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.     

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.     

Polymorphism of human thyroxine binding serum globulin (TBG); existence of a new form of serum protein without detectable triiodothyronine binding power

Two kinds of TBG polymorphism are described in human, one found in deglycosylated TBG from individual blood donors, the other is a genetically determined polymorphism. TBG from plasma samples from a patient with toxic goiter, not autoimmune, (p)TBG, from the patient's mother (m)TBG and from individual donors (n)TBG, were labeled with [125I]T4 or [125I]T3 and submitted to isoelectric focusing (IEF), followed by autoradiography. Three faint [125I]T4 radiolabeled bands were detectable in (p)TBG while four strong [125I]T4 radiolabeled bands were detectable in (m)TBG and (n)TBG), respectively. IEF of the [125I]T3 incubated serum samples resulted in no detectable isoelectric radiolabeled band for (p)TBG while a normal pattern was found in (m)TBG and in (n)TBG, respectively. These data suggest a new intraindividual not linked to sexual chromosome X polymorphism characterized by a loss in hormone binding.     

Population genetics of the vitamin D binding protein (GC) subtypes in the Asian-Pacific area: Description of new alleles at the GC locus

Isoelectric focussing (IEF) in thin layer polyacrylamide gels pH range 4-6.5 has been used to analyse the GC phenotypes of 4233 individuals from 28 different population groups in the Asian, Pacific, and Australian area. Because this technique reveals subtypes of the common GC*1 allele, there is almost a two-fold increase in the mean heterozygosity at the GC locus using IEF compared with conventional electrophoresis. The highest frequency (above 50%) of the GC*1S allele was encountered in Indian populations, reflecting genetic affinities with Europeans. By comparison, east and south east Asians are unique offing maximum values of the GC*1F allele (50%). With the exception of a few Pacific populations which show similar frequencies to east Asians, all other groups in the Pacific area, including Australia, have values of GC*1F similar to GC*1S ranging from 27% to 40%. The GC*2 frequency in most populations varies from 20% to 30%. However, some Polynesian groups have values up to 40% and Australian Aborigines less than 10%. Among other alleles, GC*1A1 is found to be widely distributed among Australian Aborigines and Melanesians and occurs sporadically in Polynesians, Micronesians, and in the Lesser Sunda Islands. Four new alleles, GC*1C24, GC*1C35 Aborigine, GC*1A21, and GC*1A22 are described. The gene frequency data at the GC locus has been used to calculate Nei genetic distances between the populations studied.     

Human thyroxine-binding globulin (TBG): Heterogeneity within individuals and among individuals demonstrated by isoelectric focusing

Isoelectric focusing of human plasma samples labeled in vitro with [¹²⁵I]-thyroxine reveals considerable biochemical and genetic variation in thyroxine-binding globulin. (1) In all individuals tested, at least three primary isoelectric bands are seen in the pH range of 4.2 to 4.5, with additional bands at lower pH ranges. Similar patterns are produced by plasma from nonhuman primates. These band differences appear to be the result of differences in sialic acid content. TBG produces a single electrophoretic band on standard polyacrylamide gel electrophoresis. (2) Genetically determined, X-linked differences in electrophoretic mobility of TBG are observed in several human populations. Female homozygotes or male hemizygotes for the TBG slow variant (TBG-S) produce band patterns shifted by 0.5 pH unit cathodal to the common pattern (TBG-C). Female heterozygotes produce patterns with six-plus bands, representing the simple sum of the common and slow types. This difference is not the result of differences in sialic acid content. The gene frequency of this variant is 10% in American Blacks. (3) In pregnant women additional anodal bands are observed, giving the impression of a shift, by integral steps, in the pattern relative to the nonpregnant type. This shift is abolished by mild neuraminidase treatment.This work was supported by a grant from the O'Brien family of Houston, Texas.     

Tooth size and morphology in a recent Australian Aboriginal population from Broadbeach,South East Queensland

Odontometric and morphologic observations were made of the dentition of skeletal remains of Australian aborigines from Boradbeach, S.E. Queensland. Tooth size, especially of the molars, was found to be significantly larger than that reported for other recent Aboriginal populations. Tooth morphology also differed, with a higher frequency of five cusped second molars, and a lower frequency of shoveling and Carabelli's cusp than previously reported as typical of Australian aborigines.     

