Download Consalez, GG, Stayton, CL, Freimer, NB, Goonewardena, Brown, WT, Gilliam, TC and Warren, ST: Isolation and characterization of a highly polymorphic human locus (DXS 455) in proximal Xq28. Genomics 12:710-714 (1992).

GENOMICS
12, 710-714
(1992)
Isolation and Characterization
of a Highly Polymorphic
Human Locus (DXS455) in Proximal Xq28
G. GIACOMO CONSALEZ,*J CAROL L. STAYTON,* NELSONB. FREiMER,t’2 PONMANI GOONEWARDENA,$
W. TED BROWN,* T. CONRAD GILuAM,t AND STEPHENT. WARREN**~
*Howard Hughes Medical
Institute and Departments of Biochemistry and Pediatrics, Emory University School of Medicine,
Atlanta, Georgia 30322; tDepartments
of Psychiatry and Genetics and Development, College of Physicians and Surgeons
of Columbia University, New York, New York 70032; and *Department of Pediatrics, North Shore University Hospital,
Cornell University Medical College, Manhasset, New York 7 1030
ReceivedJuly
18, 1991;revised
Press, Inc.
INTRODUCTION
Genetic mapping by the cosegregation of marker and
disease alleles through families has allowed the positioning of genes involved in hereditary disease throughout
the human genome (White and Caskey, 1988). The
marker genes, most often distinguished by restriction
fragment length polymorphisms (RFLPs) following
probing of Southern blots, not only have permitted the
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1991
initial mapping of a given disease gene but have subsequently proved vital in carrier detection and prenatal
diagnosis as well as in the actual cloning of the disease
gene itself (Riordan et aZ., 1989; Verkerk et al., 1991).
Paramount to the utility of marker loci is the frequency
with which they are informative in families (Botstein et
al., 1980). Most two-allele RFLPs have an average heterozygosity of ~35%, limiting their usefulness in a number of families that could otherwise benefit from diagnosis by genetic linkage. New classes of marker loci recently identified, such as those classified as variable
number tandem repeats (VNTRs; Nakamura et al.,
1987) as well as mini- and microsatellite repeats (Jeffreys et al., 1985; Weber and May, 1989), reveal much
higher levels of heterozygosities and are quite useful for
linkage analyses since most families are informative.
One region of the human genome where genetic mapping of disease loci has been particularly fruitful is the
terminal band of the human X chromosome long arm.
Band Xq28 is one of the more gene-dense regions of the
human genome yet recognized, with over 27 loci identified (Davies et at., 1990). Many of these loci have alleles
responsible for genetic disease, such as adrenoleukodystrophy, chrondrodysplasia punctata, dyskeratosis congenita, Emery-Dreifuss muscular dystrophy, GGPZ deficiency, hemophilia A, Hunter syndrome, X-linked hydrocephalus, myotubular myopathy, and nephrogenic
diabetes insipidus, (McKusick, 1991).
A major component involved in the prenatal and carrier diagnosis of diseases mapping to Xq28 is linkage to
polymorphic markers (Willems et al., 1990; Consalez et
al., 1991). Although a number of polymorphic loci have
been identified within Xq28, only one, DXS52 (detected
by probe St14), is highly polymorphic and therefore informative in most families (Oberle et al., 1985). However, several disorders in Xq28 map significant distances
from DXS52, lowering the accuracy of diagnosis. Additional highly polymorphic loci within Xq28 would therefore significantly enhance the accuracy of linkage diagnostics as well as allow an improved assessment of the
genetic map of this region.
Human Xq28 is highly gene dense with over 27 loci. Because
most of these genes have been mapped by linkage to polymorphic loci, only one of which (DXS52) is informative
in most
families, a search was conducted for new, highly polymorphic
Xq28 markers. From a cosmid library constructed using a somatic cell hybrid containing
human Xq27.3+qter
as the sole
human DNA, a human-insert
cosmid (~346) was identified and
found to reveal variation
on Southern blot analyses with female DNA digested with any of several different restriction
endonucleases.
