Hemoglobinopathies

Barbara Karczeski, MS DNA Diagnostic Lab Board Review Lecture Series 2007

Agenda

Brief overview of hemoglobin The globin genes The Thalassemias Structural hemoglobinopathies Testing

Hemoglobin

Delivers oxygen to the cells Tetramer (4 subunits 2 A and 2 B) plus Heme groups A = Alpha like genes and pseudogenes (Chromosome 16); , , B = Beta like genes and pseudo genes (Chromosome 11); ,,,

The Globin Genes

Hemoglobin structure

A B

B A

heme

Heme

Globin

Hemoglobin Type Adult Fetal Embryonic

Name A A2 F Portland Gower 1

Components 22 22 22 22 22

Gower 2
Abnormal H Barts

22
4 4

Hemoglobins in normal adults

A B A G G A A D D A

B A

HbA 98%

HbF ~1%

HbA2 <3.5%

Hemoglobinopathy
definition

An inherited mutation of the globin genes leading to a quantitative or qualitative abnormality of globin synthesis

Thalassemia - Defined

A family of genetic anemias characterized by a reduced rate of production of 1 or more globin subunits of hemoglobin (Hb) Symptoms are caused by the deleterious effects of the normally produced subunits that are now in excess

Structural variant - Defined

Abnormal globin protein that is produced at a normal rate, with varying consequences Oxygen affinity, stability and function

Normal

Normal

Thalassemia

Normal

Structural Variant

- and -thalassemia
Alpha Thalassemia Deletions of alphaglobin gene(s) Symptoms can begin in fetal life Complicated inheritance 4 alpha genes
Beta Thalassemia Nonsense, splice and frameshift mutations in beta-globin gene Symptoms begin in infancy/childhood Simple AR inheritance; genotype-phenotype correlation

Structural Variants

Missense mutations in any globin gene that cause amino acid substitutions Symptoms begin in childhood Simple AR inheritance, though the sheer number of possible combinations makes it a more complex issue.

Some terminology

Thal minor and Thal trait carrier state; generally asymptomatic Thal major affected, full blown disease Thal intermedia somewhere in between stay tuned

Beta Thalassemia

-thalassemia

One of the most common single gene disorders in the world Occurs worldwide, but more common in SE Asia, Mediterranean, India, Central/Northern regions of Africa, Middle East, Caribbean Carrier frequencies 1/100 to 1/2 Worldwide frequency: 1-2%

Pathophysiology

Excess alpha chains precipitate and form inclusion bodies that associate with the RBC cell membrane Cause membrane damage and shortened cell survival Large scale destruction of precursor cells in bone marrow Decreased B production causes increased production and an elevated A2 (22)

Normal

Carrier

Affected

Factors that affect severity

Specific B-globin mutations inherited Co-inheritance of alpha thalassemia Fetal hemoglobin production

Clinical Findings: Carrier State

Generally asymptomatic May have mild anemia (weakness, pallor, mild unexplained fever) Low MCV (measurement of cell size), elevated A2 Can see some bone marrow changes Anemia worsens during pregnancy

Identification of the Carrier State

Important for risk assessment and prenatal diagnosis Also important to avoid treating with iron

Affected Individuals Presenting Symptoms

Failure to thrive Anemia Pallor Feeding difficulties Diarrhea Recurrent infections Abdominal enlargement

Clinical Symptoms Untreated Child

Appear prior to age 10 Variable anemia Hepatosplenomegaly Recurrent infections Spontaneous bone fractures Extra-medullary hematopoesis Gallstones Leg ulcers

B-thalassemia Intermedia?

