Download Sequence Analysis of the y-Globin Gene Locus from

Sequence Analysis of the y-Globin Gene Locus from a Patient with the Deletion
Form of Hereditary Persistence of Fetal Hemoglobin
By Catherine A. Stolle, Laura A. Penny, Sharlene Ivory, Bernard G. Forget, and Edward J. Benz, Jr.
The 7-globin genes from a patient homozygous for a
deletion form of hereditary persistence of fetal hemoglobin
(HPFH-1) have been cloned and sequenced. The DNA
sequence of the patient‘s 7-globin genes corresponds to a
previously identified sequence framework (chromosomeA)
with the exception of 10 base changes. Seven of these
base changes can be attributed to normal allelic variation
generated by small gene conversion events. The remaining
three base changes are present in a 0.76 kb Hindlll
fragment containing a putative enhancer located 3’ to the
“7-globin gene. The same three base changes have also
been described in the Seattle variant of nondeletion HPFH.
We have analyzed 16 alleles from non-HPFH individuals and
five alleles from individuals with nondeletion or deletion
HPFH for the presence of these base changes by polymerase chain reaction amplification of cloned or chromosomal DNA and hybridization to allele-specific oligonucleotide probes. Although these base changes were found in
an individualwith HPFH-2, they were not found in the DNA
from two patients with nondeletion HPFH. More importantly, all three base changes were detected in DNA from
five non-HPFH individuals and appear to be common in
blacks. We conclude that these base changes do not
correlate with an HPFH phenotype and that the significant
mutation in HPFH-1 is the deletion of over 100 kb of
genomic DNA.
0 1990 by The American Society of Hematology.
H
from a patient homozygous for HPFH-1. Of the 10 base
changes identified, 7 correspond to known sequence polymorphisms, and 3 base changes located in the *y gene 3‘
enhancer” have been described previously in the Seattle
variant of nondeletion HPFH.26Further analysis of D N A
from non-HPFH individuals indicates that these three base
changes are present in normal y-globin gene sequences and
are unlikely to contribute to the increase in y-globin gene
expression observed in HPFH-1.
EREDITARY PERSISTENCE of fetal hemoglobin
(HPFH) is a benign condition in which fetal hemoglobin expression persists into adulthood at levels greater than
1% in the absence of erythropietic stress or thala~semia.’-~
Because this condition can be viewed as a failure to switch
from fetal (HbF, a2y2)to adult (HbA, ad2)hemoglobin
synthesis, it has been studied as a model for the developmental control of gene expression.
Two major categories of HPFH have been delineated
based on mutations associated with these disorders. Nondeletion HPFH is commonly associated with single base changes
in the 5’ flanking region of the y-globin genes. These
mutations may alter promoter sequences or binding sites for
trans-acting factors, resulting in a corresponding over expression of either the ‘7-globin or *y-globin gene.5-’2Deletion
HPFH is characterized by large (= 105 kb) deletions in the
&globin gene cluster that remove the adult (6 and @)globin
genes and result in increased expression of both Gy- and
*y-gl~bin.’~-’’
One hypothesis to explain the increase in y-globin gene
expression in the deletion form of HPFH described in
American blacks (HPFH-1) is that the sequences juxtaposed
to the y-globin genes as a result of the deletion contain an
enhancer-like element that serves to maintain a transcriptionally active domain. A small fragment of D N A from the
region immediately 3’ to the breakpoint in HPFH-1 has been
found to have enhancer-like activity when transfected into
erythroid cells.20.2’This region of D N A is also hypomethylated and DNAse I hypersensitive in erythroid tissues and
contains a large open-reading frame (ref 20-22; Elder and
Groudine, personal communication, March, 1989). Furthermore, deletions that extend beyond the enhancer-like element result in SPo thalassemia instead of HPFH.2’323However, no single model has been proposed that successfully
explains the mechanism underlying all forms of deletion
HPFH.24
The sequences of the y-globin genes from patients with
deletion HPFH have never been directly examined for
mutations analogous to those present in nondeletion HPFH.
