Download Mutations in EKLF/KLF1 form the molecular basis of

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Blood First Edition Paper, prepublished online May 16, 2008; DOI 10.1182/blood-2008-03-145672
Mutations in EKLF/KLF1 form the molecular basis of the rare blood
group In(Lu) phenotype.
Belinda K. Singleton1, Nicholas M. Burton2, Carole Green1, R. Leo Brady2, David J. Anstee1.
1
Bristol Institute for Transfusion Sciences (BITS), National Blood Service, Bristol, United
Kingdom; and 2Department of Biochemistry, University of Bristol, Bristol, United Kingdom
Corresponding author: Belinda K. Singleton, Bristol Institute for Transfusion Sciences, Southmead
Road, Bristol, BS10 5ND, United Kingdom.
E-mail: [email protected]
Telephone: +44 117 9912126
Fax: +44 117 9591660
Keywords: Transcription factor, erythropoiesis, Lutheran Blood Group, transcriptomics,
EKLF
Copyright © 2008 American Society of Hematology
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Abstract
Comparison of normal erythroblasts and erythroblasts from individuals with the rare In(Lu) type of
Lu(a-b-) blood group phenotype revealed increased transcription levels for 314 genes and reduced
levels for 354 genes in In(Lu) cells. Many erythroid-specific genes (including ALAS2, SLC4A1) had
reduced transcript levels suggesting the phenotype resulted from a transcription factor abnormality.
A search for mutations in erythroid transcription factors revealed mutations in the promoter or
coding sequence of EKLF in 21 of 24 individuals with the In(Lu) phenotype. In all cases the mutant
EKLF allele occurred in the presence of a normal EKLF allele. Nine different loss of function
mutations were identified. One mutation abolished a GATA1 binding site in the EKLF promoter (124T>C). Two mutations (Leu127X; Lys292X) resulted in premature termination codons, two
(Pro190LeufsX47; Arg319GlufsX34) in frameshifts and four in amino acid substitution of
conserved residues in zinc finger domain 1 (His299Tyr) or domain 2 (Arg328Leu; Arg328His;
Arg331Gly). Individuals with the In(Lu) phenotype have no reported pathology indicating that one
functional EKLF allele is sufficient to sustain human erythropoiesis. These data provide the first
description of inactivating mutations in human EKLF and the first demonstration of a blood group
phenotype resulting from mutations in a transcription factor.
Introduction
Erythroid differentiation is a highly regulated process involving numerous transcription factors
including GATA1 and EKLF.1-3 GATA1 binds to EKLF to form a functional unit.4,5 Absence of
GATA1 in mice causes embryonic death from severe anemia due to maturation arrest and apoptosis
of erythroid progenitor cells.6 In mice, EKLF is not required for the proliferation of erythroid
progenitors but is essential for terminal differentiation of erythroid cells because it activates
essential erythroid genes.7,8 Mice lacking EKLF die in utero around embryonic day 14 from severe
anemia associated with beta globin deficiency whereas heterozygous EKLF +/- mice develop
normally.9 These data suggest homozygous inheritance of inactivating mutations affecting EKLF
expression in man would be incompatible with post-embryonic life although heterozygous
inheritance of inactivating EKLF mutations would be viable. Nevertheless, inactivating mutations in
human EKLF have not been previously reported.
The blood group phenotype Lu(a-b-) is known to arise from three distinct genetic backgrounds:
homozygosity for inactivating mutations in the Lutheran gene,10,11 inheritance of an X-linked
suppressor gene (XS2)12 and inheritance of an autosomal suppressor gene InLu.13 Inheritance of XS2
has been reported in only one family.12 In contrast, inheritance of InLu is relatively common,
occurring with a frequency of about 1 in 3,000 blood donors in South-East England14 and 1 in 5,000
of South Wales blood donors.15 In order to elucidate the molecular mechanism giving rise to the
In(Lu) type of Lu(a-b-) we compared the transcriptome of normal and In(Lu) erythroblasts at
different stages of maturation and hypothesised that the differences observed could result from a
mutated erythroid transcription factor. Sequencing of several erythroid transcription factor genes
revealed heterozygous inheritance of loss of function mutations in EKLF in 21 of 24 In(Lu) donors.
This report identifies the first mutations in human EKLF, explores their likely molecular
consequences and demonstrates the viability of the EKLF+/- genotype in man.
Materials and Methods
Cell culture and flow cytometry
Waste buffy coat material from anonymous blood donors was made available from the National
Blood Service (Tooting, United Kingdom) and the Welsh Blood Service (Cardiff, United
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Kingdom). This provision complies with the Nuffield Council on Bioethics Guidance on Human
Tissue Ethical and Legal Issues 1954, the Medical Research Council's Operational and Ethical
Guidance on Human Tissue and Biological Samples for use in Research 2005, and the Royal
College of Pathologists Transitional Guidelines to facilitate changes in procedures for handling
"surplus" and archival material from human biological samples. Informed consent was provided
according to the Declaration of Helsinki.
Waste buffy coat material was provided with informed consent from four blood donors with the
In(Lu) phenotype (InLu1-4) and four controls (Con1-4) matched for age, sex and ethnicity by the
Welsh Blood Service, Cardiff and the National Blood Service, Tooting, and anonymised for the
purposes of this study. CD34+ cells were isolated by positive selection with the MiniMACS
magnetic bead system according to the manufacturer’s instructions (Miltenyi Biotec, Bisley, UK).
Erythroid progenitor cells were obtained by ex vivo culture of CD34+ cells, as described in Fricke et
al.16 In(Lu) and matched control cells were grown in parallel. Cells were harvested during culture
for analysis by flow cytometry and for freezing and storage at -70oC in RNAlater (Ambion,
Huntingdon, UK). Cells from an additional In(Lu) sample (InLu5) and an unmatched control
(Con5) had previously been cultured and stored in RNAlater.
