SAJAD AHMAD

Introduction
The inherited diseases of hemoglobin are the most common single-gene disorders About 7 % population of the world are carriers This group of diseases has a particularly high frequency in a broad belt extending from the Mediterranean basin, Middle East, The Subcontinent all the way to the islands of the Pacific

Introduction (contd)
First recognized by Thomas B. Cooley in 1925 Pathological changes first described by Whipple and Bradford in 1936

Globin Chains
Tetrameric structure HbA 22 HbA2 22 Adults HbF 22 Hb Portland 22 Hb Gower 1 22 Embryo Hb Gower 2 22

Globin genes

globin genes - Ch 11
Spread over 60 kb and arranged in the order of

5--G-A----3

globin genes - Ch 16
Arranged in the order of 5-'- 1-2-3

, and are pseudogenes

Classification

Clinical
Hydrops fetalis Four gene deletion -

thalassemia Thalassemia Major transfusion dependent, homozygous 0-thalassemia or other combinations of -thalassemia trait Thalassemia intermedia Thalassemia minor 0-thalassemia trait, +thalassemia trait, HPFH, -thalassemia trait, 0-thalassemia trait, +-thalassemia trait

Classification (contd)
Genetic

-Thalassemia
0 + Deletion (-) Non-deletion (T)

-Thalassemia
0 + Normal HbA2 Silent

Classification (contd)

-Thalassemia
()0
(A)0 ()+

Hereditary persistence of fetal hemoglobin

Deletion ()0 (A)0 Non-deletion Linked to -globin genes G+ A+ Unlinked to -globin genes

-Thalassemia -Thalassemia
0 +

-Thalassemia

-Thalassemia
The gene located on the short arm of Ch 11 Appropriate expression is dependent upon LCR located 5 25 kb upstream Complete absence of -globin chain 0 Largely variable reduction of -globin output + More than 200 mutations have been found most of which are point mutations Gene deletion is a rare cause of -thalassemia except for the Sub-continent

Classes of -thalassemia mutations

Transcription Deletions Promoters Splice junction Consensus sequence Internal IVS Cryptic splice sites in exons Cleavage and polyadenylation site CAP site

Processing of mRNA

Translation

Nonsense Frameshift Initiation site Exon 3 mutations Other unstable -chains

Post-translational instability

Non deletional -thalassemia

(1) Transcription mutations Promoter mutations
Involve the TATA box and CACCC box
Reduce the binding of RNA polymerase Reduce the rate of mRNA transcriptions by 20

30 %
Result in +-thalassemia and a mild

phenotype

5 Untranslated region mutations

This is 50-nucleotide region Mutations result in a mild phenotype

(2) Mutations Affecting mRNA Processing

Splice junction and consensus sequence mutations

Mutations of 5-GT- and 3-AG- completely

abolish normal splicing, thus 0-thalassemia Misspliced RNA cannot be translated Efficiency of normal splicing may be decreased by mutations within the consensus sequences immediately adjacent to the splice junctions e.g. mutations at position 5 of IVS-1 severe +-thalassemia phenotype

Cryptic site mutations in introns and exons

2 cryptic splice site mutations identified in IVS-1

and four in IVS-2 IVS-1-110 GA IVS-1-116 TG Severe +- or 0-thalassemia phenotype Mutations at codons 19, 26 and 27 result in abnormal hemoglobins e.g. cd 26 HbE (Glu Lys)

Poly (A) and other 3 Untranslated region mutations

The AAUAAA sequence represents a signal for

the cleavage and polyadenylation reaction. Polyadenylation is important in the stability of mRNA and mutations in this region affect the efficacy of translation resulting in +-thalassemia of mild severity

(3) Mutations Affecting mRNA Translation

Initiation codon mutations

The initiator codon ATG codes for methionine

and is a signal for starting translation 7 different point mutations identified 0-thalassemia

Nonsense mutations
Formation of stop codons TAA, TAG or TGA Premature interruption of mRNA translation 0-thalassemia Nonsense-mediated decay

Frameshift mutations
Insertion or deletion of one or a few nucleotides

alters the reading frame of the encoded mRNA starting at the site of mutation. The new reading frame results in a novel abnormal amino acid sequence and in a premature termination downstream. 0-thalassemia

(4) Post-translational stability

Nonsense mutation in exon 3 are not subjected to nonsense-mediated decay and hence abnormal mRNA is translated thus leading to the formation of long, unstable globin gene products This is the basis for dominant -thalassemia -thalassemia intermedia

Deletional -Thalassemias
Several deletions affecting only the -globin chain has been reported the most important of them is a 619 bp deletion removing the 3 end of the -globin chain and is common in Pakistan and India. Phenotype of 0-thalassemia with unusually high levels of HbF and HbA2 in heterozygotes Total deletion of -cluster result in lack of any globin production and hence in (G-A)0 thalassemia

