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Blood, Vol. 92 No. 11 (December 1), 1998:
pp. 4415-4421
Recombination Breakpoints in the Human  -Globin Gene Cluster
By
Rachelle A. Smith,
P. Joy Ho,
John B. Clegg,
Judith R. Kidd, and
Swee Lay Thein
From MRC Molecular Haematology Unit, Institute of Molecular Medicine,
John Radcliffe Hospital, Headington, Oxford, UK; Duke University School
of Medicine, Durham, NC; and the Department of Genetics, School of
Medicine, Yale University, New Haven, CT.
 |
ABSTRACT |
The human  -globin gene complex spans a region of 70 kb and
contains numerous sequence variants. These variant sites form a 5
cluster (5 -haplotype) and a 3 cluster (3 -haplotype) with
strong linkage disequilibrium among the sites within each cluster, but
not between the two clusters. The 9-kb region between the 5 and 3
clusters has been estimated to have rates of recombination that are 3 to 30 times normal, and the region has therefore been proposed as a
`hotspot' of recombination. We describe three families with evidence
of meiotic recombination within this `hotspot' of the -globin gene
cluster and in which the cross-over breakpoints have been defined at
the sequence level. In one family, the recombination has occurred in
the maternal chromosome within a region of 361 bp between positions
 911 and 550 5 to the -globin gene. In the other two families,
the recombination has occurred in the paternal chromosome within a
region of approximately 1,100 bp between positions 542 and +568
relative to the -globin gene cap site. Both regions occur within the
2-kb region of replication initiation (IR) in the -globin gene
domain with no overlap. The IR region contains a consensus sequence for
a protein (Pur), which binds preferentially to
single-stranded DNA, a role implicated in recombination events.
© 1998 by The American Society of Hematology.
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INTRODUCTION |
RECOMBINATION BETWEEN homologous DNA
sequences plays an important role in generating genetic diversity in
all organisms. Although meiotic recombination occurs throughout the
human genome, it does not occur randomly but appears to be concentrated
in specific regions.1 Such areas of relatively increased
recombination frequency are present in the major histocompatibility
complex (MHC),2,3 where they are responsible for Ig class
switching, near or within the Duchenne muscular dystrophy, insulin,
collagen, and the -globin gene loci.4-8 The human
-globin gene region on chromosome 11p is one of the most intensively
studied of all human loci; mutations here cause -thalassemia and
sickle cell anemia, which are among the most common genetic diseases in
the world.9 The earliest prenatal diagnosis of these
disorders using DNA analysis relied on linkage disequilibrium of
-globin gene mutations with adjacent restriction fragment length
polymorphisms (RFLPs).10,11 This procedure always carries
some degree of risk of recombination between the mutant allele and the
RFLP. Indeed a meiotic recombination in the paternal chromosome in one family has caused an error in the prenatal diagnosis of
-thalassemia using this approach.12
Over 20 RFLPs have now been identified in the -globin
locus.13 These polymorphisms fall into two groups: a 34-kb
5 cluster that includes the HindII- ,
HindIII-G ,
HindIII-A, HindII- ,
HindII-3, and Taq I-5 polymorphic
sites, and a 19-kb 3 cluster that includes the Hgi AI-,
AvaII- and BamHI- sites14 (see
Fig 1). Population genetic analysis showed that while sites within the
two clusters show strong linkage disequilibrium to each other, no
linkage disequilibrium exists between these two clusters and the
polymorphic sites within the 9-kb region separating the two clusters
are randomly associated.15 Indeed, a recombination rate of
3 to 30 times greater than expected was found in the region, which is
the site of 75% of the recombination events in the entire -globin
cluster.8 Thus, this region has been implicated as a
`hotspot' for genetic recombination. Studies have identified four
separate families with evidence of recombination events within this
`hotspot'.12,16-18 The localization of the crossover
events in these families has been limited by the number of
informative RFLPs available, and the narrowest localization was
approximately 10 kb.18
Several sequence elements have been proposed as candidate recombination
signals, some of which have been identified in the 9-kb `hotspot.'