Two subclones of ~346, ~346.8 and p346.T,
that respectively identify a multiallelic
VNTR locus and a frequent two-allele
TaqI polymorphism
were isolated. Examination of 21 unrelated females showed heterozygosity
of 76 and
5’7%, respectively.
These two markers appeared to be in linkage equilibrium,
and a combined analysis revealed heterozygosity in 91% of unrelated
females. Families segregating
the
fragile X syndrome with key Xq28 crossovers position this
locus (designated DXS455) between the proximal Xq28 locus
DXS296
(VK21) and the more distal locus DXS374
(lAl),
which is proximal
to DXS52. DXS455
is therefore the most
polymorphic
locus identified in Xq28 and will be useful in the
genetic analysis of this gene dense region, including
the diagnosis of nearby genetic disease loci by linkage.
o 1%~ Academic
‘Present
address:
Laboratory
of Molecular
Genetics,
Gaslini,
Genoa, Italy.
’ Present address: Department
of Psychiatry,
University
nia, San Francisco,
CA 94143.
3 To whom correspondence
should be addressed.
November6.
HIGHLY
POLYMORPHIC
Xq28
711
LOCUS
Below, we show an approach to identifying
cosmids
that reveal highly polymorphic
loci, and we describe and
characterize one such cosmid from Xq28 that detects the
DXS455 locus, which is multiallelic and heterozygous
in
greater than 90% of unrelated females.
MATERIALS
AND
METHODS
Library
screening
and subcloning.
A cosmid
library
was constructed
from a somatic
cell hybrid
containing
Xq27.3+qter
as the
sole human DNA (Warren
et al., 1990) using the vector Lorist 6 (Little
and Cross, 1985). Human-insert
cosmids were identified
by screening
with 32P-labeled
human DNA, as described
(Gusella
et al., 1980). Following secondary
screening
of duplicate
filters with human and rodent
DNA as probes, random
human cosmids were used to screen for polymorphisms.
Subcloning
was performed
into the vector pBluescript
I or II (Stratagene),
with subsequent
screening
of Southern
blots with radiolabeled human
DNA to assess the presence
of repetitive
sequences.
Southern
analysis and polymorphism
screening.
DNA was isolated
from peripheral
blood as described
by Longmire
et al. (1985). DNA
samples were digested according
to the manufacturer’s
recommendations
and electrophoresed,
blotted,
and hybridized
as described
(Southern,
1975). To use intact cosmids as probes, cosmid DNA, prepared by alkaline
lysis, was nick-translated
and preassociated
to sonicated human placental
DNA to C,, t values of 20 as described
by Sealey
et al. (1985). Unique sequence inserts were excised from plasmid
subclones and separated
electrophoretically
through
low-melting-point
agarose, and the insert fragment
was labeled by random primer synthesis (Feinberg
and Vogelstein,
1983).
To screen for cosmids that detect polymorphisms,
a Southern
blot
was prepared
from electrophoresed
DNA where each of three lanes of
a set contained
a mixture
of four unrelated
female DNAs
and the
fourth
lane a single unrelated
male DNA. Each set of four lanes, containing
DNA of a total of 25 unrelated
X chromosomes,
was digested
with MspI, TaqI, EcoRI, HindIII,
BglII, PstI, RsaI, or BclI. Following
hybridization
of preannealed
labeled
cosmid
DNA,
the autoradiograms were analyzed
for variation
between
the fourth
lane of a set,
containing
a single X of a male, against the other three lanes, composed of 12 female DNAs.
