Genetics of the Hemoglobinopathies & Newborn Screening for the Hemoglobinopathies

1. Genetics of the Hemoglobinopathies & Newborn Screening for the Hemoglobinopathies 张咸宁 zhangxianning@zju.edu.cn Tel：13105819271; 88208367 Office: A705, Research Building 2012/03

2. Required Reading Thompson &Thompson Genetics in Medicine, 7th Ed （双语版，2009） ● Pages 237-257; ● Clinical Case Studies: 37. Sickle Cell Disease 39. Thalassemia

3. Part I. Genetics of the Hemoglobinopathies

4. Learning Objectives • To review the normal structure-function relationships of hemoglobin and expression of globin genes • To examine the hemoglobinopathies as disorders of hemoglobin structure, or α- or β-globin gene expression • To explore the influences of compound heterozygosity and modifier genes on hemoglobinopathy phenotypes

5. Lecture Outline • Structure and Function of Hemoglobin • Developmental Expression of Globin Genes and Globin Switching • Sickle Cell Disease: Disorder of Hemoglobin Structure • Thalassemia: Disorder of α- or β-Globin Gene Expression • Phenotypic Consequences of Allelic Interactions and Modifier Genes

6. Molecular Disease A disease in which there is an abnormality in or a deficiency of a particular molecule, such as hemoglobin in sickle cell anemia.

7. The Effect of Mutation on Pr Function • Loss of Pr function (the great majority): is seen in (1)recessive diseases;(2)diseases involving haploinsufficiency, in which 50% of the gene product is insufficient for normal function; and (3)dominant negative mutations, in which the abnormal protein product interferes with the normal protein product.

8. The Effect of Mutation on Pr Function 2. Gain of function: are sometimes seen in dominant diseases. 3. Novel property(infrequent) 4. The expression of a gene at the wrong time (Heterochronic expression), or in the wrong place (Ectopic expression), or both. (uncommon, except in cancer)

10. Hemoglobinopathies • Disorders of the human hemoglobins • Most common single gene disorders in the world • WHO: 5% of the world’s population are carriers for clinically significant hemoglobinopatihies • Well understood at biochemical and molecular levels

11. HbA: α2β2 • Globular tetramer • MW 64.5 kD • α-Chain • Maps to chromosome 16 • Polypeptide length of 141 amino acids • β-Chain • Maps to chromosome 11 • Polypeptide length of 146 amino acids

12. Normal Human Hbs • Six including HbA • Each has a tetrameric structure • Two α or α-like genes • Clustered on chromosome 16 • Two non-α genes • Clustered on chromosome 11

13. Globin Tertiary Structure • Eight helices: A-H • Two globins highly conserved • Phe 42: wedges heme porphyrin ring into heme pocket • Mut: Hb Hammersmith • His 92: covalently links heme iron • Mut: Hb Hyde Park

14. Gene cluster: A group of adjacent genes that are identical or related. Pseudogene: DNA sequence homologous with a known gene but is non-functional.

16. Developmental Expression of Globin Genes and Globin Switching

18. Globin Gene Developmental Expression and Globin Switching • Classical example of ordered regulation of developmental gene expression • Genes in each cluster arranged in • Same transcriptional orientation • Same sequential order as developmental expression • Equimolar production of α-like and β-like globin chains

19. Human Hemoglobins: Prenatal • Embryonic • 22 • Fetal: HbF • α22 • Predominates 5 wks gestation to birth • Approx 70% of total Hb at birth • <1% of total Hb in adulthood

20. Human Hemoglobins: Postnatal • Adult: HbA • 22 •  chain synthesis increases through birth • Nearly all Hb is HbA by 3 mos of age • HbA2 • 22 • ≤2% of adult Hb • Consequence of continuing synthesis of  chains

21. Clinical Disease: Influences of Gene Dosage and Developmental Expression • Dosage • 4 - vs. 2 -globin genes per diploid genome • Therefore, mutations required in 4 -globin genes compared with 2 -globin genes for same 100% loss of function • Ontogeny •  expressed before vs.  expressed after birth • Therefore, -chain mutations have prenatal consequences, but -chain mutations are not evidenced even in the immediate postnatal period

22. The normal human hemoglobins at different stages of development

23. Genetic disorders of hemoglobin 1. Structural variants: alter the globin polypeptide without affecting its rate of synthesis. 2. Thalassemias: reduced rate of production of one or more globin chains. 3. Hereditary persistence of fetal hemoglobin (HPFH) : a group of clinically benign conditions, impairing the perinatal switch from γ- toβ-globin synthesis.

