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Polymorphisms of multiple cis-acting elements in the beta-globin locus are associated with variable fetal haemoglobin (HbF) level in sickle cell disease. We developed a multiplex assay permitting simultaneous analysis of three polymorphic cis elements spanning 53 kb of the beta-globin locus. We identified concordance between polymorphic alleles in gamma- and beta-globin promoters however a significant number of betaS-chromosomes were identified with polymorphisms in hypersensitive site 2 (HS2) of the beta-globin locus control region juxtaposed to atypical cis alleles in the gamma-promoter. Analysis of an unusually large number of such hybrid haplotype chromosomes provided unique insight into HbF level associated with specific cis alleles. Associations between cis alleles and HbF level in patients were verified by in vitro functional analysis. Our findings indicate that compared to HS2, polymorphism in the gamma-promoter exerts a dominant influence on HbF level in sickle cell disease.

The beta-globin locus control region hypersensitive site 2 (HS2) enhancer possesses a unique property for stimulating high-level globin gene expression. Although the deletion of cis-acting motifs influences the level of enhancement conferred by HS2, there is controversy on whether polymorphism of the same elements contributes to variation of the fetal hemoglobin (HbF) level among patients with sickle cell anemia. We analyzed reporter gene activity of constructs containing variant HS2 enhancers derived from beta(S) chromosomes to directly test the effect of polymorphism on enhancer activity. Constructs containing four enhancer variants linked to an identical gamma-globin promoter showed markedly different levels of reporter gene activity. Juxtaposition of HS2 derived from the Asian and Senegal chromosomes, which are associated with similarly high levels of HbF, to cognate sequence extending to -1500 of the (G)gamma globin gene showed significantly different levels of reporter gene activity. Our findings indicate that nucleotide variation regulates the level of enhancement conferred by HS2; however, the reporter activities showed no correlation with the level of Hb F associated with the common beta(S) chromosomes., ((c)2001 Elsevier Science.)

The precise identification of human hemoglobin variants, over 700 human hemoglobin variants are known, is essential for prediction of their clinical and genetic significance. A systematic approach to their rapid identification is described. Traditionally this requires protein or DNA characterization which entails lengthy analytical procedures. To overcome these obstacles a rapid approach to variant hemoglobin identification has been developed using conventional phenotypic methods combined with electrospray ionization-mass spectrometry (ESI-MS). The latter requires only a small amount of whole blood (10 microl) but in most cases 2 microl would have been sufficient and no preanalytical steps, such as separation of red cells or globin chains, are necessary. Aged, hemolyzed blood samples can also be analyzed. This approach has been used to positively identify 95% of the variants in over 250 samples. The remaining 5% in which a variant was detected by phenotypic techniques were not resolved by mass spectrometry. Ninety-nine different abnormalities comprising 36 alpha-chain variants, 59 beta-chain variants (including 2 extensions), and 4 hybrid hemoglobins were identified. These include 15 novel variants. The application of ESI-MS described requires approximately 1 h to prepare and analyze each sample and has minimal reagent costs. The turnaround time on a single sample can be as little as 2 h. This technique can now be considered a useful additional tool for reference laboratories., (Copyright 2001 Academic Press.)

Little is known about the heterozygous frequency of factor VIII gene markers in the Asian Indian population. The objective of this study was to establish the heterozygous frequency of polymorphic markers within and flanking the factor VIII gene in Indians and identify those most informative for carrier screening and prenatal diagnosis. Factor VIII gene polymorphism analysis at intragenic and extragenic sites was carried out by the polymerase chain reaction (PCR) method and Southern blot procedure. Sixty-three Asian Indian haemophiliacs and their families were screened. A control group of 150 women from nonhaemophilic families were screened for two markers, HindIII and BclI. Among the intragenic markers studied, the HindIII restriction fragment length polymorphism (RFLP) showed the highest heterozygous frequency (0.52) followed by the intron 13 (0.47) and intron 22 (0. 44) short tandem repeats (STRs). Among extragenic markers, TaqI had the highest heterozygous frequency (0.75) followed by BglII (0.54). The intron 22 inversion mutation was observed in eight (40%) of 20 severe cases. In the population studied the most diagnostic polymorphisms were the intragenic markers, intron 22 (70%) STR followed by the intron 13 (52%) STR and HindIII (52%) RFLP, and the TaqI (50%) extragenic marker. Application of HindIII, BclI and the intron 22 dinucleotide repeat combined were diagnostic in 87.2% of haemophilia A families studied.

Inherited deficiency of the housekeeping enzyme triosephosphate isomerase (TPI) is the most severe clinical disorder of glycolysis. Homozygotes manifest congenital hemolytic anemia and progressive neuromuscular impairment, which in most cases pursues an inexorable course with fatal outcome in early childhood. No effective therapy is available. Hitherto specific enzyme replacement has not been attempted in disorders of glycolysis. Primary skeletal muscle myoblasts and Epstein-Barr virus (EBV)-transformed lymphoblastoid cell lines generated from homozygous TPI-deficient patients were cultured in the presence of exogenous enzyme or cocultured with human K562 erythroleukemia cells as an exogenous source of TPI. Uptake of active enzyme by TPI-deficient cells resulted in reversal of intracellular substrate accumulation, with a reduction in dihydroxyacetone phosphate (DHAP) concentration to levels seen in TPI-competent cells. Evidence of successful metabolic correction of TPI deficiency in vitro establishes the feasibility of enzyme replacement therapy, and has important implications for the potential role of allogeneic bone marrow transplantation and gene therapy as a means of sustained delivery of functional enzyme in vivo.

