 3.12 at theta = .10. For D1S160 and CMM alone, Zmax = 1.76 at theta = .10. PND showed no evidence for linkage to CMM alone. Models II and III showed strong evidence for linkage to D1S47, D1S160, and PND in the set A pedigrees but not in the set B families. We tested for homogeneity of CMM/DN (model II) by splitting families into two groups on the basis of (1) the proportion of CMM/DN cases and (2) the occurrence of immune-related tumors. In group 1 there was significant evidence of heterogeneity with both D1S47 and D1S160, and in group 2 there was significant evidence of heterogeneity with D1S160. Thus, diagnostic, clinical, and genetic heterogeneity are the likely reasons that previous studies have failed to confirm linkage of CMM/DN to chromosome 1p. The results showed significant evidence for a CMM locus linked to D1S47, as well as significant evidence for heterogeneity with only a subset of the families appearing linked to chromosome 1p.     

Molecular basis for secretor type alpha(1,2)-fucosyltransferase gene deficiency in a Japanese population: a fusion gene generated by unequal crossover responsible for the enzyme deficiency.

About 20%-25% of Caucasian individuals are nonsecretors who fail to express soluble A, B, H, and Lewis b histo-blood group antigens in secretory organs and secretory fluids because of the absence of the Secretor gene (FUT2)-encoded alpha(1,2)-fucosyltransferase (Se enzyme) activity. Recently, the FUT2 and a pseudogene have been isolated, and an Se enzyme-deficient allele (se) caused by a nonsense mutation (G428A, se1) in Caucasians has also been reported. Although we were unable to find the se1 allele, we have found a missense mutation (A385T, se2) and two nonsense mutations (C571T, se3 and C628T, se4) in the Japanese Se enzyme-deficient alleles. In addition, we have found a fusion gene, which consisted of the 5'-region of the pseudogene and the 3'-region of the functional FUT2, as a Se enzyme-deficient allele (se5). The DNA sequence analysis of the fusion gene indicated that the crossover region corresponded to regions between bases 253 and 313 of the pseudogene and between bases 211 and 271 of the FUT2. This finding suggested that the fusion gene was generated by homologous but unequal crossover. A population study on 141 randomly selected Japanese has indicated that the se2 is a common Se enzyme-deficient allele in the Japanese population. The results suggest that Se enzyme-deficient alleles are race specific.     

Linkage of cutaneous malignant melanoma/dysplastic nevi to chromosome 9p, and evidence for genetic heterogeneity.

We examined the relationship between cutaneous malignant melanoma/dysplastic nevi (CMM/DN) and chromosome 9p in 13 pedigrees with two or more living cases of invasive melanoma. We used two highly informative (CA)n repeats, D9S126 and IFNA, previously implicated in familial malignant melanoma (MLM), to conduct linkage analysis. Three analyses were performed: (1) CMM alone--all individuals without either confirmed melanoma or borderline lesions were considered unaffected (model A); (2) CMM/DN with both variable age at onset and sporadics (model B); and (3) CMM affecteds only--all individuals either without confirmed melanoma or with borderline lesions were designated "unknown" (model C). There was significant evidence for linkage to IFNA in all three models. For CMM alone, the maximum lod score (Zmax) was 4.36 at theta = .10 for model A and 3.39 at theta = .10 for model C. For CMM/DN (model B), Zmax = 3.05 at theta = .20. There was no significant evidence for linkage between CMM alone or CMM/DN and chromosome 9p marker D9S126. In addition, there was significant evidence for heterogeneity when a homogeneity test allowing for linkage to chromosome 9p or chromosome 1p or neither region was used. These results suggest that there is an MLM susceptibility locus on chromosome 9p but that familial melanoma is heterogeneous and not all families with CMM/DN are linked to a locus in this region.     

