1. Molecular Basis Of Hemoglobin Disorders
Dr. Nabil Bashir
HLS, 2018
BAU.EDU.JO

2. Structural features of human hemoglobins
Human globin genes
Development expression of human globin genes
Clinically important hemoglobinopathies
Oxygen affinity of abnormal hemoglobin variants
Know what is Hb S and its clinical correlation
Know what is Hb C and its clinical correlation
Know molecular basis of beta thalassemia & types including Hb E
Know what is hemoglobin Lepore and its clinical correlation
Know the molecular basis of delta-beta thalassemia
Know the molecular basis of High Persistance of Fetal Hemoglobin
Know molecular basis of Alpha thalassemia and its types

11. β-Thalassemia
About 200 mutations worldwide but only about 30 of these reach a frequency of 1% or more in the at‐risk groups .
the common mutations are all point mutations because of single base substitutions, insertion or deletion of a few bases
Point mutations in the promoter.
Mutations in the translational initiation codon.
Point mutation in the polyadenylation signal.
Array of mutations leading to splicing abnormalities.

12. β-Thalassemia
In β-thalassemia, where the β-globins are deficient, the α-globins are in excess and will form α-globin homotetramers.
The α-globin homotetramers are extremely insoluble which leads to premature red cell destruction in the bone marrow and spleen
β 0-thalassemia : A complete lack of HbA
β +-thalassemia : Production of a small amount of functional β –globin
Individuals homozygous for β-thalassemia have what is termed thalassemia major.
Individuals heterozygous for β-thalassemia have what is termed thalassemia minor.

13. Mutations that disrupt splicing bo-thalassemia - no b-chain synthesis
b+-thalassemia - some b-chain synthesis
Normal splice pattern:
Exon 1
Exon 2
Exon 3
Intron 1
Intron 2
Donor site: /GU
Acceptor site: AG/
Intron 2 acceptor site bo mutation: no use of mutant site; use of cryptic splice site in intron 2
Translation of the retained portion of intron 2 results in premature termination of translation due to a stop codon within the intron, 15 codons from
the cryptic splice site
The next two slides illustrate how mutation of splice sites (eliminating or creating them) can lead to genetic disease. Mutations in the beta-globin gene that cause decreased beta-chain synthesis or no beta-chain synthesis give rise to beta(plus)-thalassemia or beta(zero)-thalassemia. Some of these mutations affect splicing. Mutation of the acceptor splice site in intron 2 from the invariant dinucleotide AG to GG would prevent splicing at that site altogether. However, in the absence of a recognizable splice site there, the splicing machinery looks for the next best site and finds one just upstream within the intron. This is the intron 2 cryptic acceptor splice site. As shown, it has the invariant AG and other nucleotides comprising the consensus sequence. While it is present there all the time, it is not normally selected for use. Apparently the downstream site is better. However, when the downstream site is mutated, the machinery is forced to utilize the upstream site. Splicing there results in a somewhat longer than normal mRNA that contains some of the intron. Unfortunately, however, there is a translation stop codon within this additional sequence, and a functional protein cannot be made, giving rise to beta(zero)-thalassemia.
Exon 1
Exon 2
Intron 1
Intron 2 cryptic acceptor site: UUUCUUUCAG/G
mutant site: GG/
9/20/2018