Cultured erythroid progenitor cells were analysed by flow cytometry after labelling with mouse
monoclonal antibodies: anti-RhAG (LA1818), anti-GPA (BRIC256), anti-Band 3 (BRIC6) and a
blend of anti-Lub (BRIC108) and anti-Lu (BRIC221), detecting epitopes on domain 1 and domain 4
of the Lutheran glycoprotein respectively. All BRIC antibodies were provided by the International
Blood Group Reference Laboratory (IBGRL, Bristol, UK). Isotype controls (Dako, Ely, UK) were
tested in parallel. Bound antibody was detected by adding RPE-conjugated F(ab′)2 anti-mouse
globulin (Dako) and the geometric mean (FL2) was recorded for each test. Flow cytometry was
performed on a FC500 flow cytometer and analysed with CXP software (Beckman Coulter, High
Wycombe, UK).
RNA isolationf
A minimum of 1.5 x106 erythroid cells were harvested every few days from day 4 to day 14 of
culture and frozen in RNALater (Ambion). Total RNA was isolated using the RNeasy Mini Kit
(Qiagen, Crawley, UK), including an on-column DNaseI digestion, according to the manufacturer’s
instructions. RNA yield was determined using the RiboGreen RNA Quantitation Kit (Molecular
Probes, Invitrogen Ltd, Paisley, UK).
Transcriptomics
Two In(Lu) samples (InLu3 and InLu4) and their matched controls (Con3 and Con4) were analysed
by microarrays using cells from day 6 and day 11 of culture. Cells from day 4 from InLu3 and Con3
were also analysed.
Total RNA (8 - 10µg) was supplied to the University of Bristol Transcriptomics Facility. Quality of
the total RNA samples and the resulting cRNA was checked using a 2100 Bioanalyser (Agilent
Technologies, Stockport, UK). Fragmented biotinylated cRNA was prepared and 15µg hybridised
to each of two HG-U133A GeneChips  (i.e., two technical replicates of each) according to the
manufacturer’s recommended protocols (Affymetrix, Santa Clara, CA, USA). Single Array
Expression Analysis was performed using the Affymetrix GeneChip Operating Software (GCOS).
A global scaling strategy was used to give a target intensity of 500 for each array. Arrays were only
included for further analysis if they complied with the quality control metrics defined by
Affymetrix.17
Data from all 20 arrays were filtered in Microsoft Excel (Microsoft, Seattle, WA, USA) to exclude
all probe sets called either Absent or Marginal in all arrays. Control probe sets with the prefix
AFFX were also removed prior to subsequent data analysis. Filtered data were imported into
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GeneSifter software (VizX Labs, Seattle, WA, USA), transformed to a log2 scale and analysed to
determine differentially expressed genes. Day 6 and day 11 data were analysed by balanced 2-way
ANOVA. Day 4 data were analysed by unpaired t-test. A 1.5-fold change threshold and test statistic
of P<.05 were used for both data sets. Gene Ontology terms showing enrichment were identified
with the Z-score Report function of the software.
Quantitative Real-Time PCR (Q-PCR)
Total RNA (1ug) from each harvested erythroid cell sample was converted to cDNA using
SuperScript II reverse transcriptase according to the manufacturer’s instructions (Invitrogen,
Paisley, UK).
Differentially expressed genes were analysed by quantitative real-time PCR (Q-PCR).
Amplification of LU, CD44H, AHSP and SLC4A1 used primers designed with Primer Express
software v2.0 (Applied Biosystems, Warrington, UK). Sequences were as follows: LU 5′CCCCCAACAAAGGGACACT-3′ and 5′-TTGCGATACCACGTGATCTTG-3′; CD44H 5′CACAGACAGAATCCCTGCTACCA-3′ and 5′-TCCCATGTGAGTCCATCTGAT-3′; AHSP 5′GTTAGACCTGAAGGCAGATGGC-3′ and 5′-GAGGATCATTGAAGACCTGCTGA-3′; SLC4A1
5′-GGATTTCTTCATTCAGGATACCTACAC-3′ and 5′-CCCCGGGCTGAGGAGTT-3′, and
probe 5′FAM-AAACTCTCGGTGCCTGATGGCTTCAAG-3′TAMRA. Amplification of SLC4A1
required TaqMan Universal PCR Master Mix whereas the remaining amplifications used SYBR
Green PCR Master Mix (Applied Biosystems). Primers for AHSP and SLC4A1 were kindly
provided by Dr. K. Finning (IBGRL). ALAS2, HBB and PABPC1 gene amplifications were
performed using TaqMan Universal PCR Master Mix and inventoried TaqMan Gene Expression
Assays (Applied Biosystems).
Q-PCR was performed on the ABI 7000 Sequence Detection System (Applied Biosystems) with the
following conditions: 50oC for 2 minutes and 95oC for 10 minutes, followed by 45 cycles of 95oC
for 15 seconds and 60oC for 60 seconds. All reactions were performed in triplicate.
Target gene expression was normalised to PABPC1 expression and compared to a calibrator sample
(UR/FL; cDNA prepared from equal amounts of human Universal Reference RNA and fetal liver
RNA (Clontech, Mountain View, CA, USA)) using the the Pfaffl method of relative quantification
as described in the Bio-Rad Real-Time PCR Applications Guide.18 PABPC1 was used as a control
for gene amplification as it has been shown by microarrays to have consistent expression in our
erythroid cultures (B.K.S., unpublished data, July 1, 2005).
Genomic DNA
Genomic DNA (gDNA) was extracted from 1 x 106 cells from each erythroid culture using the
QIAamp DNA Blood Mini Kit (Qiagen, Crawley, UK). Additional samples from individuals with
the In(Lu) phenotype (InLu6-24) were obtained from archived gDNA or prepared from archived
cryopreserved red blood cells (available in-house). Control gDNA from 32 random blood donors
(Con6-37) was kindly provided by Dr. V. Karamatic Crew (BITS).
Polymerase Chain Reaction (PCR)
PCR was used to amplify human EKLF cDNA (Entrez Accession number NM_006563.2) and
exons from gDNA (Entrez Accession number NT_086897.1: complement of region 4090501 –
4093981). PCR primers were designed with Primer Express software v2.0 (Applied Biosystems).
