Variations in the Human Serum Albumin Gene: Molecular and Functional Aspects.

The human albumin gene, the most abundant serum protein, is located in the long arm of chromosome 4, near the centromere, position 4q11-3. It is divided by 14 intervening introns into 15 exons, the last of which is untranslated. To date, 74 nucleotide substitutions (mainly missense) have been reported, determining the circulating variants of albumin or pre-albumin. In a heterozygous state, this condition is known as alloalbuminaemia or bisalbuminaemia (OMIM # 103600). The genetic variants are not associated with disease, neither in the heterozygous nor in the homozygous form. Only the variants resulting in familial dysalbuminaemic hyperthyroxinaemia and hypertriiodothyroninaemia are of clinical relevance because affected individuals are at risk of inappropriate treatment or may have adverse drug effects. In 28 other cases, the pathogenic variants (mainly affecting splicing, nonsense, and deletions), mostly in the homozygous form, cause a premature stop in the synthesis of the protein and lead to the condition known as congenital analbuminaemia. In this review, we will summarize the current knowledge of genetic and molecular aspects, functional consequences and potential therapeutic uses of the variants. We will also discuss the molecular defects resulting in congenital analbuminaemia, as well as the biochemical and clinical features of this rare condition.

Structure and properties of the oyster mushroom (Pleurotus ostreatus) lectin.

Pleurotus ostreatus Lectin (POL) is a 353 amino acid chain lectin that can be purified from the fruiting bodies of the very well-known and widely diffused edible oyster mushrooms (P. ostreatus). The lectin has been partially characterized by different groups and, although it was crystallized about 20 years ago, its 3D structure and the details of its interactions with carbohydrates are still unknown. This paper reports the 3D structure and ligand-binding properties of POL. We have determined the X-ray structure of the apo-protein purified from the fruiting bodies of the mushroom and that of the recombinant protein in complex with melibiose to a resolution of about 2 Å. The lectin is a homodimer in which the two polypeptide chains are linked by a disulfide bridge. A POL monomer is composed of two highly homologous ß-jellyroll domains each of which containing a calcium-dependent carbohydrate-binding site. A high degree of sequence similarity is observed between the two carbohydrate-binding modules present in each monomer. The structure of the lectin in complex with melibiose reveals that a POL dimer has four calcium-dependent carbohydrate-binding sites. The interaction with sugars in solution has been characterized by isothermal titration calorimetry and saturation transfer difference NMR and it sheds new light on the molecular determinants of POL specificity. The lectin exhibits in vitro antiproliferative effects against human cancer cell lines and presents structural similarity with the prototype member of the CBM67 family, the noncatalytic domain of Streptomyces avermitilis α-rhamnosidase.

Human plasma retinol-binding protein (RBP4) is also a fatty acid-binding protein.

RBP4 (plasma retinol-binding protein) is the 21 kDa transporter of all-trans retinol that circulates in plasma as a moderately tight 1:1 molar complex of the vitamin with the protein. RBP4 is primarily synthesized in the liver but is also produced by adipose tissue and circulates bound to a larger protein, transthyretin, TTR, that serves to increase its molecular mass and thus avoid its elimination by glomerular filtration. This paper reports the high resolution three-dimensional structures of human RBP4 naturally lacking bound retinol purified from plasma, urine and amniotic fluid. In all these crystals we found a fatty acid molecule bound in the hydrophobic ligand-binding site, a result confirmed by mass spectrometry measurements. In addition we also report the 1.5 Šresolution structures of human holo-RBP4 and of the protein saturated with palmitic and lauric acid and discuss the interaction of the fatty acids and retinol with the protein.

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.

BACKGROUND: Natural 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. METHODS: Binding was studied by equilibrium dialysis and computer modeling. RESULTS: Ten 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. CONCLUSIONS: It 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 SIGNIFICANCE: Placing 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.

Three-dimensional structure and ligand-binding site of carp fishelectin (FEL).