The `hotspot' encompasses the 2-kb region of replication origin in
the -globin gene domain within which is a 16-bp consensus sequence
for the Pur element.19,20 In addition, upstream
of the initiation replication region, but still within the `hotspot,' lies a 21-bp consensus sequence (with two base mismatches) that is
unique to potential initiation regions.21 It is proposed that both the Pur and the 21-bp element facilitates DNA
replication and recombination by initiating the unwinding of duplex DNA
or maintaining an open duplex. Here we report on three families with evidence of recombination events within the `hotspot' in the
-globin cluster. Two (Greek Cypriot and Asian Indian) are previously
unreported, while the third family of Amish origin was previously
reported by Gerherd et al.16 In an attempt to address the
role of the various putative recombination signals in the `hotspot,'
we have undertaken direct sequence analysis to delineate the crossover region. Two distinct regions of crossover within the 9-kb `hotspot' were defined, which suggests that recombination sites in the -globin complex show a clustering pattern also observed in the human HLA class
II region.3 The breakpoint in each family occurs within the
2 kb of replication initiation in the -globin gene domain.
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FAMILIES AND METHODS |
Recombinant families.
Three families (Greek Cypriot, Asian Indian, and Amish) were previously
identified by typing of the RFLPs (-haplotype analysis) in the
-globin gene cluster. All families analyzed included both parents
and at least three offspring. The Amish family is part of
a Centre d'Etude du Polymorphisme Humain (CEPH) family.
Informed consent was obtained in all cases before the collection of
blood samples.
-Haplotype analysis.
DNA was extracted from peripheral blood leukocytes or lymphoblastoid
cell lines using standard procedures and analyzed by restriction enzyme
digestion. Haplotypes were derived from the following RFLPs in the
-globin gene cluster HindII-,
HindIII-G,
HindIII-A, HindII-,
HindI-3 , AvaII-, BamHI-,
Taq I-5 , Pst I-3 , HinfI-5
, Rsa I-5 , and HgiAI-. The RFLPs were
analyzed using a combination of standard Southern blot
hybridization15 and restriction enzyme analysis of DNA that
was specifically amplified by the polymerase chain reaction (PCR) (see
Fig 1). PCR amplification of the various polymorphic sites was
performed in a total volume of 100 µL containing 10 mmol/L Tris-HCl
pH 8.3, 50 mmol/L KCl, 1.5 to 2.5 mmol/L MgCl2, 0.2 mmol/L
each of dTTP, dGTP, dCTP, and dATP, 10 pmol of each primer, and 2.5 U
of Taq polymerase (Cetus, Perkin Elmer,
Warrington, UK). The sequences of the primers used and details of the
conditions of amplification are available on request.
DNA sequence analysis.
A 4.5-kb DNA fragment encompassing the 3 - globin gene region,
exons 1 and 2, and intron 1 of the -globin gene was enzymatically amplified using two sets of primers, 5R3K and 3R5K and 5422 and
39. Primers 5 R 3K (5-AAT CTG TAC ATC AAG ACC CAG TGA TAT G-3)
and 3 R5K (5-GAC ATC TAA CTG TTT CTG CCT GGA CT-3) corresponding to
GenBank (HUMHBB U01317) coordinates 58299-58326 and 61315-61290, respectively, direct the amplification of a 3,016-bp fragment in the
- region (Fig 1). PCR amplification
was performed in a 100-µL reaction volume containing 0.2 mmol/L of
each dNTP, 50 mmol/L KCl, 10 mmol/L Tris-HCl pH 8.3, 2.0 mmol/L
MgCl2, 2.5 U Taq polymerase (Cetus), and 20 pmol
of each primer 5 R3K and 3 R5K. After an initial denaturation of 4 minutes at 95°C, 30 cycles of denaturation at 95°C for 1 minute,
annealing at 64°C for 2 minutes, and extension at 72°C for 3 minutes were performed, the last extension reaction was prolonged for 8 minutes. An aliquot of the PCR product was examined on a 1.0% agarose
gel. The 3.0-kb amplification product was isolated and purified using a
QIAEX II Gel extraction kit (Qiagen Ltd, Surrey, UK) after
electrophoresis in a 1.0% agarose gel. The purified PCR product was
resuspended in 40 µL 10 mmol/L Tris-HCl pH 8.3. A small aliquot (3/40
µL) of the DNA was examined in a 1% agarose gel to check for purity and concentration. Fifty to 500 ng (generally 6 of the 40 µL) of the
product was directly sequenced using the thermal cycle sequencing
technique with 33P-labeled terminators and
Thermosequenase (Amersham, Buckinghamshire, UK). A total sequence of 3,016 nucleotides was determined
using a series of forward and reverse primers.
