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Genetics of the Hemoglobinopathies & Newborn Screening for the Hemoglobinopathies 张咸宁 [email protected] Tel:13105819271; 88208367 Office: A705, Research Building 2013/03 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 Part I. Genetics of the Hemoglobinopathies Learning Objectives 1. To review the normal structure-function relationships of hemoglobin and expression of globin genes 2. To examine the hemoglobinopathies as disorders of hemoglobin structure, or αor β-globin gene expression 3. To explore the influences of compound heterozygosity and modifier genes on hemoglobinopathy phenotypes 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. The Effect of Mutation on Pr Function 1. 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. 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) 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 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 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 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 Gene cluster: A group of adjacent genes that are identical or related. Pseudogene: DNA sequence homologous with a known gene but is non-functional. Developmental Expression of Globin Genes and Globin Switching 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 βlike globin chains Human Hemoglobins: Prenatal • Embryonic – 22 • Fetal: HbF – – – – α22 Predominates 5 wks gestation to birth ~70% of total Hb at birth <1% of total Hb in adulthood Human Hemoglobins: Postnatal • Adult: HbA – 2 2 – chain synthesis increases through birth – Nearly all Hb is HbA by 3 mos of age • HbA2 – 22 – ≤2% of adult Hb – Consequence of continuing synthesis of chains 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 The normal human Hbs at different stages of development Stage in development Embryonic Fetal Adult Hb Gower I Gower II Portland I F A Structure ζ2ε2 α2ε2 ζ2γ2 α2γ2 Proportion in normal adult (%) <1 α2β2 97-98 α2δ2 2-3 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. There are >400 structural variants of normal Hb. 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. 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 AfricanAmericans will carry one sickle gene. 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. 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 Varius forms of α-Thalassemia Hb Bart’s (hydrops fetalis) β-thalassemia:underproduction of the β-chain. β-thal trait (β+/ β or β0 /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 ● Thalassemias can arise in the following ways: 1. 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 coding regions. β- globin gene andβ-thalassemia 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 reticuloendothelial cells – Membranes damaged – RBCs destroyed Phenotypic Consequences of Allelic Interactions and Modifier Genes Allelic Interactions • Relatively high frequency of alleles in populations • Example – thalS • If 0 then may be like sickle cell disease • If + then may be much milder 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 Part II. Newborn Screening for the Hemoglobinopathies Learning Objectives 1. To review the evolving principles of newborn screening 2. To examine newborn screening (NBS) for the hemoglobinopathies 3. To understand the appropriate response to a positive hemoglobinopathy NBS 4. To appreciate the role of clinical followup for the hemoglobinopathies Population-Based Screening 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 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 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 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 Principles of Newborn Screening (NBS) 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 Criteria for Effective NBS Programs 1. Treatment is available. 2. Early institution of treatment before symptoms become manifest has been shown to reduce or eliminate the severity of the illness. 3. Routine observation and physical examination will not reveal the disorder in the newborn – a test is required. Criteria for Effective NBS Programs 4. A rapid and economical laboratory test is available that is highly sensitive (no false- negatives) and reasonably specific (few false-positives). 5. The condition is frequent and serious enough to justify the expense of screening; that is, screening is costeffective. Criteria for Effective NBS Programs 6. 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. Evolving NBS Criteria 1. Treatment available – Not always • • Example: Tandem Mass Spectrometry (MS/MS) Analogy: Childhood cancer (75% survival) and protocol-driven iterative improvements 2. Pre-symptomatic treatment effective – No • Example: For rarer hemoglobinopathies may not have accurate knowledge of natural hx Evolving NBS Criteria 3. Clinical ascertainment not effective, so test required – Not always • • Example: G6-PD deficiency and kernicterus Problem: Clinical ascertainment is never 100% 4. 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 Evolving NBS Criteria 5. Screening is cost-effective – Not always • • Examples: All but PKU and congenital hypothyroidism Problems: Standard not required or met for adult-onset disorders 6. 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 Informed Decision-Making in NBS • NBS developed in state public health departments – “Public health imperative” • Informed dissent – Majority of states (all but two) • Informed consent debated for all genetic testing, but costly, time consuming to implement and too many will refuse • NBS represents the largest volume of genetic testing: 550,000 babies/yr in CA, each with a recommended core panel of 29 and secondary targets of 25 – >225M disease-tests/year nationwide Role for Federal Government in NBS System Oversight • All states and DC screen for PKU, congenital hypothyroidism, galactosemia and hemoglobinopathies, but that is the only disease-target uniformity • National agenda for NBS – Recommended by NBS Taskforce in 1999 – A specific agenda recommended by American College of Medical Genetics in 2004 NBS for the Hemoglobinopathies Hemoglobinopathy NBS • Originally designed for sickle cell disease • Utilizes hemoglobin protein analysis, e.g., – Electrophoresis – HPLC • Developmental expression of -globin gene originally required confirmatory testing at 3-4 months of age NORMAL NEWBORN NORMAL ADULT Hb F Hb A Hb F Hb A FAS S/ β+ Thal(FSa) DNA Follow-up for Hemoglobinopathy NBS • PCR-amplified DNA directly from initial NBS specimen • Reduced time to diagnosis for SCD by >50% from >4 to <2 months of age • Identified transfused infants with FAS NBS • Demonstrated DNA stable in dried blood specimens and now available for virtually all screened disorders Two-Tiered Screening • Carried out on initial dried blood specimen without need to recall patient for repeat specimen • Sickle Cell Disease – Protein phenotype – DNA genotype • General strategy in genetic screening to improve – Specificity – Cost-effectiveness Current Status of Hemoglobinopathy NBS • Sickle Cell Disease – As of 2006, all 50 states and the District of Columbia have universal NBS for SCD • Other Hemoglobinopathies – Highly variable and incomplete – Reasons • • • • Technical Financial Systems’ limitations Population demands Responding to a Positive Result in Newborn Screening System Response to a Positive Screen • Inform a followup center • Inform the physician of record to contact family and ascertain patient health • Inform family if primary physician cannot be ascertained • Obtain appropriate followup studies • Meet with family as appropriate, especially if followup test is confirmatory for a disorder Physician Response to a Positive Followup Test • Patient to specialty program as soon as acuity and psychology demand • Institute appropriate therapy • Make sure that family is appropriately educated • Make sure that family is appropriately supported psychologically • Outcome should be entered into data-base • Clinical care and outcome recording as appropriate