Known and new delta globin gene mutations and their diagnostic significance.

Abstract

Mutations in the delta-globin gene (HBD, MIM# 142000) are not pathologically relevant. However, since high HbA2 levels are diagnostic for beta-thalassemia trait and a lowered level for an alpha- or delta-mutation, co-inheritance of delta- and beta-gene defects may lead to misinterpretation of diagnostic results. We examined 29 cases with low HbA2 level diagnosed in our laboratory, in the presence or absence of a second HbA2 fraction. We found a delta globin gene mutation in 20 cases. In total four different known mutations were found, three structural and one expressional. Moreover, two new defects were observed, one causing a structural abnormality and one a beta-thalassemia. The structural abnormality HBD c.431A->G (p.His144Arg)(dcd 143 CAC->CGC) was homologous to the beta-globin gene variant called Hb-Abruzzo and we have named this mutation HbA2 -Abruzzo. The new delta-thalassemia defect HBD c.-118C->T (d -68 C->T) has no homology on the beta-globin gene (HBB, MIM# 141900). All mutations caused a low HbA2 level and through this could lead to misdiagnosis when inherited together with a beta-thalassemia.

Full text

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haematologica/the hematology journal | 2006; 91(1) | 129 |
Marelle J.Bouva
Cornelis L.Harteveld
Peter van Delft
Piero C.Giordano
Dept.of Human and Clinical
Genetics,Leiden University
Medical Centre (LUMC),Leiden,
The Netherlands
Known and new δ δ globin gene mutations and
their diagnostic significance
I
bin. Although some silent β-thalassemia
traits do not present with elevated HbA2
fractions, the finding of a slightly to clear-
ly elevated HbA2 fraction (3.5-8%) is the
classic parameter associated with β-tha-
lassemia trait. Conversely, low-normal
HbA2 levels may be associated with α-tha-
lassemia and iron deficiency.1Since HbA2
consists of two α and two δ polypeptide
chains, defects on the δ-gene (HBD, MIM#
142000) may also modify the expression
of the HbA2 fraction. Co-inheritance of δ-
and β-thalassemia results in a less elevated
HbA2 level and may therefore lead to a
wrong diagnostic conclusion. Cases in
which a δ-gene mutation is present are
often observed during diagnostic investi-
gations in our reference laboratory. Here
we report the suspected cases of δ globin
gene mutations observed during about 2
years of diagnostic activity.
n normal individuals over 2 years old
the HbA2 fraction accounts for between
2.5% and 3.4% of the total hemoglo-
Materials and Methods
We examined a total of 29 cases of
which 13 were suspected to have expres-
sion defects and 16 to have structural
abnormalities. Analytical methods includ-
ed alkaline starch gel electrophoresis and
automatic high performance liquid chro-
matography (HPLC) analysis (Figure 1).
DNA was either extracted automatically
using the Autopure LS extractor (Gentra
System, Minneapolis, Minnesota, USA) or
isolated by selective lysis2and high salt
extraction.3Polymerase chain reactions
(PCR) were carried out in the GeneAmp
PCR system 9700 (Applied Biosystems,
Perkin Elmer Corporation, Foster City/Ca,
USA), using three different primer sets.
Primer set 1 (5'agaacagccaatctcagggc3'/5'
gagcctctcttataaccttg3') gives a fragment of
330 bp. The primers for exon 2
(5'caaggttataagagaggctc3'/5'agagaaaagtgaa
gcatctc3') overlap the end of fragment 1
and give a 373 bp fragment. The last
primer set (5'tgtaaaacgacggccagtgttaac-
catatgcatgtatctgcc3'/5'caggaaacagctatgacc
gaaattaatcaggaagttgagctg3') gives a 347 bp
fragment including exon 3. The PCR were
conducted with an annealing temperature
of 65°C for 1 min and 35 cycles. The DNA
was sequenced using an ABI Prism 3730
DNA sequencer (Applied Biosystems,
Perkin Elmer Corporation, Foster City,
CA, USA).
Results and Discussion
Out of the 29 suspected cases, 20 were
confirmed to have either a known or a
new point mutation defect.
Known mutations
HbA'2 or HbB2. Ten patients were found
to be carrying the common HbB2 mutation
[HBD c.49G→C (p.Gly17Arg) (δcd 16
GGC→CGC)]4(Figure 1C). In two of these
cases with low HbA2 levels (1.6%), het-
erozygosity for -α3.7α+-thalassemia was
found. In one case with HbA2 = 1.5%, the
same α-deletion was present in the
homozygous state. In another, heterozy-
Correspondence:
Piero C.Giordano,Ph.D.,
Hemoglobinopathies,Laboratory
Human and Clinical Genetics,
Leiden University Medical Center,
Wassenaarseweg 72,2333 AL
Leiden,The Netherlands.
E-mail: p.c.giordano@lumc.nl
Disorders of Erythropoiesis • Brief Report
Mutations in the δ-globin gene (HBD, MIM# 142000) are not pathologically relevant. However,
since high HbA2 levels are diagnostic for β-thalassemia trait and a lowered level for an α- or δ-
mutation, co-inheritance of δ- and β-gene defects may lead to misinterpretation of diagnostic
results. We examined 29 cases with low HbA2 level diagnosed in our laboratory, in the presence
or absence of a second HbA2 fraction. We found a δ globin gene mutation in 20 cases.
In total four different known mutations were found, three structural and one expressional.
Moreover, two new defects were observed, one causing a structural abnormality and one a δ-
thalassemia.
The structural abnormality HBD c.431A→G (p.His144Arg)(δcd 143 CAC→CGC) was homologous
to the β-globin gene variant called Hb-Abruzzo and we have named this mutation HbA2 -Abruzzo.
The new δ-thalassemia defect HBD c.-118C→T (δ -68 C→T) has no homology on the β-globin
gene (HBB, MIM# 141900). All mutations caused a low HbA2 level and through this could lead
to misdiagnosis when inherited together with a β-thalassemia.
Key words: HDB, HbA2,δ-thalassemia, δ-variant.
Haematologica 2006; 91:129-132
©Ferrata Storti Foundation
©2006 Ferrata Storti Foundation