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| Fig 1.
Map of the -globin gene cluster with the 9-kb
`hotspot' delineated by the TaqI-5 (5) and the
HgiAI- (3) sites. RFLP sites are indicated by the
arrows. The 4.5-kb fragment encompassing the 5 flanking region is
amplified using two sets of primers: 5R3K versus 3R5K and 5422
and 39. The other polymorphic sites are indicated by vertical
lines. The pur, chi, and the 21-bp element at position
355021 are represented by thick horizontal lines. The
crossover regions and the region of initiation replication (IR) are
indicated below. A total of 11 and 14 primers were used to sequence the
forward and reverse strand of the 4.5-kb region, respectively. The
sequences and locations of these primers are available on request.
|
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Primers 5 422 (5-TCC AGG CAG AAA CAG TTA GAT GTC-3) and 3 9
(5-CAT TCG TCT GTT TCC CAT TCT A-3) corresponding to GenBank coordinates 61292-61315 and 62745-62724, respectively, direct the
amplification of a 1,453-bp fragment encompassing the 5 end of the
-globin gene from position 845 through to position 609 relative
to the cap site including exon 1, intron 1, and exon 2 of the
-globin gene (Fig 1). PCR amplification was performed in a 100-µL
reaction volume containing 0.2 mmol/L of each dNTP, 10 mmol/L Tris-HCl
pH 8.3, 50 mmol/L KCl, 2.5 mmol/L MgCl2, 2.5 U of
Taq polymerase (Cetus), and 20 pmol of each primers 5422 and 39. After an initial denaturation of 4 minutes at 94°C, 30 cycles of denaturation at 94°C for 1 minute, annealing at 59°C for
2 minutes, and extension at 72°C for 3 minutes were performed, the
last extension reaction was prolonged for 7 minutes. After checking for
amplification, the PCR product was isolated using a QIAEXII Gel
extraction kit (Qiagen Ltd, Surrey, UK) as before. The purified DNA
fragment was directly sequenced by the thermal cycle sequencing
reaction using 33P-labeled terminators and a series of
forward and reverse primers.
DNA fingerprinting.
To exclude false parentage, DNA from each member of the families was
digested with HinfI, Southern blotted, and hybridized with a
panel of seven probes known to be specific for hypervariable minisatellites (MS1, MS8, MS29, MS31, MS43, MS51,
P g3).22 These hypervariable loci are extremely variable
with heterozygosities ranging from 90% for MS8 to 99% for MS31 and
are dispersed over four autosomes. The locus-specific minisatellites
act as very sensitive hybridization probes for these loci and can be
pooled to detect the hypervariable (HVR) loci simultaneously giving
rise to multilocus Southern blot patterns that are highly
individual-specific (DNA fingerprints). Based on the heterozygosities
and the average number of DNA fragments (8.4) resolved using a mixture
of five probes, the chance that all of the fragments in one individual are present in a second randomly selected individual has been estimated
at <6 × 10 7.22 Therefore, these patterns
provide a high level of individual specificity.
| |
RESULTS |
Greek Cypriot family.
In this family, both parents were heterozygous for different
-thalassemia mutations (+ 33 C  G in I1 and
o 39 C T in I2), and all three offspring were
compound heterozygotes23 (Fig
2).
