Genetic studies on human thyroxine-binding globulin (TBG)

Summary An enzyme immunoassay technique combined with Western blotting is described to demonstrate thyroxine-binding globulin (TBG) by isoelectric focusing in thin-layer polyacrylamide gels with 8mol/l urea. Quantitative evaluation was by laser densitometry. No genetic charge variants of TBG were encountered in a sample of 840 unrelated individuals from southwestern Germany. There was no correlation between structural and quantitative variations in the TBG protein. Results from a family with quantitative TBG deficiency strongly support the postulated X-linked mode of inheritance. The method described can be considered as an additional diagnostic tool in thyroid evaluation.     

A mutation causing reduced biological activity and stability of thyroxine-binding globulin probably as a result of abnormal glycosylation of the molecule

T4-binding globulin (TBG), a 54-kilodalton glycoprotein, is the major thyroid hormone transport protein in man. The exact nature of the mutations causing X chromosome-linked TBG deficiency, which affect about 1 in 2,500 newborn males, is unknown. Here we report the sequence of a unique variant TBG (TBG-Gary) encoding a protein with severely impaired T4 binding as well as decreased stability at 37 C, resulting in its rapid in vivo denaturation. A single nucleotide substitution in the codon for residue 96 of the mature protein replaces isoleucine with asparagine; this replacement creates an additional site for N-linked glycosylation. The anodal shift of TBG-Gary on isoelectric focusing gel electrophoresis suggests that this new site is likely glycosylated. Since glycosylated is required for TBG to assume its correct tertiary structure, but is not subsequently necessary for maintenance of the biological properties or stability of the molecule, we believe that the likely presence of additional carbohydrate probably affects a higher order structure of the molecule and is thus responsible for the reduced stability and hormone binding activity of TBG-Gary (TBGASN-96).     

A new polymorphism of thyroxin-binding globulin in three African groups (Mali) with endemic nodular goitre

Summary The thyroxin-binding globulin (TBG) polymorphism was investigated in three African groups: two belonged to the Bwa villages of Mali, and the third was a Dogon group living in the same area. The Bwa groups were characterized by the occurrence of nodular goitres, whereas the Dogon population did not show similar pathological symptoms. Females were more affected by goitre than males in the affected villages. The TBG polymorphism enabled us to demonstrate the presence of an undescribed allele (TBG C1) in these populations. The frequency of the TBG S allele was also higher than previously published in other African groups. We observed a disequilibrium in the distribution of the C and S alleles in the population, with an excess of homozygous TBG S individuals. No clear relationship between the TBG polymorphism and the number of nodules can be drawn.     

Thyroxine-binding globulin synthesis by hepatocarcinoma cells in continuous culture: Effect of physiological concentrations of thyroxine

Thyroxine-binding globulin (TBG) synthesis and secretion were demonstrated in a continuous cell culture line, NCLP-6-E, of Rhesus monkey hepatocarcinoma cells. The cells were shown to survive and grow normally for up to 5 days in the absence of serum, thus permitting study of TBG production in chemically defined media. TBG was identified by its ability to bind thyroxine (T₄) and by immunoelectrophoresis, and quantitated by radioimmunoassay. TBG accumulation in the media was linear for up to 48 hours. Physiological concentrations of T₄ induced a biphasic response in TBG secretion. There was a progressive increase in TBG accumulation from 10^?14M to 10^?10M T₄. TBG accumulation decreased from the maximum at T₄ greater than 10^?10M, and was depressed below control at T₄ greater than 10^?8M. These results indicate that T₄ regulates the synthesis and secretion of TBG in hepatocarcinoma cells in culture.     

Thyroxine-binding globulin variant (TBG-Kumamoto): identification of a point mutation and genotype analysis of its family.

Thyroxine-binding globulin (TBG) is the major thyroid hormone transport protein. Several inherited TBG variants resulting in partial or complete TBG deficiencies have been shown to be caused by either one or two nucleotide substitutions, or one nucleotide deletion in the coding regions of the TBG gene. In this report, a Japanese female patient (proband) with hyperthyroid state, whose lower TBG levels did not return to normal under the euthyroid state after treatment was examined. Genomic DNA samples from the proband with thyroxine-binding globulin deficiency (termed TBG-Kumamoto) and her family were subjected to the polymerase chain reaction, and the generated DNA fragments were sequenced. A single nucleotide substitution in the codon for the amino acid 363 of native TBG molecule (CCT to CTT) was found, resulting in the replacement of proline by leucine. It was revealed that the proband was a heterozygote and her father was a hemizygote. The mutation was confirmed by the allele-specific amplification of genomic DNAs from the proband and her father using oligonucleotide primers of normal or mutant residues at the 3' position in the polymerase chain reaction. These results indicate that the abnormality of TBG-Kumamoto is the consequence of this mutation. Genetically, this point mutation observed in TBG-Kumamoto might be classified as a new type of TBG deficiency.     

