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Blood, Vol. 94 No. 6 (September 15), 1999:
pp. 2039-2047
The Human Platelet  IIb Gene Is Not Closely Linked to Its Integrin
Partner  3
By
M.A. Thornton,
M. Poncz,
M. Korostishevsky,
E. Yakobson,
S. Usher,
U. Seligsohn, and
H. Peretz
From the Department of Pediatrics, University of Pennsylvania School
of Medicine, Philadelphia, PA; and the Department of Human Genetics,
Sackler School of Medicine and Clinical Biochemistry Laboratory,
Sourasky Medical Center, Institute of Thrombosis and Hemostasis, Sheba
Medical Center, Tel-Aviv University, Israel.
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ABSTRACT |
 IIbb3 integrin is a heterodimeric receptor facilitating platelet
aggregation. Both genes are on chromosome 17q21.32. Intergenic distance
between them has been reported to be 125 to 260 kilobasepairs (kb) by
pulsed-field gel electrophoresis (PFGE) genomic analysis, suggesting
that they may be regulated coordinately during megakaryopoiesis. In
contrast, other studies suggest these genes are greater than 2.0 megabasepairs (mb) apart. Because of the potential biological implications of having these two megakaryocytic-specific genes contiguous, we attempted to resolve this discrepancy. Taking advantage of large kindreds with mutations in either IIb or  3, we have developed a genetic linkage map between the thyroid receptor hormone-1 gene (THRA1) and 3 as follows:
cen-THRA1-BRCA1-D17S579/IIb-3-qter, with a distance of 1.3 centiMorgans (cM) between IIb and 3 and the two genes being
oriented in the same direction. PFGE genomic and YAC clone analysis
showed that the 3 gene is distal and  365 kb upstream of IIb.
Additional restriction mapping shows IIb is linked to the
erythrocyte band 3 (EPB3) gene, and 3 to the homeobox HOX2b gene.
Analysis of IIb+-BAC and P1 clones confirm that the
EPB3 gene is ~110 kb downstream of the IIb gene. Sequencing the
region surrounding the human IIb locus showed the Granulin gene
~18 kb downstream to IIb, and the KIAA0553 gene ~5.7 kb
upstream. This organization is conserved in the murine sequence. These
studies show that IIb and 3 are not closely linked, with IIb
flanked by nonmegakaryocytic genes, and imply that they are unlikely to
share common regulatory domains during megakaryopoiesis.
© 1999 by The American Society of Hematology.
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INTRODUCTION |
THE  IIb3 (glycoprotein IIb/IIIa or
CD41) receptor is a megakaryocyte-specific integrin critical for
platelet aggregation.1 There are ~80,000 copies of this
heteroduplex per platelet,2 making it the most densely
represented protein on the platelet surface. The genes for both IIb
and 3 have been cloned and characterized, and the
molecular basis for their high levels of expression in megakaryocytes
has begun to be determined.3 Both genes have been localized
to chromosome 17q21.32.4,5 PFGE genomic studies have
indicated that these two genes are closely linked at a maximum distance
between 125 to 260 kilobasepairs (kb),6,7 suggesting that
these genes may be regulated coordinately during megakaryopoiesis.
This region of chromosome 17 also contains the breast cancer
predisposing gene, BRCA1 and the linked anonymous marker
D17S579.8 Sequence tagged site (STS) content mapping of
YAC, P1, and cosmid contigs across this region, placed the genetic
marker D17S579, the IIb gene and the erythroid-specific gene EPB3 in
close proximity with each other spanning a range of only 100 to 200 kb.9,10 Multicolor FISH mapping of several genes and
markers in this region established a physical map with the following
order: cen-EPB3-D17S579(mfd188)-WNT3-HOX2-3-qter.11 Because IIb is very proximal to the D17S579/EPB3 loci and given the
low resolution of FISH mapping, this suggests that there may be at
least two genes separating IIb from its integrin partner 3.
D17S579 was also used in the construction of a radiation hybrid map of
the BRCA1 region.12 This map also places IIb, EPB3, and
D17S579 as a tightly linked group that could not be ordered because no
obligate breaks occurred between them. Conversely, 3 was mapped at a
distance of over 42 centiRad(cR) 8000 units from the
D17S579/llb/EPB3 locus, and proximal to the marker for the gastric
inhibitory polypeptide gene (GIP). With a conversion ratio of 50 kb/cR(8000) units, this places IIb distal and centromeric to 3 by
more than 2.1 megabasepairs (mb). Anderson et al,13 using
genetic linkage analysis, also placed the 3 gene proximal to the GIP
gene and the Homeobox (HOX) gene cluster at a genetic distance of at
least 1.0 cM telomeric to the D17S579/llb/EPB3 locus. This region of
chromosome 17, also shows high syntenic homology in mice, and in fact,
GIP, HOX, and 3 are similarly clustered, within a region distal and
telomeric to the erythroid murine protein band 3 and IIb gene locus,
on the orthologous murine chromosome 11.14,15 Collectively,
these mapping studies suggest that 3 is more likely to be more
closely physically linked to genes near the GIP or HOX loci than to the
D17S579/llb/EPB3 region.
