Abstract
Objective: The aim of this study was to define the spectrum of α-thalassemia
determinants existing in Jordan.
Methods: A total 286 suspected α-thalassemia subjects including 29
hemoglobin Hb (Hb H) patients were examined by polymerase chain reaction and
restriction enzyme digestion. Polymerase chain reaction product was examined
by agarose gel electrophoresis.
Results: Five different
α-thalassemia determinants were characterized in 336 chromosomes. The
most prevalent α-thalassemia determinant was the single gene deletion -a3.7 (45%). The non-gene deletion a5nt accounted for 27% of thalassemic
chromosomes, followed by the non-gene deletional determinant aT-Saudi (23%). The two-gene deletional
determinant --MED was characterized only in 4% of thalassemic chromosomes. Triplicated α-gene
determinant was observed in two heterozygous individuals (aaa/aa). Four different genotypes were
found to be responsible for Hb H disease. Homozygosity for the non-deletional
determinant a T-Saudi (a T-Saudia/a T-Saudia) was
observed in the majority of those patients (76%) and was found to be associated
with high Hb H levels. Less commonly, Hb H disease occurred as a result
of compound hterozygosity between --MED
determinant with other determinants; (--MED / aT-Saudia), (--MED
/ a5nt a ), (--MED
/ -a3.7 a).
Conclusion: The outcome of this pilot study provides valuable and basic information
about the spectrum of α-thalassemia mutations in Jordan that might be useful in
setting a strategy for molecular diagnosis of α-thalassemia carrier status and
Hb H disease in this country.
Key
words: Alpha thalassemia,
Hemoglobin H , Molecular
JRMS August 2008; 15(2): 23-27
Introduction
Alpha thalassemia is one of the
most common single gene disorders in humans.(1) Most
frequently alpha thalassemia results from the loss of one (α+) or
both of the duplicated α genes (αo) from chromosome 16.(2) Carriers of the deletional forms of α thalassemia (-α/αα),
(-α/-α) or (--/αα) are clinically normal but have a mild hypochromic,
microcytic anemia. Loss of three α genes (--/-α) results in Hb H disease, a
hemolytic anemia with variable clinical course. Infants who inherit no α genes
develop severe anemia , which results in death at or shortly after the time of
birth (Hb Bart's hydrops fetalis syndrome). The most widely occurring single
gene deletions (α+) are the -a3.7
and the -a4.2. The double a-gene
deletions in cis, such as the --SEA, --FIL, and --THAI
are most common in Southeast Asia, while the
--MED and the --20.5 double gene deletions (αo)
occur most frequently in the Mediterranean area.(3) Less commonly, a-thalassemia
results from point mutations and small deletions within the a-globin
genes or in the regulatory sequences.(4,5) The most common non-gene
deletional mutations (aT/aT), existing in Mediterranean
populations are two-point mutations in the polyadenylation site (Poly A) of the
a2 globin gene; aT-Saudi also known as aPA
Saudi (AATAAA>AATAAG),
and aPA (AATAAA> AAGAAA).(6-8) The other important mutation existing in the region is the
pentanucleotide (a5nt) deletion of the first splice
donor site of the a2-globin gene.(9)
In Jordan, like in other Mediterranean
countries various hemoglobinopathies are common. Although several studies have
been carried out to define the frequency and the spectrum of b-thalassemia,(10-12) the incidence and spectrum of a-thalassemia
are still unknown in this country. This
study was carried out in order to acquire the spectrum of a-thalassemia
variants in Jordan.
Methods
A total of 286 subjects (572
chromosomes) were studied among which 29 were Hb H disease patients. Patients
were selected upon having red cell indices suggestive of thalassemia carrier
status (MCV<80 fl and MCH<25 pg) after β- thalassemia and iron deficiency
were excluded.
Blood samples were vacuum collected at Princess Iman Center
for Research and Laboratory Sciences using Na-EDTA as anticoagulant.
