Globin gene haplotype analysis in a ghanian population

Sifuma Andrew NjengaHEMIS 363677

Introduction

• Sickle cell disease is a major public health problem in several countries particularly in LEDC’s

• gives significant morbidity and mortality in particular with young children aged 1 to 3 years of age

• Life expectancy is also reduced with SCD sufferers having a life expectancy of 35 or less.

• Sickle cell anaemia is caused by inheritance of two Hb S alleles, which are caused by a mutation on the β Globin gene cluster

• Different haplotypes occur on the βs gene and they occur in different frequencies in different populations

• SCD include vasco occlusive episodes, aplastic Anaemia, chronic pain and Ischaemia

• The main objective of this study was to analyze dried blood samples for haemoglobin haplotypes

Haemoglobin: structure and function

• The haemoglobin (Hb) molecule is made up of four polypeptide chains

• Each polypeptide has a single haem group with a Fe atom serving as the O2 binding site

• molecule is held together in its quaternary structure by bonds between the opposite polar chains

Haemoglobin gene clusters- classification and expression

• The genes that code for haemoglobin in humans are found in two clusters on chromosome 16 chromosome 11

• The β-like gene cluster is located on the short arm of chromosome 11

• Contains five functional genes (€, Gγ, Aγ, δ and β)

• The γ chains are mainly coded in foetal life by Gγ and Aγ when they combine with α chains to form foetal haemoglobin (Hb F)

• βGlobin gene synthesis begins after birth.

Single nucleotide polymorphisms (SNP’s)

• commonly referred to as “snips” (SNP’s) • show a difference in a single (full name) (DNA)

nucleotide, where one nucleotide is replaced by another

• SNP’s occur throughout DNA and are normally exhibited approximately every 300 nucleotides

• 10 million (SNP’s) occurring on the human genome

• found on DNA in between functional genes • when they occur on a functional gene SNP’S

may have a role in gene abnormality and disease• SNP’s usually have no effect on health and

development • Studies conducted have found that they may

affect people’s responses to drug metabolism and the risk of developing diseases

Haemoglobin Abnormalities

• inherited disorders in the Globin chains when the haem group is in the normal state

• autosomal recessive abnormalities and common worldwide, particularly within the malarial regions in the southern hemisphere

• It is believed that some of the heterozygous carrier states with abnormal Hb afford protection against malaria

• When a haemoglobin abnormality causes a blood disorder it is known as a haemoglobinopathy

• Haemoglobinpathies are described as any blood disorder caused by defects in Globin gene chain synthesis

• most common type of haemoglobinopathies are the thalassaemias, and sickle cell anaemia

Haemoglobin S and Sickle cell anaemia

• one of the most common hereditary diseases in the world and mainly affects people whose ancestry is from sub-Saharan Africa

• shows autosomal recessive inheritance• The gene mutation causing the formation of Hb S

is: GAG GTG• results in substitution of valine (Val) for glutamic

acid (Glu) in the sixth position of the β- Globin chain (α2 /β2

6val).

• Hb S in sickle cell patients manifests itself by causing sickling in red blood cells

• Hb S is insoluble and crystallises under low O2

partial pressure• Sickled Red blood cells are less elastic to

movement inside capillaries. • Haemoglobin gives up oxygen more readily than

normal haemoglobin: the oxygen dissociation curve is shifted to the right.

Clinical complications of SCD

• Shortened life of RBC’s• Haemolytic anaemia• Ischaemia• Aplastic crisis• Organ failure• death

β-Globin cluster haplotypes

• There are 5 different haplotypes of the βs genea) Beninb) Bantuc) Cameroond) Senegale) Arab-indian• Arab-Indian Haplotype and Senegal Haplotype

exhibit less clinical symptoms than the other 3

Management of Haemoglobinpathies

• Health education• Screening programmes• Pharmacological therapy

Malaria and sickle cell disease

• Malaria is a disease caused by the parasite Plasmodium falciparum

• transmitted to people through the bite of the female anopheles mosquito

• HbAS genotype protects against malaria has been the subject of debate for more than 50 years

• thought to relate to the physical characteristics of HbAS erythrocytes

• in vitro inhibition of parasite growth in sickled RBC’s is shown.

Methodology

• Objectives1. Purify dried blood samples on FTA cards and

Amplify DNA SNP’S using RFLP with different primers specific for Hb gene cluster

2. Run amplified samples on Agarose gel using electrophoresis

• Conduct restriction enzyme digest on amplified samples and run the samples again on Agarose gel

• Create a Haplotype map of Ghanaian population

• Hypothesis “the population shall exhibit a higher

proportion of cameroon, bantu, benin haplotypes”

Restriction fragment length polymorphism analysis

• method normally used for diagnosis of haemoglobinopathies

• Polymerase Chain Reaction amplifies individual alleles on the dried blood samples

• restriction enzyme digestion shows the researcher which specific allele is present in each sample

• Hb S gene is associated to certain DNA structures by specific restriction endonuclease positions in the β-Globin cluster

• PCR products were treated by suitable enzymes and then Agarose gel electrophoresis will be used to separate the fragments

• The bands are stained with Ethidium bromide and can be visualized by ultra violet (UV) light box

• Different βs haplotypes are differentiated in RFLP by providing different profiles with restriction enzymes