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J. Biol. Chem., Vol. 276, Issue 45, 41683-41689, November 9, 2001

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Erythrocyte Ankyrin Promoter Mutations Associated with Recessive Hereditary Spherocytosis Cause Significant Abnormalities in Ankyrin Expression^*

Patrick G. Gallagher^§, Denise E. Sabatino^¶, Daniela S. Basseres^**, Douglas M. Nilson^¶, Clara Wong, Amanda P. Cline^¶, Lisa J. Garrett^¶, and David M. Bodine^¶

From the  Department of Pediatrics, Yale University School of Medicine, New Haven, Connecticut 06520, the ^¶ Hematopoiesis Section, Genetics and Molecular Biology Branch, NHGRI, National Institutes of Health, Bethesda, Maryland 20892, and ^** Hemocentro, University of Campinas, Campinas, Brazil

Received for publication, June 22, 2001, and in revised form, August 7, 2001

	ABSTRACT

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

Ankyrin defects are the most common cause of hereditary spherocytosis (HS). In several kindreds with recessive, ankyrin-deficient HS, mutations have been identified in the ankyrin promoter that have been proposed to decrease ankyrin synthesis. We analyzed the effects of two mutations, 108T to C and 108T to C in cis with 153G to A, on ankyrin expression. No difference between wild type and mutant promoters was demonstrated in transfection or gel shift assays in vitro. Transgenic mice with a wild type ankyrin promoter linked to a human ^A-globin gene expressed -globin in 100% of erythrocytes in a copy number-dependent, position-independent manner. Transgenic mice with the mutant 108 promoter demonstrated variegated -globin expression, but showed copy number-dependent and position-independent expression similar to wild type. Severe effects in ankyrin expression were seen in mice with the linked 108/153 mutations. Three transgenic lines had undetectable levels of ^A-globin mRNA, indicating position-dependent expression, and four lines expressed significantly lower levels of ^A-globin mRNA than wild type. Two of four expressing lines showed variegated -globin expression, and there was no correlation between transgene copy number and RNA level, indicating copy number-independent expression. These data are the first demonstration of functional defects caused by HS-related, ankyrin gene promoter mutations.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

Hereditary spherocytosis (HS)¹ is a common hemolytic anemia characterized by the presence of spherically shaped erythrocytes on peripheral blood smear. The principal cellular defect in HS is loss of erythrocyte membrane surface area relative to intracellular volume, accounting for the spherical shape as well as decreased deformability of the erythrocyte (1). The primary biochemical defects in HS reside in the proteins of the erythrocyte membrane, particularly those proteins involved in the interactions between the membrane skeleton and the lipid bilayer: ankyrin,  and  spectrin, band 3, and protein 4.2 (2, 3). In two thirds to three quarters of cases, HS is inherited in an autosomal dominant fashion (1-4). In the remaining patients, HS is inherited in a recessive fashion or is the result of a de novo mutation.

Ankyrin-1 (ANK1, Mendelian Inheritance in Man 182900) deficiency is one of the most common abnormalities found in the erythrocyte membranes of HS patients (5, 6). First identified in preparations of erythrocyte membranes, ankyrin provides the primary linkage between the spectrin-actin-based erythrocyte membrane skeleton and the plasma membrane by attaching tetramers of spectrin to the cytoplasmic domain of band 3 (7, 8). Studies have revealed that abnormalities of the ankyrin gene, primarily frameshift or nonsense mutations, are the most common cause of typical, dominant HS (3, 9-11).

Ankyrin-1 is transcribed in erythroid cells from a compact, erythroid-specific promoter (12). One molecular mechanism that could lead to ankyrin deficiency is a mutation in the ankyrin erythroid promoter leading to decreased ankyrin synthesis. Several reports have identified sequence variations in the ankyrin gene promoter in individuals with recessively inherited, ankyrin-deficient HS (4, 9, 11). Whether these are disease causing mutations or are merely polymorphisms in linkage disequilibrium with as yet unidentified mutations is unknown, as it is difficult to assess the effect of promoter mutations on ankyrin gene expression in affected HS patients.

