Filtration Markers, Cardiovascular Disease, Mortality, and Kidney Outcomes in Stable Kidney Transplant Recipients: The FAVORIT Trial.

Cystatin C and beta-2-microglobulin (B2M) are filtration markers associated with adverse outcomes in nontransplant populations, sometimes with stronger associations than for creatinine. We evaluated associations of estimated glomerular filtration rate from cystatin C (eGFRcys ), B2M (eGFRB2M ), and creatinine (eGFRcr ) with cardiovascular outcomes, mortality, and kidney failure in stable kidney transplant recipients using a case-cohort study nested within the Folic Acid for Vascular Outcome Reduction in Transplantation (FAVORIT) Trial. A random subcohort was selected (N = 508; mean age 51.6 years, median transplant vintage 4 years, 38% women, 23.6% nonwhite race) with enrichment for cardiovascular events (N = 306; 54 within the subcohort), mortality (N = 208; 68 within the subcohort), and kidney failure (N = 208; 52 within the subcohort). Mean eGFRcr , eGFRcys , and eGFRB2M were 46.0, 43.8, and 48.8 mL/min/1.73m2 , respectively. After multivariable adjustment, hazard ratios for eGFRcys and eGFRB2M <30 versus 60+ were 2.02 (95% confidence interval [CI] 1.09-3.76; p = 0.03) and 2.56 (1.35-4.88; p = 0.004) for cardiovascular events; 3.92 (2.11-7.31) and 4.09 (2.21-7.54; both p < 0.001) for mortality; and 9.49 (4.28-21.00) and 15.53 (6.99-34.51; both p < 0.001) for kidney failure. Associations persisted with additional adjustment for baseline eGFRcr . We conclude that cystatin C and B2M are strongly associated with cardiovascular events, mortality, and kidney failure in stable kidney transplant recipients.

Multicenter analytical evaluation of a high-sensitivity troponin T assay.

BACKGROUND: High-sensitivity cardiac troponin assays are being introduced clinically for earlier diagnosis of acute myocardial infarction (AMI). We evaluated the analytical performance of a high-sensitivity cardiac troponin T assay (hscTnT, Roche Diagnostics) in a multicenter, international trial. METHODS: Three US and 5 European sites evaluated hscTnT on the Modular® Analytics E170, cobas® 6000, Elecsys 2010, and cobas® e 411. Precision, accuracy, reportable range, an inter-laboratory comparison trial, and the 99th percentile of a reference population were assessed. RESULTS: Total imprecision (CVs) were 4.6-36.8% between 3.4 and 10.3 ng/L hscTnT. Assay linearity was up to 10,000 ng/L and the limit of blank and detection were 3 and 5 ng/L, respectively. The 99th percentile reference limit was 14.2 ng/L (n=533). No significant differences between specimen types, assay incubation time, or reagent lots existed. A substantial positive bias (76%) exists between the 4th generation and hscTnT assays at the low end of the measuring range (<50 ng/L). hscTnT serum pool concentrations were within 2SD limits of the mean of means in the comparison trial, indicating comparable results across multiple platforms and laboratories. CONCLUSION: The Roche hscTnT assay conforms to guideline precision requirements and will likely identify additional patients with myocardial injury suspicious for AMI.

[Increased risk of malignancies in heterozygotes in families of patients with Nijmegen breakage syndrome]. / Zvýsené riziko malignit u heterozygotu v rodinách pacientu s Nijmegen breakage syndromem.

