Familial Dysalbuminemic Hyperthyroxinemia Research Articles

Assessing the clinical and molecular diagnosis of inherited forms of impaired sensitivity to thyroid hormone from a single tertiary center

Resistance to thyroid hormone (RTH), characterized by persistent hyperthyroxinemia with non-suppressed thyrotropin (TSH), is mostly caused by mutations in thyroid hormone receptor beta gene (THRB). Two differential diagnoses should be considered due to similar clinical and laboratory findings: TSH-producing pituitary adenoma (TPA) and Familial Dysalbuminemic Hyperthyroxinemia (FDH). The aim of this study is to describe our single tertiary center experience in the molecular diagnosis of RTH in Brazilian patients, analyzing their clinical and laboratory characteristics and the most common differential diagnosis. We enrolled 30 subjects with clinical and laboratory features of RTH. Patient´s evaluations included clinical examination, thyroid hormone profile and imaging tests. Sequencing analysis for THRB hot spot region was conducted on all patients, and those without mutations in beta isoform of the thyroid hormone receptor (TRβ) (non-TR-RTH) were investigated for albumin gene (ALB) mutation. Seventeen patients presented mutations in TRβ (RTHβ); six were non-TR-RTH, three had a diagnosis of FDH with a mutation in ALB, and four were diagnosed with TPA. Two characteristics were different to what is commonly described in the literature: higher serum TSH levels in RTHβ patients when compared to the non-TR-RTH group, but this difference did not extend to free T4 (FT4) level; also the percentage of non-TR-RTH was higher than what was reported in other series. In the present series, most cases were RTHβ with higher levels of TSH. We described three novel mutations in THRB (p.M313V, p.R320G and p.R438P) and the first patients with FDH molecular diagnosis (p.R242H) documented in Brazil.

Seven Familial Dysalbuminemic Hyperthyroxinemia Cases in Three Unrelated Japanese Families and High-Performance Liquid Chromatography Analysis of the Thyroxine Binding Profile

Familial dysalbuminemic hyperthyroxinemia (FDH) is caused by abnormal human serum albumin (HSA) with an increased thyroxine (T4) affinity leading to euthyroid hyperthyroxinemia. One- and 2-step immunoassays of serum samples from FDH patients (e.g., Japanese patients) with the HSA R218P mutation can yield false-positive free thyroxine (FT4) results. Therefore, it is difficult to distinguish FDH from syndrome of inappropriate secretion of thyroid-stimulating hormone (TSH) (e.g., syndrome of resistance to thyroid hormone, TSH-producing pituitary adenoma), even when multiple assays are used. To investigate T4 to HSA binding, we examined serum samples from 7 patients from 3 Japanese families with FDH. Clinically, abnormal thyroid function tests were noted in pregnant Patient 1. Patients 2 and 3 had histories of inappropriate treatment with antithyroid drugs and surgery. All patients and affected family members were diagnosed with FDH using direct sequencing analysis. Gel filtration high-performance liquid chromatography was used for the biochemical analyses. The genomic analysis revealed a heterozygous missense mutation in HSA (R218P). In FDH patient sera, the albumin effluent corresponded to the peaks for total T4 (TT4); approximately 60% of the T4 in the effluent was detected as FT4. The results for the albumin effluent from healthy volunteer and TSHoma patient sera showed no corresponding TT4 peak. In the FDH patients, a relatively larger quantity of T4 was bound to abnormal HSA. This bound T4 was measured as FT4 during the analysis. F = free; FDH = familial dysalbuminemic hyperthyroxinemia; HPLC = high-performance liquid chromatography; HSA = human serum albumin; PCR = polymerase chain reaction; SITSH = syndrome of inappropriate secretion of TSH; T = total; T3 = triiodothyronine; T4 = thyroxine; TSH = thyroid-stimulating hormone; WT = wild-type.

Clinical, Genetic, and Protein Structural Aspects of Familial Dysalbuminemic Hyperthyroxinemia and Hypertriiodothyroninemia.