14. Intron 1 b+ mutation creates a new acceptor splice site: use of both sites
Exon 1
Exon 2
Exon 3
Intron 2
Donor site: /GU
AG/: Normal acceptor site (used 10% of the time in b+ mutant)
CCUAUUAG/U: b+ mutant site (used 90%of the time)
CCUAUUGG U: Normal intron sequence (never used because it does not conform to a splice site)
Translation of the retained portion of intron 1 results in termination at a stop codon in intron 1
Exon 1 b+ (Hb E) mutation creates a new donor splice site: use of both sites
Exon 2
Exon 3
Intron 2
This figure shows two examples in which splicing mutations have given rise to beta(plus)-thalassemia. In these cases there is some remaining beta-globin synthesis because the normal splice sites were not mutated - they are still functional. Instead, new splice sites were created from sequences that closely resembled splice sites but did not normally function as such. (For an understanding of these mutations, it would be instructive to view the slide showing the frequency of bases in each position of the splice sites.)
In the top case, a G to A mutation has occurred in intron 1 creating an AG dinucleotide, making that site more dominant over the normal acceptor site. In the bottom case, a G to A mutation has occurred in exon 1 creating a somewhat better overall donor splice site than was already there (and normally unused). It is important to see that there is a region in exon 1 that already has a GU dinucleotide, but that the G in the third position (while tolerable in a splice site) was not good enough in the overall context of that sequence to render it functional under normal circumstances. However, mutating the G (which is found in only 29% of donor sites) to an A (which is found in 62% of donor sites) was able to "tip to balance" and make it functional (unfortunately for patients with disease). The Hb E mutation is very common – it is estimated that there are 30 million heterozygotes for the Hb E amino acid variant in Southeast Asia.
/GU: Normal donor site (used 60% of the time when exon 1 site is mutated)
GGUG/GUAAGGCC: b+ mutant site (used 40%of the time)
GGUG GUGAGGCC: Normal sequence (never used because it does not conform to a splice site)
The GAG glutamate codon is mutated to an AAG lysine codon in Hb E
The incorrect splicing results in a frameshift and translation terminates at a stop codon in exon 2
9/20/2018

15. Thalassemia Intermedia
Both β -globin genes express reduced amounts of protein or where one gene makes none and the other makes a mildly reduced amount.
A person who is a compound heterozygote with α-thalassemia and β +-thalassemia will also manifest as thalassemia intermedia
Anemia
Do not require transfusions.

16. Hemoglobin lepore

17. DELTA-BETA THALASSEMIA, OR TO HEREDITARY PERSISTENCE OF FETAL HEMOGLOBIN (HPFH)
Large deletions of the delta-beta region of chromosome 11 can give rise to delta-beta thalassemia, or to hereditary persistence of fetal hemoglobin (HPFH), which is a benign disorder with no apparent disease symptoms. 
in delta-beta thalassemia there is some compensation by continued (or persistent) expression of the gamma genes,
in HPFH there is complete compensation by this persistent gamma chain expression. 
It has been hypothesized that these large HPFH mutations delete regulatory sequences that control the decreased expression of the gamma chain genes that occurs during hemoglobin switching shortly after birth.  

18. DELTA-BETA THALASSEMIA, OR TO HEREDITARY PERSISTENCE OF FETAL HEMOGLOBIN (HPFH)

19. α-THALASSEMIA
Unequal crossing over between the alpha1 and alpha2 genes. 
Crossing over results in the fusion of the two genes and therefore in essence the deletion of one of the genes. 
This event by itself would not give rise to alpha thalassemia because in diploid cells there would still be three alpha genes, which is sufficient. 
However, if two individuals who are heterozygous for the deleted gene have children, there is a 25% chance that a child will inherit two of these deletion chromosomes.

21. Molecular Diagnosis of Hemoglobin Disorders. B
Molecular Diagnosis of Hemoglobin Disorders B. Examples on Molecular Basis of Hemophilia and Thrombophilia

22. Objectives A.
Be able to identify different hemoglobin types by hemoglobin electrophoresis
Interpret hemoglobin electrogram to diagnose of hemoglobin disorders
Understand some examples on Molecular diagnosis of hemoglobin disorders; RFLP, PCR----- B.
Understand how mutation of factor IX gene causes two different types of hemophilia
Understand how mutation in the 3' UTR of thrombin gene causes Hereditary thrombophilia
Correlate Pulmonary embolism of maternal death during pregnancy or in the period following delivery and thrombophilia

23. Hemoglobin electrophoresis
Hemoglobin electrophoresis is used as a screening test to identify variant and abnormal hemoglobins, including hemoglobin A1 (HbA1), hemoglobin A2 (HbA2), hemoglobin F (HbF; fetal hemoglobin), hemoglobin C (HbC), and hemoglobin S (HbS).
Separation of hemoglobins is based on variable rates of migration of charged hemoglobin molecules in an electrical field.