EKLF cDNA was amplified using 2 overlapping pairs of primers: 5′AGTTCACGAGGCAGCCGAG-3′ and 5′-CCGGGTCCCAAACAACTCA-3′; 5′GCACTTCCAGCTCTTCCGC-3′ and 5′-CTTGTCCCATCCCCAGTCACT-3′. Two additional
primers were also used for sequencing: 5′-CCGAGACTCTGGGCGCATA-3′ and 5′ACCCAAAAGCCCAGCCAC-3′. PCR reactions contained 1U Expand DNA polymerase with 1.5
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mM MgCl2 and 1 x buffer (Expand High Fidelity PCR system, Roche, Mannheim, Germany), 200
mM dNTPs (GE Healthcare, Amersham, UK), 500 mM each primer (Sigma-Aldrich, Haverhill,
UK) and 1 x PCRx Enhancer solution (Invitrogen). Amplification was performed using the
GeneAmp PCR system 9700 (Applied Biosystems) with a Touchdown PCR profile as follows:
95oC for 5 minutes then 9 cycles of 95oC for 30 seconds, 70oC for 30 seconds, 72oC for1 minute,
with a drop in annealing temperature of 2oC per cycle (to 54oC). This was followed by 35 cycles of
95oC for 30 seconds, 52oC for 30 seconds, 72oC for1 minute with a final extension at 72oC for 10
minutes.
EKLF exon 1 and 286 bp of promoter sequence was amplified from gDNA using primers 5′TTACCCAGCACCTGGACCCT-3′ and 5′-GAACCTCAAACCCCTAGACCACC-3′ with the
same conditions as for cDNA plus the addition of 2 x PCRx Enhancer solution (Invitrogen). EKLF
exon 2 was amplified with 2 overlapping pairs of primers: 5′-GTGTCCAGCCCGCGATGT-3′ and
5′-CCGGGTCCCAAACAACTCA-3′; 5′-CCGAGACTCTGGGCGCATA-3′ and 5′GCCCTCTGCAACCCTTCTTC-3′ with the same conditions as described for cDNA amplification.
Exon 3 was amplified using primers 5′-TGCGGCAAGAGCTACACCA-3′ and 5′CTTGTCCCATCCCCAGTCACT-3′ and required the addition of 2 x PCRx Enhancer solution
(Invitrogen). Primers described for the amplification of EKLF cDNA were also used as gDNA
sequencing primers, as appropriate.
gDNA derived from random blood donors was amplified as described except for the following
modifications: exon 1 amplification used 0.625 U GoTaq Flexi DNA polymerase with its own
buffer (Promega, Southampton, UK) and without PCRx Enhancer solution, and exons 2 and 3 used
1 U AccuPrime GC-Rich DNA polymerase with Buffer A (Invitrogen), also without PCRx
Enhancer solution.
PCR products were purified using either the Qiaex II Gel Extraction Kit or QiaQuick PCR
Purification Kit (Qiagen) and sequenced on both strands using the ABI-PRISM 3100 Applied
Biosystem automated DNA sequencer (Applied Biosystems).
Details of primer sequences and PCR conditions used for the amplification and sequencing of other
erythroid transcription factor genes (GATA1, FOG1, GFI1B, TAL1, SPI1, MYB, MZF1) are
available on request.
Homology modeling
An homology model was constructed of the EKLF zinc finger domains in complex with a duplex
oligodeoxynucleotide (5'-TTCCACACCCT-3'), based on the crystal structure of a Zif268-DNA
complex (PDB ID 1AAY).19 The protein sequences are 44% amino acid identical with one insertion
of two residues in EKLF. The initial protein model was constructed using Modeller20 to mutate the
Zif268 sequence to EKLF; all other molecular modeling operations were performed with
SYBYL7.3 (Tripos, St Louis, MO). The initial DNA model was constructed by manual mutation of
DNA from the Zif268 structure. The two initial models were merged and steric clashes between
protein and DNA were relieved by manual alteration of amino acid sidechain conformations. Three
layers of water were then added to the complex and subsequently iteratively minimized to
convergence using the Amber99 force-field21 while constraining the co-ordinates of the
macromolecular atoms. During subsequent rounds of energy minimization the positional constraints
on the macromolecular components were incrementally removed from the protein sidechains, then
the protein backbone, and finally the DNA. Molecular geometry was assessed with MolProbity22
and NUCheck.23 Areas with poor geometry were manually adjusted and the solvation and energy
minimization procedures repeated. Models of three EKLF variants (Arg328Leu, Arg328His and
Arg331Gly) were constructed by mutation of the native EKLF model and repetition of the
minimization process. All final models did not contain any Ramachandran outliers and were ranked
by MolProbity within the 85th percentile of crystallographic structures determined at approximately
2 Å resolution.
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Results
Transcription is altered in erythroblasts from cultures of In(Lu) cells
Transcriptional differences between control and In(Lu) erythroid cells at days 6 (pronormoblasts)
and 11 (normoblasts) of ex vivo culture were determined by microarray analysis using RNA
obtained from cultured erythroblasts. A balanced 2-way ANOVA was used to look for genes
differentially expressed between the two cell types and between the two days tested. The data
discussed in this publication have been deposited in NCBI’s Gene Expression Omnibus (GEO,
http://www.ncbi.nlm.nih.gov/geo/) and are accessible through GEO Series accession number
GSE10584.
