Assessing the Role of APNH, a Gene Encoding for a Human Amiloride
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Assessing the Role of APNH, a Gene Encoding for a Human Amiloride-Sensitive Na/H Antiporter, on the Interindividual Variation in Red Cell Na/Li Count ert ransport1 Christopher R.K. Dudley,2 Luis A. Giuffra, Anthony E.G. Rome, and Stephen T. Reeders correction of SIC values for known covariotes. There C.R.K. Dudley, S.T. Reeders, Department of Internal was no significant association between mean SIC Medicine. Yale University School of Medicine, New- values and any of the three possible genotypes of Haven, CT the APNH locus either before or after regressing out L.A. Giuffra, ST. Reeders, Department of Human Ge- covariates (F= 0.64 and P> 0.52; F= 0.63 and P> netics, Yale University School of Medicine, New- 0.53, respectively). Linkage analysis similarly foiled Haven, CT to demonstrate a relationship between the squared A.E.G. Rome, Department of Nephrology, The Royal difference In SIC values and the identity by descent Hospital of St. Bartholomew, London, England status for APNH as well as other loci that map close to APNH (D1S57, RH, and ALPI). Taking these results (J. Am. Soc. Nephrol. 1991; 2:937-943) together, we conclude that mutations at the APNH locus are not responsible for the observed variation in SIC values. ABSTRACT Key Words: Blood pressure, intermediate phenotype, associ- The “reverse genetic” approach to essential hyper- ation study, sib pair method, linkage analysis tension Is complicated by the tact that blood pres- sure is a heterogeneous, quantitative, complex trait. One strategy is to use Intermediate phenotypes” that are not only associated with hypertension but S ince the original observation by Canessa et at. that also have a simple mode of inheritance, com- demonstrating an association between red cell patible with the action of a single gene. Red cell sodium-lIthium countertransport (SLC) and essential sodium-lithium countertransport (SIC) is one of the hypertension (1), a number of other groups have best characterized intermediate phenotypes for hy- reported similar findings (2.3). Genetic studies of pertension. The similarity in stoichiometry and kinet- countertransport levels have shown familial aggre- ics between SIC and Na/H exchange has led to gation and a higher concordance rate for monozygotic than for dizygotic twins (4.5). These findings suggest the proposal that the gene encoding the Na/H that SLC has a major heritable component and estI- antiporter (APNH) may be responsible for the individ- mates that up to 80 to 90% of the individual variance ual variance in SIC. We have tested this hypothesis in SLC is accounted for by inherited factors (5.6). by both an association study and Haseman and Complex segregation analysis has been performed to Elston’s sib pair method of linkage analysis, by using determine which of a variety of genetic models best a polymorphism at the APNH locus detected by de- explains the familial pattern of SLC values, Two such naturing gradient gel electrophoresis. Both analyti- studies provide evidence for the action of a single cal techniques were performed before and after major gene on a polygenic background (7,8). These results have important implications, not the least of which is that the same genes that determine SLC values may also be involved in the pathogenesis of ‘ReceIved December 18, 1990. Accepted June I, 1991. 2Correspondence to Df. C.RK. Dudley. Nuffteld Department of Clinical Medi- hypertension. The identification of such genes, cine, John Radcliffe Hospital, Headinglon, Oxford 0X3 9DU. England. therefore. may provide important insights into the l0466673/0204-0937$03.O0/0 mechanisms determining blood pressure variation Journal of the Ameflcan Society of Nephrology Copyright 0 1991 by the American Society of Nephroiogy and control. Journal of the American Society of Nephromogy 937 APNH and Na/Li Countertransporf The physIological role of the transport system that 7.4) at 4#{176}C.SLC activity was determined by measur- mediates In vitro erythrocyte SLC is not established. ing, in parallel and in duplicate, lithium efflux from The similarity In kinetics and stoichiometry between loaded cells into a sodium-contaIning medium (150 SLC and Na/H countertransport has led to the pro- mM NaC1, 10 mM glucose, 0.1 mM ouabain, and 10 posal that red cell SLC is carried out by the ubiqui- mM Tris-MOPS [pH 7.4J at 37#{176}C)and into a sodium- tously distributed Na/H antiporter (9,10). In sup- free medium (110 mM magnesium chloride, 10 mM port of this argument is the demonstration that both glucose, 0.1 mM ouabain, and 10 mM Tris-MOPS [pH SLC and Na/H exchange in sarcolemmal vesicles 7.4] at 37#{176}C)incubated at 37#{176}C.After 0, 20, 40 and from bovine artery are mediated by a single ethyli- 60 mm of