Because the published data suggest a conflict between the reported
physical distance on PFGE blot and the genetic and radiation studies,
the work presented here was conducted to determine the position and the
distance of the IIb gene relative to 3 gene and other informative
markers mapped to the same chromosomal region. In this study, we have
used both more detailed linkage studies and detailed physical mapping.
For the linkage studies, we took advantage of a 13 bp mutation at the
intron 3/exon 4 junction in the IIb gene occurring in four Arab
kindreds,16,17 which we used as a polymorphic marker to
improve linkage analysis in these large pedigrees. Based on our linkage
analysis, we estimated the recombination distance between the IIb
and 3 genes. Additionally, using actual and historical
recombinations and the linkage disequilibrium observed in Arab and
Iraqi-Jewish Glanzmann thrombasthenic (GT) families, we positioned the
IIb and 3 genes relative to THRA1, BRCA1 gene, and D17S579. We
also predict the orientation of the IIb and 3 genes relative to
each other by analyzing our haplotype data using an algorithm for
ordering tightly linked markers. This analysis was done concurrently to
a detailed PFGE analysis of genomic DNA of this region, and mapping and
sequencing of YAC, BAC, and P1 clones containing these two genes. These
studies provide a detailed map of the IIb region and suggest that
the IIb and 3 genes are not closely linked, and may be 1 mb
apart. Further, we have identified several nonmegakaryocytic genes both
5' and 3' of IIb that separate this gene from its
partner 3 in both humans and mice. Collectively, our results suggest
that these genes are not likely to function as a tightly linked,
centrally regulated, megakaryocyte-specific gene locus and define the
maximum size of the IIb gene locus.
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MATERIALS AND METHODS |
Linkage analysis.
Blood specimens for genomic DNA preparations were collected from 254 individuals in 28 families with Glanzmann thrombasthenia. The origin of
these thrombasthenic families, the type of mutation, and the number of
individuals analyzed are detailed in Table
1. Genomic DNA was prepared by the
desalting method.19 The polymorphic and mutation markers
analyzed in these families are listed in Table 2.
Dinucleotide repeat and restriction fragment length polymorphisms
(RFLP) were analyzed.26
Pairwise linkage analysis of the polymorphisms was performed using the
LIPED software program (Rockefeller University, New York, NY) under the
assumption of no sex difference. The markers analyzed are shown in
Table 2. Haplotypes consisting of allele combinations of markers M4 and
M5 and markers M6 to M10 were used as single alleles for IIb and
3, respectively. The X2 test with a 2×K
contingency table, in which K is the number of marker alleles present
in the sample, was used. Significance of the association was determined
by a right-side X2 test with K-1 degrees of freedom.
Calculations were performed in each ethnic group separately. Chromosomes identical by descent were considered as one chromosome. Two-sided P values of  .05 were considered to be significant.
Haplotype resemblance analysis (HRA).
A method for ordering linked markers on a short chromosomal interval
termed HRA was used to orient the IIb and 3 genes. This method is based on a comparison of multilocus haplotypes that
share identical alleles in most loci, representing tightly linked
polymorphic markers that show a high level of resemblance that
descended from a common ancestral haplotype, and that has yet to
undergo many recombinations. In such pairs of haplotypes, loci located
away from the center should differ more frequently than loci located
near the center. In a set of pairs of multilocus haplotypes with a high
level of resemblance, the number of pairs that differ by alleles in any
particular locus divided by the locus heterozygosity may be considered
an estimate of the relative distance of the locus from the chromosomal
interval center.
The algorithm of HRA involves:
| 1. |
Comparison between all pairs of haplotypes and counting the identical
alleles in each pair.
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| 2. |
Choosing high-score pairs, eg, those with a number of identical alleles
that exceed half the number of the analyzed markers.
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| 3. |
For every marker, counting the number of high-score haplotype pairs in
which the markers are not the same.
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| 4. |
Dividing the number obtained by the marker's heterozygosity.