Family
studies
were performed
using DNAs
obtained
from the
CEPH reference
pedigrees
(Dausset
et al., 1990) or from selected fragile X syndrome
families
previously
found to contain crossover
points
between
key loci. All Southern
blots were performed
as described
above except for those analyzing
the BcZI, BglII,
or PstI polymorphisms, which required
very long electrophoretic
runs to achieve sufficient resolution
to distinguish
the alleles. For these studies, electrophoresis was carried out through
a 20 X 25.cm 0.7% agarose gel in 1X
TAE
(0.04 M Tris-acetate,
pH 8.0, 0.004 M EDTA)
uncirculated
buffer at 30 V for ~65 h with fresh buffer added every 24 h. Under
these conditions,
the 4.4-kb HindIII-cleaved
X marker
will have run
222 cm.
RESULTS
To identify cosmids that are likely to reveal highly
polymorphic
loci in human Xq28, human cosmids constructed from a somatic cell hybrid containing human
Xq27.3+qter
were chosen randomly for screening. Cosmids were radiolabeled, blocked by preassociation
with
excess sonicated human DNA, and hybridized to Southern blots of human DNA. For rapid screening of many
independent
X chromosomes
with a variety of restriction endonucleases,
gels were prepared with four lanes
per endonuclease where the first three lanes each represented equal amounts of DNA from four unrelated fe-
-b
..
.I
-a -‘*
blot analysis
of DNA derived from 12 unrelated
FIG. 1. Southern
females and 1 unrelated
male cleaved with one of four restriction
endonucleases
and probed with cosmid 346. DNA samples of females l-4,
5-8, and 9-12 were pooled (3 pg of each female DNA),
and 12 pg of
male DNA was cleaved to completion
with the indicated
enzymes,
electrophoresed,
blotted,
and hybridized
with radiolabeled
and preannealed ~346 as described
above. Arrows
indicate
differences
between
1
or more female lanes and the male lane, suggesting
polymorphism.
males, and the fourth lane represented a single unrelated
male. Hence, a banding difference(s)
between the male
lane (lane 4) and any of the three female lanes suggests a
polymorphism
among 1 or more of the 25 unrelated X
chromosomes.
In this fashion, a single filter of 32 lanes
can assess, within
resolution
limits due to multiple
bands, polymorphisms
with 8 enzymes.
Of four cosmids initially studied, three showed banding variations,
with one exhibiting variant bands with
several restriction
endonucleases.
This cosmid, designated ~346 and detecting locus DXS455, showed obvious
banding changes with Z’aqI and BgZII (Fig. l), as well as
more subtle variation with Z&I, P&I, and BcZI. Since
variation
with multiple enzymes may reflect a highly
polymorphic
locus, this cosmid was studied further.
Restriction
analysis of ~346 showed an approximate
34-kb insert that was further subcloned as shown in Fig.
2. The DXS455 locus detected by fragments of ~346 was
shown to segregate in an X-linked Mendelian fashion in
CEPH families (data not shown) and to map to human
Xq27.3+qter
using a somatic cell hybrid panel (Warren
et al., 1990) independent
of the hybrid used to prepare
the cosmid library (data not shown).
Two distinct polymorphic
regions of ~346 were found
by analysis of subclones. The two-allele
Z’aqI polymorphism, shown in Fig. 3, was observed with p346.T, which
maps approximately
15 kb from ~346.2 (Fig. 2). Subclone ~346.2 was found to detect a multiallelic polymorphism observed with BclI-cleaved
DNA that was also
712
CONSALEZ
ET
AL.
23.0 kb +
p346.T
9.4 kb
+
6.6kb
+
~346.8
FIG. 2. Restriction
map of cosmid
346 (33.5 kb) and subclone
~346.2
(8 kb). Shaded
areas indicate
the restriction
fragments
to
which various subclones
map. The asterisk
indicates
the BamHI
sites
of the cosmid vector
that were destroyed
during
cloning.