Analysis of control and In(Lu) transcript levels showed 373 genes with increased levels between
day 6 and day 11 and 389 with decreased levels. The same genes were affected in control and
In(Lu) erythroblasts. Selected results are displayed in Supplemental Table S1. As expected, many
genes involved in hemoglobin synthesis or encoding blood group antigens were found to increase
their expression. Genes involved in antigen processing and presentation showed decreased
expression, in keeping with the flow cytometric findings of Southcott et al.24 Further inspection of
the data revealed significant differences between control and In(Lu) erythroblasts with respect to
the magnitude of transcript levels. In In(Lu) samples, 314 genes had higher expression than controls
and 354 had lower expression at day 6 and/or at day 11. Gene expression between days 6 and 11 of
cultured In(Lu) cells showed the same qualitative pattern seen with controls but marked differences
regarding the levels of gene expression (Table 1). Genes with decreased expression during
erythropoiesis (e.g., the major histocompatibility complex (HLA) genes) showed higher expression
in the In(Lu) cells while erythroid-specific genes (e.g. delta-aminolevulinate synthase 2 (ALAS2),
alpha hemoglobin stablising protein (AHSP), and glycophorin A (GPA)) showed lower expression,
suggesting that the pattern of gene expression in In(Lu) cells is less capable of promoting erythroid
maturation. Flow cytometric analysis of In(Lu) cells (donor InLu2) from day 8 of culture
demonstrated reduced surface expression of GPA, Band 3 and Lutheran (mean fluorescence
intensity 106.4, 1.9 and 0.6 respectively compared with control (Con2) values of 172.5, 5.6 and
11.6 respectively) suggesting reduced transcript levels are reflected in the cell surface phenotype.
The differences between In(Lu) and control cells were most apparent on day 6 where almost all
erythroid-specific genes show a difference between the In(Lu) and the control cells. The only
exceptions are the genes for the basal cell adhesion molecule (BCAM) which carries the Lutheran
blood group antigens and CD44 which carries the Indian blood group antigens. These genes show a
greater difference change at day 11. This is of particular interest since the expression of these blood
group antigens on the surface of red blood cells is known to be suppressed in individuals with the
In(Lu) phenotype.13,25,26
We reasoned that if gene expression profiles at day 6 revealed a relative reduction in transcription
of erythroid genes in In(Lu) cells then this effect might be even more apparent earlier in the
cultures. cRNA derived from day 4 cells from one pair of control and In(Lu) cultures were
hybridised to arrays (2 technical replicates of each). Microarray data were analysed by unpaired ttest and the results revealed 378 genes showing at least a 1.5-fold difference in expression between
the two cell types (P<.05). Selected results are shown in Supplemental Table S2. The 159 genes upregulated in In(Lu) cells at day 4 again involved the defense response and antigen processing and
presentation. Two hundred and nineteen genes were down-regulated and, as at day 6, these
contained genes for hemoglobin synthesis and erythroid-specific factors. The transcript for ALAS2
showed a greater reduction at day 4 than at the later days (12.5-fold at day 4 compared to 2-fold at
day 6 and no change at day 11). ALAS2 is an enzyme that catalyses the first step of heme synthesis.
This first step is rate-limiting and so the reduction in expression may be expected to have severe
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consequences for heme and hence hemoglobin production. At day 4 reduced expression of alpha
and beta globin transcripts is also apparent. This was not seen at the later timepoints. Furthermore,
expression of RNA encoding the erythrocyte anion exchanger (SLC4A1, also known as Band 3)
also shows a much greater reduction at day 4 compared with later days (i.e. 18.8-fold down at day
4, 3.1-fold at day 6 and only 1.1-fold at day 11). The greater differences in expression of these
erythroid-specific genes at the earlier timepoint are consistent with our initial observation that
erythroid cells from individuals with the In(Lu) phenotype are less able to promote erythroid
maturation at the transcriptional level.
Validation of microarray data by quantitative real-time PCR (Q-PCR)
In order to validate our microarray findings and to obtain more comprehensive data regarding
temporal changes in expression of selected genes, we performed Q-PCR on cDNA from four pairs
of control and In(Lu) cultures, and included a greater number of timepoints (Figure 1 and
Supplemental Figure S1).
In control cells, LU RNA is expressed at very low levels during early days of culture, rising by day
8 and peaking at day 11. The Lutheran antigens can be detected on the surface of cultured
erythroblasts at around day 7 to 8.24 In In(Lu) cells, LU appears to be transcriptionally repressed (a
maximum of 40-fold at day 8; Figure 1 and Supplemental Figure S1), although some expression is
detected at all days with a slight increase at day 11 suggesting that the gene is not completely
silenced. Although red blood cells from individuals with the In(Lu) phenotype type serologically as
Lu(a-b-), in many cases adsorption/elution techniques have shown the presence of very weak
Lutheran antigen expression.27
The hemopoietic form of CD4426,28 shows consistently higher expression in the control cells than in
the In(Lu) cells but the difference is much smaller than for LU (a maximum of 3-fold at day 14).
Four additional erythroid genes tested by Q-PCR (AHSP, SLC4A1, ALAS2 and beta globin (HBB))
share similar patterns of expression. In control cells there is an increase in expression from day 4 to
day 8 or 11 and then a reduction at day 14. The In(Lu) cells show a similar pattern but expression is
lower at all but the last timepoint. For HBB, expression is always lower in the In(Lu) cells. For all 4
genes, the difference in expression between control and In(Lu) cells is greatest at day 4: 3.2-fold for
AHSP, 17.1-fold for SLC4A1, 4.4-fold for ALAS2 and 5.2-fold for HBB (Figure 1 and Supplemental
Figure S1). These results are consistent with the microarray findings and our hypothesis that In(Lu)
erythroblasts have a general abnormality of transcription with respect to erythroid genes.
In(Lu) erythroblasts have mutations in erythroid transcription factor EKLF.
Since abnormal transcript levels were observed for multiple erythroid genes in In(Lu) type
erythroblasts we looked for evidence of altered erythroid transcription factors. We amplified and
sequenced exons from two control and two In(Lu) samples (gDNA or cDNA) for genes encoding
several transcription factors functioning in erythropoiesis (GATA1, FOG1 (90% covered), EKLF,
GFI1B, MYB, MZF1 (80% covered), TAL1, SPI1 (PU.1)). Only EKLF showed the presence of
mutations unique to In(Lu) samples. We sequenced the three EKLF exons from 3 more cDNA
samples (derived from In(Lu) erythroid cultures),18 more gDNA samples typed as Lu(a-b-) In(Lu)
type and one unusual Lu(a-b-) (InLu24), derived from archived gDNA or archived blood samples
(available in house). Mutations were detected in all but three of the samples (Table 2). In all cases
the mutated allele was expressed with a normal EKLF allele.
