From www.bloodjournal.org by guest on June 16, 2017. For personal use only.
CONCISE
REPORT
Unequal
Crossing-Over:
in Asians
and American
By John
A.
Phillips
A Common
Blacks
Terry A. Vik, Alan F. Scott,
Ill,
Virgil
The
a-globin
which
to
genes
there
have
one
T
patterns
mechanism
by
two
were
lacking
DNA
both
obtained
of the
single
a
3700
Chinese,
and
genes.
and
in these
that
tional a-globin
genes occur in 3%-7%
Malaysians
and 20%-3
I % of Thais.5
with
HbH
Jr.
disease
endonuclease
a single
identical
femtoliters);
(3.7
HbH
kb).’3
and
(2)
(0.2-0.4);
(3)
(an
a
in fresh
consistent
Nuclear
phoresis
DNA
be
in
For
but most of the
retained,
thereby
having
blacks,
three
clease
were
HbH
1066
the common
genes, we
of subjects
with
HbH
selected
mechanism(s)
studied
the
solely
on
a
was
with
a-globin
analyses
identical
and
from
gels.
a-globin
(I)
amount
were
these
fragment
of the
probe
the
basis
mispaired
a
of their
as
Md.;
Rochester.
of
their
Diego.
AND
was confirmed
by at least
(mean
corpuscular
four
of
Medical
a genes
at saturation.
and
DNA
with
per
Under
complementary
of hybridization
3 a-globin
and
genes
School
Laboratory
Naval
conducted
for
and
the
Biochemical
ofMedicine,
Baltimore,
Medicine.
Regional
at
guidelines.
Medicine
Laboratory
University
the
were
the NIH
Pediatrics
Institute
of
and
of a probe
four
0, 1 , 2 and
recombinant
Hopkins
amount
percentages
with
individuals
of quanti-
respectively.7
in accordance
Minn.;
relative
a
with
Mayo
Clinic,
Medical
Center.
San
of Health
Grants
AM
Calif
Supported
in part
by National
(J.A.P.),
AM
13983
/5026
(K.D.S.)
and
a grant
Institutes
(H.H.K.),
from
RR
the
05378
National
(J.A.P.),
Foundation,
and
HL
6-194
(H.H.K.).
Submitted
February
reprint
of
Pediatrics,
1980
by Grune
1 9. 1 980;
requests
Johns
accepted
to John
March
A. Phillips
Hopkins
Hospital,
10, 1980.
III,
M.D.,
Baltimore,
DepartMd.
21205.
of the following
volume
for genomes
and 43%,
Department
controls
and
and method
containing
the
gene
and
to completion
of probe
and
equal;
00423
ment
METHODS
The
translaFor
1.710
was prepared,
hybridized
DNA,
gene
by the “nick
polymerase
50% of the probe
Departments
Johns
the
normal
involving
Hughes
Genetics,
was
from
Forget.9
of approximately
a probe
concentrations
are
33%,
the
Address
MATERIALS
microcytosis
genes
the
containment
Howard
probe
obtained
a-globin
activity
DNA
coli
hybridize
expected
are 0%, 20%,
Thereafter,
Bernard
containing
and cs32P-dCTP
reported.7
so that
sequences
then
was
by Dr.
to a specific
as previously
conditions,
genomic
by electrotransferred
sequences
DNA
(3 mg/mI)
from
Hybridization
conditions
cell, would
diploid
restric-
was
the a probe.
provided
the
of this
chosen
with
meth-
with
separated
DNA
single-stranded
genomic
disease.
was
(6)
electrophoresis,
digested
were
The
gene
a-32P-dATP
constant
used
of
and
by standard
blood,
hybridized
of Escherichia
experiments,
tation
ratios
presence
and autoradiographed.7’
generously
counting
From
a
genes. Furthermore,
restriction
endonuof the single
remaining
a-globin
gene
in all subjects
and compatible
with
between
of origin.
disease
function
sonicated
with HbH
(4)
deficiency;
determined
fragments
radiolabeled
*Experiments
producing
genes
in
Hbl-I
disease.