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| Fig 2.
The Greek Cypriot family illustrating segregation of the
haplotypes and informative recombination events. Both parents are
heterozygotes (+ 33 C G in I1 and
o39 C T in I2) and all three offspring, compound
heterozygotes for -thalassemia. Haplotypes P1 and M1 are
associated with the paternal and maternal -thalassemia alleles,
respectively. While individuals II2 and II3 have inherited the P1 and
M1 haplotypes intact, individual II1 has inherited P1 and a maternal
recombinant chromosome, as identified by the haplotype M1/M2. The site
of recombination is located within the 361-bp interval between
positions 911 and 550 relative to the -globin gene cap site.
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The haplotypes for each member of the family were derived from the
results of 14 RFLPs (HindII-,
HindIII-G,
HindIII-A, HindII-,
HindII-3 , AvaII-,
BamHI-, Taq I-5, Pst I-3, HinfI-5 at positions 1823 and 990, Rsa
I-5, HgiAI-, and HinfI-3 (see Fig 2).
There are four haplotypes in this family, the phase of the RFLPs
was established using the two virtually homozygous offspring, II2 and
II3. Both II2 and II3 have inherited the -thalassemia alleles from
the father (I1) and the mother (I2) associated with haplotypes P1 and
M1, respectively. II1, who is also a compound heterozygote for
-thalassemia, has inherited an intact paternal chromosome, P1.
However, while II1 has inherited the maternal -thalassemia mutation,
the -thalassemia allele in II1 is associated with the 5 haplotype
of M2 and the 3 haplotype of M1. On the basis of RFLP analysis, the M2
chromosome is intact 5 up to the Taq I-5 site, and the
M1 chromosome is intact 3 up to the Rsa I-5 site, but
the origin of the variant sites in the 10.4-kb segment between these
two sites, including the Pst I-3 and
HinfI-5, is not clear.
Direct sequence analysis of the area between the Taq I-5
and Rsa I-5 site was undertaken to search for additional
sequence polymorphisms. The analysis identified several polymorphisms; the T/C polymorphism at position 1866, G/A polymorphism at position 1069, deletion of a single C at position 911 (a novel
polymorphism), and A/C polymorphism at position 704 from the cap
site of the -globin gene (see Fig 2). Analysis of the
(TG)n repeat24 was not attempted as it lay
outside the area of interest.
The direct sequence analysis established that the proband, II1, had
inherited an intact maternal M2 chromosome from HindII- to the C deletion at position 911 (5 to 3), and an intact M1 chromosome from BamHI- to the Rsa I-5 site
at position 550 relative to the -globin gene cap site (3 to 5)
(Fig 3). The segment bordered by these two
sites is 361 bp; no informative polymorphisms were found within this
segment.
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| Fig 3.
DNA sequence immediately 5 of the -globin gene in the
Greek Cypriot family. The individuals II1, I1, I2, II2, and II3 (as
shown in Fig 2) are represented by lanes 1, 2, 3, 4, and 5. The
termination reactions are loaded in consecutive lanes in a block, ddGTP
(G), ddATP (A), ddTTP (T), and ddCTP (C). (Panel A) The 5 breakpoint
indicated by the 1-bp deletion (arrowed) at position 911; (Panel B)
the 3 breakpoint indicated by the arrow (Rsa I site) at
position 550.
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Asian Indian family.
The haplotypes of both parents and the three offspring were
generated from 13 RFLPs: HindII-,
HindIII-G,
HindIII-A, HindII-,
HindII-3 , Taq I-5,
HinfI-5 at position 1823, HinfI-5 at
position 990, Rsa I-5, HgiI-,
AvaII-, HinfI-3, and BamHI-
(see Fig 4). In this family, the
inheritance of the -alleles is not associated with thalassemia.