incubation, duplicate samples were taken sopropyl amiloride-sensitive cation exchanger with from duplicate tubes, cooled quickly to 4#{176}C,and cen- affinity for sodium, lithium, and hydrogen ions (11). trifuged. Furthermore, Na/H antiport activity has been re- The lithium concentration in the supernatant was ported to be elevated in leukocytes and platelets from determined by atomic-absorption spectrophotome- patients with hypertension (12.13) as well as in ge- try. The hematocrit of each efflux medium was meas- netically hypertensive rats (14,15). ured at the end of the experiment by using a Coulter A flbroblast Na7H antiporter cDNA has been counter. The lithium efflux rates in the sodium-free cloned by complementation (16) and mapped by in and sodium-containing media were taken as the situ hybridization to chromosome lp (17). Linkage slopes of the linear regression lines calculated with studies have placed the genomic locus for this Na/ lithium efflux as a function of time. Correlation coef- H antiporter (designated APNH) close to the rhesus ficients greater than 0.98 were considered accepta- locus (RH) (18,19). It has been proposed that the ble. Net SLC activity was calculated by subtracting APNH gene product carries out SLC and is responsi- the lithium efflux rate in the sodium-free medium ble for the variation observed in SLC values (20,21). from that In the sodium-containing medium and was We have tested this hypothesis by association studies expressed as millimoles of lithium released from one and by the sib pair method of linkage analysis by liter of red cells per hour. The coefficient of variation using members from six large pedigrees in whom of repeated assays was 10%. SLC was measured. Because the power of the sib pair method is limited by a requirement for a highly in- APNH Polymorphism formative locus, we have also performed linkage analysis between SLC and other polymorphic loci A polymorphism in the APNH gene was detected by (namely D1S57, RH, and ALPL, the gene for alkaline denaturing gradient gel electrophoresis (DGGE). A phosphatase), which map close to APNH. 376-base-pair product of the polymerase chain re- action (PCR). corresponding to nucleotides -25 .e METHODS +351 of the coding sequence for APNH, (where + 1 is the first nucleotide of the translation initiation co- Subjects don) (16), was generated by using genomic DNA from As part of a broader study on the genetic determi- each individual as a template. This product was then nants of blood pressure, 190 individuals from six subjected to DOGE on a 35 to 65% gradient as de- large English kindreds were evaluated. Subjects were scribed previously (18). seen by a single investigator, weight was measured DGCIE is a powerful technique allowing two DNA in a standardized way, and blood was drawn for DNA fragments differing by as little as a single base sub- analysis and assay of SLC activity. Thirty-six sub- stitution to be physically separated from each other jects were receiving one or more antihypertensive (23). The method can be further enhanced by allow- agents at the time of the investigation. ing denatured PCR products to reanneal. creating heteroduplex DNA in regions where sequence poly- SIC Activity morphism exists as shown (Figure 1). PCR products SLC was measured as the rate of sodium-dependent from individuals homozygous for each allele were lithium efflux from lithium loaded red blood cells as subcloned into pBSII (Stratagene) and sequenced. Se- described by Canessa et at. (1) and as modified by quence comparison revealed that a single base-pair Smith et at. (22). In brief, washed red cells were substitution of cytosine for thymine at nucleotide 69 suspended at a hematocrit of approximately 20% In distinguishes two alleles, designated Cl and C2 (Fig- a loading solution consisting of 150 mM lithium chlo- ure 2). Although this substitution does not alter the ride, 10 mM glucose, and 10 mM Tris-morpholine- amino acid sequence, It is responsible for the poly- propanesulfonic acid (MOPS) (pH 7.4) and were In- morphism detected by DOGE. The cytosine at posi- cubated in a shaking water bath at 37#{176}Cfor 3 h. tIon 69 in the C2 allele confers greater stability on After loading, external lithium was removed by five the DNA duplex under denaturing conditions and washings with 75 mM magnesium chloride, 85 mM allows the DNA fragment to migrate further down sucrose, 10 mM glucose, and 10 mM Tris-MOPS (pH the denaturing gradient before “melting” occurs. 938 Volume 2 ‘ Number 4 #{149}1991 Dudley et al Statistical Methods The possible genetic role of APNH in SLC variation was tested in two ways. Association Studies. Mean values for SLC were compared by analysis of variance (ANOVA) across the three genotypes (C1C1, C1C2, and C2C2) that 23 4 56 7 could be detected at the APNH locus by using the DGGE polymorphism described above.