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Two Novel P-Thalassemia Mutations in the 5’ and 3’ Noncoding Regions of the
f3-Globin Gene
By Shi-Ping Cai, Barry Eng, William H. Francombe, Nancy F. Olivieri, Alan G. Kendall, John S.Waye, and David H.K. Chui
Two novel pthalassemia mutations are described. The first
mutation, found in an Italian family, is a G+A substitution in
nucleotide (nt) +22 relative to the B-globin gene Cap site.
This mutation creates a cryptic ATG initiation codon, the
utilizationof which for translation would result in premature
termination 36 bp 3’ downstream. The second mutation,
found in an Irish family, is a T - 4 substitution in nt +1570, or
12 bp 5’ upstream of the AATAAA polyadenylationsignal in
the 3’ noncoding region. It is postulated that this mutation
leads to destabilizationof the encoded p-globin mRNA.
o 1992 by The American Society of Hematology.
T
the a-,c-, p-, and y-globin genes were labeled and hybridized as
previously described.4
Polymerase chain reaction (PCR) and direct nucleotide sequence
analysis. The p-globin gene was amplified by PCR using primers
Al, 5‘-TCCTA AGCCA GTGCC AGAAG-3’ (nucleotide positions -161 to -142 relative to the P-globin gene Cap site), and
primer A2, 5’-GAC(=T CCCAC ATTCC CITIT-3’ (nucleotide
positions +1662 to +1643). The PCR amplified DNA was electrophoresed on aqueous 8% polyacrylamide gel as previously described! For direct nucleotide sequencing of the PCR product, the
dideoxy termination method was used?
Dot bloning using allele-specific oligonucleotideprobes. The PCRamplified DNA was blotted onto nylon filter and hybridized with
’*P-labeled allele-specific oligonucleotide probes as has been described!
HE P-THALASSEMIAS are hereditary disorders due
to mutations in the P-globin gene on chromosome 11,
leading to either decreased or absent P-globin chain synthesis. The most common genetic defects in P-thalassemias are
due t o point mutations or mutations involving small deletions or insertions in the @-globingene. More than 100 such
naturally occurring mutations are known.’ The characterization of these mutations is essential for performing the
prenatal diagnosis of fetuses at risk. Furthermore, they
provide additional insight into the identification or confirmation of nucleotide sequences that are important in the
regulation and expression of the p-globin gene?
In this investigation, we have identified two new P-thalassemia mutations in the 5‘ and 3’ noncoding regions of the
P-globin gene. The proposed mechanisms whereby these
two mutations cause a decrease in normal P-globin chain
synthesis are novel.
MATERIALS AND METHODS
Hematologic studies. Peripheral blood counts and erythrocyte
indices were determined using an electronic cell counter. Hemoglobin (Hb) electrophoresis was performed on cellulose acetate
membranes at pH 8.6. Hb F was measured with a modification of
the alkali-denaturation method of Chui et a1 as has been previously
described.’
Gene mappingstudies. Genomic DNA was isolated from peripheral blood, digested with restriction endonucleases, separated by
electrophoresis through 0.8% agarose slab gel, and transferred to
nylon membranes using standard procedures. Probes specific for
From the Provincial Hemoglobinopathy DNA Diagnostic Laboratory and the Department of Pathology, McMaster University School of
Medicine, Hamilton, Ontario; the Depament of Medicine, The
Toronto Hospital; the Division of HematologylOncology, Hospital for
Sick Children, Toronto, Ontario; and the Division of Hematology,
Royal Mctoria Hospital, Montreal, Quebec, Canada.
Submitted May 30, 1991; accepted October 23, 1991.
Supported in part by research grants from the Medical Research
Council of Canada (MT-5004) and the US National Institutes of
Health (HL-37652). N.F.O. is a Career Scientist of the Ontario
Ministry of Health.
Address reprint requests to David H.K. Chui, MD, Department of
Pathology, McMaster University Medical Centre, Hamilton, Ontario,
Canada L8N 325.
The publication costs of this article were defrayed 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 this fact.
0 1992 by TheAmerican Society of Hematology.
0006-4971I921 7905-0002$3.00/0
1342
RESULTS
Family M. The proband is a 23-year-old man of Italian
ancestry. His clinical course has been that of P-thalassemia
intermedia. He was found to have P-thalassemia at age 5.