A high frequency of nucleotide substitutions -5A/G, -8G/A, -24T/G in the triosephosphate isomerase (TPI) gene promoter has been demonstrated in African-Americans. The biological significance of these promoter variants, two of which, -8G/A and -24T/G, occur within regulatory elements essential for transcription, is controversial. The geographical distribution and frequency of allelic variation in the TPI promoter was determined in 378 unrelated normal subjects from sub-Saharan African (n = 103), Caribbean (n = 26), Northern European (n = 57), Mediterranean (n = 55), Middle Eastern (n = 42), Asian Indian (n = 48) and Oriental (n = 47) populations. Five haplotypes were identified: the common haplotype, -5A-8G-24T, -5G, -8A, -5G-8A, and -5G-8A-24G. All, with the exception of the -8A haplotype, were present in geographically dispersed populations. The -5G allele, which was found at varying frequency in all groups, has attained high frequency in the African, Caribbean and Oriental populations. Phylogenetic comparison suggests this may represent the ancestral promoter haplotype. Homozygosity for the -5G-8A haplotype identified in four subjects confirms that these variants are not responsible for a null allele as formerly postulated. Linkage disequilibrium between related TPI promoter haplotypes, -5G, -5G-8A and -5G-8A-24G, and a single nucleotide polymorphism at nt2262 of the TPI gene supports a single ancestral origin for these mutations which precedes the separation of African and non-African populations.

Wiskott-Aldrich syndrome (WAS) and X-linked thrombocytopenia (XLT) are allelic phenotypes caused by defects of the WAS gene. Fourteen distinct mutations including seven novel gene defects in 16 WAS and four XLT patients were identified by single strand conformation polymorphism analysis and DNA sequencing of the WAS gene. Eleven (79%) of these mutations are located within exons 1 to 4 with clustering in exon 2. Carrier detection in 33 at-risk females and prenatal diagnosis at 12 weeks gestation in one family with a novel WAS mutation was performed by direct mutation analysis. A remarkably high frequency (72%) of point mutations involved CpG dinucleotides. C-->T or G-->A transitions at CpG sites were identified in all isolated WAS cases (n = 7). Allele frequencies for the dinucleotide repeat at locus DXS6940 were determined in Northern European, African and Asian populations. Mutation screening alone or in combination with analysis of polymorphic loci DXS6940 and DXS255 delineated the germline origin of a unique insertion mutation and four recurrent CpG mutations, three of which arose spontaneously during maternal gametogenesis.

Several cis elements at the beta-globin gene cluster and the upstream locus control region (LCR) have been implicated in modulation of fetal haemoglobin (Hb F) level in beta-globin disorders. To determine the role of elements at the LCR and the beta-globin gene cluster on HbF level among sickle cell anaemia (SCA) patients, hybrid haplotype betaS chromosomes exhibiting variation in the association of alleles of LCR hypersensitive site 2 (HS2) and the beta-globin gene cluster restriction fragment length polymorphosim (RFLP) haplotypes were identified in an unselected population of 100 patients. On 15 chromosomes the polymorphic HS2 short tandem repeat(TA)xN10-12(TA)y containing a Hox2 binding motif differed from that typically associated with the corresponding beta-globin gene cluster RFLP haplotype. Among patients homozygous for the Benin RFLP haplotype, in whom one chromosome carried the (TA)9N10(TA)10 allele, no effect on HbF level was observed. Polymorphism of the pre-Ggamma framework, an enhancer located 25 kb downstream of HS2 localised the breakpoint for each of these 'hybrid' haplotype chromosomes upstream of this element. Previously described hybrid haplotype chromosomes with the (TA)9N10(TA)10 HS2 allele associated with raised HbF by contrast arise by recombination 1 kb downstream of the pre-Ggamma framework. This study suggests that variability in HbF level associated with polymorphisn of the HS2 enhancer depend on downstream determinant (s) in tight linkage disequilibrium with HS2. The pre-Ggamma framework is the only known polymorphic cis-active determinant in this region., (Copyright 1999 Academic Press.)

Familial hemophagocytic lymphohistiocytosis (FHL), also known as familial erythrophagocytic lymphohistiocytosis and familial histiocytic reticulosis, is a rare autosomal recessive disorder of early childhood characterized by excessive immune activation. Linkage of the disease gene to an approximately 7.8-cM region between markers D9S1867 and D9S1790 at 9q21.3-22 was identified by homozygosity mapping in four inbred FHL families of Pakistani descent with a combined maximum multipoint LOD score of 6.05. This is the first genetic locus to be described in FHL. However, homozygosity by descent across this interval could not be demonstrated in an additional affected kindred of Arab origin, whose maximum multipoint LOD score was -0.12. The combined sample revealed significant evidence for linkage to 9q markers (LOD score with heterogeneity, 5.00). Identification of the gene(s) involved in the pathogenesis of FHL will contribute to an understanding of the control of T-lymphocyte and macrophage activation, which is central to homeostasis in the immune system.

