Questions for further study and thinking
1.
How does g gene expression protect against b-thalassemia?
2.
What is the most likely phenotype of a child with a genotype / , b/-? How about a child having the genotype
/ , b/-?
3.
(Optional!!) Describe mechanisms that could account for the
high frequencies of -thal and b-thal mutations in local
population. (Hints: selection, genetic drift and founder effects)
Ref: Thompson and Thompson Genetics in Medicine, 6th ed., 2004,
chapter 7 (Genetic variation in populations)
4.
Suppose the incidence of b-thalassemia major is 0.01% in the
local population. What is the carrier rate of b-thalassemia
mutation? (Hint: Hardy-Weinberg equilibrium: p2 + 2pq + q2 = 1)
(See slide 48 for answer.)
39
Suggested textbooks:
•
Nussbaum, R.L. et al.: Thompson & Thompson Genetics in
Medicine, 6th ed. Saunders, 2004 (chap. 11: Principles of
Molecular Disease: Lesson from the Hemoglobinopathies)
•
Gelehrter, T.D. et al.: The Principles of Medical Genetics, 2nd
ed. Williams and Wilkins,1998 (chap. 6: Molecular Genetics
of Human Disease: Hemoglobinopathies)
•
NCBI Books: GeneReviews ( and b thalassemia); Human
Molecular Genetics 2 (chap. 16)
Go back to basic (NCBI Books have earlier version):
Lewin’s Gene XI 2014
Molecular Biology of the Cell 2014
Stryer Biochemistry 7th ed 2012 (chaps. 7 and 29)
Molecular Cell Biology 7th ed 2013
40
References and further reading
•
1)
2)
3)
4)
Recent review articles
N. Eng. J. Med. 353:1135-1146, 2005 (“b-thalassemia”)
N. Eng. J. Med. 371:1908-1916, 2014 (“ -thalassemia”)
Lancet 391:155-167 2018 (“Thalassaemia”)
Blood 112:3927-3938, 2008 (“Hemoglobin research and the origins of
molecular medicine”)
•
1)
Books for specialists (extended reading for your own interest only)
Stamatoyannopoulos G et al.: The Molecular Basis of Blood Diseases, 3rd ed.,
Saunders, 2001
2)
Weatherall, D.J. and Clegg, J.B.: The Thalassaemia Syndromes, 4th ed.,
Blackwell, 2001
3)
Steinberg M.H. et al.: Disorders of Hemoglobin: Genetics, Pathophysiology
and Clinical Management, Cambridge, 2001
•
Websites
Globin Gene Server: http://globin.cse.psu.edu/
NCBI OMIM: http://www.ncbi.nlm.nih.gov/
Bloodline: http://www.bloodline.net/
41
Learning Outcomes
By the end of the lecture (and after the Biochemistry
Practical, PBL and your study), you should be able to:
•
Define the organisation of the α- and β- globin gene
clusters
•
Outline the molecular and genetic basis of α- and βthalassemia
•
Correlate genotypes with phenotypes in α- and βthalassemia
Discuss the molecular mechanisms of β-thalassemia
Understand the scientific basis of its diagnosis and
treatment
•
•
- and b-globin gene clusters
The genomic structure of the clusters of -like and β-like globin genes, on
chromosomes 16 and 11, in human beings. The functional -like genes are
shown in dark blue and the pseudogenes are in light blue; 2 of these (µ and -1)
code for small amounts of RNA. The functional β-like genes are shown in light green.
The important control elements, HS-40 and the LCR, are also shown at their
approximate locations. The -gene cluster is approximately two thirds of the length
of the β-gene cluster; it is transcribed from telomere toward centromere, the opposite
of the β cluster. The various hemoglobin species that are formed from these genes,
with their prime developmental stages, are shown in the lower part of the figure.
Illustration by Alice Y. Chen. (Legend to slide 5)
- thalassemias: genotypes and phenotypes
Genotype-phenotype relationships in -thalassemias: In rare cases, hemoglobin H (HbH)
disease or hemoglobin Bart's hydrops fetalis can result from homozygosity for
nondeletional forms of α-thalassemia. Although HbH disease is usually symptomatic,
some patients, particularly those with deletional variants of α-thalassemia, are
asymptomatic. Furthermore, three α-globin gene deletions involving severe nondeletional
mutations (e.g., hemoglobin Constant Spring) can also cause the hemoglobin Bart's
hydrops fetalis syndrome. With regard to the lethality of hemoglobin Bart's hydrops fetalis,
four α-globin gene deletions with persistence of an intact embryonic ζ-globin gene — for
example, –(α)20.5 in the Mediterranean area — may be associated with neonatal survival.
(NEJM 2014; Legend to slide 8)
43
Developmental switches of globin expression
The timeline of the expression of the human globin genes from early
stages of fetal development to the changes that occur at birth and in
the first year of life. Also shown are the major sites of erythropoiesis and
the types of hemoglobin-containing cells during these periods. These
analyses are largely based on observations of clinical samples made by
Huehns et al. in the 1960s; the figure is reprinted from Wood (Br Med Bull.