At age 7, he was transfused for the first time; subsequently,
he required transfusions once or twice a year. At age 18, his
transfusion requirement increased. At this time his spleen
was markedly enlarged. Splenectomy was performed and he
has since been relatively asymptomatic without the need for
transfusions during the past 5 years. The relevant hematologic results and P-thalassemia mutations found in the
proband and his parents and two twin sisters are tabulated
in Table 1.
The p-globin gene region of the proband was amplified
by PCR and sequenced. He was found to be a carrier of
IVSI-110 G+A P’-thalassemia mutation (data not shown).
In addition, a novel mutation, G+A in nt +22 relative to
the P-globin gene Cap site in the 5‘ noncoding region, was
also detected by direct nucleotide sequencing (Fig 1). Gene
mapping of the y-P globin gene cluster showed that the
proband has the normal complement of four y-globin
genes, and that he did not have the C+T substitution at nt
- 158 5‘ to the ‘y-globin gene, otherwise known as the Xmn
I site (data not shown).’ Similarly, the a-globin gene cluster
was found to be normal.
The other four immediate family members were similarly
studied. The proband’s father and two sisters were P-thalassemia carriers for the IVSI-110 G+A p’-thalassemia
mutation. His mother also had a typical P-thalassemia trait
phenotype, and was found to have the nt +22 G+A
mutation (Table 1). Figure 2 shows the dot blotting results,
using oligonucleotide probes specific for the nt +22 G+A
mutation and its normal counterpart.
Blood, Vol79, No 5 (March l),1992: pp 1342-1346
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TWO NOVEL @-THALASSEMIAMUTATIONS
1343
Table 1. Hematologic Results and @-ThalassemiaMutations in Family M of Italian Ancestry
Family Members
Father
Mother
Sister
Sister
Proband
Age (vrl
Hb (glLI
MCV ( f L)
HbA, (Yo)
HbF (%)
Serum ferritin (WglL)
@-Thalassemiamutation
68
137
66
5.1
Normal
134
Carrier of IVSI-110
G-A p'-thalassemia mutation
65
128
74
4.0
Normal
77
Carrier of nt 22 G+A
mutation
29
123
65
5.2
3.5
60
Carrier of IVSI-110
G+A @'-thalassemia mutation
29
129
64
5.2
Normal
27
Carrier of IVSI-110
G-A @'-thaiassemia mutation
23
102
71
5.4
20.3
1,812
Carrier of IVSI-110
G-A P'-thalassemia mutation and nt
22 G+A mutation
a-Globin gene cluster
aalaa
aalaa
Family K. The proband is a 29-year-old man of Irish
ancestry who was found to have hypochromic microcytic
anemia with elevated HbAz (5.4%). The p-globin gene
region was amplified by PCR and sequenced. The only
abnormality detccted was a T - C mutation in nt +1570
relative to the p-globin gene Cap site, or 12 bp 5' upstream
of the AATAAA polyadcnylation signal in the 3' noncoding
region (Fig 3). The rest of the p-globin gene from nt -161
Antisense
sequence
Sense
sequence
sr
G A T C
aalaa
aalua
aalaa
to nt +1662 was found to be normal as assessed by direct
nucleotide sequencing of the PCR-amplified DNA of the
P-globin gene. The a-globin gene cluster was found by gene
mapping to be normal. The pedigree of this Irish family is
shown in Fig 4.
The T - C substitution in nt +I570 creates a new
restriction enzyme Bsp El recognition site, TCCGGA. The
3' @-globingene regions from members of this family were
amplified by PCR using primers 5'-CAATC CAGCT ACCAT TCTG-3' (nt +1232 to 1250 3' to the f3-globin gene
Cap site) and 5'-GGGCC TATGA TAGGG TAATA-3'
(nt +721 to +702 3' to the AATAAA polyadenylation
signal of the p-globin gene). The amplified product was
~~
C
T
G
T
G
T
T
G
A
CT
A
C
A
A
G
T
G
A
T
C
1
+
G
A
C
A
C
A
A
C
T
GA
T
G
T
T
C
A
C
T
A
G
3'
Fig 1. Direct nucleotide sequencing of the antisense strand of the
PCR product, showing the G+A substitution in nt +22,found infamily
M of Italian ancestry.
I
M
N
0.0..