Nine different mutations were detected. Eight UK individuals had a single base insertion in exon 3
(Arg319GlufsX34). Six individuals from two Spanish families had a single base deletion in exon 2
(Pro190LeufsX47). Six of the nine mutations occur in that part of the EKLF gene encoding the zinc
finger domains (Figure 2). One (Arg319GlufsX34) is predicted to cause a frameshift and another
(Lys292X ) encodes a premature termination codon; all others result in single amino acid
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substitutions (His299Tyr, Arg328Leu, Arg328His and Arg331Gly). The three mutations outside of
the zinc finger domains result in a premature termination codon (Leu127X), a frameshift
(Pro190LeufsX47) and an altered GATA1 binding site in the EKLF promoter (-124T>C).
Two known single nucleotide polymorphisms (SNPs; dbSNP rs2072597 and dbSNP rs2072596)
were also detected (Table 2). Interestingly, a third reported SNP (dbSNP rs35891202) has a single
base deletion at the beginning of exon 3 which would be predicted to result in a frameshift affecting
the entire zinc finger region. This SNP may therefore represent an EKLF mutation in another
individual with the In(Lu) phenotype.
We also sequenced all EKLF exons and the promoter region in 5 cDNA samples from control
erythroid cultures and 32 control gDNAs from random blood donors. None of the mutations found
in the In(Lu) samples were seen in the controls.
EKLF mutations in InLu create a non-functional allele
Mutation 1 disrupts a GATA1 binding site in the EKLF promoter (Table 2). The other mutations
identified are located in the EKLF coding region. Several mutations must render the affected EKLF
allele non-functional. Mutations 2 and 4 convert Leu127 and Lys292 respectively to termination
codons resulting in translated proteins lacking all zinc finger (ZF) domains. Mutation 3 is a base
deletion in the codon for Pro190 while mutation 6 is a base insertion in the codon for Arg319. Both
mutations would alter the subsequent reading frame with consequent loss of all ZF domains
(mutation 3) or loss of ZF domains 2 and 3 (mutation 6). Mutations 5, 7, 8 and 9 cause amino acid
changes in or near the consensus sequence of a ZF domain. Mutation 5, which changes His299
(ZF1) to Tyr, is expected to diminish the binding of zinc which is integral to the overall protein
conformation. Mutations 7 and 8 lead to substitution of Arg328 (ZF2) by His and Leu respectively.
These changes are expected to disrupt the normal mode of DNA binding in which Arg328 is
predicted to form two central hydrogen bonds with a guanine base (Figure 3). Mutation 9
substitutes Arg331 with Gly. In the model, Arg331makes a water-mediated interaction with a
phosphate of the DNA backbone; this interaction is not possible when Gly is incorporated at this
position (Figure 3).
Discussion
The molecular basis of the In(Lu) type of Lu(a-b-) blood group phenotype has not previously been
established. Many families exhibiting this phenotype have been studied using serological methods
and apparent dominant inheritance observed - hence InLu (inhibitor of Lutheran) to describe the
gene responsible.14,15,33,34 The phenotype is of interest because its characteristic reduced red cell
surface expression of antigens from several genetically independent blood group systems (i, P1 and
Inb) in addition to Lutheran system antigens suggests abnormality in a pathway common to the
biosynthesis of these blood group-active molecules (reviewed in Daniels35). The Inb antigen is
carried on CD44 and defined by its amino acid sequence.25,26 In contrast the i and P1 antigens are
carbohydrate antigens.36,37
In order to explore the molecular basis of this phenotype we compared gene expression profiles of
normal and In(Lu) cultured erythroblasts. The results showed a clear difference between the two
cell types with respect to transcript levels of LU and CD44 (Table 1, Figure 1), consistent with the
serological profile of In(Lu) red cells. Lutheran expression occurs late during ex vivo
erythropoiesis24 and the markedly reduced levels of LU transcript appear indicative of incomplete
erythroid maturation. Further inspection of the gene expression data revealed a reduction in
transcript levels of erythroid genes in general. This was particularly noticeable when erythroblasts
from day 4 of culture were examined where marked reductions in transcript levels for ALAS2,
globin genes and SLC4A1 (Band 3) were apparent (Supplemental Table S2, Figure 1 and
Supplemental Figure S1). These data prompted us to search for mutations in erythroid transcription
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factor genes in DNA from blood donors with the In(Lu) phenotype. Initial analysis of DNA from
two individuals of the In(Lu) type revealed mutations in EKLF. Subsequently, we were able to
obtain In(Lu) DNA from 24 individuals. Our data showing the occurrence of mutations in the
promoter or coding region of EKLF in all but 3 of these individuals and the absence of any of these
mutations in individuals of normal Lutheran phenotype provide persuasive evidence that the EKLF
mutations are responsible for the In(Lu) phenotype in these individuals. All 21 In(Lu) samples in
which we found EKLF mutations had the mutations on one allele whereas the other EKLF allele
had a normal sequence (Table 2).
EKLF comprises a proline-rich N-terminal region (amino acids 1-275) containing a transactivation
domain and a C-terminal region (amino acids 276-358) containing three zinc finger domains
essential for binding to the DNA consensus sequence 5'-CCACACCCT-3' (Figure 2).29,38,39 In mice,
EKLF is expressed at high levels only in erythroid cells and abundant evidence supports the view
that it is a key regulator of erythroid-specific gene expression (reviewed in Bieker40). EKLF knockout mice die at around embryonic day 14 due to severe anemia resulting from the failure to switch
from embryonic to adult beta-globin expression.2,3 Murine EKLF is also required for the
transcription of heme biosynthesis genes such as ALAS2 and AHSP, and for genes encoding
erythroid membrane proteins including EPB49, DARC and ERMAP.7,8 Comparison of the gene
expression profiles observed for In(Lu) with those reported for mice deficient in EKLF7,8 reveals
numerous similarities with respect to reduced expression of EKLF-dependent erythroid genes
(Table 1, Figure 1). Less information is available regarding transcript levels in EKLF +/- mice but
beta globin and TER119 (murine glycophorin A) are expressed at intermediate levels and in this
respect are similar to human In(Lu) cells (Figure 1).8,9
The 21 In(Lu) individuals studied exhibited 9 different EKLF mutations. Mutation 1 (-124T>C;
Table 2) causes the loss of one of three possible GATA1 binding sites in the human EKLF promoter.
