J Community Genet DOI 10.1007/s12687-013-0171-z

SHORT COMMUNICATION

Carrier screening for inherited haemoglobin disorders among secondary school students and young adults in Latium, Italy Antonio Amato & Maria Pia Cappabianca & Maria Lerone & Alessia Colosimo & Paola Grisanti & Donatella Ponzini & Paola Di Biagio & Maria Perri & Debora Gianni & Silvana Rinaldi & Roberta Piscitelli

Received: 7 May 2013 / Accepted: 3 October 2013 # Springer-Verlag Berlin Heidelberg 2013

Abstract To reduce the incidence of β-thalassaemia major and other severe haemoglobin-related disorders by the early identification of healthy carriers, the Centro Studi Microcitemie Roma has been organising since 1975 a prevention programme in Latium, an Italian central region. This programme entails two different types of carrier screening on a voluntary basis: a universal screening offered to secondary school students and a screening offered to young adults. In 36 years of scholastic screening (from 1975 until 2011), 1,466,100 students have been examined and 26,786 (1.8 %) carriers of non-α thalassaemia have been identified. In the extra-scholastic screening, 388, 690 adult subjects (including the carriers' relatives) have been examined and a total of 38,457 (9.9 %) carriers of non-α thalassaemia have been detected. These results demonstrate that the precocious identification of healthy carriers allowed the identification of at-risk couples and reduced to zero the birth of affected babies in the Latium native population. This programme does not involve huge resources and is relatively inexpensive and, as such, it is essential to be offered to the total Latium scholastic and extra-scholastic population, which is epidemiologically changing due to migratory fluxes from countries in which haemoglobin disorders are common. Keywords Haemoglobin disorders . Prevention programme . Carrier screening . Epidemiological changes A. Amato (*) : M. P. Cappabianca : M. Lerone : P. Grisanti : D. Ponzini : P. Di Biagio : M. Perri : D. Gianni : S. Rinaldi : R. Piscitelli Centro Studi Microcitemie Roma, Associazione Nazionale per la lotta contro le Microcitemie in Italia (ANMI Onlus), Rome, Italy e-mail: [email protected] A. Colosimo Department of Comparative Biomedical Sciences, University of Teramo, Teramo, Italy

Introduction The inherited haemoglobin disorders are a group of haematological diseases caused by different molecular defects in the α- and β-globin loci, leading to either structural haemoglobin variants (including HbS, HbC, HbE) or defective globin chain synthesis (α-, β-, δβ- and εγδβ-thalassaemia) (Weatherall and Clegg 2011) Regarding thalassaemia, the reduced or absent synthesis of one or more of the globin chains leads to an imbalance in the α/non α-globin synthesis ratio, that ultimately determines the severity of the diseases. The heterozygous subjects are generally asymptomatic but can be identified by simple and relatively inexpensive haematological analysis; for this reason, the main purpose of the carrier screening programmes for thalassaemia is prevention. Healthy thalassaemia carriers, when properly informed, may decide in full awareness how to deal with the risk of an eventual pregnancy with a partner carrier and make an informed reproductive choice either taking prevention measures or accepting the risk of an affected child. It is evident that more options of choice are available if the screening is performed earlier in life. Carrier screening for β-thalassaemia and sickle cell disease by genetic testing is the most widely performed strategy in the prevention of β-thalassaemia major and sickle cell disease (Cousens et al. 2010). Differently from other countries (including Iran, Saudi Arabia, Palestine and Cyprus) (Abolghasemi et al. 2007; Alswaidi et al. 2012; Tarazi et al. 2007; Papasavva et al. 2008) in which β-thalassaemia screening programmes are mandatory before marriage, in Italy, voluntary screenings are offered (Amato et al. 2011; Cao and Galanello 2010). This last type of screening includes educational sessions, population and family screening, genetic counselling and the eventual choice of performing prenatal diagnosis analysis. This is the case of the scholastic screening programme organised by the Centro Studi

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Microcitemie Roma (CSMR), offered since 1975 to students (13–14 years old) attending the third year of the secondary school in the Italian Latium region (Amato et al. 2009). Early information may help young carriers in the choosing a partner, in considering adoption, donor gametes, in vitro fertilisation after pre-implantation diagnosis or in asking for prenatal diagnosis.