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The result obtained is an estimate of the marker's distance from the
center of the chromosomal interval. The higher the number obtained for
a locus, the greater its distance from the center.
YAC, P1, BAC, and lambda clones studies.
YAC containing the human IIb and 3 genes were isolated by
polymerase chain reaction (PCR) screening of a YAC library (Human Genomic Library at the Center for Genetics in Medicine, Washington University, St Louis, MO). The library was screened using primers for
exon 21 of IIb (sense-5'-CAGACCTTCCAAGGGCAG-3'
antisense-5'-AGACTATGTGGCTCTAA-3')27 and for exon 5 of 3
(sense-5'-CTCTACCAGTGACATGGCTGA-3'
antisense-5'-GCAAGCTGAAACCGAGCCC TG-3').28
IIb and 3 P1 clones were obtained by screening of a genomic P1
library (DuPont Co, Glenolden, PA).29,30 A PCR-based
screening was used with the same IIb and 3 primers detailed above
for the YAC library screen. An IIb BAC clone was obtained by
hybridization screening of a human BAC library (Genome Systems, St
Louis, MO) using a random primer labeled probe for the 3.1 kb IIb
complementary DNA (cDNA).31
The IIb+-P1 clone, P1147 E2, was partially digested with
Sau 3a. Fragments ranging from 10 to 20 kb were isolated via
electroelution and ligated into the BamHI arms of a
 DASH vector (Stratagene, La Jolla, CA). A library of
phage was created following the manufacturer's directions. DNA was
extracted from PCR-positive clones,32 and used for routine
and PFGE Southern blots and sequencing as described below. Murine
IIb lambda phage were obtained by filter hybridization of a mouse
genomic library 129SV in FIX vector (Stratagene), using
a rat IIb genomic fragment as a probe.27 This clone was sequenced (see below), and primers for murine 129SV IIb exon 12 were
then used in the PCR screening of a murine BAC library (Genome Systems)
to isolate a corresponding BAC clone.
Southern, PFGE, and field inversion gel electrophoresis (FIGE)
blots.
High molecular weight genomic DNA for routine and PFGE analyses was
extracted from HEL and HeLa cells (American Type Culture Collection,
Rockville, MD)33,34 for routine Southern blotting as
previously described.32 For PFGE, the cells were split to a
density of 0.4 × 106 cells/mL 24 hours
before making the plugs. 2.0 × 106 cells were used
for each 200 µL plug. Cells were embedded in 1% Seakem GTG agarose
gel (FMC, Rockland, ME) and protease digested with proteinase K (Sigma
Co, St Louis, MO) as detailed elsewhere.35
Plugs containing either digested genomic DNA or undigested whole
chromosome YAC DNA were placed in the wells of 1% Seakem GTG agarose
gels containing 0.5× filtered TBE (22.5 mmol/L Tris-HCl, 10 mmol/L Boric acid, and 5 mmol/L EDTA-NaCl, pH 7.4) and then sealed into
the wells with 1% agarose. The gel was run in this buffer at 14°C
on a CHEF-DR II (BioRad, Richmond, CA). PFGE conditions were as
follows: 160V, 60-second pulse, for 20 hours followed by 180V,
90-second pulse for 12 hours. BAC and P1 clones were also restricted
and analyzed by both standard electrophoresis and FIGE. Running
conditions for FIGE involved a forward voltage of 180V and a reverse of
120V, and each ramped linearly for time periods from 0.2 to 1.8 seconds
over an 18-hour period. After standard electrophoresis, the gels were
stained with ethidium bromide and photographed. The DNA was then nicked
and transferred to Gene Screen Plus membrane (New England Nuclear,
Boston, MA) by salt transfer using 10 × sodium citrate chloride
(SSC).32 Filters were prehybridized in a 50%
formamide solution for 12 hours and the probe of interest (see below)
was random primer labeled with -32P-dCTPs,32
denatured at 100°C, and then added to the filter at a specific
activity of 1 × 107 cpm/mL. Hybridization was for 24 hours at 42°C. Filters were washed at various salt stringencies in
SSC as previously described,32 and then exposed to Kodak
X-OMAT film (Eastman Kodak, Rochester, NY). Filters were stripped of
probe in a 50% formamide, 1% sodium dodecyl sulfate (SDS), 0.1 SSC
solution at 75°C for 1 hour before reuse.
cDNA and PCR probes.
Full-length cDNA fragments, either the 3.3 kb IIb31 or
the 3.9kb 336, were random-primer labeled and used as
probes on routine PFGE and FIGE genomic blots. The EPB3 5' and
3' cDNA fragments were generated using Superscript RT-PCR (Life
Technologies, GIBCO/BRL) of human reticulocyte RNA (generously provided
by Dr Christian Stoeckert, The Children's Hospital of Philadelphia, Philadelphia, PA), and cloned into the Invitrogen TA cloning vector pCR.