Only the
Tag1 site in brackets
is shown on the map, although
there are several
other such sites within
~346.
responsible for variation seen with all other enzymes except TuqI. Further subcloning of ~346.2 to a single-copy
fragment (~346.8, Fig. 2) results in a probe that exhibits
marked variation with DNA cleaved by BcZI, BglII,
BstYI, or P&I. Figure 4 shows a Southern blot of BclIdigested DNA from 21 unrelated females. Of these females, 76% are heterozygous, showing two bands, each
representing one allele. At least 10 alleles have been
identified on BcZI-digested DNA probed with ~346.8. All
currently identified alleles, with the exception of a 6.9kb allele (lane 3, Fig. 4), fall within an SOO-bp window
differing by approximately 100 bp with an average size of
8.2 kb (7.8-8.6 kb). Since the allele size variation is relatively small, enhanced resolution of the fragment sizes is
achieved by long electrophoresis times, as described
above.
As shown in Table 1, the combined heterozygosity for
both DXS455 probes (~346.8 and p346.T) in 21 unrelated females of mixed ethnic background is 91%, which
is not significantly different from the expected heterozygosity calculated from the individual frequencies for
each probe. The polymorphisms observed using both
probes appear to be in linkage equilibrium, as can be
1
2
3
4
5
6
7
0
9
FIG. 4. Southern
blot analysis of BclI-digested
DNA of 21 females
hybridized
with ~346.8. The same DNA samples are used, in the same
order, as in Fig. 3. Most polymorphic
bands have an average size of 8.2
kb with the exception
of the DNA in lane 3, which displays
a less
frequent
6.9-kb allele. Lanes 1, 7, 14, 17, and 19 are homozygous
for
this polymorphism,
although
only females 1 and 17 are homozygous
for the TuqI polymorphism
(Fig. 3).
appreciated from inspection of Fig. 3 and 4, where each
lane number corresponds to the same female subject.
TO map DXS455 within Xq28 more precisely, meiotic
crossovers between established Xq28 marker loci were
used as map indicators. As shown in Fig. 5, two families
segregating fragile X syndrome that exhibited crossovers with four flanking markers were analyzed (RN1
detecting DXS369; VK21 detecting DXS296; 1Al detecting DXS374; St14 detecting DXS52). In family 69,
DXS455 maps distal to DXS369 and proximal to
DXS497 and DXS52, while in family 44, DXS455 is distal to DXS296. Since the order of the marker loci
has been established as DXS369-FRAXA-DXS296DXS497-DXS52
(Davies and Craig, 1991), these data
place DXS455 within the interval flanked by DXS296
and DXS497. By inference with the genetic distance of
these two loci from the FRAXA locus (Rousseau et al.,
1991; Dahl et al., 1989), DXS455 may be estimated to be
between 2 and 5 CM distal to the fragile X mutation, with
inferred lod scores in excess of 10.
10 11 12 13 14 15 16 17 18 19 20 21
TABLE
Observed
4.1 kb -+
3.6kb -+
FIG. 3. Southern
blot analysis of Z&$-digested
DNA of 21 females
hybridized
with p346.T.
The 4.1- and 3.4-kb
alleles are indicated.
Lanes 1,2,11-13,
15-17,
and 21 are homozygous
for this polymorphism of DXS455.
The 21 females are unrelated
and represent
three
ethnic groups (Caucasian,
Asian, and American
Black).
Probe
~346.8
p346.T
Combined
1
Heterozygosity
in Unrelated
Using Two DXS455
Probes
Enzyme
BclI
TaqI
D Survey
of 21 unrelated
females.
* Not significantly
different
from
gosity of 89.7%.
Females
Heterozygosity”
Percentage
16/21
12/21
19/21
the expected
76
57
91*
combined
heterozy-
HIGHLY
Normal phenotype
Family 69
POLYMORPHIC
.6 cytogenetiis
21
I
:;
l
22
I
53
6
21
1
II
;,
2
12
2
21
36
5
21
56
2
4
111
RN,
FRAX
346
1.4,
ST14
22
t.
