Laboratory of Molecular Genetics
Department of Physiology and Biochemistry
Biomedical Science Building
University of Malta
Alpha and Alpha-Like Globin Genes
Chromosome 16
5’
Cis-Trans Interplay
at the β-globin locus
ζ
ψζ ψα2 ψα1 α2 α1
θ
3’
β
3’
Beta and Beta-Like Globin Genes
Chromosome 11
5’
ε
Gγ Aγ
ψβ δ
Joseph Borg B.Sc(Hons)
Research Objectives
• In vivo Globin Gene Expression
• Relate DNA sequence variations within the
beta globin locus to globin chain protein
quantification.
• Model Conditions
Haemoglobinopathies
Beta Globin Gene Model
Epidemiology in the Maltese Islands
Haemoglobin F Malta I
(or α2Gγ2 117(G19) His Æ Arg)
Beta Thalassaemia
1.5% carrier rate
Other Silent Abnormal Haemoglobin Variants
1.8% carrier rate
Alpha Thalassaemia
5’ ε
G γ0
5’ ε
GγFMI
5’ ε
G γ0
Hb F
ψβ
δ
βA 3’
AγI
Hb F Malta I
ψβ
δ
βV 3’
AγT
Hb F Sardinia
ψβ
δ
βA 3’
AγI
(very rare)
Sickle Cell Disease
Hereditary Persistence of Foetal Haemoglobin (HPFH)
-158 CÆT
XmnI site enhancer
-530bp (AT)xTy
repressor
1
XmnI
Regulation of the Gamma Globin Gene Promoter and
Transcription
Gγ -158 C Æ T
•
Co
(N up T
F- F
E3 II
)
Linked with a determinant that raises
Gγ globin level and maintains a foetal
Gγ:Aγ ratio (Gilman & Huisman, 1985).
FKLF
•
DNase HS site -50 to -150bp 5’ to the
Gγ mRNA CAP site (Groudine et al., 1983).
•
Site may act by keeping DNA in an
open conformation
•
DRED
GATA-1
GATA-1
p2
2
NF
-E
4
SSP
TATA
CP1 G
IID
1 TF
CP
s
TF
RNA Pol II
More accessible to TFs in vivo
BP-1 Protein Binding DNA Sequences 5’ β Globin Gene
(Silencer I at -530bp & Silencer II at -300bp)
Chase et al., 2002 Molecular and Cellular Biology 22:8
8 2505-2514
1 tgtatttatt ctatttttag acataattta ttagcatgca tgagcaaatt aagaaaaaca
-530 69758T/C
(AC)n
(AT)x
61 acaacaaatg aatgcatata tatgtatatg tatgtgtgta tatatacaca tatatatata
(T)y
121 tatttttttt tcttttctta ccagaaggtt ttaatccaaa taaggagaag atatgcttag
69840T/C
181 aactgaggta gagttttcat ccattctgtc ctgtaagtat tttgcatatt ctggagacgc
241 aggaagagat ccatctacat atcccaaagc tgaattatgg tagacaaaac tcttccactt
69969C/T
301 ttagtgcatc aacttcttat ttgtgtaata agaaaattgg gaaaacgatc ttcaatatgc
-300
361 ttaccaagct gtgattccaa atattacgta aatacacttg caaaggagga tgtttttagt
421 agcaatttgt actgatggta tggggccaag agatatatct tagagggagg gctgagggtt
481 tgaagtccaa ctcctaagcc agtgccagaa gagccaagga caggtacggc
Six polymorphisms analyzed in this study. The sequence is
described on GenBank sequence entry DQ306876 [GI:83588747]
Regulation of the Beta Globin Gene Promoter and
Transcription
5’ ε
G γ0
AγI
ψβ
δ
βA 3’
EKLF
FKLF
EKLF
GATA
FKLF
EKLF
EKLF
ta
Be ein I
ot
Pr
C PB
0
p 30
1
GFO
NF
-E
2
GATA-1
TATA
XmnI negative (C nucleotide)
Short (AT)xTy sequence
XmnI positive (T nucleotide)
Long (AT)xTy sequence
HMG2
FKLF
G
CP
1
G1
HM
s TF
TF
IIB
FKLF
GATA
EKLF
RNA Pol II
EKLF
5’ ε
G γ0
AγI
ψβ
δ
βA 3’
2
Haemoglobin F Malta I
(or α2Gγ2 117(G19) His Æ Arg)
Genotype – Phenotype Correlation
Hb F Malta I heterozygotes
Hb F Malta I heterozygote
Hb F Malta I homozygote