To address this question, we have analyzed the effect of two HS-associated, ankyrin gene promoter mutations in vitro and in vivo. The first mutation, a T to C substitution at position 108, was discovered in the heterozygous state in four of seven German families with ankyrin-deficient, recessive HS (9). In two of the four kindreds, mutations in the coding region of the ankyrin gene were discovered in trans, and the mutation was silent in the heterozygous state. In this mostly German HS population, the allele frequency was estimated to be 29% in HS patients and 2.0% in normal individuals. This mutation has also been associated with ankyrin-deficient, nondominantly inherited HS in Italy (4). The second mutation, a G to A substitution at position 153, was discovered in the heterozygous state in a Brazilian kindred with ankyrin-deficient, recessive HS (11). The 153 substitution was always found in cis to the previously reported 108T to C ankyrin promoter mutation. These linked substitutions were silent in the heterozygous state. No individuals with the 153 mutation alone were detected. In a control Brazilian population, the allelic frequency of the 108/153 allele was 2.4% and the 108 allele was 1.4%.

Previously, we have shown the wild type ankyrin promoter directed expression of a linked ^A-globin reporter gene in an erythroid-specific, position-independent, copy number-dependent fashion in transgenic mice (13). Levels of ^A-globin mRNA correlated directly with levels of functional -globin protein in erythrocytes of these transgenic mice (14). We used this ankyrin promoter/human ^A-globin reporter transgenic mouse model as a system to study the effect of the spherocytosis-associated promoter mutations in vivo. Significant abnormalities in reporter gene mRNA and protein expression were seen, the first demonstration of functional defects caused by HS-related ankyrin gene promoter mutations. These promoter mutations may represent a common pathogenetic mechanism of recessive HS in ankyrin-deficient patients.

	MATERIALS AND METHODS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

Mutations-- The mutations described in this report have been reported previously and are schematically shown in Fig. 1 (4, 9, 11).

View larger version (20K): [in this window] [in a new window]   Fig. 1.   The human ankyrin-1 erythroid promoter. The top line shows a map of the minimal ankyrin promoter with the positions of critical CACCC-, CGCC-, and GATA-binding sites. The locations of mutations associated with hereditary spherocytosis and a control used in experimental studies are shown in the lower lines.

Cell Culture-- The tissue culture cell lines K562 (chronic myelogenous leukemia in blast crisis with erythroid characteristics, ATCC CCl243) and HEL (human erythroleukemia, ATCC TIB 180) were used to study expression of the mutant ankyrin promoters. Cells were maintained in RPMI 1640 medium containing 10% fetal calf serum.

Preparation of Mutant Promoter-Reporter Plasmids for Transfection Assays-- We have shown previously that a 286-bp minimal ankyrin gene promoter fragment directs high level expression of a luciferase reporter gene in erythroid cells (plasmid p296 in Ref. 12). Mutant ankyrin promoter fragments corresponding to this 286-bp promoter fragment were generated by PCR amplification and subcloning into the firefly luciferase reporter plasmid pGL2B (Promega Corp., Madison, WI). Test plasmids were sequenced to exclude cloning or PCR-generated artifacts.

Transient Transfection Analyses-- All plasmids tested were purified using Qiagen columns (Qiagen, Inc., Chatsworth, CA) or cesium chloride plasmid purification, and at least two preparations of each plasmid were tested in triplicate. 10⁷ K562 or HEL cells were transfected by electroporation with a single pulse of 300 V at 960 microfarads with 20 µg of test plasmid and 0.5 µg of pCMV, a mammalian reporter plasmid expressing -galactosidase driven by the human cytomegalovirus immediate early gene promoter (CLONTECH, Palo Alto, CA) to normalize for transfection efficiency. Twenty-four hours after transfection, cells were harvested and lysed, and the activity of both luciferase and -galactosidase activity determined in cell extracts. All assays were performed in triplicate.

Preparation of Nuclear Extracts and Gel Mobility Shift Analyses-- Nuclear extracts were prepared by hypotonic lysis followed by high salt extraction of nuclei as described by Andrews and Faller (15). Oligonucleotide primers used in gel mobility shift assays are shown in Table I. Gel mobility shift binding reactions were carried out as described (16, 17). Competitor oligonucleotides were added at molar excesses of 100-fold. Resulting complexes were separated by electrophoresis through 6% polyacrylamide gels in 0.5× Tris borate-EDTA at 21 °C at 200 watts for 2 h. Gels were dried and subjected to autoradiography.