Phase of the RFLPs was established in the children based on the phase
in the mother, I2, who was homozygous for most of the RFLPs. Two of the
children, II2 and II3, have inherited intact chromosomes, P2 and M2,
from their father (I1) and mother (I2), respectively. The proband, II1,
has inherited an intact chromosome (M1) from the mother but the
paternally inherited chromosome comprised of the 5 end of P1 (up to
Hind-3) and the 3 end of P2 (up to
HinfI-3). The origin of the chromosome in the 15-kb
segment between the HindII-3 and
HinfI-3 sites is not clear from RFLP analysis because
the sites within it are uninformative both in the father and proband.
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| Fig 4.
The Asian Indian family showing segregation of the haplotypes and informative recombination events. Individual II1 has
inherited a paternal recombinant chromosome, as identified by the
haplotype P1/P2. The site of recombination is located within a region
of ~1,100 bp between the (AT)xTy repeats at
position 542 and the G/T polymorphism at position +568.
(AT)xTy at position 542 is represented by A:
(AT)7T7 and B:
(AT)9T5.
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Sequence analysis of the 5 of the -globin gene showed several
polymorphisms, including the T/C polymorphism at position 1866, (ATTTT)n at position 1411, G/A polymorphism at
position 1069, A/C polymorphism at position 704,
(AT)xTy at positions 542 to 522, and G/T
polymorphism in intron 2 (at position +568 from the -globin gene cap
site).
The sequence analysis established that II1 had inherited the 5 P1
chromosome intact up to the (AT)xTy region and
that the 3 P2 chromosome was inherited intact up to the G/T
polymorphism in intron 2 of the -globin gene (Fig
5).
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| Fig 5.
DNA sequence of the 5 region of the -globin gene in
the Asian Indian family. The individuals I1, I2, II1, II2, and II3 (as
in Fig 4) are represented by lanes 1, 2, 3, 4, and 5, respectively. The
termination reactions are loaded in consecutive lanes in a block, ddGTP
(G), ddATP (A), ddTTP (T), and ddCTP (C). (a) The 5 breakpoint of the
crossover region is located in the region of the
(AT)xTy repeats; (b) the 3 breakpoint is
located at the G/T polymorphic site at position +568 (arrowed).
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Amish family.
Previous study on the third family, of Amish descent, by Gerhard et
al,16 had established that a single recombination event occurred in the paternal chromosome of II2 between the Taq
I-5 and BamHI-3 sites, within a region of 15 kb (see
Fig 6), which was confirmed by our RFLP
analysis of the same sites. Our analysis of additional RFLPs suggested
that the putative recombination occurred within a 2.5-kb segment
between the Rsa I-5 and the HinfI-3
sites. There are three haplotypes in this family; the parents I1 and I2
share a common haplotype (M1 and P1).
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| Fig 6.
The Amish family showing segregation of the haplotypes and informative recombination events. Individual II2 has
inherited a paternal recombinant chromosome as identified by the
haplotype P1/P2. The site of recombination is located within a region
of ~1,100 bp between the (AT)xTy repeats at
position 542 and the G/T polymorphic site at position +568.
(AT)xTy is represented by A: (AT)
7T7 and B: (AT) 9T5.
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Direct sequence analysis proved that the family was informative at the
same sequence polymorphisms as in the Indian family with similar
recombination breakpoints, ie, the (AT)xTy site
at positions 542 to 522 and the G/T polymorphism at position
+568.
There are four possible explanations for the recombinant chromosomes in
the three families: (1) a meiotic recombination event has occurred
within the -globin cluster; (2) false parentage; (3) point mutations
at multiple sites; or (4) gene conversion. False parentage had been
previously excluded in the Amish family.16 In the
Greek-Cypriot family, Southern blot hybridization of HinfI digest with a pool of seven hypervariable minisatellite probes showed
10 bands in the father, 14 in the mother, 13 in II1, 9 in II2, and 11 in II3. Of the 13 bands in I1, 6 were shared with father and 7 with
mother. Of the 9 bands in II2, 5 were shared with father and 4 with
mother. Of the 11 bands in II3, 6 were shared with father and 5 with
mother. Similarly, in the Asian Indian family, 12 bands could be scored
in the father, 10 in the mother, 11 in II1, 10 in II2, and 12 in II3.
