The Genetics of Sickling
By Shaikha Al Arrayed*
It is known that Bahrain is one of the countries where haemoglobinopathies (especially sickling) form real health problems. This drains the health drastically affects family and personal life. It is essential that there is far more understanding of sickle cell disease.
The sickle cell disease patient differs from the normal human only in the structure of the haemoglobin is a tetramer with a molecular weight of 64,500. it consists of two non-alpha globin polypeptide chains, each of which has a single covalently bound haem groups. Each of the four haem groups is made of an iron atom within a protoporphyrin ring.
In sickle cell disease, when oxygenated, the cells are biconcave discs like red blood cells of a normal individual. However, when they are deoxygenated, they become elongated, filamentous and sickle shaped.
Heterozygous individuals who receive the abnormal gene from one parent but its normal allele from the other, would be expected to have sickle cell trait. homozygous individuals who receive the abnormal gene from each parent, would develop sickle cell anemia. When mating occurs between two individuals with sickle cell trait, the ratio between normal children, children with sickle cell anemia is 1:2:1:
Hb S differs in its electrophoresis from Hb A. The difference lies in the protein part. The nature of the difference was found by Ingram (1957-1959). That a particular position in the amino acid sequence of the B-polypeptide chain, which is occupied by a glutamic acid residue in Hb A, is occupied by a valine residue in HB S. If normal Hb a is written as Alpha 2 Beta 2, the structure of Hb S may be written as Alpha 2 Beta 2 6 glu→ val. And it was found to be due to a single mutational step which is the smallest unit difference in the primary structure of a specific protein.
At the chromosomes level, there are 6 globin genes coding for the 6 globin chains, Alpha, Beta, Gamma, Delta, Epsilon, Zeta. Epsilon, Gamma, Delta chains are similar to a Beta chain. While Zeta is similar to an alpha chain. There are 9 different genetic loci which code for the 6 globin genes. In addition, there are at least 3 pseudo genes which have sequences similar to other globin genes, but which differ in that they are not expressed into globin protein.
The region of chromosome 11 coding for Beta and Beta like genes (Epsilon, Gamma A, Gamma G, Delta) has been thoroughly mapped by restriction endonuclease analysis, and these genes have been sequenced.
At the DNA level, the full base sequence messenger RNA fro the polypeptide chain has now been determined. Starting from the 5 end it consists of a sequence of 50 bases which are not translated into amino-acid sequence, the initiator codon (AUG) which codes for methionine and is later removed from the polypeptide chain, the sequence of 438 bases which code in triolets for the sequence of the 146 amino-acid of the normal Beta polypeptide, the chain termination codon (UAA), a sequence which is also not translated.
The DNA base sequence is even longer because it includes two intervening sequences (IVS). These IVS in the DNA are transcribed into RNA but are removed during processing. So, they are not represented in the final messenger RNA molecule which binde to the ribosomes and serves as a template for polypeptide synthesis.
Sickle cell mutation which results in substitution of glutamic acid by valine at position No. 6 in the polypeptide chine can be attributed to a change from RNA codon GAG to GUG. This change, resulting from the substitution of A (Adenine) by U (Uridine) at position No. 20 in the messenger RNA sequence, can in turn be attributed to a mutational sequence of the gene itself. So, all the sickling phenomena is a result of a single base change at the level of DNA.
The antenatal diagnosis of sickle cell disease
The antenatal diagnosis of sickle cell disease can be used to detect the status of the fetus early in pregnancy. The parents then know the exact condition of the fetus, whether it is normal, a carrier or diseased. Provided with this information, they have the right to decide whether to continue the pregnancy or not.
Fetal blood sampling: The antenatal diagnosis of sickling has been carried out by fetoscopy, fetal blood sampling and analyzing the globin chain synthesis since 1975. It requires a very specialized technique carried out in very few medical centers. It can only be done after 18 weeks of gestation and it has a 5-10%b risk of fetal mortality.
DNA analysis of amniotic fluid cell : a ) By using HpaI endonuclease. In 1978 Kan and Dozy described polymorphism located extremely close to the Beta globin gene on chromosome 11. the DNA was digested with restricted endonuclease HpaI, Beta globin gene was contained in a fragment of DNA 7.6 kilobase in length. We expect the person with Hb A to have only the 7 or 7 Kb fragments, while persons with sickle cell trait will have the 7 Kb and 13 Kb fragments. Those with sickle cell disease will have only the 13 Kb fragment.
b) By using mst II endonuclease. It was mentioned that Hb S results from a single base mutation from an A to a T. this base change happens to occur in the middle of a recognition site for the restriction endonuclease Mst II. This enzyme recognizes the sequence CCTNAGG ( N is any base ) in the normal Beta globin chain. Mst II normally cuts 5 to the B globin gene and at the GAG codon to generate a 1.1 Kb fragment. The base change from A to T destroys the recognition site resulting in a larger fragment of 1.3 Kb. Therefore, Mst II can be used to diagnose sickle cell anemia directly, and we can expect to see the 1.15 Kb fragment in the AA genotype. The 1.35 Kb fragment in the SS and both the 1,15 Kb and the 1.35 Kb fragment in the HbAS.
Chorionic villa biopsies and the first trimester diagnosis of the sickling 8. Dr. J.M. old developed a method of extraction of a small amount of fetal DNA from chronic villi biopsies which can provide a first trimester (8-10 weeks) antenatal diagnosis by DNA analysis. The DNA was best prepared from a fresh chorionic villi biopsy or one that had been quickly frozen and transported in dry ice. This DNA is then subjected to endonuclear Mst II for analysis.
From this we can see that the genetic clinic has a major role to play in preventing this disorder. It's aim is to provide genetic counseling to all those who need it. Carry out prenatal diagnosis. Administer the new technology in investigating and treating this disorder. Perform new born screening and carrier detection. And increase the awareness of the public regarding methods to avoid bringing sick children into their families and the community.
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