Widespread pyrethroid resistance in Australian diamondback moth, Plutella xylostella (L.), is related to multiple mutations in the para sodium channel gene

Populations of Plutella xylostella, extending over 3800 km in southern Australia, show no genetic structure as assessed by microsatellite markers; yet outbreaks of pyrethroid resistance occur sporadically in cropping areas. Since mutations in the para voltage-gated sodium channel gene have been implicated in pyrethroid resistance, we looked for DNA sequence variation at this target among Australian moths. We found two resistance mutations previously reported for this species (L1014F and T929I), as well as a novel substitution (F1020S). Of the eight possible haplotypes formed by combinations of these three biallelic polymorphisms, only four were found in Australian populations: the wild-type allele (w), the kdr mutation allele (kdr) with only L1014F, the super-kdr-like combination of L1014F and T929I (skdrl), and the crashdown allele with only F1020S (cdr). Comparison of genotype frequencies among survivors of permethrin assays with those from untreated controls identified three resistant genotypes: skdrl homozygotes, cdr homozygotes and the corresponding heterozygote, cdr/skrdl - the heterozygote being at least as resistant as either homozygote. Spatial heterogeneity of allele frequencies was conspicuous, both across the continent and among local collections, consistent with reported spatial heterogeneity of pyrethroid resistance. Further, high resistance samples were sometimes associated with high frequency of cdr, sometimes high frequency of skdrl, or sometimes with a high combined cdr+skdrl frequency. The skdrl and cdr alleles explain a high proportion of the Australia-wide resistance variation. These data add to evidence that nerve insensitivity by mutations in the para-sodium channel gene is a common pyrethroid resistance mechanism in P. xylostella.     

Low Frequency Variants,Collapsed Based on Biological Knowledge,Uncover Complexity of Population Stratification in 1000 Genomes Project Data

Analyses investigating low frequency variants have the potential for explaining additional genetic heritability of many complex human traits. However, the natural frequencies of rare variation between human populations strongly confound genetic analyses. We have applied a novel collapsing method to identify biological features with low frequency variant burden differences in thirteen populations sequenced by the 1000 Genomes Project. Our flexible collapsing tool utilizes expert biological knowledge from multiple publicly available database sources to direct feature selection. Variants were collapsed according to genetically driven features, such as evolutionary conserved regions, regulatory regions genes, and pathways. We have conducted an extensive comparison of low frequency variant burden differences (MAF<0.03) between populations from 1000 Genomes Project Phase I data. We found that on average 26.87% of gene bins, 35.47% of intergenic bins, 42.85% of pathway bins, 14.86% of ORegAnno regulatory bins, and 5.97% of evolutionary conserved regions show statistically significant differences in low frequency variant burden across populations from the 1000 Genomes Project. The proportion of bins with significant differences in low frequency burden depends on the ancestral similarity of the two populations compared and types of features tested. Even closely related populations had notable differences in low frequency burden, but fewer differences than populations from different continents. Furthermore, conserved or functionally relevant regions had fewer significant differences in low frequency burden than regions under less evolutionary constraint. This degree of low frequency variant differentiation across diverse populations and feature elements highlights the critical importance of considering population stratification in the new era of DNA sequencing and low frequency variant genomic analyses.     

Population cytology of the genus Phaulacridium

Samples were taken from twenty three Australian mainland populations of the shorthorned grasshopper, Phaulacridium vittatum. Five of these populations were found to be polymorphic for B-chromosomes. The anaphase I segregation of the B-chromosome appeared to be random with respect to the X (contra Jackson and Cheung, 1967). The B-chromosomes did not appear to influence chiasma frequency in the population in which they were found.     