Familial dysalbuminemic hyperthyroxinemia (FDH-T4) and hypertriiodothyroninemia (FDH-T3) are dominantly inherited syndromes characterized by a high concentration of thyroid hormone in the blood stream. The syndromes do not cause disease, because the concentration of free hormone is normal, but affected individuals are at risk of erroneous treatment. FDH-T4 is the most common cause of euthyroid hyperthyroxinemia in Caucasian populations in which its prevalence is about 1 in 10,000 individuals, but the prevalence can be much higher in some ethnic groups. The condition is caused by a genetic variant of human serum albumin (HSA); Arg218 is mutated to histidine, proline, or serine or Arg222 is changed to isoleucine. The disorder is characterized by greater elevation in serum l-thyroxine (T4) than in serum triiodothyronine (T3); T4 can be increased by a factor 8–15. The high serum concentration of T4 is due to modification of a binding site located in the N-terminal half of HSA (in subdomain IIA). Thus, mutating Arg218 or Arg222 for a smaller amino acid reduces the steric restrictions in the site and creates a high-affinity binding site. The mutations can also affect binding of other ligands and can perhaps cause modified pharmacokinetics of albumin-binding drugs. In normal HSA, the high-affinity site has another location (in subdomain IIIB). Different locations of these sites imply that persons with and without FDH-T4 can have different types of interactions, and thereby complications, when given albumin-binding drugs. FDH-T3 is caused by a leucine to proline mutation in position 66 of HSA, which results in a large increment of the binding affinity for T3 but not for T4. For avoiding unwanted treatment of euthyroid persons with hyperthyroxinemia or hypertriiodothyroninemia, protein sequencing and/or sequencing of the albumin gene should be performed.

A case of familial dysalbuminemic hyperthyroxinemia (FDH) in Japan: FDH as a possible differential diagnosis of syndrome of inappropriate secretion of thyroid-stimulating hormone (SITSH)

Familial dysalbuminemic hyperthyroxinemia (FDH) is an autosomal dominant condition and is the most commonly inherited euthyroid hyperthyroxinemia in Caucasians. However, it is extremely rare in Asian populations. A 30-year-old Japanese woman, who was incidentally found to have apparent thyroid dysfunction, was admitted to our hospital in 2004. She had extremely elevated serum free thyroxine (FT4), moderately elevated free triiodothyronine (FT3), and normal thyroid-stimulating hormone (TSH). Clinical thyroid examination revealed no abnormalities other than small goiter. Anti-thyroglobulin antibody titer was positive, but titers of other anti-thyroid antibodies, including antithyroid peroxidase antibody, TSH receptor antibodies, and thyroid-stimulating antibody, were negative. Levels of FT3, FT4, and TSH were similar when measured by three different laboratory kits, and FT4 was still high when measured by equilibrium dialysis. By affinity chromatography, FT4, TT4, and albumin were extracted to the same fraction, and the levels of FT4 and TT4 were extremely high. By combination of reversed phase liquid chromatography and mass spectrometry techniques, the amino acid sequence of human serum albumin was determined. The patient was found to be a heterozygote for p.R218P mutation in the gene for human serum albumin and was diagnosed as FDH. This patient, who harbored the p.R218P mutation in the albumin gene, is the fifth case report of FDH in Japan. This condition is characterized by extremely high serum FT4 and moderately high serum FT3 levels. Although rare, FDH should be considered in the differential diagnosis for syndrome of inappropriate secretion of TSH (SITSH) in Japan.

Mutants and molecular dockings reveal that the primary L-thyroxine binding site in human serum albumin is not the one which can cause familial dysalbuminemic hyperthyroxinemia