24. Indications/Applications
Indications and applications of hemoglobin electrophoresis include the following:
Evaluation of unexplained hemolytic anemia
Microcytic anemia unrelated to iron deficiency, chronic disease, or lead toxicity
A peripheral smear with abnormal red cell features (eg, target cells or sickle cells)
Positive family history of hemoglobinopathy
Positive neonatal screen results
Positive results on sickle cell or solubility test

25. HB ELECTROPHORESIS

26. HbS + ( HbA > HbS):
-Sickle cell trait (HbAS) or Sickle α-thalassemia
HbS + HbF, but no HbA:
Sickle cell anemia (HbSS), Sickle beta 0 -thalassemia Sickle–HPFH
HbS > HbA and HbS> HbF: Sickle beta + -thalassemia

27. HbC < HbA :
HbC trait (HbAC)
HbC and HbF, but no HbA: HbC disease (HbCC),
HbC –beta 0 -thalassemia (HbC-HPFH)
HbC > HbA:
HbC beta + -thalassemia
HbS and HbC:
HbSC disease

28. HbH:
HbH disease
Increased HbA 2:
Beta-thalassemia minor
Increased HbF: Hereditary persistence of fetal hemoglobin, Sickle cell anemia, Beta-thalassemia,
HbC disease,
HbE disease

29. DNA analysis Sources of DNA
The main source of DNA is peripheral leucocytes obtained from peripheral blood anticoagulated, preferably with EDTA.
Fetal DNA is mainly isolated from chorionic villi
Fetal DNA can also be prepared from amniotic fluid cells directly or after culture.
Noninvasive methods of prenatal diagnosis utilize DNA from fetal cells in maternal circulation (Cheung, Goldberg & Kan, 1996) .

30. Overview of techniques and methodology
Almost all the methods for DNA analysis of the hemoglobinopathies used today are based on the polymerase chain reaction (PCR) (Saiki et al., 1985).
Therefore whether a mutation is a deletion, a rearrangement or a point mutation a similar test will be performed with the variability and specificity coming from the primers used.
The sensitivity and specificity of PCR has revolutionized the molecular diagnostic field.

31. The PCR‐Based Techniques used in Hemoglobin Diagnostics include :
A. Known mutations
Allele‐specific oligonucleotide (ASO) hybridization or dot‐blot analysis,
Allele‐specific priming or amplification refractory mutation system (ARMS),
Restriction enzyme analysis, amplification created restriction analysis,( RFLP)
Gap‐PCR.
The ultimate method of mutation identification is by direct sequence analysis of specifically amplified DNA.
B. Unknown mutations: (SSCP)

32. GLOBIN GENES

33. Direct sequence analysis of beta globin genes
The majority of the mutations causing β‐thalassaemia are concentrated in two regions –
5′ promoter and 5′ untranslated region (UTR), exon 1, intron 1, exon 2 and the flanking intron 2 sequences
3′ intron 2 sequences, exon 3 and the 3′ UTR
The arrow indicates the location of a heterozygote where two signals are read at the same location with half the signal intensity of a homozygous signal.

34. Detection of the α2 IVS1–5 bp del (αHphα/) thalassaemia variant.
Line diagram illustrating the α2 and α1 globin genes and positions of the amplification primers.
Sequence analysis of the α2‐globin gene at the exon 1/intron 1 junction. One base (G) after the end of exon 1 the sequencing peaks of the chromatogram are reduced in height with double peaks in some locations.
This picture is typical of a frameshift due either to an insertion or deletion.
Comparison of the frameshifted sequence with that of the wild type indicates a 5 bp deletion from IVS1‐1 position (α2 IVS1‐5 bp). The mutation is confirmed by Hph I restriction analysis.