A mitochondrial cytochrome b mutation but no mutations of nuclearly encoded subunits in ubiquinol cytochrome c reductase (complex III) deficiency

Ubiquinol cytochrome c reductase (complex III) deficiency represents a clinically heterogeneous group of mitochondrial respiratory chain disorders that can theoretically be subject to either a nuclear or a mitochondrial mode of inheritance. In an attempt to elucidate the molecular bases of the disease, we first determined the nucleotide sequence of three unknown subunits (9.5 kDa, 7.2 kDa, 6.4 kDa) by cyberscreening of human expressed sequence tag data bases and sequenced the 11 cDNA subunits encoding complex III in five patients with isolated complex III deficiency. No mutation in the nuclearly encoded complex III subunits was observed, but a mutation in the cd2 helix of the mitochondrial (mt) cytochrome b gene was found to alter the conformation of the bc ₁ complex in one patient with severe hypertrophic cardiomyopathy. The present study is highly relevant to genetic counseling as the absence of mtDNA mutations in all but one patient in our series strongly supports autosomal rather than maternal inheritance in the majority of patients with complex III deficiency. Received: 15 January 1999 / Accepted: 31 March 1999     

Analysis of transgene(s) (psy+crtI) inheritance and its stability over generations in the genetic background of indica rice cultivar Swarna

Rice the major staple food crop which feeds more than half of the world’s population but, lacks pathway to synthesize and accumulate provitamin A in endosperm therefore rice eaters particularly children, and pregnant women suffer due to vitamin A deficiency. The pathway for provitamin A synthesis in rice endosperm has been engineered and transgenic rice lines have yellow endosperm, called ‘Golden Rice’. The present study aimed at studying the inheritance of transgene(s) in six transgenic events of ‘Golden Rice’ and transfer of provitamin A trait from transgenic lines to a widely grown mega rice variety Swarna. The events E1, R1 and W1 showed normal Mendelian inheritance in F₂, BC₁F₁ and BC₁F₂ generations. The event W1 was studied in BC₁F₃ as well and showed normal Mendelian inheritance of 3:1. The inheritance pattern in L1 event in BC₁F₁ and BC₁F₂ showed normal Mendelian inheritance following expected ratio 1:1 and 3:1 respectively. The two events G1 and T1 showed distorted segregation in BC₁F₂ and BC₂F₂ respectively in Swarna genetic background. In G1 event, transgene inheritance showed segregation distortion in BC₁F₂ in favour of transgene negative plants. In T1 event, inheritance followed expected Mendelian segregation in BC₁F₁, BC₂F₁ and BC₂F₂, generations. However, when tested against co-dominant inheritance 1:2:1 pattern in BC₂F₂, segregation distortion was observed with less than the expected transgene homozygotes. While against 3:1 ratio, it showed the expected segregation pattern in BC₂F₂ generation. Segregation distortion probably due to differential transmission of transgene positive/negative gametes through either/both parents which needs further study.     

Radioimmunoelectrophoretic analysis of proteins binding 3, 3', 5'-triiodothyronine in human serum

The binding of purified 131I-3, 3', 5'-triiodothyronine (reverse T3) (rT3) to normal human serum components was investigated by a radioimmunoelectrophoretic technique. When anti-whole human serum was used, five distinct arcs of radioactivity were observed. Evidence was obtained that five of these radioactive arcs were not artifacts, but were due to components binding rT3. From the radioimmunoelectrophoretic patterns with specific antisera, five of these components were identified as thyroxine binding prealbumin, albumin, thyroxine binding globulin (TBG) and alpha 1-and beta-lipoproteins. No radioactive arc of TBG was detected in serum from a patient with TBG deficiency.     

Mode of inheritance of azinphosmethyl resistance in a laboratory-selected strain of Trioxys pallidus

Homogeneity in azinphosmethyl resistance was assessed in males of a laboratory-selected (Select-17) and susceptible (Yolo) colony of Trioxys pallidus Haliday (Hymenoptera: Aphidiidae) using a time response assay. No evidence of heterogeneity within the two colonies was found. Reciprocal crosses between the Yolo and the Select-19 (the Select-17 colony following two additional selections) colonies resulted in F₁ females that exhibited a semidominant response to azinphosmethyl with a dominance value (D) of 0.32, as well as no evidence of maternal effects or sex linkage. Responses of F₂ progeny to azinphosmethyl suggest that more than one gene may be involved because no inflection was observed in the time response lines of F₂ males. Additional research is required to fully elucidate the mode of inheritance.     

