Hindawi BioMed Research International Volume 2019, Article ID 2080352, 7 pages https://doi.org/10.1155/2019/2080352 Research Article Alpha-Thalassemia in North Morocco: Prevalence and Molecular Spectrum Achraf Laghmich ,1 Fatima Zahra Alaoui Ismaili ,1 Amina Barakat,1 Naima Ghailani Nourouti,1 Mohamed Khattab,2 and Mohcine Bennani Mechita1 1 Biomedical Genomics and Oncogenetics Research Laboratory, Faculty of Sciences and Techniques of Tangier, University Abdelmalek Essaadi,ˆ Tangier 90000, Morocco 2Pediatric Hemato-Oncology Service, Children’s Hospital, CHU Rabat 10010, Morocco Correspondence should be addressed to Achraf Laghmich; [email protected] Received 14 October 2018; Revised 9 January 2019; Accepted 24 February 2019; Published 13 March 2019 Academic Editor: Pedro Cabrales Copyright © 2019 Achraf Laghmich et al. Tis is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Unlike the other hemoglobinopathies, few researches have been published concerning �-thalassemia in Morocco. Te epidemi- ological features and the mutation spectrum of this disease are still unknown. Tis regional newborn screening is the frst to study �-thalassemia in the north of Morocco. During the period from January 2015 to December 2016, 1658 newborns umbilical blood samples were investigated. Suspected newborns were screened for �-globin defects using Gap-PCR and Multiplex Ligation- dependent Probe Amplifcation technique. Te prevalence of �-thalassemia, its mutation spectrum, and its allelic frequencies were described for the frst time in Morocco. Six diferent �-globin genetic disorders were detected in 16 neonates. Tis screening valued the prevalence of �-thalassemia in the studied population at 0.96% and showed the wide mutation spectrum and the heterogeneous geographical distribution of the disease. A high rate of carriers was observed in Laouamra, a rural commune in Larache province. Heterogeneity of �-globin alleles in Morocco explains the high variability of �-thalassemia severity. Tis diversity refects the anthropological history of the country. Tese results would contribute to the prevention of thalassemia in Morocco directing the design of a nationwide screening strategy and awareness campaign. 1. Introduction of these defects. Some abnormal Hb variants, once specifc to particular ethnic groups, are now worldwide spread [7, 8]. �-Talassemia represents a group of recessively inherited Morocco, a Mediterranean country, is a potential predilection hemoglobin disorders due to defcient or absent synthesis zone of hemoglobinopathies due to its geographical position. of �-globin genes. Afecting 5% of the world’s population, Te frequency of the �-mutated genes is believed to be �-thalassemia is the most common monogenic disorder [1]. increased due to the country’s socioeconomic and cultural Te clinical phenotype of �-thalassemia varies according to system encouraging consanguinity, the selective pressure of the number of afected genes from almost asymptomatic to malaria in endemic close countries, and its history as a a lethal hemolytic anemia. More than 80 diferent genetic migration crossroad between sub-Saharan Africa and Europe defects have been described; most of them are deletions [9, 10]. Te World Health Organization estimates the rate and less commonly nondeletional defects involving �-globin of hemoglobinopathies carriers in Morocco at 6.5%, with genes[2,3].Tisleadstothewidespectrumofhematological more than 30 000 cases of major forms [11]. A previous and clinical phenotypes. Genotype-phenotype correlation is regional study highlighted the region of North Morocco as a still not well documented [4]. geographical hot-spot for hemoglobinopathies due to its high �-Talassemia is prevalent in populations of Southeast consanguinity rate already estimated and its location in the Asia,CentralAfrica,andtheMiddleEast[5,6].Inthelastfew malaria belt [10, 12]. Nevertheless, very scarce data about the decades, massive population migrations favored the spread occurrence and epidemiology of �-thalassemia is available. 