35. lane 2 is a heterozygous individual and lane 3 is a wild type control.
(c) Confirmation of the α2 IVS1‐5 bp deletion by Hph I restriction analysis.
The deletion destroys the Hph I site resulting in a larger restriction fragment of 676 bp in the affected individual. Lanes 1 and 4 are DNA size markers (markers X and IX respectively, Roche),
lane 2 is a heterozygous individual and
lane 3 is a wild type control.

36. Allele‐specific priming – ARMS‐PCR
Principle: a perfectly matched primer is much more efficient at annealing and directing primer extension than a mismatched primer.
allele‐specific amplification relies on the specificity of the 3′ terminal nucleotide.
two reverse allele‐specific primers, one complementary to the mutant sequence, the other to the normal DNA sequence.
Presence of the mutant allele will generate a PCR product in the tube containing the mutation‐specific primer, and vice versa.

37. ARMS‐PCR analysis ARMS‐PCR analysis for : (a)Hb S (b) Hb C
Conventional ARMS‐PCR is performed in two separate reactions using a common primer with a primer specific either for the wild type (N) or mutant (Mt) sequence.
All PCRs are monitored by a control reaction (C) which amplifies a different part of the genome. Lanes labelled Mr are DNA size markers (Marker IX, Roche).

38. Gap‐PCR
Principle:
PCR primer pairs are designed to flank a known deletion generating a unique amplicon that will be smaller in the mutant sequence compared with the wild type (Faa et al., 1992).
For small deletions such as the 619 bp deletion, a common cause of β‐thalassaemia in Asian Indians, differential amplification products are generated in the mutant and wild type.

39. Gap-PCR for a-thal 1
Normal
314 bp
Agarose gel
a-thal 1
188 bp

40. PCR for a -thal 1 1 2 3 4 5 M 1. Father = Negative (aa/aa)
314 bp
188 bp
1. Father = Negative (aa/aa)
2. Mother = Heterozygous (--/aa)
3. Sister = Heterozygous (--/aa)
4. Daughter = Heterozygous (--/aa)
5. Positive control (--/aa)
M = f X 174 Hae III digest

41. Restriction enzyme analysis of PCR product
Restriction enzymes cut double‐stranded DNA at specific recognition sequences;
some mutations naturally create or abolish restriction enzyme sites..
Genomic DNA containing the mutation, the ‘target’, is amplified by PCR and the product is then digested by the diagnostic restriction enzyme and the resulting DNA fragments separated on gels.
The presence or absence of the recognition site is determined from the pattern of the PCR digest hence an alternative term for this technique is restriction fragment length polymorphism (RFLP) analysis.).

42. Restriction enzyme analysis of PCR product
Detection of βS mutation by Dde I restriction analysis.
Line diagram of the β globin gene showing locations of the PCR primers (horizontal arrows) and the βS mutation (in red) in codon 6, exon 1. Exons are shown in striped boxes and introns in open boxes.
Several Dde I restriction sites are found in this PCR amplicon, the Dde I site in codon 6 is removed by the βS mutation, creating the largest restriction fragment of 308 bp. The additional and constant Dde I sites act as internal control for complete Dde I digestion.
Electrophoresis of the Dde I restriction digest. Lane 1 is the DNA size marker, marker V Roche; lane 2 is a homozygous affected control (Hb SS), lane 3 is the heterozygous control (Hb AS) and lane 4, the wild type control (Hb AA). Lanes 5, 6 and 7, are patient samples found to be Hb SS, Hb SS and Hb AS, respectively.

43. Diagrammatic respresentation of an RFLP analysis for the presence of the sickle-cell locus. Genomic DNA is isolated and digested with the restriction enzyme MstII. One MstII site is lost at the sickle-cell locus. The DNA is then Southern blotted and analyzed with a b-globin-specific probe corresponding to sequences at the 5'-end of the gene. Individuals homozygous for the normal globin genes will exhibit a single hybridization band since both maternal and paternal genes are unaffected. Heterozygotes will exhibit the normal band and the larger sickle-cell gene band. Homozygous sickle-cell individuals will exhibit a single larger hybridization band.

