Prevention of hemoglobinopathies in Turkey

Hemoglobinopathies are the most common genetic disorders in Turkey. The incidence of beta thalassemia and sickle cell trait (HbAS) is 2.0% and 0.3% respectively. In addition to HbS, 51 abnormal hemoglobins and 42 different beta thalassemia mutations have been detected by DNA analysis. In Turkey, beta thalassemia and sickle cell anemia cause major health problems. For thirty years, screening programs for carriers, genetic counseling and prenatal diagnosis have sought to prevent hemoglobinopathies. In 1983, the first prenatal diagnosis center was established for sickle cell anemia and beta thalassemia at Hacettepe University, Ankara. After many populationscreening studies, a law was passed in 1993 by the Turkish Parliament for the eradication of hemoglobinopathies. Forty-one premarital screening centers were set up by the Ministry of Health in the 33 provinces where most of the transfusion-dependent thalassemic patients live. The mothers at risk for hemoglobinopathies were given genetic counseling and directed to prenatal diagnosis centers. Since 1990, four prenatal diagnosis centers have been established at university hospitals in Adana, Antalya, stanbul and zmir. A total of 5255 prenatal diagnoses have been made for sickle cell anemia and beta thalassemia in 5 centers; 1338 fetuses have been diagnosed as homozygous or compound heterozygotes for hemoglobinopathies. Prenatal diagnosis was performed on families who had decided to terminate the pregnancy if it were to be found that the fetus was affected.


Introduction
Turkey is a large country located on the European and Asian continents.The country has a population of 73,722,988 and covers a total area of 783,562 km².Due to the presence of various ancient civilizations, there is great genetic diversity.[3][4][5][6][7][8][9][10][11] Beta thalassemia is seen all over the country but sickle cell anemia is peculiar to the Çukurova region in southern Turkey.The incidence of beta thalassemia and sickle cell trait for the country as a whole is 2% and 0.3%, respectively. 4,7,9Different regions report different percentages of beta thalassemia trait.The west and south of the country are at risk for beta thalassemia.3][14] Although the overall frequency of beta thalassemia is 2%, it reaches as high as 10% in the Thrace region.Sickle cell hemoglobin (HbS) is common in southern Turkey.Average HbAS is approximately 8.2% in the Çukurova region (Adana, Hatay and Mersin).3][14][15][16][17][18] In 1983, the first prenatal diagnosis center was established at Hacettepe University in Ankara.Over eight years, a total of 101 prenatal diagnoses were made by in vitro globin chain analysis to prevent sickle cell anemia and beta thalassemia. 19This method was used at Hacettepe University for some couples whose beta globin gene mutation had been unknown until 2000. 20This was the main diagnostic procedure adopted and was performed by using fetal blood samples taken during the 18 th -20 th weeks of gestation.In the Gurgey 1991 study, the majority of the pregnant women had given birth to one or more affected offspring.Since the beginning of the 1990s, amplification of genomic DNA by polymerase chain reaction (PCR) techniques has been adopted in prenatal diagnosis. 21,22These PCR techniques are still being adopted by using DNA isolated from chorionic villus sampling (CVS) at the first trimester of pregnancy.Later, 2 prenatal diagnosis centers were established in Turkey, in 1990 in stanbul and in 1992 in Adana.While many prenatal diagnoses were performed by PCR, molecular diversity was well documented in hemoglobinopathies. 23,24t was found that affected births had not been eradicated completely, despite the studies carried out between 1983 and 1992.Due to the high burden on the state of the medical costs of thalassemic patients, in 1993 the Turkish Parliament passed a law for the prevention and eradication of human genetic disorders.6][27] Given the success of the pilot studies, a hemoglobinopathy scientific board was set up, and in 2002 the Ministry of Health published regulations to govern the centers' activities.Since 2003, premarital screening tests have been carried out in the 33 provinces with the highest incidence of carriers in Turkey.[30]

Materials and Methods
Prenatal diagnosis is still the only option that can prevent hemoglobinopathies.After screening couples who manifest the sickle cell or beta thalassemia trait, prenatal diagnosis is performed at the first trimester of pregnancy, and affected fetuses are aborted as early as possible.

Blood samples and hematologic procedures for premarital screening
Many population-screening studies were carried out in the regions in which beta thalassemia and sickle cell anemia were reported between 1993 and 2002.The main screening tests carried out were complete blood count and analysis of hemoglobin variants.Blood samples (5 mL) with EDTA as anticoagulant were taken for hematologic and hemoglobin analysis.A complete blood count (CBC) was taken using a cell counter.Hemoglobin variants were characterized by cellulose acetate electrophoresis. 31HbA 2 was quantified by micro column chromatography for detection of beta thalassemia trait. 32ver the last ten years, the fully automated high performance liquid chromatography (HPLC) systems have been used for both determination of hemoglobin variants and quantification of the Hb levels. 32,33These HPLC systems are used for hemoglobin analysis and have made it easier to set up premarital screening centers.Forty-one centers have been set up in the 33 provinces with the highest incidence of beta thalassemia trait over the last decade (Figure 1). 34