Primer pairs were synthesized based on published sequences for the
IIb, 3, EPB3, and HOX2B genes to PCR to amplify desired subregions of these genes using genomic DNA as template. A list of the
primer sets are given here:
IIb-exon 227.
sense 5'-TGA TGT TTG AAC TGA ATT-3';
antisense 5'-AAG GCA GTA TGT GTA AAG CAT-3'.
IIb exon 3027.
sense 5'-TGG TCC AGG GAG GTG CTC AT-3';
antisense 5'-CTC AGT CTC TTT ATT AGG CA-3'.
5'EPB337.
sense 5'-GAG GAG AAT CTG GAG CAG GAG-3; antisense
5'-TAT GCG GAA CAC CCT CTC TGA-3'.
3'EPB338.
sense 5'-ACG CTG ATT GTC AGC AAA CCT-3';
antisense 5'-ACA GGC ATG GCC ACT TCG TCG-3'.
HOX 2B39.
sense 5'-GCT CTA TAG GAG GCC CTG AG-3'; antisense
5'-GAG GCT GTT TAG ATG AGA CA-3.
Each PCR-amplified genomic fragment used for probing the above
blots was sequenced to confirm its identity.
Sequence analysis in the IIb gene locus.
The above IIb-containing clones were end sequenced beginning with
the appropriate primers (eg, T7, SP6, and T3
primers29,32,40). In addition, the region from  500
bp upstream of the transcriptional start site of the human and murine
IIb gene to 12000 bp was sequenced beginning with primers
based on the published 5'-flanking region of the IIb
gene.27 Subsequent primers were generated based on the new
data and used to prime the next round of sequencing reaction. All
sequencing used fluorescenated dNTPs and Sequenase41 and an
ABI 373A automated sequencer.
New sequences determined in the IIb locus were analyzed using the
databases at the National Center for Biotechnology, which host an
internet site at URL: http://www.nlm.nih.gov/. Using the sequence
similarity program Basic Local Alignment Search Tool (BLAST),42 sequences from IIb containing
clones were entered into a Basic BLAST search using the BLASTn program
and the nr, month, or high throughput genomic sequence (htgs)
databases.43 Values greater than 500 with E values of 0.0 were evaluated further with the expressed sequence tag (dbEST), and
mouse and human EST databases.43
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RESULTS |
Linkage analysis in Glanzmann thrombasthenia pedigrees.
We have genotyped 254 members in 28 thrombasthenic families (Table 1)
for 10 polymorphic markers (Table 2). The results of such an analysis
in 1 Arab thrombasthenic family is presented in Fig
1. The pedigree illustrates the large
number of offsprings in each generation, the increased informativity in
the IIb gene due to heterozygosity for the thrombasthenia mutation,
and the presence of two recombination events. Nine recombination events were observed in the 28 families analyzed, and these were as follows: 2 paternal, 2 maternal, and one of unknown origin between THRA1 and the
other loci (BRCA1, D17S579, IIb, 3), 2 maternal recombinations between (THRA1, BRCA1) and (D17S579, IIb, 3), and 2 additional maternal recombinations between the 3 gene and the other loci (THRA1, BRCA1, D175579, IIb). These recombination events are consistent with the following order:
THRA1-BRCA1-(D17S579/IIb)-3.The results of pairwise linkage
analysis between the loci are presented in Table
3. The largest recombination distance of
4.6 cM (maximum Lod score, 17.85) was obtained between the THRA1 and
3 genes. The most probable distance between the IIb and 3
genes was 1.3 cM (Lod score 27.87). However, the odds were not
significantly different for distances of 1 to 4 cM. The location of the
IIb gene respective to the anonymous probe D17S579 could not be
resolved by linkage analysis because no recombinations have been
observed between these two loci in our families. We took another
approach to determine the order of the markers in this region by
comparing the haplotypes of the apparently independent chromosomes
bearing the common Iraqi-Jewish (IJ)-1 mutation in our families (Table 4). Nineteen different haplotypes have been
identified. A common, most probable founder haplotype, extending over
the whole interval was observed in 9 chromosomes. Using this apparent
haplotype for comparison, the type and number of the apparent
historical recombinations (denoted by  ) are summarized:[THRA1] [BRCA1, D17S579, IIb, 3] 14 times; [THRA1, BRCA1] [D17S579, IIb, 3] 10 or 11 times; [THRA1, BRCA1, D17S579] [IIb, 3] 4 or 5 times; and [THRA1, BRCA1, D17S579, IIb]
[3] 3 times. This pattern of historical recombinations
suggests the following order: THRA1-BRCA1-D17S579-IIb-3.