Carp FEL (fishelectin or fish-egg lectin) is a 238-amino-acid lectin that can be purified from fish eggs by exploiting its selective binding to Sepharose followed by elution with N-acetylglucosamine. Its amino-acid sequence and other biochemical properties have previously been reported. The glycoprotein has four disulfide bridges and the structure of the oligosaccharides linked to Asn27 has been described. Here, the three-dimensional structures of apo carp FEL (cFEL) and of its complex with N-acetylglucosamine determined by X-ray crystallography at resolutions of 1.35 and 1.70 Å, respectively, are reported. The molecule folds as a six-bladed ß-propeller and internal short consensus amino-acid sequences have been identified in all of the blades. A calcium atom binds at the bottom of the funnel-shaped tunnel located in the centre of the propeller. Two ligand-binding sites, α and ß, are present in each of the two protomers in the dimer. The first site, α, is closer to the N-terminus of the chain and is located in the crevice between the second and the third blades, while the second site, ß, is located between the fourth and the fifth blades. The amino acids that participate in the contacts have been identified, as well as the conserved water molecules in all of the sites. Both sites can bind the two anomers, α and ß, of N-acetylglucosamine, as is clearly recognizable in the electron-density maps. The lectin presents sequence homology to members of the tachylectin family, which are known to have a function in the innate immune system of arthropods, and homologous genes are present in the genomes of other fish and amphibians. This structure is the first of a protein of this group and, given the degree of homology with other members of the family, it is expected that it will be useful to experimentally determine other crystal structures using the coordinates of cFEL as a search probe in molecular replacement.

Human serum albumin isoforms: genetic and molecular aspects and functional consequences.

BACKGROUND: At present, 67 different genetic variants of human serum albumin and proalbumin have been molecularly characterized at the protein and/or gene level. SCOPE OF REVIEW: This review summarizes present knowledge about genetic and molecular aspects, functional consequences and potential uses of the variants. MAJOR CONCLUSIONS: The frequency of bisalbuminemia in the general population is probably about 1:1000, but it can be much higher in isolated populations. Mutations are often due to hypermutable CpG dinucleotides, and in addition to single-amino acid substitutions, glycosylated variants and C-terminally modified alloalbumins have been found. Some mutants show altered stability in vivo and/or in vitro. High-affinity binding of Ni(++) and Cu(++) is blocked, or almost so, by amino acid changes at the N-terminus. In contrast, substitution of Leu90 and Arg242 leads to strong binding of triiodothyronine and l-thyroxine, respectively, resulting in two clinically important syndromes. Variants often have modified plasma half-lives and organ uptakes when studied in mice. GENERAL SIGNIFICANCE: Because alloalbumins do not seem to be associated with disease, they can be used as markers of migration and provide a model for study of neutral molecular evolution. They can also give valuable molecular information about albumins binding sites, antioxidant and enzymatic properties, as well as stability. Mutants with increased affinity for endogenous or exogenous ligands could be therapeutically relevant as antidotes, both for in vivo and extracorporeal treatment. Variants with modified biodistribution could be used for drug targeting. In most cases, the desired function can be further elaborated by producing site-directed, recombinant mutants. This article is part of a Special Issue entitled Serum Albumin.

Congenital analbuminaemia: molecular defects and biochemical and clinical aspects.

BACKGROUND: DNA and mRNA sequencing of the coding regions of the human albumin gene (ALB) and of its intron/exon junctions has revealed twenty-one different molecular defects causing congenital analbuminaemia (CAA). SCOPE OF REVIEW: To describe the mutations in molecular terms and to present the current knowledge about the most important biochemical and clinical effects of CAA. MAJOR CONCLUSIONS: CAA is rare, but its frequency seems to be significantly higher in restricted and minimally admixed populations. The condition affects especially the lipid metabolism but apart from a possible increased risk for atherosclerotic complications, it is generally associated with mild clinical symptoms in adults. By contrast, several reports indicate that analbuminaemic individuals may be at risk during the perinatal and childhood periods, in which they seem to show increased morbidity and mortality. The twenty-one causative defects include seven nonsense mutations, seven changes affecting splicing, five frame-shift/deletions, one frame-shift/insertion and one mutation in the start codon. These results indicate that the trait is an allelic heterogeneous disorder caused by homozygous (nineteen cases) or compound heterozygous (single case) inheritance of defects. Most mutations are unique, but one, named Kayseri, is responsible for about half of the known cases. GENERAL SIGNIFICANCE: Study of the defects in the ALB resulting in CAA allows the identification of "hot spot" regions and contributes to understanding the molecular mechanism underlying the trait. Such studies could also give molecular information about different aspects of ALB regulation and shed light on the regulatory mechanisms involved in the synthesis of the protein. This article is part of a Special Issue entitled Serum Albumin.