Effect of thyroxine-binding globulin (TBG) on thyroxine (T4) uptake by human peripheral mononuclear cells: evidence of TBG-dependent uptake of T4

The effect of sialylated TBG and desialylated TBG on thyroxine (T4) uptake by human peripheral mononuclear cells was investigated in vitro. [125I]-T4 uptake was observed when the cells were incubated with free [125I]-T4. The uptake was inhibited in a concentration dependent manner when TBG was added. During the incubation, [125I]-T4 binding to TBG was observed. [125I]-T4 incorporation into cells was also observed when the cells were incubated with [125I]-T4-sialylated TBG or with [125I]-T4-desialylated TBG complex. The uptake was related to the temperature and length of time of the incubation. The amount of [125I]-T4 incorporated into the cells incubated with [125I]-T4-sialylated TBG was greater than that into the cells incubated with [125I]-T4-desialylated TBG during the early 0-20 min. incubation, whereas the amount of [125I]-T4 incorporated into the cells incubated with [125I]-T4-desialylated TBG became greater than that into the cells incubated with [125I]-T4-sialylated TBG after 20 min. of incubation. Pretreatment of the cells with methylamine blocked [125I]-T4 uptake in both cases, i.e. incubated with [125I]-T4-sialylated TBG and incubated with [125I]-T4-desialylated TBG. The results suggest that TBG plays a role not only as a carrier protein for T4 in circulation but also as a protein which can transport T4 from the extracellular into the intracellular space, so that the mechanism of T4 transport mediated by desialylated TBG is different from that mediated by sialylated TBG, and that the T4 transport system in both cases, mediated by sialylated TBG and by desialylated TBG, may be related to the internalization of T4-TBG-TBG receptor complex or of T4-T4 receptor complex if TBG receptors are present in the outer surface of the cell membrane.     

The Association Between HLA-A Alleles and Young Alu Dimorphisms Near the HLA-J, -H,and -F Genes in Workshop Cell Lines and Japanese and Australian Populations

At least two polymorphic Alu insertions have been previously identified and characterized within the class I region of the major histocompatibility complex (MHC). We have identified another two new polymorphic Alu insertions, AluyHJ and AluyHF, located near HLA-J and HLA-F, respectively, within the a block of the MHC. Here we report on (1) the haplotypic relationships between the Alu dimorphisms and the HLA-A locus within a panel of 51 IHW homozygous cell lines representing at least 36 HLA class I haplotypes, (2) the Alu genotype, allele, and haplotype frequencies present in the Australian Caucasians and Japanese populations, and (3) the frequency of association between the different Alu dimorphisms and the HLA-A alleles in 109 Australian Caucasians and 99 Japanese. PCR was used to detect the presence or absence of insertion for AluyHJ, AluyHG, and AluyHF within the DNA samples prepared from the cell lines and the two population groups that had been previously typed for HLA-A. In the homozygous cell lines, all three Alu insertions were found in only one HLA class I haplotype (HLA-A1, -B57, -Cw6), no Alu insertions were detected in six HLA class I haplotypes and one or more of the Alu insertions were found in 29 HLA class I haplotypes. At least one of the Alu insertions was found in about 86% of the Japanese and Australian individuals, with the AluyHJ generally related inversely to AluyHG and/or AluyHF. The gene frequency of the AluyHJ and AluyHF insertions was significantly different (p <0.05) BETWEEN JAPANESE AND AUSTRALIANS, WHEREAS THERE WAS NO DIFFERENCE (P > 0.05) between the frequencies of AluyHG in the two populations. The Alu haplotype frequencies were also significantly different between the Japanese and the Australians. In the cell lines and the population groups, the AluyHJ insertion was most frequently found associated with HLA-A1 or A24, AluyHG with HLA-A2, and AluyHF with HLA-A2, -A10, or -A26. This study suggests that the three polymorphic Alu elements have been inserted into the a block of the MHC in different progenitor groups and therefore will be useful lineage and linkage markers in human population studies and for elucidating the evolution of HLA class I haplotypes.     

An additional carbohydrate chain in the variant thyroxine-binding globulin-Gary (TBGAsn-96) impairs its secretion.