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| Fig 1.
Marker segregation in an Arab GT family near the
IIb-3 genomic locus. The extensive family tree for one of the
studied families with the determined inherited markers (Table 2) are
shown. Crossover regions are shown on a black background. The inherited
thrombasthenia gene is also indicated by the grayed regions in the
family tree over the affected chromosome. The polymorphic markers
studied are indicated at the top left of the fig and refer to Table
2.
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Another way of obtaining information about the position of the IIb
gene with respect to the neighboring markers, is to compare the
tightness of the linkage disequilibrium between IIb and the other
markers in the studied populations. In the Arab chromosomes, the 3
gene was the only locus for which a significant linkage disequilibrium
with IIb was found (P = .038). In the Iraqi-Jewish chromosomes, linkage disequilibrium was found to D17S855 (P = .076), D17S579 (P = .0004), and 3 (P = .00002).
These results support the map order inferred from the comparison of the
historical recombinations.
Orientation of the IIb and 3 genes.
To orient the two genes respective to their 5' to 3'
direction, HRA was applied to markers within the IIb and 3 genes.
The analysis was performed for independent normal chromosomes in the Arab GT families using the M4 and M5 markers in IIb and the M6 and
M8 markers in 3. Eleven distinct haplotypes and 32 high score haplotype pairs (those sharing 3 identical alleles) were identified in
our pedigrees. The relative distances of the markers from an arbitrary
center between the IIb and 3 genes computed by the HRA algorithm
were calculated. The markers M4 at the intron 3/exon 4 junction of the
IIb gene and M8 in exon 9 of the 3 gene were closer to this
center of the interval between the two genes (3.47 and 20.21, respectively). Markers M5 in exon 26 of the IIb gene and M6 in
intron 6 of the 3 gene were distant from the center (29.68 and
43.91, respectively). These results suggest that the 5' end
marker (exon 3) of the IIb gene is close to the 3' end marker
(exon 9) of the 3 gene, and that the 3 gene is upstream and in
the same orientation as the IIb gene.
Characterization of the physical distance between the IIb and 3
genes.
The 3 gene has a naturally occurring Sfi I site in it that splits
the gene into a 125 kb 5' fragment and a 260 kb 3'
fragment, and the 260 kb 3' 3 fragment was
reported to cohybridize with both 5' and 3' IIb cDNA
probes on FIGE genomic blots.6 This would suggest that the
5' end of the 17 kb IIb gene might be within 180 to 260 kb of
the 3' end of the 63 kb 3 gene. We set out to more precisely
determine the exact physical distance between these loci, using rare
enzymes that would split the IIb locus into 5' and 3'
oriented fragments. PFGE gels of human genomic DNA digested with rare
restriction enzymes were sequentially hybridized to IIb and 3
cDNA probes (top, Fig 2). IIb shared no
common restriction bands with 3. The closest in size were two Sfi I bands (arrows 4 and 5), which are clearly different in size on close
observation.
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| Fig 2.
Genomic and YAC PFGE hybridized to the IIb and 3
probes. Sequential rehybridization of the same filter to the IIb and
3 cDNAs, the EPB3 5' genomic fragment, a HOX 2b genomic
fragment, and the IIb exon 2 5' and exon 30 3' genomic
fragments. Numbered arrows indicate bands of interest.
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Because the IIb and 3 cDNA probes have no common bands on the
PFGE genomic blots, it was not possible to estimate the exact distance
between them. Instead we opted to determine the minimal distance
between them, by using 5' and 3' IIb-specific probes. The same PFGE blots were sequentially hybridized with a 5'
genomic IIb exon 2 probe and then with a 3' IIb exon 30 probe (bottom, Fig 2). The 5' IIb exon 2 probe, hybridized to
Nru I bands of 600 and 365 kb (arrows 2 and 3). In contrast, the
3' IIb exon 30 probe detected the identical 600 kb fragment
and a 235 kb band (arrows 1 and 3). Given that the full-length IIb
cDNA probe detected the same 600, 365, and 235 kb Nru I fragments, and
that the Nru I sites in the IIb gene were previously
defined,27 it appears that the 365 kb band contains the
5' end of IIb, the 235 kb band contains the 3' end of
IIb, and the 600 kb band is a partially-digested band containing
both bands. A restriction map, derived from the fragments produced on
the above PFGE blots and from sequence and restriction analysis of BAC
and P1 clones containing this region (data not shown, but see below) is
provided in Fig 3. This figure illustrates
the orientation of the three Nru I fragments. Genetic recombination
data presented above, suggest that the 3' end of the 3 gene is
closest to the 5' end of the IIb gene. Given that 3 is
telomeric to the IIb gene whose 3' end is oriented towards the
centromere,9-12 3 must be oriented in the same direction with its 3' end directly upstream and telomeric to the 5'
end of IIb (Fig 3). Because none of the 3 bands cohybridized to any of the three Nru I fragments, and given the fact that the upstream
Nru I site in the IIb gene is in exon 4, this suggests that the 3
gene cannot be less than ~365 kb 5' to the IIb gene.