BEL ß-trefoil: a novel lectin with antineoplastic properties in king bolete (Boletus edulis) mushrooms.

A novel lectin was purified from the fruiting bodies of king bolete mushrooms (Boletus edulis, also called porcino, cep or penny bun). The lectin was structurally characterized i.e its amino acid sequence and three-dimensional structure were determined. The new protein is a homodimer and each protomer folds as ß-trefoil domain and therefore we propose the name Boletus edulis lectin (BEL) ß-trefoil to distinguish it from the other lectin that has been described in these mushrooms. The lectin has potent anti-proliferative effects on human cancer cells, which confers to it an interesting therapeutic potential as an antineoplastic agent. Several crystal forms of the apoprotein and of complexes with different carbohydrates were studied by X-ray diffraction. The structure of the apoprotein was solved at 1.12 Å resolution. The interaction of the lectin with lactose, galactose, N-acetylgalactosamine and T-antigen disaccharide, Galß1-3GalNAc, was examined in detail. All the three potential binding sites present in the ß-trefoil fold are occupied in at least one crystal form and are described in detail in this paper. No important conformational changes are observed in the lectin when comparing its co-crystals with carbohydrates with those of the ligand-free protein.

A novel mutation in the albumin gene (c.1A>C) resulting in analbuminemia.

BACKGROUND: Analbuminemia (OMIM # 103600) is a rare autosomal recessive disorder manifested by the absence or severe reduction of circulating serum albumin in homozygous or compound heterozygous subjects. The trait is caused by a variety of mutations within the albumin gene. DESIGN: We report here the clinical and molecular characterisation of two new cases of congenital analbuminemia diagnosed in two members of the Druze population living in a Galilean village (Northern Israel) on the basis of their low level of circulating albumin. The albumin gene was screened by single-strand conformation polymorphism and heteroduplex analysis, and the mutated region was submitted to DNA sequencing. RESULTS: Both the analbuminemic subjects resulted homozygous for a previously unreported c.1 A>C transversion, for which we suggest the name Afula from the hospital where the two cases were investigated. This mutation causes the loss of the primary start codon ATG for Met1, which is replaced by a - then untranslated - triplet CTG for Leu. (p.Met1Leu). The use of an alternative downstream ATG codon would probably give rise to a completely aberrant polypeptide chain, leading to a misrouted intracellular transport and a premature degradation. CONCLUSIONS: The discovery of this new ALB mutation, probably inherited from a common ancestor, sheds light on the molecular mechanism underlying the analbuminemic trait and may serve in the development of a rapid genetic test for the identification of a-symptomatic heterozygous carriers in the Druze population in the Galilee.

A novel splicing mutation causes analbuminemia in a Portuguese boy.

Analbuminemia is a rare autosomal recessive disorder manifested by the absence or severe reduction of circulating serum albumin in homozygous or compound heterozygous subjects. It is an allelic heterogeneous defect, caused by a variety of mutations within the albumin gene. The analbuminemic condition was suspected in a Portuguese boy who presented with low albumin level (about 3.8 g/L) and a significant hypercholesterolemia, but with no clinical findings. The albumin gene was screened by single strand conformational polymorphism and heteroduplex analysis and submitted to direct DNA sequencing. The proband was found to be homozygous for a previously unreported G>A change at position c.1289+1, the first base of intron 10, which inactivates the strongly conserved GT dinucleotide at the 5' splice site consensus sequence of the intron. The effect of this mutation was evaluated by examining the cDNA obtained by RT-PCR from the albumin mRNA extracted from proband's leukocytes. The splicing defect results in the skipping of the preceding exon. The subsequent reading frame-shift in exon 11 produces a premature stop codon located 33 codons downstream the 5' end of the exon. This extensive cDNA alteration is responsible for the analbuminemic trait. Both parents were found to be heterozygous for the same mutation. DNA and cDNA sequence analysis established the diagnosis of congenital analbuminemia in the proband. The effects of the so far identified splice-site mutations in the albumin gene are discussed.

