Genetics of the Hemoglobinopathies & Newborn Screening for the

Hemoglobinopathies

张咸宁zhangxianning@zju.edu.cn

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

Part I. Genetics of the Hemoglobinopathies

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.

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 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

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 reticulo-

endothelial 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 follow-up 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 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

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 A

Hb F

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