Infection-associated FUT2 (Fucosyltransferase 2) genetic variation and impact on functionality assessed by in vivo studies

The secretor (Se)/nonsecretor (se) histo-blood group variation depends on the action of the FUT2 enzyme and has major implications for human susceptibility to infections. To characterize the functionality of FUT2 variants, we assessed the correlation between saliva phenotypes and sequence variation at the FUT2 gene in sixty seven individuals from northern Portugal. While most non-secretor haplotypes were found to carry the 428G > A nonsense mutation in association with a 739G > A missense substitution, we have also identified a recombinant haplotype carrying the 739*A allele together with the efficient 428*G variant in individuals with the Se phenotype. This finding suggested, in contrast to previous results, that the 739*A allele encodes an efficient Se allele. To test this hypothesis we evaluated the in vivo enzyme activity of full coding expression constructs in transient transfection of CHO-K1 cells using FACS (fluorescence-activated cell sorting) analysis and expression of type 2 and type 3 chain H structures as read out. We detected FUT2 activity for the 739*A expression construct, demonstrating that the 739G > A substitution is indeed not inactivating. In accordance with the hypothesis that FUT2 is under long standing balancing selection, we estimated that the time depth of FUT2 global genetic variation is as old as 3 million years. Age estimates of specific variants suggest that the 428G > A mutation occurred at least 1.87 million years ago while the 739G > A substitution is about 816,000 years old. The 385A > T missense mutation underlying the non-secretor phenotype in East Asians appears to be more recent and is likely to have occurred about 256,000 years ago.     

Red cell H-deficient,salivary ABH secretor phenotype of Reunion island. Genetic control of the expression of H antigen in the skin

Based on the genetic model proposing thatH andSe are two structural genes, we predicted that the red cell H-deficient, salivary ABH secretor phenotype should be found on Reunion island, where a large series of H-deficient non-secretor families have been previously described. Two such Reunion individuals are now reported. POU [A_h, Le(a–b+), secretor of A, H, Le^a and Le^b in saliva] and SOU [O_h, Le(a–b+), secretor of H, Le^a and Le^b in saliva]. Both are devoid of H -2-fucosyltransferase activity in serum. In addition, the preparation of total non-acid glycosphingolipids from plasma and red cells of POU revealed the type 1ALe^b heptaglycosylceramide and small amounts of the monofucosylated type 1 A hexaglycosylceramide. Both glycolipids possess an H structure probably synthesised by the product of theSe gene. No other blood group A glycolipids, with types 2, 3 or 4 chains, normally present in the presence of the product of theH gene, were found on red cells or plasma of POU.TheH,Se andLe genetic control of the expression of ABH and related antigens in different tissue structures of the skin is described in 54 H-normal individuals of known ABO, secretor and Lewis phenotypes; in one red cell H-deficient salivary secretor (SOU); and in one H-deficient non-secretor (FRA). Sweat glands express ABH under the control of theSe gene. Sweat ducts express ABH under the control of bothH andSe genes and Lewis antigens under the control ofLe and bothH andSe genes. Epidermis, vascular endothelium and red cells express ABH under the control of theH gene. The products ofH andSe genes are usually expressed in different cells. However, the results illustrate that in some structures, like the epithelial cells of sweat ducts, both the products ofH andSe genes can contribute to the synthesis of the same Le^b structure.     

Interactions of radiolabelled ligands with specific receptors: an analysis

The binding and displacement of beta-adrenoceptor blockers, [3H]propranolol ([3H]PRP) and [3H]dihydroalprenolol ([3H]DHA), were studied on isolated rat erythrocytes, their membranes and ghosts; the binding of [3H]DHA and a M-cholinoceptor blocker, [3H]quinuclidinylbenzylate ([3H]QNB), on cerebral cortex membranes. In all experiments, ligand-receptor interactions conformed to a model of two pools of receptors in the same effector system and the binding of two ligand molecules to the receptor. The results were similar for the displacement of [3H]PRP, [3H]DHA and [3H]QNB with propranolol, dihydroalprenolol and quinuclidinyl-benzylate, respectively. The parameters of [3H]PRP to beta-adrenoceptor binding for intact erythrocytes were: Kd1 = 0.74+/-0.07 nM, Kd2 = 14.40+/-0.41 nM, B1 = 24+/-2 unit/cell, B2 = 263+/-5 unit/cell; for ghosts, Kd1 = 0.70+/-0.17 nM, Kd2 = 19.59+/-2.59 nM, B1 = 9+/-1 fmol/mg protein, B2 = 39+/-4 fmol/mg protein. Receptor affinities were similar in erythrocytes and ghosts; on the ghost membrane, the number of receptors was considerably lower (B1 = 2 unit/cell, B2 = 6 unit/cell). The parameters of [3H]QNB to M-cholinoceptor binding of the cerebral cortex membrane were the following: Kd1 = 0.43 nM, Kd2 = 2.83 nM, B1 = 712 fmol/mg, B2 = 677 fmol/mg.     