Elucidation of the molecular basis for persistent fetal haemoglobin (Hb F) production in adult life has important implications for the pathophysiology and treatment of human beta haemoglobinopathies. Electrospray ionisation mass spectrometry (ESMS) was applied to analyse the pattern of gamma-globin expression in patients with hereditary persistence of fetal haemoglobin (HPFH) and sickle cell anaemia (SCA). Ggamma and Agamma-globin chains were identified by their measured molecular masses and distinguished by mass difference (14 Da) following deconvolution of ESMS spectra using maximum entropy based software. Prediction of HPFH type by ESMS was confirmed by molecular analysis. Direct determination of Ggamma:Agamma globin chain ratio from whole blood by the novel application of ESMS provides a rapid and sensitive approach to characterisation of gamma-globins and facilitates correlation of gamma-globin level and polymorphism of cis-active elements at the beta-globin locus.

Screening of the factor VIII (FVIII) gene which spans 186 kb and codes for 26 exons, was originally hampered by its size but is now feasible because rapid DNA scanning methodologies have been developed. The present study for the first time directly compares the three most widely applied screening methods, denaturing gradient gel electrophoresis (DGGE), single-stranded conformational polymorphism (SSCP) and chemical mismatch cleavage (CMC) for their sensitivity of mutation detection in a selected group of ten haemophilia A patients. Nine of these patients are known to be cross-reacting material positive and eight exhibited a mild to moderate phenotype. Of the ten patients screened, we identified mutations in nine by all three screening methods. Of the mutations characterised, two are previously unpublished. T to C (S373P) and G to A (D525N). In one mildly affected haemophiliac, we identified a second T to C sequence change in the 5' untranslated region at -601 bp, probably having no effect on FVIII gene expression. Modelling studies were performed on those mutations lying within the A domains of FVIII (D525N, R527W, I566T) to study the possible effect of these mutations on structure and/or function. When the three methods are performing optimally and have been standardised, our experience is that CMC and DGGE are equally efficient at sequence variation detection while SSCP is slightly less sensitive.

Background/aims: Genetic haemochromatosis is the most common autosomal recessive disorder in Northern European populations. A major histocompatibility complex class I-like gene, HLA-H, has been proposed to be responsible for genetic haemochromatosis. The prevalence of HLA-H gene mutations 282(TGC; Cys/TAC; Tyr) and 63(CAT; His/GAT; Asp) was determined in patients of Austrian origin., Methods: DNA extracted from the blood of 40 Austrian patients and 271 controls was used to amplify HLA-H gene fragments by the polymerase chain reaction method. The base changes responsible for mutations Cys282Tyr and His63Asp alter recognition sites for restriction enzymes SnaB I and Bcl I, respectively. Digestion products were separated by agarose gel electrophoresis and visualised by ethidium bromide staining., Results: Thirty-one (77.5%) genetic haemochromatosis patients were homozygous for mutation Cys282Tyr and three compound heterozygous for mutations Cys282Tyr and His63Asp. One patient was homozygous for mutation His63Asp but normal for mutation Cys282Tyr. Four patients were normal at both genetic loci and one patient was heterozygous for mutation His63Asp. One control subject homozygous for mutation Cys282Tyr was found on investigation to fulfill diagnostic criteria for haemochromatosis. Eight control subjects homozygous for mutation His63Asp showed no biochemical or clinical evidence of haemochromatosis indicating that this variant is not directly responsible for haemochromatosis. Absence of the Cys282Tyr mutation in six genetic haemochromatosis patients with distinct haplotypes indicates mutations within the HLA-H gene or at alternative genetic loci are the cause of genetic haemochromatosis in these patients., Conclusions: The HLA-H Cys282Tyr defect is likely to play a key role in the pathogenesis of haemochromatosis in most patients. Predominance of a single HLA-H gene mutation in haemochromatosis allows presymptomatic screening by genotypic analysis.

Triosephosphate isomerase (TPI) deficiency is an autosomal recessive disorder of glycolysis characterized by multisystem disease and lethality in early childhood. Among seven unrelated Northern European kindreds with clinical TPI deficiency studied, a single missense mutation at codon 104 (GAG;Glu-->GAC;Asp) predominated, accounting for 11/14 (79%) mutant alleles. In three families molecular analysis revealed compound heterozygosity for Glu104Asp and novel missense mutations. In two cases the second mutation was a Cys to Tyr substitution at codon 41 (TGT-->TAT) and in one an Ile to Val substitution at codon 170(ATT-->GTT). The origin of the Glu104Asp mutation was defined by haplotype analysis using a novel G/A polymorphism at nucleotide 2898 of the TPI gene. Cosegregation of the low frequency 2898A allele with the G-->C base change at nucleotide 315 supports a single origin for the Glu104Asp mutation in a common ancestor.