BACKGROUND: The autosomal recessive chromosomal instability and hyperradiosensitivity Nijmegen breakage syndrome (NBS) in consequence of a mutation in the NBSI gene at 8q21 is associated with high occurrence of lymphoreticular malignancies due to deficient DNA reparation (double strand breaks). In the Slavic population the majority of patients are homozygotes of the so-called "Slavic mutation" 657de15 in exon 6. Increased occurrence of malignant solid tumors (1) in families of NBS patients has been described already prior to the identification of the responsible gene, and the increased risk of malignancies in heterozygotes was thus hypothetical. METHODS AND RESULTS: The possibility of discerning mutation carriers in families from normal homozygotes enables verification of that hypothesis. Through molecular genetics investigations of grandparents and immediate relatives, we have been successful in determining the genotype in 79 of 112 grandparents in 28 families of our 39 patients and 54 their parents and siblings. A single family had affected children in consequence of compound heterozygosity of the 657de15 and R215W mutations in the same exon of the NBSI gene. The proband's families were investigated genealogically and data on relatives were obtained over four generations. Obtained data were repeatedly supplemented and objectively verified in church books and in healthcare documentation. Seven families have been followed up for 20-30 years, six families for 10-20 years, and 15 families for 1-10 years. Out of 28 families we were successful in examining the genotype of both grandparents in 18 families, there having been revealed one non-paternity; in five families only one of the grandparents has been examined; in five families we were not successful in examining any grandparent. Among 40 grandparents - normal homozygotes, there has appeared a malignancy in three (7.4 %), while among 39 heterozygotes of mutation 657de15 in the NBSI gene malignancies were documented in 15 (38,2 %). Mean age of NBS heterozygotes at manifestation of malignancy was 59.3 year (range 47-72 years), in the group of homozygotes it was 52.6 years (range 44-62 years). Nine grandparents died of malignancy prior to the discovery of the NBSI gene and their genotype has been deduced genealogically in seven on the basis of the genotype in the sponse and children, in two from preserved DNA. Out of that number, from three grandparents that had died of malignancies we were successful in obtaining neoplastic tissue for molecular genetics investigation, aimed at LOH or amplification of the NBS1 gene. In another seven grandparents - heterozygotes, malignancies were manifested after determination of their genotype by DNA analysis, and consequently also from tumor tissue that has been obtained from three of them for molecular genetic investigation. CONCLUSIONS: The age distribution and socio-economic status of both groups of grandparents did not differ, the sex ratio was slightly shifted towards females in the group of homozygotic grandparents (22 females and 18 males), and in the group of heterozygotes it was towards males (21 males and 18 females). The sex ratio between heterozygotic grandparents with malignancies was likewise shifted towards the male gender (11 males and 4 females), in the group of homozygotic grandparents malignancy affected one male and two females. As verified in healthcare and church books documentation, the occurrence of malignancies was significantly more frequent among grandparents heterozygotic for NBS1 mutation than in healthy homozygotes. Among sibs of grandparents and great-grandparents was found significant difference in frequency of malignancies in heterozygotes (5/18 = 27,7 %) and healthy homozygotes (2/36 = 5,5 %), too.

The dynamics of gene expression in human lung microvascular endothelial cells after stimulation with inflammatory cytokines.

Vascular endothelium plays an essential role in the pathogenesis of vasoocclusion. The changes in the endothelial cell function can be triggered by changes in gene expression caused by interaction with cytokines and blood cells. Using cDNA arrays, we have recently reported complex patterns of gene expression after stimulation of endothelial cells with TNFalpha and IL-1beta. Better understanding of the time course of gene expression changes, their concentration dependence and reversibility after withdrawal of the offending cytokine is essential for successful prevention and therapy of vasoocclusion. Here we present a detailed study of the concentration dependence and time course of gene expression in endothelial cells after their exposure to TNFalpha and IL-1beta. We focus on the adhesion molecules (VCAM-1, ICAM-1, E-selectin) and cytokines (IL-6, GCP-2, MCP-1) that are likely to contribute to vasoocclusion. We report differences in the time course and intensity of their expression and in their response to TNFalpha and IL-1beta stimulation. We demonstrate that expression of the studied genes is upregulated by low TNFalpha concentrations that better reflect the TNFalpha levels detected in the plasma of patients developing vasoocclusion. These results help to understand the changes in the endothelium and to design rational prevention and therapy of vasoocclusion.

[Chromosome instability syndromes]. / Význam syndromu chromozomální instability.

We refer 55 cases of the chromosomal instability syndromes (SCI), diagnosed in patients of our genetical clinics. Problems of early diagnosis can be documented by a discrepancy between the expected number of patients and their relative advanced age at the time when SCI was ascertained. We have also shown that NBS patients can be diagnosed earlier and the disease sufficiently confirmed on the basis of congenital microcephaly and on the direct detection of 657de15 mutation in NBS1 gene. Genealogical analysis of families with SCI revealed a low risk of prenatal selection of affected homozygotes and high cancer prevalence in relative (in NBS families recognized heterozygotes) at young adult age. Due to severe DNA repair disorder and hyperradiosensitivity of affected homozygotes as well as unaffected heterozygotes, conventional diagnostics and treatment protocols of lymphoreticular malignancies in affected homozygotes are prohibited. The use of Nijmegen treatment protocol improved in our patients dramatically their clinical prognosis, which is documented by 6 NBS patients surviving one or two malignancies. Early diagnose of SCI and information for families and their doctors about consequences of DNA repair disorder and about their hyperradiosensitivity is essential for improving the clinical prognosis of SCI patients.