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| 130 | haematologica/the hematology journal | 2006; 91(1)
M. J. Bouva et al.
gosity for the HbC [HBB c.19G→A (p.Glu7Lys)] was
present (HbC; 38.7%, HbA2; 1.8%).
HbA2-Yialousa. The δ globin gene mutation HbA2 -
Yialousa [HBD
c.82G→T (p.Ala28Ser)(δcd 27
GCC→TCC)],5was found in four independent
patients. In one the HbA2 level was 1.7% and het-
erozygosity for -α3.7α+-thalassemia was present.
Another patient (HbA2=1.6%) was heterozygous for
--Med Iα°-thalassemia.
HbA2-Etolia. One patient with an HbA2 level of
1.4% had the rare mutation called HbA2-Etolia [HBD
c.257T→C (p.Phe86Ser)(δcd 85 TTT→TCT)].6
HBD c.275dupT. The frame shift HBD c.275dupT
(p.Leu92fs)(δcd 91 +T)7was detected in one patient
with an HbA2 level of 1.5% and an elevated HbF frac-
tion (2.3%). This patient also had a new mutation on
the
90A→T (γ -37 A→T).8These data are summarized in
Table 1.
©Ferrata Storti Foundation
G-γ globin gene (HBG2, MIM# 142250), c.-
New mutations
HbA2-Abruzzo. In three patients from a single fami-
ly (father and two children), all carriers of sickle cell
trait [HBB c.20A→T] with normal levels of HbS
(41.9%; 40.4% and 41.4% respectively), a new δ glo-
bin gene mutation was observed at HBD c.431A→G
(p.His144Arg)(δcd 143 CAC→CGC). This mutation,
new on the δ gene, is described at the same position
on the β-globin gene as Hb-Abruzzo9and was named
HbA2-Abruzzo.
HBD c.-118C→T. A new δ-thalassemia was found
in a patient with a low HbA2 level (2.0%). The muta-
tion (HBD c.-118C→T)(δ -68 C→T) is localized on
the AACCAAC sequence [HBD from c.-120 to -114
(δ -70 to -64)], where another mutation, also causing
δ-thalassemia, was described by Papadakis et al. in
1997.10 These data are summarized in Table 1.
In nine out of 29 cases no association between the
low HbA2 levels and δ-globin genes mutations was
found. Further investigation revealed iron deficiency
in three out of these nine cases. A low mean cell
hemoglobin as a consequence of iron deficiency may
cause a fractional decrease of the HbA2 level due to
an artifact related to the lower amount of total hemo-
globin loaded on the HPLC column.1In the remaining
six cases particular integration conditions of the
HPLC diagram could explain the lower estimation
(data not shown). The mutations found in the 20
remaining cases are discussed below.
HbA'2 or HbB2. In ten out of 20 samples HbA'2
(Figure 2A) was found in a heterozygous form. This
polymorphism, inducing the substitution of the
amino acid glycine with an arginine, also called HbB2,
is the most common δ chain variant, frequently
found in Africans. The -α3.7α+-thalassemia rightward
deletion was found in three of these ten cases (one
homozygous, two heterozygous). The rightward
deletion in the homozygous state did reduce the total
HbA2 level (HbA2 +HbB2) to 2.2%, while the right-
ward deletion in the heterozygous state apparently
did not (2.9%). One patient was a carrier of the HbC
variant and had an HbA2 level of 1.8%, which is sig-
nificantly lower than in normal carriers of HbC. Due
to the division of the HbA2 into two fractions in the
presence of HbB2 the automatic estimation of the
HbA2 fraction is always substantially reduced
(≈1.65%). One should always be alert to additional
HbA2 peaks (Figure 1C) when the diagnosis of β-tha-
lassemia trait is based only on the estimation of the
HbA2 fraction.
HbA2-Yialousa. In the three patients found to be
heterozygous for the HbA2-Yialousa mutation, no
abnormal HbA2 fraction was observed. This variant is
caused by an alanine to serine substitution as a result
of a G→T transversion at c.82 (δcd 27)(Figure 2B) and
results in a δ+-thalassemia phenotype.5Besides hav-
ing the δ-globin gene mutation, one of the patients
was heterozygous for -α3.7α+-thalassemia deletion
and another one for --αMed-Iα°-thalassemia deletion.
HbA2-Yialousa could compromise the diagnosis of
Figure 1. Results of high performance liquid chromatography. A.
HPLC results of a normal sample. B. HPLC results of the sample
with HbA2-Etolia. There is less HbA2 and no variant is visible. This
supports the assumption that HbA2 -Etolia is unstable. C. HPLC
results of a sample with HbB2. Besides a lowered HbA2 level, the
variant is also visible.
0
123456
Time (min.)
0
123456
Time (min.)
0123456
Time (min.)
HbA
HbA2
HbA
HbA
HbA2
HbA2
HbB2
F - 1.07
- F - 1.06
- F - 1.07
- 1.18
- - 1.27
- 2.29
- - 4.57
- A2 - 3.60
- - 0.94
- - 0.94
- - 1.18
- - 1.67
- 2.30
- 2.30 - 3.58
-
-
-
-
-
-
- - 1.27
- - 1.66
- 2.35
- A2 - 3.60
45%
37.5%
30%
22.5%
15.0%
7.5%
0.0%
45%
37.5%
30%
22.5%
15.0%
7.5%
0.0%
45%
37.5%
30%
22.5%
15.0%
7.5%
0.0%
A
B
C

