Proc. Nati. Acad. Sci. USA Vol. 91, pp. 6476-6480, July 1994 Biochemistry Genetic variants of human serum albumin in Italy: Point mutants and a carboxyl-terminal variant (alloalbumin/genetic polynorpUhM/r shlt mutation/amIno add substitution/DNA sequence) JEANNE MADISON*, MONICA GALLIANOtt, SCOTT WATKINS*, LORENZO MINCHIOTTI*, FRANCO PORTA§, AGOSTINO Rossi§, AND FRANK W. PUTNAM*¶ *Department of Biology, Indiana University, Bloomington, IN 47405; tInstitute of Biology, University of Sassari, Sassari, Italy; *Department of Biochemistry, University of Pavia, 27100 Pavia, Italy; and 1Ospedale di Circolo, 21100 Varese, Italy Contributed by Frank W. Putnam, March 28, 1994 ABSTRACT Of the >50 different genetic variants of hu- of clinical laboratories throughout Italy (21). CISMEL has man serum albumin (afloalbumins) that have been character- thus far uncovered 634 instances ofinherited bisalbuminemia ized by amino acd or DNA sequence analysis, almost half have in unrelated individuals, including several homozygotes; been Identified in Italy through a long-term electrophoretic CISMEL also identified about 100 cases of transient bisal- survey of serum. Previously we have reported structural stud- buminemia induced by pancreatic disease or penicillin ther- ies of 11 Italian afloalbinins with point mutations, 2 dfferent apy and 4 cases of analbuminemia (an inherited condition in carboxyl-terminal variants, and 1 case of analbumia in an which little or no albumin is produced in the homozygous Italian family. This article describes cnimation by DNA state). Some 26 different Italian alloalbumins were distin- sequendung of mutations previously inferred from protein guished in electrophoresis by use oftwobuffers ofpH 8.6 and sequencing of 4 of the above aoalbun; it ao reports the 5.0. Our laboratories have identified the molecular defect in mutations identied by protein and DNA sequence analysis of 18 of the 26, including the 4 variants reported here (2, 4-11). 4 other It aflolbns not previously recorded: albumin Two ofthe latter have amino acid substitutions in a structural Larino, His3 -l Tyr; Tradate-2 (protein sequencing only), region (subdomain IB-subdomain HA) for which few changes Lys= -- Gln; Caserta, Lys"" -- Asn; and Ba ngo, a car- have previously been reported. In one instance (albumin boxyl-terminal variant. The first 3 have point mutations that Bazzano) a nucleotide deletion produces a frameshift that produce a single amino acid substitution, but a nucleotide results in an altered and truncated carboxyl-terminal se- deletin causes a frameshift and an altered and truncated quence. carboxyl-terminal sequence in albumin Bazno. In these 4 instances the expression ofthe alloalbumin is variable, r MATERIALS AND METHODS from 10% to 70% of the total albumin, in contrast to the usual 50% each for the normal and mutant albumin. The distribution Protein Strutua Studies. Fresh sera with albumins having of point mutations in the albumin gene is nonrandom; most of altered electrophoretic mobility were designated geographi- the 47 reported point bstitutions involve charged amino acid cally as follows: albumin Larino, Tradate-2, Caserta, and residues on the surface ofthe molecule that are not concerned Bazzano. Whole blood for DNA preparation was received in sites. all cases except Tradate-2. The alloalbumins were typed by with lignd-binding cellulose acetate electrophoresis at pH 8.6 and 5.0 by com- parison with known variants, and the ratio of variant to Many different, benign genetic variants of human serum normal albumin (albumin A) was determined by densitomet- albumin (HSA) (alloalbumins) have been identified electro- ric analysis (2, 21). All the subjects were heterozygotes, and phoretically during population genetics surveys or routine inheritance ofthe traits had been demonstrated. In this work clinical analysis (1-20). Because the cumulative frequency of a family study was done only for albumin Caserta. alloalbumins in most populations is only -1 in 3000, most The normal and variant albumins were separated on a carriers are heterozygous and have nearly equal amounts of DEAE-Sephadex column, and the purity of the proteins was normal and mutant albumin, a condition called bisalbumin- checked by SDS/PAGE (11). The reduced and carboxymeth- emia. Most alloalbumins are given provisional names- ylated albumins were cleaved with CNBr, and the CNBr geographical, ethnic, or tribal-until they have been charac- digests were compared by analytical isoelectric focusing to terized structurally. To date, the structural change in >50 identify the site ofmutation (5). The variant CNBrfriagments different alloalbumins has been determined by amino acid were isolated preparatively either by gel filtration on a TSK and/or DNA sequence analysis (1-20). Although several G3000 SW column with isocratic elution orby reversed-phase proalbumins and some point mutants, such as albumin B, are HPLC (2, 15). Tryptic digests ofthe purified CNBr fragments found in diverse populations (1-3, 12, 19), many alloalbumins were mapped by reversed-phase HPLC on a Vydac C15 appear