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| Fig 3.
Schematic organization of the IIb (and 3) gene
locus region. The heavy line near the top indicates the gene locus
drawn to scale with the various identified genes indicated. A partial
restriction map for several rare restriction enzymes around the IIb
gene is also indicated. The restriction sites shown on this map were
generated using data from both pulsed-field genomic fragments (above)
and BAC, P1, and YAC sequence and restriction analysis (data not
shown). A summary of all clones that have been characterized is shown
below with the sequenced regions shown matching the genome or in white
rather than in gray. This map only focuses on the region surrounding
the IIb locus. A complete map of the 3 locus has been previously
reported.6,7,49 (All sequences reported here have been
registered with GenBank accession no. AF160252).
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Defining the genes closely linked to the IIb and 3 genes.
Additional candidate genes that colocalize to this region of chromosome
17 were studied by PFGE. Hybridization of PFGE Southern blots with both
5'- and 3'-specific probes for the EPB3 gene resulted in
bands that cohybridized with the IIb bands (Fig 2, arrows 1, 3, 7, and 8, for the 5' probe; 3' probe not shown). The 235 kb
Nru I fragment is the identical fragment that bound to the 3'
IIb exon 30 probe but not the 5' IIb exon 2 probe. These data reconfirm and expand the studies that had previously genetically and physically linked the IIb and EPB3 genes, and suggest that the
EPB3 gene is less than 235 kb downstream of the IIb gene.
Radiation hybrid maps, place the 3 gene near a cluster of genes that
includes the GIP and HOX 2B gene.12,13 Using a Hox 2B
genomic probe,39 we rehybridized the above PFGE blot and showed cohybridization of its bands with a number of 3 bands (Fig 2,
top and middle right panels, arrows 5 and 6), but with no IIb or
EPB3 bands, suggesting that the HOX 2B gene is more closely linked to
3 than either of the other two genes.
The last lane in each of the PFGE blots shown in Fig 2 contain DNA from
an IIb+-YAC clone (A206C3), which contains a genomic
insert of 200 kb.10 Both the IIb and EPB3 probes
hybridize to this YAC clone, but neither the HOX 2b nor the 3 probe
hybridize to it (Fig 2). Two other IIb+-YAC clones
(A75H8 and A247E2), containing insert sizes of 800 and 350 kb,
respectively, do not hybridize with 3, but A247E2, which extends 300 kb beyond the 3' end of IIb does hybridize with an EPB3 probe
(data not shown). Additionally, a BAC clone positive for IIb that
contains an insert of 220 kb also crosshybridizes to the EPB3 5'
and 3' probes but not the 3 probe (data not shown, but see Fig
3). Two 3+-YAC clones (A144B1 and B160F6), with insert
sizes 500 and 650 kb, respectively, do not crosshybridize with IIb
or EPB3 probes, but do crosshybridize to the HOX 2B probe (data not
shown). These data further suggest that the IIb gene is closely
linked to the EPB3 gene, and the 3 gene is closely linked to HOX 2B,
but that IIb and 3 do not appear to be closely linked to each other.
Further characterization of the genes immediately 5' and
3' of the IIb gene.
Screening of human BAC and P1 genomic libraries provided one BAC and
two P1 IIb+ clones. FIGE restriction mapping and
Southern blotting suggested that these clones overlapped and spanned
from 50 kb 5' of the IIb gene to 150 kb 3' of its
last exon (data not shown, but see Fig 3).
End sequence analysis of YAC A206C3 followed by a BLASTn nr
search42 showed the 5' end of the EPB3 gene (Fig 3).
Southern blot mapping and sequencing of our IIb+-BAC
clone with EPB3 primers, also confirmed its presence on the IIb+-BAC. The combination of this sequence analysis and
restriction mapping, not only showed that EPB3 is ~110 kb downstream
of the IIb gene, but that the two genes are oriented in the same
5' to 3' direction.