Hematological parameters were obtained from an automated counter, Sysmex
XE-2100 (Sysmex-Toa Medical Electronics Co. Kobe, Japan). Red cell lysates were examined on cellulose
acetate electrophoresis at pH 8.6. Hb A2, Hb F and Hb H fractions
were measured by high performance liquid chromatography (HPLC) using BioRad
Variant II. Hb H disease was confirmed by electrophoresis and the demonstration
of Hb H inclusion bodies by supravital staining.
Genomic DNA was isolated by DNA isolation kit
(Instagene genomic isolation kit. BioRad. USA). Detection of α-thalassemia mutations –α3.7, triplicated a-gene,
-a4.2, --MED, --20.5
was achieved by gap polymerase chain reaction (PCR) using published primer
sequences with some modifications to the original technique.(13) Positivity for deletional α-thalassemia was confirmed by
hybridization technique using (mDx a1 gene & mDx a2 gene, BioRad) gene amplification
product. Restriction enzymes used were, Stu I for the (aT
Saudi) mutation,(14) Nla III for the (aPA
) mutation(15) and Hph I for the (a5
nt) mutation.(9) Restriction enzymes
were obtained from (NE Biolabs, Beverly; MA, USA) 10 units
of restriction enzyme were added to 25 μl of the PCR product and incubated at 37 Cο
overnight. For Gap PCR detection of deletional α-thalassemia mutations the following
conditions were used; each 50 μl reaction contained 200 μM of each dNTP, 1.5 mM MgCl2,
2.5 μg
BSA, 10% DMSO, 250- 500ng of genomic DNA and two unit of Taq DNA polymerase
(Ampli Taq Gold polymerase, Perkin Elmer) in the supplied reaction buffer. Reactions
were performed in icycler BioRad. USA. The program was initiated with
denaturation at 96οC for five minutes followed by 32 cycles of 95οC denaturation for one minute, 62οC for 75 sec, and 72οC extension for 135 sec. The
reaction was completed with final extension at 72οC for 10 minutes. After
amplification, 10 μl
of product were electrophoresed through 1% agarose gel in 1x Tris-EDTA-Borate-buffer
at 10 volts/cm for one hour. The ethidium bromide-stained gel was visualized
and photographed under a UV transilluminater. a-globin
gene sequence analysis was performed for 16 samples and was carried out at the Weatherall
Institute of Molecular Medicine (John Radcliffe Hospital,
Oxford, UK).
Results
Five different a-thalassemia
determinants were observed including deletional, non- deletional and triplicated
alpha gene. The gene-deletional determinants were the α+ thalassemia
deletion (-a3.7) and αo thalassemia
deletion(--MED). The non-gene deletional determinants were the (aT
Saudi), sometimes
referred to as aPA1, and the (a 5nt) mutation. The -a3.7 mutation was detected in 45%
Jordanian thalassemic chromosome. The a5nt determinant accounts for 27% of a-thalassemic
chromosomes and the aT Saudi was characterized in23% of a-thalassemic
chromosomes. This mutation affects the poly A addition site of the a2 gene and was first described in patients from Saudi Arabia.(16) The αo (--MED) determinant was detected in 4% and only two individuals were heterozygotes for triplicated a-gene (aaa) determinant (1%). Homozygosity for aT-Saudi (aT-Saudi a/aT-Saudi a) was observed in 76% (22 out of 29). Compound heterozygosity for αo -- MED deletion with other determinants (--MED / - a 3.7), (--MED / aT-Saudi) or (--MED / a5nt) were responsible for the remaining 24% of the total number of Hb H disease genotypes. In one patient, a 28-years old pregnant woman with the genotype (--MED / a5nt). Hb Barts was detected in measurable amounts (4%).
