Download Phenotypic Effect of Heterozygous a and $#{176

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Phenotypic
Effect
By Maria
In
this
study.
mapping
10
normal
carriers
one
with
with
(
the
-a),
(
a
I
and
aa)
-
a)
type.
-THALASSEMIA
genotype
we
basis
of these
heterogeneity,
found
populations
where
incorporate
the
the
defect
show
in
found
remarkable
with
hematologic
mani-
ranging
from an isolated
a condition
of moderately
increase
severe
of HbA2
thalasse-
manifestations.2’4
This
confirmed
by cr-globin
restriction
thalassemia
endonuclease
of different
Another
coinheritance
nase (G6PD)
suggestion
has
gene
analysis
mapping
in both
racial backgrounds.5
already
known
ameliorating
of the glucose-6-phosphate
defect
of the Mediterranean
has also been seen to produce
larger
and more completely
cells than seen in the majority
#{176}
analysis
ping
we report
carried
in a group
zygotes,
the results
out by restriction
endonuclease
of 1 0 Sardinian
fl#{176}-thalassemia
maphetero-
showed
acteristics
and whose
an increase
of HbA2
normal
hematologic
only distinguishing
levels.
In 8 of these
the Institute
Cagliari
Medical
University.
Institute
University
Supported
in part
(HL-241
(Rome,
Italy)
of
San
Biology
Francisco,
della
study
73-03),
Project
Program
MEE
Thalassemias
and
the
of
Preventive
81.361.83.
and
ratios
Mediterrean
Anemias,
Address
reprint
Biologia
del/Eta
(Case/la
Postale
© 1983
by Grune
27. 1982;
requests
Evolutiva,
251)
09100
& Stratton,
0006-4971/83/6106-0018$O1.OO/O
226
accepted
to
December
Professor
Cagliari.
Inc.
Sardinia,
Cao,
Cagliari,
Italy.
in the
first
set
of
(-a/-a)
a-globin
Mediterranean
type.
we may conclude
that
may be the cause
of
that have been seen in
From
factor
our data,
contributing
effect
could
defect
of the
MATERIALS
AND
METHODS
ji#{176}-thalasscmia
on the basis
balanced
I
Seven
).
it
to
the
be
the
Mediterranean
or
heterozygotes
of normal
can
Sardinian
All
synthesis
of a Ii#{176}-thalassemia
children
remainder,
be
f-thaIassemia
chain
major/intermedia
The
also
in this
cell indices.
globin
carriers
of thalassemia
values,
since
cc/si
are obligatory
included
red blood
reduced
3#{176}-thaIassemia.
Southern
S regularly
Counter
was quantitated
assumed
is almost
detected
to
be
by
carriers
exclusively
of
of the
population.6
according
grams
e
to
WHO.9
of DNA
II and Bam
hr
under
restricted
from
a-globin
G6PD
white
and
with
in
cells
West
the
electrophoresed
in
0.8%
It
Vol.
was
prepared
62.
No.
blood
by phenolchloroform
by
Blood,
peripheral
Ten
30 U of restriction
probe
F
was determined
precipitation.”
Mannheim,
filters.’2
(4C,
Hemoglobin-A2
et al.’#{176}
recommended
gene
Counter
and hemoglobin
to Kan
blood
Coulter
standard
activity
synthesis
ethanol
(Boehringer,
was
to nitrocellulose
32P-labeled
Diagnostic).
chain
was digested
DNA
Coulter
according
extraction
HI
the
a commercial
microchromatography7
Globin
conditions
with
with
method.8
was prepared
alcohol
measured
Control,
was performed
Bgl
ferred
Cell
denaturation
reticulocytes
DNA
were
calibrated
by DE-52
by an alkali
Sar-
C/mica
the
genes
a-globin
the (-a/aa)
genotype
we
or the hemizygous
state of
phenotypic
Red blood cell indices
the
Via Jenner,
structural
of a single
G6PD
HbA2
in the
model
13. 1982.
Antonio
Universit#{224} Studi
this
homozygous
isoamyl
April
HbA2
should
Methods
CNR
Cagliari,
of
fl#{176}-thalassemia,
dinia.
