Proc. Nati. Acad. Sci. USA Vol. 91, pp. 3999-4003, April 1994 Medical Sciences Two point mutations within the adducin genes are involved in blood pressure variation GIUSEPPE BIANCHI*t, GRAZIA TRIPODIt, GIORGIO CASARIt, SERGIO SALARDII, BARRY R. BARBERt, RODOLFO GARCIA§, PATRICIA LEONI§, LUCIA TORIELLIf, DANIELE CUSI*, MARA FERRANDI*, LORENZO A. PINNA¶, FRANCISCO E. BARALLE§, AND PATRIZIA FERRARII *Nephrology Clinic and Department of Sciences and Biomedical Tecnologies, University of Milan, San Raffaele Hospital, Via Olgettina 60, 20132 Milan, Italy; 4Prassis-Sigma Tau Research Institute, Via Forlanini 3, 20019 Settimo Milanese, Milan, Italy; International Centre for Genetic Engineering and Biotechnology, Padriciano 99, 34012 Trieste, Italy; and IDepartment of Biological Chemistry, University of Padua, Via Marzolo 3, Padua, Italy Communicated by David Weatherall, January 7, 1994 ABSTRACT The Milan hypertensive strain of rats (MHS) develops a genetic form of renal hypertension that, when compared to its normotensive control (MNS), shows renal dysfunction similar to that of a subset of human patients with primary hypertension. MHS and MNS were shown to be homozygous by multilocus miisatellite analysis and monolocus microsatellite markers. We show here that one point mutation in each of two genes coding for the membrane skeleton protein adducin is associated with blood pressure in the Milan strain of rats. Adducin is a heterodimer formed by a and 13 subunits that promotes the assembly of actin with spectrin. MHS and MNS differ, respectively, by the amino acids Y and F at position 316 of the a subunit. In the ,&adducin locus, MRS is always homozygous for R at position 529 while in MNS either R or Q occurs in that position. The R/Q heterozygotes showed lower blood pressure than any of the homozygotes. In vitro phosphorylation studies suggest that both of these amino acid substitutions occur within protein kinase recognition sites. Analysis of an F2 generation demonstrated that Y alleles segregated with a significant increment in blood pressure. This effect is modulated by the presence of the R allele of the 1 subunit. Taken together, these dings strongly support a role for adducin polymorphisms in causing variation of blood pressure in the Milan strain of rats. Primary or essential hypertension is a heterogeneous disease affecting 15-20%o of the adult population (1). Different genetic mechanisms causing renal, endocrine, humoral, or nervous dysfunction have been suggested (2). Different rat models of genetic hypertension have been developed, and for three of them a genetic association between a DNA polymorphism and hypertension has been suggested (3-8). The Milan hy- pertensive strain of rats (MHS) was developed by selection for hereditary hypertension in divergence to its normotensive control (MNS), which was selected for low blood pressure. At present, 85 generations of inbreeding have been reached (9, 10). Compared to MNS, MHS shows a greater pressor effect of the kidney after transplantation (9, 11), a faster glomerular filtration rate and tubular reabsorption (9, 12), a lower kidney weight (9) and plasma renin activity (13), and a lower volume of erythrocytes (14) and tubular cells (15), both of which show faster Na transport across their plasma membrane (14, 16, 17). Erythrocyte functional differences are genetically determined within the stem cells and are genetically associated with hypertension in F2 hybrids (18). Thus, a generalized and genetically determined cellular de- fect involving faster Na transport across the renal cell mem- branes (19) was considered a probable cause of hypertension in MHS. Some of the erythrocyte and kidney dysfunctions seen in these rats have also been found in a subset (=25%) of human patients with primary hypertension (10, 20-22). In the rat model, the difference in membrane ion transport disap- peared after elimination of the membrane skeleton, which indicated the involvement of some of its components (23, 24). Cross-immunizations between MHS and MNS raised an antibody against a membrane skeleton protein subsequently identified as adducin (25). As this was the only cytoskeletal difference found that could be associated with membrane ion transport differences, adducin was considered a candidate for genetic studies in hypertension. Adducin is an af3 heterodimer with subunits of Mr 103,000 (a) and 97,000 (/3). It promotes the organization of a spectrin- actin lattice, a function regulated by phosphorylation and Ca-calmodulin interactions (26-28). Furthermore, a- and (3-adducin have similarities with the MARCKS group of proteins, which are involved in cellular signal transduction mechanisms (29). We characterized the rat full-length (- and a-adducin cDNAs (ref. 30; G.T., G.C., G.B., and F.E.B., unpublished data) and localized the genes on chromosomes 4 and 14, respectively (G.C., G.T., G.B. and F.E.B., unpub- lished data). Corresponding human a- and (3-adducin cDNAs were isolated from an erythroblastoid cell line (31) and, recently, by positional cloning from yeast artificial chromo- some clones (32). The aims of this study were to investigate the eventual structural differences of adducin subunits in MHS and MNS rats and their functional significance regarding blood pres- sure variation. MATERIALS AND METHODS Animal Procurement and Housing. All MNS and MHS rats were bred in our own facilities and maintained in conditions described elsewhere (9), in agreement with Directive 86/609/ CEE of the Council of the European Community and Italian Law no. 116, 22/1/1992. The experimental F2 population was produced as described in the text. Blood Pressure Measurements. In the foundation colonies, selection for blood pressure levels was carried out each generation on awake animals restrained by wrapping lightly in a small cloth (33), using an indirect tail-cuff method. Measurements were made on a W + W BP recorder (Ugo Basile, Varese, Italy) with piezoelectric pickup. For genetic analysis of the F2 population, a cannula was inserted in the carotid artery of the rat under light halothane anesthesia and externalized at the back of the neck through- out a subcutaneous tunnel. The animals recovered within 3-5 min. Four hours later, the rats were connected by catheter to Abbreviations: MHS, Milan hypertensive strain; MNS, normoten- sive control. tTo whom reprint requests should be addressed. 3999 The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.