All I 2 subjects
(5 American
2 Chinese,
and 5 Filipinos)
had deletions
crossing-over
mechanism
criteria:
by Orkin’s
a genes in
Israelis
Restriction
synthetic
HbF;
hemoglobin
were
slab
washed
JWIO1,
lO cpm/zg
and
of iron
(MCV),
DNA
hybridizations,
P2-EK2
To determine
single
a-globin
series
and
in
found
crossing-over
a/13-globin
absence
isolated
agarose
for human
filter
(5)
was
filters
were
sequences
tion”
precluding
unequal
crossing-over
as a mechanism
for
the deletion.3
Unequal
crossing-over
as a common
Turks,
a).
unequal
of HbA2
ratios
and
in 0.8%
A probe
decreased
volume
synthetic
endonucleases,
(ion
and
single
a gene could
have originated
by unequal
crossing-over
between
a genes.6
However,
Embury
et al.
have reported
another
Asian
with a single
a gene in
Mediterraneans,
disease.6
I
-
were
studies.
corpuscular
the plasmid
is also challenged
but nondeleted,
state
subjects
-
with
amounts
hemolysates;
and a/3-globin
ads.7
markedly
family
Mean
the filters
genome.3’5’6
The data
of Orkin
et al. on a
subject
with HbH disease
suggest
that his
for single a genes
of dysfunctional,
(
gene
(-
basis
report
Smith,
a-thalassemia
All
compatible
normal
to nitrocellulose
or nonfunc-
of Chinese
whom
the 5’ a gene was deleted
sequences
between
the genes were
D.
individuals.
Chromosome
16s with a single a-globin
gene can
studied
in HbH
disease,
an a-thalassemia
state
which
there
is only
one functional
a gene
-/--a)
per diploid
single Asian
Kirby
,
techniques.
containing
were
16 contains
of which are
deleted
Haig H. Kazazian,
Filipinos
another
all 1 2 subjects
a gene
nucleotides
suggest
newborns
five
a-Globin
Genes
Disease
M. Koenig
by restriction
Abnormal
chromosome
I 6s bearing
a single functional
a-globin
gene lead to mild a-thalassemia
syndromes.
For example,
Dozy et al. have reported
that 16% of
chromosome
l6s in American
blacks
have a single a
gene ( -a).4
Furthermore,
hemoglobin
screening
studies of Asian
E. Young,
and Harold
analyzed
from
human
chromosome
genes (aa), the centers
about
Keith
F. Fairbanks,
Americans.
a gene)
1 6.
of the
of origin
HE NORMAL
two a-globin
separated
black
functional
chromosome
endonuclease
as the
of five
is a single
Basis
of Single
With
Hemoglobin-H
of
less
than
c Stratton,
six
©
60
0006-4971/80/5506-000l
Inc.
$01 .00/0
Blood,
Vol.
55. No. 6
(June),
1980
From www.bloodjournal.org by guest on June 16, 2017. For personal use only.
UNEQUAL
CROSSING-OVER
IN a-GLOBIN
1067
GENES
RESULTS
The
number
HbH
the
their
med
their
disease
percentage
genomic
annealed
having
of
a-globin
subjects
genes
was
of single-stranded
in
the
‘4.4
by determining
a probe
annealed
0.4
by
DNAs.
DNAs
from all subjects
examan amount
of a probe
consistent
with
a single a-globin
gene per diploid
genome
(Fig.
1). Thus,
all subjects
had
lacking
both a genes and another
gene
-/-a).
Restriction
remaining
ascertained
#{174}
A,
B,
B3
F,
F2
F3
C,
F.
A2 F5 B2
-7.4
one chromosome
16
containing
a single a
3.0
(-
endonuclease
analysis
was
used
normal
DNA
that contained
©
to deter-
mine the origin
of the deletions
on these chromosome
l6s bearing
the single
remaining
a gene. Following
Barn
HI digestion
and agarose
gel electrophoresis,
yielded
a single
fragment
jj4.4
kb)2’7
both a loci (Fig. 2A).
In contrast,
all
HbH
disease
DNA
samples
had a 10.4-kb
fragment,
and DNA
from a black a-thal-2
(aa/-a)
individual
had 1 4.4- and 1 0.4-kb
fragments.
This implies
that the
C
0
4-
0
C-)
0
Cl)
U)
B2
B3F,
©A,82
5.l,,
3
4.84.l”’
I
0
Fig. 2.
Autoradiograms
of a-globin
gene containing
DNA fragments
following
restriction
endonuclease
digestion
of normal
control
(aa/aa)
(Al ). a black a-thal-2
individual
(aa/ - a) (A2),
and black (B). Chinese
(C). and Filipino (F) individuals
(Fig. 1 ) with
HbH disease
( - - I - a). Individuals
84. B5. and C2 had identical
restriction
patterns
that are not shown
here. Fragment
sizes are
given in kilobases
(kb). Restriction
endonuclease
digestions
are
shown:
(A) Barn HI; (B) Barn HI plus Hind Ill; (C) Hpa I plus Eco RI;
and (D) Hind II.
gene
than
fragment
in these
normal
containing
individuals
(Fig.