BackgroundNatural mutations of R218 in human serum albumin (HSA) result in an increased affinity for L-thyroxine and lead to the autosomal dominant condition of familial dysalbuminemic hyperthyroxinemia. MethodsBinding was studied by equilibrium dialysis and computer modeling. ResultsTen of 32 other isoforms tested had modified high-affinity hormone binding. L-thyroxine has been reported to bind to four sites (Tr) in HSA; Tr1 and Tr4 are placed in the N-terminal and C-terminal part of the protein, respectively. Site-directed mutagenesis gave new information about all the sites. ConclusionsIt is widely assumed that Tr1 is the primary hormone site, and that this site, on a modified form, is responsible for the above syndrome, but the binding experiments with the genetic variants and displacement studies with marker ligands indicated that the primary site is Tr4. This new assignment of the high-affinity site was strongly supported by results of MM-PBSA analyses and by molecular docking performed on relaxed protein structure. However, dockings also revealed that mutating R218 for a smaller amino acid increases the affinity of Tr1 to such an extent that it can become the high-affinity site. General significancePlacing the high-affinity binding site (Tr4) and the one which can result in familial dysalbuminemic hyperthyroxinemia (Tr1) in two very different parts of HSA is not trivial, because in this way persons with and without the syndrome can have different types of interactions, and thereby complications, when given albumin-bound drugs. The molecular information is also useful when designing drugs based on L-thyroxine analogues.

Familial dysalbuminaemic hyperthyroxinaemia: a novel mass spectrometry approach to diagnosis

Background and aims Familial dysalbuminaemic hyperthyroxinaemia (FDH) is an important cause of discordant thyroid function tests (TFT). Free thyroxine (T4) levels are falsely elevated on some assays and current diagnostic tests for FDH are laborious. We aimed to investigate a novel, mass spectrometry approach to diagnosis of FDH in a 51-year-old man with persistently elevated free T4 levels, discordant normal TSH and normal free triiodothyronine. He was clinically euthyroid and had a daughter with similar TFT profile. Methods Serum albumin was assessed using electrospray time-of-flight (TOF) liquid chromatography/mass spectrometry (LC/MS). Targeted sequencing of exons 3 and 7 of the albumin gene was used to confirm mutations and to screen 15 archived DNA samples that were negative for thyroid hormone receptor mutations. Results LC/MS demonstrated heterozygosity for an albumin variant with a 19 Da decrease in mass, indicative of an Arg→His substitution. This was confirmed by DNA sequencing which showed a guanine to adenine transition at codon 218 of exon 7, a mutation known to increase T4 binding. Discussion TOF LC/MS is a novel procedure for diagnosing FDH. The test is rapid (taking

A Novel Mutation in theAlbuminGene (R218S) Causing Familial Dysalbuminemic Hyperthyroxinemia in a Family of Bangladeshi Extraction

Familial dysalbuminemic hyperthyroxinemia (FDH) is a common cause of euthyroid hyperthyroxinemia. Clinical recognition of FDH is crucial for preventing unnecessary therapy in clinically euthyroid patients with abnormal thyroid function tests. Our goal was to identify the cause of abnormal serum tests of thyroid function in a Canadian family of Bangladeshi extraction. The proposita was found to have elevated free thyroxine (fT4) and free triiodothyronine (fT3) with nonsuppressed thyrotropin (TSH) on screening blood work. After detailed studies excluding hyperthyroidism and resistance to thyroid hormone, blood was obtained from all members of her immediate family for further investigation. We conducted laboratory analyses and sequencing of candidate genes. Two members of this family have FDH, caused by a not previously identified mutation in the albumin gene. This mutation, located in exon 7 of the gene (652A>C), produces a single amino acid substitution in the protein molecule (R218S). The mutant albumin is associated with a ninefold increase in serum total T4 and a twofold increase in serum total reverse T3 compared to patients with normal albumin. Modeling data for the R218S variant are compatible with the increased binding affinity of this variant albumin for T4. The R218S substitution reported here causes FDH that, in terms of the magnitude of serum iodothyronine elevation, is intermediate to the two previously reported mutations at codon 218 FDH: R218H being more mild and R218P more severe.

