Hemoglobinopathies

What is the role of molecular diagnostics in healthcare?

Molecular diagnostics play an important role in the pillars of health care which include diagnosis of disease, detection of MRD and monitoring patients with AML, ALL, or CML

What are some widely used methods in molecular diagnostics in detection of MRD

PCR, MFC, NGS

How can molecular diagnostics help with therapy selection

They help predict the outcome prediction like state or cure or state or relapse

Which hemoglobinopathies are diagnosed with the help of molecular methods?

1. Thalassemias.

2. Structural hemoglobin variants.

3. Myeloid malignancies.

4. Lymphoid malignancies.

What samples are used in MRD detection in leukemia?

Bone marrow (BM) and leukocytes.

In what type of disease is MRD detection performed using lymph nodes and lymphocytes?

Lymphomas.

What conditions are included in the pathophysiology of hemoglobinopathy?

thalassemia and hemoglobin variant (mutant form of Hgb).

What do hemoglobin variants affect?

Globin chains.

What types of changes do these genetic disorders cause?

Quantitative or qualitative changes.

What is an example of a condition caused by quantitative changes?

Thalassemia.

What types of genetic alterations lead to quantitative changes in thalassemia?

Amino acid insertions, deletions, or mutations in the intervening sequences (introns).

What process is affected by mutants that alter RNA processing?

Splicing and processing of the primary mRNA transcript.

What is the consequence of these RNA processing defects?

Decreased globin chain production.

What imbalance occurs in quantitative changes such as thalassemia?

A decrease in β-chain and an increase in α-chain.

What causes qualitative changes in hemoglobin disorders?

Amino acid substitutions (point mutation).

Give examples of hemoglobin variants caused by qualitative changes.

HbS (E6V) mutation

HbC (E6K) mutation

HbE (E26K) mutation.

What do qualitative changes result in?

Structural hemoglobin variants that lose the ability to carry oxygen.

How is a hemoglobin molecule formed?

Two distinct globin chains (each with its individual heme molecule) combine to form hemoglobin.

What are the types of globin chains involved?

One chain is designated alpha; the second chain is called non-alpha (β, δ, γ).

What produces a complete hemoglobin molecule?

The combination of two alpha chains and two non-alpha chains (a total of four chains per molecule).

What is the structure of HbA?

HbA is a tetramer of α₂β₂.

What kind of disorder is thalassemia?

An inherited blood disorder.

What does thalassemia result in?

Abnormal formation of hemoglobin.

What are the two types of thalassemia?

Alpha thalassemia and beta thalassemia.

What makes up adult hemoglobin?

Adult hemoglobin is a tetramer made up of alpha globin and beta globin.

How many genes encode the alpha globin chain?

Four genes.

How many genes encode the beta globin chain?

Two genes.

What is the tetrameric composition of HbA₂?

HbA₂ is a tetramer of α₂δ₂.

What is the tetrameric composition of HbF?

HbF is a tetramer of α₂γ₂.

What causes α-thalassemia disease, trait, and silent carrier states?

They are due to deletion in the alpha globin.

What does the severity of α-thalassemia correlate with?

The number of α-globin gene deletions.

What is the phenotype of a person with one α-globin gene deletion?

Unremarkable phenotype.

What is another term for individuals with one α-globin gene deletion?

Silent carrier.

What condition results from two α-globin gene deletions?

α-thalassemia trait.

What condition results from three α-globin gene deletions?

HbH disease.

What are the clinical features of HbH disease?

Varied clinical and hematological features.

Can individuals with HbH disease reach adulthood?

Yes, they can make it to adulthood.

What condition results from four α-globin gene deletions?

Hb Bart’s hydrops fetalis. Very SEVERE

Is Hb Bart’s hydrops fetalis compatible with life?

NO

When does death usually occur in Hb Bart’s hydrops fetalis?

Death occurs in utero.

On which chromosome are the alpha globin genes located?

Chromosome 16.

What happens to beta or gamma chains when alpha genes are deleted?

There is an excess of beta or gamma chains = imbalance

What is formed as a result of the imbalance in globin chains?

Unstable tetramer.

What is an example of a disease with 4 beta chains forming an unstable tetramer?

Hb H disease.

What happens to HbF (α2γ2) in Hb Bart -Hydrops Fetalis?

No HbF is formed.

What does the fetus inherit in Hb Bart’s-Hydrops Fetalis?

The fetus inherits 2 chromosome 16, both lacking any alpha gene.

What forms as a compensatory mechanism for the lost alpha chains?

Excess gamma chain tetramer.