to be unique to a particular ethnic group (i-3, 13, 14, column (2), and the purified variant peptides were submitted 20). Some of the latter variants are extremely rare, whereas to amino acid analysis and sequence analysis (1-4, 15, 22). others, such as albumins Naskapi and Yanomama, are poly- Intact albumin Larino was also submitted to protein sequence morphic, having an allele frequency -1% in certain Amer- analysis. indian tribes (14), as does albumin Ortonovo in a small village DNA Struetural Studies. High molecular weight DNA was in Italy (8). prepared by proteinase K treatment and phenol extraction of Since 1971 the Italian Committee for Standardization of leukocytes (23). Primer sets (designated AO1A, etc.) that Electrophoretic Laboratory Methods (CISMEL) has carried encompassed specific exons of HSA and their intron-exon on an extensive survey for alloalbumins in serum with the aid junctions (24) were used to PCR amplify regions of the HSA The publication costs ofthis article were defrayed in part by page charge Abbreviations: dsDNA, double-stranded DNA; HSA, human serum payment. This article must therefore be hereby marked "advertisement" albumin. in accordance with 18 U.S.C. §1734 solely to indicate this fact. ITo whom reprint requests should be addressed. 6476 Downloaded by guest on September 24, 2021 Biochemistry: Madison et al. Proc. Nadl. Acad. Sci. USA 91 (1994) 6477 gene ranging from 288 to 464 bp in length (25). PCRs were Albumin Tradate-2 (Lys225 -* Gln). AlloalbuminTradate-2, performed as described (7, 23). The DNA samples were present in equimolar ratio with albumin A in a person from denatured and reannealed slowly to form heteroduplexes (23, Tradate (Lombardy region), had a -1 (fast) mobility. Iso- 26), and the products were resolved in a mutation-detection- electric focusing of a CNBr digest of the purified variant enhancement gel (AT Biochem, Malvern, PA) (23). For albumin indicated that the substitution was in CB3 (residues subcloning and DNA sequencing the PCR product was di- 124-298). The variant CB3 was purified by HPLC of a gested with appropriate restriction enzymes and the resulting preparative CNBr digest ofthe alloalbumin and was digested fagment was ligated into pBluescript (Stratagene) cut with with trypsin. The HPLC tryptic peptide map showed the compatible ends. Transformation, screening, and double- absence of normal peptides T33 and T34 (residues 223-225 stranded DNA (dsDNA) sequencing were performed as de- and 226-233, respectively) and the presence ofa new peptide, scribed (7). T33-34'. Amino acid sequence analysis of the latter estab- lished the substitution Lys225 -- Gin in the variant peptide; this had the sequence Phe-Pro-Gin-Ala-Glu-Phe-Ala-Glu- RESULTS AND DISCUSSION Val-Ser-Lys, corresponding to residues 223-233 (Table 1). This substitution accords with the change in mobility. The DNA and Protein Sequendng of Previously Unreported nucleotide mutation AAA -- CAA was inferred from the Point Mutants. In our continuing structural study of Italian amino acid exchange but could not be confirmed by DNA alloalbumins, DNA and/or protein sequence analysis was sequencing because a specimen of whole blood was not used to identify the site and type of mutation in four previ- available. ously unreported albumin variants described below and listed Albumin Caserta (Lys276 -- Asn). This alloalbumin was in Table 1. identified by its fast (-1) mobility in the mother, daughter, Albumin Larino (His3-. Tyr). Albumin Larino comprised and son in a family in Caserta, a city near Naples. The three only 10-12% of the total albumin in a mother and daughter subjects were heterozygous, butthe variant/normal ratio was from Larino (Campobasso province, Molise region). The 1.5:1 in the serum of the mother, whereas it was about 2: 1 total albumin content was also decreased, but there was a in both siblings. In all three cases an increased total albumin compensatory increase in other plasma proteins. Larino was content was observed. Isoelectric focusing of a CNBr digest initially thought to be a variant ai-antitrypsin because of its of the purified alloalbumin suggested a point substitution in slow mobility, but reaction with antisera showed that it was CB3 (encoded by exons 4-7 and part of exons 3 and 8). an albumin variant. Isoelectric focusing of a CNBr digest of Protein sequence analysis was done on a variant peptide the purified variant indicated that the substitution was in (T39'-T40) that had been purified by HPLC from a tryptic CNBr fragment CB1 (residues 1-87; encoded by exons 1-3 digest of CB3 prepared from purified alloalbumin of the son. and part of exon 4); all the other CNBr fiagments appeared This established the substitution Lys276 -+ Asn in the se- to be normal. Exons 1, 2, 3, and 4 were PCR amplified, and quence Leu-Asn-Glu-Cys-Cys-Glu-Lys-Pro-Leu-Leu-Glu- heteroduplex analysis showed that the change was in exon 1. Lys (residues 275-286). Automated amino acid sequence analysis of the purified Because Lys276 is in exon 8, 250-bp Xmn I restriction alloalbumin established the substitution His3 -* Tyr.