Preparation of Mutant Promoter-Reporter Plasmids for Transgenic Mice-- To generate mutant Ank/^A-globin transgenes, 276-bp SmaI/BglII fragments containing mutant ankyrin promoters (291 to 20 plus polylinker sequence) were excised from the pGL2B luciferase reporter vector (see above). A 1938-bp BglII/HindIII ^A-globin fragment was excised from plasmid 72sp+^A (described in Sabatino et al. in Ref 18). Triple ligations consisting of the ankyrin promoter, the ^A-globin gene, and SmaI/HindIII-digested pSP72 were used to generate the mutant Ank/^A-globin plasmids. The ankyrin/^A-globin gene fragments (2244 bp) were excised from the plasmids with EcoRV and HindIII for microinjection (Fig. 2A). All plasmid constructs were sequenced to confirm that the appropriate mutant ankyrin promoter was correctly fused to the ^A-globin gene.

Generation of Transgenic Mice-- Transgenic mice were generated as described by Hogan et al. (19) and Sabatino et al. (18). Fertilized eggs were collected from superovulated FVB/N female mice (Taconic Farms, Germantown, NY) ~9 h after mating to CBy6F1 male mice (Jackson Laboratory, Bar Harbor, ME). Fragments for microinjection were separated on an agarose gel, electroeluted, and purified with an Elutip-d minicolumn (Schleicher & Schuell). The fragments were diluted to a concentration of 2 ng/µl in 10 mM Tris, 0.25 mM EDTA (pH 7.5), and ~ 2 pl was injected into the male pronucleus of fertilized eggs. The injected eggs were transferred to psuedopregnant CByB6/F1 foster mothers. Founder animals were identified by Southern analysis of DNA extracted from tail biopsies by probing with an Ank/^A-globin probe. Founder animals were crossed to FVB/N mice for propagation.

Transgene Copy Number Analysis-- Copy number was determined by comparing the -globin signals from Southern blot analysis of transgenic mouse and K562 DNA using a Molecular Dynamics PhosphorImager. Statistical analysis of copy number and expression data was analyzed by linear regression using GraphPad Prism^® version 2.0 software.

Isolation of RNA-- Total cellular RNA was extracted from adult reticulocytes, obtained by collecting 200 µl of blood from phlebotomized animals, using TRIZOL reagent according to the manufacturer's specifications (Life Technologies, Inc.).

RNase Protection Assays-- Linear DNA templates for RNase protection assays were prepared by restriction enzyme digestion of cesium chloride purified plasmid preparations. Riboprobe one, which contains sequences for both exon 2 of the human ^A-globin gene and exon 2 of the murine -globin gene, was linearized with BglII (Fig. 2B). This riboprobe ensures that both the human ^A-globin and murine -globin sequences are labeled to equal specific activity, allowing direct comparison of human ^A-globin and murine -globin mRNA levels. Riboprobe two, which includes part of ^A-globin exon 2, all of exon 1, and the ankyrin transcription initiation site, was linearized with EcoRI (Fig. 2C) (18). This riboprobe allows detection of the transcription initiation site directed by the mutant ankyrin promoters. Templates were purified by agarose gel electrophoresis and purified using a Geneclean^® II kit (Bio 101, Inc.). Linear DNA template for the mouse -actin gene was obtained from the MAXIscript^TM in vitro transcription kit (Ambion, Inc.). ³²P-Labeled RNA probes were transcribed using the MAXIscript^TM in vitro transcription kit (Ambion, Inc.). Hybridization of the probe and RNA (0.1-0.25 µg) was carried out overnight according to standard procedures (RPA II, Ambion, Inc.). RNase digestion was performed using an RNase A/RNase T1 mixture in RNase digestion buffer and the protected fragments separated on an 8% nondenaturing polyacrylamide gel.