Hb F Malta I*Hb F Sardinia
Discussion…
• The post-natal decline in total Hb F may result from
interplay between 5’ gamma globin genes and 5’ beta
globin (AT)xTy sequences
One-way ANOVA p = 0.003
Hb F Malta I * Hb F Sardinia
Two-way ANOVA p = 0.038
Haplotypes including the BP1 binding site detected in
Maltese patients with β-thalassaemia
Haplotype
Total
• Longer (AT)xTy sequences together with an XmnI
positive site have a delayed gamma to beta globin gene
switching
• This research shall now also be applied to adults with
Hb F Malta I/Beta Valletta. Generating antibodies to the
HbF and HbFMaltaI to quantify the gamma globins in
vivo. Aim is to determine whether XmnI, (AT)xTy and
others… are active in healthy adults.
In vivo Expression of HbF among β+IVSIIVSI-6C and β0IVSIIVSI-110 Homozygotes
and Double Heterozygotes
Discussion…
• Under erythropoetic stress, XmnI site
plays a pivotal role in gamma globin gene
expression and overrides the (AT)xTy
regulatory function
• (AT)9T5 beta thalassaemic patients have a
slight increase in total HbF than (AT)7T7
3
Cis- and trans- acting elements of the human γ-globin gene promoters
and point mutations associated with nondeletion forms of HPFH
Currently
XmnI and (AT)xTy are cis-acting sequence elements.
What about trans- acting proteins and their effect on
gene expression?
A three-generation family with HPFH has been picked
up through our screening program.
-175 Sardinian & black Gγ HPFH
black Aγ HPFH
TCTTG GGGGC CCCTTCCCCACA TATCT CA ATGCAAATAT CT GT
Black Aγ HPFH
deletion
-202 black
Gγ
HPFH
-202 black
Aγ
HPFH
-198 British
-195 Brazilian
γ HPFH
A
-196 Italian and
Chinese Aγ HPFH
TTGCTTGA CCAAT AGCCT TGACA AGGCAAACTTGA CCAAT AGTCTT
γ HPFH
A
-114 Japanese
-117 Greek
Aγ
γ HPFH
G
HPFH
γ-globin
Extensive molecular haplotyping throughout the beta
globin locus has been carried out.
This is one of the most informative families thus far, as it
carries no known mutations.
Microarray
• Phase I
500K GeneChip analysis on all family members and
narrow down significant areas. Re-type such areas with
a better SNP profile to narrow at manageable levels of
DNA
• Phase II
Scan using molecular biology
techniques to Compare/Contrast
with healthy controls having
<1% HbF
Conclusion
γ to β globin gene switching still not fully elucidated
Acknowledgements
Prof. A. E. Felice (Supervisor)
Prof. G. J. Hunter (DNA sequencing facility)
Ms. R. Galdies (Protein Chemistry)
Dr .G. Patrinos
Dr. C. A. Scerri
Ms. W. Cassar
Ms. S. Bezzina-Wettinger
Dr. S. Philipsen
Ms. M. Pizzuto
Mr. A. Al-Ashtar
Dr. A. G. Fenech
Dr. P. Schembri-Wismayer
Ms. S. Schembri-Wismayer
References
Further research work is necessary for the rational
design of drug therapeutics for a variety of
haemoglobinopathies.
In addition, studies of gene control and regulation using
β-globin locus as a model remains one of the leading
paradigms in which new molecular mechanisms will
be discovered and existing ones better characterized.