Milder form of B-thal Transfusion requiring (to aid quality of life); not transfusion dependent More likely to have B+ mutations than B0 mutations A clinical classification more than a disease

Treatment

Transfusions of packed RBC as needed (up to every 3-4 weeks) Iron chelation therapy (desferroxamine) Splenectomy Vitamin Supplementation Prevention of Infection Psychosocial issues of chronic disease Bone marrow transplant functions as cure

Treatment - Risks

Blood born infections (HIV, Hepatitis, etc) Transfusion reactions (errors) Failure of compliance with chelation regimen

Clinical Symptoms - Treated

Usually due to iron overload Liver disease Endocrine dysfunction (diabetes, growth retardation, failure of sexual maturation, Cardiac complications (infections, right sided failure) No direct effect on behavior or intelligence

Genetic / Molecular Aspects

Mutations cause decreases in chain production, not normal production of abnormal B-globin Nonsense, small insertions; splice mutations Some deletions

Types of B-globin mutations

B0 No B-globin chains are produced Nonsense - Nt169C>T Frame shifts Nt176delCTTT Destruction of splice site Nt142+1G>A
B+ - some beta chains produced Decreased splice efficiency Nt142+6T>C Cryptic splice site Nt365+654C>T

Types of B-globin mutations

B+mild subset of B+ with higher rates of Bglobin production Poly A mutation Nt494+110T>C Promoter mutations Nt -88C>T Bsilent - ??, some promoter, deep intronic, 5 or 3 UTR mutations Nt-101C>T, 5UTR+10delT

Alpha Thalassemia

Alpha thalassemia
AA / AA
AA / A AA / - A-/AA-/---/--

Normal
Mild microcytosis (Silent Carrier) Mild microcytosis (Trait or Carrier) (cis vs trans) Hemoglobin H diseaseclinically variable Hydrops Fetalis (Alpha Thal Major)

Since alpha chains are synthesized in fetal life, symptoms of AlphaThalassemia Major begin in fetal life

Hydrops Fetalis: Ultrasound

Hydrops Fetalis: Path photo

Death occurs in utero or shortly after delivery About 17% of these fetuses also have developmental abnormalities Maternal complications: anemia, preeclampsia, hemorrhage, sepsis, retained hydropic placenta

Pathophysiology

Excess of Beta chains leads to Hb Barts (gamma 4) and Hb H (beta 4) These Hbs have high O2 affinity and are unstable useless! Lead to early RBC death Hb H precipitates out and damages membranes

Hb H Disease

Presence, in a person more than 6 years old, of significant amount (1-2%) of Hb H Sx variable; but can include acute or chronic microcytic anemia, hepatosplenomegaly, mild bone changes, gallstones, leg ulcers, venous thrombosis, transient aplasia May have Hydrops Fetalis

Management

Hb H Hydrops Fetalis
Blood transfusions, iron chelation may have sequelae of hypoxia (congenital anomalies; developmental delay)

Hb H Disease
Folate supplementation Splenectomy Transfusions as needed

Molecular / Genetic Aspects

Most common molecular cause is one of several deletions of one or both alpha genes of a chromosome Some point mutations described (Hb Constant Spring) Hb variants of alpha also described

Alpha Deletions

Alpha Deletions

Deletions named according to their size

3.7kb; 4.2kb; 5.2kb; 20.5kb

Deletions named according to their origin

Med, Fil, Thai, SE Asian

Double gene deletions more common in SE Asia

These are the populations at higher risk for severe alpha-thalassemia

Single gene deletions more common in African, Indian, Caribbean populations

Not at usually at risk for severe disease

Non-Deletional Alpha Thal

Usually represented as /T
May be more severe than deletional form there is an abnormal product made; can cause more cellular damage Most common is Hb Constant Spring

Other forms of Alpha-Thalassemia

ATR-16: contiguous gene deletion syndrome, diverse clinical features ATR-X: mutations in an X-linked gene having some regulatory effect on alpha gene expression. Sx include hypotonia, seizures, short stature, skeletal and GU abnormalities, cardiac defects

Structural Hemoglobin Variants

More than 750 variants described by the late 90s Theoretically, could have multiple missense mutations for every amino acid Alpha chain variants are individually rare or of little clinical consequence