Materials and methods The present screening, operating within the Regional Health Service and funded by the Latium Region Government, involves two distinct pathways: (1) a universal screening proposed to students and (2) an outpatient screening proposed to young adults that may include also molecular analyses (with information and therapies for families having an affected child). At present, this last cohort of subjects mainly involves the immigrant individuals, which are usually never preventively informed about procreative risks. Scholastic screening The prevention plan that was elaborated in 1975 by Silvestroni and Bianco, the founders of Associazione Nazionale Microcitemie in Italia (ANMI)-CSMR, consists of the following phases: (1) Health education activities to clarify, at school, the hereditary mechanisms leading to the diseases and the essential concepts of “β-thalassaemia healthy carrier” and “homozygous patient”. (2) Drawing of blood samples After obtaining the parental informed consents, the medical doctors of CSMR collect a venous blood sample (2 ml) from each individual during a normal school morning. (3) Diagnostic tests All the tests are carried out at the CSMR laboratories within 24 h from drawing blood, following a three-stage track. First stage: all samples are subjected to the single tube osmotic fragility test (in 0.36 % NaCl) and blood smear screening (to identify eventual morphological alterations of red cells). All samples that result to be positive for one or both tests undergo the second stage, consisting of complete blood count on an automatic counter, followed by separation and measurement of the haemoglobin fractions by high-performance liquid chromatography (Stephens et al. 2012; Van Delft et al. 2009). Samples showing typical alterations are recorded as positive cases (carriers of β-thalassaemia, δβ-thalassaemia or HbE, etc.) or as suspect cases that need the third stage of examination. These suspect cases may include carriers of

α-thalassaemia, of β-thalassaemia associated with δthalassaemia, carriers of hereditary persistence of foetal haemoglobin and of rare haemoglobin variants with a thalassaemia phenotype. All these subjects and their family members are asked to undergo an additional more complete haematological examination, including ferritin and bilirubin measurements to exclude iron deficiency. For unsolved cases, a fourth stage that consists of in vitro globin chain synthesis analysis is performed (Amato et al. 2007). (4) Communication of results and family examination The collective results are mailed to the schools and then delivered to the students' parents by the school medical doctor or by the school principal. All subjects who were found to be carriers are asked by CSMR to undergo a second complete examination together with all their family members. This procedure confirms the preliminary diagnosis and provides more detailed information to all family members. (5) Genetic information and genetic counselling Families of student carriers are given general information about thalassaemia, prevention, and counselling when discussing the definitive results with a geneticist. Eventual at-risk couples, who are seeking to start a family, are referred to public structures specialised in thalassaemia prenatal diagnosis. Extra-scholastic screening Differently from countries in which universal screening is carried out, this voluntary outpatient screening includes selected individuals: (1) parents and other family members of the adolescents (who were found to be carriers following the scholastic screening), who come to the Centre to get their own diagnosis; (2) anaemic children who are sent by paediatricians for suspected microcythemia; (3) couples who are sent by gynaecologists, requiring preconception or prenatal tests; (4) women who come for anaemia due to gynaecological causes (i.e. metrorrhagia) and (5) people who come to solve different types of anaemia. For all these motivations, they represent a population in which the probability of finding carriers is greater than expected. All these subjects are offered the entire diagnostic programme for thalassaemia and haemoglobinopathies (as described above) while a service of genetic counselling is offered only to subjects having a positive screening result.

Results The scholastic screening programme started in 1975, and since 1993, the birth of affected babies in Latium native

J Community Genet

population was reduced to zero. Before 1975, an average of 10 affected babies/year was expected in Latium (Bianco et al. 1986), with a dramatic physical and emotional burden for patients and families and considerable financial consequences for the National Sanitary Service. In 36 years of scholastic screening, 1,466,100 students have been examined (at the rate of 400–500/day for 8 months/year) and 26,786 (1.8 %) carriers of beta gene defects have been identified. In the extra-scholastic screening, 388,690 subjects (including the carriers' relatives) have been examined and a total of 38,457 (9.9 %) carriers of beta gene defects have been detected. Considering the two screening programmes during the years 1975–2011, a total of 1,854,790 subjects have been examined, 65,243 (3.5 %) of which were found to be carriers (Table 1). The second stage of tests was offered to about 44 % of the subjects, the third stage to about 15 %, and the fourth stage to less than 1 %. The applied protocols were also able to distinguish several forms of anaemia and to identify rare haematological diseases, such as hereditary spherocytosis and constitutional elliptocytosis that, interacting with thalassaemia, may modify the resulting phenotype. In the last 9 years, the scholastic screening allowed the identification of 11 foreign students, who were found to be homozygous for HbS, HbE variants or were affected with HbH disease, that are pathologies requiring continuous medical care. Since the first year of implementation, this scholastic screening programme has been characterised by a high compliance rate by both native and foreign populations, with an adhesion rate of 67 and 73 %, respectively (Amato et al. 2012). More recently, due to the relatively high percentage of immigrant students (8.7 %) in our school population, a different protocol including a complete haematological and haemoglobin analysis has been carried out to detect also HbS and HbC carriers, which are usually missed by the single tube osmotic fragility test. The extra-scholastic screening in the young and adult population allowed the identification of the most common thalassaemia syndromes (α-, β-, δ- and γ-thalassaemias) and haemoglobinopathies, particularly among the immigrants (13.8 % were carriers for HbS, 4 % for HbE, 1.8 % for HbC, and 4.3 % were carriers of rare or novel haemoglobin