1.
Cauchi, M.N., Clegg, J.B., and Weatherall, D.J. (1969) Haemoglobin F(Malta): a new foetal haemoglobin variant with a high incidence in Maltese infants. Nature
223(203):311-3
2.
Bank, A. (2006) Regulation of human fetal hemoglobin: new players, new complexities. Blood 107(2): 435-443.
3.
Kutlar, F., Felice, A.E., Grech, J.L., Bannister, W.H., Kutlar, A., Wilson, J.B., Webber, B.B., Hu, H.Y., Huisman, T.H. (1991) The linkage of Hb Valletta [α2β2 287(f3)
ThrÆPro] and Hb F-Malta-I [α2γ2 2117(G19) HisÆArg] in the Maltese population. Human Genetics 86(6): 591-4.
4.
Altay, G., Garver, F., Bannister, W.H., Grech, J.L., Felice, A.E., and Huisman, T.H.J. (1977) Detection and quantification of the fetal hemoglobin variant Hb F-Malta-I in
adults. Biochemical Genetics 15(9-10): 915-23.
5.
Miller, S.A., Dykes, D.D., and Polesky, H.F. (1988) A simple salting out procedure for extracting DNA from human nucleated cells. Nucleic Acid Research 16, 1215.
6.
Kalotychou, V., Kolloa, P., Voskaridou, E., Patargias, T., Anagnou, N.P., and Loukopoulos, D. (2002) Functional role of the four different types of (AT)xTy motifs 5’ to
the β-globin gene and their distribution in the Greek population. Blood Cells, Molecules and Diseases 28(1): 39-46
7.
Pulis, S. In vivo expression of the Gγ globin gene of foetal haemoglobin. (2000) M.Phil thesis. Faculty of Medicine and Surgery, University of Malta.
8.
Scerri, C.A. Abela, W., Galdies, R., Pizzuto, M.L. Grech, J.L., and Felice, A.E. (1993) The β+ IVS, I-NT no. 6 (T - C) thalassaemia in heterozygotes with an associated
Hb Valletta or Hb S heterozygosity, and in homozygotes from Malta. British Journal of Haematology 83, 669 – 671.
9.
Borg, J., Scerri, C.A., Galdies, R., Cassar, W., Pizzuto, M., Caruana, M., Bezzina Wettinger, S., and Felice, A.E. (2006) Haemoglobin F Malta I: Interactions in vivo that
Regulate Gamma to Beta Globin Gene Switching. Malta Medical Journal 18: Supplement 1, O-131.
10.
Borg, J., Galdies, R., Scerri, C. A., and Felice, A. E. (2006) Linkage analysis of the (AT)xTy genotype on Haemoglobin F Malta I in the Maltese Population. European
Journal of Human Genetics 14 (5): Supplement 1, P0938.
11.
Felice, A. E., Borg, J., Cassar, W., Galdies, R., Pizzuto, M., Caruana, M., and Scerri, C. A. (2006) HbF MaltaI in Association with HbF Sardinia (AyT) and HbValletta in
Heterozygotes: Quantification of the Six Globins Suggests Developmental Control of the XMN-I site and Interplay with the (AT)xTy Sequence in Connection with
Globin Gene Switching. (Blood 108(11): 34b 3830. ).
12.
Felice, A.E., Borg, J., Pizzuto, M., Cassar, W., Galdies, R., Bezzina Wettinger, S., Pulis, S., Hunter, G.J., Caruana, M.R., Farrugia, M., and Scerri, C.A. (2007) A
Review of Cis-Trans Interplay Between DNA Sequences 5’ to the Gγ and the β Globin Genes Among Hb-F-Malta-I Heterozygotes / Homozygotes and β Thalassemia
Homozygotes / Compound Heterozygotes, and the Effects of Hydroxyurea on the Hb F / F-Erythrocyte; The Need for Large Multi-Center Trials. (HEMOGLOBIN
submitted)
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