Submitted
in
prevalent.
heterozygotes.
additional
showed
increased
of
Institutefor
of the
an
selected
(Table
with
e Sanit#{224}
Medicine
CNR
with
gene, as they are parents
Hughes
Medicine,
Institute
In three
10 Sardinian
of them
Age.
Igiene
National
therefore.
$-thalassemia
ct-globin
deletion
of the
were
Coulter
the Assessorato
the
may
Subjects
CA.
Sardegna,
two
f3-thalassemia
association
type.
was
we
of Developmental
Italy,
and
the Howard
and the Department
of
by grantsfrom
Autonoma
Health
and
Sardinia,
Laboratory
of california.
Regione
Study
of Clinic
of
defect
seems
that
production
type
From
of
in 2, the
(-a/aa).
several
char-
feature
carriers,
suggest,
is also
On the basis of these findings,
the association
of a-thalassemia
the normal
red blood cell indices
picture
of
red blood
heterogene
deletion
the G6PD
f3-
ofct-globin
all of whom
We
of
complex
heterozygous
determination
of red
association
gene
effect.
for
one
production
The
a-thalassemia
and
The
article,
to this
genotype
and in one with
also found the homozygous
been
with
and
the
gene
zygotes.3
In this
the
thalassemia
programs
(-a/-a),
factor
is the
dehydrogetype, which
a hematologic
hemoglobinized
offl-thalassemia
to
of either
may
mia.’4
Genetic
studies
and globin
chain
synthesis
analysis
have provided
evidence
that the coinheritance
of a-thalassemia
may be a cause
of the less severe
phenotypic
recently
lead
fl#{176}-thalas-
deletion
tests.
of the
we
heterozygous
to the
indices.
this
Cao
Antonio
of
may
contribute
screening
and
due
normal
with
that
and
findings.
heterozygotes
phenotypic
also
(G6PD)
with
of
Interaction
interaction
genes.
defect
eventually
two
In three
cells
G6PD
of
the
TuVeri,
a-thalassemia.
a-globin
blood
increased
Teresa
that
with
or two
whom
remaining
a-globin
Furbetta,
semia
of
deletion
(-a/act).
genotype.
On the
the
in the
gene
of
and
found
dehydrogenase
Mediterranean
festations
levels
to
I
a
-
all
Mario
$#{176}-Thalassemia
conclude
genotype
indices
we
Galanello,
endonuclease
a-globin
cell
a-globin
the
-
blood
( -al
glucose-6-phosphate
tJ
the
a and
Renzo
restriction
subjects,
of a single
Pirastu,
heterozygotes,
red
genes
deletion
these
out
In 8 of these
a-globin
the
carried
to characterize
with
levels.
two
Mario
$#{176}-thalassemia
presented
HbA2
A. Melis,
we
in order
Sardinian
of Heterozygous
micro-
endonucleases
Germany)
for 24
manufacturers.
agarose
hybridized
by
1 (July),
The
and
with
trans-
a specific
nick-translation,
1983:
pp.
as
226-229
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fl’-THALASSEMIA
a AND
Table
INTERACTION
1 . Hematologic
227
Fee tures
and Gb bin Chain
RBC
Sex
Sub,ects
(x 1O’2/L,tec)
Sy nthesis
Anal ysis in Alpha
Hb
MCH
MCV
HbA,
(g/dl)
(pa)
fl)
(%)
and Beta
Th alassemia
HbF
(%)
Do uble Heterozygotes
a/
G6PD
(Ratio)
a-Glob,n
(Ul/gHb(
Genes
Arrangement
L.G.
M
5.67
17.1
30.3
88
5.24
0.88
0
-cs/-n
S.D.
M
4.92
14.4
29.4
90
5.00
-
0.86
0
-a/-sn
PT.
F
5.25
13.6
26.0
79
5.51
0.73
-
0
-a/-a
cL.
M
5.70
14.9
26.2
79
5.15
0.62
0.73
8.4
-a/-a
P.S.
M
5.50
15.0
27.2
80
5.21
0.50
0.83
7.2
-a/-a
R.G.