Haemophilia A is a recessive X linked bleeding disorder caused by deficiency or functional abnormality of coagulation factor VIII. This disease usually has no visible phenotype in female carriers; hence, great efforts are made to offer all haemophilia A families accurate carrier diagnosis. Significant progress in this direction was made with the identification of the intron 13 variable number tandem repeat (VNTR), which is hitherto the most informative single marker within the factor VIII gene. The authors have established intron 13 VNTR detection in their laboratory by adapting its analysis to an automated sequencer using different primers of which one is fluorescent dye labelled. With this method, which is more rapid and convenient than that originally described, 67 haemophilia A families of German origin were screened and two new alleles (alleles 17 and 25) were identified. The informativeness of the VNTR in these families based on the patients maternal X chromosomes (134) is about 67%.

Haemophilia A is a bleeding disorder caused by defects in the gene coding for the co-factor, factor VIII (FVIII). The few available intragenic restriction fragment length polymorphisms (RFLPs) currently used in carrier detection and prenatal diagnosis of haemophilia A are informative in only about 65% of cases. We earlier reported a multi-allelic dinucleotide tandem repeat, (CA)n, specific to intron 13, which remains the single most informative marker within the FVIII gene. We here report a second informative dinucleotide repeat of the form (GT)n (AG)n, located to intron 22 of the FVIII gene. The polymerase chain reaction (PCR) method was used to examine the variability of the repeat in 60 individuals (75 X-chromosomes) and revealed four alleles. The calculated heterozygosity rate is 45%, and family studies showed X-linked mendelian inheritance. The intron 22 dinucleotide repeat is tightly linked with established RFLPs and tracks with haemophilia A in family studies. We now show that by simultaneous amplification of the intron 13 and 22 repeats using PCR all alleles for both markers are detectable on a single polyacrylamide gel. The information thus obtained from a single multiplexed analysis is greater than from multiple RFLP analyses. Hence, rapid haplotype determination by simultaneous amplification and detection of two intragenic dinucleotide repeats should supersede less informative RFLP analysis.

We report the analysis by single-stranded conformation polymorphism of the essential sequences of the factor VIII(FVIII) gene (total length about 14 kb) including the entire coding sequence, flanking intronic sequences and the putative regulatory sequences 5' to the gene, in twelve unselected haemophilia A patients of Portuguese origin. Direct sequencing of the fragments with an altered migration pattern led to the identification of the disease-producing mutations in five patients. Three of these mutations, namely a 1 bp insertion in a motif of eight consecutive A residues at codon 1439 (FVIIIPorto3); a C to T transition at codon 1966 (Arg-->Stop), found in an inhibitor-positive patient (FVIIIMontijo); and a G to A transition at codon 479 (Gly-->Arg; FVIIIPorto1), have been reported in other ethnic groups. The two novel mutations are the substitution of AG by GG at the 3' end of intron 4 (FVIIILisboa1) destroying the invariant splice acceptor sequence, and a G to A transition at codon 1948 resulting in an aspartic acid substitution for glycine (FVIIIPorto2).

In this study we determined the activity of the rat luteinising hormone-beta gene promoter in a heterologous rat pituitary cell line (GH3 cells). 1.7 kb of LH-beta 5' flanking sequence and the first 5 bp of the 5' untranslated region were ligated to the chloramphenicol acetyltransferase (CAT) receptor gene (LH-beta-CAT) and transiently transfected by calcium phosphate precipitation into subconfluent cultures of GH3 cells. Basal low-level CAT activity was only detected in GH3 cells, being absent in two non-pituitary cell lines (BeWo and HeLa) RNase analysis revealed that mRNA from transfected GH3 cells protected a fragment of labelled antisense probe of correct size for transcription initiation from the LH-beta CAP site, confirming that promoter activity reflected correctly initiated LH-beta-CAT fusion gene transcripts. CAT activity was consistently induced by an average of 3-5-fold from the full-length 1.7 kb promoter, in a dose- and time-dependent manner, by forskolin, dibutyryl cAMP, and 8-bromo cAMP implying presence of a cAMP-responsive cis-acting domain in the LH-beta promoter region. Transfection of deletion mutants delta-615-CAT, delta-385-CAT and delta-250-CAT each reduced forskolin inducibility to 1.7-fold but did not abolish induction completely suggesting a domain between -1.7 and -0.6 kb contained a cAMP-responsive element(s) (CRE). Further deletion of LH-beta 5' flanking sequences to delta-85-CAT restored forskolin induction to wild-type levels (3-5-fold), suggesting the presence of a weak inhibitory element between -600 and -85 kb, and a cAMP-responsive domain in the proximal promoter region. The LH-beta promoter does not contain perfect tandem repeat palindromic CRE DNA sequences, though there are several octanucleotide sequences differing by only 1 bp from AP-2 binding sites, the consensus CRE, and the vasointestinal peptide gene CRE. Although these data suggest that the LH-beta gene is cAMP responsive this is likely mediated by several and complex protein interactions with multiple DNA sequences in the proximal and distal LH-beta promoter enhancer.