Rescue of a telomere length defect of Nijmegen breakage syndrome cells requires NBS and telomerase catalytic subunit.

Nijmegen breakage syndrome (NBS) is a rare human disease displaying chromosome instability, radiosensitivity, cancer predisposition, immunodeficiency, and other defects [1, 2]. NBS is complexed with MRE11 and RAD50 in a DNA repair complex [3-5] and is localized to telomere ends in association with TRF proteins [6, 7]. We show that blood cells from NBS patients have shortened telomere DNA ends. Likewise, cultured NBS fibroblasts that exhibit a premature growth cessation were observed with correspondingly shortened telomeres. Introduction of the catalytic subunit of telomerase, TERT, was alone sufficient to increase the proliferative capacity of NBS fibroblasts. However, NBS, but not TERT, restores the capacity of NBS cells to survive gamma irradiation damage. Strikingly, NBS promotes telomere elongation in conjunction with TERT in NBS fibroblasts. These results suggest that NBS is a required accessory protein for telomere extension. Since NBS patients have shortened telomeres, these defects may contribute to the chromosome instability and disease associated with NBS patients.

[Silent forms of hereditary spherocytosis]. / Nemé formy hereditární sférocytózy.

We report 7 cases of "silent" form of hereditary spherocytosis observed among members of 4 different families. Silent form of hereditary spherocytosis occurred in 5.4% of all patients with hereditary spherocytosis treated in our institute. The patients with silent form featured normal Hb level and red blood cell count, normal or slightly elevated reticulocyte count and bilirubin level. Osmotic resistance of red blood cells was decreased and autohemolysis was increased, nevertheless, the differences from normal range were less prominent than in patients with manifest form of the disease. Analysis of red cell membrane revealed deficiency of band 3 protein in all cases of silent form of hereditary spherocytosis.

[Pregnancy in patients with hereditary spherocytosis]. / Tehotenství u nemocných s dedicnou sférocytózou.

The influence of pregnancy on the course of hereditary spherocytosis was investigated in 21 women during their 44 pregnancies. Fourteen pregnancies were followed up directly, 30 were evaluated from anamnestic data. In the majority of investigated women with hereditary spherocytosis pregnancy caused no problems. When complications developed, they were not serious as a rule. Only about one third of pregnancies in non-splenectomized women developed anaemia or anaemia deteriorated. In the latter enhanced haemolysis participated. In splenectomized patients the incidence of complaints was minimal.

Characterization of seven low incidence blood group antigens carried by erythrocyte band 3 protein.

Recent studies have demonstrated that band 3 carries antigens of the Diego blood group system and have elucidated the molecular basis of several previously unassigned low incidence and high incidence antigens. Because the available serological data suggested that band 3 may carry additional low incidence blood group antigens, we screened band 3 genomic DNA encoding the membrane domain of band 3 for single-strand conformational polymorphisms. We found that the putative first ectoplasmic loop of band 3 carries blood group antigen ELO, 432 Arg-->Trp; the third putative loop harbors antigens Vga (Van Vugt), 555 Tyr-->His, BOW 561 Pro-->Ser, Wu (Wulfsberg), 565 Gly-->Ala, and Bpa (Bishop), 569 Asn-->Lys; and the putative fourth ectoplasmic loop carries antigens Hga (Hughes), 656 Arg-->Cys, and Moa (Moen), 656 Arg-->His. We studied erythrocytes from carriers of five of these blood group antigens. We found similar levels of reticulocyte mRNA corresponding to the two band 3 gene alleles, normal content and glycosylation of band 3 in the red blood cell membrane, and normal band 3-mediated sulfate influx into red blood cells, suggesting that the mutations do not have major effect on band 3 structure and function. In addition to elucidating the molecular basis of seven low incidence blood group antigens, these results help to create a more accurate structural model of band 3.

Autosomal dominant distal renal tubular acidosis is associated in three families with heterozygosity for the R589H mutation in the AE1 (band 3) Cl-/HCO3- exchanger.