Such mutations might provide an additional explanation for
the persistence in expression of the y-globin genes in deletion
HPFH. We have cloned and sequenced the y-globin genes
Blood, Vol 75, No 2 (January 15). 1990: pp 499-504
METHODS
Cloning and sequencing of HPFH-I y-globin genes. Genomic
DNA from a lymphoblastoid cell line, LAZ-149;’ obtained from a
black patient homozygous for HPFH-1,I5was digested to completion
with BglII (New England Biolabs, Beverly, MA). EMBL 3 phage
arms (Promega Biotec, Madison, WI) were prepared by digestion of
the vector with BamHl and EcoR1, followed by precipitation with
isopropanol, as described in the manufacturer’s protocol. Phage
arms and digested genomic DNA were ligated in a 10 pL reaction
containing 66 mmol/L Tris-HC1 (pH 7.5), 5 mmol/L MgCl,, 5
mmol/L DTT, 1 mmol/L adenosinetriphosphate (ATP), and 3 units
of T4 DNA ligase (Pharmacia, Piscataway, NJ). Ligation reactions
were incubated at 16OC for 16 hours. A portion of the ligation
mixture was then packaged using Gigapack packaging extracts
(Stratagene, San Diego, CA) according to the manufacturer’s
~
From the Departments of Internal Medicine and Human Genetics, Yale University School of Medicine. New Haven, CT.
Submitted June 9,1989; accepted September 8. 1989.
Supported by a Research Career Development Award to E.J.B.
and grants from the NIH to E.J.B. and B.G.F. C.A.S. was supported
by a National Research Service Award.
Presented in part at the annual meeting of the American Society
for Clinical Research in Washington, DC, May, 1988, and has been
published in abstract form (Clin Res 36:569,1988).
Address reprint requests to Edward J. Benz, Jr, MD, Department
of Internal Medicine, Yale University School of Medicine. 333
Cedar St, New Haven, CT 0651 0.
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.
6 1990 by The American Society of Hematology.
0006-4971/90/7502-0017$3.00/0
499
STOLLE ET AL
500
NaOH and 25 mmol/L EDTA and blotted onto a Nytran membrane
(Schleicher and Schuell, Keene, NH) using a slot blot manifold.
Oligonucleotide probes corresponding to the *y-globin gene published sequence (TTGGAAGGTTTACTTAA; AGGTTTAGC TGAGCTCA; AGCACCTTACACTGAGT)32.33
or the Seattle variant (TTGGAAGGCTTACTTAA; AGGTTTAGATGAGCTCA;
AGCACCTTGCACTGAGT)26 were 5' end-labelled to a specific
activity of 1 x lo7 cpmlpg using T4 polynucleotide kinase (Boehringer, Mannheim, W Germany) and y- [32P]dATP(Amersham,
Chicago, IL). Each probe was hybridized to the membrane-bound
amplified DNA at 5OC below its calculated melting temperature.
The membrane was then washed at 2OC below the probe melting
temperature to remove nonspecifically bound probe.34 The filters
were exposed to X-ray film (Kodak, Rochester, NY) at -7OOC with
an intensifying screen.
suggestions. VCS 257 bacteria were infected with the packaged
phage DNA and plated using standard methods." Approximately
lo5recombinant phage plaques were screened by hybridization with
a nick translated 2.6 kb EcoRI fragment from the 5' end of the
Ay-globingene. Potential positive clones were counter screened with
a 1.6 kb EcoRI fragment from the 3' end of the Gy-globin gene.
Positive identification was achieved by restriction enzyme digestion,
and Southern blot analysis of insert DNA was compared with a 13
kb BglII fragment of normal DNA containing the Gy-and *y-globin
genes.
Insert DNA containing the y-globin genes from one of the
patient's chromosomes was excised from the vector by digestion with
SalI. A Gy-globinfragment (2.6 kb StuIISphI) digested with AluI
and an Ay-globinfragment (2.2 kb RsaI) digested with HaeIII were
blunt end ligated into M13mpl0, which had been linearized with
SmaI. In addition, a 0.76 kb Hind111 fragment from the region 3' to
the patient's Ay-globingene was similarly subcloned. Single stranded
or double stranded M13 templates were sequenced using the dideoxy
sequencing method and Sequenase (US Biochemicals, Cleveland,
Each fragment was sequenced at least twice using several
independent isolates of M13 subclones (Fig 1).
Amplification of genomic DNA and hybridization with allelespecijc oligonucleotide probes. Genomic DNA (1 p g ) was amplified by the polymerase chain reaction technique for 30 cycles with
1.5 minute denaturations at 94OC, primer annealing for 2 minutes at
5S0C, and extensions for 3 minutes at 720C3' Amplified DNA (1%
to 2% of reaction product) was denatured in a solution of 0.4 N
GY
- 4
Sal I/ Bgl II
n.