A Novel Albumin Gene Mutation (R222I) in Familial Dysalbuminemic Hyperthyroxinemia

Context:Familial dysalbuminemic hyperthyroxinemia, characterized by abnormal circulating albumin with increased T4 affinity, causes artefactual elevation of free T4 concentrations in euthyroid individuals.Objective:Four unrelated index cases with discordant thyroid function tests in different assay platforms were investigated.Design and Results:Laboratory biochemical assessment, radiolabeled T4 binding studies, and ALB sequencing were undertaken. 125I-T4 binding to both serum and albumin in affected individuals was markedly increased, comparable with known familial dysalbuminemic hyperthyroxinemia cases. Sequencing showed heterozygosity for a novel ALB mutation (arginine to isoleucine at codon 222, R222I) in all four cases and segregation of the genetic defect with abnormal biochemical phenotype in one family. Molecular modeling indicates that arginine 222 is located within a high-affinity T4 binding site in albumin, with substitution by isoleucine, which has a smaller side chain predicted to reduce steric hindrance, thereby facilitating T4 and rT3 binding. When tested in current immunoassays, serum free T4 values from R222I heterozygotes were more measurably abnormal in one-step vs two-step assay architectures. Total rT3 measurements were also abnormally elevated.Conclusions:A novel mutation (R222I) in the ALB gene mediates dominantly inherited dysalbuminemic hyperthyroxinemia. Susceptibility of current free T4 immunoassays to interference by this mutant albumin suggests likely future identification of individuals with this variant binding protein.

Rare thyroid diseases

In general, rare thyroid diseases can be divided into neoplastic and non-neoplastic. Neoplastic rare thyroid diseases include the following: 1. primary thyroid lymphoma (1-5% all malignant neoplasms of the thyroid; 2:1 000 000 per year); 2. metastases to the thyroid gland (2-3% malignant thyroid neoplasms; 0.5-2.8% in autopsy) from various primary cancers such as breast carcinoma, renal carcinoma, colon carcinoma, melanoma and others; 3. others (ie: sarcoma). On the contrary, non-neoplastic rare thyroid diseases include the following: 1. congenital defects of thyroxine binding proteins, particularly partial and total congenital TBG deficiency (1:5 000), congenital TBG excess (1: 25 000) (both caused by TBG gene mutations) and familial dysalbuminemic hyperthyroxinemia (1:25 000, more frequent in some populations, such as the Spanish and Portuguese); 2. congenital resistance to thyroid hormones (with frequency about 1:40 000) as a result of THRB gene mutations; 3. rare cases of congenital thyroid hormones biosynthesis defects, for instance: Pendred syndrome (with frequency of 7.5-10:100 000) resulting from PDS gene mutations, total iodide organification defects (1:66 000; TPO and THOX2 gene mutations); 4. congenital defects of the thyroid gland development, which constitute about 85-90% causes of primary congenital hypothyroidism. About 2% of these are associated with the presence of other nonthyroid congenital anomalies and are caused by genetic mutations: for example, in the PAX, TTF1, TTF2, GNAS 1 genes; 5. congenital nonautoimmune hyperthyroidism as a result of TSH receptor gene mutation (TSHR) (until now 55 various mutations and over a dozen cases of de novo germinal mutations have been described); 6. hyperthyroidism associated with McCune-Albright syndrome (in about 30% of patients with this syndrome; McCune-Albright syndrome frequency of occurrence is estimated - 1:10 000-1:1 000 000; background - mutation in the GNAS 1 gene); 7. an association of the thyroid autonomous nodule and Graves’ disease (4.1%), and the thyroid cancer and Graves’ disease (1.1%-6.5%). The latest data on the etiopathogenesis of rare thyroid diseases, diagnostic criteria and possibilities for their treatment will be discussed. In addition, information obtained from PUBMED database concerning patients with rare thyroid diseases detected in the Polish population, particularly the data on molecular-genetic results, will be presented.

Serum electrophoresis with a bifid albumin peak.

