Sickle Cell Gene Flashcards
What is hemoglobin?
A heme protein: a specialized protein with heme as a tightly bound prosthetic group
The heme group of hemoglobin reversibly binds oxygen (O2)
Hemoglobin is found exclusively in red blood cells (RBC s) where it transports O2 from the lungs to the capillaries of tissues
What is hemoglobin tetramer?
Made up of four subunits or two identical dimers (aB)1 and (aB)2. Each subunit is composed of a polypeptide chain
Explain the binds between hemoglobin bonds
For T conformation: Low-oxygen-affinity form IgG Hb. Taut, structure if deoxyhemoglobin:
Weak ionic and hydrogen bonds occur between two aB dimers in the deoxygenated state
String interctions, primarily hydrophobic, between a and B chains firm stable aB dimers
R conformation: High-oxygen-affinity form of Hb:
Some ionic and hydrogen bonds between two aB dimers are broken in the oxygenated state
Where are globin genes located?
The gene where the a-globin gene is located, is on the short arm of chromosome 16, while the B-globin gene cluster is on the short arm of chromosome 11
In addition to the adult form of Hb, encoded within the N Hb cluster are the Hb forms that substitute for the adult B Hb during the various stages of development
Summarize Globin gene Organization
a-globin gene family (chromosome 16)
Hemoglobins are formed by combinations of chains from each gene family
B-globin family (chromosome 11)
The two copies of the a-globin gene are designated a1 and a2. Each can provide a-globin chains that combine with B-globin chains
Summarize globin gene synthesis
a-globin and B-globin gene family members contain three exons(coding regions) separated by two noncoding introns
Leads to transcription, splicing in nucleus and then translation
Describe hemoglobinopsthies
Hemoglobinopwthies are a group of genetic disorders:
-production of a structurally Abnormal hemoglobin mol3cule, e.g. sickle cell anemia(Hb SS), hemoglobin C disease (Hb CC) and hemoglobin SC disease (Hb SC)
Synthesis of insufficient quantities of normal hemoglobin e.g. Thalassemias.
Rarely both
Hemogloinopwthies may have serious clinical implications
What is sickle cell disease ?
A group of inherited red blood cell disorders.
People with SCD have variant with hemoglobin, called hemoglobin S or sickle hemoglobin, in their red blood cells.
People who have SCD inherit two variant hemoglobin genes, one from each parent
Some for,s of sickle cell disease:
- hemoglobin SS
- hemoglobin SC
- hemoglobin SB^0 thalassemia
- hemoglobin SB+ thalassemia
- hemoglobin SD
- Hemoglobin SE
Explain hemoglobin S
Hemoglobin S(Hb S) is the variant form of the normal adult hemoglobin A(Hb A)
This ( Hb S) is due to a single nucleotide substitution (a point mutation)
-This results in an amino acid substitution in the B polypeptide (Glu6val)
Describe Glu6Val in HbS
In individuals who suffer from certain blood diseases like sickle cell diseases like sickle cell disease(SCD), the binding and subsequent transport of oxygen is compromised due to a single nucleotide mutation and eventually amino acid substitution
Summarize sickle cell anemia
Autosomal recessive anemia
The substitution of the polar side chain (Glu) with a nonpolar hydrophobic side chain (Val) results in polymerization of the deoxygenated form of Hb S in RBCs and subsequent precipitation of such polymers stiffen and distort the RBC into a sickle cell shape
- Sickle-shaped RBCs die prematurely, leading to shortage of RBCs (anemia)
- Sickled RBCs can also block small blood vessels, causing pain and ischemic death of nearby cells or organ damage
What are the signs and symptoms of SCD?
- tiredness and Lethargy (lack of energy)
- Acute and chronic pain in any body part
- priapism in males
- bone pain
- anemia
- ulcers
- aplastic crisis
- infection
- growth retardation, delayed sexual maturation, being underweight
- Hand foot syndrome: dactylitis-painful and swollen hands and/or feet in children
- Acute chest syndrome
- Stroke
- Renal dysfunction
Give the history of SCD
- SCD is worlds largest genetic disorder
- Mainly affects people of African, Caribbean, middle eastern, eastern Mediterranean and Asian origin
- Thousands of years old, but identified as a chronic condition in 1910 by Dr. Herrick who described the banana shaped cells found in the blood of a young Grenadian dental student (Walter Clement Nobel) studying at the University of Chicago
- Dr. Walter Clement Noel(1884-1916)
- The treatment of and research into SCD has progressed over the years
- The expectations and lives of many who struggle with SCD have been much improved
What are the managements for the SCD?