1976;32:282) with permission. Illustration by Alice Y. Chen. (Legend to
slide 6)
Normal developmental switches in globin expression. (A) The
structure of the α-like and β-like globin gene clusters. IVS=intervening
segments (or introns). (B) The sites of hemopoiesis at different stages of
development and the levels of expression of the embryonic, fetal, and
adult globin chains at various gestational ages are shown. ψ designates
non-expressing pseudogenes. The three exons of the globin genes are
shown in pale blue. (Lancet 2018; 391:155-167; Legend to slide 17)
44
Genetic basis of the phenotypic diversity of b-thal
Primary and secondary modifiers of the b thalassemia phenotype include variable output from
the b globin (b genotype), variable output from the globin genes ( genotype) and variable
HbF response (co-inheritance of different QTLs (quantitative trait loci) controlling HbF and Fcell levels). The consequences of these factors is the degree of chain imbalance ( / nonglobin ratio) and severity of ineffective erythropoiesis. bsilent: have minimal effect on b globin
production. bN: Normal. (Legend to slide 21)
The primary cause lies in the -, b- and g-globin genes, as shown at the top of the figure,
and they affect the magnitude of the excess of -chains. The secondary modifiers such as
AHSP ( hemoglobin stabilizing protein; a chaperone that binds and stabilizes
globin,
thereby reducing 4 precipitation) are shown in the middle. HPFH: hereditary persistence of
fetal hemoglobin. Tertiary modifiers are shown at the bottom of the figure: VDR, vitamin D
receptor; ESR1, estrogen receptor; collagen, several genes determined in collagen synthesis;
HFE, the locus for hereditary hemochromatosis; HAMP, hepcidin antimicrobial peptide (a
peptide hormone that is the master regulator of iron homeostasis); UGT1, UGT
glucuronyltransferase involved in bilirubin glucuronidation; HLA-DR, major histocompatibility
complex locus; TNF, tumour-necrosis factor- ; ICAM1, intercellular adhesion molecule 1;
GDF11, growth differentiation factor 11 (a member of transforming growth factor β
superfamily); JAK2, Janus kinase 2 (disease gene of myeloproliferative disorders); TMPRSS6,
transmembrane protease serine 6 (a metalloprotease governing hepcidin expression)
(Legend to slide 23)
From SNP to mechanism and potential therapy. Hemoglobin genes on human
chromosome 11 are differentially expressed in the embryo, fetus, and adult. A SNP in the
BCL11A gene is associated with varying amounts of fetal hemoglobin in human populations.
Because there is an inverse correlation between BCL11A and fetal hemoglobin expression,
inhibiting BCL11A is a potential therapy for adult hemoglobinopathies. LCR HSS: locus
control region hypersensitive sites. The asterisk indicates a DNA region that, when deleted, is
associated with increased fetal hemoglobin production. (Legend to slide 24)
45
Nonsense-mediated mRNA decay (NMD)
Genes contain exons and introns. Genes are transcribed into a precursor mRNA, called pre-mRNA.
Pre-mRNA is processed co- and post-transcriptionally in a series of temporally and mechanistically
connected steps. One such step is 5′-end capping (m7Gppp). The presence of a 5′ cap enables the
association of the cap binding protein (CBP) heterodimer. Another step is 3′-end polyadenylation. A
third step is splicing, which results in intron removal and concomitant ligation of exons. Introns are
recognized in part because they begin with a 5′-splice site and end with a 3′-splice site. Translation
will terminate at the normal termination codon and the mRNA will not be targeted for NMD. However,
if intron 1 contains a nonsense codon that resides in frame with the AUG initiation codon or if it
lacks such a nonsense codon but is not a multiple of 3 nt and, thus, shifts the reading frame, then
translation could terminate prematurely and elicit NMD. NMD involves three distinct processes. In
the first, NMD substrates are detected by the NMD machinery. In the case of 3′ untranslated region
(UTR) exon junction complex (EJC)-mediated NMD, if translation terminates, for example, at a
premature termination codon (PTC), more than ∼50–55-nt upstream of an exon–exon junction
(depicted as “^”), then termination is detected as aberrant. This is because a proteinaceous EJC,
situated ∼20–24-nt upstream of the exon–exon junction, lies too far downstream from the PTC to be
removed by the terminating ribosome. At the EJC, UPF2, anchored by UPF3X (also called UPF3B),
interacts with UPF1 and SMG1 at the site of termination. Alternatively, on substrates with unusually
long 3′UTRs, a large amount of UPF1 can promiscuously bind to the 3′UTR. The second phase of
NMD is the commitment phase, where UPF1 is phosphorylated by its associated kinase, SMG1. This
occurs efficiently during a series of regulated events on 3′UTR EJC-mediated NMD and less
efficiently on 3′UTR EJC-independent NMD substrates. UPF1 phosphorylation represents a
commitment to NMD. During the third phase of NMD, that is, mRNA degradation, phosphorylated
UPF1 recruits RNA degradation activities either directly, by recruiting the SMG6 endonuclease (solid
line with arrow to the scissors, which represent SMG6 itself) and/or the SMG5–SMG7 heterodimer,
which recruits (dotted lines with arrow to the scissors) decapping and deadenylation enzymes
(scissors) that produce unstable RNAs that are targeted for further degradation by exonucleases
(red pacmen). CBP, cap-binding protein(s); 5′ dot, 7-methylguanine 5′ cap; AUG, translation initiation
codon; AA(A)n, 3′ poly(A) tail; P, phosphate.