I
nm
II
-,
-AY
RESULTS
The LAZ-149 cells used in this study were derived from a
patient who was the subject of previous report^.'^^'"^*^ The
patient is homozygous for a form of HPFH common among
American blacks (HPFH-1) as determined by Southern blot
analy~is.'~-'~
The two y-globin gene loci were indistinguishable by restriction enzyme mapping and presumed to contribute identically to the HPFH phenotype. The '7- and *yglobin genes from one of the patient's two chromosomes
e+
n n
II / s a I
I
stu I
stu-TGi?l
I
SI" I
50kb
Rsa I
R s I~
--ET---
Hind 111
Hind 111
b
1
2
. 1 L
Sph I
I
SI" I
AA
C
St" I
L.-.
Sph I
A
A
A A A A
A
A
AAAA
AA
A
Fig 1. Sequencing strategy
for HPFH-1 7-globin gene
clones. (a) Diagramatic representation of '7- and 'y-globin
gene fragments isolated from a
13 kb Sa/I/Bg/ll cloned DNA
insert from a patient with
HPFH-1. The solid bar indicates
the portions of the gene fragments sequenced. lb) Sequencing strategy for the '7-globin
gene and (c) sequencing strategy for the '7-globin gene and
putative 3' enhancer. The location of the A M (A) or Haelll (HI
restriction enzyme sites in the
y
' or ' 7 globin genes (respectively) used to prepare subclones for sequencing in M13
are illustrated below the diagram of each gene. Sequencing
was obtained using singlestranded clones and universal
primer ( 4 1 , double-stranded
clones and reverse primer
(h),
or synthetic oligonucleotide primers (I+).
Differences in the nucleotide sequence compared w i t h
published sequence are indicated by a numbered arrow
above the diagram of each gene.
The numbers refer to sequence
differences listed in Table 1.
501
y-GLOBULIN GENE SEQUENCE IN HPFH
'y-globin gene and the 'yAy inter-genic region derived from
the patient's cloned D N A was subcloned into M13 and
sequenced as described above using a universal primer. Three
Sequence Change
Position
Normal Sequence Fw
additional
base changes were detected: a deletion of one A in
Gy-Globin
a string of four at -601 to -603; an insertion of an A at
-T
+1,611 to +1.618
Gr(B)
-605; and a deletion of a T at -61 1 (Fig 1C and Table 1).
CG+T
+ 1,800 and + 1,801
G7(B)
The three base changes support the notion that a gene
"7-Globin
-T
-61 1
*Y(B)
conversion event has occurred in which the 5' end of the
+A
- 605
"y(B)
Ay(A) gene has been converted to an Ay(B) gene. However,
-A
-603 to -600
"y(B)
the predicted base change at position -588 (A-G), which
C+G
-369
"-AB)
would have been consistent with a gene conversion event
"7 3' Enhancer
involving
the entire sequence from -369 to -61 1, was not
c
T-C
+2,276
observed.
GG+M
+2,357 and +2,358
"y(B)
Small regions of converted gene sequence interspaced by
C-+A
+2,451
c
nonconverted regions have been identified previously in the
A+G
+2,667
I
y-globin genes and have been called "patchy" gene conThe position of each base change is relative to the cap site for the "7version~.~'.~*
Such gene conversions are relatively common in
or Ay-globingenes, respectively.
y-globin gene sequences and frequently result in an Ay-globin
*Base changes not identifiedin normal y-globin gene sequence.
gene converted to a 'y-globin gene sequence. The base
changes
described here clearly suggest conversion of an Ay
bearing the deletion form of HPFH-1 were sequenced (Fig 1)
(A)-type chromosome to an Ay (B)-type sequence, and not
and compared with the sequence of previously reported '7conversion by a 'y-globin gene (Fig 2). In any event, the
and Ay-globin
conversion of the sequences of a normal y-globin gene to that
The sequence of the '7-globin gene from -383 to +2,209
of another normal y-globin gene cannot explain the phenobp (relative to the cap site) was identical to 'y-globin from
the previously reported chromosome A sequence f r a m e ~ o r k , ' ~ type of y-globin gene expression in this patient.
