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CORRESPONDENCE
RAPID DETECTION OF A 13.4-kb DELETION CAUSING S/3 THALASSEMIA IN AN EGYPTIAN
FAMILY BY POLYMERASE CHAIN REACTION
To the Editor:
At least 35 deletions involving the p globin cluster on chromosome
1 1 have been described.’ Analysis of the phenotypes associated with
the different deletions has provided much insight into the normal
regulation of expression of the human /3 globin gene cluster, leading
to the identificationof several important regulatory elements including
the major upstream locus control region (LCR) 5‘ to the c
Deletions of similar size but affectingdifferent regions ofthe p cluster
may give rise to different clinical entities such as Sp thalassemia and
deletional hereditary persistence of fetal hemoglobin (HPFH); the
distinction between the two conditions is subtle and is determined
by the degree to which HbF is elevated and the red blood cell indices!
Arguments based on genotype/phenotype correlations are strengthened by the accumulation of unrelated individuals of different ethnic
origin with identical deletions, presenting with very similar phenotypes.
We describe here an Egyptian family with So thalassemia caused
by a 13.4-kb deletion that removes the 3’ region of the 6 gene, the
entire p gene, and its 3’ flanking sequences extending into the 6.4kb LI repeat element. We have used the polymerase chain reaction
(PCR)5 and direct sequencing to characterize the endpoints of this
deletion which proved to be identical to those of the Sicilian (Sp)”
thalassemia.6Subsequently, an Italian individual with a similar phenotype was shown to be heterozygous for this deletion. The phenotypes of both the Egyptian and the Italian individuals are strikingly
similar to those of the Sicilian individuals heterozygous for the same
deletion.
The hematologic parameters of individuals in the Egyptian family
and the Italian patient are summarized in Table I and are compared
with the average values reported for the previous cases of Sicilian Sp
thalassemia.’ All individuals affected have hypochromic microcytic
indices with substantially elevated HbF (4.8% to 9.6%) and normal
hemoglobin A2 (HbA2)levels.
The @ globin complex in the propositus of the Egyptian family
was analyzed with a series of restriction enzymes and Southern blot
hybridizations with specific globin gene probes. Hybridization with
the $p probe proved to be informative. In addition to the expected
normal fragments, abnormal-sized fragments were observed when
DNA digested with Xmn I, BstE 11, HindII, and HindIII was hybridized with the $@probe, but BamHI-$P hybridization produced the
normal 15.3-kb band. The probe pRK29, which is located 19 kb 3’
to the /3 gene, was used to obtain data pertaining to the 3’ end of the
deletion. When pRK29 was hybridized to DNA digested with a range
of restriction enzymes, no abnormal bands were observed, with the
exception of HindIII, which produced a band of approximately 30
kb that appeared to be identical in size to the HindIII-$(3 fragment.
On the basis of the restriction mapping data (not shown), the 5‘ end
ofthe deletion was localized to between the BamHI site at coordinate
55232 (Genbank HUMHBB), and the Hind111 site at coordinate
59607. The 3’ deletion breakpoint was shown to lie downstream of
the BstE I1 site at coordinate 68829 by considering the data obtained
from the relevant $0 probe hybridizations. PCR primers chosen to
flank the putative breakpoints were synthesized and used to amplify
a deletion specific fragment (Fig 1) containing the deletion breakpoints. A PCR product of approximately 1.8 kb was obtained, which
predicted a deletion size of 13.4 kb as the primers are 15,210 bp
apart in normal DNA. The amplification primers were biotinylated
to facilitate the separation of single-stranded DNA for direct sequencing as previously described.8 The noncoding strand was sequenced using the sequencing primer SI (Fig 1).
