Genetic variants of serum albumin in Americans and Japanese.

A collaborative search for albumin genetic variants (alloalbumins) was undertaken by cellulose acetate and agarose electrophoresis at pH 8.6 of the sera of patients at two major medical centers in the United States and of nearly 20,000 blood donors in Japan. Seventeen instances of alloalbuminemia were ascertained, and seven different alloalbumin types were characterized by structural study. Two previously unreported alloalbumin types were identified. In one type, which was present in a Caucasian family and designated Iowa City-1, aspartic acid at position 365 was replaced by valine (365 Asp----Val); this is the second reported mutation at this position. The other type present in a Japanese blood donor had the mutation 128 His----Arg. An unexpected finding was the presence in a single Japanese of a Naskapi-type alloalbumin (372 Lys----Glu), a variant that had previously been described only for certain Amerindian tribes in whom it occurs with a polymorphic frequency (greater than 1%) and in Eti Turks. An arginyl-albumin (-1 Arg, 1 Asp----Val) occurred in an American family. The other alloalbumin types identified were proalbumins Lille and Christchurch and albumin B that have a cumulative frequency of about 1:3500 in Caucasians probably because of the hypermutability of CpG dinucleotides at the mutated sites. All of the variants characterized in this study are point mutants, and the sites are spread throughout the albumin gene. However, about one-fourth of all known albumin mutations are clustered in the sequence segment from position 354 through 382.     

Point substitutions in albumin genetic variants from Asia.

Despite their rarity and physiologically neutral character, more inherited structural variants of serum albumin (alloalbumins) are known than for any other human protein except hemoglobin. Including three previously unreported examples described here, we have identified 13 different point substitutions in alloalbumins of Japanese origin. Of these only albumin B and two proalbumins have been reported in other ethnic groups, and these are the most common variants of European origin. Some alloalbumins of Asiatic origin, but not yet identified in Japanese, are present in diverse ethnic groups. An alloalbumin found in indigenes of New Guinea (lysine----asparagine at position 313) is also present in Caucasians of various European descents. Albumin Lambadi, occurring in a tribal group in south India, has a mutation (glutamic acid----lysine at position 501) also found as a rare variant in individuals of diverse ethnic origin resident on four continents. These results suggest that some alloalbumins with the same substitution may have originated by independent mutations in various populations. This, together with the apparent clustering of point substitutions in the protein structure, may reflect hypermutability of the albumin gene.     

Mutations in genetic variants of human serum albumin found in Italy.

A long-term electrophoretic survey of genetic variants of serum albumin has identified an alloalbumin in 589 unrelated individuals in Italy. The alloalbumins were classified electrophoretically into 17 types. The number of unrelated carriers for each type varied from 1 for several variants reported here to 103 for albumin B. The structural change in 8 of these types has previously been determined, and the amino acid substitutions in 3 additional types are reported here. Albumin Varese has a substitution, -2 arginine to histidine (-2 Arg----His), the same as that reported for proalbumin Lille; albumin Torino has the substitution 60 Glu----Lys; and albumin Vibo Valentia has the substitution 82 Glu----Lys. The ability to distinguish so many alloalbumin types by electrophoresis at several pH values indicates that similar substitutions at different sites produce variants with different electrophoretic mobilities. Except for chain terminations in two Italian variants, all the mutations thus far determined for alloalbumins are attributable to a single-base change in the structural gene, and there is a preponderance of transitions and purine mutations. Seven alloalbumins for which the structural change has been established have been ascertained only in Italy. Several of these are clustered in specific geographic regions of Italy, which suggests an origin through a founder individual. Other variants that occur worldwide are nonetheless clustered in geographic regions within Italy. In these cases an independent mutation probably occurred at a hypermutable site such as a CpG dinucleotide.     

Point substitutions in Japanese alloalbumins.