In II1, 5 of the 11 bands were shared with father, 6 with mother; in
II2, 5 of the 10 bands were shared with the father and 5 with the
mother, while in II3, 6 of the 12 bands were shared with father and the other 6 with mother. In all cases, all the alleles present in the
offspring could be traced to either of the parents, indicating that the
genetic relationships have been correctly assigned with no evidence of
false paternity or maternity. The third explanation is unlikely because
it requires several independent point mutations at the different RFLP
sites and, in the case of the Greek family, including the C-T mutation
at codon 39 of the -globin gene to create a -thalassemia mutation
identical to the one in the mother. The fourth explanation is not ruled
out but seems unlikely as it would involve gene conversion of a large
stretch of DNA (~22 kb) in the Amish and Asian Indian families. In
the Greek Cypriot family a conversion of ~1 kb of M2 by M1, from 3
of the A/C at position 704 to 3 of the thalassemia mutation at
codon 39, could explain the composite maternal chromosome in II1.
The most likely explanation is that a meiotic recombination has
occurred within the -globin gene cluster, between the C deletion at
position 911 and the Rsa I site at position 550 in the
Greek family, and between the (AT)xTy repeats
at position 542 and the G/T site at position +518 in the Asian
Indian and Amish families.
| |
DISCUSSION |
Haplotypes generated from RFLPs have led to the identification of a
recombination event in the -globin gene cluster in two families,
bringing the total reported to six. We then used DNA sequence analysis
to delineate the region of crossover to 361 bp in one family and 1,100 bp in the other family. The region of crossover was also sequenced in a
previously reported family and this corresponded to the same region of
1,100 bp. These represent the most precisely defined region of
recombinations within the human -globin gene cluster.
The 9-kb region immediately 5 to the -globin gene has been proposed
as a recombination `hotspot' with recombination rates of 3 to 30 times higher than those of the surrounding regions. The `hotspot,' as
identified by Chakravarti et al,8 extends from the
Taq I-5 site to the HgiAI- site (Fig 1).
Although the crossover region falls within this `hotspot' in one
family (Greek Cypriot), the 3 boundary of the crossover region in the other two families extends ~500 bp 3 beyond the `hotspot.'
However, in the absence of any further polymorphisms within the
1,100-bp segment, it is quite possible that the cross-over region in
the Amish and Asian Indian families falls within the boundaries of the
9 kb `hotspot.'
Previous studies have suggested several potential signal sequences that
could enhance recombination within the `hotspot.'25 These
included the (ATTT)n, (TG)n, and
(AT)xTy repeats. In all three families, the
crossover regions are located 3 of the (TG)n24 and (ATTTT)n repeats which lie ~1,800 bp and ~500 bp,
respectively, upstream of position 911 (the 5 crossover breakpoint
in the Greek family) which makes these repeats as unlikely candidates initiating recombination, at least in these three families.
Interestingly, a previous study of sequence-specific meiotic
recombination in Saccharomyces cerevisiae comparing three
adjacent restriction fragments immediately 5 of the human -globin
gene showed that deletion of the (TG.CA)n sequences at the
2678 to 2648 interval does not significantly reduce the high
frequency of genetic recombination in this region.26
It is of note that in all three families, the crossover region lies
within the small well-defined 2-kb fragment (between positions 1461
and +476) which contains the replication origin of the -globin cluster.19,27 This initiation region (IR) contains a 16-bp consensus sequence (5-GGNNGAGGGAGARRRR-3 at positions 63 to 48)
for the Pur protein, which has been shown to have
preferential binding for single-stranded DNA.20 On this
basis it has been suggested that Pur would be expected to
function as a helix-destabilizing protein, especially at the position
of the Pur element, a role implicated in initiation of
replication and recombination. The Pur element is located
within the 1,100-bp crossover region. Although the Pur
element lies outside the crossover region in the Greek family, it is
still possible that it is involved in the initiation of
recombination/replication as initial duplex opening is not restricted
to a single site but can occur throughout within each initiation
zone.28 Evidence to support this possibility includes, firstly: in many mammalian loci where the Pur element has
been identified, it is located at least 1 kb away from the actual start site of replication.20 Secondly, it has been observed in
primate cells that the binding of replication initiator protein dnaA, for oric of Escherichia coli,
destabilizes the DNA helix to promote duplex opening at a
region removed from the original recognition site.29
Frequently, this region is a reiterated AT-sequence,30 such
as the (AT)xTy repeats, which forms the 5
breakpoint of the 1,100-bp crossover regions. Furthermore, the 3
breakpoint of the 361-bp crossover region also lies within an AT-rich
sequence.