The deletion is 13,377 bp in length the 5’ breakpoint of the deletion
lies within IVS-2 of the 6 gene at coordinate 55961 (Genbank
HUMHBB), and the 3’ breakpoint lies at position 69338 within the
full-length 6.4-kb L1 repeat located downstream of the p gene. Two
orphan nucleotides, AC (sense) or TG (antisense), of unknown origin
lie at the breakpoint making it identical to the Sicilian Sp thalassemia
breakpoint as determined from cloned DNA by Henthorn et aI6 (Fig
2). DNA from the Italian individual (who is adopted) was subjected
to similar restriction mapping and abnormal bands of the same size
as in the Egyptian family were found. PCR using the same primers
and conditions as described above and in Fig 1 produced an abnormal
band of similar size to that observed for the affected Egyptian individuals. Furthermore, direct sequencing of the PCR product showed
changes identical to those found in the Egyptian and Sicilian cases.
In addition to the two orphan nucleotides, two single-base variations
Table 1.
Subject
Egyptian family
Father
Mother
Propositus
Brother
Brother
Adopted Italian individual
Sicilian Sg thalassemia
MCV (fL)
MI72
F/62
MI29
MI32
MI20
MI22
11.5
14.7
12.7
14.4
14.2
12.1
12.4
68.5
92.1
65.7
90.1
92.8
72.1
7 1 .O
21.6
31.8
21.2
30.7
30.7
22.4
23.0
HbA, (%)
HbF (%)
F Cells (%)
2.8
4.8
0.4
8.0
0.3
0.5
9.6
9.0
58
5
70
4.4
1.5
70
NA
2.4
2.2
2.6
2.8
2.5
2.1
Hematologic parameters of the Egyptian family, the unrelated Italian individual, and the previously reported cases of Sicilian Sp thalassemia’ (for
which the average values are shown). F-cell percentage was determined on smears as previously described; in a survey of 138 normal blood donors,
it was found that 89.2% had less than 4.3% F cells.” In the three cases of Sg thalassemia described in this report, the F-cell distribution was found
to be markedly heterocellular.
Abbreviation: NA, not available.
Blood, Vol81, No 3 (February 1). 1993: pp 861-870
861
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862
Psi I-delta
A f i
U
-
CORRESPONDENCE
pRK29
U
PSt I-beta
NORMAL
3'
I
Deletion 13377 bp
BamHl
DELETION
MUTANT
3'
Breakpoint
-1.8
kb +
1 2 3 4 5 6 7 MNiN2N3MB
4.9 k b - b
1.8 kb
+
Fig 1. (A) Restriction map showing the location and extent of the deletion. The positions of probes Pst 16, Pst 1 B, and pRK 29 used in
the restriction mapping of the deletion are shown. Restriction endonuclease sites are shown for EcoRI, BemHI, Hindlll, and BstEll. The
location of a Bg/ II site specific for the deletion allele and the lass of a 7eg Isite present in normal DNA are highlighted. Primers P1 and P2
were used to amplify a deletion specific fragment. A sequencing primer S1 was used to sequence across the breakpoint. The fragment
amplified by primers P3 and P4 was digested with 7aq Iand Bg/ II t o examine for the presence/absence of restriction sites in a series of
normal individuals. (B) Amplification of a 1.8-kb deletion-specific PCR product across the deletion breakpoints in the Egyptianfamily members
(lanes 1 through 5). an adopted Italian individual (lane 6), and an individual with a 10.3-kb deletion* as a positive control for the amplification
primers P1 and P2 (B). Lanes N1, N2, and N3 represent three normal individuals who demonstrate lack of amplification under identical
conditions. P1 represents the forward primer 5'-TTGGGTITCTGATAGGCACTG-3 and P2 the reverse primer, 5'TGTGTITGCTCCTGCTTCTCTAG-3, which are 15.2 kb apart in normal DNA. Lane M represents the A-Hindlll DNA markers. Lane B
represents a water blank, included as a negative control for the PCR.
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CORRESPONDENCE
863
A3'
C
T
C
C
T
--
\
viously described in the Sicilian thalassemia are also found in the
two cases under discussion, suggesting that the deletion is probably
of a single origin. Amplification of the deletion-specific fragment encompassing the deletion junction should be useful for the rapid detection of this (bB)o-thalassemia, which appears to be common in
the Mediterranean region.