12
2 1
35
22
t12
2 1
35
2
+
1
2
3
2
+
“1‘
1
6
2
a
II
. .
&
11
2
L
46
5
*
3
46
6
Family 44
II
FIG. 5. Results of crossover
mapping
of DXS455
in fragile X syndrome families
showing
recombination
with key Xq28 loci. Family
69
shows a key crossover
in individual
111-4, which positions
DXS455
(probe 346) proximal
to DXS347
(probe 1Al). Family
44 shows a key
crossover
in individual
111-5, which
positions
DXS455
distal
to
DXS296
(probe VK21).
These and other crossovers
are indicated
by
the hatched
bar. Other
indicated
loci are DXS369
(probe
RNl),
DXS52
(probe St14), and FRAX
(fragile X syndrome,
FMR-1
{19)).
DISCUSSION
Human cosmids, isolated from a somatic cell hybrid
containing Xq27.3*qter
as the sole human DNA, were
used to identify new polymorphic
loci within the metaphase band Xq28. Cosmid 346 was found to exhibit variable bands among the female and male DNAs digested
with each of several enzymes. The map location of this
cosmid insert was confirmed to be Xq28 by somatic cell
hybrid analysis, and the polymorphism
detected by ~346
(DXS 455) was found, as expected, to segregate in an
X-linked fashion in CEPH reference pedigrees.
Two unique subclones of ~346 were isolated (~346.8
and p346.T) and placed on the 34-kb restriction map of
the human DNA insert separated by 22 kb (Fig, 2). Both
probes were found to detect polymorphisms;
~346.8 detects a frequent multiallelic
polymorphism
(PIC = 0.76),
and p346.T detects a two-allele TuqI polymorphism
with
the useful PIC value of 0.57. This latter probe is valuable
to those investigators who already have TagI-digested
family DNA electrophoresed
and blotted. The ~346.8
polymorphism
exhibits at least 10 alleles, with most differing in size by lOO-bp increments.
Xq28
713
LOCUS
When DXS455 is analyzed by both ~346.8 andp346.T,
over 90% of females are heterozygous. Thus, this locus
represents one of the more polymorphic loci identified in
the human genome to date. The high frequency of polymorphism detected by DXS455, coupled with its location in Xq28, makes this locus particularly
useful for
linkage studies. By meiotic crossover mapping, DXS455
was shown to be between 2 and 5 CM distal to the fragile
X mutation within the interval defined by DXS296 and
DXS374.
Xq28, with over 27 loci assigned, is a highly genedense region. Thus, DXS455 would be useful both for
diagnostic studies in families segregating these disorders
and for positioning
these mutations within Xq28. The
latter effort may be readily performed in conjunction
with DXS52, a well-studied, highly polymorphic
locus
previously mapped to Xq28. Our positioning of DXS455
between DXS296 and DXS374 indicates that DXS455 is
proximal to DXS52 and would define, by genetic studies,
three regions within Xq28: that proximal to DXS455 or
distal to DXS52 or the interval between these loci. Recent physical mapping studies indicate that Xq28 is approximately 9 Mb in size, with DXS52 about 3 Mb from
Xqter (Poustka
et al., 1991). Since DXS296
and
DXS374 appear to be approximately
5 and 3 Mb proximal to DXS52, respectively, the three intervals defined
by DXS455 and DXS52 may evenly divide 3-Mh regions
of Xq28. Given the high level of heterozygosity exhibited
by these two loci, it should be possibIe to readily position
mutations responsible for Xq28 genetic diseases within
these three genomic intervals as a prelude to positional
cloning efforts.
ACKNOWLEDGMENTS
We thank
F. Zhang
for assistance.
This work was supported
by
grants from the W. M. Keck Foundation
(T.C.G.),
the NIH (HD20521
and HG00038),
and the Muscular
Dystrophy
Association
(S.T.W.).
S.T.W.
is an investigator
of the Howard
Hughes Medical
Institute.
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