Table I: α-thalassemia chromosome frequency
observed in 286 Jordanian individuals (572 chromosomes) carrying a-thalassemia
|
Type
of chromosomes
|
No. of
chromosomes
|
Frequency
|
|
-a3.7
|
151
|
45%
|
|
a5nt
|
91
|
27%
|
|
aT-Saudi
|
79
|
23%
|
|
--MED
|
15
|
4%
|
|
aaa
|
2
|
1%
|
|
Total No. of (thalassemic chromosomes)
|
338
|
|
|
aa (normal chromosomes)
|
234
|
|
Table
II: a-thalassemia genotypes observed in Hb H disease
patients
|
Genotype
|
No. of
patients
|
Hb(g/dl)
|
MCV(fl)
|
MCH(pg)
|
Hb A2(%)
|
Hb H(%)
|
|
a
T-Saudi a/aT-Saudi
|
22
|
9.2±1.0
|
59.6±7.6
|
18.7±1.4
|
1.2±0.4
|
16.3±5.2
|
|
--MED/a
T-Saudi a
|
3
|
9.5±0.9
|
61.1±2.8
|
18.6±0.3
|
1.1±0.2
|
18.7±6.9
|
|
--MED /-a3.7a
|
2
|
9.6, 9.4
|
62.4, 60.8
|
18.1, 18.9
|
1.2, 1.3
|
8.5, 5.5
|
|
--MED/a5nt a
|
2
|
8.1, 8.4
|
60.0, 60.4
|
18.6, 17.9
|
0.9, 0.8
|
13.8,Hb Barts 4.5% 18.7
|
Discussion
It is believed that a-thalassemia
in Jordan
has been underestimated because Hb H disease is relatively uncommon. Initially, the evaluation
of the possible occurrence of a-thalassemia
determinants was based only on screening methods such as hematological indices
and supravital staining of Hb H bodies. None of these methods is reliable or
sensitive to detect a-thalassemia trait carriers. Other
electrophoretic and immunological methods detecting the small amounts of Hb
Bart’s in newborn babies underestimate the frequency of a-thalassemia
trait carriers.(16) This problem was overcome by the application
of PCR approach for the various a-
thalassemia determinants.(17-19)
In this study we defined the spectrum
of a-thalassemia mutations in Jordan. We reported only the data
in which molecular characterization was achieved and the a-thalassemia
determinants were clearly defined. Since only the most common Mediterranean
determinants have been investigated, we cannot entirely exclude the occurrence
of other rare determinants in few suspected a-thalassemia
cases. These cases were not included in the study. However, the outcome of DNA sequence analysis for these samples is
not expected to affect significantly the data obtained in this study.
As expected, the -a3.7 determinant was the most common (45%),
which is in consistence with the high incidence of -a3.7 genotype and its remarkable
frequency in the Mediterranean region.(1) The second most frequent
determinant was the a 5nt (27%). Fortunately,
even in the homozygous state, both determinants are not associated with severe
clinical phenotype. The frequency of the severe non- deletional determinant aT
Saudi was
strikingly higher than expected (23%), while
the two-gene deletion -- MED was not as
common as reported in other countries in the region.(20) It was observed only
in 4% of thalassemic chromosomes. Heterozygosity
for triplicated α gene (ααα) was observed in two sporadic cases. Despite the rare occurrence of this determinant,
the coinheritance of triplicated alpha
gene with heterozygous β thalassemia
might result in severe clinical phenotype which has important
implications for genetic counseling and prenatal diagnosis.(21) Surprisingly, the a-thalassemia
determinants --20.5, and aPA2 (AATAAA>AAGAAA), believed to be quiet common in
the Mediterranean, were not detected in our
patients.(20,22) More importantly is the lack of -a =4.2
in Jordan
although it was found to be quite common in other Arab countries in the Gulf
region,(23) probably because of the high numbers of immigrants of non-Arab ethnicities to these countries.
Hb H disease patients of the sample
analyzed, 29 individuals were Hb H disease patients. The most prevalent genotype
was homozygosity for a T Saudi (aT
Saudi aT
Saudi /aT
Saudi a) as
many Jordanians are descendants from Saudi origin and because of the high rate
of consanguinity among Jordanians.(24) This genotype accounted for 76% (22/29) of Hb
H genotypes. The average Hb H fraction
for this group was 16.3% (Table II).