Prevent tissue deoxygenation
Promote proper hydration
Avoid sources of infection
Proper, balanced diet
Regular medical check up
Prompt medical attention when sick
How can SCD be treated?
Medication
Transfusion
Bone marrow transplantation
Gene therapy
Explain electrophoresis of Hb from lysed RBC
Hemoglobins are negatively charged and migrate toward the anode
Mobility of Hb S is altered during agarose gel electrophoresis at alkaline pH
-due to absence of negatively charged glutamate in the B-globin chain, making Hb S less negative than Hb A
Describe Moleculaar Detection of B^s-globin mutation
Targeted mutation analysis-
- ASO
- RFLP: mutation can be identified directly in DNA by use of other of 2 restriction endonucleases- Ddel or MstII. The. Nucleotide substitution alters a specific cleavage site recognized B6 each of these 2 enzymes
-PCR
Prenatal diagnosis
-FISH
Preimplantation genetic analysis
-PCR, FISH
DNA sequencing
Describe ASO probe detection of Hb S allele
A. DNA from a patient with sickle cell disease
Portion of the gene for the B^S-chain of hemoglobin S. DNA codes for valine (Val) instead of glutamate (Glu) in the sixth position of B-globin
Oligonucleotide probe hybridizes with a DNA fragment from the gene for the B chain of HbS
B. DNA that encodes Hb A
Oligonucleotide probe fails to hybridizes with the DNA fragment from the gene for the B chain of Hb A
Two samples of DNA from each individual are applied to the membrane
Explain RFLP Analysus for detection of Bs-globin mutation is Sickle Cell Anemia(SCA)
- The basis of the test is the recognition by restriction enzymes of specific sequences in DNA
- In the normal B-globin gene, the sequence of nucleotides that specifies amino acids 5, 6 and 7 (Pro-Glu-Glu) are CCT-GAG-GAG
The point mutation in codon 6 converting the A to T changing the sequence CCT-GTG-GAG in SCA
The recognition site of the restriction enzyme Mstll is CCTNAGG, where N can be any of the four nucleotides
Close examination of the sequence shows that Mstll will recognize the normal B globin CCT-GAG-G where N is a G, but not the mutated form
Explain RFLP and Southern Blotting fir detection of B^s-globin mutation
Cleavage of 5he gene for B-globin with a restriction endonuclease
Restriction site is present in normal DNA and sickle cell DNA
Electrophoresis of reaction fragments from the DNA of normal individuals yields a 1.15 kB fragment using a probe specific for the B-globin gene
Electrophoresis of restriction fragments from the DNA of a patient with sickle cell disease yields a 1.35kb fragment because of the loss of a cleavage site
Electrophoresis of restriction fragments from the DNA of a heteroxygote yields both 1.15 kB and 1.35 kB fragments
Describe e,ectrophoresis and staining of Mstll restriction fragments
Restriction site is present in normal DNA and sickle cell DNA
Restriction site is present in normal DNA but missing in sickle cell DNA
Describe experimental procedure for identification of sickle cell gene from genomic DNA
Electrophoresis of digested and amplified genomic DNA
Place the gel (still on the tray ) into the electrophoresis chamber
COVER the gel with 1X electrophoresis buffer. The gel should be completely submerged.
PUNCTURE the foil overlay of each sample (genomic DNA samples previously digested with Mstll and amplified by PCR) with a pipet tip
Experiment overview:
1. Prepare agarose gel in casting grey
- Remove and blocks & comb, then submerge gel under buffer in electrophoresis chamber
- Puncture the foil overlay of each sample with a pipet tip
When identifying sickle cell Gene electrophoresis of DNA samples?
- After loading samples into wells in gel, place safety cover on the unit
- CHECK that the gel is properly oriented. Remember, the DNA samples will migrate toward the positive (red) electrode.
- CONNECT leads to the power source
- PERFORM electrophoresis of samples at 150 volts for 30 to 45 minutes. Allow the tracking dye to migrate at least 3.5 cm from the wells
4. Attach safety cover, connect leads to power source and conduct electrophoresis
How can DNA be stained and visual9zed?
After electrophoresis is complete, REMOVE the gel and casting tray from the electrophoresis chamber and proceed to Stain the gel and/or if stain was included in buffer, view results
Strained gel may be viewed in a gel doc (documentation)
- After electrophoresis, transfer gel for staining
How can DNA be stained on agarose Gel after electrophoresis ?
Ethidium bromide:
Fast and sensitive. Requires UV trans illuminator. A potential mutagen
SYBR safe:
-very sensitive. Requires UV transilluminator. Expensive. Non-mutagenic
Methylene Blue:
-Less sensitive