M
4.90
13.2
27.2
83
5.13
0.73
0.98
9.0
-a/-a
S.M.B.
F
4.63
12.1
26.9
77
5.17
0.51
-
6.1
-a/-a
GS.
M
5.86
15.7
27.5
80
4.44
0.55
0.89
5.8
-a/-a
V.A.
F
5.05
13.7
27.1
84
5.66
-
-
0
-a/aa
A.F.
M
5.31
14.9
28.2
83
4.76
2.7
1.30
7.2
-a/aa
M
5.18
Normal
080’
±
15.9
(3.65-6.69)
F
14.0
(3.88-5.81)
Obligate
M
6.12
f(-ThaI.
F
5.44
+
2 SD
described
plasmid
by
JW
probe
and
ranges
prepared
30.2
16.3)
±
21.9
+
4.3
87.6
(9.
1-
±
4.6
66.5
21.9
14)
et al.,’3
The
filters
from
were
66.2
(18.5-25.8)
a-globin
gene
also hybridized
by nick-translation
±
ofa
In normal
DNA
digested
ized with
an a-globin-speciflc
Hinfl
cloned
with
the
in
a c-specific
fragment
with
ofthe
pBR
HI and hybridthe duplicated
Barn
probe,
loci reside
in a DNA
fragment
(Kb)
in length.’5
Restriction
maps
of
/3#{176}-thalassemia carriers
showed
that they
shortened
10.5-Kb
fragment,
characteristic
zygous
a-thalassemia-2
(-a/-a).
The
a-globin
both the 14.5- and 10.5-Kb
the silent
carrier
state
semia-2
enzyme
probe,
the
by Bgl II digestion.
and
hybridized
DNA
that
contain
respectively.’6
can
be
14.5 kilobase
8 of the
10
had only the
of homoremaining
2
fragments,
which
or heterozygous
( -a/aa).
characterized
normal
±
0.44
(1.86-2.98)
11.0
2.52
±
4.99
±
0.52
0.36
1.02
0.98
4.98
±
0.56
±
±
1.02
0.54
±
0.12
(0.92-1.14)
(0.14-0.98)
(4.09-6.57)
8.7
0.37
(0.11-1.13)
(1.53-3.09)
11.5
(55-78)
RESULTS
a-thalassemia-2
We further
2.55
100)
±
plasmid.
showed
indicates
10.2
(59-85)
3.1
±
±
(77-
(19.5-25.1)
1.8
±
(79-102)
(25.4-34.6)
1.6
11.8
1)
89.1
#{247}
13.3
0.90
3.9
±
±
1.12
(0.16-2.5)
1.04
(4. 19-6.29)
0.94
±
1.83
1.18
±
0.52
(1.40-2.32)
(0.12-3.35)
in parentheses.
Maniatis
l0I.’
1.8
(11.5-15.3)
(4.02-6.7
‘Means
30.9
(26.9-35.0)
±
(10.51.06
±
(4.99-7.38)
Heteroz.
2.1
±
(12.5-19.1)
4.69+0.86
‘
080
yields
nature
When
digested
with
with
an a-globin-specific
1 2.5-
and
7.0-Kb
the a, and a, globin
The a-thalassemia-2
produced
by
two
of a-thalas-
chromosome
the rearranged
and
unequal
I 6.0-Kb
fragment
from
while
in the homozy-
an additional
chromosome,
gous
state
for this
lesion,
only
the
1 1 .0-Kb
and
16.0-Kb
fragments
can
be seen.
Mediterranean
a-thalassemia-l
heterozygotes
(--/aa)
gave a threefragment
pattern
with the normal
bands,
1 1 .0 and I 2.0
Kb in length,
and
an additional
14.0-Kb
deleted
chromosome.
specific
probe,
we
have the rightward
With
confirmed
deletion
in the homozygous
hetenozygous
state.
state and
Moreover,
did we detect
band
from
the
Bgl
II and the -globinthat
8 of our subjects
a-thalassemia-2
genotype
in the
cases
2 the same
lesion
in none of these
the a-thalassemia-
I haplotype.