View larger version (21K): [in this window] [in a new window]   Fig. 2.   Human ankyrin promoter/A-globin transgene constructs and riboprobes. A, human ankyrin promoter/A-globin transgenes. Wild type or mutant human ankyrin promoter fragments (296 to 20) were fused to the human A-globin gene (4 to +1902) to create the transgene construct shown. Although the 153G to A mutation has not been described without the 108T to C mutation in cis, the corresponding transgenic construct was prepared as an experimental control. B, riboprobe one, a hybrid human A-globin/mouse -globin riboprobe. An Sp6 riboprobe containing sequences for both exon 2 of the human A-globin gene and exon 2 of the murine -globin gene was prepared to ensure that both the human A-globin and murine -globin sequences are labeled to equal specific activity in ribonuclease protection assays. C, riboprobe two. A second riboprobe transcribed by Sp6 that protects part of exon 2 and all of exon 1 of the human A-globin reporter gene as well as the transcription initiation site of the transgene directed by the promoter region of ankyrin.

Quantitation of mRNA Levels-- To quantitate the levels of mRNA, the gel was exposed to a PhosphorImager screen and scanned on a Molecular Dynamics PhosphorImager (Amersham Pharmacia Biotech). The relative amounts of the bands human ^A-globin exon 2 (223 bp) and mouse -globin exon 2 (186 bp) were estimated by the following formula: (^A-globin RNA/transgene copy number) × (1/mouse -globin RNA).

Expression of Human -Globin Protein in the Erythrocytes of Transgenic Mice-- Detection and measurement of -globin protein in red blood cells was performed as described by Thorpe et al. (20). Blood cells collected by phlebotomy from the retro-orbital sinus of transgenic mice were washed in cold (4 °C) phosphate-buffered saline and then fixed in ice-cold (4 °C) 4% paraformaldehyde solution. The cells were washed with 1:1 acetone/water (20 °C), acetone (20 °C), and 1:1 acetone/water (20 °C) before resuspension in phosphate-buffered saline plus 2% fetal bovine serum (4 °C). Hemoglobin tetramers containing human -globin were identified with a fluorescein isothiocyanate-conjugated human hemoglobin F antibody (PerkinElmer Life Sciences). Analysis was performed on a FACStar instrument (Becton Dickinson, Franklin Lakes, NJ). To prevent leaching of hemoglobin, cells were maintained at 4 °C or lower throughout the procedure.

Computer Analyses-- Computer-assisted analyses of mutant ankyrin promoter nucleotide sequences were performed utilizing the sequence analysis software package of the University of Wisconsin Genetics Computer Group (Madison, WI) (21) and the BLAST algorithm (National Center for Biotechnology Information, Bethesda, MD) (22).

	RESULTS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

Computer Analyses-- Our previous in vitro studies showed that expression of a minimal human ankyrin-1 promoter in erythroid cells was mediated by GATA-1-, CACCC-, and CGCCC-binding proteins (12). Inspection of the ankyrin promoter sequence reveals that neither the 108 nor 153 mutations are located in these binding sites, nor are they located in regions protected during in vitro DNase I footprinting experiments using erythroid nuclear extracts (12). The 108 mutation disrupts a potential AP-2 binding site; it does not create a novel site for any known DNA-binding proteins. The 153 mutation is not contained in a known DNA binding protein site, nor does it create a site for any known DNA-binding proteins.

Transient Transfection Analyses-- Plasmids containing wild type and mutant 108 and 108/153 ankyrin promoter fragments linked to a firefly luciferase reporter gene transiently transfected into K562 and HEL cells and luciferase expression assayed. There was no significant difference in luciferase activity directed by wild type or mutant plasmids in either K562 or HEL cells (Fig. 3).

View larger version (17K): [in this window] [in a new window]   Fig. 3.   Activity of mutant ankyrin gene promoters in erythroid cells in transfection assays. Wild type or mutant ankyrin promoter fragments generated by PCR amplification were subcloned upstream of a firefly luciferase reporter gene. These mutant ankyrin promoter/luciferase reporter plasmids were transiently transfected into K562 or HEL cells and relative luciferase activity determined as described. The data are means ± S.D. of at least six independent transfection experiments. A, K562 cells. B, HEL cells.