We have completed the structural study of five rare types of inherited albumin variants (alloalbumins) discovered in the Biochemical Genetics Study of 15,581 unrelated children in Hiroshima and Nagasaki. We have also identified the structural change in five other alloalbumin specimens detected during clinical electrophoresis of sera from Japanese living near Tokyo. Each of the five albumin variants from Nagasaki and Hiroshima has a single amino acid substitution. All of these substitutions differ, and none has been reported in non-Japanese populations. No instances of proalbumin variants or of albumin B (the most frequent alloalbumins in Caucasians) were detected in the children in Hiroshima and Nagasaki. However, one instance of a variant proalbumin and two examples of albumin B occurred in Japanese from the vicinity of Tokyo. In addition a previously unreported point substitution was found in albumin Tochigi, which is present in two unrelated persons from Tochigi prefecture. Four of the point mutations in the Japanese alloalbumins are in close proximity in a short segment of the polypeptide chain (residues 354-382) in which three additional point substitutions have been reported in diverse populations. These results, combined with earlier data, suggest that point substitutions are grouped in certain segments of the albumin molecule.     

Amino acid substitutions in genetic variants of human serum albumin and in sequences inferred from molecular cloning.

The structural changes in four genetic variants of human serum albumin were analyzed by tandem high-pressure liquid chromatography (HPLC) of the tryptic peptides, HPLC mapping and isoelectric focusing of the CNBr fragments, and amino acid sequence analysis of the purified peptides. Lysine-372 of normal (common) albumin A was changed to glutamic acid both in albumin Naskapi, a widespread polymorphic variant of North American Indians, and in albumin Mersin found in Eti Turks. The two variants also exhibited anomalous migration in NaDodSO4/PAGE, which is attributed to a conformational change. The identity of albumins Naskapi and Mersin may have originated through descent from a common mid-Asiatic founder of the two migrating ethnic groups, or it may represent identical but independent mutations of the albumin gene. In albumin Adana, from Eti Turks, the substitution site was not identified but was localized to the region from positions 447 through 548. The substitution of aspartic acid-550 by glycine was found in albumin Mexico-2 from four individuals of the Pima tribe. Although only single-point substitutions have been found in these and in certain other genetic variants of human albumin, five differences exist in the amino acid sequences inferred from cDNA sequences by workers in three other laboratories. However, our results on albumin A and on 14 different genetic variants accord with the amino acid sequence of albumin deduced from the genomic sequence. The apparent amino acid substitutions inferred from comparison of individual cDNA sequences probably reflect artifacts in cloning or in cDNA sequence analysis rather than polymorphism of the coding sections of the albumin gene.     

Amino acid substitutions in albumin variants found in Brazil.

Conventional horizontal starch-gel electrophoresis in four buffer systems and structural studies were performed on four albumin variants, and the findings were compared with similar previous data. Albumins Coari I and Porto Alegre I have a previously unreported amino acid substitution (glutamic acid replaced by lysine at position 358, denoted 358 Glu----Lys). The alteration in albumin Porto Alegre II (501 Glu----Lys) is the same as that found for three alloalbumins of Asiatic origin, designated Vancouver, Birmingham, and Adana. Albumin Oriximiná I has the same exchange as albumin Maku (541 Lys----Glu). Some of these findings can be explained only by the occurrence of independent mutations at the same site in the albumin gene. They also point to a third cluster of mutations in that gene, indicating hypermutability in some of its segments.     

Amino acid substitutions in inherited albumin variants from Amerindian and Japanese populations.

We report an effort to determine the basis for the altered migration of seven inherited albumin variants detected by one-dimensional electrophoresis in population surveys involving tribal Amerindians and Japanese children. An amino acid substitution has thus far been determined for four of the variants. The randomness in the albumin polypeptide of these and the other sixteen independently ascertained amino acid substitutions of albumin and proalbumin thus far established was analyzed; the clustering of eight of these at two positions in the six-amino acid propeptide sequence seems noteworthy. By comparison with other proteins studied by electrophoresis, albumin exhibits "average" variability. It is a paradox that individuals who, for genetic reasons, lack albumin exhibit no obvious ill effects; yet, electrophoretic variants of albumin are no more numerous than are variants of proteins, the absence of which results in severe disease.     

Genetic variants of human serum albumin in Italy: point mutants and a carboxyl-terminal variant.