Analysis of mouse EKLF promoter activity by transient transfection in MEL cells of promoter
constructs in which one or other of three GATA1 binding sites were disrupted showed that one site
was critical for EKLF expression while inactivation of the other sites had a relatively minor effect.41
In this context it is interesting to note that red cells from the individual with the promoter mutation
(InLu24) had Lutheran antigens and Inb antigen expression weaker than normal red cells but
noticeably stronger than most In(Lu) samples (J. Poole, IBGRL, e-mail, February 4, 2008)
suggesting that some expression of EKLF occurs from the mutant allele in the absence of this
GATA1 site.
Eight different mutations were found in the EKLF coding sequence of individuals with the In(Lu)
phenotype. Eight individuals had the Arg319GlufsX34 mutation and 6 had the Pro190LeufsX47
mutation; it seems likely these groups share common ancestors in the UK and Spain respectively.
All other mutations were single examples. The In(Lu) phenotype appears to be benign with no
associated pathology and little evidence of erythroid abnormality reported. The red cells of two
In(Lu) siblings with normal hematological indices had a normal half life although they showed
increased hemolysis on storage at 4oC.42 In one family the In(Lu) phenotype was associated with
acanthocytosis.43 It would be of interest to examine EKLF in the latter family to see if the reported
acanthocytosis is linked to a unique mutation in EKLF.
By analogy with EKLF “knock-out” mice it would be predicted that inheritance of two InLu alleles
would result in embryonic death and in this context it is interesting to note that none of the many
reported families with In(Lu) type red cells have progeny of proven homozygosity with respect to
InLu. Of particular interest is the NEA family described by Gibson34 in which a first cousin
marriage produced 5 children all of whom had the In(Lu) phenotype. Four of these children had
children of their own (who were not of the In(Lu) phenotype), proving that they were heterozygous.
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The presence of a mutated EKLF allele is easily detected by observing the almost complete absence
of Lutheran blood group antigens on the red cells of affected individuals since most individuals
with the Lu(a-b-) phenotype will be of the In(Lu) type. The dramatic reduction in Lutheran
expression observed in In(Lu) cells may be related to its transcriptional activation being at a later
stage in erythropoiesis than other erythroid-specific genes. This suggests that EKLF availability in
In(Lu) becomes limiting at later stages of erythroid maturation (Figure 1, Supplemental Figure S1).
In this context it is interesting to note that EKLF protein content declines as J2E cells mature in
response to erythropoietin under conditions where globin synthesis is increasing rapidly.44
Spadaccini et al44 suggest lower levels of EKLF are needed to maintain transcription once initial
activation has occurred. If this observation also applies to human erythropoeisis it may be that
levels of EKLF in In(Lu) are too low for transcriptional activation of LU at day 8 of culture. Several
potential EKLF binding sites have been identified in the LU promoter.45
We conclude that the In(Lu) phenotype is most likely caused by inheritance of a loss of function
mutation on one allele of EKLF. Transcription of EKLF from the single normal allele results in
reduced levels of EKLF and as a consequence, a general reduction in transcription of erythroid
genes ensues. The absence of mutation in the EKLF coding sequence in 3 In(Lu) samples (InLu8, 9
and 23) suggests either the presence of a mutation in part of the gene not sequenced (enhancer or
distal promoter regions), or the possibility of other causes of the In(Lu) phenotype. In several
In(Lu) individuals the mutated copy of EKLF can only produce non-functional protein and so they
are representative of the EKLF +/- phenotype. It can be inferred that this phenotype is
physiologically benign in man as it is in mice. In other cases mutations change single amino acids
in the region of zinc finger domains 1 and 2 and so, by inference, alter residues critical for normal
EKLF function. The diversity of mutations within a small number of In(Lu) individuals is consistent
with sporadic mutation. The In(Lu) phenotype therefore results from reduced transcription (as
previously postulated42) rather than the action of a dominant inhibitor gene. These data provide the
first evidence of naturally occuring mutations affecting human EKLF and the first description of
transcription factor mutations directly responsible for a specific blood group phenotype.
Acknowledgements
This work was supported by the Department of Health (England).
We thank Heather Davies and Alan Gray for the Lu(a-b-) samples and their controls, Peter Martin
and Joanna Summers-Yeo for DNA sequencing, Jane Coghill for the microarray hybridisation
service, and Vanja Karamatic Crew and Kirstin Finning for providing useful reagents. We are
grateful to Geoff Daniels, Frances Spring and Anna Ribera for providing archived samples. We also
thank Joyce Poole and Nicole Warke for serology data and Kevin Gaston and Lucio Luzzatto for
helpful advice and discussions.
Contribution: B.K.S. designed and conducted experiments and wrote the paper; N.M.B. and C.G.
conducted experiments; R.L.B. designed experiments; D.J.A. designed experiments and wrote the
paper.
Conflict of interest disclosure: The authors declare no competing financial interests.
Correspondence: Belinda K. Singleton, Bristol Institute for Transfusion Sciences, Southmead Road,
Bristol, BS10 5ND, United Kingdom; e-mail: [email protected]
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antigens. Hum Hered. 1976;26:171-174.
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cells from persons with the In(Lu) gene. Transfusion. 1992;32:607-611.