Electrophoretic Mobility Shift Assays-- Oligonucleotides containing the wild type and mutant ankyrin promoter sequences were synthesized, and protein binding activity was analyzed in mobility shift assays using K562 cell nuclear extracts. Wild type and mutant 108 oligonucleotides formed complexes that migrated at the same mobility as a control AP-2 oligonucleotide. In competition assays, the 108 mutant oligonucleotide competed away both the corresponding wild type ankyrin and control AP-2 complexes (data not shown). Neither wild type nor mutant 153 oligonucleotides formed complexes with K562 extracts (data not shown).

Transgenic Mice with Human Ankyrin Promoter/^A-Globin Reporter Genes-- Previously, we have shown the wild type ankyrin promoter-directed expression of a linked ^A-globin reporter gene in an erythroid-specific, position-independent, copy number-dependent (p = 0.004) fashion in transgenic mice (18). We used this ankyrin promoter/human ^A-globin reporter transgenic mouse model as a system to study the effect of the spherocytosis-associated promoter mutations in vivo. To dissect the effect of the 108 mutation from the linked 153 mutation, we created mutant ankyrin promoter transgenes with the 108 mutation, the linked 108 and 153 mutations, and an experimental control with the 153 mutation (Fig. 1), even though the 153 mutation has not been reported solely without the 108 mutation.

Wild type or mutant minimal ankyrin-1 promoter fragments were fused to the human ^A-globin sequence immediately upstream of the ATG initiation codon (Ank WT, 108, 108/153, or 153, respectively/^A-globin; Figs. 1 and 2). Sixteen wild type, eight 108, seven 108/153, and eight 153 transgenic mouse lines were generated. Southern blot analysis determined that the transgene copy number in these mice ranged between 1 and 15 (Table II).

View this table: [in this window] [in a new window]   Table II Expression of ankyrin/A-globin mRNA in transgenic mice

^A-Globin Gene Expression in Transgenic Animals-- The levels of Ank/^A-globin mRNA and endogenous murine -globin mRNA in reticulocytes were analyzed by ribonuclease protection. Riboprobe one (Fig. 2), which ensures that both the human ^A-globin and murine -globin sequences are labeled to equal specific activity, allowing comparison of human ^A-globin and murine -globin mRNA levels, was used. Wild type mice demonstrated position-independent ^A-globin gene expression in all 16 lines (Fig. 4 and Table II). Transgenic mice with the 108 mutation demonstrated position-independent expression (eight of eight transgenic lines expressed -globin with Ank/^A-globin mRNA levels similar to wild type, p = 0.6533). Severe effects on Ank/^A-globin mRNA levels were seen in seven lines of mice with the linked 108/153 mutations (Fig. 5 and Table II). Three lines had undetectable levels of Ank/^A-globin mRNA, indicating position-dependent expression, and in four lines the level of Ank/^A-globin mRNA was significantly lower than wild type (108/153 mean 0.006 ± 0.002% versus wild type 0.041 ± 0.004% ^A-globin RNA/copy number, p < 0.0001). Transgenic mice with the 153 mutation demonstrated position-independent expression (8 of 8 transgenic lines expressed -globin with Ank/^A-globin mRNA levels similar to wild type, p = 0.5907).

View larger version (70K): [in this window] [in a new window]   Fig. 4.   Detection of Ank/A-globin mRNA in reticulocytes of transgenic mice. 0.1 µg of RNA from adult reticulocytes was hybridized to 32P-labeled antisense riboprobe one, which protects exon 2 of the Ank/A-globin transgene (top band) and exon 2 of the mouse -globin gene (lower band), and digested with RNase. The numbers indicate transgene copy number. Samples from individual strains are shown from left to right in alphabetical order, similar to Table II. A, wild type ankyrin promoter transgenic mice. B, 108T to C mutant ankyrin promoter transgenic mice. C, experimental mutant promoter control, 153G to A, transgenic mice. D, linked 108T to C/153G to A mutant promoter transgenic mice.

View larger version (26K): [in this window] [in a new window]   Fig. 5.   Correlation of transgene copy number with the levels of Ank/A-globin mRNA. Linear regression analysis of the transgene copy number with the corrected mRNA expression level was performed. A, there is a linear relationship in wild type mice, indicating copy number-dependent expression. Correlation of corrected mRNA expression versus copy number in 108/153 mutant transgenic mice (r2 = 0.1505, p = not significant) demonstrates complete loss of copy number-dependent expression. B and C, expression is nearly identical to wild type in 108 (r2 = 0.6533, p = 0.0028) and 153 (r2 = 0.8755, p = 0.0006) transgenic mice.