Confirmation of maternal transmission ratio distortion at Om and direct evidence that the maternal and paternal “DDK syndrome” genes are linked

The polar, preimplantation-embryo lethal phenotype known as the ``DDK syndrome' in the mouse is the result of the complex interaction of genetic factors and a parental-origin effect. We previously observed a modest degree of transmission-ratio distortion in favor of the inheritance of DDK alleles in the Ovum mutant (Om) region of Chromosome (Chr) 11, among offspring of reciprocal F<sub>1</sub>-hybrid females and C57BL/6 males. In this study, we confirm that a significant excess of offspring inherit DDK alleles from F<sub>1</sub> mothers and demonstrate that the preference for the inheritance of DDK alleles is not a specific bias against the C57BL/6 allele or a simple preference for offspring that are heterozygous at Om. Because none of the previous genetic models for the inheritance of the ``DDK syndrome' predicted transmission-ratio distortion through F₁ females, we reconsidered the possibility that the genes encoding the maternal and paternal components of this phenotype were not linked. We have examined the fertility phenotype of N₂ females and demonstrate that the inter-strain fertility of these females is correlated with their genotype in the Om region. This result establishes, directly, that the genes encoding the maternal and paternal components of the DDK syndrome are genetically linked. Received: 1 February 1997 / Accepted: 26 April 1997     

Surface plasmon resonance assay of inhibition by pharmaceuticals for thyroxine hormone binging to transport proteins

We developed a surface plasmon resonance (SPR) assay to estimate the competitive inhibition by pharmaceuticals for thyroxine (T4) binding to thyroid hormone transport proteins, transthyretin (TTR) and thyroxine binding globulin (TBG). In this SPR assay, the competitive inhibition of pharmaceuticals for introducing T4 into immobilized TTR or TBG on the sensor chip can be estimated using a running buffer containing pharmaceuticals. The SPR assay showed reproducible immobilization of TTR and TBG, and the kinetic binding parameters of T4 to TTR or TBG were estimated. The equilibrium dissociation constants of TTR or TBG measured by SPR did not clearly differ from data reported for other binding assays. To estimate the competitive inhibition of tetraiodothyroacetic acid, diclofenac, genistein, ibuprofen, carbamazepine, and furosemide, reported to be competitive or noncompetitive pharmaceuticals for T4 binding to TTR or TBG, their 50% inhibition concentrations (IC₅₀) (or 80% inhibition concentration, IC₈₀) were calculated from the change of T4 responses in sensorgrams obtained with various concentrations of the pharmaceuticals. Our SPR method should be a useful tool for predicting the potential of thyroid toxicity of pharmaceuticals by evaluating the competitive inhibition of T4 binding to thyroid hormone binding proteins, TTR and TBG.     

Serum thyrotropin response to thyrotropin-releasing hormone and free thyroid hormone indices in patients with familial thyroxine-binding globulin deficiency.

The response in serum thyrotropin (TSH) to synthetic thyrotropin-releasing hormone (TRH) as well as serum free thyroxine index (FT4I) and free triiodothyronine index (FT3I) was investigated in six patients with familial thyroxine-binding-globulin (TBG) deficiency. The total serum thyroxine (T4) and triiodothyronine (T3) concentrations were significantly decreased, compared with those of normal subjects (3.4 +/- 0.9 microgram/dl, mean +/- SD. vs. 9.0 +/- 1.5 microgram/dl, p less than 0.01 and 87 +/- 27 ng/dl vs. 153 +/- 37 ng/dl, p less than 0.01, respectively). FT4I was lower than the normal range in all but one (5.3 +/- 1.5 vs. 8.9 +/- 1.6, p less than 0.01), whereas FT3I was all in the normal range and of no significant difference from the normal control (132 +/- 22 vs. 148 +/- 25). Serum TSH concentrations in TBG deficiency were all in the normal range (1.0-4.2 muU/ml) and the maximum TSH increments following TRH 500 microgram iv were 8.9 +/- 2.0 muU/ml and of no significant difference from the normal control (10.2 +/- 4.5 muU/ml). These results indicate that the euthyroid state in familial TBG deficiency is more clearly defined by TRH-test and the normal response to TRH in familial TBG deficiency is presumably under the control of the serum free T3 level rather than the serum free T4 level.     