In addition, a DASH bacteriophage sublibrary was made
from one of the P1 clones (P1 1147 E2). Several of these clones
were mapped against each other, and the original P1 and BAC clones using both PCR and Southern blotting. The resulting contig is shown in
Fig 3. These BAC, P1, and lambda clones were end sequenced using the
natural T3, T7, and Sp6 sequences that flank the polylinker site.29,32,40 BLAST analysis of the sequences on the T7 end of P1 1147 showed that this clone ends near the Granulin or Epithelin gene.44 The Granulin gene ends ~18 kb 3' to exon 30 of the IIb gene and is in opposite 5' to 3' orientation
(Fig 3).
End sequence analysis followed by extensive overlap sequencing of clone
5'- IIb-1, showed that it began 9 kb upstream of
the transcriptional start site of the IIb gene and extended to exon
12 of the IIb coding region. At its 5' end, and continuing upstream into the sequence of P1 1147 (Fig 3), there is a 6 kb region
containing the gene for the previously defined human brain cDNA named
the KIAA0553 gene.45 This gene spans from ~ 12 kb to 5.8 upstream to the IIb gene transcriptional start site.
Additionally, FIX 129SV clones for the mouse IIb gene
were isolated initially by crosshybridization with a previously
described rat IIb 5' genomic region.27 In turn,
DNA sequence data for the murine IIb was used to isolate a murine
IIb+-BAC clone. Southern blot and sequence analysis of
these murine IIb clones showed that both the KIAA0553 and Granulin
genes are highly conserved in sequence, orientation, and relative
position. This further supports other reports that the murine and human syntenic relationships amongst genes in this region on chromosomes 11 and 17, respectively, are unparalleled in their degree of
conservation.13,14
| |
DISCUSSION |
The recent positional cloning of the BRCA1 gene within the 17q21 region
of chromosome 17 uncovered many useful polymorphic markers and
genes.46 Other markers within this region have been found
through the efforts of the Human Genome Project.11-13
Taking advantage of this high density of chromosomal markers, we have genetically mapped the locations and relative distances of several markers surrounding the platelet-specific IIb gene, to determine its
position relative to the gene of its integrin partner 3. Using
haplotype studies of 254 members in 28 thrombasthenic families of Arab
and Iraqi-Jewish descent, as well as historic recombination and linkage
disequilibrium data, we have determined the relative order of five
markers as follows: cen-THRA1-BRCA1-(D17S579-IIb)-3-qter. Pairwise linkage studies and HRA studies suggest a probable distance of
4.6 cM between THRA1 and the 3 gene and a distance of 1.3 cM between
the IIb and 3 genes, with the two genes being in the same
5' to 3' direction.
The determination of these linkage distances may also be useful in the
care of families with Glanzmann thrombasthenia. This bleeding diathesis
is due to qualitative or quantitative defects in the platelet
IIb3 receptor. Often, the bleeding is sufficiently severe that
families are interested in determining the at-risk status of
potentially affected fetuses. Unfortunately, the number of mutations
causing Glanzmann thrombasthenia is large. At present, over 40 distinct
mutations have been identified.47 Except in some
well-characterized, in-breed populations, the defect must be determined
for each affected family, and this can be labor and time intensive.
Therefore, to make a prenatal determination of risk for a fetus may
require linkage analysis of such polymorphisms as listed in Table
2.26,48 The likelihood of crossover between a mutation and
these polymorphisms decreases the ability to use linkage studies. Our
data shows the risk factor for a crossover between any two markers. We
propose that multiple markers in this region be used when examining the
linkage of a thrombasthenic mutation in a potentially affected fetus to
decrease the chance of recombination affecting the conclusion.
An earlier report based on PFGE suggested that the 3' end of the
3 gene was linked to the IIb gene on a 260 kb Sfi I fragment and
may have been as little as 125 kb apart6,49 although the orientation of the IIb gene was not defined. Our results support the
close physical and genetic linkage in that earlier report, except that
the actual distance between the two genes is greater than was initially
concluded. We suggest that the IIb and 3 genes are oriented in
the same direction with the 3' end of 3 closest to the
5' end of IIb, placing the IIb gene at a minimum distance
of 365 kb downstream of the 3 gene. Whether 5' or 3' of
IIb, the 3 gene, by our Nru I studies, is still greater than 235 kb away from its integrin partner. Furthermore, other
nonmegakaryocyte-specific genes are located between them.
