Transcript Clinical Case Studies

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
– 22
• Fetal: HbF
–
–
–
–
α22
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
– 22
– ≤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
– thalS
• 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