Identifizierung der genetischen Geschlechtschromosomen bei der monogenen Schmeißfliege Chrysomya rufifacies (Calliphoridae,Diptera)

Previous investigations have shown the sex determination in the monogenic blowfly Chrysomya rufifacies to be controlled by a cytologically not discernible homogamety-heterogamety mechanism in the female. Female-producing (thelygenic) females are assumed to be heterozygous for a dominant female sex realizer (F′) with sex-predetermining properties, while male-producing (arrhenogenic) females as well as males are supposed to be homozygous for the recessive allele (f). In order to identify the genetic sex chromosomes of C. rufifacies among its five pairs of long euchromatic chromosomes (nos.1–5) plus one pair of small heterochromatic ones (no. 6), all chromosomes were marked by reciprocal translocations induced by X-ray treatment of adult males. The inheritance of thirteen different heterozygous translocations has been analyzed. All of the translocations (eleven) between two of the four longer chromosomes did not show sex-linked inheritance, thus demonstrating the autosomal character of the chromosomes nos 1, 2, 3 and 4. The same is true for the translocation T6 (2/6). Therefore the small heterochromatic chromosome no. 6, corresponding to the morphologically differentiated sex chromosomes within the amphogenic calliphorid species, remains without sex determining function in the monogenic fly. This could be confirmed by the analysis of monosomic (monosomy-6) and trisomic (trisomy-6) individuals, which resulted from meiotic non-disjunction in T6/+ translocation heterozygotes. Contrary to these translocations, the heterozygous 5/2 translocation (T14) exhibited sex-linked inheritance: There was but a very low frequency (0,76 per cent) of recombinants resulting from crossing-over between F′/f and the translocation breakage point in thelygenic F₁ T14/+ females. The sex-linked inheritance of T14 was confirmed by the progeny of a thelygenic F₁ T14/+ female crossed to a homozygous T14/T14 translocation male. Among the offspring of that F₁ T14/+ female, which had received the translocation from its father, all of the F₂ T14/+ females were thelygenic compared to their arrhenogenic T14/T14 sisters. These results prove that the chromosomes of pair no. 5 genetically act as X′X-XX sex chromosomes in C. rufifacies.     

TBG-dependency of age related variations of thyroxine and triiodothyronine.

Thyroxine (T4), triiodothyronine (T3) and thyroxine-binding globulin (TBG) were determined in healthy individuals ranging in age from newborn to 95 years. T4: 10.25 +/- 1.62 microng/100 ml, T3: 1.62 +/- 0.35 ng/ml and TBG: 1.34 +/- 0.15 mg/100 ml, were found elevated until puberty compared to a middle age group with T4: 7.27 +/- 2.26 microng/100 ml, T3: 1.15 +/- 0.24 ng/ml and TBG: 0.98 +/- 14 mg/100 ml. T4 and T3 followed almost TBG concentration. In old age is dissociation between T4: 5.79 +/- 1.56 microng/100 ml, T3: 0.79 +/- 0.21 ng/ml and TBG: 1.28 +/- 0.15 mg/100 ml was found. Except for old age the ratio T4/TBG and T3/TBG minimized the age dependent variation of T4 and T3 and reduced the coefficient of variance from 26% to 17.7% for T4 and from 26.5 to 25% for T3. Age reduction of T4/TBG is 15% and of T3/TBG 13% respectively more pronounced than for T4 and T3 alone. These data indicate: 1) age related variations of T4 and T3 due to age dependency of TBG, 2) deviation of T4 and T3 values in old age from that expected by their TBG levels and 3) the importance of the routine use of hormone/TBG ratio.     

Mitochondrial genes inPodospora anserina: Recombination and linkage

Summary A fifth cytoplasmic mutation (cap ^r 1) obtained inPodospora anserina is described. In addition to chloramphenicol resistance it confers a strong deficiency in cytochrome aa₃ and impairs the germination of ascospores. Genetic analysis shows: 1) strict maternal inheritance of (cap ^r 1) allele; 2) selection against the (cap ^r 1) allele as well in sexual crosses as during vegetative growth; 3) complete reversion of this selection by even low concentration of CAP. On the basis of their cytoplasmic inheritance and altered cytochrome spectra the five cytoplasmic mutations are assumed to be mitochondrial. Analysis of crosses between them allows to class them in 3 loci, 2 of which being closely linked.     