Another consensus DNA sequence implicated in the initiation of DNA
replication and recombination is the 21-bp 5-WAWTTDDWWWDHWGWHMAWTT-3 where M = A or C, W = A or T, D = A or G or T, and H = A or C or
T.21 This consensus is found, with two base mismatches,
within the `hotspot' at position 3550 upstream of the -globin
gene. However, it is located ~1 kb outside the IR region and
certainly outside the crossover regions in the three families. The
1,100-bp crossover region also contains a chi ( ) sequence
(5-GCTGGTGG-3) at ~300 bp 5 of the G/T polymorphic site in exon 2 of the -globin gene. The sites are known to be hotspots for
homologous recombination in E coli, increasing recombination
by 5- to 10-fold.25,31,32 However, the exact role of these
elements in recombination remains unclear.
Although all the three crossover regions fall within the broad 9-kb
`hotspot,' sequence analysis indicates that the crossover events
occurred at distinct sites within this area. It is possible that this
9-kb hotspot contains several smaller areas with increased rates of
recombination that are nonrandomly distributed. This suggests a
clustering of recombination breakpoints such as has been observed in
the MHC complex where the crossover breakpoints occurred within several
defined areas associated with an increased frequency of recombination,
all located within the larger `hotspot.'3 Despite
sequence analysis, only an estimate of the minimal crossover regions
can be obtained, the limiting factor being the presence of informative
polymorphisms. In one family this region is 361 bp and 1,100 bp in the
other two families. In contrast, sequence analysis of a crossover site
was defined to a 138-bp segment in a recombination event in the HLA
class II region.33
Although only six families with recombination events within the
-globin cluster have been reported to date, in all six the crossovers occurred within the region immediately 5 to the -globin gene, which suggests that the frequency of recombination in this area
is higher than those in the rest of the complex. These observations
support the results of a recent study of allelic sequence diversity at
the human -globin locus which concluded that recombination and gene
conversion in the 5 region of the -globin gene has contributed to
the -haplotype diversity, which was higher than expected on the
basis of the observed nucleotide polymorphisms.34 However,
to fully appreciate the frequency of recombination, ie, to establish if
this is truly a `hotspot' or just a `warm patch' in cold
surroundings, a systematic study of the -globin cluster compared
with its flanking regions on chromosome 11p involving sperm DNA typing
or a larger number of families (such as the other CEPH pedigrees) is
needed.
| |
ACKNOWLEDGMENT |
We thank Dr John Old for the gift of some of the PCR primers; Prof Sir
D.J. Weatherall for encouragement and support; and Liz Rose and Milly
Graver for preparation of the manuscript.
| |
FOOTNOTES |
Submitted June 1, 1998;
accepted July 28, 1998.
P.J.H. was a Nuffield Dominions Fellow; R.A.S. was supported by the
Medical Research Fund, Oxford; and J.R.K. is supported in part by a
grant (SBR-96 32509) from the National Science Foundation (USA). We
thank the MRC, UK, for support.
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 Swee Lay Thein, MD, Medical
Research Council Molecular Haematology Unit, Institute of
Molecular Medicine, John Radcliffe Hospital, Headington, Oxford, OX3
9DS UK; e-mail: swee.thein{at}imm.ox.ac.uk.
| |
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His and hers recombinational hot-spots.
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Abstract
Introduction