Page 3

δ globin gene mutations
haematologica/the hematology journal | 2006; 91(1) | 131 |
β-thalassemia heterozygosity when this is based on
the HbA2 level only.
HbA2-Etolia. The
δ-thalassemic
observed in one patient was caused by HbA2-Etolia
heterozygosity [HBD
TTT→TCT) (Figure 2C).6Because of the instability
of HbA2-Etolia, HbA2 expression is decreased in the
heterozygous state (1.4%) and no HbA2 variant is vis-
ible on HPLC (Figure 1B). This may compromise the
diagnosis of a β-thalassemia carrier.
HBD c.275dupT. One of the samples showed the
heterozygous presence of a frame shift. We found
the insertion of thymidine at c.275 (δcd 91) in the sec-
ond exon of the δ-globin gene (Figure 2D). This
mutation was reported once before in a Belgian fam-
ily with δ0-thalassemia in 1989.7The frame shift
results in a premature stop of δ-globin synthesis at
c.278 (δcd 94). In the present case the frame shift was
found in a Dutch patient and resulted in a lowered
HbA2 level (1.5%) and an elevated level of HbF
(2.3%). This patient was also found to be carrier of a
new polymorphism on the G-γ globin gene [HBG2 c.-
90A→T](γ -37 A→T) apparently associated with ele-
vated HbF expression.8
The presence of this δ-globin gene mutation,
resulting in decreased HbA2 expression, could com-
promise the diagnosis of β-thalassemia carriership.
HbA2-Abruzzo. In one family HbS occurred with
slightly decreased levels of HbA2 (2.0%) and an addi-
tional slow moving HbA2 fraction on electrophoresis.
After sequencing we found a heterozygous mutation
at c.431 (δcd 143). Histidine replaced arginine due to
a CAC→CGC transition (Figure 2E). Further family
research revealed that the δ-variant was not localized
on the same chromosome as the HbS mutation. The
mutation has an equivalent on the β-globin gene,
phenotype
c.257T→C] (δcd85
called Hb-Abruzzo, which has been found in a few
Italian and Italian-American families.9This δ-variant
also causes a risk of overlooking β-thalassemia carri-
er ship when a diagnosis is based on the HbA2 level.
HBD c.-118C→T. In one of the samples we found
a new mutation at position c.-118 (δ -68) (Figure 2F).
The mutation C→T is located in an AACCAAC
sequence (c.-120 to -114)(δ -70 to -64) in which
another δ-thalassemia mutation has been reported
(HBD c.-115A→G)(δ -65A→G).10The sequence is
considered to be a regulatory element, like the AAC-
Table 1. Patients’ data.
Sample no. Suspected δ
HbA2%
HbF %% HbA2/X
on HPLC
Electrophoresis
δ gene mutation*
(HUGO nomenclature)
Other mutations
(HUGO nomenclature)
Ethnic origin
gene defect
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
Hb variant
Hb variant
Hb variant
Hb variant
Hb variant
Hb variant
Hb variant
Hb variant
Hb variant
Hb variant
Thalassemia
Thalassemia
Thalassemia
Thalassemia
Thalassemia
Thalassemia
Hb variant
Hb variant
Hb variant
Thalassemia
2.2
1.5
1.9
1.4
1.8
1.3
1.5
1.6
1.6
1.7
1.3
1.7
1.8
1.6
1.4
1.5
2.1
2.0
2.1
2.0
0.8
0.7
0.3
0.3
0.6
0.5
0.7
0.2
0.2
1.6
0.8
0.5
0.5
0.5
0.4
2.3
0.4
0.7
0.4
1.3
2.5 HbS-like
0.8 HbS-like
1.6 HbS-like
1.5 HbS-like
1.5 HbS-like
1.3 HbS-like
1.2 HbS-like
1.3 HbS-like
1.5 HbS-like
1.5 HbS-like
Not visible
Not visible
