Cytogenetic & molecular Investigations of hemoglobinopathies

1. Cytogenetic & molecular Investigations of hemoglobinopathies

2. inherited haemolytic disorders can be classified into three major groups: (i) genetic disorders of haemoglobin (ii) abnormal membrane (including the cytoskeleton) (iii) abnormal metabolism (enzymopathies)

3. the inherited disorders of globin synthesis The inherited disorders of haemoglobin are the commonest single gene disorders, with an estimated carrier rate of 7% among the world population. They occur at particularly high frequencies in populations of the tropical and subtropical belt, and consist mainly of the α and β thalassaemias, and the haemoglobin variants S, C and E.

4. The clinical syndromes produced by haemoglobin abnormalities.

5. The geographical distribution of the thalassaemias and the more common, inherited,structural haemoglobin abnormalities.

6. The structure, genetic control and synthesis of haemoglobin Different haemoglobins are synthesized in the embryo, fetus and adult, each adapted to their particular oxygen requirements. They all have a tetrameric structure made up of two different pairs (one α - like and one β - like) of globin chains, each attached to one haem molecule, the moiety responsible for the reversible binding and transfer of oxygen

7. The embryonic haemoglobins include Hb Portland ( ζ 2 γ 2 ), Hb Gower 1 ( ζ 2 ε 2 ), and Hb Gower 2 ( α 2 ε 2 ). In the fetus, HbF( α2 γ2 ) predominates in adults, HbA ( α 2 β 2) comprises over 95% of the total haemoglobin, with a minor component of HbA 2( α2 δ2 ) in the red blood cells.

8. There are two kinds of HbF composed of γ - chains that differ in their amino acid composition at position 136, where they have either glycine or alanine; those with glycine are called G γ - chains and those with alanine A γ -chains.

9. The G γ and A γ chains are the products of separate globin gene loci ( G γ and A γ ). These different types of haemoglobin are adapted to the changes in physiological requirements that occur during development.

10. Fetal haemoglobin (HbF) exhibits a higher oxygen affinity than adult haemoglobins in vivo ; The higher oxygen affinity of HbF relative to adult haemoglobin facilitates the transfer of oxygen across the placenta from the maternal to the fetal circulation.

12. Each of the α - like and β - like globin chains is encoded by genetically distinct loci, the α - like cluster on the tip of chromosome 16p and the β - like cluster on chromosome 11p15.5 In both clusters, the genes are arranged along the chromosome in the order in which they are expressed during development

13. the genetic control of globin chainsynthesis & Levels of action of mutations

14. Examples of mutations that produce beta thalassaemia. single base changes, Small deletions and insertions of one or two bases affecting introns, exons or the flanking regions of the beta globin gene. FS, 'frameshifts': deletion of nucleotide(s) that places the reading frame out of phase downstream of the lesion; NS,'non-sense': premature chain termination as a result of a new translational stop codon (e.g. UAA);

15. SPL, 'splicing': inactivation of splicing or new splice sites generated (aberrant splicing) in exons or introns; promoter, CAP, initiation: reduction of transcription or translation as a result of lesion in promoter, CAP or initiation regions; Poly A, mutations on the poly A addition signal resulting in failure of poly A addition and an unstable mRNA.

17. Distribution of different mutations of beta thalassaemia major in the Mediterranean area.

18. Classification of the disorders of haemoglobin β Thalassaemia β 0 Deletion Non - deletion β + ‘ Silent ’ Normal HbA 2 Dominant α Thalassaemia α 0 α + Deletion (/ − α ) Non - deletion (/α T α ) δ β Thalassaemia ( δ β ) 0 ( δ β ) + γ Thalassaemia δ Thalassaemia ε γ δ β Thalassaemia Hereditary persistence of fetal haemoglobin Deletion Non - deletion A γ G γ

19. Suggested scheme of investigation for thalassaemia.

21. β Thalassaemia There are more than 100 β thalassaemia mutations are known, each ethnic groups has its own subset of mutations, so that as few as five different mutations may account for more than 90% of the affected individuals in a population. This makes the direct detection of β thalassaemia mutations a reasonable possibility and it has become the method of choice where it is most important: in prenatal diagnosis

22. The majority of mutations causing β thalassaemia are point mutations affecting the coding sequence, splice sites or promoter of the β globin gene. they are detected by direct DNA sequence analysis. Unstable and other unusual haemoglobins may also cause disease and can also be identified by direct DNA sequence analysis.

