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1992 80: 1582-1585
Rapid detection of Spanish (delta beta)zero-thalassemia deletion by
polymerase chain reaction
JL Vives-Corrons, MA Pujades, A Miguel-Garcia, A Miguel-Sosa and S Cambiazzo
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Rapid Detection of Spanish (S@)O-ThalassemiaDeletion by
Polymerase Chain Reaction
By J.L. Vives-Corrons, M.A. Pujades, A. Miguel-Garcia, A. MigueCSosa, and S. Cambiazzo
8pThalassemia and hereditary persistence of fetal hemoglobin (HPFH) are inherited disorders characterized by the
persistent synthesis of fetal hemoglobin (HbF) during adult
life. The Spanish type of Gpthalassemiais a mild thalassemic
condition due to a large deletion starting at the Alu I repeat
between the 4 and &-globingenes immediately 3‘ to the RIH
probe and extending 11 and 17 kb downstream of the 3‘
endpoints of HPFH 1 and HPFH 2, respectively.Using probes
from the Spanish (Sp)”-thalassemic DNA, the 3’ breakpoint
region has been mapped to a point approximately 8.5 to 9.0
kb downstream from that of HPFH type 1 and, as we know
the restriction sites 3’ to this breakpoint,the presence of the
deletion can be identifiedwith the polymerase chain reaction
(PCR). In the present study, a PCR method using three
specific oligonucleotideshas been developedfor the identification of the Spanish (8p)”-thalassemia in 100 patients with
Sp-thalassemia (99 heterozygotes with mild anemia, decreased mean corpuscular volume, and 5% to 15% HbF, and
one homozygotewith 100% HbF and thalassemia intermedia
phenotype).We conclude that the finding of the Spanish type
of (Sp)”-thalassemia in all the patients studied here suggests
Spain as the most probable origin of this thalassemic phenotype. Moreover,the amplificationof the fragment encompassing the deletion junction and normal sequence is useful for
the rapid molecular detection of Spanish (8p)”-thalassemia.
8 1992by The American Society of Hematology.
ELETIONS in the p globin gene cluster cause a
variety of hematologic phenotypes including Spthalassemia and hereditary persistence of fetal hemoglobin
(HPFH) (Fig 1). Both conditions are characterized by
increased levels of fetal hemoglobin (HbF)during adulthood, ranging from 5% to 15% in SP-thalassemia carriers
and 20% to 30% in the heterozygous state of HPFH. HPFH
consists of a heterogeneous group of conditions in which
the switch from the synthesis of HbF to that of adult
hemoglobin (HbA)fails to occur or it is i n c o m ~ l e t e .The
~-~
precise breakpoint location of the four known HPFH
deletions (HPFH-1, -2, -3, and -4) have been identified and
in all cases both the globin genes S and p are deleted?v5 The
GP-thalassemias are a related group of deletion mutants in
which the expression of HbF is considerably lower and, in
heterozygotes, is distributed in a heterogeneous or heterocellular fashion among red blood cells (RBCs), in contrast
to the fairly uniform or pancellular distribution observed in
HPFH. Recently, the nucleotide sequence in the region
corresponding to the “3’ end” of the deletion causing
Spanish (GP)”-thalassemia has been reported,6g7 permitting
the development of a rapid screening method for the
detection of this deletion by amplifymg the 3‘ breakpoint in
this condition. Taking advantage of this possibility we used
the polymerase chain reaction (PCR) method and three
different oligonucleotide primers (two for the normal gene
and one for the deletion) for the rapid molecular study of
100 subjects from 42 different families diagnosed as having
SP-thalassemia according to clinical and hematologic data.
In all 99 patients with heterozygous SP-thalassemia phenotype, two fragments of 685 bp and 299 bp were obtained,
whereas single fragments of 685 bp and 299 bp, respectively,
were observed in normal subjects and the patient with
homozygous SP-thalassemia.
D
From the Haematology Laboratory Department, Hospital Clinic i
Provincial, University of Barcelona, Barcelona; and the Department of
Haematology and Haemotherapy, Hospital Provincial de Valencia,
University of Valencia, Valencia, Spain.