Since no base changes similar to the mutations that give
except for a deletion of one of eight T's (+ 1,611 to + 1,618)
rise to nondeletion HPFH were detected, the sequence of the
and a CG-T substitution (+ 1,800 and + 1,801; Fig 1B and
putative enhancer located 3' to the Ay-globin gene was
Table 1). Both of these nucleotide base changes are present
examined. A 0.76 kb HindIII fragment from the 3' end of the
in the normal '7-globin gene of the B chromosome frameAy-globingene (Fig 1) was isolated from a digest of the 13 kb
work (Slightom, personal communication, February 1988)
Sal1HPFH- 1 fragment by gel electrophoresis and electroeluand, as such, are not likely to be responsible for the increase
tion. The HindIII ends were filled using a mixture of all four
in expression of both '7- and Ay-globin genes characteristic
dNTPs
and the large fragment of D N A polymerase. The
of this condition. A T at - 158 (which creates a restriction
fragment was then blunt end ligated into M13mplO and
enzyme cleavage site for XmnI) is normal for a 'y-globin
D N A templates were sequenced as described above. Four
gene from chromosome A and is present in the patient's
sequence differences were detected:( 1) T-C at +2,276; (2)
D N A sequence. The presence of a T a t -158 has been
GG-AA a t +2,357 and +2358; (3) C-A at +2,451; and
associated with a 'yAy ratio of 60:40 in nonanemic adults
(4) A-G a t +2,667 relative to the Ay-globin gene cap site
and a small increase in H b F (of the Gy variety) in patients
is
(Fig 1C and Table 1). One of these changes (GG-AA)
with sickle cell anemia or B thala~semia,~'
but cannot explain
characteristic of a normal Ay-globin gene of the B chromothe large increase in both '7- and Ay-globin observed in this
patient. The presence of a T at - 158 of the 'y gene has also
been demonstrated in other individuals with HPFH-1 .36
-611 -605 -602
-588
-369
+25 +65
The sequence of the Ay-globin gene from -383 bp to
___
+ 1,760 bp (relative to the cap site) was also identical to the Ay(A)
T
(-)
A
A
C
published sequence for a chromosome A y-globin gene,
___
except for a single base change (C-G) at position -369
(-)
A
(-)
G
Ay (B)
-__
__
(Fig 1C and Table 1). A G a t position -369 is characteristic
C
A
C
c (4 A
A
Gy (A)
of a normal Ay-globin gene from chromosome B33 and is
therefore not a candidate for the mutation underlying HPFH.
Ar (HPFH-I) (-)
A
(-)
A
G
A
C
The C to G base change a t -369 could be the result of a
Fig 2. Sequence variation in the 6' flanking region of the
random mutation at a single base or a gene conversion event
"y-globin gene from a patient with HPFH-1. The sequence obinvolving the 5' end of the Ay-globingene. If indeed a gene
tained from the 5' flanking region of the "7-globin gene from a
conversion had occurred, one might expect to see additional
patient with HPFH-1 ("y[HPFH-l]) is compared with the normal
base changes a t positions where the Ay(A) and Ay(B) genes
"'y-globin gene sequence of the chromosome A ("y[A]) or chromosome B("'Y[B]) frameworks and the "y-globin gene sequence
differ (ie, -588, -603, -605, and -61 l ) , depending on the
("y[A]). The "y(HPFH-1 1 gene sequence can be accounted for by a
size of the gene segment involved in the conversion (ref 33;
"patchy" gene conversion event(s) involving the "y(A) and *y(B)
Slightom, personal communication, February 1988).
genes, most simply illustrated as the sequence between the solid
To obtain sequences from the area further 5' of the
bars above. The exact boundaries of the gene conversion event(s)
Ay-globin gene, a 5.0 kb StuI fragment containing the
are unknown (dashed lines).
