8th Postgraduate Course for Training in Reproductive Medicine and Reproductive Biology

Antenatal Screening of Sickle Cell Disease

M. Tchana Sinou
Cameroon

Tutors
S.E. Antonarakis, C. Delozier-Blanchet
Division of Medical Genetics
Geneva University Hospital

Introduction

Pauling and associates (Pauling, Itano, and al) identified the abnormality of sickle cell hemoglobin in 1949 (1). Twenty years ago, Kan and Dozy carried out the first prenatal DNA diagnosis in 1978 for sickle cell anemia (Kan Y.W., Dozy A.M. et al)(2). Today, the DNA-based diagnosis of monogenetic disorders by the use of restriction enzyme or the nucleic acid hybridization is considered as a routine service of Medical Genetic. This is a consequence of rapidly increasing knowledge in the field of human molecular genetic (De Lozier-Blanchet CD, Old JM et all.). In the most recent comprehensive review about DNA testing, Connor (4) listed forty seven diseases for which DNA-based prenatal diagnosis has been reported and a further sixty nine for which it would technically be possible if desired (Connor J.M., 1992). In the last few years, this list has expanded by perhaps one quarter.

In some regions of the world, sickle cell disease is almost endemic and the highest mortality rate is among children from one to ten years. (5). There is no more controversy about the sensitivity and the specificity of which laboratory tests yield the highest efficiency. The result of a screening test is a risk estimate and parental counseling is essential to ensure informed consent for further investigation and for any termination of pregnancy that may result.

Cultural and moral standards as well as practical considerations affect the decisions made on the basis of screening results. A number of factors influence the accuracy of screening, including gestation dating methods, maternal weight and number of fetuses.

Epidemiology

2.1 Worldwide

Sickle cell disease affects approximately 50,000 Americans of all racial and ethnic backgrounds (31).

Among infants born in the US, sickle cells occur in 1 in every 375 Africans Americans, 1 in 3,000 Native Americans, 1 in 20,000 Hispanics, and 1 in 60,000 Whites (1). According to Platt OS et al, (10), the average life expectancy of patients with sickle cell anemia is decreased by 25- 30 years. Symptoms severity and life expectancy vary considerably with patients surviving beyond middle age and others dying during infancy and childhood. Mortality in children with sickle cell disease peaks between 1 and 3 years of age, and is mainly due to sepsis caused by Streptococcus pneumonia (8). Pneumococcal septicemia occurs at the rate of approximately 8 episodes per 100 persons per year in children under the age of 3 with sickle cell disease(11). In a study among black Americans, carried out by Wong and Powers (12), the case fatality rate can be as high as 35%.

One in every 150 African American couples in the US is at risk of giving birth to a child with sickle cell disease (13).

Mediterranean HbS disease occurs especially in Sicily, Calabria and some parts of Greece. On the Chalkidiki peninsula, the heterozygote frequency may reach 30 %. The gene is quite common in the South Indian population and has also been found in the Arabic population. In fact, there is an unequal distribution.

Table 1. Geographic distribution of Sickle Cell disease (frequency among total population): Summary from US Task force Preventive Service and American Academy of Family Physicians, 1994 (Reprint N° 510)

* West Africa and Nigeria

2.2. Situtation in Cameroon

Geographically, Cameroon is situated in Central Africa. The country’s name derives from« camaröes » a Portuguese name, meaning Shrimps, so called by the 15^th Century Portuguese explorer Fernando Po, who named the Douala’s river Wouri ‘Rio dos Camaröes’ (Shrimps river), after the many shrimps. Having the Atlantic coast in the south-west (Gulf of Guinea), Cameroon has borders with Nigeria in the West region, with Chad and Central African Republic in the north-east and east region and with Equatorial Guinea, Gabon and Congo-Brazzaville in the South.

The surface area is about 475 442 square kilometers and it has both, Sahelian and Equatorial climate.

The estimated population is about 14, 320 000 inhabitants (in 1996) among which are about 23% of women of reproductive age. The fertility rate is among the highest in the sub-region, and the crude mortality rate is estimated at about 17,5%o. Perinatal and infant mortality increased during the past ten years, reaching 56% live births in 1985. Over a 10 years period, about 157 deaths were recorded (average 15/year).