The T4-binding globulin-Gary (TBG-G) variant has severely impaired T4 binding, is unstable at 37 C, and presents an apparent anodal shift of all isoforms when submitted to isoelectric focusing. Inheritance of this abnormal TBG produces a profound decrease in the serum levels of native TBG with reciprocal changes in its denatured form, causing thyroid hormone concentrations to be as low as those found in complete TBG deficiency. The TBG-G gene possesses a single nucleotide substitution replacing the normal IIe96 (ATC) with Asn (AAC), thus creating a new site for N-linked glycosylation. In order to determine whether TBG-G contains an additional carbohydrate chain as indirectly suggested by the isoelectric focusing results, cDNAs containing the normal TBG (TBG-N), and TBG-G were inserted in the appropriate vectors to allow their expression in mammalian cells (COS-1) and in amphibian (Xenopus) oocytes. In both systems, expression of TBG-G yielded a larger molecule than TBG-N when analyzed by polyacrylamide gel electrophoresis under denaturing conditions. However, both were identical in size when synthesized in COS-1 cells in the presence of tunicamycin or when deglycosylated after their synthesis in Xenopus oocytes. Pulse chase experiments revealed impaired secretion and excessive overall intracellular degradation of TBG-G relative to TBG-N. As expected from studies on serum from affected subjects, in vitro expressed TBG-G had a 10-fold lower affinity for T4. These studies prove that the new site for potential glycosylation created by the point mutation in TBG-G is indeed glycosylated.(ABSTRACT TRUNCATED AT 250 WORDS)     

A sensitive immunoblotting technique to identify thyroxin-binding globulin protein heterogeneity after isoelectric focusing

An enzyme-linked immunoblotting technique is described to detect thyroxin-binding globulin (TBG) variation in human plasma samples after isoelectric focusing in thin-layer polyacrylamide gels followed by transfer to a nitrocellulose membrane. The technique is used to demonstrate the polymorphic variation in thyroxin-binding globulin present in populations of African and Mongoloid origin and has enabled the detection of a new TBG variant in Eskimos. This technique eliminates the use of radioactive material and related expensive equipment. The technique is specific, sensitive, and reliable for the routine detection of genetic and epigenetic variation in thyroxin-binding globulin.This work was supported by NIH Grants RR05451-24, CA29614, and CA34836.     

Prevalence of p.V37I Variant of GJB2 in Mild or Moderate Hearing Loss in a Pediatric Population and the Interpretation of Its Pathogenicity

A p.V37I variant of GJB2 has been reported from subjects with moderate or slight hearing loss especially in East Asian populations. This study aimed to estimate the prevalence of the p.V37I variant among such subjects and prove, epidemiologically, its pathogenic potential to cause mild hearing loss. A total of 380 subjects from 201 families with hearing loss were enrolled. From them, 103 families were selected who had autosomal recessive inheritance or sporadic occurrence of hearing loss and who were younger than 15 years old. GJB2 sequencing was carried out for the probands of all 103 families. The prevalence of the p.V37I variant was compared between the subtle, mild or moderate hearing loss (group I) and the severe or profound hearing loss (group II) groups. Where possible, a targeted next generation sequencing of 82 deafness genes was performed from the p.V37I carrier to exclude the existence of other pathogenic genes. Five (4.8%) of 103 probands were found to carry p.V37I. The carrier frequency of p.V37I among group I (18.2%) was significantly higher than that of group II (1.2%) or the reported Korean normal hearing control group (1.0%). Detection of the p.V37I variant of GJB2 in 18.2% of Koreans with mild hearing loss strongly suggests its contribution to the pathogenesis of milder hearing loss, which might justify sequencing of GJB2 from these subjects in the Korean population.     

Angiotensin-converting enzyme gene (ACE) insertion/deletion polymorphism in Mexican populations

The angiotensin-converting enzyme gene (ACE) insertion/deletion polymorphism was determined in 211 Mexican healthy individuals belonging to different Mexican ethnic groups (98 Mestizos, 64 Teenek, and 49 Nahuas). ACE polymorphism differed among Mexicans with a high frequency of the D allele and the D/D genotype in Mexican Mestizos. The D/D genotype was absent in Teenek and present in only one Nahua individual (2.0%). When comparisons were made, we observed that Caucasian, African, and Asian populations presented the highest frequencies of the D allele, whereas Amerindian (Teenek and Pima) and Australian Aboriginals showed the highest frequencies of the I allele. The distribution of I/D genotype was heterogeneous in all populations: Australian Aboriginals presented the lowest frequency (4.9%), whereas Nahuas presented the highest (73.4%). The present study shows the frequencies of a polymorphism not analyzed previously in Mexican populations and establishes that this polymorphism distinguishes the Amerindian populations of other groups. On the other hand, since ACE alleles have been associated with genetic susceptibility to developing cardiovascular diseases and hypertension, knowledge of the distribution of these alleles could help to define the true significance of ACE polymorphism as a genetic susceptibility marker in the Amerindian populations.     