3).
the
ci
I-.
a-gene-cOntaining
subjects
0
which
the
4-
C
a)
C.)
fragments
corresponds
to sequences
ci
ment
in HbH
and
3.0
between
DNAs
(Fig.
2C).
-
2
3
Number
Fig. 1 .
Percentage
of single-stranded
a probe
annealed
by
DNA from
black
American,
Chinese,
and Filipino
HbH disease
subjects
(shown
by circles).
Horizontal
and vertical
lines represent
observed
means
and ranges
for percentage
annealed
by DNAs
from other
individuals
with
one (HbH disease).
two
(a-thal-l).
three
(a-thal-2).
and four (normal
controls)
a genes.
The average
percentages
of HbH disease
individuals
reported
here (.) were:
black (Bi -3). 27%. 24%. and 22%; Chinese
(Cl ). 20%; and Filipino
(Fl -.5). 20%. 24%. 1 7%. 23%, and 27%. Three
individuals.
blacks
(B4. B5) and Chinese
(C2). were not studied
by gene counting,
but
were studied
by restriction
endonuclease
mapping.
simple
deletion
of either
the 5’ (such
Embury3)
or 3’ a gene (Fig. 3).
All
the centers
of
I showed
fragments
in
5.1-kb
fragThis
compatible
with loss of the intergenic
Hpa
deletion
of 3.8 kb (4.8 + 4. 1
5. 1 kb) and
Gene
kb).
3.8 kb,
(Fig. 3#{149})2.6
RI and Hpa
4. 1 kb a-gene-containing
were replaced
by a single
disease
a
4 kb smaller
with Barn
HI
of approximately
two normal
a-globin
genes
Double
digestion
with Eco
that the 4.8 and
normal
DNA2’3
remaining
DNA
yielded
fragDNA
from all HbH
two
of the
normal
(7.4
the fragment
lacked
single
is approximately
After
digestion
and Hind
III (Fig.
2B), normal
ments
of 7.4, 3.8, and 3.0 kb.2’6
disease
subjects
yielded
only
.0
0
F5A2
3
DNA
a
4,8,
pattern
is
I site and
precludes
a
a
as reported
by
To further
verify
the location
of this 3.8-kb
deletion
in HbH disease DNAs,
Hind
II digestions
were done.
The
normal
3.6- and
1.5-kb
fragments,
containing
sequences
5’ to the 5’ a gene and 3’ to the 3’ a gene,
respectively,2
and
an
additional
1 .2-kb
fragment
unknown
origin
were seen in both control
and HbH
disease
DNAs
(Fig. 2D). However,
the two fragments
(3. 1 and 0.8 kb) that correspond
to sequences
between
the center
of the 5’ and 3’ a genes in normal
DNA2
of
From www.bloodjournal.org by guest on June 16, 2017. For personal use only.
1068
PHILLIPS
NORMAL
ENZYME
4
HI
DNA
HbH
DELETION
DNA
PROBABLE
ORIGIN
OF
SINGLE
ET AL.
-GLOBIN
GENE
4
3’
5’
0 4
40
5,
HI
JN.P
‘
74
41
Hpa I
+
Eco
83.1
35
51
3.8
36+15
39
35+45
39
15
EL
E21
3 8
48
RI
36
HIND
30
74t
831
45
‘
,
BAM
a
HpaI
a
Eco RI
0
HINDU
Fig. 4.
Diagrammatic
representation
of an unequal
crossingover
event
compatible
with
the deletion
patterns
observed
in
DNAs from 1 2 HbH disease
individuals
with a single a-globin
gene.
Homologous
normal
chromosome
1 6s are
shown
mispairing
above, and the resulting
chromosome
with a single a-globin
gene
is shown
below.
Symbols
are as in Fig.
3. Crossing-over
is
arbitrarily
shown
within
the 5’ and the 3’ a loci of homologous
chromosome
1 6s. The arrow
represents
the bounds
of the crossing-over
site. and intergenic
sequences
deleted
by such crossingover are represented
by the dashed
line.
HI
Fig. 3.
Map of normal
restriction
endonuclease
sites in the
DNA region
containing
the a-globin
genes.2’’’7
The 5’ and 3’
orientation
is from
left to right.
and the a-globin
genes
are
diagrammatically
represented
by rectangles
of arbitrary
size.