Distal renal tubular acidosis (dRTA) is characterized by defective urinary acidification by the distal nephron. Cl-/HCO3- exchange mediated by the AE1 anion exchanger in the basolateral membrane of type A intercalated cells is thought to be an essential component of lumenal H+ secretion by collecting duct intercalated cells. We evaluated the AE1 gene as a possible candidate gene for familial dRTA. We found in three unrelated families with autosomal dominant dRTA that all clinically affected individuals were heterozygous for a single missense mutation encoding the mutant AE1 polypeptide R589H. Patient red cells showed approximately 20% reduction in sulfate influx of normal 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid sensitivity and pH dependence. Recombinant kidney AE1 R589H expressed in Xenopus oocytes showed 20-50% reduction in Cl-/Cl- and Cl-/HCO3- exchange, but did not display a dominant negative phenotype for anion transport when coexpressed with wild-type AE1. One apparently unaffected individual for whom acid-loading data were unavailable also was heterozygous for the mutation. Thus, in contrast to previously described heterozygous loss-of-function mutations in AE1 associated with red cell abnormalities and apparently normal renal acidification, the heterozygous hypomorphic AE1 mutation R589H is associated with dominant dRTA and normal red cells.

Incomplete distal renal tubular acidosis coinherited with a mutation in the band 3 (AE1) gene.

BACKGROUND: Band 3 (anion exchanger 1, AE1) is one of the most abundant proteins of the erythrocyte membrane. We have previously characterized twenty AE1 gene defects underlying spherocytic haemolytic anaemia with band 3 deficiency. Since AE1 is also expressed in the intercalated cells of renal cortical collecting ducts where it is thought to participate in urine acidification, we asked whether the spherocytogenic AE1 mutations also affect the regulation of urine acidity. METHODS: We examined 10 patients from seven unrelated families with hereditary spherocytosis with band 3 deficiency using the short urine acidification test with CaCl2 administration at a dose of 0.2 g/kg b.w. To asses the ability of the nephron to secrete protons, 400 ml of NaHCO3 were infused over a period of 2 h. RESULTS: While we detected no significant abnormalities in eight patients, we have diagnosed incomplete distal renal tubular acidosis (dRTA) in two patients from one family whose urinary pH 5 h after CaCl2 administration were 6.56 and 6.89. Administration of bicarbonate in these two patients resulted in high urinary HCO3- concentration. The patients carry the previously characterized mutation band 3PRIBRAM that encodes a C-terminally truncated band 3 containing only the cytoplasmic domain and the first three putative transmembrane segments. CONCLUSIONS: This finding shows an association of a band 3 defect with abnormal urinary acidification perhaps secondary to Cl-/HCO3- exchange in the basolateral membrane of alpha-intercalated cells of cortical collecting ducts.

Characterization of the underlying molecular defect in hereditary spherocytosis associated with spectrin deficiency.

Several subsets of patients with hereditary spherocytosis (HS) have been defined based on the specific red blood cell membrane protein deficiencies involving spectrin, ankyrin, band 3, and protein 4.2. Mutations of the genes encoding these proteins are currently being uncovered. Regarding spectrin, only three isolated cases of beta-spectrin gene mutations were recently reported in association with HS and spectrin deficiency. We have screened the coding region of the beta-spectrin gene using the SSCP technique, in 40 families with HS associated with spectrin deficiency or combined spectrin and ankyrin deficiencies. In this report we describe six frameshift and nonsense mutations and four missense mutations of the beta-spectrin gene in 11 unrelated families. Taking advantage of modifications in the restriction enzyme recognition sequences introduced by the mutations, we show, in all cases of frameshift and nonsense mutations, the loss of heterozygosity at the cDNA level when compared to genomic DNA, reflecting the absence of the mutant mRNA transcripts. In one family with a large pedigree including six generations and 112 members, we firmly establish the autosomal dominant inheritance of one of the beta-spectrin null mutations. Most of the mutations described are responsible for a phenotype of mild to moderate autosomal dominant form of HS associated with a conspicuous spherocytosis with frequent spiculated cells (8% to 15% acanthocytes). One missense mutation appears to be associated with a recessive form of the disease. Five common restriction enzyme polymorphisms of the coding region of the beta-spectrin gene are also described. Overall, these findings underscore the importance of the beta-spectrin gene mutations in the pathogenesis of HS and reemphasizes the extreme heterogeneity of the underlying molecular basis of this condition.