A 51-year old female patient with newly diagnosed chronic kidney disease (CKD) presented to the hospital with an acute exacerbation of chronic obstructive pulmonary disease (COPD). On admission her serum creatinine was 247 µmol/L and urea was 15 mmol/L. The patient was a known hypertensive and was on irregular treatment. The CKD was probably a result of hypertensive nephropathy. As the liver function test showed a reversal of the albumin globulin ratio, a serum protein electrophoresis was requested. The overall electrophoresis was normal, except that the gamma globulin was elevated and a bifid peak was seen in the albumin region. The electrophoretic pattern of serum in Figure 1 shows that the albumin peak has a bifid mountain where albumin has two heads; this condition is commonly known as Bisalbuminemia. It is a result of two types of serum albumin that differ in their electrophoretic mobility as a result of splitting of the serum albumin into two components, of which one represents a variant on account of genetically inherited or acquired determinant [1]. Fig. 1. The top serum protein electrophoresis shows the presence of bisalbuminemia. Below is a normal serum protein electrophoresis. Bisalbuminemia is of two types, genetic and acquired. Genetic bisalbuminemia is quite rare and is inherited in autosomal dominant form. Bisalbuminemia is of little diagnostic or therapeutic significance, except that in some cases it might result in altered binding of steroid hormones and thyroxine. Familial dysalbuminemic hyperthyroxinemia (Arg 218 AEHis and Arg 218 AE Pro mutations) and hypertriiodothyroninemia (Leu 66 AEPro mutation) have been directly linked to the presence of inherited bisalbuminemia [2]. The acquired type may result from long-term, aggressive beta lactam antibiotic therapy or in acute pancreatitis. Physicians should be aware of this condition and interpret such findings with caution as well as in the context of the clinical scenario.

Spuriously High Free Thyroxine Values in Familial Dysalbuminemic Hyperthyroxinemia

To the Editor: Cartwright et al. recently showed that current commercially available free thyroxine (FT4)1 methods yield spuriously high results in individuals with familial dysalbuminemic hyperthyroxinemia (FDH) (1). In particular, variants of the “one-step labeled analog” technique have been notorious in this respect, whereas the 2-step approach, in which the sample is washed away before the antibody-bound T4 is brought into contact with the labeled T4 analog, is thought to eliminate the problem of tracer binding to albumin and especially albumin in FDH patients. Some 2-step assays, however, also yield spuriously high values in individuals with this condition; thus, interference is not simply a problem of 1-step assays. Inhibitors of T4 and T4 analog binding to the albumin included in the incubation buffer have been suggested to play a role for both the 1- and 2-step designs. In addition, the report of Cartwright et al. referred to similarly increased results obtained by symmetric dialysis when the incubation buffer of the Beckman Coulter Access 2-step assay (known to give falsely increased results for FDH samples) was used. We …

Elevated Free Thyroxine Levels Detected by a Neonatal Screening System

In Sapporo city of Japan, neonatal screening for congenital hypothyroidism has used the measurement of free thyroxine (T4) and thyroid-stimulating hormone (TSH) in the filter-paper blood spot. This system has enabled us to identify hyperthyroxinemic diseases. Filter papers were collected from neonatal infants born at 4-6 d of age and neonates who showed elevated free T4 (>4.0 ng/dL, 4 SD above the mean) were studied. Between January 2000 and December 2006, 83,232 newborns were screened. Eleven infants demonstrated persistent hyperthyroxinemia. One patient with slightly elevated free T4 and normal TSH was diagnosed as having familial dysalbuminemic hyperthyroxinemia (FDH). The other two patients with elevated free T4 without suppressed TSH were considered as having resistance of thyroid hormone (RTH), and analysis of thyroid hormone receptor (TR) beta gene confirmed the diagnosis. The remaining eight patients were diagnosed as having neonatal Graves' disease (NGD). Seven of eight pregnant women were treated with antithyroid drug and thus only one unrecognized NGD during pregnancy was detected by screening. Our screening system enables for early awareness of RTH and FDH. Regarding Graves' disease, the benefit of elevated free T4 screening is small, because most pregnant women with Graves' disease were managed.