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Table 1: Selected genes that are differentially expressed between In(Lu) and control cells
Gene Symbol
Gene Title
Antigen processing and presentation
ARTS-1
Type 1 tumor necrosis factor receptor shedding aminopeptidase regulator
HLA-A
Major histocompatibility complex, class I, A
HLA-B
Major histocompatibility complex, class I, B
HLA-DPB1
Major histocompatibility complex, class II, DP beta 1
HLA-DRA
Major histocompatibility complex, class II, DR alpha
HLA-DRB1
Major histocompatibility complex, class II, DR beta 1
HLA-DRB4
Major histocompatibility complex, class II, DR beta 4
HLA-G
HLA-G histocompatibility antigen, class I, G
Defense response
ECRP
Eosinophil cationic related protein
ELA2
Elastase 2, neutrophil
FCER1A
Fc fragment of IgE, high affinity I, receptor for; alpha polypeptide
IL4R
Interleukin 4 receptor
MPO
Myeloperoxidase
NCF4
Neutrophil cytosolic factor 4, 40kDa
PRG3
Proteoglycan 3
RNASE2
Ribonuclease, RNase A family, 2 (liver, eosinophil-derived neurotoxin)
TPSAB1
Tryptase alpha/beta 1
TPSAB1 /
Tryptase alpha/beta 1 / tryptase beta 2
TPSB2
TPSB2
Tryptase beta 2
Heme / globin synthesis
AHSP
Alpha hemoglobin stabilising protein
ALAD
Aminolevulinate, delta-, dehydratase
ALAS2
Aminolevulinate, delta-, synthase 2 (sideroblastic/hypochromic anemia)
HBE1
Hemoglobin, epsilon 1
UROD
Uroporphyrinogen decarboxylase
Blood group antigens / erythroid factors
ANK1
Ankyrin 1, erythrocytic
AQP1
Aquaporin 1 (Colton blood group)
ARG1
Arginase, liver
ART4
ADP-ribosyltransferase 4 (Dombrock blood group)
BCAM
Basal cell adhesion molecule (Lutheran blood group)
BSG
Basigin (Ok blood group)
CD44
CD44 molecule (Indian blood group)
DARC
Duffy blood group, chemokine receptor
EPB42
Erythrocyte membrane protein band 4.2
EPOR
Erythropoietin receptor
ERMAP
Erythroblast membrane-associated protein (Scianna blood group)
GYPA
Glycophorin A (MNS blood group)
NFE2
Nuclear factor (erythroid-derived 2), 45kDa
PKLR
Pyruvate kinase, liver and RBC
SLC4A1
Solute carrier family 4, anion exchanger, member 1 (erythrocyte membrane
protein band 3, Diego blood group)
TMOD1
Tropomodulin 1
Day 6
fold change
Day 11
fold change
4.6
1.5
1.1
1.8
2.0
1.9
1.7
1.2
2.4
1.6
2.1
1.6
1.8
1.3
1.0
1.8
2.5
3.4
1.8
2.2
3.0
2.1
1.5
1.7
4.6
4.5
3.0
2.8
1.2
2.2
1.6
1.5
1.6
1.8
3.9
4.0
5.0
3.5
-1.8
-2.1
-2.1
-1.8
-1.6
-1.1
-1.3
-1.0
-1.3
-1.2
-1.6
-3.0
-2.3
-2.6
-1.5
-1.7
-1.7
-1.7
-1.8
-1.9
-1.5
-1.5
-1.5
-2.6
-3.1
-1.2
-1.7
-1.6
-1.3
-3.5
-1.3
-1.9
-1.3
-1.1
-1.3
-1.4
-1.1
-1.2
-1.7
-1.1
-2.0
-1.3
Differentially expressed genes are grouped according to gene ontology terms showing enrichment.
Separate values are shown for fold differences in expression (In(Lu) value divided by control value)
in cells at day 6 and at day 11. Genes showing higher expression in In(Lu) cells than in controls
have positive values; genes showing lower expression in In(Lu) cells than in controls have negative
values. Average values have been taken for genes that are represented more than once on the arrays.
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Table 2: Sequence variants detected in the EKLF gene in erythroid cells of the In(Lu)
phenotype
Variant Sample
type
name
1
InLu24
2
InLu10
3
InLu17
InLu18
InLu19
InLu20
InLu21
InLu22
4
InLu6
5
InLu16
6
InLu1
InLu3
InLu4
InLu5
InLu7
InLu13
InLu14
InLu15
7
InLu11
8
InLu12
9
InLu2
10
InLu8
InLu9
InLu23
□
DNA change□
Deduced protein change†
[=]+[-124T>C] ‡
[=]+[380T>A]
[=]+[569delC]
[=]+[?]
[=]+[Leu127X]
[=]+[Pro190LeufsX47]
[=]+[874A>T]
[=]+[895C>T]
[=]+[954dupG]
[=]+[Lys292X]
[=]+[His299Tyr]
[=]+[Arg319GlufsX34]
[=]+[983G>T]
[=]+[983G>A]
[=]+[991C>G]
[=]+[?]
[=]+[Arg328Leu]
[=]+[Arg328His]
[=]+[Arg331Gly]
[=]+[?]
dbSNP
rs2072597
T/C
T/C
C/C
C/C
T/C
T/C
C/C
T/C
T/T
T/C
T/T
T/T
T/T
T/T
T/C
T/T
T/C
T/C
T/T
C/C
T/C
T/C
T/C
T/T
dbSNP
rs2072596
T/T
T/T
T/T
T/T
T/T
T/T
T/T
T/T
T/T
T/T
T/T
T/T
T/T
T/T
T/T
T/T
T/T
T/T
T/T
T/C
T/T
T/C
T/T
T/T
DNA changes are numbered relative to the translation initiation codon (where A is +1) in the
reference cDNA sequence (Entrez Gene Accession number NM_006563.2). [=] indicates the
presence of the wild-type allele. [?] indicates an unknown change.
†Deduced protein changes are numbered relative to the translation initiator (where Met is +1) in the
reference protein sequence (Entrez Protein Accession number NP_006554.1). [=] indicates presence
of wild-type protein. [?] indicates unknown effect.
‡Promoter sequence can be obtained from the reference gDNA sequence (Entrez Gene Accession
number NT_086897.1: complement of region 4090501 – 4093981).
