Sickle cell anemia and thalassemias Paul R. Earl Facultad de Ciencias Biológicas Universidad Autónoma de Nuevo León San Nicolás, NL, Mexico

Genetic explanations. Thalassemia is caused by impaired production of either the  or  hemoglobin chain. Alpha (  ) thalassemia occurs when one or more of the 4 alpha chain genes fails to function.

A) The loss of one gene diminishes the production of the alpha protein only slightly. This condition is so close to normal that it can be detected only by specialized laboratory techniques. A person with this condition is called a silent carrier because of the difficulty in detection.

B) The loss of 2 genes (2-gene deletion alpha (  ) thalassemia) produces a condition with small RBCs, and at most a mild anemia. People with this condition look and feel normal. The condition can be detected by routine blood testing, however.

C) The loss of 3 alpha genes produces a serious hematological problem (3-gene deletion  thalassemia). Patients with this condition have a severe anemia, and often require blood transfusions to survive. The severe imbalance between the alpha chain production (now powered by one gene, instead of 4) and beta chain production (which is normal) causes an accumulation of beta chains inside the RBCs. Normally, beta chains pair only with alpha chains.

D) The loss of all 4 alpha genes during fetal life causes death in utero or shortly after birth. Rarely, 4 gene deletion alpha thalassemia has been detected in utero, usually in a family where the disorder occured in an earlier child. Repeated transfusions can keep victims alive.

E) There are 2 genes for the beta chain of hemoglobin in  - thalassemia. Unlike  -thalassemia,  -thalassemia rarely arises from the complete loss of a beta globin gene that is present, but produces little beta globin protein. The types of genes can be analyzed in each case.

Even when the affected gene produces no beta chain, the condition is mild since one of the 2 beta genes functions normally. The red cells are small and a mild anemia may exist. People with the condition generally have no symptoms. The condition can be detected by a routine laboratory blood evaluation. (Note that in many ways, the one-gene beta thalassemia and the two-gene alpha thalassemia are very similar, from a clinical point of view. Each results in small red cells and a mild anemia).

The sickle cell disorders usually result from S mutation homozygocity which is an A for T oligonucleotide substitution at codon 6 of the beta globin gene leading to the nonsynchronous replacement of valine for glutamic acid.

Clinical classification of the thalassemias. Alpha thalassemia has 4 manifesta-tions (a-d) according to the number of defective genes. a) Silent carrier state. This is the one- gene deletion alpha thalassemia condition. People with this condition are hematologically normal. They are detected only by sophisticated laboratory methods.

 -Thalessia has 3 states (a-c). a) Thalassemia minor (known as thalassemia trait) in people who have small red cells and mild or even no anemia. These patients are usually only detected through routine blood testing. b) Thalassemia intermedia in people with anemia able to survive without blood transfusions. c) Thalassemia major patients require chronic transfusions.

Carrier detection. The carrier detection procedure of a preventive program should be designed to be precise enough to secure all couples at risk. Heterozygous  -thalassemia, either the  0 or  + type, is characterized by high RBC counts, microcytosis, hypochromia, increased hemoglobin A 2 (HbA 2 ) levels and unbalanced  -globin/non-  -globin chain synthesis. However, this phenotype can be modified, causing problems in carrier identification.

Heterozygous  -thalassemia: Phenotypic modifications Normal RBC indices  and  interactions Normal HbA 2 level Iron deficiency Coinheritance of  and  thalassemias Some mild  -thalassemia mutations  -thalassemia Normal RBC indices and HbA 2 Silent  -thalassemia mutations  -Globin gene triplication

The preliminary selection of individuals at risk of being heterozygous for a form of thalassemia is based on the determination of mean corpuscular volume (MCV) and mean corpuscular hemoglobin (MCH) values. However, double heterozygotes for both  and  anemias could have normal MCV and MCH values, and thus could be missed. Quantitaion of the HbA 2 level should also be tested for.

Molecular diagnosis. To date at least 150 molecular defects have been defined in  -thalassemias. The common polymerase chain reaction (PCR) procedures used are given in the table:

Reserve oligonucleotide hybridization uses membrane-bound allele specific probes that hybridize to the complementary PCR sequence prepared by using the patient’s DNA as the starting template. Up to mutations have been screened in one step! Primer-specific amplification of the target DNA can detect mutants. Only the normal primer amplifies normal DNA,while DNA from homozygotes is amplified only by the  -thalassemia primer and DNA from heterozygotes by both primers.

 -Globin gene analysis. This analysis is carried out to define double heterozygotes for  and  thalassemias with normal HbA 2 that can be confused with  -thalassemia.

Sickle cell anemia. It commonly results from homozygosity for the HbS. Sometimes it is caused by compound heterozygosity for the HbS mutation, and HbC and other variants like HbO Arab. Dot blot analysis with allele specific probes or primer specific amplification may be particularly useful.

Prenatal diagnosis. For some years, the diagnosis of thalassemia was obtained either indirectly by polymorphism analysis or direcly by oligonucleotide hybridization on electrophoretically separated DNA fragments. Nowadays, thalassemias are detected directly by the analysis of amplified DNA from fetal trophoblasts and amniotic fluid cells.

Counselling for hemoglobin disorders. Couples at risk for hemoglobin disorders may be identified retrospectively after the birth of an affected child, or prospectively by analyzing childless spouses. Prospective identification allows parents to have a disease-free family. Education of the public by mass media, posters, lectures, etc. was carried out in Sardinia, Italy. Special meetings were held with physicians and especially with pediatricians and obstetricians, family planning associations, nurses and social workers.

Future prospects. Simplification and automation of some PCR procedures such as primer-specific amplification for carrier screening and prenatal diagnosis are expected. Oligonucleotide microchip assay is a new application for detecting mutations in medicine. If mutation detection is used, all hematologic steps would be skipped. A strong advance would be fetal diagnosis by analysis of fetal cells in the maternal circulation. Point mutations responsible for  -thalassemia and sickle cell anemia can be successfully identified in fetal cells involving magnetically activated cell sorting using anti-transferrin receptor antibody.