Familial Dysalbuminemic Hyperthyroxinemia: A Persistent Diagnostic Challenge

Familial dysalbuminemic hyperthyroxinemia (FDH)1 is a well-characterized condition associated with increased circulating total thyroxine (T4) concentrations and normal physiological thyroid function. It is caused by mutations in the ALB (albumin) gene that increase the affinity of albumin for T4 by approximately 60-fold. When measured by a technique that minimally disturbs the equilibria between T4 and its serum binding proteins, such as equilibrium or symmetric dialysis (SyD) performed in a near-physiological medium, the free T4 (FT4) value is characteristically within the reference interval. Assays that rely on the competition of a T4 analog with unbound T4 in the sample can give spuriously high results in FDH patients, because albumin binding of the T4 analog is enhanced by the FDH mutation. “Two step” methods, in which the T4 analog never comes into contact with serum albumin owing to a wash step immediately after capture, avoid this problem. Such assay methods are expected to give FT4 results within the reference interval in FDH patients, but this expectation has been questioned (1)(2). Thyroid-function tests, including 1- and 2-step methodologies, were examined in 4 affected individuals from different families who had their FDH diagnoses proved genetically …

Structural basis of albumin-thyroxine interactions and familial dysalbuminemic hyperthyroxinemia.

Human serum albumin (HSA) is the major protein component of blood plasma and serves as a transporter for thyroxine and other hydrophobic compounds such as fatty acids and bilirubin. We report here a structural characterization of HSA-thyroxine interactions. Using crystallographic analyses we have identified four binding sites for thyroxine on HSA distributed in subdomains IIA, IIIA, and IIIB. Mutation of residue R218 within subdomain IIA greatly enhances the affinity for thyroxine and causes the elevated serum thyroxine levels associated with familial dysalbuminemic hyperthyroxinemia (FDH). Structural analysis of two FDH mutants of HSA (R218H and R218P) shows that this effect arises because substitution of R218, which contacts the hormone bound in subdomain IIA, produces localized conformational changes to relax steric restrictions on thyroxine binding at this site. We have also found that, although fatty acid binding competes with thyroxine at all four sites, it induces conformational changes that create a fifth hormone-binding site in the cleft between domains I and III, at least 9 A from R218. These structural observations are consistent with binding data showing that HSA retains a high-affinity site for thyroxine in the presence of excess fatty acid that is insensitive to FDH mutations.

Congenital hypothyroidism in a child with unsuspected familial dysalbuminemic hyperthyroxinemia caused by a mutation (R218H) in the human albumin gene

We found familial dysalbuminemic hyperthyroxinemia (FDH) in a 5-month-old boy with congenital hypothyroidism (CH) who had a blood thyrotropin (TSH) level of 479 mU/L but normal total serum thyroxine (T 4) and higher than normal total triiodothyronine (T 3) levels. Thyroid hormone substitution began at 5 weeks of age when T 4 and T 3 concentrations were below normal. Until the age of 5 months, treatment with levothyroxine was suboptimal on the basis of high serum TSH levels despite above-normal T 4 levels. FDH was confirmed by isoelectric focusing and testing of other family members. DNA analysis of the patient revealed R218H, a mutation in the serum albumin gene associated with FDH, which was also present in the patient's euthyroid father and brother. Thyroid scans, serum thyroglobulin measurements, and free T 4 measurements using equilibrium dialysis or 2-step immunoassay methods can identify thyroid hormone-binding protein defects and simplify the diagnosis and treatment of infants with CH. (J Pediatr 2001;139:887–91)

Familial Dysalbuminemic Hyperthyroxinemia in a Swiss Family Caused by a Mutant Albumin (R218P) Shows an Apparent Discrepancy between Serum Concentration and Affinity for Thyroxine

Familial Dysalbuminemic Hyperthyroxinemia in a Swiss Family Caused by a Mutant Albumin (R218P) Shows an Apparent Discrepancy between Serum Concentration and Affinity for Thyroxine

Familial dysalbuminemic hyperthyroxinemia may result in altered warfarin pharmacokinetics