Small symbols
represent
the normal
locations
of restriction
endonuclease
cleavage
sites. and the numbers
indicate
fragment
sizes
in kb observed
in control
(Al ) and HbH (Bl -5, Cl -2, Fl -5) DNAs
(Fig. 2). The approximate
deletion
sizes in HbH DNAs are shown
on the right.
were
data
absent in all HbH
disease DNAs.
The Hind
II
were confirmed
by patterns
observed
following
normal
restriction
single
Hpa
remaining
I and
Hind
deletion
restriction
patterns
(data
a single
gene
simple
would
Taken
patterns
as
in
a
all
whole,
the
I 2 subjects
indicate
single
a-gene-containing
deletion
of approximately
indistinguishable
by
restriction
chromosome
3.8 kb
this
analysis
endonuclease
that
have
a
(Fig.
1) with
of DNA
that
they
a
is
from
the
distance
between
the centers
of the two a-globin
structural
genes in normal
DNA
(Figs.
2 and 3)2.3.6
We found no
examples
of differences
in a gene number
as reported
in Mediterraneans,
Turks,
Israelis,
and recently
in
Cypriots
with HbH
disease6”
and
for clinical
differences
in severity
different
racial
groups.
no molecular
basis
of HbH
disease
in
Furthermore,
the
presence
of
with
Asian
HbH
are
the
disease6
the
excluded
by
restriction
recent
chromodeletions
or the 3’
patterns
seen
Hpa I plus Eco RI, Hind
II, and Hpa I plus
digestions
(Figs.
3 and 4). For example,
a
5’ a gene
deletion,
yield the following
Eco
1.5-kb
4.5-kb
DISCUSSION
between
of individuals
bearing
the reciprocal
16 that has three
a genes.’2
Disparate
the 5’ a gene (as reported
by Embury3)
following
Hind
III
plus
not shown).
sequences
5’ and 3’ a genes
(Fig.
3). This
with an unequal
or nonhomolo-
and
a
identical
of the
from
sequences
between
the centers
of the genes
(3. 1 and
0.8-kb
fragments).
These
data are summarized
in Fig.
3. In addition,
recent
data
on three
more
unrelated
HbH
disease
subjects
(two American
blacks
and one
yielded
the
center
the intergenic
that
the 3.8-kb
in Fig. 4. This
data
of DNA
report
some
ofjust
Chinese)
to
of the normal
is compatible
of
gous cross-over
mechanism,
as shown
mechanism
agrees
with
both
Orkin’s
double
digestion
with
Hpa
I and Hind
III,
which
showed
the
normal
5’ (3.5
kb)
and
3’ (4.5
kb)
a-gene-containing
fragments2’3
but
absence
of the
-
3’ to the
gene
with
loss
II sites suggests
corresponds
centers
pattern
5’ and
sites
a
RI,
4.8-kb
fragment;
and
fragments;
fragments.
as reported
by Embury,3
restriction
patterns:
Hpa
Hpa
Hind
II,
I plus Hind
3.1-
III,
plus
3.1-
plus
The compatibility
of our results
from
12 subjects
(unselected
for gene
number
or deletion
pattern)
representing
two different
racial
groups
(American
blacks
suggests
deletion
and
Asians)
with
unequal
that this may be a common
of one of the normally
linked
However,
spontaneous
these data
deletions
cannot
in our
exclude
subjects,
crossing-over
mechanism
for
a-globin
genes.
the possibility
of
which
are equal
in size to the distance
between
the centers
of the two a
genes
and include
the intergenic
Hpa
I and Hind
II
sites and the 5’ a gene
(Fig.
3). To eliminate
this
unlikely
sequences
possibility,
derived
from
probes
genomic
for
DNA
the
are
intergenic
required.
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I
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a-globin
by molecular
structural
gene
hybridization
to chromosome
assay.
Cell
I 6 in somatic
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CROSSING-OVER
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in cellular
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1980 55: 1066-1069
Unequal crossing-over: a common basis of single alpha-globin genes in
Asians and American blacks with hemoglobin-H disease
JA 3d Phillips, TA Vik, AF Scott, KE Young, HH Jr Kazazian, KD Smith, VF Fairbanks and HM Koenig
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Blood (print ISSN 0006-4971, online ISSN 1528-0020), is published weekly by the American Society of
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Copyright 2011 by The American Society of Hematology; all rights reserved.