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 2014/03

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. 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. 先天性代谢缺陷（Inborn Error of Metabolism）：Any of a group of congenital disorders caused by an inherited defect in a single specific enzyme that results in a disruption or abnormality in a specific metabolic pathway.

6. 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.

7. 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)

8. 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

9. 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

10. 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

11. 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

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

14. Developmental Expression of Globin Genes and Globin Switching

16. 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

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

18. 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

19. Clinic Disease: Influences of Gene Dosage and Developmental Expression • Dosage • 4 - vs. 2 -globin alleles per diploid genome • Therefore, mutations required in 4 -globin alleles compared with 2 -globin alleles 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

20. The normal human Hbs at different stages of development

21. Genetic disorders of Hb 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.

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

25. 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.

26. 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.

28. 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

29. Varius forms of α-Thalassemia

31. Hb Bart’s (hydrops fetalis水肿胎儿)

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

34. 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.

35. β-globin gene andβ-thalassemia

36. Part II. Newborn Screening for the Hemoglobinopathies

37. Learning Objectives • To review the evolving principles of newborn screening • To examine newborn screening (NBS) for the hemoglobinopathies • To understand the appropriate response to a positive hemoglobinopathy NBS • To appreciate the role of clinical follow-up for the hemoglobinopathies

38. Population-Based Screening

39. Genomic Medicine • Principles • Predictive • Preventive • Personalized • Change from current paradigm with emphasis on acute intervention • Will rely on strategies from preventive medicine and public health

40. Genetic Screening • Population-based approach to identify individuals with certain genotypes known to be • Associated with a genetic disease, or • Predisposition to a genetic disease • Disorder targeted may affect • Individuals being screened, or • Their descendents

41. Objective of Population Screening • To examine all members of the population designated for screening • Carried out without regard for family history • Should not be confused with testing for affected individuals or carriers within families ascertained because of a positive family history

42. Genetic Screening • Important public health activity • Will have increasingly significant role with availability of more and better screening tests for • Genetic diseases • Diseases with an identifiable genetic component • Critical strategic hurdle for implementation • Venue in which to capture 100% of target population

43. Principles of Newborn Screening (NBS)

44. NBS • Public health governmental programs • Population screening for all neonates • Intervention • Prevents or at least ameliorates consequences of targeted disease • Cost-effective • Controversial • Not simply a test, but a system

45. Criteria for Effective NBS Programs • Treatment is available. • Early institution of treatment before symptoms become manifest has been shown to reduce or eliminate the severity of the illness. • Routine observation and physical examination will not reveal the disorder in the newborn – a test is required.

46. Criteria for Effective NBS Programs • A rapid and economical laboratory test is available that is highly sensitive (no false- negatives) and reasonably specific (few false-positives). • The condition is frequent and serious enough to justify the expense of screening; that is, screening is cost-effective.

47. Criteria for Effective NBS Programs • The societal infrastructure is in place • To inform the newborn’s parents and physicians of the results of the screening test, • To confirm the test results, and • To institute appropriate treatment and counseling.

48. Evolving NBS Criteria • Treatment available – Not always • Example: Tandem Mass Spectrometry (MS/MS) • Analogy: Childhood cancer (75% survival) and protocol-driven iterative improvements • Pre-symptomatic treatment effective – No • Example: For rarer hemoglobinopathies may not have accurate knowledge of natural hx

49. Evolving NBS Criteria • Clinical ascertainment not effective, so test required – Not always • Example: G6-PD deficiency and kernicterus（核黄疸） • Problem: Clinical ascertainment is never 100% • Rapid and effective lab test available – No • Example: Severe combined immunodeficiency (SCID) • Problems: Limited federal funding for test development until recently, and low cost and margin limit corporate interest

50. Evolving NBS Criteria • Screening is cost-effective – Not always • Examples: All but PKU and congenital hypothyroidism • Problems: Standard not required or met for adult-onset disorders • System infrastructure in place – Variable • Example: Practitioner- and state-based • Problems: Some states fund only the test and not the follow-up, and sub-specialists not available in every state