Homozygotes for aT
Saudi mostly have severe Hb H disease phenotype and detectable amounts
of Hb H. It has been reported that homozygosity for aT
Saudi might be very severe and in one instant, it caused
fetal loss.(8,25)
This is the contrary of homozygotes
for the other non deletional mutation a5nt detected in Jordan, those
patients have thalassemia trait carrier
phenotype with only a mild hypochromic microcytic anemia and no detectable Hb H
fraction as shown in Table III.
Table
III: a-thalassemia
genotypes observed in asymptomatic carriers
a thalgenotypes | No. of patients | Frequency | Hb H % |
-a3.7a/aa | 117 | 41% | ND* |
a5nt a/ aa | 75 | 26% | ND |
aT-Saudi a/ aa | 31 | 11% | ND |
--MED / aa | 8 | 3% | ND |
-a3.7a/-a3.7a | 16 | 6% | ND |
aaa/aa | 2 | <1% | ND |
a5nt a/a5nt a | 7 | 2% | ND |
a5nt a/aT-Saudi | 1 | <1% | ND
|
ND*: Not detected by cellulose acetate electrophoresis and
HPLC
The other Hb H disease patients showed
compound heterozygous genotypes; (--MED / -a3.7a), (--MED
/ aT Saudia) and
(--MED / a5nta), together they accounted for 24% of Hb H
genotypes. Compound heterozygosity for deletional a-thalassemia
with non deletional type a-thalassemia (--/aTa) have
Hb H disease, which is more severe than deletional type Hb H disease (--/-a). Those
patients might require blood transfusion, which is unusual in patients with the
deletional type Hb H disease.(26) Table II shows that higher levels
of Hb H are associated with these genotypes. However, the small number of
patients with these genotypes might not be sufficient to draw conclusions.
Despite the high frequency of -a3.7 and a5nt
we believe that the absence of compound heterozygous cases with the
genotype (-a3.7 / a5nta)
might be due to the fact that positive samples for -a3.7 with thalassemia trait phenotype
were not further examined for other mutations.
The low incidence of ao thalassemia in Mediterranean
populations accounts for the rare occurrence of Hb Bart’s hydrops fetalis in
the region.(27) However, the occurrence of Hb H disease associated with
aT Saudi in
Jordan might impose
the requirement of specific screening and prevention also molecular
characterization is also useful for the purpose of definitive diagnosis and
counseling.
Based on our results, a suggested strategy
for molecular diagnosis of a-thalassemia trait carriers among
Jordanians should begin with the detection of -a3.7, a5nt, a TSaudi,
and --MED determinants, respectively, while Hb H disease patients
should first be examined for a T Saudi and --MED
determinants, rather then for other determinants.
References
1. Weatherall
DJ, and Clegg JB.
The Thalassemia Syndromes. 3rd edition. Oxford: Blackwell Scientific Publication, 1981; 51-40
2. Huisman
THG, Caver MFH.
The β
and α-Thalassemia
repository. 8th edition. Hemoglobin 1998; 19: 219-236
3.
Higgs DR, Vickers MA, Wilkie AO, et al. A review of the molecular genetics
of the human Alpha- globin gene cluster. Blood 1989; 73: 1081-1104.
4. Paglietti
E, Galanello R, Moi P, et al. Molecular pathology of Hb H diease in Sardinians. British
Journal of Haematology 1986; 63: 485-496.
5.
Pirastu
M, Saglio G, Chang GC, et al. Initiation codon mutation as a cause of alpha –
thalassemia. Journal of Biological Chemistry 1984; 259: 12315-12317.
6. Higgs
DR, Goodboun SF, Lamb J, et al. a-thalassemia
caused by a polyadenylation signal mutation. Nature 1981; 306: 398-400.
7.
Thein
SL, Wallace RB, Pressly L, et al. The
polyadenylation site mutation in the alpha- globin
gene cluster. Blood 1988; 71: 313-319.
8.