The diagnosis of haemophilia A and the identification of carriers has greatly improved with knowledge of the structure of the gene for factor VIII. This has permitted the defect to be tracked in families by the study of restriction fragment length polymorphisms (RFLPs), irrespective of the nature of the molecular defect. However, this approach is time-consuming and the information yielded falls away as more polymorphisms are added. Within the factor VIII gene lies another source of polymorphism, a dinucleotide repeat sequence of varying length known as (CA)n. Conventional mapping localised this (CA)n repeat to intron 13. The polymerase chain reaction, used to examine (CA)n variability in genomic DNA from 25 males and 67 females, revealed eight allelic bands between n = 16 and n = 24. 91% of females were heterozygous for this repeat, and family studies showed X-linked mendelian inheritance with allelic frequencies ranging from 1% to 45%. The intron 13 (CA)n repeat is tightly linked with established RFLPs and tracks with haemophilia A in family studies. The analysis requires DNA from other family members, and relatives of sporadic cases of haemophilia A are only amenable to exclusion. Nonetheless, this intron 13 (CA)n repeat provides the most highly informative marker so far available for factor VIII gene tracking studies in haemophilia A kindreds and a result can be available within a day.

Free thyroid hormone concentrations measured by Amerlex assays were studied in subjects with inherited disorders of thyroxine-binding globulin (TBG) synthesis, variant albumins with a high avidity for T4, and iodothyronine-binding autoantibodies. Free T4 (fT4) and free T3 (fT3) levels were normal in euthyroid subjects with TBG deficiency and excess. Free T3 concentration was in the low normal range in subjects having a variant albumin but fT4 (Amerlex) was erroneously elevated because of the enhanced affinity of the 125I-T4 analogue employed in the assay for the abnormal albumin. The 125I-T3 analogue used in the fT3 assay does not bind more strongly to this variant albumin than to normal albumin. Amerlex assays for fT4 and fT3 in patients with iodothyronine-binding autoantibodies to thyroglobulin give variable results according to the specificity of the autoantibodies: non-specific antibodies cause extraordinarily high values even in hypothyroid patients; fT3 measurements may be appropriate in patients with T4-specific antibodies and even in some with T3-specific antibodies. The presence of such antibodies should be suspected if the results of Amerlex assays for fT4 and fT3 are discordant or are inconsistent with the clinical picture or TSH levels.

Patients with hypogammaglobulinaemia have been regularly found to have abnormal conventional thyroid function test results. The abnormality is due to an increased plasma concentration of T4-binding globulin (TBG). As the prevalence of autoimmune thyroid disease is probably increased in hypogammaglobulinaemia this further abnormality in the plasma proteins may lead to diagnostic confusion. Administration of gammaglobulin by infusion causes a rapid but transient fall in plasma concentrations of TBG and T4 which is probably due to a temporary redistribution of the plasma proteins.

Serum thyroxine was consistently unmeasurable by radioimmunoassay in an elderly patient with myxoedema after successful treatment with oral thyroxine. Abnormal binding of thyroxine was suspected and shown to be due to the presence in serum of antibodies of the IgG variety. The characteristics of these antibodies with respect to their binding of thyroxine (T4), triiodothyronine (T3), reverse triiodothyronine (rT3) and human thyroglobulin (Tg) were systematically studied. Three preparations of Tg, and t4, T3 and rT3 were examined for their ability to compete with 125I-Tg, 125I-T4, 125I-T3 and 125I-rT3 for binding to the antibodies. For each tracer used the order of competitive efficiency was Tg greater than T4 greater than T3 greater than rT3. This provides for the first time direct evidence that iodothyronine reacting antibodies occurring in man are generated against Tg. All three iodothyronines were able to inhibit tracer binding of labelled iodothyronines completely, the order of effectiveness being T4 greater than T3 greater than rT3, suggesting antibodies with one type of binding site and that these were probably raised against a Tg sequence incorporating T4, although there was some evidence for the existence of a minor subpopulation of antibodies with higher specificity for T3. Complete displacement of labelled Tg by cold iodothyronines, however, was not possible. The experimental evidence suggests two classes of Tg antibodies, 70% of which were directed towards the T4 containing region, and 30% directed against other part(s) of the Tg molecule. Despite the presence of such Tg antibodies conventional haemagglutination tests of the patient's serum for Tg antibodies were negative.

A three generation family study was carried out after inappropriate treatment with radioactive iodine of a 50 year old woman with a raised serum total thyroxine concentration and free thyroxine index. Subsequent investigations showed that she and five members of her family had raised thyroxine binding globulin concentrations. Free thyroxine and free triiodothyronine concentrations were normal. Problems encountered in the recognition of this thyroxine binding protein disorder are discussed. Clinicians and clinical biochemists should be aware of these pitfalls and thus avoid further incorrect treatment on the basis of biochemical findings, even though free hormone estimations are now becoming readily available.

