Hum Genet (1985) 71 : 49-52 © Springer-Vertag 1985 Molecular evidence of triplication in the haptoglobin Johnson variant gene Salvatore Oiiviero 1, Mario DeMarchi 1, Angelo O. Carbonara 1, Luigi F. Bernini 2, Giuliano Bensi 3, and Giovanni Raugei 3 1 Istituto di Genetica medica, and CNR Centro Immunogenetiea ed Istocompatibilit~, Via Santena 19, 10126 Torino, Italy 2State University Department of Human Genetics, Wassenasserweg 72, Leiden, The Netherlands 3 European Molecular Biology Laboratory, Meyerofsstrasse 1, D-6900 Heidelberg, Federal Republic of Germany Summary. The protein and gene structure of the Hp Johnson variant (Hp3) were analyzed in two related heterozygous indi- viduals. The molecular weight (23 kd) and amino acid compo- sition of Hp3 alpha chain were in agreement with the triplicat- ed structure first suggested by Smithies in 1964. Direct gene analysis by Southern blotting showed a three-fold tandem repeat of the same 1.7 kb DNA segment implicated in the Hp2 gene duplication. On the basis of these data a nine exon model for the Hp3 gene is proposed. Introduction Haptoglobin (Hp) is a haemoglobin-binding, polymorphic plasma protein composed of disulphide-linked alpha2-beta2 tetramers. In man, its most common allele (Hp2) bears a duplication of 59 alpha-chain residues responsible for the protein's molecular weight polymorphism (Bowman and Kurosky 1982; Smithies et al. 1962). The alpha and beta chains are both coded by the same discontinuous gene, com- posed of five exons in Hpl and seven in Hp2 (Bensi et al. 1985; Maeda et al. 1984; Raugei et al. 1983; vander Straten et al. 1983; Yang et al. 1983); the duplicated stretch corresponds to exons 3 and 4, within a larger 1.7 kb DNA duplication at precise, non-homologous sites in the second and fourth Hpl introns (Maeda et al. 1984). Other rarer alleles, such as the triplicated Johnson variant (Hp3), are thought to have origi- nated from Hp2 through unequal crossing over in the duplicat- ed region (Giblett and Brooks 1963; Smithies 1964). We here offer direct confirmation at the DNA level of intragenic triplication in a ease of Hp Johnson. Since the same 1.7kb segment as in Hp2 is again implicated, it may be sup- posed that the Hp3 gene has a nine exon structure. Materials and methods Individuals of the healthy, Caucasian Family I.G., living in Holland, have the following Hp genotypes: father Hp 1/3, mother Hp 1/2, son Hp 2/3. The father provided the serum sample for studies of molecular weight and amino acid compo- Offprint requests to: Salvatore Oliviero, Istituto di Genetica medica, and CNR Centro Immunogenetica ed Istocompatibilit~t, Via Santena 19, 10126 Torino, Italy sition. Briefly, Hp was isolated from serum as previously de- srcibed (Bernini and Borri-Voltattorni 1970), reduced and cyanoethylated, and run on a G-75 Sephadex column, using the alpha-1 and beta chain fractions as internal mol. wt. stand- ards. The isolated, 23 kdalton alpha-3 chain was hydrolysed at 110°C for 72h, and used for amino acid composition analysis. The son was analyzed at the Hp gene level. DNA was extracted from the buffy coat as described previously (Oli- viero et al. 1985), digested with a three-fold excess of restric- tion endonucleases (Boehringer Mannheim) following the manufacturer's instructions, electrophoresed on 0.8% agarose gel, transferred to nitrocellulose filters (Schleicher and Schuell), and hybridized to 32p nick-translated probes (South- ern 1975). Probes were (i) Hp2 cDNA from liver, cloned in the pMBL 9 plasmid (Raugei et al. 1983), and excised as a 1.1kb PstI-HindIII fragment; (ii) Hp-alpha, the 480bp PstI- BamHI fragment from the Hp2 clone; (iii) Hp-beta, the 1017bp Barn HI-PstI fragment from the same clone. Filters were then washed twice at 65°C with 2 x SSPE, 0.1% sodium dodecyl sulphate (SDS) for 10rain, twice with 0.1% x SSPE, 0.1% SDS for 30min, and exposed for autoradiography on Kodak XoMat films for 3-5 days at -80°C. Results As shown in Fig. 1, the gel filtration profile of the reduced and alkylated father's haptoglobin (Hp 1/3) showed the alpha-3 peak in front of the alpha-2 and alpha-1 chains, with an esti- mated mol. wt. of 23 kdaltons. This value is in close agreement with the expected mol. wt. of 22,683, calculated on the basis of the number (= 201) and composition of amino acid residues (Table 1). Haptoglobin genotype restriction patterns of the Hp 2/3 heterozygous child were compared with those of the common Hp genotypes. As previously described (Maeda et al. 1984; Oliviero et al. 1985; Raugei et al. 1983), HindIII digested genomic DNA, hybridized with a Hp2 cDNA probe, displays Hpl and Hp2 as 3.2kb and 4.9kb fragments respectively (Fig. 2). The 1.7kb difference, due to the increased length of Hp2, can be recognised with Eco RI (Hpl = 10.1kb, Hp2 = ll.Skb), PstI (4.2kb and 5.9kb) (not shown), and several other enzymes, unless polymorphic sites are present. In all individuals, cross-hybridization with the same probe also re- veals the Hp-related gene (Hpr) as an additional band of 3.4kb with Hind III, 8.3kb with Eco RI, 2.4kb with BamHI, and 3.7kb with SacI. In the Hp 2/3 heterozygote the Hp2 and