Sex determination of the onion fly,Hylemya antiqua (Meigen)

In testes of many XXY₂ and XXY₂Y₂ males of Hylemya antiqua a numerical variation for Y₂, a consequence of mitotic nondisjunction, has been established. In 22 of 79 progenies of XXY₂ (including some unidentified XXY₂Y₂) males the ratio of females to males was significantly (p<0.05) different from 11, 9 with excess females and 13 with excess males. It could be concluded, firstly, that in progenies of XXY₂ males sex ratio distortion results from a numerical variation of Y₂ between the primordial germ cells of the male parent and secondly, that probably at least three primordial germ cells are involved in the formation of the male germ cells of the onion fly. Besides, the occurrence of some highly distorted sex ratios in male direction could be attributed to the presence of XXY₂Y₂ males. The frequency of M II cells with 2 Y₂ chromosomes established in XXY₂ males amounted to 2.9%. In XXY₂Y₂ males this percentage was higher and occasionally M IIs with 3 Y₂ were found. In the progeny of one cross a chromosome morphologically identical with Y₂ but lacking its holandric inheritance was found. From its inheritance, it could be concluded that it was a nonfunctional (mutated) Y₂ chromosome (Y_m). Interesting phenomena were the reduced transmission of Y_m through the female, and the observation of very high numbers of Y_m in some ovarian cells. A possible causal relationship between these two phenomena is discussed.     

Evidence of doubly uniparental inheritance of the mitochondrial DNA in Polititapes rhomboides (Bivalvia,Veneridae): Evolutionary and population genetic analysis of F and M mitotypes

Doubly uniparental inheritance (DUI) is a particular mitochondrial DNA inheritance mode reported in a number of bivalves. DUI species show two types of mtDNA, one transmitted from females to daughters and sons (F mitotype) and another one from males to sons (M mitotype). In Veneridae, the existence of DUI has been investigated in several species but it was found in only two of them. In this study, we obtained partial sequences of rrnL, cytb and cox1 genes of males and females of Polititapes rhomboides from NW Spain and we demonstrated the existence of heteroplasmy in males, as expected under DUI. F and M mitotypes showed a taxon-specific phylogenetic pattern and similar evolutionary rates. We focused on cox1 for population genetic analysis, examining separately F and M mitotypes, but also F mitotypes from females (F_♀) and males (F_♂). In all cases, cox1 bears signs of strong purifying selection, with no apparent evidence of relaxed selection in the M genome, while the divergence between F and M genomes is in agreement with the neutral model of evolution. The cox1 polymorphism, higher at the M than at the F genome, also shows clear footprints of genetic hitchhiking with favourable mutations at other mtDNA loci, except for F_♂. In terms of population structure, results suggest that the pattern depends on the examined mitotype (F, F_♀, F_♂ or M).     

In vitro expression of thyroxine-binding globulin (TBG) variants. Impaired secretion of TBGPRO-227 but not TBGPRO-113.

Thyroxine-binding globulin (TBG) is a glycoprotein that transports thyroid hormones in blood. Of two naturally occurring variants in man that harbor single proline substitutions (TBG-CD5 and TBG-Montreal), only TBG-CD5 manifests as complete TBG deficiency. In order to determine the pathophysiology of these TBG disorders, we expressed TBG-CD5 and TBG-Montreal (TBG-M), as well as the common type TBG (TBG-C) in reticulocyte lysate and Xenopus oocytes. Vectors encoding the three TBG types were constructed, transcribed in vitro, and their products of cell-free translation and processing by canine microsomal membranes were analyzed. TBG-C and TBG-M had identical mobility on denaturing polyacrylamide gel electrophoresis but could be distinguished by differences in thyroxine (T4) binding. TBG-CD5 had altered electrophoretic mobility and did not bind T4. TBG-C and TBG-M expressed in microinjected Xenopus oocytes showed properties similar to their respective serum forms, whereas TBG-CD5 was found in small amounts only intracellularly. Our results confirm that the previously described alanine 113 to proline substitution is responsible for the altered properties of TBG-M. The substitution of leucine 227 by proline in TBG-CD5 appears to impair its cotranslational processing and secretion.     

Detection of genetic variation with radioactive ligands. IV. X-linked, polymorphic genetic variation of thyroxin-binding globulin (TBG).

A genetically determined, polymorphic electrophoretic variant of thyroxin-binding alpha-globulin (TBG) is found in sera from populations of African and Oceania origin, although not in Caucasians nor Orientals. The TBG polymorphism is inherited in X-linked fashion, based on data from American blacks, and thus provides an X-chromosome marker with a relatively high gene frequency in this ethnic group (frequency of the slow allele, TBGs, is 11%). This slow variant should prove valuable in expanding the map of the X chromosome and in linkage studies. An additional family exhibiting X-linked TBG deficiency is also described.