A euthyroid adult female (LC) was found to have persistently raised concentrations of total T4 (159 nmol/l) and rT3 (500 pmol/l) in her serum in association with a normal T3 (2.1 nmol/l). Serum concentrations of all three T4-binding proteins were within normal limits. A variant prealbumin with an increased affinity for T4 was found to be responsible for the raised serum level of T4. Unlike normal prealbumin, the variant also bound appreciable amounts of rT3. The affinity constant for T4 binding to prealbumin LC was 5.5 X 10(8) l/mole which is sevenfold higher than that obtained for normal prealbumin (8.5 X 10(7) l/mole). The affinity constant for the binding of rT3 to prealbumin LC was 2.0 X 10(6) l/mole while that for the normal protein was unmeasurable by our method. The T4-binding capacity of prealbumin LC in serum was within the normal range indicating that there is no new additional T4-binding site on the protein. Prealbumin LC has the same molecular size as the normal protein, hence it is likely that the tetrameric structure has been preserved. The electrophoretic mobility of prealbumin LC was normal indicating no alteration in charge. It is postulated that the increased affinity for T4 (and presumably for rT3) results from a hydrophobic amino-acid substitution in the prealbumin monomer.

The serum concentrations of thyroxine (T4), 3,5,3'-triiodothyronine (T3) and 3,3'5'-triiodothyronine (reverse T3) in 10 massively obese women were studied before and at intervals for one year after jejuno-ileal bypass operation. A significant rise in T3 (P less than 0.01) over the immediately preoperative value, a significant fall in rT3 (P less than 0.01) but no significant change in T4 was found during the post-recovery follow-up period. It is concluded that maintaining a relatively high T3 level following jejuno-ileal bypass surgery may contribute to the substantial weight reduction achieved by patients after this operation.

Pre- and postcastration changes in LH beta and common alpha mRNAs were correlated with pituitary and serum LH levels in two different species after abolition of pituitary stimulation by GnRH. A GnRH antagonist (GnRH-ANT) was used to block gonadotroph GnRH receptors in male rats, and a GnRH antiserum (GnRH-AS) was used to inhibit GnRH stimulation of female and male mouse and male rat pituitaries. The postcastration increases in LH beta and common alpha mRNA levels (2- and 3.5-fold, respectively) were abolished in male rats after 7 days of continuous GnRH-ANT infusion. The postcastration increases in LH beta and common alpha mRNA in female (1.9- and 2.2-fold respectively) and male mice (1.4- and 3.6-fold, respectively) were also prevented after daily sc injection of GnRH-AS, as were the rises in LH beta (3-fold) and common alpha (4-fold) in castrated male rats. The pituitary LH content (postgonadectomy) was no different from intact control levels in all experimental animals regardless of treatment, while the increase in serum LH concentration in rats (7- and 8-fold) and in female (4.8-fold) and male mice (9.8-fold) was prevented by both GnRH-ANT and GnRH-AS administration. In intact rats treated with GnRH-ANT the LH beta mRNA level decreased (57%) while the common alpha mRNA level was unaffected after 7 days. Neither pituitary nor serum LH levels were altered in intact rats or mice after appropriate treatments. We conclude that endogenous GnRH is required for the postcastration rise of both LH beta and common alpha-subunit mRNA levels in rats and mice.

A new variant albumin with increased affinities for iodothyronines has been identified. A euthyroid woman had raised total serum concentrations of T4 (155 nmol/l), T3 (3.0 nmol/l) and rT3 (700 pmol/l) but normal levels of all three iodothyronine-binding proteins. The affinity constant for T3 binding to the albumin was substantially raised (2.2 x 10(5) l/mole; normal immeasurable), that for rT3 (1.4 x 10(6) l/mole) was increased three-fold. This new albumin binds the analogues of T4 and T3 used in Amerlex free-hormone assays more strongly than does normal albumin, resulting in erroneously elevated estimates of serum free-T4 and free-T3 by this method. The new variant albumin was indistinguishable from normal albumin in molecular size and by electrophoretic and immunological techniques. Three distinct variant albumins exhibiting differential binding of iodothyronines have now been defined: Type I causes a raised total serum T4 only; Type II produces increased total T4 and rT3; Type III (the present example) results in elevated total T4, rT3 and T3. All three variants have normal free-T4 by dialysis but spuriously raised results by the Amerlex free-T4 method. Type III also causes an artificial increase in Amerlex free-T3. The pattern of thyroid function test results in Type III can readily be confused with both hyperthyroidism and with partial peripheral resistance to thyroid hormones.