Not visible
Not visible
Not visible
Not visible
Not visible
Not visible
Not visible
Not visible
Slow HbX
Slow HbX
Slow HbX
Slow HbX
Slow HbX
Slow HbX
Slow HbX
Slow HbX
Slow HbX
Slow HbX
Not visible
Not visible
Not visible
Not visible
Not visible
Not visible
Slow HbX
Slow HbX
Slow HbX
Not visible
HBD c.49G→C
HBD c.49G→C
HBD c.49G→C
HBD c.49G→C
HBD c.49G→C
HBD c.49G→C
HBD c.49G→C
HBD c.49G→C
HBD c.49G→C
HBD c.49G→C
HBD c.82G→T
HBD c.82G→T
HBD c.82G→T
HBD c.82G→T
HBD c.257T→C
HBD c.275dupT
HBD c.431A→G
HBD c.431A→G
HBD c.431A→G
HBD c.-118C→T
West African
Surinam
Surinam
West African
Surinam
Morocco
Surinam
Surinam
West African
Surinam
Surinam
Iran
Turkey
Turkey
Turkey
The Netherlands
Turkey
Turkey
Turkey
Surinam
-α3.7/-α3.7
HBB c.19G→A
-α3.7
-α3.7
-α3.7
--αMed I
HBG2 c.-90A→T
HBB c.20A→T
HBB c.20A→T
HBB c.20A→T
(*:HbVar nomenclature is reported in the text).
Figure 2. Sequence results of the δ-globin gene mutations found.
A HBD c.49G→C
C HBD c.257T→C
E HBD c.431A→G
F HBD c.-118C→T
D HBD c.275dupT
B HBD c.82G→T
T G
T G G N G C A A A
C A C T T N T T C
T C
G C T N G N N N G G G
T GG C TC N C A AG
C T T A A A T
C A A C
G T G A G N C C C T G
©Ferrata Storti Foundation

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| 132 | haematologica/the hematology journal | 2006; 91(1)
M. J. Bouva et al.
CAAT sequence, which normally exists in the β-glo-
bin gene. CCAAT sequences are critical in all globin
genes, and many tissue-specific genes, for the correct
initiation point and a high level of transcription. The
modification of CCAAT to CCAAC is considered to
be responsible for the lower transcription level of the
δ-globin gene in respect to the β gene, which empha-
sizes the remarkable effect of a single nucleotide sub-
stitution in these conserved sequences.11Due to the
A→G substitution at position -115 (δ -65)(resulting in
AACCAGC) the transcription factor GATA-1 has a
decreased binding ability. Accordingly, our patient
had a slightly decreased HbA2 level (2.0%). The pres-
ence of the HBD c.-118C→T mutation could com-
promise the diagnosis of β-thalassemia carriership,
because of the lower HbA2 expression.
In conclusion, over 600 β- and only about 60 δ-glo-
©Ferrata Storti Foundation
bin gene mutations have been reported so far
[http://globin.cse.psu.edu/hbvar/menu.html]. It is quite
likely that the number of mutations on the δ-gene is
comparable to the number of mutations on the β-
gene. Therefore, more attention should be paid dur-
ing diagnostics to a possible (co-) inheritance of a δ
mutation, because, though not pathologically signifi-
cant, the knowledge of these mutations could avoid
a misdiagnosis of β-thalassemia minor based on
HbA2 levels.
MJB, a graduate in Biomedical Science, is participating in sever-
al ongoing projects at the Hemoglobinopathies Laboratory and was
in charge of this study; CLH, Staff Member and Molecular
Geneticist was consultant in this. PVD, Senior Technician provided
practical assistance. PCG, Head of the Laboratory, coordinated the
study and supervised the writing.
Manuscript received June 20, 2005, Accepted September 21,
2005.
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