Table 1. Summary of Sequence Differences in the "7- and "7Globin Genes from a Patient With HPFH-1
l--T--u--
I
F
-
STOLLE ET AL
502
some framework. The remaining three base changes have
been identified in a variant of nondeletion HPFH in Seattle.26
To determine whether these three base changes represent
mutations that underlie HPFH, cloned or genomic DNAs
from patients with deletion or nondeletion HPFH. other
unrelated disorders (HbS, @ thalassemia and HbE), and
without hemoglobinopathies were amplified using the polymerase chain reaction, applied to a nylon membrane, and
hybridized to allele-specificoligonucleotideprobes. Representative results for the sequence variation found at position
+2,667 are shown in Fig 3. The results of this analysis
(Table 2) indicate that the three base changes are ( I ) usually
inherited as a cluster; (2) not found in two nondeletion
HPFH alleles; (3) present in a patient homozygous for
another major form of deletion HPFH (HPFH-2); but (4)
also present in patients with hemoglobinopathiesunrelated to
HPFH (ie, HbS) as well as in four individuals without
hemoglobinopathies, all of whom had normal HbF levels.
Interestingly, sequences corresponding to the Seattle variant
were detected in the genomic DNA from 9 of 1 I black
individuals, suggesting that these sequences may represent a
cluster of polymorphisms common among blacks. However,
the presence of the variant sequence in individuals with
normal HbF levels indicates that these base changes alone
are insufficient to increase Gy- and *y-globin levels and are
not likely to contribute to the phenotype of HPFH.
DISCUSSION
The phenotypic expression of HPFH is variable.'" The
distribution of HbF may be heterocellular or pancellular in
erythrocytes. The composition of HbF may reflect an increase in predominantly Gy-globin, 'y-globin, or both. The
increase in HbF may be associated with large deletions in the
@-globin gene cluster, point mutations in the promoter
regions of the Gy- or '?-globin genes.' or as yet undefined
mutations either linked to the @-globingene cluster or at
some distant l o c u ~ . ~ ~ ~ " O
A common form of HPFH in American blacks (HPFH-I)
is associated with a deletion of approximately 105 kb in the
@-globingene cluster, which removes the 6- and @-globin
genes and extends over 80 kb 3' to the @-globingene."."
HPFH-I is characterized by an increase in both Gy-and "7-
Table 2. Hybridization of Allele-Specific Oligonucleotide Probes
Corresponding t o the Normal or Variant "7-Globin Gene
Sequence t o Clones or Genomic DNA
3
4
5
C
A
C
A
G
2276 2451 2667 2276 2451 2667
Single Allele
HPFH-1
-
-
-
Nondeletion HPFH
( 202 Oy)
+
+
+
HbS
GreekBthalassemia
+
+
+
Greek HPFH
(- 117 A ~
+
+
-
-
-
)
Genomic DNA
66 thalassemia
heterozygote
HbS heterozygote
HbE heterozygote
HPFH-2 homozygote
normal
normal
normal
normal
+
+
-
-
+
+
+
+
-
-
+
-
+
-
+
-
-
-
-
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
-
+
+
Cloned DNA corresponding to a single allele and genomic DNA was
polymerase chain reaction-amplifiedfor the region corresponding to the
enhancer 3' to the Ay-globin gene and bound to a nylon filter for
hybridization to allele-specificoligonucleotide probes. A ( 1 represents
hybridizationto an oligonucleotide probe containing the normal or variant
base: a ( - 1 represents lack of hybridizationto the probe. The position of
the normal or variant base relative to the 'y-globin gene cap site is
indicated. With the exception of Greek &thalassemia and Greek HPFH,
the DNA examined was that of black individuals.
+
globin expression (Gy, 50.7% f 4.3%)," an HbF level of
approximately 30% in heterozygotes, and pancellular distribution. Feingold and Forget" have identified an enhancerlike sequence in the region 3' to the deletion breakpoint in
HPFH-I, which is brought to within 8 to 10 kb of the
y-globin genes as a result of the deletion. The enhancer-like
region increased the activity of a linked CAT gene when
plasmid constructs were transfected into K562 cells. The
DNA from this region was also hypomethylated and DNase I
hypersensitiveonly in erythroid tissues and contained a large
VA R IA NT
2
T
Base Number
NORMAL
1
Variant SeqUaKa
Namal Sequencs
DNA
Fig 3. Hybridizationof allde-mpdfic ofie0nUd.otides t o polymerasechain raaction amplifik DNA.
Cloned DNAs were amplified using primers flanking
the 0.75 kb Hindlll fragment located 3' t o the
"y-globin gene. The top row was hybridized t o the
normal oligonuceotide spanning the +2,667 region, and the bottom row was hybridized t o the
variant oligonuceotide spanning the
2,667 region. DNA samples are as follows: Lane 1, HPFH-1;
lane 2, nondeletion HPFH (-202 'y1: lane 3, HbS
lane 4, Greek &thalassemia; lane 6, Greek nondeletion HPFH ( - 117 "y).