Named for how they migrate on electrophoreses: Hb E Named for place in which they were discovered: Hb Rio Grande Both: Hb D-Iran; Hb D-Ibidan; Hb D-Los Angeles

Some rare, only described in a single family Some very common and occurring at high frequency in certain populations
Hb E in SE Asia Hb S and Hb C in Africa

Hb C

Found mainly in West Africa Lower oxygen affinity than Hb A Crystallizes, reduced deformability of RBC; hemolysis Milder than Hb S

Hb E

The most common Hb variant in the world Nearly exclusively in SE Asia and India Has Beta thal properties (An amino acid substitution AND activates a cryptic splice site) Homozyotes extremely mild Compound het with Beta thal can be very severe

Sickle Cell Anemia

What is Sickle Cell Anemia?

Disorder affecting hemoglobin (HBB gene) Inherited as autosomal recessive pattern Characterized by low number of red blood cells Life span of red blood cell -Normal cells 120 days -Sickle cells 10-15 days Affects millions through the world: Sub-saharan Africa, Spanish speaking region, Mediterranean countries Most common inherited disorder in USA - 70000 to 80000 American 1 in 500 African- American 1 in 1000 to 1400 Hispanic-American

Symptoms of SCD

Hand-foot syndrome Fatigue, paleness, and shortness of breath. Pain that occurs unpredictably in any body organ or joint. Eye problems. Yellowing of skin and eyes. Delayed growth and puberty in children and often a slight build in adults.

Complications: Infections. Stroke Acute chest syndrome.

Pathophysiology
1.

2.

Hemolysis Occlusion of blood vessels

a. b. c. d. e. f. bone (painful crisis) lung (acute chest syndrome) brain heart spleen (Acute splenic sequestration) hands (dactylitis in children)

Sickle Cell Anemia blood film

Sickle Cells

Erythroblasts HowellJolly Body

Normal vs. Sickle Red Cells

Normal cell

Normal cell release oxygen and maintain it biconcave shape

Deformation of the cell due to the polymerization of hemoglobin in sickle red cells when they release oxygen
Sickle cell

Sickle Cell Anemia EM of red cell showing tactoids

Fibers of Sickle Hemoglobin

Fibers of Sickle Hemoglobin cross section

Nature of the problem

Substitution of valine residue for glutamic acid at position 6 in the beta-subunit of hemoglobin.

HB A

HB S

Adenine

Thymine

Testing
Hemoglobin electrophoresis: Hemoglobin electrophoresis is a test that measures the different types of hemoglobin in the blood. The presence of significant levels of abnormal hemoglobins may indicate:
Hemoglobin C disease Rare hemoglobinopathy Sickle cell anemia

Modulators of SCD

Fetal Hemoglobin

High level of fetal hemoglobin protects the infants during the first year and declines between one and two year of age. Patient with hereditary persistence of fetal hemoglobin (HPFH) have few symptoms. In HPFH, Hb F usually comprise 20% of the hemoglobin in the erythrocytes.
Fetal Hb disrupts the polymerization of dexoxyHb-S

Modulators of SCD (Cont.)

Alpha-thalassemia

The rate of SCA is lower in people who also have two-gene deletion The mechanism by which alpha-thalassemia ameliorates red cell destruction is unknown

Sickle Cell Anemia - treatment

Opiates and hydration for painful crises Transfusion for serious manifestations (eg stroke) Bone Marrow Transplantation: can cure Sickle Cell Disease The optimal time of transplantation is during childhood

Treatment

Hydroxyurea: Used mainly to enhance fetal hemoglobin production -Blocks DNA synthesis and cell division by inhibiting ribonucleotide reductase - Induce fetal Hb production -Reduces the number of dense cells and irreversibly sickled cells in the circulation