Fig 2. Pedigree and dot blotting of the family M. The hatched
symbol represents carrier of the IVSI-110 G-rA @'-thalassemiamutation. The dotted symbol represents carrier of the novel G+A substitution in nt +22. M represents dot blotting using =P-labeled probe
complementary t o the G-A substitution in nt 22, 5'-ACACA ACTAT
GlTCA CTAGC-3'. N represents dot blotting using =P-labeled normal
probe complementary t o the normal n t +22 region, 5'-ACACA ACTGT
GlTCA CTAGC-3'.
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CAI ET AL
1344
other family members, including the proband's father and
sistcr and onc of his matcrnal cousins, wcre not carricrs of
thc T+C mutation, and had normal peripheral blood
counts other than iron deficiency detected in thc proband's
sister (Table 2). Furthcrmorc, thc P-globin gene was
amplificd by PCR and thc 3' untranslatcd region was
scqucnccd in 25 additional unrelated individuals. The T+C
substitution in nt +1570 was not observcd in any of these 25
individuals (data not shown).
3'
A
C
A
A
A
A
A
G A T C
A
T
A
DISCUSSION
A
T
I
I
t
0
-
0
C
C
G
T
C
T
T
A
G
G
TC
C
T
A
C
G
A
G
E
,
5
Fig 3. Direct nucleotide sequencing of the sense strand of
the PCR product showing the
T-C substitution in nt +1570,
found in family K of Irish ancestry.
1080 bp long. It was digestcd with Bsp El rcstriction cnzymc,
and the prcsencc of two fragments. 743 bp and 337 bp,
is indicativc of thc T+C substitution in nt + 1570 (Fig 5).
Four othcr family membcrs, including the proband's
brothcr, matcrnal grandmother, matcrnal aunt, and onc of
hcr daughters had 6-thalasscmia trait and all were carriers
of thc T+C mutation in nt + 1570, confirmed by nuclcotidc
scqucncing and rcstriction cnzymc Rsp El digcstion. Thrcc
I
w
"
w
Fig 4. Pedigreeof family K. Blackenedsymbols representcarrier of
the novel T-C substitution in nt +1570. Note that I1 and 112 are
deceased, and that 114 was not tested. See Table 2.
This study shows two novcl P-thalassemia mutations, onc
in thc 5' and the othcr in the 3' transcribed but noncoding
regions of the P-globin gene. The G+A substitution in nt
+22 rclativc to thc P-globin gene Cap site, found in the
Italian family, creates a cryptic initiation codon ATG
(Table 3), which is 26 bp 5' upstream of the normal ATG
initiation codon. Utilization of the cryptic ATG codon
would lead to a framc shift abnormality, in phase with a
TGA termination codon 36 bp 3' downstream of the cryptic
initiation codon. It is generally accepted that the minimal
distance bctwccn two adjaccnt ribosomcs on the samc
mRNA strand is approximately cighty nuclcotidcs long.'
Therefore, it is unlikcly that both the cryptic and the
normal ATG codons on thc samc mRNA strand can be
uscd simultancously. The fact that the proband who is a
compound heterozygote for this novel mutation as well as
thc IVSI-110 G-A @+-thalassemiamutation has a P-thalasscmia intermcdia phenotypc is highly suggestive that the
G+A substitution in nt +22 alone confers a relatively mild
P'-thalasscmia phcnotype. Additional experiments are
planncd to dcterminc thc rclativc utilization of thc normal
and thc cryptic ATG initiation codons for translation of the
mutant P-globin mRNA.
It is also conccivablc that the G+A substitution in nt
+22 could affcct thc transcription of the P-globin genc. In
the gfa gcnc that codcs for thc glial fibrillary acidic protein,
an initiation clemcnt, located bctwccn 10 and 50 bp
downstrcam from thc transcription initiation sitc, has been
shown to be necessary for the proper binding of the TATA
box binding factor, TFIID, to enhance efficient transcription." Thus, the G+A substitution in nt +22 in the @-globin
gene may altcr the binding efficiency of the TFIID, lcading
to a decrease in P-globin genc transcription. This hypothcsis rcquircs furthcr cxpcrimcntal confirmation.
In thc Irish family, thc proband has a typical P-thalassemia trait phenotypc with mild anemia, and the only molecular abnormality dctcctcd in the P-globin gcnc is a T+C
substitution in nt +1570 rclative to thc P-globin gcnc Cap
sitc, or 12 bp 5' upstream of the AATAAA polyadcnylation
signal in thc 3' noncoding region. This is not a known
polymorphism. Furthermore, all five family members with
the p-thalassemia trait phenotype have the same novel
mutation, whereas the othcr three normal family members
havc not inherited this mutation. Thcreforc, this T+C
substitution is either linked to thc putative @-thalassemia
mutation in this family or is, in fact, the causc of the
P-thalassemia trait phenotype.
