genetics of the hemoglobinopathies & newborn screening for the hemoglobinopathies
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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, 7 th Ed (双语版, 2009 ) ● Pages 237-257; - PowerPoint PPT PresentationTRANSCRIPT
Genetics of the Hemoglobinopathies & Newborn Screening for the
Hemoglobinopathies
Tel : 13105819271; 88208367 Office: A705, Research Building
2013/03
Required ReadingThompson &Thompson Genetics
in Medicine, 7th Ed (双语版, 2009)
● Pages 237-257; ● Clinical Case Studies:
37. Sickle Cell Disease 39. Thalassemia
Learning Objectives1. 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 α- or 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.
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
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 22
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
Hb Structure Proportion in normal adult
(%)
Embryonic Gower I
Gower II
Portland I
ζ2ε2
α2ε2
ζ2γ2
-
-
-
Fetal F α2γ2 <1
Adult A 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 African-Americans 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
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.
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
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
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 follow-up for the hemoglobinopathies
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
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 cost-effective.
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
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
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
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