Binding of rabbit hemorrhagic disease virus to antigens of the ABH histo-blood group family

The ability of rabbit hemorrhagic disease virus to agglutinate human erythrocytes and to attach to rabbit epithelial cells of the upper respiratory and digestive tracts was shown to depend on the presence of ABH blood group antigens. Indeed, agglutination was inhibited by saliva from secretor individuals but not from nonsecretors, the latter being devoid of H antigen. In addition, erythrocytes of the rare Bombay phenotype, which completely lack ABH antigens, were not agglutinated. Native viral particles from extracts of infected rabbit liver as well as virus-like particles from the recombinant virus capsid protein specifically bound to synthetic A and H type 2 blood group oligosaccharides. Both types of particles could attach to adult rabbit epithelial cells of the upper respiratory and digestive tracts. This binding paralleled that of anti-H type 2 blood group reagents and was inhibited by the H type 2-specific lectin UEA-I and polyacrylamide-conjugated H type 2 trisaccharide. Young rabbit tissues were almost devoid of A and H type 2 antigens, and only very weak binding of virus particles could be obtained on these tissues.     

Studies on selenium-related compounds. V. Cytogenetic effect and reactivity with DNA.

Five selenium compounds, Na2Se04, H2Se04, Na2Se03, H2Se03 and Se02, were tested for their capacity to induce chromosome aberrations in cultured human leukocytes and for their reactivity with DNA by a rec-assay system and inactivation of transforming activity in Bacillus subtilis. Chromosome-breaking activity was significantly higher for the compounds with four-valent than with six-valent selenium, the efficiency being in the decreasing order H2S03 greater than Na2Se03 greater than Se02 greater than H2Se04 greater than Na2Se04. Rec assay using B. subtilis with different recombination capacities suggested that damage to DNA was produced by selenites but not by selenates. The reactivity of selenites with DNA was also indicated by a significant loss of transformation of the tryptophan marker of B. subtilis DNA treated with H2Se03 and Se02.     

In vitro metabolism of selenite in sheep blood: factors controlling the distribution of selenium and the labelling of plasma protein.

Some factors controlling the distribution of Na275SeO3 in sheep blood were studied in vitro. After centrifuging Na275SeO3-incubated blood most of the radioactivity was found in the plasma. The labelling of plasma protein by 75Se was dependent on the presence of erythrocytes. The degree of labelling of plasma protein increased with erythrocyte concentration. When phosphate-buffered saline-washed erythrocytes were suspended in phosphate-buffered saline and incubated with Na275SeO3 the majority of the 75Se was detected in the erythrocytes. On incubating these labelled erythrocytes with unlabelled plasma there was a transfer of radioactivity to the plasma. The calculated activation energy for the labelling of plasma was 107.52 kJ/mol. Albumin was shown not to be a principal acceptor of 75Se from the erythrocytes by ammonium sulphate precipitation of radioactive plasma. Addition of Na2SeO3 to the labelled blood resulted in the transfer of 75Se from plasma to the erythrocytes. Radioactive plasma incubated at 37 degrees C was thermolabile with respect to its 75Se content whereas in whole blood the degree of 75Se binding to plasma protein did not vary suggesting that a recycling of selenium was occurring in blood. From the results presented an in vitro model of selenium metabolism in blood is postulated.     

The Lewis blood group system among Chinese in Taiwan.

The nonsecretor gene se is absent (or very rare) among Chinese in Taiwan and the previously reported Le(a+b-) phenotype in this population is in fact Le(a+b+) as proven by the presence of small amounts of Leb antigen on red blood cells. Salivary ABH substances in this phenotype are usually (although not always) markedly reduced. The Chinese Le(a+b+) phenotype is postulated to be the result of a weak secretor gene Se omega. Although the Le(a+b+) phenotype is very rare in Caucasians, it has a frequency of 25% in Chinese. All Le(a-b-) Chinese are ABH secretors and have varying amounts of Lea and/or Leb substances in saliva.     