Experimental Therapies

Erythropoietin: stimulate red cell production and increase Hb F level Clotrimazole: reduces the number of dense cells and the number of irreversibly sickled cells in patients with sickle cell disease Nitric oxide: An inhaled gas that can reduce the polymerization tendency of sickle hemoglobin Gene therapy: replacing the defective gene with a normal one

Sickle Cell Trait

Heterozygous state for Hb S (Hb AS) No serious clinical consequences Sudden death during intensive training Hematuria, isosthenuria (renal papillary necrosis)

Hereditary Persistence of Fetal Hemoglobin

Significant Hb F production into adulthood Molecular heterogeneity

Several deletions of all or part of the delta and beta globin genes Point mutations in delta globin Mutations in genes outside the beta gene cluster

Frequency and Distribution of Hemoglobinopathies

Ethnic Group Mediterranean African American Non-Hispanic Caribbean West African Hispanic Caribbean Mexican/Central Amer. Asian SE Asian India/Pakistan Middle Eastern

Beta Thal 1/20-30 1/75 1/50-75 1/50 1/75 1/30-50 1/50 1/30 1/30-50 1/50

Alpha Thal 1/30-50 t 1/30 t 1/30 t 1/30 t Variable Variable 1/20 c >1/20 c Variable Variable

Ethnic Group Mediterranean African American Non-Hispanic Caribbean West African Hispanic Caribbean Mexican/Central Amer. Asian SE Asian India/Pakistan Middle Eastern

HbS 1/30-50 1/12 1/12 1/6 1/30 1/30-200 Rare Rare 1/50-100 1/50-100

HbC Rare 1/50 1/30 1/20-30 Rare Rare Rare Rare Rare Rare

Ethnic Group Mediterranean African American Non-Hispanic Caribbean West African Hispanic Caribbean Mexican/Central Amer. Asian SE Asian India/Pakistan Middle Eastern

Other Hb D,G Lepore O, D O, D O, D Variable J, E E E D, O, E D, O, E, J

Hemoglobinopathies and Malaria

Why is thalassemia so common?

The thalassemias and other hemoglobinopathies are so common that, unless you want to entertain a ridiculously high new mutation rate, there must be some selection for the carrier state The Malaria Hypothesis

The Malaria Hypothesis

When mapped out, the global distribution of malaria and populations with high carrier frequencies for hemoglobinopathies are essentially the same Being a carrier protects you from Malaria so you have an evolutionary advantage over a non-carrier. How does this work? Hasnt been pinned down yet, but theories abound

Hb S reduces oxygen tension in cells retarding growth of the parasite A thal confers susceptibility to a milder form of malaria; once contracted, the patient has immunity to the more severe form and increased survival B-thal - ???; possibly a role in decreasing cell adhesions in the deadly cerebral form of malaria

Screening and Testing

Screening

CBC to look for MCV (mean corpuscular volume) and MCH (mean corpuscular hemoglobin)
Will be reduced in both thalassemias (microcytic anemia)

Hb Electrophresis to look for A2: Will be elevated in B-thal, normal on Athal. May also be elevated in HbS

Screening

Hb Electrophresis may also identify abnormal hemoglobins

Structural Hb Variants Some Hb Barts or Hb H Wont find unstable variants except in exceedingly small quantities may be missed

Iron studies to rule out iron deficiency

Mutation Identification

Not usually a diagnostic tool. You can narrow down the diagnosis well with non-molecular blood testing, smears, etc. Necessary for prenatal diagnosis Helpful in estimating severity

Just when you thought you understood Hemoglobinopathies

Other causes of a + carrier screen (low MCV and high A2) Multiple hemoglobinopathies can interact and alter the results of screening; produce lots of undescribed phenotypes Non-classic: B: mutations in LCR, upstream promoter; A: point mutations, novel/rare deletions Other Deletions (thalassemia, Hb Lepore) Dominant B-thal (Irish family) Thal plus structural hemoglobinopathies (HbS, HbC, HbE and any one of the >300 other abnormal Bglobins)