Mahmoud Sirdah, PhDAssociate Professor of Blood Pathophysiology

Al-Azhar University Gaza

Why Genetics is important in Gaza?

Overall Consanguinity is high 39.9 %Consanguineous marriages are significantly higher in

semi-urban areas (41.6%) than in urban areas (39.1%).Compound consanguinity (two generation) is 20.7%.The average age of those with:first-cousin marriages 22.4 ± 4.4 years) second-cousin marriages (24.3 ± 6.1 years) non-consanguineous (26.5 ± 8.2 years).

• Thalassemias reflect quantitative abnormalities in Hb production.

•The term thalassemia is derived from the Greek words: thalassa (sea) and eima (blood), so it means sea in the blood.

•Also known as Cooley's Anemia, Mediterranean Anemia

• Heterogeneous proteins packed inside the RBCs.

• About 270 millions of molecules of hemoglobin/ RBC.

• Tetrameric in structure, made up of two different pairs of globin polypeptide chains:

• 2 like (141 a.a), controlled by genes on chrs 16• 2 like (146 a.a), controlled by genes on chrs 11.

• This heterogeneity is expressed at all stages of development, during which different hemoglobin forms are synthesized during human life.

Hemoglobin Type Structure Developmental Stage

Hb Gower 1 2 2embryo

Hb Gower 2 2 2embryo

Hb Portland 2 2embryo

HbF 2 2fetal and adult

HbA2 2 2adult

HbA 2 2adult

Heterogeneity of Human Hemoglobin Heterogeneity of Human Hemoglobin

• After birth, the adult and globin chains begin gradually to replace the -globin chain.

• This results in a major switch from HbF (2 2) to the adult hemoglobin HbA (2 2) synthesis.

• This switch occurs about the time of birth and ends 6 -9 months later.

• After the HbF (2 2) to HbA (2 2) switch, 97-98% of the hemoglobin is HbA, while HbA2 (2 2) accounts for approximately 2%.

• Small amounts (1%) of HbF are also found in adults blood.

Hb F (Hb F (22 22) to Hb A () to Hb A (22 22) Switch) Switch

• The inherited disorders of hemoglobin are known as hemoglobinopathies:

• QuantitativeQuantitative: defect in the rate of production of one or more of the globin chains, known as thalassemias.

• QualitativeQualitative: structural change in the globin chain, known as Hb variants which alter the Hb stability or function of Hb, e.g. HbS, HbC, HbD, HbE, etc..

• A group of recessively autosomal inherited conditions, leads to hereditary anemias.

• Characterized by decreased or absence of synthesis of one or more polypeptide chains (α-like or β-like ) that form Hb.

• The commonest genetic diseases of mankind.

•WHO estimates : 1.5 % of the world’s population might be carriers of β-thalassaemia, 60,000 thalassemic births /year

• Affect men and women equally.

• Thalassemias have been encountered practically in every racial group and geographic location of the world.

• Most common in the Mediterranean region and equatorial or near equatorial regions of Africa and Asia.

• The high frequencies of thalassemia are found in those areas historically afflicted with endemic malaria

Worldwide Distribution of Thalassemias

• Thalassemias are classified according to which particular globin chain(s) is produced in a reduced amount.

• The main types which have now been defined with certainty are the , , , , , and -thalassemias.

• and -thalassemias are the most common classes

• The most important type of thalassemias.

• So common, widely distributed and result in severe anemia in the homozygous and compound heterozygous states.

• High prevalence in a broad belt extending from the Mediterranean basin and parts of north and west Africa through the Middle East and Far East.

• In some regions of Italy, Greece, Cyprus, Sardinia and South Eastern Asia carriers frequencies ranges between 15-20 %

• -thalassemia: defect in -chain production alone, is a due mainly to point mutations, and it is the most common type.

• -thalassemia: reduced/ absence of and chain synthesis resulting usually from deletions in the and globin genes, , it is a less common type.• -thalassemia and -thalassemias : are rare forms of thalassemia which results from a long deletion in the -like globin cluster, and so removing the, , and globin gene

The three main clinical phenotypes (Severity Classification) in subjects with β thalassaemia are major, intermedia, and minor•-Thalassemia major, Cooley’s anemia or homozygous -thalassemia, double heterozygous -thalassemia is a severe, transfusion dependent disorder• -thalassemia intermedia: is applied to a less severe clinical phenotype in which significant anemia occurs but regular transfusion therapy is not required• -thalassemia minor or trait or heterozygous -thalassemia is the symptomless carrier state.