Electrostatics of folded and unfolded bovine ß-lactoglobulin.

We report on electrophoretic, spectroscopic, and computational studies aimed at clarifying, at atomic resolution, the electrostatics of folded and unfolded bovine ß-lactoglobulin (BLG) with a detailed characterization of the specific aminoacids involved. The procedures we used involved denaturant gradient gel electrophoresis, isoelectric focusing, electrophoretic titration curves, circular dichroism and fluorescence spectra in the presence of increasing concentrations of urea (up to 8 M), electrostatics computations and low-mode molecular dynamics. Discrepancy between electrophoretic and spectroscopic evidence suggests that changes in mobility induced by urea are not just the result of changes in gyration radius upon unfolding. Electrophoretic titration curves run across a pH range of 3.5-9 in the presence of urea suggest that more than one aminoacid residue may have anomalous pKa value in native BLG. Detailed computational studies indicate a shift in pKa of Glu44, Glu89, and Glu114, mainly due to changes in global and local desolvation. For His161, the formation of hydrogen bond(s) could add up to desolvation contributions. However, since His161 is at the C terminus, the end-effect associated to the solvated form strongly influences its pKa value with extreme variation between crystal structures on one side and NMR or low-mode molecular dynamics structures on the other. The urea concentration effective in BLG unfolding depends on pH, with higher stability of the protein at lower pH.

Wards in the keyway: amino acids with anomalous pK(a)s in calycins.

As a follow-up to our recent analysis of the electrostatics of bovine ß-lactoglobulin (Eberini et al. in Amino Acids 42:2019-2030, 2011), we investigated whether the occurrence in the native structure of calycins-the superfamily to which ß-lactoglobulin belongs-of amino acids with anomalous pK (a)s is an infrequent or, on the contrary, a common occurrence, and whether or not a general pattern may be recognized. To this aim, we randomly selected four calycins we had either purified from natural sources or prepared with recombinant DNA technologies during our previous and current structural and functional studies on this family. Their pIs vary over several pH units and their known functions are as diverse as carriers, enzymes, immunomodulators and/or extracellular chaperones. In our survey, we used both in silico prediction methods and in vitro procedures, such as isoelectric focusing, electrophoretic titration curves and spectroscopic techniques. By comparing the results under native conditions (no exposure of the proteins to chaotropic agents) to those after protein unfolding (in the presence of 8 M urea), a shift is observed in the pK (a) of at least one amino acid per protein, which results in a measurable change in pI. Three types of amino acids are involved: Cys, Glu, and His, their position varies along the calycin sequence. Although no common mechanism may thus be recognized, we hypothesize that the 'normalization' of anomalous pK (a)s may be the phenomenon that accompanies, and favors, structural rearrangements such as those involved in ligand binding by these proteins. An interesting, if anecdotal, validation to this view comes from the behavior of human retinol binding protein, for which the pI of the folded and liganded protein is intermediate between those of the folded and unliganded and of the unfolded protein forms. Likewise, both solid (from crystallography) and solution state (from CD spectroscopy) data confirm that the protein undergoes structural rearrangement upon retinol binding.

Structure of a lectin with antitumoral properties in king bolete (Boletus edulis) mushrooms.

A novel lectin has been isolated from the fruiting bodies of the common edible mushroom Boletus edulis (king bolete, penny bun, porcino or cep) by affinity chromatography on a chitin column. We propose for the lectin the name BEL (B. edulis lectin). BEL inhibits selectively the proliferation of several malignant cell lines and binds the neoplastic cell-specific T-antigen disaccharide, Galß1-3GalNAc. The lectin was structurally characterized: the molecule is a homotetramer and the 142-amino acid sequence of the chains was determined. The protein belongs to the saline-soluble family of mushroom fruiting body-specific lectins. BEL was also crystallized and its three-dimensional structure was determined by X-ray diffraction to 1.15 Å resolution. The structure is similar to that of Agaricus bisporus lectin. Using the appropriate co-crystals, the interactions of BEL with specific mono- and disaccharides were also studied by X-ray diffraction. The six structures of carbohydrate complexes reported here provide details of the interactions of the ligands with the lectin and shed light on the selectivity of the two distinct binding sites present in each protomer.