Fetal samples and molecular procedures for prenatal diagnosis
The reason for setting up the first prenatal diagnosis center for sickle cell anemia and beta thalassemia at Hacettepe University, Ankara, in 1983, was mainly to help multiparous pregnant women at risk of having a baby affected with hemoglobinopathies.Fetal blood samples were taken between the 18 th -20 th weeks of gestation.By using fetal blood samples, in vitro hemoglobin chain analysis was performed using 3 H-Leucine by conventional methods. 19This proved to be a safe and reliable procedure for prenatal diagnosis for sickle cell anemia and beta thalassemia.The method was also used for some couples whose mutation had been unknown until 2000.After detection of the beta globin gene mutations of the parents, PCR-based methods are used for prenatal diagnosis.
Genomic DNA is isolated from leukocytes by conventional methods. 35Fetal DNA is isolated from CVS obtained in the 10 th -11 th weeks of pregnancy.Beta globin gene mutations are detected by PCR-based techniques, such as amplification refractory mutation systems (ARMS) and restriction fragment length polymorphism (RFLP). 22,36Some rare or unknown beta thalassemia mutations were characterized by DNA sequencing with the dideoxy chain termination reaction procedure. 37
Forty-one premarital screening centers have been set up in 33 provinces by the Ministry of Health over the last ten years.The couples at risk for hemoglobinopathies have been given genetic counseling and directed to prenatal diagnosis centers.The percentage of the cou- A total of 5255 prenatal diagnoses have been made for sickle cell anemia and beta thalassemia in five centers at university hospitals since 1983.A summary of the results is presented in Table 2. 20,23,29,30 A total of 1338 affected fetuses were homozygous or compound heterozgotes for hemoglobinopathies. 20,23,29,30Two hundred and seventeen fetuses were diagnosed by in vitro globin chain analysis at Hacettepe University Hospital in Ankara. 20The rest of the results (3.739) were detected by DNA analysis in 5 prenatal diagnosis centers.

Article
Frequency and type of beta thalassemia mutations in couples at risk are shown in Table 3. 20,23,29,30 The IVS1-110 G>A mutation was the most common.The ten most common mutations account for 77.1-95.2% of all the beta thalassemia mutations.
In Turkey, genetic heterogeneity of hemoglobinopathies was well documented during the prenatal diagnosis and population screening.In addition to HbS, 51 hemoglobin variants and 42 different beta thalassemia mutations were found in the country as a whole.,30,40,[47][48][49][50]

Discussion
Hemoglobinopathies are the most common genetic diseases causing health problems in the world.Sickle cell anemia and beta thalassemia constitute the majority of the hemoglobin disorders in Turkey.Unfortunately, there is no ultimate cure for these diseases, and prevention is the best option for families at risk.Since 1983, prenatal diagnosis has been a well-accepted solution in Turkey for prevention of the hemoglobinopathies.
Hemoglobinopathies are quite heterogeneous in the Turkish population.Up to now, more than 42 different point mutations in the beta globin gene and 52 hemoglobin variants have been characterized in the country.The seven most common beta thalassemia mutations alone account for approximately 72% of the total number of mutations found in the Çukurova region.In addition to HbS, there are some rare Hb variants, such as HbC, HbD and HbE, and also, more rarely, Hb Sarrebourg. 50ue to molecular diversity, in some couples at risk for hemoglobinopathies prenatal diagnosis requires time.For this reason, it is strongly recommended that mutation analysis should be detected before pregnancy.
All premarital screening centers were certified over nine years by the Turkish Ministry of Health.][43][44][45][51][52][53][54] Although Erzurum is included in the 33 provinces, here the incidence of beta thalassemia trait was only 0.68 per cent. 41ouples at risk for hemoglobinopathies have been directed by the centers to university hospitals for prenatal diagnosis.A large number of fetuses are diagnosed each year.It became apparent that some of the couples did not fully understand the importance of screening and genetic counseling, and so did not go to university hospitals for prenatal diagnosis.Approximately 100 affected babies are born annually because the couples had married before 2000.Consanguineous marriage also contributes significantly to affected births all over Turkey.Overall, consanguinity is approxi-mately 21%, but rates may be as high as 46-63% in some regions. 30,40n conclusion, in Turkey, a national hemoglobinopathy screening program has played a major role in lowering the rate of affected births.Last year, the Turkish Ministry of Health adopted a policy of Family Medicine

Figure 1 .
Figure 1.Provinces of premarital screening centers in Turkey.

Table 2 . Results of prenatal diagnosis centers in Turkey.
ples screened at the centers has reached 81 per cent at six years.46
IVS, intervening sequence; Cod, codon; Fsc, frameshift codon.throughout the country.Family Medicine is the medical speciality that provides continuing, comprehensive health care for individuals and their families.The scope of Family Medicine encompasses all ages, both sexes and every disease entity.If physicians examine and obtain blood samples for CBC and HPLC analysis from all pregnant women, it will be possible to eradicate hemoglobin disorders in Turkey.Article