Since the role of GnRH in the control of FSH release and synthesis is controversial, we have examined the effect of elimination of GnRH action on gonadotropes on FSH beta gene expression, FSH release, and synthesis. GnRH stimulation of the pituitary was abolished by continuous infusion of either a GnRH antagonist or a GnRH antiserum. We also examined the effects of gonadotrope desensitization, using a continuous infusion of GnRH or GnRH agonist analog. FSH beta mRNA levels were determined by dot blot hybridization using rat FSH beta cDNA, and changes were related to pituitary and serum FSH concentrations. FSH beta mRNA levels increased after orchidectomy and correlated well with serum FSH concentrations. Overall FSH synthesis was increased after castration, as judged by elevated serum FSH and unchanged pituitary FSH content. In orchidectomized rats, continuous GnRH antagonist infusion prevented the postcastration rise in FSH beta mRNA levels and serum FSH. Pituitary FSH content was reduced at 7 days, but not at 14 days. In intact rats, GnRH antagonist infusion for 7 days had no effect on FSH beta mRNA levels, but after 14 days, there was a 33% reduction, and serum FSH was suppressed. Pituitary FSH content was decreased after GnRH antagonist treatment for 7 or 14 days. Daily injection of GnRH antiserum for 6 days abolished the increases in FSH beta mRNA levels and serum FSH in orchidectomized rats, but pituitary FSH content was unaffected. In intact rats, GnRH antiserum treatment reduced FSH beta mRNA levels by 38%, suppressed serum FSH, and decreased pituitary FSH content. When gonadotropes were desensitized by a continuous infusion of GnRH for 14 days or GnRH agonist analog for 28 days, FSH beta mRNA levels and pituitary FSH content were markedly reduced, and serum FSH was suppressed to undetectable levels. We concluded that 1) endogenous GnRH is required for the maintenance of FSH beta mRNA levels in both intact and orchidectomized rats; 2) FSH beta mRNA levels are coupled to the level of FSH biosynthesis, indicating the physiological importance of this pretranslational regulation; 3) desensitization is more effective at inhibiting FSH beta gene expression and FSH synthesis than preventing gonadotrope stimulation using the GnRH antagonist or antiserum; and 4) the actions of GnRH on FSH beta mRNA levels are paralleled by its effects on LH beta mRNA levels, suggesting that GnRH provides a common primary stimulus for the induction of both beta-subunit genes in vivo. These data provide further evidence for the crucial stimulatory role of GnRH in the control of FSH synthesis.

The effect of castration and gonadal steroid replacement on the concentrations of LH-beta and alpha subunit and prolactin mRNA was examined in mice. Mouse LH-beta, alpha and prolactin mRNAs were approximately 0.8, 0.7 and 1.1 kb in size respectively. After ovariectomy, LH-beta mRNA levels increased 2- to 2.5-fold, while alpha mRNA levels increased 2.5-fold 6 and 10 days after ovariectomy. Serum LH rose after 2 days to reach six times control values at 10 days. Pituitary LH content doubled by 8 days after ovariectomy. Prolactin mRNA levels decreased to 50-60% of control at 3, 6, 8 and 10 days after ovariectomy and parallelled the fall in serum prolactin. Pituitary prolactin content fell more slowly, to 50% of intact control values by 10 days. The increase in both LH-beta and alpha subunit mRNA, and decrease in prolactin mRNA, and serum and pituitary hormone changes, after ovariectomy were prevented by oestradiol or oestradiol plus progesterone replacement. Levels of LH-beta mRNA increased more quickly in male than in female mice, the earliest change being seen 24 h after orchidectomy. Maximum values (two- to threefold) were found on day 6 after orchidectomy. Concentrations of alpha mRNA increased by 12 h to between 2 and 2.5 times control from 3 to 10 days after orchidectomy. Serum LH doubled by 12 h and was three to five times greater than control values up to 10 days. Pituitary LH content fell by 48 h before gradually increasing to intact values after 10 days. Prolactin mRNA levels decreased progressively from 2 days after orchidectomy, and this decrease was preceded by a fall in serum and pituitary prolactin which remained low throughout the experiment. Testosterone treatment attenuated the rise in alpha mRNA, prevented the rise in LH-beta mRNA and serum LH and partially restored the decrease in prolactin mRNA seen after orchidectomy. We conclude that in mice, as in rats and ewes, both LH-beta and alpha subunit mRNAs are negatively regulated by gonadal steroids, whereas prolactin mRNA is positively regulated, although there are temporal differences in patterns of mRNA responses between males and females. By comparison with female rats the rise in LH-beta mRNA after ovariectomy was slower in mice. Moreover, the discordant changes in pituitary LH content and LH subunit mRNAs seen in mice after castration were not observed in rats. Furthermore, pituitary prolactin and prolactin mRNA do not fall after orchidectomy of rats.(ABSTRACT TRUNCATED AT 400 WORDS)

The T4-binding proteins of a euthyroid subject with persistent hyperthyroxinemia (T4, greater than 20 micrograms/dl) were present in normal concentrations. Abnormal transport of both T4 and rT3 was demonstrated by reverse flow paper electrophoresis; excess T4 was bound to albumin and prealbumin, while increased binding of rT3 was confined to prealbumin. The three T4-binding proteins in the serum of the subject were isolated by affinity chromatography and characterized. Equilibrium dialysis experiments demonstrated a 20-fold increase in affinity of the albumin for T4 (Ka, 5.1 X 10(6) M-1) and a 4-fold increase in affinity of prealbumin for T4 (Ka, 3.0 X 10(8) M-1); T4-binding globulin affinity was normal. Nine other members of the family were also studied. Two sisters of the propositus have both the abnormal albumin and the variant prealbumin, while a brother has normal T4-binding proteins. The mother has the abnormal albumin alone. The father, his sister, and one of his three brothers have the variant prealbumin only. Despite the presence of the variant prealbumin in some of the paternal relatives of the propositus, their total iodothyronine concentrations were within the normal ranges; the condition may, therefore, often go undetected. The characteristics of the albumin found in the affected members of this kindred are those we have defined for familial dysalbuminemic hyperthyroxinemia type I, which is inherited as an autosomal dominant trait. The pattern of inheritance of the variant prealbumin is also consistent with a dominant mode with strong penetrance. The presence of two separately inherited abnormal T4 transport proteins in the same family suggests that both conditions may be more common than has been thought.