DISCUSSION
fragments
structural
haplotype
different
this
taming
the 2
and a 12.0-Kb
fragment,
containing the -,,
and a, genes’7
(Fig.
I). DNA
from the
nightwa rd heterozygous
a-thalassemia-2
genotype
yields
the two normal
fragments
from
the normal
loci,
(-a)
crossover
the
In this study,
restriction
endonuclease
analysis
of
a-globin
structural
genes
in 10 /3#{176}-thalassemia
heterozygotes
selected
on the basis
of normal
red
blood
mechanisms,
indicated
as the nightward
or leftward
deletion.’6
Digestion
of the DNA
from
the leftward
deletion
a-thalassemia-2
haplotype
produces
only the
cell characteristics
more
frequently,
This
association
7.0-Kb
the two
study,’8
the frequency
of the single
a-gene
deletion
( -a)
in a random
group
of /3-thalassemia
heterozygotes from the same racial
background
was found to be
0.18.
The
coinheritance
of heterozygous
/3-thalassemia
with the deletion
of two a-globin
loci reduces
both a
and /3 globin
chain
production
to half that observed
in
fragment,
normal
while
fragments
a 15.8-Kb
in DNA
fragment
from the
replaces
rightward
deletion.’6
We found the nightward
deletion
in all the
carriers
analyzed,
both in the homozygous
and heterozygous
We
state.
also analyzed
haplotype
hybridizing
by
(--)
with
our
samples
for a-thalassemia-l
digesting
DNA
a nick-translated
with
Bgl II
-globin-speciflc
probe.
When
normal
DNA
is cleaved
with
hybridized
with
a -globin-specific
probe,
ments
are
produced,
i.e.,
an
1 1.0-Kb
and
Bgl II and
two frag-
fragment,
con-
showed
either the deletion
ofone
or,
of two a-structural
globin
gene loci.
is highly
significant
as, in a previous
the unaffected
subject.
This results
in an almost
balanced
globin
chain
synthesis,
which
could,
in turn,
explain
the quasi-normal
volume
and hemoglobinization
of the red
blood
cells.
However,
the
same
amelio-
From www.bloodjournal.org by guest on June 17, 2017. For personal use only.
MELIS
228
I
2
ET AL.
3
i6.okb
12.0
11.0
S
-a/-a
-a/aa
aa/aa
Fig.
1.
Autoradiogram
of Bgl-ll-digested
DNA
hybridized
with
a ‘-gIobin-specific
probe.
Lane
1:
a-thalassemia-2
heterozygote;
lane 2: a-thalassemia2 homozygote;
lane 3: normal a-globin
gene genotype.
A map of the Bgl II restriction
sites (B) in the ‘-a
globin
gene
cluster
is shown
at the bottom.
The
‘.
?.i
B
(L2
tpal
(Li
B
B
11.0-Kb
fragment
Kb fragment
i’,,
the
gene
‘2
and the
12.0-
a2 genes.
The 16.0-Kb
fragment
containing
the i’,. ‘,l’a, and only one a-globin
locus,
is the
product
of an unequal
crossing
over.
deleting
a 3.7-Kb fragment
between
the two a-genes.
3k
5’
contains
5K;-’
the
and
a,.
rating
effect
was also found
in two /3#{176}-thalassemia
heterozygotes
with only one a-structural
gene deleted.
A possible
explanation
for one of these cases might
be
previous
study,4
the finding
of normal
red blood
cell
indices
in several
/3-thalassemia
heterozygotes
seems to
represent
the maximum
expression
of the ameliorating
the association
of the G6PD
defect
of the Mediterranean type. This defect,
when associated
with
heterozygous /3-thalassemia,
has already
been found
to lead to
tendency
due to the coinheritance
This extreme
effect
is relatively
of a-thalassemia.
frequent,
since in the
Southern
Sardinian
semia heterozygotes
3.5%
to have
greater
volume
was never seen
indices.
mean
This
red
chronic
ever, may
nondeletion
and increased
to produce
red
effect
blood
slight
has
cell
Hb
blood
content/cell,3
cells with
but
normal
been attributed
to a younger
population,
resulting
from
hemolysis.’9
Another
be the undetected
a-thalassemia
presence
resulting
possibility,
tologic
a
how-
may
light
(aat/_a)
genotype.