24. There are over 400 structural variants of normal hemoglobin. The 4 most common structural variants are: • Hb S(Sickle cell anemia): β chain: Glu6Val • Hb C: β chain: Glu6Lys • Hb E: β chain: Glu26Lys • Hb M(Methemoglobin):An oxidizing form of Hb containing ferric iron that is produced by the action of oxidizing poisons. Non-functional.

27. Clinical Features of SS Disease • Presentation typically in first 2 years of life • Infections • Anemia • Failure to thrive (FTT) • Splenomegally • Dactylitis: Extremely painful swelling of hands and feet from capillary occlusion in small bones

28. Vaso-Occlusive Infarctions • Strokes • Acute chest syndrome • Renal papillary necrosis • Autosplenectomy • Leg ulcers • Priapism • Bone aseptic necrosis • Visual loss

29. Bone Crises • Vaso-occlusion of bones • Extremely painful • Persist for days or weeks if untreated • Treatment • Pain management • Hydration • Oxygen • Prevention • Hypertransfusion

30. Functional Asplenia • Due to splenic infarction and other poorly understood factors • Increases susceptibility to • Pneumococcal sepsis • Salmonella osteomyelitis • Infection • Major cause of death at all ages

32. HbS is the first variant to be discovered (1949). Its main reservoir is Central Africa where the carrier rate approximates 20%. (Heterozygous advantage) Approximately 8% of African-Americans will carry one sickle gene.

33. Heterozygote Advantage • Mutant allele has a high frequency despite reduced fitness in affected individuals. • Heterozygote has increased fitness over both homozygous genotypes e.g. Sickle cell anemia.

35. Multiple Origins of S Allele

36. Multiple Origins of S Allele • HpaI restriction fragment length polymorphism (RFLP) • S allele associated with 13 kb fragment originated in West Africa • S allele with 7.6 kb fragment originated elsewhere and probably more than once • Selective pressure for multiple origins • Malaria resistance among AS heterozygotes

37. Thalassemia: An imbalance of globin-chain synthesis • Hemoglobin synthesis characterized by the absence or reduced amount of one or more of the globin chains of hemoglobin. • α-thalassemia • β-thalassemia

38. Varius forms of α-Thalassemia

40. Hb Bart’s (hydrops fetalis)

41. β-thalassemia:underproduction of the β-chain. ●β-thalassemia trait (β+/ β orβ0 /β) : .asymptomatic (β+:reduced;β0:absent) ●β-thalassemia intermedia (β+/ β+ ): . moderate anemia ●β-thalassemia major (β0 /β0 orβ+/β0 or β+/ β+ ) : . severe anemia during the first two years of life . hepatosplenomegaly . growth failure . jaundice . thalassemic facies

43. Thalassemias can arise in the following ways: • One or more of the genes coding for hemoglobin chains is deleted. • 2. A nonsense mutation that produces a shortened chain. • 3. A frameshift mutation that produces a nonfunctional chain. • 4. A mutation may have occurred outside the codingregions.

44. β-globin gene andβ-thalassemia

45. Thalassemias: Pathological Effect of Globin Chain Excess • Thalassemia • Spleen from -thal homozygote • Excess -chains form a Heinz body inclusion (seen also in -thal) • Inclusions • Removed by reticulo-endothelial cells • Membranes damaged • RBCs destroyed

46. Phenotypic Consequences of Allelic Interactions and Modifier Genes

47. Allelic Interactions • Relatively high frequency of alleles in populations • Example • thalS • If 0 then may be like sickle cell disease • If + then may be much milder

48. Modifier Genes: Locus Interactions • These would involve mutations in the  and  loci • Example • -thal homozygotes who also inherit an -thal allele may have less severe -thalassemia, due to less imbalance or reduced excess -globin chains

49. The most famous authority on Hb: David Weatherall

50. Part II. Newborn Screening for the Hemoglobinopathies