Blood group antigens Rb(a), Tr(a), and Wd(a) are located in the third ectoplasmic loop of erythroid band 3.

BACKGROUND: Rb(a), Tr(a), and Wd(a) are three low-incidence blood group antigens that have not been assigned to a particular structure of the red cell membrane. Recent genetic and serologic data suggested erythroid band 3 as a possible carrier of these three antigens. STUDY DESIGN AND METHODS: Ten band 3 gene exons that encode the membrane domain of band 3 were screened for single strand conformation polymorphism (SSCP). Exons displaying SSCP were cloned and sequenced, and the presence of the mutations was verified by restriction digestion. RESULTS: Substitutions 548 Pro-->Leu, 551 Lys-->Asn, and 557 Val-->Met, all located in the third ectoplasmic loop of band 3, were detected in the subjects with Rb(a+), Tr(a+), and Wd(a+) red cells, respectively. The presence of the Rb(a) and Wd(a) mutations was confirmed in additional carriers of these blood group antigens. Chymotryptic cleavage at Tyr 553 and Tyr 555 abolished the agglutinability of Tr(a+) and Wd(a+) cells with the corresponding antisera, further demonstrating that the epitopes are located in the third ectoplasmic loop of band 3. Similar quantities of mRNA corresponding of the two band 3 alleles, a normal pattern of red cell membrane proteins, and normal DIDS (4,4'-diisothiocyanatostilbene-2,2'-disulphonic acid, disodium salt)-inhibitable sulfate flux were detected, which suggests that the mutations do not affect band 3 mRNA stability or band 3 protein expression and transport function. CONCLUSION: Wd(a) and Rb(a), and tentatively Tr(a), can be assigned to the Diego blood group system.

A Thr552 -->Ile substitution in erythroid band 3 gives rise to the Warrior blood group antigen.

BACKGROUND: Recent family studies established that the low-incidence red cell antigen WARR is not part of the MNS, Lutheran, Lewis, Duffy, Kidd, Xg, Chido/ Rodgers, Kx, or Gerbich blood group systems. Continued serologic and genetic studies of WARR suggest that it is carried on erythroid band 3. STUDY DESIGN AND METHODS: To test the hypothesis that expression of WARR is controlled by the anion exchanger 1 gene (AE1), AE1 intronic primers that flank the exons encoding the membrane domain of band 3 were prepared. Polymerase chain reaction-amplified products corresponding to exons 11-20 of AE1 were analyzed for single-strand conformational polymorphism (SSCP) in DNA from WARR-positive and WARR-negative individuals. RESULTS: An SSCP was detected in exon 14. Subsequent sequencing revealed a C-->T mutation in codon 552 that leads to a Thr-->Ile substitution. Because the C-->T mutation eliminates a Bbs I restriction site, it was possible to confirm the phenotypes of all family members. To study the possible effect of the Thr552-->Ile substitution on the expression and function of band 3, polymerase chain reaction-amplified reverse-transcribed reticulocyte mRNA was digested with Bbs I. Both alleles of band 3 mRNA were detected in similar quantities, which suggests that the substitution underlying WARR did not interfere with mRNA stability. Comparison of sodium dodecyl sulfate-polyacrylamide gel electrophoretic mobility and size patterns revealed no difference between proteins isolated from WARR-positive and WARR-negative red cells. Further, the presence of WARR did not alter the di-isothiocyano-dihydrostilbene disulfonate (DIDS)-inhibitable influx of radiolabeled sulfate. CONCLUSION: Although it appears inconsequential to the function of band 3, the red cell polymorphism known as WARR is controlled by AE1. WARR should be therefore included in the Diego blood group system.

[Relation between types of erythrocyte membrane defects and the clinical picture in hereditary spherocytosis]. / Vztah mezi typem membránového defektu erytrocytu a klinickým obrazem u hereditární sférocytózy.

From total number of 130 patients with hereditary spherocytosis from 75 families in 119 patients from 69 families a defect of membrane proteins was detected. In 23 families (33.3%) a spectrin defect was involved, in 32 families (46.3%) a combined defect of spectrin and ancyrine and in 14 families (20.3%) a defect of band 3 proteins. Investigation of the membrane defect and the clinical and laboratory picture revealed that the band 3 protein defect of spectrin and ancyrine. There are significant differences in the clinical picture of the two latter defects.