In our population,
such nondeletion defects
have been observed
with a frequency
of
25% of all the a-thalassemia-2
haplotypes.’8
We conclude,
therefore,
that the interaction
of heterozygous
/3#{176}-thalassemia with the deletion
of either
one or two a-globin
genes
blood
cell indices.
Moreover,
that
the
contribute
Based
association
of
to this effect.
on
the
evidence
the
may produce
normal
red
from our data
it seems
G6PD
defect
may
also
in
this
and
carrier
screening
due
based
miss a significant
of these
results,
incorporate
to the
coinheri-
testing
on
MCV/MCH
proportion
it would
for
HbA2
in popula/3-thalasvalues
of carriers.
In the
seem
advisable
to
in the
first
set of tests
for heterozygous
a-thalassemia
in
/3-thalasis also prey-
alent.
ACKNOWLEDGME
We
Rita
Forget
a
probably
of /3#{176}-thalasnormal
hema-
ofa-thalassemia
gene(s).2G
Therefore,
where a-thalassemia
is also prevalent,
genetic
screening
programs
semia
in populations
where
plasmid
produced
characteristics,
tance
tions
semia
of an associated
in an effective
population,
were found
are grateful
Loi
to Dr.
for editorial
for
the
pBR,
to us before
plasmid
and
Y. W.
assistance.
Dr.
publication.
JW
101,
E. Kanavakis
Kan
We
Drs.
NT
for
his continuing
would
also like
J. Lauer
for making
and
advice
to thank
T.
and
Dr.
Maniatis
his results
available
B.
for
From www.bloodjournal.org by guest on June 17, 2017. For personal use only.
a AND
f3’-THALASSEMIA
INTERACTION
229
REFERE NCES
1. Weatherall
3). Oxford,
2.
Di,
Blackwell
Bianco
Thalassaemia
Syndromes
(ed
1981
for
I, Salvini
$
a and
P. Silvestroni
microcytemia.
Hum
3. Sanna
ciency
F, Melis
between
MA,
Galanello
R, De Virgiliis
the glucose-6-phosphate
thalassemia
genes
at
5, Cao
dehydrogenase
4.
R, Melis
E,
Cao
#{176}-thalassemia
A2 levels.
5.
Rev
Kanavakis
Furbetta
phenotype
Furbetta
Globin
chain
level.
Galanello
with
M,
Br
isolated
23:193,
JS,
R,
Angius
synthesis
with
Fr Hematol
E, Wainscoat
M,
a-thalassaemia
6.
A:
MA,
heterozygotes
Nouv
J Haematol
Cao
MT.
A, Scalas
analysis
types
increase
in
obligate
Huisman
of hemoglobin
Weatherall
T:
The
AM,
THJ,
A, Angius
Maccioni
DJ,
for
86:700,
1975
9.
A, Rosatelli
MA,
J Med
of
10.
WA,
Brodie
of hemoglobin
ME,
small
J Clin
Scientific
McWade
Galanello
AN,
R:
HE,
Lie-Injo
LE,
a-thalassemia
1974
sequences
electrophoresis.
among
J Mol
DNA
Biol
98:503,
A, Kleid
DG:
Proc
Nucleotide
NatI
sequence
Acad
Sci
USA
of the
72:1 184,
SM,
Clin
Embury
Pathol
of
25:738,
Group:
DJ:
foetal
Reliable
hemoglobin
routine
by
gene
deletions.
1972
of procedures
dehydrogenase.
WHO
Tech
for the
trait:
Schwartz
thalassemia
F, Nathan
syndromes.
DG:
J Clin
Cell
Haematol
Globin
Invest
chain
synthesis
in
Maccioni
47:25
1 2, 1968
JA,
Dozy
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3#{176}-
From www.bloodjournal.org by guest on June 17, 2017. For personal use only.
1983 62: 226-229
Phenotypic effect of heterozygous alpha and beta 0-thalassemia interaction
MA Melis, M Pirastu, R Galanello, M Furbetta, T Tuveri and A Cao
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