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Figure legends
Figure 1. Validation of selected microarray results by Q-PCR. Changes in target gene
expression during ex vivo erythropoiesis are depicted. The vertical axis represents the target gene
expression normalised to PABPC1 expression and relative to the UR/FL calibrator (as described in
“Materials and Methods”). Black lines indicate average expression in control erythroblasts (n=4);
gray lines indicate average expression in In(Lu) erythroblasts (n=4).
Figure 2. Sequence variants located in the zinc finger domains of EKLF. Alignment of the zinc
finger domains (ZF1 – ZF3) from EKLF is compared with ZIF268 (based on Feng et al29(Fig4)).
Protein sequences of genes identified as putative homologs of EKLF by NCBI’s HomoloGene30 are
from Homo sapiens (h), Pan troglodytes (p), Canis lupus familiaris (c), Mus musculus (m) and
Rattus norvegicus (r). Boxed residues indicate XYZ amino acids required for DNA sequence
recognition;31 bold residues show matches to the classical CC/HH motif;32 and circled numbers
indicate positions of sequence variant types as described in Table 2.
Figure 3. Homology model of the EKLF-DNA complex. (A) Overall structure of the complex.
DNA is shown as an orange molecular surface; protein is shown as a gray Cα cartoon. The amino
terminus (Ala278) is labelled N; the carboxy terminus (Leu362) is labelled C. The side chains of
mutated residues are highlighted as sticks with the carbon atoms of Arg328 and Arg331 colored
green and cyan, respectively. (B) Close-up of the ZF2 domain highlighting protein-DNA
interactions involving mutated residues. DNA is shown as sticks colored by atom type. Protein is
displayed as in (A). The bound water molecule is shown as a red sphere, and putative hydrogen
bonds are shown as dashed yellow lines.
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Figure 1
CD44H
60
Expression relative to
UR/FL (x100)
Expression relative to
UR/FL (x1000)
LU
90
80
70
60
50
40
30
20
10
0
50
40
30
20
10
0
4
6
8
10
12
14
4
6
Days in culture
AHSP
120
Expression relative to
UR/FL (x100)
Expression relative to
UR/FL (x10)
14
SLC4A1
25
20
15
10
5
0
100
80
60
40
20
0
4
6
8
10
12
Days in culture
14
4
6
ALAS2
8
10
12
Days in culture
14
HBB
14
80
12
70
Expression relative to
UR/FL (x10)
Expression relative to
UR/FL (x10)
8
10
12
Days in culture
10
8
6
4
2
0
60
50
40
30
20
10
0
4
6
8
10
Days in culture
12
14
4
6
8
10
Days in culture
12
14
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Figure 2
4
5
278
ZF1
A
A
A
A
A
P
H
H
H
H
H
Y
T
T
T
T
T
A
C
C
C
C
C
C
A
A
T
G
G
P
H
H
H
H
H
V
P
P
P
E
E
E
G
G
G
G
G
S
C
C
C
C
C
C
G
G
G
G
G
D
K
K
K
K
K
R
S
S
S
S
S
R
Y
Y
Y
Y
Y
F
T
T
T
S
T
S
K
K
K
K
K
R
S
S
S
S
S
S
S
S
S
S
S
D
H
H
H
H
H
E
L
L
L
L
L
L
K
K
K
K
K
T
A
A
A
A
A
R
L
L
L
L
L
I
8
7
6
H
H
H
H
H
H
R
R
R
R
R
R
T
T
T
T
T
I
H
H
H
H
H
H
T
T
T
T
T
T
G
G
G
G
G
G
E
E
E
E
E
Q
K
K
K
K
K
K
hEKLF
pEKLF
cEKLF
mEKLF
rEKLF
ZIF268
K
K
K
K
K
T
H
H
H
H
H
H
T
T
T
T
T
T
G
G
G
G
G
G
Q
Q
Q
H
H
E
R
R
R
R
R
K
hEKLF
pEKLF
cEKLF
mEKLF
rEKLF
ZIF268
9
308
ZF2
P
P
P
P
P
P
Y
Y
Y
Y
Y
F
A
A
A
A
A
Q
C
C
C
C
C
C
T
T
T
S
S
W
W
W
W
W
R
E
E
D
D
D
I
G
G
G
G
G
C
C
C
C
C
C
G
G
G
D
N
M
W
W
W
W
W
R
R
R
R
R
R
N
F
F
F
F
F
F
A
A
A
A
A
S
R
R
R
R
R
R
S
S
S
S
S
S
D
D
D
D
D
F
E
E
E
E
E
H
L
L
L
L
L
L
T
T
T
T
T
T
R
R
R
R
R
T
H
H
H
H
H
H
Y
Y
Y
Y
Y
I
R
R
R
R
R
R
F
F
F
F
F
F
R
R
R
C
C
A
C
C
C
C
C
C
Q
Q
Q
G
G
D
L
L
L
L
L
I
C
C
C
C
C
C
P
P
P
P
P
G
R
R
R
R
R
R
A
A
A
A
A
K
F
F
F
F
F
F
S
S
S
S
S
A
R
R
R
R
R
R
S
S
S
S
S
S
D
D
D
D
D
D
H
H
H
H
H
E
L
L
L
L
L
R
A
A
A
A
A
K
L
L
L
L
L
R
H
H
H
H
H
H
M
M
M
M
M
T
K
K
K
K
K
K
R
R
R
R
R
I
H
H
H
H
H
H
338
ZF3
P
P
P
P
P
P
362
L
L
L
L
L
hEKLF
pEKLF
cEKLF
mEKLF
rEKLF
ZIF268
From www.bloodjournal.org by guest on June 15, 2017. For personal use only.
Figure 3
From www.bloodjournal.org by guest on June 15, 2017. For personal use only.
Prepublished online May 16, 2008;
doi:10.1182/blood-2008-03-145672
Mutations in EKLF/KLF1 form the molecular basis of the rare blood group
In(Lu) phenotype
Belinda K. Singleton, Nicholas M. Burton, Carole Green, R. Leo Brady and David J. Anstee
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