Submitted August I , 1991; accepted May 21,1992.
Supported in part by a Grant of Scientific Cooperation between the
National Health Services of Spain and Argentina. S.C. is supported by
a grant fiom the Argentina Natwnal Health Service.
Address reprint requests to J.L. Eves- Comns, MD, Haematology
Laboratory Department, Hospital Clinic i Provincial, Universiv of
Barcelona, c / EllaroelI70, 08036 Barcelona, Spain.
The publication costs of this article were defiayed 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 thisfact.
6 1992 by TheAmerican Sociev of Hematology.
0006-4971/92/8006-OOO5$3.00/0
1582
MATERIAL AND METHODS
Ninety-nine 6p-thalassemia carriers belonging to 41 unrelated
families with HbF levels ranging from 3.5% to 10.5% and with
normal or decreased HbA2 were identified during survey studies
performed in Spain. Forty of these patients were studied, before
the standardization of the PCR method, by restriction endonuclease digestion of genomic DNA and blot hybridization (Southern
blot method) and tested later by PCR. The remaining 59 patients
were only tested by the PCR method. For comparison purposes, a
patient with homozygous tip-thalassemia was also included in the
study.
Basic hematologic parameters were obtained by the Technicon
H*2 System (Bayer Diagnostics). HbA2 levels were calculated by
microchromatography and HbF percentage by alkali denaturatioa8
Restrictwn endonuclease mapping of DNA. DNA was prepared
from peripheral blood bu@ coat by phenol-chloroform extraction
and digested with appropriate restriction enzymes (BamHI, Bgl 11,
Pvu I, EcoRI, HindIII, and Pst I)? According to the manufacturer’s
recommendations, DNA fragments were separated on agarose gel
(0.8% to 1.2%) and transferred to nitrocellulose filters. The filters
were hybridized to the genomic probes corresponding to the G,(a
2.7-kb fragment of EcoRI containing the G,gene), pseudo-p gene
(a 1.8-kb fragment of Bgl 111% I), and H p S (a 4.4-kb fragment of
Pst IIHindIII containing the p gene). The DNA probe for Spanish
(6p)”-thalassemia was kindly supplied by the INSERM (Hopital
Henry Mondor, Paris, France).
PCR method. PCR reaction of the 3’ breakpoint region was
performed using Taq I DNA polymerase and three specific
oligonucleotide primers obtained from normal 5’ DNA sequence
within RIH region (N,), normal 3‘ DNA complementarysequence
(Nz), and DNA complementary sequence to the Spanish (6p)”thalassemic 3‘ breakpoint region (Sppe) (Fig 2). The DNA se-
Blood, Vol80, No 6 (September 15). 1992: pp 1582-1585
From www.bloodjournal.org by guest on October 15, 2014. For personal use only.
SPANISH GF-THALASSEMIA DETECTION BY PCR
=v
fg
AIJ
(6p)- thal Spanish
(6s).thal Sicilian
0
kb
10
-40 kh
I
I
(6s)" thal Black
-a p
1583
30
I
D
( 6 p ) O thal Turkish
(ab)" thal
Japanese
II
IIII
I
(6s)"thal Chinese
UPFU-1
Fig 1. Map of the human
@-globin gene cluster showing
the location and extent of various deletions associated with
HPFH and a@-thalassemia.
BpFu-4
the 5' breakpoint of the deletion is situated between the 3'
Bgl I1 restriction site of the normal p gene and the 5' Pvu I
restriction site of the deleted ti gene.
PCR analysis. One fragment of 685 bp was obtained in
all cases when the oligonucleotides N1 and N2 were used,
and a second fragment of 299 bp was also apparent when
the DNA amplification was performed in the presence of
the N1 and SpsPoligonucleotides. In normal controls and in
the case of homozygous Spanish (tip)"-thalassemia (one
case), only single fragments of 685 and 299 bp, respectively,
were obtained (Fig 3).
quences of the three oligonucleotides N1, Nz, and Spsp used in the
present study are: N1,5'-ATG GGT A l T TCA CTT GTT AT-3';
Nz, 5'-ACT TTG TCT GTT AAT TCC AA-3'; Spsp, 5'-ACT GTG
GGA GCC CCT TTC TG-3'.