Two euthyroid subjects with high total concentrations of T4 in their sera have been studied (J.D.: T4, 170; T3, 1.90; rT3, 0.54 nmol/l. E.T.: T4, 185; T3, 1.63; rT3, 0.37 nmol/l). Concentrations of all three T4-binding proteins were within normal limits in both cases. However, on reverse-flow electrophoresis an abnormally large amount of thyroxine was found to travel with albumin. The three T4-binding proteins in the sera of both patients were separated from each other by a novel affinity chromatographic method using dye-Sepharose conjugates. Affinity constants for T4 binding to TBG and to prealbumin from both patients were normal. The albumin preparations were further purified and shown by physical and immunochemical techniques to be uncontaminated by other proteins. Scatchard plots of the binding of T4 to each of these pure albumins revealed two components, one having a normal affinity constant (J.D., 1.8 x 10(5) lmol-1 and E.T., 2.3 x 10(5) lmol-1), the other having a raised affinity constant (J.D., 5.4 x 10(6) lmol-1 and E.T., 5.8 x 10(6) lmol-1). Extrapolation of the plots showed that the high affinity components comprised 66% (J.D.) and 54% (E.T.) of the total purified albumin. The raised affinity and high concentrations of the variants thus account for the raised total T4 concentrations in the patients. The presumed amino acid substitution in the albumins may be different in the two patients since the affinities for rT3 differ. Some methods for the estimation of free T4 levels give misleading results in the presence of these albumin variants. In the course of two episodes of illness, patient J.D. manifested large falls in serum T4 levels which could only be accounted for by reduced carriage of T4 by the abnormal and conventional binding proteins. Many cases reported in the literature as partial peripheral resistance to thyroid hormone may be examples of similar albumin variants.

In this study we investigated changes in steady state cytoplasmic mRNA levels for LH subunits in pituitaries of male rats desensitized by continuous infusion of GnRH in vivo. Seven days of GnRH infusion (340 micrograms/day) reduced (P less than 0.01) LH beta mRNA levels in intact adult male rats and prevented the LH beta mRNA rise observed after castration. In contrast, common alpha mRNA doubled (P less than 0.05) in intact rats, and the elevated alpha mRNA after 7 days castration was unchanged. Serum and pituitary LH levels were suppressed below values of intact controls. Fourteen days of GnRH infusion (290 micrograms/day) further reduced LH beta mRNA levels in both intact and castrated male rat pituitaries. alpha mRNA levels in intact rat pituitaries were unchanged by 14 days of GnRH infusion, while in castrated rats there was a 23% (P less than 0.05) decrease, though values were still twice those of intact controls. As at 7 days, serum and pituitary LH were suppressed. Infusion of a superagonist analog (Buserelin) at a dose of 14 micrograms/day for 28 days reduced LH beta mRNA to 15% of intact control values in both castrated and intact rats. Common alpha mRNA was significantly (P less than 0.05) increased in intact rats and reduced by 13% (P less than 0.05) in castrates by superagonist infusion. These results were similar to those produced by 20- to 30-fold higher doses of native GnRH. GnRH and agonist analog effects were specific since no changes were observed in other mRNA species (GH, PRL, actin). These results indicate that in GnRH-desensitized gonadotropes LH beta gene expression is inhibited, and this may largely explain the reduced LH biosynthesis. However, there is a differential effect of continuous GnRH or agonist analog treatment on LH subunit gene expression, with a time-dependent stimulation of common alpha gene expression in intact rats. This may be caused by a stimulatory interaction between GnRH and progestagens at the level of the gonadotrope. Thus, common alpha gene expression is less tightly coupled than that of LH beta to GnRH action.

Amiodarone is chiefly bound to albumin (62.1%) and much of the remainder (33.5%) is carried on a high molecular weight protein, probably beta-lipoprotein. Analysis of data for amiodarone binding to albumin revealed a high affinity primary binding site (Ka 5.6 X 10(6) litre mol-1) with about four secondary sites (average Ka 1.9 X 10(5) litre mol-1). Studies of the binding of amiodarone in serum revealed one type of binding site only with an affinity constant (Ka 4.2 X 10(6) litre mol-1) similar to that of the primary site on albumin. The secondary albumin binding sites do not seem therefore to be utilized in whole serum and the affinity of the lipoprotein must be similar to that of the primary amiodarone binding site on albumin. The effects of a wide range of compounds on albumin binding of amiodarone were examined by equilibrium dialysis. Quinidine, amitriptyline, cephazolin and palmitate decreased albumin-bound [125I]amiodarone. Neither warfarin nor digoxin affected the binding of amiodarone by albumin, thus of the three drugs known to be potentiated by concomitant amiodarone administration, only potentiation of quinidine could be explained by displacement from serum albumin. Rifampicin, frusemide, phenytoin, (-)-adrenaline, bromocresol green, (-)-noradrenaline and bromocresol purple were found to increase binding of [125I]amiodarone by albumin.(ABSTRACT TRUNCATED AT 250 WORDS)