23. moderate anaemia is seen in the heterozygotedue to the highly unstable and electrophoretically silentvariant, haemoglobin, Durham, NC.

24. α Thalassaemia In contrast to the b thalassaemias, the most common α thalassaemia mutations are deletions. Two categories exist: those that remove only one of the two alpha globin genes and those that remove both of the alpha genes from one chromosome Although PCR amplification around the alpha globin locus has proved to be rather difficult, the common deletions can now be identified by a reasonably robust Gap-PCR

25. More than 30 non-deletional forms of α thalassaemia have been described. Of these, Hb Constant Spring and it is relatively common in South-east Asian Unlike the β thalassaemias, α thalassaemias are not easily diagnosed using routine haematological techniques. The diagnosis of α thalassaemias is often made following exclusion of β thalassaemia and iron deficiency. the only definitive diagnostic test is DNA analysis.

26. The diagnosis of a thalassaemia is particularly relevant if prenatal diagnosis is to be offered to a couple who are at risk of having a fetus with hydrops, where there is an increased risk of maternal death at delivery.

27. Sickle Cell Disease The presence of a sickle cell gene can be determined by haemoglobin cellulose acetate electrophoresis or a sickling test. However, there are occasions when it is beneficial to make this diagnosis by DNA analysis e.g. in prenatal diagnosis, which can be performed at 10 weeks of pregnancy, in distinguishing HbS/S from HbS/β thalassaemia or in confirming the diagnosis of sickle cell anaemia in a neonate).

28. The sickle cell mutation in codon 6 of the b globin gene (GAG ! GTG) results in the loss of a restriction enzyme site that is present in the normal gene. It is therefore possible to detect the mutation directly by restriction enzyme analysis of a DNA fragment generated by the PCR. A pair of primers are used to amplify exons 1 and 2 of theβ globin gene and the products of the PCR are digested with restriction enzyme in the sickle cell gene gives rise to an abnormally large restriction fragment that is not seen in normal individuals

29. Studies in Iraq A total of 591 couples (1182 individuals) attending health centres for premarital health screening were tested; 44 (3.7%) were found to be carriers of β-thalassaemia, 14 (1.2%) of the sickle-cell gene and 1 (0.1%) of δβ-thalassaemia. A total of 3 couples (i.e. 5/1000) were at risk of having a child with β-thalassaemia major, and the estimated number of affected children with a major haemoglobinopathy was 39 per year.

30. A random 123 carriers of β-thalassemia (β-thal), A total of 11 different β-thal mutations was identified in the studied samples, of which eight represented 96% of the mutated β-globin genes. These were : IVS-II-1 (G>A), IVS-I-110 (G>A), codon8 (–AA), codons 8/9 (+G), IVS-I-5 (G>C), codon 5 (–CT), IVS-I-6 (T>C) and IVS-I-1 (G>A). Other mutations were less common or sporadic.

31. Molecular defects responsible for β-thalassemias (thal) were investigated from 127 transfusion dependent. Among fourteen identified mutations, the seven most common found in 88.2% of the thal chromosomes were: IVS-II-1 (G → A), IVS-I-1 (G → A), codon 8 (−AA), codon 39 (G → T), codon 8/9 (+G), codon 44 (−C), and codon 5 (−CT). There were some notable differences in frequencies of various mutations in comparison to other Eastern Mediterranean populations There is relative heterogeneity of the mutations distribution in Iraq