+
503
-/-GLOBULIN GENE SEQUENCE IN HPFH
open reading frame. The enhancer-like region may serve to
maintain the y-globin genes in a transcriptionally active
configuration, and, as a result, contribute to the phenotype of
HPFH.
However, it is also possible that as yet undetected mutations linked to the @-globin gene cluster or at some distant
locus may be responsible (in part or in whole) for the increase
in y-globin gene expression in HPFH-1. Unlike the mutations identified in nondeletion HPFH, such a mutation(s)
would have to account for the increase in both ‘y- and
Ay-globin gene expression observed in the deletion form of
HPFH. We have examined the sequence of the ‘7- and
Ay-globingenes from a patient homozygous for HPFH-I for
mutations that might contribute to the phenotype.
The ‘7- and Ay-globin genes from an HPFH-1 patient
revealed a total of 10 sequence differences compared with
published sequences.’,32333
Two of these base changes are
located within 230 bp of the ‘7-globin gene poly A signal,
and both are present in normal allelic variants of ‘y-globin.
Four base changes are located 5’ of the Ay-globin gene and
appear to be vestiges of a “patchy” gene conversion event in
which an Ay (A)-chromosome was converted to an *y
(B)-type gene sequence. Four sequence differences were
identified in the putative enhancer located 3‘ to the Ay-globin
gene. One sequence change (G-AA)
is found in normal
Ay-globin genes of a B chromosome framework (Fig 1 and
Table 1). The remaining three base changes have also been
reported by Gelinas et a126in the 3’ enhancer region in cis to
the y-globin genes of a patient heterozygous for nondeletion
HPFH (Seattle variant).
The presence of an identical cassette of base changes in
both deletion and nondeletion forms of HPFH raised the
possibility that one or all of these base changes may contribute to the increase in y-globin gene expression observed in
these conditions. However, analysis of amplified genomic
D N A from a variety of patients with normal or elevated H b F
by hybridization to allele-specific oligonucleotide probes
failed to detect a correlation between elevated y-globin gene
expression and the presence of the three base changes.
Furthermore, the presence of these base changes in patients
unaffected by HPFH argues against a role for these base
changes in increasing y-globin gene expression.
While this manuscript was in preparation, two other
groups reported similar findings. Han et a142sequenced the 3‘
Ay-globingene enhancer from four individuals with various
levels of H b F expression (including one patient with nondeletion HPFH) and found that the presence of the Seattle
variant sequence did not necessarily correlate with high H b F
levels. Bouhassira et a143analyzed 196 chromosomes from
normal individuals of various ethnic groups for the presence
or absence of the variant sequence by polymerase amplification of the 3’ Ay-globin gene region followed by allele-specific
oligonucleotide hybridization or restriction enzyme digestion. They observed the three base changes in every ethnic
group studied. In particular, the changes were present in
100% of native Africans homozygous for HbS, and 93% of
American blacks with HbA. I t is of interest to note that our
black patient with ‘y -202 nondeletion HPFH does not have
these variant sequences. Thus, our data and that of other^^^,^^
suggest that these base changes are not causally related to
HPFH, but instead represent clustered polymorphic sites
common in the black population. Such a clustering of
polymorphic sites has been described previously in the 5’
flanking region of the &globin gene.44
In summary, sequence analysis of the promoter regions of
the ‘7- and Ay-globin genes and the 3’ Ay-globin enhancer
element from a patient with deletion HPFH failed to reveal a
mutation(s) likely to increase both ‘7- and Ay-globin expression. It is possible that as yet unidentified mutations within
the /3-globin gene cluster or at some distant site (such as in
the gene for a trans-acting factor) contribute to the HPFH
phenotype. However, in the absence of other detectable
mutations that correlate with increased ‘7- and Ay-globin
levels, we conclude that the significant mutation in HPFH-1
is the deletion of over 100 kb of genomic DNA.
ACKNOWLEDGMENT
The authors gratefully acknowledge Dr Jerry L. Slightom for
comparison of the sequence data with chromosome A and chromosome B sequence frameworks, Sina Nasri-Chenijani for assistance
with the EMBL-3 genomic cloning, and Nicole E. Sykes for
preparation of the manuscript.
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