Since the introduction of a survey on reproductive health in Cameroon, in 1996, maternal mortality among SS pregnant patients is decreasing. Still, children among 1 to 10 years are those who pay a high tribute to sickle cell anemia (4 to 6 hospital visits per month). The frequency of homozygous cases in the total population varies from 1 to 5%. Almost all the clinical forms described in the literature and 4 haptotypes are encountered in Cameroon (Benin, Senegal, Bantu, and Kenya). The emergency cases that result from complications are severe anemia, strokes, pneumococcal infections, painful crisis and maternal death during delivery.

Two third of people suffering from the disease come from a very low socio-economic level. However, in certain parts of the country, better living conditions, nutrition and medical care have resulted in significant reduction in mortality.

The effective treatment for Sickle Cell disease and other hemoglobinopathies, the allogenic bone marrow transplantation remains inaccessible. For all these patients, the only approach for reducing the number of being born affected is preventive (14). This depends upon education, the detection of carriers, genetic counseling and on prenatal diagnosis, done in couples who are both carriers.

2.3. Sickle Cell Disease and Malaria

The distribution of HbS gene in Africa corresponds closely to that of Plasmodium falciparum, the organism causing the most severe form of human malaria.

J.B.S. Haldane, in 1949, first suggested that individuals with various red cell disorders might be protected against malaria infection.

The protection of sickle cell trait against Plasmodium falciparum applies almost entirely to infants. Infants with sickle cell trait become infected with plasmodium falciparum but their infections occur less frequent and are milder than those in AA infants. This difference was not observed in older children. It is likely that the acquisition of immunity against the parasite blunts differences in susceptibility between older AS and AA individuals. Even though, the cellular mechanisms underlying this phenomenon are not all clear.

Pathophysiology

3.1. Normal hemoglobin

The structures of human hemoglobin vary from embryo to adult.

The various globin genes with their respective globin chains and the various normal hemoglobin are shown in table 2.

Table 2

Stage	Hemoglobin	Structure
Embryonic	Gower I	d 2 e 2 (d 2 e 2)
	Gower II	a 2 e 2
	Portland I	d 2 g 2
Fetal	F	a 2 g 2
Adult	A A2	a 2 b 2 a 2 d 2

The a gene cluster is located on the short arm of chromosome 16, on the 25-kb region. It is composed of 1431 Amino Acid.

The gamma, beta, delta family is situated on the short arm of chromosome 11, on the 60-kb region. It is composed of 146 Amino Acid.

The genetic mechanisms that regulate coordinated gene function of the two different chromosomes to allow equal output of a and nona genes remain unknown.

3.2. Hemoglobin Structural Variants

Most of the variant hemoglobin results from point mutations in one of the structural genes, but a few are formed by other more complex, molecular mechanisms. More than 400 abnormal hemoglobin structures have been described and approximately half of these are clinically significant. The structural hemoglobin variants can be divided into three classes, depending on the clinical phenotype.

3.2.1. Variants that cause hemolytic anemia.

The great majority of hemoglobin mutants that cause hemolytic anemia are unstable, but 2 of the best known variants that cause hemolysis, sickle cell globin and Hb C, do so because they have unusual rigid structures.

3.2.2. Mutants with altered oxygen transport,

are able to increase or decrease oxygen affinity or form methemoglobin, which is incapable of reversible oxygenation.

3.2.3. Structurally abnormal globins

that cause thalassemia, because the mutation in the coding region also impairs the rate of synthesis or the stability.

3.3. Pathogenesis of sickle cell disease

Sickle cell anemia is a hemoglobinopathy, a monogenic autonomic recessive gene disorder. Hemoglobin S is formed as the result of a single gene defect, causing substitution of valine for glutamic acid in the cordon 6 of the b chain of adult hemoglobin.

Glutamic acid has two COOH groups and one NH₂ group. This charge difference explains the electrophoretic differences between normal and sickle hemoglobin.

Under conditions of low oxygen tension, hemoglobin S polymerizes, causing the red blood cell to take on <sickle> shape. The deformity of red blood cells leads to blockage of the blood flow causing the symptoms of sickle cell disease.