-Thalassemia
Much less common than -thalassemia Some cases are due to deletions of - and - gobin genes, others are due to unequal crossing over between the homologous and - genes thus forming hybrid genes called Lepore and -genes called antiLepore The Lepore Hb contains N-terminal amino acid sequence of the normal -chain and the C-terminal sequence of normal -chain

Three variants of Hb Lepore have been described

Boston or Washington ( 87/ IVS-2-8)

Baltimore
Hollandia

Depending upon the point of transition from to sequence

()0-thalassemia results from different length deletions of the - and -globin genes

Based on the presence of one (G-)or both (G- and A-) genes and synthesis of only G- or both (G- and A-) globin genes, two groups of ()0-thalassemia have been identified
G(A)0 GA()0

Hereditary Persistence of Fetal Hemoglobin (HPFH)

Heterogenous group of diseases Little clinical importance but may change the phenotype of -hemoglobinopathies Some forms result from long deletions of globin gene cluster. Homozygotes have 100 % HbF Another type of HPFH results from point mutations in the promoter regions upstream from either the G- or A-globin genes which allows these to be active in adult life

The linked -genes also remain active and hence are called G+ and A+ HPFH The third type consists of low HbF and is called Swiss HPFH. Genetic determinant seems to be located on Ch 6

-Thalassemia Intermedia

The term is used to describe patients with the clinical picture of thalassemia which, although not transfusion dependent, is associated with much more severe degree of anemia than found in carriers

-Thalassemia Intermedia

Mild forms of -thalassemia

Homozygosity for mild +-thalassemia alleles

heterozygosity for two mild +thalassemia alleles Compound heterozygosity for a mild and more severe -thalassemia allele
Compound

Inheritance of - and -thalassemia

+-thalassemia with 0-thalassemia (- -/) or +-

thalassemia (- / or - /- ) +-thalassemia with genotype of HbH disease (- -/- )

-Thalassemia with elevated -chain synthesis

Homozygous -thalassemia with heterocellular

HPFH Homozygous -thalassemia with G or A promoter mutations Compound heterozygosity for -thalassemia and deletion forms of HPFH

Compound heterozygosity for -thalassemia and - chain variants

HbE/ -thalassemia Other interactions with rare -chain variants

Heterozygous -thalassemia with triplicated -chain genes () Dominant forms of -thalassemia Interactions of and ()+ or ()0 -thalassemia

The Pakistani perspective

The five most common mutations

IVS 1-5 (G-C) (Most common in South Pakistan)
Fr 8-9 (+G) (Most common in North Pakistan) del 619

Fr 41-42 (-TTCT)
IVS 1-1 (G-T)

These five constitute about 82 % of all the mutations

Phenotype-Genotype relationships
Remarkable phenotypic variability Molecular basis for this diversity partly understood Genetic modifiers of -thalassemia

Primary mutations Secondary reduce degree of imbalance Tertiary complications of disease

-Thalassemia
More common than -thalassemia Located in telomeric region of Ch 16 1 and 2 only differ in IVS-2 and in 3 noncoding region Level of transcription of 2 is two to three times more than 1 i.e. 2 produces more -globin than 1 The expression of -globin genes is controlled by the sequences in and around the structural genes and by a region located 40 kb upstream called Hypersensitive site (HS)-40.

Deletional -Thalassemia Nondeletional -Thalassemia

Deletional -Thalassemia
Common cause of -Thalassemia Mechanism

The

chains are embedded within 2 highly homologous regions extending approx 4 kb. 3 homologous subsegments (X, Y and Z) separated by non-homologous elements have been defined.

Reciprocal recombination between Z boxes which are 3.7 kb apart and between X boxes which are 4.2 kb apart gives rise to chromosomes with only one gene These are referred as 3.7-kb rightward deletion and 4.2-kb leftward deletion Based on the exact location within the Z box where the crossover took place, the 3.7kb deletion is further divided into 3.7 I, 3.7 II and 3.7 III

Deletions that remove all or part of the globin gene cluster including both genes and sometimes the embryonic gene result in 0-thalassemia 0-thalassemia also occurs from deletions of the -globin regulatory element HS-40 and twelve such deletions have been reported

Nondeletional -Thalassemia
Less common Constant Spring mutation is common in South-East Asia Majority of the nondeletional mutants so far reported occur in the 2 gene and have a severe effect on -globin gene expression Hb Constant Spring ( 142 TAA CAA, Stop Glu) results from change of stop codon to an amino acid resulting in very low amount of an -chain variant elongated by 31 amino acids

Phenotype-Genotype relationships

The homozygous states for the nondeletional forms of +-thalassemia often have a more severe phenotype than those for the deletion forms.