Amplification of normal DNA with N1 and NZprimers gives a
single band of 685 bp, whereas in DNA with homozygous Spanish
(tip)"-thalassemia deletion, no bands are obtained by using N1 and
Nz primers and a single fragment of 299 bp is obtained with N1 and
[email protected]
RESULTS
HematoZogicfindings. The results (mean 2 SD) relative
to the 99 subjects heterozygous for (tip)"-thalassemia are
shown in Table 1. These values were compared with those
of the p-thalassemia trait and the a-thalassemia trait
(homozygous and heterozygous) also obtained in our laboratory. The single case with homozygous Spanish (tip)"thalassemia showed the following hematologic data: RBC,,
4.3 x 1OlZ/L; Hb, 109 g/L; mean corpuscular volume
(MCV), 80 fL; RDW, 28.8%; reticulocytes, 6%; HbA2,
0.65%; HbF, 86.7%. The Kleihauer Test showed a homogenous pattern.
Southem blot analysis. Digestion of patients' DNA with
HindIII and EcoRI restriction enzymes and hybridization
to the 4 genomic probe showed, in all cases, a normal
band. The same pattern was obtained by digesting with
BumHI enzyme and hybridizing with HPS. Hybridization
with p probe provided normal fragments when Pst I and Bgl
I1 restriction enzymes were used, whereas abnormal bands
appeared with Pvu I and HindIII digestion, indicating that
50
1-
L
upFu-2
UppH-3
60
70
DISCUSSION
Spanish (tip)"-thalassemia is a mild thalassemic condition
characterized by increased levels of HbF production during
adult life. It is known that the 5' breakpoint of the Spanish
(tip)"-thalassemia deletion lies within the Alu I repeat,
between the 4 and ti-globin genes immediately 3' to the
RIH probe,ll and extends at 11 and 17 kb downstream to
the 3' endpoints of HPFH-1 and HPFH-2, respectively (Fig
1).Up to now, only the Japanese (tip)"-thalassemia deletion
has been found to be at least 16 kb longer than the Spanish
(tip)"-thalassemia and its 3' breakpoint extends further
downstream to the 3' end of the Spanish (tiP)"-thalassemia.l2,l3Knowing the 3' restriction sites of this breakpoint has allowed us to identify the presence of the specific
deletion by using the PCR.
In the present study, a PCR screening method for the
Spanish (tip)"-thalassemia deletion was undertaken in a
90
80
100
Worral 5'DWA
TG-CGGTGGC TCAGGCTTGT AAACCCAGCA CTTTGGGAGG CCAAGGCAGG CAGATCACTT
( d B ) O t h a l Sp
GGGAGCTGGC TCCGGCCTTG GCCAGC4AG AAAGGGGCTC CCACAG+CA
Worm61 3'DWA
GGGAGCTGGC TCCGGCCTTG GCCAGCCCAG AAAGGGGCTC
I I Ill1 II Ill I
I
IIIIIIIIII IIIIIIIIII IIIIIIIIII I I I I I I I I I I
GTGGCGGGCT
IIIIIIIIII IIIIIIIIII
CCACAGTGCA GTGGCGGGCT
Spw: ACT GTG GGA GCC CCT TTC TG
Fig 2. Sequence of normal DNA in the
region corresponding to the 3' end of the
deletion causing Spanish (Sf.%)"-thalassemia.
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VIVESCORRONS ET AL
1584
Table 1. Comparhon of General Hematologk Data Between PatientsWith @3-ThrlauemIa,other HeterozygoteThalas"la8,
and Nonnal Controls
RBC (x1012/L)
Hb (glLI
MCV(n)
MCHC (glL)
RDW (%)
HDW (glL)
WBC (XlOolL)
Platelets (xlOo1L)
Reticulocytes(%)
HbA2 (%)
HbF (%)
No. of cases
N m l
(8@Y-Thel
Trait
@-ThSl
Trait
a*-Thal
Homozygous
a'-Thsl
Heterozygous
Controls
5.5 f 0.7
117 f 12
67.4 f 5.4
314 f 32
18.6 f 1.42
31.4 f 2.86
6.9 f 1.9
253 f 65.6
1.73 f 0.71
2.75 f 0.41
6.7 f 3.29
99.