Characterization of 13 novel band 3 gene defects in hereditary spherocytosis with band 3 deficiency.

Hereditary spherocytosis (HS) is a common hemolytic anemia of variable clinical expression. Pathogenesis of HS has been associated with defects of several red cell membrane proteins including erythroid band 3. We have studied erythrocyte membrane proteins in 166 families with autosomal dominant HS. We have detected relative deficiency of band 3 in 38 kindred (23%). Band 3 deficiency was invariably associated with mild autosomal dominant spherocytosis and with the presence of pincered red cells in the peripheral blood smears of unsplenectomized patients. We hypothesized that this phenotype is caused by band 3 gene defects. Therefore, we screened band 3 DNA from these 38 kindred for single strand conformational polymorphisms (SSCP). In addition to five mutations detected previously by SSCP screening of cDNA, we detected 13 new band 3 gene mutations in 14 kindred coinherited with HS. These novel mutations consisted of two distinct subsets. The first subset included seven nonsense and frameshift mutations that were all associated with the absence of the mutant mRNA allele from reticulocyte RNA, implicating decreased production and/or stability of mutant mRNA as the cause of decreased band 3 synthesis. The second group included five substitutions of highly conserved amino acids and one in-frame deletion. These six mutations were associated with the presence of comparable levels of normal and mutant band 3 mRNA. We suggest that these mutations interfere with band 3 biosynthesis leading thus to the decreased accumulation of the mutant band 3 allele in the plasma membrane.

Combination of two mutant alpha spectrin alleles underlies a severe spherocytic hemolytic anemia.

We studied a patient with a severe spherocytic hemolytic anemia without family history of spherocytosis. Analysis of patient's erythrocyte membrane proteins revealed spectrin deficiency and a truncated alpha spectrin protein. We determined that the patient is a compound heterozygote with two mutations in alpha spectrin gene. Mutation in the paternal allele, designated alpha spectrin(PRAGUE), is a transition A to G in the penultimate position of intron 36 that leads to skipping of exon 37, frameshift, and production of the truncated alpha spectrin protein. The maternal allele, designated alpha spectrin(LEPRA), contains transition C-->T in position -99 of intron 30. This mutation enhances an alternative acceptor splice site 70 nucleotides upstream from the regular site. The alternative splicing causes a frameshift and premature termination of translation leading to a significant decrease in alpha spectrin production. The alpha(LEPRA) mutation is linked to a spectrin alphaIIa marker that was found to be associated with recessive or nondominant spectrin-deficient hereditary spherocytosis in approximately 50% of studied families. We conclude that the alpha(LEPRA) mutation combined in trans with the alpha(PRAGUE) mutation underlie the severe hemolytic anemia in the proband. We suggest that allele alpha spectrin(LEPRA) may be frequently involved in pathogenesis of recessive or nondominant spectrin-deficient hereditary spherocytosis.

Overexpression of AE1 Prague, but not of AE1 SAO, inhibits wild-type AE1 trafficking in Xenopus oocytes.

Mutations in the AE1 (band 3) anion exchanger of human erythrocytes have been associated with altered red cell shape and heritable disease. The Southeast Asian Ovalocytosis (SAO) AE1 mutation, a 27 nt deletion producing the delta 400-408 form of AE1, and the AE1 Prague mutation, a 10 nt insertion producing a frameshift after AE1 aa 821 leading to premature termination, are found only in the heterozygous state. We therefore examined accumulation and function of wt AE1 polypeptide in Xenopus oocytes when coexpressed with AE1 SAO and with AE1 Prague. Our SAO construct lacked the K56E (AE1 Memphis) polymorphism present in the endogenous AE1 SAO protein. Neither mutant AE1 mediated Cl- uptake into cRNA-injected Xenopus oocytes. Coinjection of mutant and wt cRNAs led to dose-dependent inhibition of wt function by AE1 Prague, but not by SAO. Though in vitro translation of the two mutants revealed little difference in their insertion into microsomal membranes, AE1 Prague accumulated in Xenopus oocytes to lower levels than did AE1 SAO or wt. Unlike AE1 SAO polypeptide, AE1 Prague polypeptide was not detectable at the oocyte surface. Moreover, overexpression of AE1 Prague, in contrast to AE1 SAO, reduced the accumulation of wt AE1, at the oocyte surface. This inhibition occurred in the absence of detectable heteromer formation between the AE1 Prague and wt AE1 polypeptides.