• -Thalassemia is very heterogeneous at the molecular level, with more than 400 different mutations (most of which are point mutations, large deletion mutations are found in rare cases) identified that causes the disease.

• The disease arise from mutations that affect every step in the pathway of -globin gene expression: transcription, mRNA processing or mRNA translation

•The basic molecular defect in -thalassemia (HBB gene) results in either absence (0) or reduced (+) beta chain production.

•Consequences of (0) or (+) beta chain production. :

• Reduced production of Hb A (2 2).

• Imbalanced globin chain synthesis : . • The excess chains are unstable and precipitate in BM RBC precursors, giving rise to a large intracellular and subsequently intramedullarly (ineffective erythropoiesis) and extramedullarly destructions of RBC become the norm.

•Anemia of -thalassemia results:

• Hb production• Ineffective Erythropoiesis• Shortened RBC survival

• Without treatment/ management, β-thalassemia major is lethal in early childhood,

• Treatment/ management with a regular transfusion program and iron chelation allows for normal growth and development and extends life expectancy into the 3rd to 5th decade.

• Most deaths are caused by the cardiac complications of iron overload

Before 1997• About 300 -Thalassemia major patients •All efforts were directed toward providing the patients with blood transfusion and iron chelating agents.

• -Thalassemia new births 15-25/ year.

•No estimation of the prevalence of the gene in the population.

•Knowledge and awareness is very limited or lacking among the population

• 1997 Establishment of the first official Palestinian Thalassemia foundation that take care of all social and medical aspects of the thalassemic patients and their families.

• 1997-1998 first screening study that estimated the prevalence (4.3%, range 3.3-7.9%) of the -Thalassemia in Gaza Strip.

•1999 -2000, a series of nationwide activities that included:• Public lectures that covered people at urban, rural,

refugee camps as well as other remote communities. • Media awareness programs and brochures• Scientific lectures to students at high School and

universities • Discussions and meetings with national policy

makers.• TV interviews with geneticist, hematologist together

with affected families and patients.

•September 2000, the superior legitimate judge adopted the implementation of the premarital tests for -thalassemia as an obligatory step before any proposed couple can be issued with a marriage certificate.

2010. Molecular characterization of thalassemia in Gaza Strip

We screened patients and carriers IVS-I-110 G>A IVS-I-1 G>AIVS-I-6 T>C Cd39 C>TCd 37 G>AThree novel HBB variants were

discovered by direct sequencing of the gene:

5′ UTR-50 (−/G) 5′ UTR-43 C>TIVS-II-26 T>G.

• The first year of the implementation of obligatory tests was considered to be the most problematic.

• After that, this process is easier and the obligatory tests procedure is going very smoothly.

•About 10,000 couples are screened /year.

• Most ( 95 %) couples at risk (carries for thalassemia) separated and selected another partner (not carrier).

• The relative success of the Palestinian thalassemia prevention program is clear through the actual and significant chronological reduction in the number of -thalassaemia major births to almost zero in the last years.

2009200820072006200520042003200220012000

Year

14

12

10

8

6

4

2

0

Nu

mb

er o

f th

alas

sem

ic b

irth

s p

er y

ear

0

1

7

5

4

10

6

10

13

15

• Mature RBC has no mitochondria.

• ATP is generated by anaerobic glycolysis.

• Glucose is anaerobically catabolize to lactic acid.

• This pathway is known as (Embden-Meyerhof pathway).

• Other shunts or pathways are also directly connected to Embden-Meyerhof pathway:• Hexose-Monophosphate Shunt (pentose phosphate pathway.• Rapoport-Luebering shunt

• Production of ATP molecules (ATP is needed for Na+/K+ ATPase pump, maintain cell shape and flexibility (deformability)

• NADPH production through hexose monophosphate shunt (NADPH used to prevent accumulation of H2O2, this shunt is dependent on G6PD enzyme).

• 2,3 diphosphoglycerate (2,3 DPG) production (generated in Rapoport-Luebering shunt).