What is a property of the gamma chain tetramer in Hb Bart’s-Hydrops Fetalis?

It has a high affinity for oxygen.

What conditions result from the high oxygen affinity of the gamma tetramer?

Anoxia and severe hypoxia.

What are some clinical features of Hb Bart’s-Hydrops Fetalis?

Total body edema and congestive heart failure (CHF).

What causes Beta thalassemia?

Mutation in the beta globin gene on chromosome 11.

How many types of mutant alleles exist in Beta thalassemia?

Two types of mutant alleles.

What is the BO allele?

Absence of expression of the beta globin gene.

What forms when the BO allele is present?

Formation of α4 tetramers.

What happens to HbA when the BO allele is present?

No HbA is formed.

What is the B+ allele?

Reduced expression of the beta globin gene.

What happens to HbA in the B+ allele?

Reduced HbA.

What typically causes the B+ allele?

Promoter mutation.

What is the effect of the promoter mutation in the B+ allele?

Weak promoter activity (weak expression).

What is Beta thalassemia major?

Severe form of Beta thalassemia.

What is the genotype of Beta thalassemia major?

BO/BO genotype.

Is any functional beta chain globin produced in Beta thalassemia major?

No.

Is HbA present in Beta thalassemia major?

No HbA.

What does the absence of β-globin cause in Beta thalassemia major?

Formation of α4 tetramers that are highly toxic to the RBC membrane.

What is Beta thalassemia intermediate?

Intermediate form of Beta thalassemia.

What are the genotypes of Beta thalassemia intermediate?

BO/B+ or B+/B+ genotype.

How much beta chain globin is produced in Beta thalassemia intermediate?

Few beta chain globin.

How much HbA is present in Beta thalassemia intermediate?

Few HbA.

What is Beta thalassemia minor?

Less severe form of Beta thalassemia.

What are the genotypes of Beta thalassemia minor?

B/B+ or B/BO genotype.

Is functional beta chain globin produced in Beta thalassemia minor?

Yes, functional beta chain globin is produced from the normal allele.

Does Beta thalassemia minor contain HbA?

Yes, it contains HbA.

What type of changes are structural hemoglobin variants?

Qualitative changes.

What causes structural hemoglobin variants?

Creation of beta globin variants due to amino acid substitution.

Give an example of a typical amino acid substitution in structural hemoglobin variants.

Glutamic acid to valine (typical change in HbS).

What happens to the tetramer in structural hemoglobin variants?

The tetramer is unstable and easily dissociates.

Name some commonly seen structural hemoglobin variants.

HbS, HbE, HbC, HbD, and HbG variants.

What statuses do these variants have?

Carrier (trait) status and disease status.

What is an example of carrier (trait) status?

Heterozygote state (HbAS).

What is the genotype of the carrier (trait) state for HbS?

HbAS.

How does the heterozygote state (HbAS) occur?

Due to combination of variant allele hemoglobin (mutated HbS) and normal allele hemoglobin (HbA).

What effect does the mutation have on Hb in structural variants?

Hb loses its tetramer state (unstable), causing major RBC morphological changes (thus, sickle cell).

When does a structural hemoglobin variant result in disease?

If mutations occur in both β-globin subunits.

What is a homozygous mutation?

Inheriting similar variants in both β-globin subunits.

Give an example of a homozygous mutation causing disease.

Sickle cell anemia (HbSS).

What is produced in sickle cell anemia (HbSS)?

HbSS.

What causes the production of HbSS?

Homozygous expression of the variant allele.

What is a compound mutation?

Inheriting different variants in the β-globin subunits.

Give examples of compound mutations.

HbSC and HbC.

Which hemoglobinopathies are classified under sickle cell disease (SCD)

HbSE, HbSC, and HbSβ-thalassemia.

What causes these SCD variants?

Heterozygous expression of variant alleles.

Give an example of a child with a compound mutation.

Child with HbSC inherits HbS (E6V) mutation from father and HbC (E6K) mutation from mother.

How does a child get HbSC?

By inheriting a mutated allele from each parent.

What causes sickle cell anemia mutations?

Single nucleotide polymorphism (SNP).

What specific nucleotide change occurs in HbS?

Change from A to T, i.e., GAG to GTG codon of the beta globin gene.

What does the SNP in HbS lead to?

Amino acid substitution.

What is another name for HbS?

E6V.

What amino acid substitution occurs in HbS (E6V)?

Glutamic acid is replaced by valine at position 6.

What commonly causes HbS?

Homozygosity for HbS, with the mutated allele inherited from both parents.

How can sickle cell disease sometimes be caused other than homozygosity?

By compound heterozygosity.