Of the > 50 different genetic variants of human serum albumin (alloalbumins) that have been characterized by amino acid or DNA sequence analysis, almost half have been identified in Italy through a long-term electrophoretic survey of serum. Previously we have reported structural studies of 11 Italian alloalbumins with point mutations, 2 different carboxyl-terminal variants, and 1 case of analbuminemia in an Italian family. This article describes confirmation by DNA sequencing of mutations previously inferred from protein sequencing of 4 of the above alloalbumins; it also reports the mutations identified by protein and DNA sequence analysis of 4 other Italian alloalbumins not previously recorded: albumin Larino, His3-->Tyr; Tradate-2 (protein sequencing only), Lys225-->Gln; Caserta, Lys276-->Asn; and Bazzano, a carboxyl-terminal variant. The first 3 have point mutations that produce a single amino acid substitution, but a nucleotide deletion causes a frameshift and an altered and truncated carboxyl-terminal sequence in albumin Bazzano. In these 4 instances the expression of the alloalbumin is variable, ranging from 10% to 70% of the total albumiN, in contrast to the usual 50% each for the normal and mutant albumin. The distribution of point mutations in the albumin gene is nonrandom; most of the 47 reported point substitutions involve charged amino acid residues on the surface of the molecule that are not concerned with ligand-binding sites.     

Absence of mutations at the activated protein C cleavage sites of factor VIII in 125 patients with venous thrombosis

Resistance to activated protein C (APC), caused by a mutation at amino acid position Arg⁵⁰⁶ of the factor V gene, has recently been identified as the most prevalent genetic defect associated with venous thrombosis. Similarly to factor V, mutations at the cleavage sites of factor VIII for APC may occur in patients with venous thrombosis. Here we have analysed 125 consecutive patients with incidental or recurrent venous thromboembolism for the presence of mutations at the cleavage sites for APC at amino acid positions Arg³³⁶ and Arg⁵⁶² of factor VIII. Our findings indicate that mutations at these amino acid positions of factor VIII do not occur in the patient group analysed.     

Glucose-6-phosphate dehydrogenase (G6PD) mutations database: review of the "old" and update of the new mutations

In the present paper we have updated the G6PD mutations database, including all the last discovered G6PD genetic variants. We underline that the last database has been published by Vulliamy et al. [1] who analytically reported 140 G6PD mutations: along with Vulliamy's database, there are two main sites, such as http://202.120.189.88/mutdb/ and www.LOVD.nl/MR, where almost all G6PD mutations can be found. Compared to the previous mutation reports, in our paper we have included for each mutation some additional information, such as: the secondary structure and the enzyme 3D position involving by mutation, the creation or abolition of a restriction site (with the enzyme involved) and the conservation score associated with each amino acid position. The mutations reported in the present tab have been divided according to the gene's region involved (coding and non-coding) and mutations affecting the coding region in: single, multiple (at least with two bases involved) and deletion. We underline that for the listed mutations, reported in italic, literature doesn't provide all the biochemical or bio-molecular information or the research data. Finally, for the "old" mutations, we tried to verify features previously reported and, when subsequently modified, we updated the specific information using the latest literature data.     

NS5A mutations predict biochemical but not virological response to interferon-α treatment of sporadic hepatitis C virus infection in European patients