Molecular diagnosis of analbuminemia: a new case caused by a nonsense mutation in the albumin gene.

Analbuminemia is a rare autosomal recessive disorder manifested by the absence, or severe reduction, of circulating serum albumin (ALB). We report here a new case diagnosed in a 45 years old man of Southwestern Asian origin, living in Switzerland, on the basis of his low ALB concentration (0.9 g/L) in the absence of renal or gastrointestinal protein loss, or liver dysfunction. The clinical diagnosis was confirmed by a mutational analysis of the albumin (ALB) gene, carried out by single-strand conformational polymorphism (SSCP), heteroduplex analysis (HA), and DNA sequencing. This screening of the ALB gene revealed that the proband is homozygous for two mutations: the insertion of a T in a stretch of eight Ts spanning positions c.1289 + 23-c.1289 + 30 of intron 10 and a c.802 G > T transversion in exon 7. Whereas the presence of an additional T in the poly-T tract has no direct deleterious effect, the latter nonsense mutation changes the codon GAA for Glu244 to the stop codon TAA, resulting in a premature termination of the polypeptide chain. The putative protein product would have a length of only 243 amino acid residues instead of the normal 585 found in the mature serum albumin, but no evidence for the presence in serum of such a truncated polypeptide chain could be obtained by two dimensional electrophoresis and western blotting analysis.

Bovine beta-lactoglobulin acts as an acid-resistant drug carrier by exploiting its diverse binding regions.

Binding of fluorine-containing drugs to bovine beta-lactoglobulin, the most abundant whey protein in bovine milk, was investigated by means of (19)F NMR and mass spectrometry. The stoichiometry of the binding and its stability in acidic medium, where beta-lactoglobulin is folded and stable, were also studied, along with competition from molecules that can be regarded as analogs of physiological ligands to bovine beta-lactoglobulin. Conditional binding data were combined with protein structural information derived from circular dichroism and limited proteolysis studies. Spectroscopic techniques were also used to assess whether the bound drugs stabilize the protein structure against denaturation by chaotropes or temperature at various pH values. The results obtained provide evidence for the presence of multiple binding regions on the protein, with a specific and different affinity for structurally different classes of hydrophobic drugs and, more generally, that bovine beta-lactoglobulin can bind and protect against low pH values various classes of drugs of pharmaceutical relevance.

Neuronal Proteins as Targets of 3-Hydroxykynurenine: Implications in Neurodegenerative Diseases.

The neurotoxic activity of the tryptophan metabolite 3-hydroxykynurenine (3OHKyn) in neurodegenerative disorders, such as Parkinson's and Alzheimer's diseases, is related to oxidative stress and 3OHKyn interaction with cellular proteins. The pattern of protein modification induced by 3OHKyn involves the nucleophilic side chains of Cys, His, and Lys residues, similarly to the one promoted by dopamine and other catecholamines. In the present work, we have analyzed the reactivity of 3OHKyn toward the neuronal targets α-synuclein (and its N-terminal fragments 1-6 and 1-15) and amyloid-ß peptides (1-16 and 1-28) and characterized the resulting conjugates through spectrometric (LC-MS/MS) and spectroscopic (UV-vis, fluorescence, NMR) techniques. The amino acid residues of α-synuclein and amyloid-ß peptides involved in derivatizations by 3OHKyn and its autoxidation products (belonging to the xanthommatin family) are Lys and His, respectively. The pattern of protein modification is expanded in the conjugates obtained in the presence of the metal ions copper(II) or iron(III), reflecting a more oxidizing environment that in addition to adducts with protein/peptide residues also favors the fragmentation of the protein. These results open the perspective to using the 3OHKyn-protein/peptide synthetic conjugates to explore their competence to activate microglia cell cultures as well as to unravel their role in neuroinflammatory conditions.

Diagnosis, Phenotype, and Molecular Genetics of Congenital Analbuminemia.