The similarity between the phenotypes of these new cases and the
previously described heterozygotes for this mutation is of interest to
those interpreting data comparing the various deletions arising within
the @ globin cluster and examining the mechanisms by which they
cause the associated phenotypes.
~
A
C
---_-.
G
E
,
-
A
T
C
C
G
**+/
H
-
ACKNOWLEDGMENT
/
We thank Liz Rose for preparation of the manuscript. J.E.C. is a
Nuffield Dominion Scholar.
T
+ /
5'
8 Normal 5' aequo"
+
TGTATCTCCTACACACACATATCTATATACMTATA
I l l
IIIIIIIIIIIII
Ill
Deletion mutant
TGTATCTCCTACAC~~~TGTGTGTATCCGAG~TAIT
Normal3' sequence
TAGNXGCACOTCTCTGTGTGTATCCGsmAm
I
I
I
I IIIIIIIIIIIIII IIIIII
JAMIE E. CRAIG
REBECCA BARNETSON
DAVID J. WEATHERALL
SWEE LAY THEIN
MRC Moleciilar Haemarology Unit
Insriliile of Moleciilar Medicine
Oxford, UK
t
Fig 2. (A) DNA sequence of the deletion breakpoint region obtained by sequence analysis of the PCR product (noncoding strand)
shown in (Fig 1 B) using the sequencing primer S1. The potential
breakpoints are indicated by arrows and the position of two orphan
nucleotides (TG) found at the deletion junction is highlighted (*). A
C
T single-base substitution (") destroys the recognition sequence for Teq 1 (underlined in B). An A + C point mutation (***)
creates a recognition site for the restriction enzyme Bg/ II. (B) The
5 normal and 3 normal sequences are compared with the deletion
mutant allele. The cleavage site for Taq 1 in the 3 normal sequence
is underlined. Vertical lines represent identical bases.
-.
from the published normal sequence (Genbank HUMHBB) immediately 3'to the breakpoint were reported in the Sicilian S@ thalassemia;
G to A at coordinate 69353 (C to T on the noncoding strand), and
T to G at coordinate 69373 (A to C on the noncoding strand). The
former change abolishes a cleavage site for the restriction enzyme
Taq 1, and the latter change creates a cleavage site for the enzyme
Bgl 11. Identical single-base changes were found in the affected individuals from Egypt and the unrelated Italian individual. We have
screened several normal individuals by the PCR using primers P3
and P4 to amplify a 4.1-kb fragment (Fig 1) that was subjected to
restriction analysis with Taq I and Bgl 11. In all these normal individuals, the Taq I site was present and the Bgl I1 site absent, supporting
the conclusion of Henthom et aI6 that these point changes are specific
to this deletion.
We have characterized the mutations responsible for a &3 thalassemia phenotype in an Egyptian family and an unrelated Italian individual. In both cases a deletion has been demonstrated by amplification of an abnormal deletion-specific PCR product. Direct
sequencing has shown the deletion breakpoints to be identical to
each other and also to the previously described Sicilian &3 thalassemia.
Two sequence variations immediately 3' to the deletion junction pre-
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1991
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The formation ofdevelopmentally stable hypersensitivesites in globinexpressing hybrids. Nucleic Acids Res 15:10159, 1987
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GT, Mullis KB, Erlich HA: Primer-directed enzymatic amplification
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9. Craig JE, Kelly SJ, Barnetson R, Thein S L Molecular characterization of a novel 10.3 kb deletion causing @-thalassaemiawith
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IO. Sampietro M, Thein SL, Contreras M, Pazmany L Variation
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From www.bloodjournal.org by guest on June 15, 2017. For personal use only.
1993 81: 861-863
Rapid detection of a 13.4-kb deletion causing delta beta thalassemia in
an Egyptian family by polymerase chain reaction [letter]
JE Craig, R Barnetson, DJ Weatherall and SL Thein
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