Fei
YJ, Öner R, Bozcunt G, et al. Hb H disease caused by homozygousity for the AATAAA>
AATAAG mutation in the polyadenylation site of the a2
globin gene. Acta Haematol 1992; 88: 82-85.
9.
Orkin
SH, Goff SC,
Hechtman RL. Mutation
in the intervening sequence splice junction in man. Proceedings of the National Academy
of Science of the United
States of America 1981; 78: 5041-5045.
10. Bashir N, Barkawi M, Sharif L. Prevalence of haemoglobinopathies
in North Jordan Valley.
Ann Trop Paediatr 1991; 11: 373-376.
11. Bashir N, Barkawi M, Sharif L, et al.
Prevalence of
haemoglobinopathies in North Jordan. Trop
Geogr Med 1991; 44: 122-125
12. Sadiq MF, Eigel A, Horst J. The spectrum of β-thalassemia in Jordan:
Identification of Two Novel Mutations. American Journal of Hematology
2001; 68:16-22.
13. Liu YT, Old JM, Miles K, et al. Rapid detection of alpha-
Thalassemia deletions and alpha-globin gene triplication by multiplex
polymerase chain reaction. British Journal of Haematology 2000; 108:
295-299.
14. Jassim N, AL-Arrayed S, Gerard N, et
al. A
Mismatched- Primer Polymerase Chain Reaction- Restriction Fragment Length
Polymorphism Strategy for Rapid Screening of the Polyadenylation Signal
Mutation aT-Saudi (AATAAA> AATAAG) in the a2
Globin Gene. Hemoglobin 1999; 23(3): 213-220.
15. Fei Y-J. Dissertation. Medical College
of Georgia, Augusta, Georgia,
1992.
16. Zorai A, Cornelis L, Harteveld C L, et
al. Molecular
Spectrum of α-thalassemia in Tunisia:
Epidemiolgy and detection at birth. Hemoglobin 2002; 26(4): 353-362.
17. Al-Qaddoumi AA. A PCR-based method to detect alpha thalassemia with Southeast Asia mutation. JRMS 2005; 2(2): 72-74.
18. Baysal E, Huisman THJ. Detection of common deletional α-thalassemia-2
determinants by PCR. Am J Hemato 1994; 46: 208-213.
19. Chong SS, Boehm CD, Higgs DR, et al.
Single-tube multiplex-PCR screen for common deletional determinants of
α-thalassemia. Blood 2000; 95: 360-362.
20. Baysal E, Kleanthous M, Bozkunt J, et
al. a-Thalassemia
in the population of Cyprus.
British Journal of Haematology 1995; 89: 496-499
21. Oron V, Filon D, Oppenheim A, et al. Severe thalassemia intermedia
caused by interaction of homozygosity for a-globin
gene triplication with hetrozygosity for βo-thalassmia. British
Journal of Haematology 1994; 86:
377-379.
22. Oner C, Gurgey A, Oner R, et al. The molecular basis of Hb H
disease in Turkey.
Hemoglobin 1997; 21(1): 41-51.
23. El Kalla S, Baysal E. Alpha-thalassemia in United Arab Emirates.
Acta Haemtol 1998; 100(1): 49-53.
24. Khoury SA, Massad D. Consanguineous marriages in Jordan.
Am Med Genet 1992; 43: 769-75.
25. Cürük, MA, Kiline Y, Evrüke C, et al. Prenatal diagnosis of Hb H disease
caused by a homozygosity for the a2 polyA (AATAAA>AATAAG) mutation.
Hemoglobin 2001; 25: 255-258.
26. Higgs DR, Bowden DK. Molecular mechanisms of a
thalassemias. In: Steinberg MH, Forget BG, Higgs DR, Nagel RL, ed. Disorders of
hemoglobin. Cambridge University
Press, Cambridge. 2001; pp. 431-469.
27. Chu DHK, Waye JS. Hydrops fetalis caused by a-thalassemia:
an emerging health problem. Blood 1998; 91: 2213-2222.