(Legend to slide 28)
46
RNA splicing: b-globin as an example
The β-globin gene contains three protein-coding exons (red) and two intervening
noncoding introns (blue). The introns interrupt the protein-coding sequence between the
codons for amino acids 31 and 32 and 105 and 106. Transcription of this and many other
genes starts slightly upstream of the 5′ exon and extends downstream of the 3′ exon,
resulting in noncoding regions (gray) at the ends of the primary transcript. These regions,
referred to as untranslated regions (UTRs), are retained during processing. The 5′ 7methylguanylate cap (m7Gppp; green dot) is added during formation of the primary RNA
transcript, which extends beyond the poly(A) site. After cleavage at the poly(A) site and
addition of multiple A residues to the 3′ end, splicing removes the introns and joins the
exons. The small numbers refer to positions in the 147-aa sequence of β-globin.
(Legend to slide 30)
Abnormal processing of the b-globin primary RNA transcript in humans with the disease b
thalassemia. In the examples shown, the disease is caused by splice-site mutations,
denoted by black arrowheads. The dark blue boxes represent the three normal exon
sequences; the red lines are used to indicate the 5 and 3 splice sites that are used in
splicing the RNA transcript. The light blue boxes depict new nucleotide sequences
included in the final mRNA molecule as a result of the mutation. Note that when a
mutation leaves a normal splice site without a partner, an exon is skipped or one or
more abnormal "cryptic" splice sites nearby is used as the partner site, as in (C) and
(D). (Adapted in part from S.H. Orkin, in G. Stamatoyannopoulos et al., eds, The
Molecular Basis of Blood Diseases, pp. 106-126. Philadelphia: Saunders, 1987.)
(Legend to slide 31)
47
New strategies in the treatment of thalassemias
• Hematopoietic stem cell transplantation
• Cellular and molecular modifiers (5-azacytidine,
hydroxyurea, etc)
• Antioxidants
• CRISPR/Cas9 (correcting mutations by targeted genome
editing)
• Gene therapy (lentiviral vectors: treatment succeeds,
with a caveat)
(Science 326:1468-1469, 2009; Nature Medicine 21:221-230, 2015)
(http://asheducationbook.hematologylibrary.org/cgi/content/full/2009/1/690)
(http://asheducationbook.hematologylibrary.org/content/2010/1/445)
If the incidence of b-thalassemia major is 0.01% in the local population, the carrier
rate of b-thalassemia mutation is 2 0.01 0.99 = 0.0198 = 1.98% 2%.
48
Hematopoietic stem/progenitor cells(HSPC) gene therapy
Hematopoietic stem/progenitor cell (HSPC) gene therapy. HSPCs are isolated
from the bone marrow (or mobilized peripheral blood) of a patient with b-thalassemia.
Following culture ex vivo in conditions that stimulate cell proliferation, the cells are
exposed to a retroviral vector expressing a functional copy of the defective gene and
then infused back into the patient after a few days. Infusion usually takes place
following administration of a pharmacological conditioning regimen that eliminates the
endogenous bone marrow progenitors and favours engraftment of the transplanted
cells. The engrafted gene-corrected progenitor cells generate functional progeny that
reconstitute all lineages and restore red blood cell functions to the patient. If the genecorrected cells have a selective growth advantage compared to the unmodified cells,
full reconstitution of the red blood cell compartments is obtained even from a few
engrafted transduced progenitor cells, as depicted in the figure, and this may occur
even without conditioning. If the engrafted progenitor cells have self-renewal capacity,
they ensure long-term correction of the disease. If the engrafted cells are multipotent
stem cells, they generate gene-marked cells in all haematopoietic lineages. NK,
natural killer cells. (Legend to slide 34)
Advantages of HSPCs: 1) Capacity to self renew and maintain specific functions
over an individual’s lifetime: HSPCs are defined by their ability to regenerate all blood
lineages (multipotency). Transfer and expression of genes added to a small number of
cells result in gene correction of much greater numbers of cells by self-renewal and
differentiation. 2) Accessibility: can be obtained easily. 3) Ability to survive and be
manipulated in ex vivo cell culture. 4) Transplantability: HSPC transplantation provides
the best chance for a cure for many diseases.
49