variants) (Amato et al. 2007; Manconi et al. 2010). In addition, complex gene rearrangements such as deletions or duplications involving the α- and β-globin gene clusters, have been detected by the use of novel technologies, including genomic DNA direct sequencing, DHPLC (Colosimo et al. 2002) and multiplex ligation-dependent probe amplification analysis (Phylipsen et al. 2009; Colosimo et al. 2011).

Conclusions Latium is the only Italian region in which a systematic plan of prevention for Mediterranean anaemia (β-thalassaemia) has been carried out in a continuous way for more than 37 years. An analogous genetic screening programme for Tay–Sachs disease and β-thalassaemia has been carried out for >20 years in Montreal (Canada) involving a high school cohort (Mitchell et al. 1996). This last study showed that students, who were found to be carriers, remembered their carrier status, were not discriminated by it and used the genetic information in useful ways. Lena-Russo et al. (2002) have verified, in a similar βthalassaemia screening programme, that the information is stored for a long period (about 15 years from the exams and the first pregnancy), even though the written result is often lost. The age of the screened students, who are younger than any other school-based screening programmes, is mainly due to the Italian scholastic system, whose attendance is obligatory until the third class of secondary school (with students aged 13–14 years). Considering the obtained results and the moderate economic cost (about €5.50/test for the complete procedure), the described prevention programme appears highly satisfactory. Besides its positive effects on public health, this programme allowed to acquire relevant scientific information on molecular defects that are rare in the native population but common in the growing immigrant ones, originating from countries where prevention programmes are not available. Nowadays, with a regional population of about 5,775,000 people and 2.5 % of carriers and with a migratory flux of about 10.6 % carrier frequency, we strongly believe in the benefits of the presented prevention programme. We also believe that it is fundamental to continue informing the entire population, which is epidemiologically changing at an increasing rate. In detail, in the Latium region, 163 different nationalities are now

Table 1 Prevention programme of inherited haemoglobin in Latium (Italy) Subjects

Scholastic screening (October 1975– June 2011)

Extra-scholastic screening (January 1975– December 2011)

Total

Tested populations Non-α thalassemia carriers diagnosed

1,466,100 26,786 (1.8 %)

388,690 38,457 (9.9 %)

1,854,790 65,243 (3.5 %)

Global results in 36 years since implementation

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represented, with Rumanians and subjects coming from East Europe being the most prevalent (32 %), followed by Africans (Central Africa, Morocco, Egypt) (27 %) and by Asians (Bangladesh and Philippines) (25 %). Most of these immigrants are coming from malaria-endemic countries, showing very high rates of haemoglobin disorders and alarming incidence figures. These individuals are expected to choose their partner within their own ethnic group for several generations, probably following customary consanguineous marriages traditions and herewith keeping the risk of affected children as high as their country of origin (Giordano 2009). Acknowledgments The authors would like to thank Prof. Cornelis Harteveld (LUMC, Center for Human and Clinical Genetics; The Hemoglobinopathies Laboratory, Leiden University Medical Center, Leiden, the Netherlands) for his help in revising the text and in the improvement of language. Funding The screening programme of ANMI Onlus–CSMR is funded by the Latium Region Government–Health Department. Compliance with Ethics Guidelines The experiments comply with the current laws of the country in which they were performed.

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Carrier screening for inherited haemoglobin disorders among secondary school students and young adults in Latium, Italy.

To reduce the incidence of β-thalassaemia major and other severe haemoglobin-related disorders by the early identification of healthy carriers, the Ce...
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