44. PCR with restriction digestion of amplified products
Mutation creating cutting site
665
C/C
445
220
T/T
PCR with restriction digestion of amplified products

45. Marker;Ø174 RF DNA / Hae III Fragment
Result of digestion of PCR products
Family 1
1.Brother (+ /-)
2.Propositus (+ /-)
3.Father (- /-)
4.Mother (+/+)
Family 2
5.Mother (- /-)
6.Brother(+ /-)
7.Father (+/+)
8.Propositus (+ /-)
Negative control (- /-)
Positive control (+/+)
665 bp
445 bp
220 bp 1 2 3 4 5 6 7 8 N P
Marker
Marker;Ø174 RF DNA / Hae III Fragment

46. Allele‐specific oligonucleotide (ASO) hybridization and reverse dot‐blot
The method is based on the principle of specific hybridization of two oligonucleotide probes, one complementary to the mutant sequence and the other to the normal sequence (Wallace et al., 1981; Conner et al., 1983). The ASO's differ from each other by a single base change designed to be in the centre of the ASO to maximize instability of any mismatch

47. normal and mutant probes
PCR with ASO-dot blot
PCR
Dot on Nylon membrane
Hybridization with
normal and mutant probes
Color development
Result
All negative

48. PCR-SSCP analysis of normal and sickle cell β-globin genes
The PCR products from the wild-type locus and the sickle cell locus will migrate differently due to sequence-specific conformations.
Normal homozygous persons will display two bands as will homozygous sickle cell persons (although of different sizes than normal).
Persons heterozygous at the sickle cell locus will display four bands.

50. The factor IX gene: located on the X chromosome
Hemophilia
The factor IX gene: located on the X chromosome
The factor IX gene promoter
there are overlapping binding sites for AR and HNF4
AR = androgen receptor
transcription factor
binds androgen
androgen levels increase at puberty
HNF4 = hepatocyte nuclear factor-4
ligand unknown - therefore an “orphan” receptor
HNF4 is expressed early in development and in adult liver
-36
-27
-22
-15 AR
HNF4
The promoter of the factor IX gene can be the site of single base-pair mutations that have quite different phenotypes. The factor IX promoter contains two overlapping DNA elements that are binding sites for the androgen receptor (AR) and hepatocyte nuclear factor-4 (HNF4), which are both transcription factors. The next slide shows the effects of mutations in these two binding sites.

51. -36 -27 -22 -15 AR HNF4 mutation at -20 results in
Hemophilia B Leyden in which
the hemophilia improves at puberty
when levels of androgen increase
-36
-27
-22
-15 AR
HNF4
Single base-pair mutations affecting sites within the promoter that are only six base-pairs apart can have vastly different phenotypes. The mutation at -20 within the HNF4 site gives rise to hemophilia B Leyden. Patients with this mutation have hemophilia at a young age, but improve upon reaching puberty. This is due to the increased levels of testosterone which activate the androgen receptor, thus turning on the factor IX gene. The mutation at -26 occurs within the overlap zone for the two transcription factors. These patients, who have hemophilia B Brandenburg, do not improve with age, since the binding of both transcription factors is prevented.
mutation at -26 results in
Hemophilia B Brandenburg
in which factor IX levels remain low even after puberty

52. Hereditary thrombophilia
Hereditary thrombophilia (increased tendency for the blood to clot) is caused by a single nucleotide substitution in the 3' UTR, which is present in 1-2% of the population.
This mutation increases the efficiency of 3‘ end processing, leading to excess production of thrombin mRNA and protein (Mendell and Dietz, Cell 107:411; 2001).
Pulmonary embolism is the most common cause of maternal death during pregnancy or in the period following delivery.
About 70% of women who present with venous thromboembolism during pregnancy are carriers of hereditary or acquired thrombophilia (Eldor, J Thromb Thrombolysis 12:23; 2001).

53. Hereditary thrombophilia
A sequence in the 3’ UTR that regulates 3’ end processing of thrombin mRNA, which when mutated causes hereditary thrombophilia.
5’ UTR
3’ UTR 5’
AUG
UGA 3’
Translated region (open reading frame)
There is also
Hereditary
thrombophilia