The apparent inconsistency between our data and the previous studies
may be due to the difficulty in discerning the two closely-sized Sfi I
bands, a 180 kb IIb Sfi I band and a 160 kb 3 Sfi I band. By
using high-percentage agarose gels, we provided sufficient clarity to
distinguish the two bands on close examination. Also there have been
reports of band inversions during FIGE runs.50 Therefore,
FIGE might make two bands close in size appear to be identical during
certain periods of the run.
Sequencing of both the human and mouse IIb gene locus showed the
presence of the Granulin gene 18 kb 3' of the IIb gene (Fig
3). Granulins are potential growth factors that were initially discovered in leukocytes and bone marrow.44 They are also
expressed in myelogenous leukemic cell lines as well as in fibroblast,
epithelial, and kidney primary cells. Therefore, this gene is not only
present in the opposite orientation to the IIb gene, but also
appears to have a different profile of tissue-specific expression.
Our sequence analysis also identified the KIAA0553 gene spanning a
region from 6 to 12 kb upstream of the IIb gene and oriented in the
same direction in both human and mouse. Database comparison of the KIAA
protein sequence showed no homologous proteins. Nucleotide BLAST
comparisons also showed no homologies. BLAST comparisons against a
human EST database showed that the brain, ovaries, and testes contained
cDNAs for the KIAA gene. Thus, it represents a novel protein of unknown
function. RT-PCR studies of the expression profile of KIAA showed that
it was fairly ubiquitous with 10 out of the 14 tissues having
transcripts.45 Our own studies with platelet RNA suggest
that it is not expressed in megakaryocytes (data not shown).
The original view of the IIb-3 locus suggested that these genes
might be very closely linked and potentially could form a
platelet-specific expression locus under the regulation of a common
cis-regulatory element, such as a locus control element (LCR).
LCR domains have been mapped to other tissue-specific genes such as the
-globin and human growth hormone loci.51,52 Because the
two integrin and heterodimeric partners were physically linked
and expressed at high levels in a coordinated fashion during megakaryopoiesis, this hypothesis seemed plausible. However, given the
greater physical distance between these two genes, a common cis-regulatory element seems less feasible. Additionally, the fact that the IIb gene is surrounded by other genes that are not
expressed in platelets, further supports a model of separate regulation. With the appreciation that 3 is actually expressed in
multiple tissues besides megakaryocytes, it becomes clearer that the
two genes are likely to only be accidentally on the same chromosomal
locus without any functional implications to this physical association.
The IIb gene has been the most carefully studied platelet-specific
gene. Often such regulation not only involves the proximal promoter,
but also more distal regulatory elements. Our paper shows that the
IIb gene is surprisingly restricted in size at both its 5' and
3' ends, consisting of less than 6 kb of upstream sequence and
less than 18 kb of downstream sequence. Thus, the 6 kb region between
the KIAA gene and the IIb gene may potentially contain important
enhancer-like regulatory regions that would direct tissue- and/or
stage-specific expression of IIb in hematopoiesis. Moreover, this
region as well as the 18 kb 3' region between IIb and the
Granulin gene might be envisioned to have insulator elements that
protect the tissue expression of IIb from the influences of its
diversely expressed neighbors. The fact that the distance between
IIb and KIAA is conserved in mouse and human also supports the idea
that this region may contain important regulatory elements. Recently,
we have found several DNase I hypersensitive sites
(unpublished observations) within the region between KIAA and IIb,
two of which are constitutive and one which is tissue specific. It will be interesting to see if any of these sites represent these putative distal regulatory elements.
| |
ACKNOWLEDGMENT |
YAC clones studied were provided by Dr Francis Collins and S.C.
Chandrasekharappa, who at the time were at the University of Michigan
in Ann Arbor, MI. The screening of the P1 library and the isolation of
the P1 clones were done in collaboration with Dr Nat Sternberg at
DuPont Co, Glenolden, PA.
| |
FOOTNOTES |
Submitted March 25, 1999; accepted May 21, 1999.
Supported in part by grant HL40387 (M.P.), a grant from the Schulman
Foundation (M.P.) and a grant from The Council for Tobacco Research-USA, Inc (#3152, M.P.).
The publication costs of this
article were defrayed in part by
page charge payment. This article
must therefore be hereby marked
"advertisement"
in accordance with 18 U.S.C. section
1734 solely to indicate this fact.
Address reprint requests to H. Peretz, Clinical Biochemistry
Laboratory, Tel Aviv Sourasky Medical Center, 6 Weizman St, 64239, Tel
Aviv, Israel; e-mail: hperetz{at}tasmc.health.gov.il
| |
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ABSTRACT
INTRODUCTION