MOLECULAR BASIS OF HEMOGLOBIN DISORDERS Dr Nabil Bashir

MOLECULAR BASIS OF HEMOGLOBIN DISORDERS Dr. Nabil Bashir HLS, 2018 BAU. EDU. JO

1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 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

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.

β-Thalassemia 1. 2. 3. 4. 5. 6. 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 Hb. A 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.

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 2 Intron 1 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 Exon 1 Exon 2 Intron 1 Intron 2 cryptic acceptor site: UUUCAG/G 12/3/2020 mutant site: GG/ 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 13

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 /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 12/3/2020 14

Thalassemia Intermedia Ø Ø 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.

Hemoglobin lepore

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.

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

α-THALASSEMIA � � Unequal crossing over between the alpha 1 and alpha 2 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.

MOLECULAR DIAGNOSIS OF HEMOGLOBIN DISORDERS B. EXAMPLES ON MOLECULAR BASIS OF HEMOPHILIA AND THROMBOPHILIA

Objectives A. 1. 2. 3. 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. 1. 2. 3. 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

Hemoglobin electrophoresis � � Hemoglobin electrophoresis is used as a screening test to identify variant and abnormal hemoglobins, including hemoglobin A 1 (Hb. A 1), hemoglobin A 2 (Hb. A 2), hemoglobin F (Hb. F; fetal hemoglobin), hemoglobin C (Hb. C), and hemoglobin S (Hb. S). Separation of hemoglobins is based on variable rates of migration of charged hemoglobin molecules in an electrical field.

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

HB ELECTROPHORESIS

Hb. S + ( Hb. A > Hb. S): -Sickle cell trait (Hb. AS) or Sickle α-thalassemia Hb. S + Hb. F, but no Hb. A: Sickle cell anemia (Hb. SS), Sickle beta 0 thalassemia Sickle– HPFH Hb. S > Hb. A and Hb. S> Hb. F: Sickle beta + thalassemia

Hb. C < Hb. A : Hb. C trait (Hb. AC) Hb. C and Hb. F, but no Hb. A: Hb. C disease (Hb. CC), Hb. C –beta 0 thalassemia (Hb. CHPFH) Hb. C > Hb. A: Hb. C beta + thalassemia Hb. S and Hb. C: Hb. SC disease

Hb. H: Hb. H disease Increased Hb. A 2: Beta-thalassemia minor Increased Hb. F: Hereditary persistence of fetal hemoglobin, Sickle cell anemia, Beta-thalassemia, Hb. C disease, Hb. E disease

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).

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.

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

GLOBIN GENES

Direct sequence analysis of beta globin genes The majority of the mutations causing β‐thalassaemia are concentrated in two regions – (i) 5′ promoter and 5′ untranslated region (UTR), exon 1, intron 1, exon 2 and the flanking intron 2 sequences (ii) 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. •

Detection of the α 2 IVS 1– 5 bp del (αHphα/) thalassaemia variant. (a) (b) • • Line diagram illustrating the α 2 and α 1 globin genes and positions of the amplification primers. Sequence analysis of the α 2‐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 IVS 1‐ 1 position (α 2 IVS 1‐ 5  bp). The mutation is confirmed by Hph I restriction analysis.

(c) Confirmation of the α 2 IVS 1‐ 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.

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‐specificamplification 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.

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).

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.

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

PCR for a -thal 1 1 2 3 4 5 M 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

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. ).

Restriction enzyme analysis of PCR product Detection of βS mutation by Dde I restriction analysis. (a) 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. (b) 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. (c) 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.

Diagrammatic respresentation of an RFLP analysis for the presence of the sickle-cell locus. Genomic DNA is isolated and digested with the restriction enzyme Mst. II. One Mst. II site is lost at the sickle-cell locus. The DNA is then Southern blotted 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 the larger sickle-cell gene band. Homozygous sickle-cell individuals will exhibit a single larger hybridization band.

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

Result of digestion of PCR products Family 1 1. Brother (+ /-) 2. Propositus (+ /-) 665 bp 3. Father (- /-) 4. Mother (+/+) 445 bp Family 2 5. Mother (- /-) 220 bp 6. Brother(+ /-) 7. Father (+/+) 8. Propositus (+ /-) Negative control (- /-) 1 2 3 4 5 6 7 8 N P Marker; Ø 174 RF DNA / Hae III Fragment Positive control (+/+)

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

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

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.

Hemophilia The factor IX gene: located on the X chromosome The factor IX gene promoter • there are overlapping binding sites for AR and HNF 4 -36 -27 AR -22 -15 HNF 4 • AR = androgen receptor • transcription factor • binds androgen • androgen levels increase at puberty • HNF 4 = hepatocyte nuclear factor-4 • transcription factor • ligand unknown - therefore an “orphan” receptor • HNF 4 is expressed early in development and in adult liver

• mutation at -20 results in Hemophilia B Leyden in which the hemophilia improves at puberty when levels of androgen increase -36 -27 AR -22 -15 HNF 4 • mutation at -26 results in Hemophilia B Brandenburg in which factor IX levels remain low even after puberty

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 m. RNA 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 Thrombolysis 12: 23; 2001).

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