Allozyme variation among Brazilian and U.S. populations ofRhyzopertha dominicaresistant to insecticides

The lesser grain borer (Rhyzopertha dominica (F.)) is an important pest of stored grain in many parts of the world (Paleartic, Ethiopian, Oriental, Australian, Neotropical, and Neartic regions) with the ability to fly long distances. These insects have been shown to be resistant to organophosphorus insecticides in several studies. Polyacrylamide gel electrophoresis was used to assess the genetic variability within and among eight Brazilian and seven United States populations of R. dominica and to determine how insecticide resistance may be spreading within both countries. Significant variation in allele frequency among populations was observed at all six polymorphic enzyme loci that were examined. The Brazilian and U.S. populations were genetically differentiated from one another; populations within the U.S. and those within Brazil were also differentiated from one another. The mean genetic similarity among the seven U.S. populations collected in a small region in northeast Kansas was smaller than that among eight Brazilian populations collected in a relatively large geographical area. These results are consistent with the resistance ratios to chlorpyriphos-methyl in R. dominica populations from Brazil and the U.S. and the information available concerning patterns of flight activity in this insect.     

New aspects of isolation and purification of thyroxine-binding globulin from human biological fluids

Based on the new data concerning the multicomponent system of thyroxine-binding proteins in human plasma, some methodological aspects of isolation and purification of thyroxine-binding globulin (TBG) were examined, and a simple two-step procedure for TBG purification was developed. Normal human blood serum, retroplacental serum and amniotic fluid were used as TBG sources. The procedure includes affinity chromatography and adsorption chromatography on a hydroxyapatite column. A biospecific adsorbent was synthesized by stepwise binding of epichlorohydrin and thyroxine to Sepharose. The yield of pure TBG varied from 60 to 80%, depending on the TBG source used. The properties of TBG preparations from retroplacental serum and amniotic fluid were identical; both preparations contained a pregnancy-associated molecular variant, TBG-1. Two novel serum thyroxine-binding proteins were detected, isolated and partly characterized.     

Variants of galactose-1-phosphate uridyl transferase in the Greek populations

Summary The frequency of variants of galactose-1-phosphate uridyl transferase was determined among the nine Greek populations by studying a sample of 1570 unselected individuals. Average frequency of normal allele GALT=0.942, galactosemia gene GALT ^G=0.0021 and the Duarte variant gene GALT ^D=0.0548 were observed. Frequency of galactosemia heterozygotes among Greeks was similar to that in other Caucasian populations, but the frequency of the Duarte variant was considerably higher. With the exception of two populations, one with low (Epirus) and one with high (Thrace) frequencies, the polymorphism of the Duarte variant displays very similar frequencies in the various Greek population groups.Supported by Grants HD 01 974 and GM 15 253 from the U.S. Public Health Service     

Red cell antigen, serum protein and red cell enzyme polymorphisms in Eastern Highlanders of New Guinea

A series of 1,187 blood samples from eight population groups in the Eastern Highlands of Papua New Guinea were tested for genetic variation in blood groups, serum proteins and red cell enzyme systems. The populations belonged to the language groups Gahuku-Asarc-Bena Bena, Kamano, Yagaria, Keiagana, Fore, Agarabe, Auyana and Tairora. Polymorphic variation was found in the ABO, MNS, P1, Rh, Hp, Tf, SEP, 6-PGD, ADA, MDH, and PGM genetic systems. East to West variation was shown in the language groups; the O, S, R2, and R0 genes increase in frequency from East to West and the A, R1, and M genes decrease in the same direction. In the East higher frequencies were found for the Du antigen, for the PGM21 gene and for a PGM second locus variant. The MDH 3 variant was found in all the populations, its highest value being in the Tairora.