5.8 f 0.7
123 f 13
68 f 6.6
313 f 12
15 f 1.2
28.4 f 2.9
7.0 f 1.5
253 f 57
1.20 f 0.60
5.3 f 1.8
0.3 f 0.10
54
5.6 f 0.5
127 c 12
72.4 f 6.6
313 f 14
14.7 f 0.62
26.8 f 1.9
7.9 f 3.2
282f56
0.98 I 0.38
2.6 f 0.40
0.58 f 0.45
27
5.1 f 0.5
137 f 15
82.9 f 5.6
324 f 11
13.7 L 0.89
24.3 f 2.25
7.7 2 2.1
246.5 f 64.8
1.06 f 0.44
2.52 f 0.33
0.53 f 0.36
79
5.4 f 1.5
150 f 20
90 10
330 f 20
13.7 f 0.4
24.8 f 2.2
7.5 f 3.5
275 f 125
1.25 I0.75
2.25 f 0.75
52
50
Abbreviations: WBC, whiie blood cell count.
.The results of the single case with homozygousSpanish (8p")thalassaemia are not included here.
large group of subjects with slightly increased HbF. Three
different oligonucleotides were used: two (NI and Nz) for
the normal DNA and two (NI and SpW)for the thalassemic
DNA. In normal subjects, the amplification of DNA in the
presence of N I and Nz provides a single fragment of 685 bp,
whereas in homozygous Spanish (SPY-thalassemia,a unique
fragment of 299 bp is observed only if NI and SpW
oligonucleotides are used. Therefore, by using the three
oligonucleotides, the genotype of heterozygous Spanish
(GPY-thalassemia is characterized by two different fragments of 685 bp and 299 bp, respectively. It is noteworthy
that in the present study all the subjects with heterozygous
Sp-thalassemia phenotype analyzed for the presence of the
Spanish (SPY-thalassemia deletion showed the presence of
the 299-bp fragment, characteristic of this deletion. Because in all the cases the normal 685-bp fragment was also
apparent when the NI and Nz primers were used, it can be
concluded that all the subjects were carriers for the Spanish
@BY-thalassemiadeletion.
Although Sp-thalassemia has been observed in different
ethnic groups, it has been described mainly in subjects of
Mediterranean extraction.Iel* In Spain, tip-thalassemia is
not a rare condition, and about 7% of thalassemia trait
carriers are heterozygous for tip-thalassemia? Spanish
(SpY-thalassemia was first described by Ottolenghi and
GiglioniIz in 1982, but its incidence among the Spanish
population is presently unknown. Except for the report of
hematologic studies performed in homozygous Spanish
(Sf3Y-thalassemiapatients by Baiget et a17in 1983, there are
no other references to this specific type of deletion. The
present study performed on a large group of tip-thalassemia
carriers shows that Spanish (Spy-thalassemia is the most
frequent trait producing high HbF in Spain and contributes
to a better definition of this type of thalassemia trait at both
the hematologic and molecular levels. Furthermore. the
fact that all of the patients studied here were from Spain
suggests that the Spanish (tip)"-thalassemia may originate
from that part of the world.
ACKNOWLEDGMENT
We are indebted to Drs Michel Vidaud and Nada Ganem for
providing us with the oligonucleotides used for the PCR method
and to Prof Goosens (Hopital Henry Mondor. Paris, France) for
the critical review of the manuscript.
Fig 3. Three percent agar080 ebctmphomb of
PCR perfonnod In DNA samples of patients wlth
Spanbh (8pr-thalassemla and in normalcontrob. M:
molecular weight marker; lanes 1 and 7: normal
controls; lanes 2,3,4,6,8,9, and 10: heterozygotes
for the Spanish (f@)"-thaIa8semia deletion; lane 5:
homozygous patient for Spanish (8pr-thalassemia.
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SPANISH SV-THALASSEMIA DETECTION BY PCR
1585
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