3.4. Clinical Manifestations

The manifestations of sickle cell disease can be grouped in four major categories:

1. Chronic hemolytic anemia
2. Systemic manifestations, including impairment of growth and development, and increased susceptibility to infections.
3. Vaso-occlusive and painful "crisis" of varying severity and frequency, affecting different parts of the body. The pain is attributed to the occlusion of small vessels by cells containing polymerized HbS.
4. Organs damage that is the consequence of multiple vaso-occlusive events and chronic anemia. Adults with severe sickle cell anemia may have impairment of a number of different organ systems: cardiac, pulmonary, renal, skeletal, cerebral and hepatic damage.

Antenatal screening

4.1. Indications

Screening for sickle cell trait has been initiated in several parts of the world among couples that are both carriers. Still, the advisability of screening programs remains a controversial issue.

If screening programs are employed, it is very important that confidentiality is preserved and that affected individuals have the benefit of adequate education and counseling.

4.2. Sampling Technics

Considerable progress has been made on antenatal diagnosis of sickle cell disease.

Initially, it was necessary to obtain a sample of fetal blood from the umbilical vein or from the placenta (if situated anteriorly) in order to measure the synthesis of radioactively labeled b globulin chains. But this invasive procedure has a risk of 5 % fetal mortality. The discovery of the linkage between the HbS gene and restriction endonuclease polymorphism enables the diagnosis to be made prenatal by analyzing genomic DNA of fetal cells obtained by amniocentesis. This procedure carries only a 0,5 % fetal risk.

4.3. Laboratory Analysis

Usually between the 14^th and 16^th week of gestation amniocentesis is performed. There are two main laboratory test performed on the amniotic fluid obtained:

Polymerase Chain Reaction (PCR) + Enzyme detection of the mutation

Polymerase Chain Reaction + Oligonucleid hybridization

The Mst II (or the Dde I) will cut normal DNA directly at the b 6 GLU site, but fails to cut b ^S DNA. The assay is sufficient sensitive so that the fetal cells do not need to be cultured prior to analysis.

Conclusion

Ironically, despite the fact that the molecular basis of sickle cell disease has been known for longer than that of any other gene defect, current treatment is only supportive. Although there is a general association of the events that lead to sickling, the molecular details have yet to be worked out, and no specific therapy that prevents or reverses the process in vivo has been identified. (Wong C, Antonarakis S.E et all 1986).

Prenatal diagnosis for couples known to be carriers for the gene can be performed with chorionic villous biopsy samples at twelve weeks of pregnancy. The restriction enzyme Mst II recognizes the specific site of the sickle mutation, thereby providing a direct diagnosis test that distinguishes between affected homozygous, carriers, and healthy individuals. (American Academy of Family Physicians, 1994, reprint n° 510)

In normal circumstances, prenatal screening of genetic disorders by DNA-based analysis is performed if it is indicated and is generally contra-indicated when the risk of performing the test exceeds the risk of the disease being present (4). Prenatal diagnosis screening is justified in sickle cell disease because of the high frequency of the disease in some populations and because of the high mortality rate among homozygous infants. Ethical considerations are of great importance in this procedure, because for the majority of disorders, termination is the intended course of action (24).

Perspectives for screening

Programs in Cameroon

So far, screening programs for sickle cell disease are not available in Cameroon. The first and most important point against making these programs a reality is poor financial resources and low economic status of the country, with:

• Lack of medical genetic divisions in our hospital
• Lack of expertise
• Lack of counselors that may be able to present the information clearly and thoughtfully, so that the prospective parents can make an informed and guilt free decision.

We also keep in mind the importance of ethical problems these programs can lead to, considering that termination of pregnancy, which is the intended course of action, remain a taboo in our society.

• Epidemiological studies to determine the incidence of the anomaly have been performed.
• We also have an idea about the financial burden of the disease on the population of Cameroon. If the screening program is seen as a priority, then a pilot study may be performed, assessing the acceptability of the method by the population.
• The main laboratory will then receive convenient equipment and kits for polymerase chain reaction plus restriction enzyme Mst II.
• Skilled geneticists or gynecologist-obstetricians could carry out sample collection.
• What is really missing is the initial fund for the amount of kits to perform the pilot study.

We hope that, with the available data from the study, proving the economical efficiency, the Cameroon government will find the possibility of funding this practice throughout the whole country, thus improving maternal and prenatal care.

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