Two distinct genotypes that result in the amino acid substitutions R218P and R218H in subdomain 2A of human serum albumin (HSA) have been identified as the cause of familial dysalbuminemic hyperthyroxinemia (FDH). These substitutions increase the affinity of subdomain 2A for thyroxine by approximately 10-fold elevating plasma thyroxine levels in affected individuals. While many studies have examined the binding of thyroxine to FDH HSA, the binding of FDH HSA to drugs has not been widely investigated. The widely administered drug warfarin was selected as a model compound to study FDH HSA/drug interactions since it binds to subdomain 2A and its pharmacokinetics are dramatically influenced by HSA binding. Using two independent methods, fluorescence spectroscopy and equilibrium dialysis with radioactive warfarin, the binding of recombinant R218P, R218H, R218M and wild type HSA to warfarin was measured. Both methods showed an approximately 5-fold decrease in the affinity of R218P, R218H and R218M HSA for warfarin relative to wild type HSA. The K d values determined by fluorescence spectroscopy for wild type, R218H, R218P and R218M HSA binding to warfarin were 1.35, 5.38, 5.61, and 8.34 μM, respectively. The values determined by equilibrium dialysis were 5.36, 29.5, 14.5, and 23.4 μM, respectively. Based on the above findings one would expect the free serum warfarin concentration in homozygous R218P and R218H FDH patients to be elevated about 5-fold, resulting in about a 5-fold reduction in the serum half-life of the drug.

家族性異常アルブミン性高サイロキシン血症 日本人家系と最近の研究成果

A Japanese family with familial dysalbuminemic hyperthyroxinemia (FDH) was presented and recent research progress concerning FDH was reviewed. Two mutations in the albumin gene have been reported as a cause of FDH; Arg 218 His in Caucasian and Chinese cases, and Arg 218 Pro in a Japanese family. In the latter, higher thyroxine levels are observed than those for the former, possibly due to a conformational change favorable to the binding with thyroxine. A recent study suggested that FDH might result in altered warfarin metabolism.

Structural Investigations of a New Familial Dysalbuminemic Hyperthyroxinemia Genotype

In a previous study, we found that the amino acid substitution R218H in human serum albumin (HSA) was the cause of familial dysalbuminemic hyperthyroxinemia (FDH) in several Caucasian patients. Subsequently the substitution R218P was shown to be the cause of FDH in several members of a Japanese family. This study attempts to resolve discrepancies in the only other study of R218P HSA and identifies two new Japanese R218P FDH patients unrelated to those described previously. Recombinant R218H, R218P, and wild-type HSA were synthesized in yeast, and the affinities of these HSA species for l- and d-thyroxine were determined using fluorescence spectroscopy. The dissociation constants for the binding of wild-type, R218P, and R218H HSA to l-thyroxine were 1.44 x 10(-6), 2.64 x 10(-7), and 2.49 x 10(-7) mol/L, respectively. The circular dichroism spectra of thyroxine bound to R218H and R218P HSA were markedly different, indicating that the structure of the thyroxine/HSA complex is different for either protein. The K(d) values for l-thyroxine bound to R218P and R218H HSA determined in this study were similar. The extremely high serum total-thyroxine concentrations reported previously for R218P FDH patients (10-fold higher than those reported for R218H FDH patients) are not consistent with the K(d) values determined in this study. Possible explanations for these discrepancies are discussed.

Mutagenesis studies of thyroxine binding to human serum albumin define an important structural characteristic of subdomain 2A.

The familial dysalbuminemic hyperthyroxinemia (FDH) phenotype results from a natural human serum albumin (HSA) mutant, with histidine instead of arginine at amino acid position 218. This mutation results in an enhanced affinity for thyroxine. In our earlier study, site-directed mutagenesis and a yeast protein expression system were used to synthesize FDH HSA and several other HSA mutants. Measurement of the binding of these HSA mutants to thyroxine and several thyroxine analogs using equilibrium dialysis and quenching of tryptophan 214 fluorescence allowed us to propose a preliminary model of thyroxine binding to the 2A subdomain of wild type and FDH HSA. In this study, we have produced several other HSA mutants. By comparing the binding affinity of these mutants for thyroxine and tetraiodothyroacetic acid to the binding affinity of other mutants, we were able to suggest a new model for thyroxine binding to the 2A subdomain of HSA. We found that the substitution of arginine at position 218 with alanine increased the binding affinity for thyroxine by 2 orders of magnitude relative to the binding affinity of wild type HSA for thyroxine. A more accurate understanding of the mechanism of thyroxine binding to HSA has allowed us to define an important structural characteristic of subdomain 2A, one of the two principal binding sites on HSA for small hydrophobic ligands.