Previously, we have shown that the ankyrin promoter/^A-globin transgene initiates at position 81 in the corresponding ankyrin promoter/5'-flanking genomic DNA sequence (12, 18). To ensure that transcription was properly initiated in transgenic mice with mutant ankyrin promoters, we analyzed ^A-globin mRNA expression in reticulocyte RNA using riboprobe two (Fig. 2). All three mutant promoter transgenes initiated transcription at the same location as wild type (data not shown).

Copy Number Correlation with mRNA Levels-- Transgenic mice with the wild type ankyrin promoter expressed the ^A-globin transgene in a copy number-dependent fashion (copy number correlation with mRNA level r² = 0.5890, p = 0.0005 for all 16 strains, linear relationship, Table II and Fig. 5). Reporter gene expression relative to copy number in the 108 mice was similar to wild type, i.e. copy number-dependent (copy number correlation with mRNA level r² = 0.7991, p = 0.0028, linear relationship). In the four 108/153 mutant-expressing mice, there was no correlation between transgene copy number and mRNA level, indicating copy number-independent expression (copy number correlation with mRNA level r² = 0.1505, p = not significant; Table II and Fig. 5). The 153 experimental control mice expressed the ^A-globin reporter gene relative to copy number in a copy number-dependent fashion (copy number correlation with mRNA level r² = 0.8755, p = 0.0006, linear relationship).

Distribution of Human ^A-Globin Protein in Erythrocytes of Transgenic Mice-- To determine whether human ^A-globin protein was present in the red cells of transgenic animals, we used an anti-human -globin monoclonal antibody for fluorescence-activated cell sorting analyses. Transgenic mice with the wild type promoter expressed human -globin in a uniform pattern, i.e. in 100% of erythrocytes (Table III, Fig. 6). Similar analyses demonstrated different results in the mutant promoter transgenic lines (Table III, Fig. 6). -Globin expression was variegated in all eight lines of 108 mice, with between 10 and 80% of erythrocytes expressing -globin. In two of the four expressing transgenic lines with the 108/153 mutation and in five of eight 153 mutant control lines, -globin gene expression was variegated (Table III, Fig. 6).

View this table: [in this window] [in a new window]   Table III -Globin protein expression in erythrocytes of transgenic mice

View larger version (50K): [in this window] [in a new window]   Fig. 6.   Expression of human -globin protein in erythrocytes of transgenic mice. Fluorescence intensity from a fluorescein isothiocyanate-conjugated monoclonal antibody against human -globin was correlated with the number of erythrocytes counted. In each panel, the thick line represents a transgenic mouse and the thin line represents a nontransgenic, littermate control. Dual peaks represent variegated expression of human -globin. a, top panels show control and wild type ankyrin promoter/A-globin transgenic mice. The left panel shows results from a positive control mouse with uniform (i.e. 100% of erythrocytes) -globin erythrocyte expression. The three other top panels (D, Q, and C), demonstrate uniform expression in transgenic mice with the wild type ankyrin promoter. a, bottom panel, 108 transgenic mice. -Globin expression was variegated in eight of eight transgenic lines, with 10-80% of erythrocytes containing -globin. b, top panel, 153 transgenic Mice. Variegated -globin expression was seen in five of eight transgenic lines, with 50-60% of erythrocytes containing -globin. b, bottom panel, 108/153 transgenic mice. -Globin expression was variegated in two of four expressing transgenic lines (B and C), with 50-80% of erythrocytes containing -globin. Panel E is an example of a nonexpressing line.

	DISCUSSION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

Nondominant inheritance is the term assigned to patients with clinical and laboratory features of HS whose parents do not demonstrate clinical features of the disease. Nondominant inheritance is relatively common, seen in one quarter to one third of HS patients (1-4). In these cases, the underlying genetic defects are heterogeneous. In some cases, true recessive inheritance as a result of abnormalities in either  spectrin or protein 4.2 has been suspected (1-3, 23-28), but precise genetic defects have been identified in only a few (1, 27, 28). In other cases, a de novo dominant mutation in ankyrin or -spectrin has been identified (29-33). However, in most cases, the underlying genetic defect is unknown (4, 9, 11, 23-25, 28). These cases present a challenging and unsolved problem in genetic counseling.