The NS5A region of the hepatitis C virus (HCV) genome has been reported by Japanese but not European investigators to be a significant factor in predicting interferon (IFN) response patients with HCV of genotype 1. We correlated the NS5A region with treatment outcome in patients with sporadic HCV infection. Twenty‐eight patients (10 men, 18 women, mean age 60 ± 2 years) with histologically proven HCV chronic hepatitis, genotype 1b, were treated with 6 MU IFN‐α for 6 months. The 6954–7073 area of the NS5A region was directly sequenced for nucleotide and amino acids mutations and the results were related to biochemical and virological response. None of the patients had a strain with nucleotide sequence identical to the Japanese HCV‐J. However, in five strains the nucleotide mutations led to synonymous amino acids and the amino acid sequences were identical to the prototype Japanese strain. Only 2/28 patients had four or more amino acid mutations (mutant strains) while 21 demonstrated an intermediate type and five belonged to the wild‐type. The most frequent non‐synonymous substitution was at position 6982 (A→G) corresponding to an amino acid change at codon 2218 (His→Arg). All patients with the wild‐type were biochemical nonresponders while the two patients with the mutant strains had a sustained biochemical response. Twenty‐three percent of the intermediate type had a sustained biochemical response. NS5A mutations predict the biochemical but not the virological response of patients. Virological response was poor and unrelated to the type of HCV strain. Biochemical responders had significantly lower amino acid mutations (1.14 ± 0.19) compared with nonresponders (2.57 ± 1.4, P < 0.003) as well as lower aminotransferase values (P < 0.01). Hence, mutational analysis of the NS5A region showed that our patients have a mutational profile similar to the European studies with a wild‐type that is slightly different from the Japanese HCV‐J sequence. The biochemical, but not the virological response to IFN‐α is similar to the Japanese studies, with no response of the patients with wild‐type sequence, a good response in the limited number of patients with mutant strains and 23% response rate in the patients with intermediate type sequences.     

Novel mutations in two Japanese cases of glycogen storage disease type IIIa and a review of the literature of the molecular basis of glycogen storage disease type III

We report two novel mutations in two Japanese patients with glycogen storage disease type IIIa (GSD IIIa). In addition, we review the literature on mutations in GSD III to understand better the molecular basis of GSD III. In our first case, the homozygous A-to-C mutation at the acceptor site of intron 5 (IVS5–2A>C) was identified. This leads to the skipping of exon 6 and the predicted mutant protein was found to be 68 amino acids shorter than normal. This is the first report of skipping exon 6, which encodes one of the putative active sites, resulting in a profoundly deleterious effect on debrancher activity. In our second case, the homozygous deletion of an A at position 4234 (4234delA) was identified; this induces a frameshift resulting in the appearance of a stop codon at amino acid position 1276 (1276X). In patients with GSD IIIa, several mutations of the debrancher gene located in the C-terminal region containing putative glycogen binding domains have been identified as well as 4234delA in our second case. On the other hand, specific localization of the mutations within exon 3 was proposed in patients with GSD IIIb.     

A Routine Sanger Sequencing Target Specific Mutation Assay for SARS-CoV-2 Variants of Concern and Interest

As SARS-CoV-2 continues to spread among human populations, genetic changes occur and accumulate in the circulating virus. Some of these genetic changes have caused amino acid mutations, including deletions, which may have a potential impact on critical SARS-CoV-2 countermeasures, including vaccines, therapeutics, and diagnostics. Considerable efforts have been made to categorize the amino acid mutations of the angiotensin-converting enzyme 2 (ACE2) receptor binding domain (RBD) of the spike (S) protein, along with certain mutations in other regions within the S protein as specific variants, in an attempt to study the relationship between these mutations and the biological behavior of the virus. However, the currently used whole genome sequencing surveillance technologies can test only a small fraction of the positive specimens with high viral loads and often generate uncertainties in nucleic acid sequencing that needs additional verification for precision determination of mutations. This article introduces a generic protocol to routinely sequence a 437-bp nested RT-PCR cDNA amplicon of the ACE2 RBD and a 490-bp nested RT-PCR cDNA amplicon of the N-terminal domain (NTD) of the S gene for detection of the amino acid mutations needed for accurate determination of all variants of concern and variants of interest according to the definitions published by the U.S. Centers for Disease Control and Prevention. This protocol was able to amplify both nucleic acid targets into cDNA amplicons to be used as templates for Sanger sequencing on all 16 clinical specimens that were positive for SARS-CoV-2.     

Hypermutability of CpG dinucleotides in the propeptide-encoding sequence of the human albumin gene.