Nonsyndromic cleft lip with or without cleft palate in west Bengal, India: evidence for an autosomal major locus.

Ninety extended families having one or more individuals affected with nonsyndromic cleft lip (CL) with or without cleft palate (CL/P) were ascertained in rural West Bengal, India. These families included 138 affected people, 64% of whom had CL alone and 66% of whom were male. Multiple-affected-member ("multiplex") pedigrees were less common than single-affected-member ("simplex") pedigrees, composing 34% of all extended pedigrees. There was no difference between multiplex and simplex pedigrees in the frequency of affected persons with CL alone, but multiplex pedigrees had a lower frequency of affected males (58%) than did simplex pedigrees (76%; P = .02). Complex segregation analysis using the POINTER computer program rejected both the hypothesis of no familial transmission (P < .0001) and the hypothesis that familiarity could be explained solely by a multifactorial/threshold model (P < .05). The hypothesis of major-locus inheritance alone could not be rejected. Among major-locus models examined, strictly recessive inheritance was rejected (P < .0001), but codominant and dominant models were not. Neither the addition of a multifactorial component nor the addition of a proportion of sporadic cases to the major-locus model improved the fit of the data. In conclusion, the results of complex segregation analysis were consistent with a dominant or codominant major-locus mode of inheritance of CL/P in these families.     

Relationship of blood trace elements to liver damage, nutritional status, and oxidative stress in chronic nonalcoholic liver disease

Trace elements are involved in chronic liver diseases because these elements may have a direct hepatic toxicity or may be decreased as a consequence of the impaired liver function, particularly in patients with alcoholic cirrhosis and/or malnutrition. In this study, we determined plasma and erythrocytes trace elements in 50 inpatients with nonalcoholic chronic liver disease (11 with biopsy-proven chronic hepatitis, 39 with cirrhosis [16 in stage A according to Child-Pugh criteria, 23 Child B+C]), and in a control group of 10 healthy subjects by the proton induced x-ray emission method. The relationship between trace element concentration and the extent of liver damage, the nutritional status (by anthropometric evaluations), and various blood markers of oxidative stress--reduced glutathione, total lipoperoxides and malonyldialdehyde--was investigated. We found that cirrhotics had a significant decrease of Fe, Zn, Se, and GSH levels in the plasma and of GSH and Se in the erythrocytes with respect to the control and chronic hepatitis groups. GSH levels were related to the degree of liver damage; a significant direct correlation was observed among Se, Zn, and GSH plasma values and between GSH and Se in the erythrocytes. The trace element decrease was, on the contrary, independent of the degree of liver function impairment and only partially affected by the nutritional status. Data indicate that liver cirrhosis, even if not alcohol related, induces a decrease of Se and Zn and that, in these patients, an oxidative stress is present, as documented by the significant correlation between Se and GSH. The plasma Br level was higher in cirrhotics with respect to the control and chronic hepatitis groups.     

In Vivo Formation and Binding of SeHg Complexes to the Erythrocyte Surface

The in vivo dynamics of selenium (Se) and mercury (Hg) interaction was studied in mouse tissues using direct visualization of individual Se, Hg, and SeHg particles on the surface of circulating erythrocytes. This high-resolution detection of Se and Hg was obtained by scanning electron microscopy coupled to X-ray microanalysis. BALB/c mice were injected in the peritoneal cavity with Se and Hg salts, and the animals were sacrificed 3 min after the Hg injection. Only a minority (9%) of the metal dots seen on mouse liver erythrocytes were SeHg complexes when Se and Hg salts were mixed together before injection. In contrast, the majority (73%) of metal dots on liver erythrocytes were SeHg complexes if Se was injected at least 5 min before Hg injection. All metal dots on liver erythrocytes were of SeHg complexes if Se was injected 9 or 12 min before the Hg injection. We conclude that the formation of stable in vivo SeHg complexes requires preliminary interaction of Se with a putative serum factor before complexes between Se and Hg are formed and are bound to the erythrocyte cell surface.