Large genetic screens of ankyrin-deficient HS patients identified the ankyrin promoter mutations studied in this report (4, 9, 11). In several of these cases, additional mutations in the coding region were found in the ankyrin gene in trans to the allele with the promoter mutation (9, 11). As the inheritance is recessive, it is anticipated that all patients heterozygous for ankyrin gene promoter mutations have an additional ankyrin gene mutation in trans. These promoter mutations are common in the general population, with a frequency of ~2-3% (9, 11). Thus, the inheritance of one of these promoter mutations could unmask other ankyrin gene mutations, making this a relatively common cause of recessive HS. It is interesting to note that osmotic fragility screening of random blood donors identified an HS "carrier state" in slightly more than 1% of donors (34, 35). It has been suggested that these donors were potential parents of patients with recessively inherited HS (4). It is tempting to speculate that some of these donors may be heterozygous for an ankyrin promoter mutation.

We attempted to determine the mechanism(s) by which these mutations exert their influence on ankyrin gene expression. The transgenes tested in these assays initiated mRNA transcription at the correct site, suggesting that a defect in transcript initiation is unlikely. The most severe effects on Ank/^A-globin mRNA and protein expression were seen in mice with the linked 108/153 promoter mutations who demonstrated significantly decreased -globin mRNA in a position-dependent, copy number-independent manner, as well as variegated distribution of -globin protein in erythrocytes. The 108 mutant promoter transgenic mice demonstrated differences only in the cellular distribution of the -globin protein. These are interesting observations, as no differences between these promoter mutations and wild type promoter were seen in in vitro studies. Together, these data suggest that intact chromatin is necessary to manifest the promoter defects.

The 153 mutation does not create or abolish consensus sequences for any known DNA-binding proteins and although the 108 mutation occurs in an AP-2 consensus binding sequence, in vitro studies suggest that this is not a functionally important site in wild type ankyrin gene expression (Ref. 12 and this report). It is possible that a previously undescribed DNA-binding protein interacts with either or both of these sites.

The upstream 153 mutation is located in a stretch of CpG dinucleotides, a region of potential DNA methylation. DNA methylation controls gene expression by modulating access of regulatory elements to a gene promoter. There is a second ankyrin-1 gene promoter, active in neural and muscle cells, located 40 kilobase pairs upstream of the erythroid promoter (36). It is possible that, in wild type cells in vivo, erythroid cell-specific methylation in the region of the 153 mutation prevents binding of a protein with negative regulatory activity or one with enhancer-blocking activity that creates a boundary element (i.e. an "insulator"). Mutation and disruption of methylation in this region could then facilitate DNA-protein binding. The latter has recently been shown at the H19/Igf2 locus, where methylation mediates the binding and enhancer blocking activity of the multifunctional zinc finger protein CTCF (37-39). Additional evidence for this hypothesis comes from the observations that removal of a long range insulator or boundary element from a region can shut down the locus and lead to position effects (40). These phenomena, i.e. decreased gene expression and position-dependent expression, were observed in the mutant 153/108 mice. Future studies will provide insight into how these mutations affect regulation of the ankyrin gene.

	FOOTNOTES

^* This work was supported in part by grants from the NHLBI, National Institutes of Health (to P. G. G.) and the March of Dimes Birth Defects Foundation (to P. G. G.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

^§ To whom correspondence should be addressed: Dept. of Pediatrics, Yale University School of Medicine, 333 Cedar St., P. O. Box 208064, New Haven, CT 06520-8064. Tel.: 203-688-2896; Fax: 203-785-6974; E-mail: patrick.gallagher@yale.edu.

Current address: Div. of Hematology, Dept. of Pediatrics, Children's Hospital, Philadelphia, PA 19104.

Published, JBC Papers in Press, August 29, 2001, DOI 10.1074/jbc.M105844200

	ABBREVIATIONS

The abbreviations used are: HS, hereditary spherocytosis; PCR, polymerase chain reaction; bp, base pair(s); Ank, ankyrin.

	REFERENCES

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

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