Congenital analbuminemia (CAA) is an inherited, autosomal recessive disorder with an incidence of 1:1,000,000 live birth. Affected individuals have a strongly decreased concentration, or complete absence, of serum albumin. The trait is usually detected by serum protein electrophoresis and immunochemistry techniques. However, due to the existence of other conditions in which the albumin concentrations are very low or null, analysis of the albumin (ALB) gene is necessary for the molecular diagnosis. CAA can lead to serious consequences in the prenatal period, because it can cause miscarriages and preterm birth, which often is due to oligohydramnios and placental abnormalities. Neonatally and in early childhood the trait is a risk factor that can lead to death, mainly from fluid retention and infections in the lower respiratory tract. By contrast, CAA is better tolerated in adulthood. Clinically, in addition to the low level of albumin, the patients almost always have hyperlipidemia, but they usually also have mild oedema, reduced blood pressure and fatigue. The fairly mild symptoms in adulthood are due to compensatory increment of other plasma proteins. The condition is rare; clinically, only about 90 cases have been detected worldwide. Among these, 53 have been studied by sequence analysis of the ALB gene, allowing the identification of 27 different loss of function (LoF) pathogenic variants. These include a variant in the start codon, frame-shift/insertions, frame-shift/deletions, nonsense variants, and variants affecting splicing. Most are unique, peculiar for each affected family, but one, a frame-shift deletion called Kayseri, has been found to cause about one third of the known cases allowing to presume a founder effect. This review provides an overview of the literature about CAA, about supportive and additional physiological and pharmacological information obtained from albumin-deficient mouse and rat models and a complete and up-to-date dataset of the pathogenic variants identified in the ALB gene.

A novel insertion (c.1098dupT) in the albumin gene causes analbuminemia in a consanguineous family.

Congenital analbuminemia (OMIM # 616000) is an extremely rare autosomal recessive disorder, caused by variations in the albumin gene (ALB), which is generally thought to be a relatively benign condition in adulthood, but seems to be potentially life threatening in the pre- and peri-natal period. The subject of our study was a consanguineous family, in which we identified two analbuminemic individuals. Mutation analysis of ALB revealed that both are homozygous for a previously unreported insertion in exon 9 (c.1098dupT), causing a subsequent frame-shift with the generation of a premature stop codon, and an aberrant truncated putative protein product, p.Val367fsTer12. This variation is present in heterozygous condition in several other members of the family. The phenotype and the molecular genetics of CAA are discussed.

Mutations and polymorphisms of the gene of the major human blood protein, serum albumin.

We have tabulated the 77 currently known mutations of the familiar human blood protein, serum albumin (ALB). A total of 65 mutations result in bisalbuminemia. Physiological and structural effects of these mutations are included where observed. Most of the changes are benign. The majority of them were detected upon clinical electrophoretic studies, as a result of a point mutation of a charged amino acid residue. Three were discovered by their strong binding of thyroxine or triiodothyronine. A total of 12 of the tabulated mutations result in analbuminemia, defined as a serum albumin concentration of <1 g/L. These were generally detected upon finding a low albumin concentration in patients with mild edema, and involve either splicing errors negating translation or premature stop codons producing truncated albumin molecules. A total of nine mutations, five of those with analbuminemia and four resulting in variants modified near the C-terminal end, cause frameshifts. Allotypes from three of the point mutations become N-glycosylated and one C-terminal frameshift mutation shows O-glycosylation.

Analbuminemia Zonguldak: case report and mutational analysis.

OBJECTIVES: To document a new case of the rare disease analbuminemia and to study the molecular defect responsible for the trait. DESIGN AND METHODS: Single-strand conformational polymorphism (SSCP), heteroduplex analysis (HA), and DNA sequencing of the 14 exons and their flanking intron regions, as well as of the 5' and 3' UTR, of the albumin gene were conducted on DNA extracted from peripheral blood samples. RESULTS: DNA sequence analysis showed that the proband was homozygous, and his parents were both heterozygous, for a previously unreported 5180 T-->A transversion. This silent mutation creates at position 5180-81 a new AG dinucleotide, the invariant sequence encountered in all eukaryotic intron acceptor splice sites. This aberrant splice site near the 3'end of exon 5 might alter the normal splicing mechanism. No other mutation was found in the examined regions of the gene. CONCLUSIONS: Our results define a new molecular defect in the albumin gene.