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TWO NOVEL p-THALASSEMIA MUTATIONS
1345
1
3
2
5
4
6
7
8
9 1 0
1080
743
Fig 5. Restriction enzyme Bsp E l digestion of the
3' @globin gene region amplified by PCR, in family K.
The normal allele is represented by the 1,080-bp
fragment, while the T-C mutant allele is represented
by the 743-bp and 337-bp fragments. See text. Lane
1, OX 174 Hae 111 digestion size markers; 2, family
member 12 as in Fig 4; 3, II 1; 4,111 1; 5,111 2; 6,111 2.7, II
3; 8,111 4; 9,111 5; 10, normal individual.
337
This report reaffirms the importance of continued scrcening for, and analysis of, other novel mutations causing thc
thalasscmias, in thc quest for a morc complete undcrstanding of thc regulation and cxprcssion of thc globin gcncs and
their products.
Thc function of the 3' noncoding rcgion between thc
termination codon and thc AATAAA polyadenylation
signal is not wcll undcrstood. The mRNAs of a numbcr of
regulatory ccllular proteins havc a common AUUUA
pcntamer in their 3' untranslated region. It was proposcd
that thc interaction of this pcntamcr with its binding
protein can lcad to rapid degradation of its mRNA."'
Similarly, it has becn shown that thc interaction bchvecn
poly (A) and its associated poly (A)-binding protein affect
mRNA stability, including that of P-globin mRNA."."
Recently, it was reported that at lcast two mutations or
clusters of mutations constructcd in the 3' untranslatcd
region of the a-2 globin gene could lcad to destabilization
of the cncoded a-globin mRNA." We plan to cxaminc if thc
T-C substitution 12 bp 5' upstream of the polyadcnylation
signal in thc @-globingcnc found in this Irish family may havc a
similar dcstabilizing cffcct on thc encoded P-globin mRNA.
NOTE ADDED IN PROOF
Since this manuscript was submitted, Oner et al" have described
independently the +22 G+A mutation in patients from Turkey
and Bulgaria.
ACKNOWLEDGMENT
We thank members of both families for their cooperation during
this investigation, Bev Tyler for help in obtaining blood samples
from family M,and Gloria Charlow for excellent assistance i n the
investigation of family K.
Table 2. Hematologic Results and @ThalassemiaMutation in Family K of Irish Anceshy
Family
Members
Age
Ivr)
12
II 1
II 3
Ill 1
111 2
111 3
Ill 4
111 5
89
60
53
35
33
29
21
19
Sex
Hb
MCV
HbA,
m Globin Gene
IfLl
( 0 4
(%I
Serum
Ferritin
Iv9/L)
nt + 1570
IalLl
T-.C Mutation
Cluster
132
134
113
114
108
102
133
106
60
90
64
78
59
60
88
63
5.5
NA
4.8
2.5
6.4
5.4
2.9
4.9
0.8
NA
1.3
0.6
1.4
0.9
0.6
1.1
NA
NA
60
<2
NA
340
11
31
Present
Absent
Present
Absent
Present
Present
Absent
Present
aalaa
HbF
aalaa
aalaa
aalaa
aalaa
aalaa
aalaa
aalaa
See Fig 4 for the relationship of family members.
Abbreviation: NA, not available.
Table 3. Nucleotide Sequences of the 5' Noncoding Region of f3-Globin Gene and the First Ten Codons
A
t
5'-AGATT TGCTT CTGAC ACAAC TGTGT TCACT AGCAA CCTCA
AACAG ACACC ATGGT
- GCACC =ACT
CCTGA GGAGA AGTCT-3'
The G-A mutation in the nt +22 found in family M creates a cryptic ATG initiation codon, which is in phase with the TGA termination codon 36
nucleotides 3' downstream. The normal ATG initiation codon is also underlined.
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1346
CAI ET AL
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1992 79: 1342-1346
Two novel beta-thalassemia mutations in the 5' and 3' noncoding
regions of the beta-globin gene [see comments]
SP Cai, B Eng, WH Francombe, NF Olivieri, AG Kendall, JS Waye and DH Chui
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