• NADH production.( Used by methemoglobin reductase to reduce MetHb to Hb)

•Under normal conditions, about 10% of the glucose utilized by RBC is catabolized via the hexose-monophosphate shunt.

• This shunt provides a critical protection against oxidative damage through generation of the NADPH NADPH which enables cells to counterbalance oxidative stress.• Since RBCs do not contain mitochondria, the HMS is their only source of NADPH; therefore, defense against oxidative damage is dependent on G6PD.

•G6PD deficient red cells are highly susceptible to oxidative damage.

• G6PD is the first and rate-controlling enzyme in the Hexose-Monophosphate Shunt .

• G6PD oxidizes glucose-6-phosphate to 6-phosphogluconate. NADP+, which serves as a cofactor in this reaction, is reduced to NADPHNADPH.

• NADPHNADPH is a cofactor for glutathione reductase, which then reduces oxidized glutathione (GSSG) to the reduced form (GSH) of glutathione.

• GSH serves as a cofactor for the enzyme glutathione peroxidase, which reduces various peroxides to water.

G6PD gene, is located near the telomeric region of the long arm of the X chromosome (Xq28)

• Consists of 13 Exons and 12 Introns, Exon 1 is noncodingExon 1 is noncoding. • Encodes 515 amino acids.• About 18.5 kilobases (kb) in length.• Intron 2 constitutes about 12 kb.• The significance of this large Intron 2 is unknown• Important for efficient transcription or for processing???

• The enzymatically active form is either a dimer or a tetramer of a single polypeptide subunit of about 59 kd.

• Because it is X-linked, • Males can be only:

• normal hemizygotes • or deficient hemizygotes.

• Females can be:• normal homozygotes,• deficient homozygotes• heterozygotes

• G6PD Mutations display the typical pattern of Mendelian X-linked recessive inheritance (crisscross pattern).

•Phenotype pedigree

• Severe G6PD deficiency is much more common in males than in females.

• Because of X-chromosome inactivation, G6PD heterozygous females exhibit mosaic somatic cells.

Class ISeverely deficient, associated with chronic non-spherocytichemolytic anemia

Class IISeverely deficient (1–10% residual activity), associated withacute hemolytic anemia

Class IIIModerately deficient (10–60% residual activity)

Class IVNormal activity (60–150%)

Class VIncreased activity (>150%)

• Almost all variants of the G6PD gene that result in enzyme deficiency affect the coding sequence.

• About 186 variants have been reported, most of which are single-base substitutions leading to amino acid replacements.

• In a few cases, two amino acid replacements are found.

• a few cases (8) of small in-frame deletions have been discovered.

• Only one mutation affecting splicing site has been discovered.

• No mutations have yet been reported in the promoter region

• G6PD) deficiency is the most common human enzyme defect, being present in more than 500 million people worldwide.

• The global distribution of G6PD deficiency is remarkably similar to that of malaria, providing a support to the so-called malaria protection hypothesis.

• G6PD deficient RBCs are more resistant to Malaria parasite Plasmodium falciparum malaria.

• Mutations within G6PD were identified and characterized by Sanger sequencing of the 13 exons using 3100 Genetic Analyser.

• Following biochemical evaluation:

• 65 out 80 (81.3%) hemolytic anemic children (60 males and 5 females, were found to be G6PD deficient.

• Hemolytic crisis in all G6PD deficient children was due to the ingestion of fava beans (either green or dried).

• Genetic analysis of G6PD deficient Palestinian children revealed three predominant and two less frequent mutations within the Gaza Strip population.

A novel missense mutation G6PD Gaza is characterized by a G > A transition at nucleotide c.536, which changes serine p.179 to asparagine

Molecular modeling and dynamics studies suggest that Gaza mutation affects key protein conformation regions, leading to decreased binding affinity of the enzyme for NADP+ due to general displacement of active site residues within the binding site

• we characterize the molecular heterogeneity of G6PD variants among Gaza Strip Palestinians, which shows a wide molecular diversity of G6PD deficient variants.

• Favism was the predominant cause of hemolytic anaemia in Palestinian children at Gaza Strip, representing 81% of patients admitted with hemolytic crisis.

• The association of these G6PD mutants with clinically significant hemolysis requiring hospital admission justifies nationwide programs of newborn screening for G6PD deficiencies