An electrophoretically slow albumin variant was detected with a phenotype frequency of about 1:1000 in Sweden and was also found in a family of Scottish descent from Kaikoura, New Zealand, and in five families in Tradate, Italy. Structural study established that the major variant component was arginyl-albumin, in which arginine at the -1 position of the propeptide is still attached to the processed albumin. A minor component with the amino-terminal sequence of proalbumin was also present as 3-6% of the total albumin. After amplification of the gene segment encoding the prepro sequence of albumin, specific hybridization of DNA to an oligonucleotide probe encoding cysteine at position -2 indicated the mutation of arginine at the -2 position to cysteine (-2 Arg----Cys). This produced the propeptide sequence Arg-Gly-Val-Phe-Cys-Arg. This was confirmed by sequence analysis after pyridylethylation of the cysteine. This mutation produces an alternate signal peptidase cleavage site in the variant proalbumin precursor of arginyl-albumin giving rise to two possible products, arginyl-albumin and the variant proalbumin. Another plasma from Bremen had an alloalbumin with a previously described substitution (1 Asp----Val), which also affects propeptide cleavage. Hypermutability of two CpG dinucleotides in the codons for the diarginyl sequence may account for the frequency of mutations in the propeptide. Mutation at these two sites results in a series of recurrent proalbumin variants that have arisen independently in diverse populations.     

Additivity of mutant effects assessed by binomial mutagenesis.

Eleven amino acid positions in the helix-turn-helix of lambda repressor have been mutagenized by using a combinatorial method in which alanine is substituted at each position with a probability of 0.5. Approximately 25% of the 2048 proteins in the resulting binomial library are active, including some variants with as many as seven alanine substitutions. The frequency of alanine substitutions in the set of active variants is a measure of the importance of the wild-type residue at each mutagenized position, and comparison of the frequencies of pairwise mutations with those expected based upon the single-position frequencies allows the additivity of mutant effects to be tested. For the positions examined here, we find that the effects of multiple substitutions are largely additive and are able to predict the activity class of the binomial mutants with 90% accuracy by using a model that simply sums penalty scores derived from the alanine substitution frequencies. We also find, however, that several residue pairs, including some that are distant in the three-dimensional structure, do display nonadditive effects that appear to be statistically significant.     

A donor splice mutation and a single-base deletion produce two carboxyl-terminal variants of human serum albumin.

At least 35 allelic variants of human serum albumin have been sequenced at the protein level. All except two COOH-terminal variants, Catania and Venezia, are readily explainable as single-point substitutions. The two chain-termination variants are clustered in certain locations in Italy and are found in numerous unrelated individuals. In order to correlate the protein change in these variants with the corresponding DNA mutation, the two variant albumin genes have been cloned, sequenced, and compared to normal albumin genomic DNA. In the Catania variant, a single base deletion and subsequent frameshift leads to a shortened and altered COOH terminus. Albumin Venezia is caused by a mutation that alters the first consensus nucleotide of the 5' donor splice junction of intron 14 and the 3' end of exon 14, which is shortened from 68 to 43 base pairs. This change leads to an exon skipping event resulting in direct splicing of exon 13 to exon 15. The predicted Venezia albumin product has a truncated amino acid sequence (580 residues instead of 585), and the COOH-terminal sequence is altered after Glu-571. The variant COOH terminus ends with the dibasic sequence Arg-Lys that is apparently removed through stepwise cleavage by serum carboxypeptidase B to yield several forms of circulating albumin.     

Effect of the substitution of critical residues on the allorecognition of HLA-B27.

The three-dimensional structure of the HLA class I molecules has highlighted the importance of the "groove" formed by the helices. We used site-directed mutagenesis to construct a series of HLA-B27 mutants with different substitutions at the sites of the conserved amino acid residues of HLA-B27 subtypes, specifically residue 77 which is thought to be critical to the binding site of the molecule, and a residue at the CD8 binding site. We formed an anti-B27 CTL line and derived six anti-B27 clones. Each of the six clones showed a different pattern of reaction, reflecting the diversity of the epitopes recognized. All nine mutants were effective in altering allorecognition by HLA-B27 specific CTL, although positions 45 and 77 caused the most drastic effect. The residue in position 77 is also the last amino acid of the peptide sequence shared with Klebsiella. Our results highlight the importance of certain epitopes in allorecognition that may have important implications for the immunotherapy of autoimmune diseases.