2 BioMed Research International Mediterranean Sea Atlantic Ocean 10 km N Tanger Assilah Fahs-Anjra Tetouan M’diq Fnideq Larache Figure 1: Geographic location of the region concerned by the study. Tus, the overarching aim of our present study is assessing Information sheets with nationality, sex, age, dialect, and theprevalenceandspectrumof�-thalassemia in the north of natives or not and written consent forms were available in Morocco. Our conclusions would help decoding the context Arabic to ensure comprehensive understanding of the study of the disease epidemiology and lead to a better formulation objectives. Tis study was approved by the Ethics Committee of country-specifc and optimized �-thalassemia policies. for Biomedical Research in the Faculty of Medicine and Pharmacy of Rabat (CERB), Morocco. Several Researches have studied the appropriate cutof 2. Population and Methods values of hematologic features for �-thalassemia. In adults, �- A total of 1658 newborns were screened for �-thalassemia thalassemia carriers’ diagnosis is based on microcytosis and in the three provincial hospitals of Tangier, Tetouan, and normal HbA2 level [14]. In newborns, an efective primary Larache cities between January 2015 and December 2016. screening with 100% accuracy could be carried out using Te studied region is located in the northwestern part of mean corpuscular volume less than 95fL, mean corpuscu- Morocco and on the southern Mediterranean coast of the lar hemoglobin less than 30 pg, and/or the presence of thalassemic belt (Area: 7098.8 km2, Population: 2386852 hemoglobin Bart’s fraction [15, 16]. Tese were the inclusion inhabitants (national census of 1st September 2014) [13]). Te criteria in our study. choice was based on the region’s history with malaria, its high All the newborns were analyzed for hematological consanguinity, and the increase in length of stay and cumu- indices, including red blood cells (RBC), hemoglobin con- lative hospitalized days due to hemoglobinopathies in the centration (Hb), mean cell volume (MCV), mean cell region hospitals. Figure 1 shows the geographic location of the hemoglobin (MCH), and mean corpuscular hemoglobin studied region divided into three provinces: Tangier Assilah concentration (MCHC) using an automated cell counter Fahs-Anjra, Tetouan M’diq-Fnideq, and Larache (Figure 1). SYSMEX XT-1800 (Kobe, Japan). Ferritin level was assessed Our study is a newborn screening, using new techniques with established protocols using the automated VIDAS- of molecular biology, for an accurate determination of the �- BIOMERIEUX Immunoanalyzer, and the Hb separation thalassemic genotypes and their frequencies. Tis can lead studies were done by capillary electrophoresis method (MIN- diagnostic centers to develop an efective genetic counseling ICAP Sebia, Paris, France). of couples and a better diagnosis. Genomic DNA was isolated from leukocytes using the Umbilical blood samples of 4ml volume were taken QIAamp5 Blood Mini Kit (Qiagen, Germany) and stored ∘ by qualifed midwives immediately afer delivery on EDTA at − 20 C. Molecular screening of most common �-globin 3.7 4.2 tubes from newborns in the hospitals. Each sample was mutations (-� ,-� ) was performed using Gap-PCR (Gap- accompanied by prior consent signed or thumb printed by the Polymerase Chain Reaction) as previously described by Dode´ mothers. All the parents were of the region descents and were and Samuel S. Chong [17]. MLPA analysis was carried out well informed about the study procedures and objectives. using the SALSA MLPA Kit, HBA140-B4 Kit (MRC- Holland, BioMed Research International 3 Table 1: Complete Blood Count and ferritin profle in the north of Morocco. Ferritin level Province population Anemic Non Anemic Microcytic hypochromic anemia Other anemia∗ Low Normal High Tangier, Assilah-Fahs-Anjra 633 163 470 79 84 52 11 16 Tetouan,M’diq,Fnideq 483 109 374 66 43 46 5 15 Larache 542 169 373 119 50 75 9 35 ∗ means other anemia: macrocytic normochromic, macrocytic hypochromic, normocytic normochromic, and normocytic hypochromic. Table 2: Mutations and their allelic frequencies in the studied region. Molecular defect Allele frequencies (%) Genotypes N 3.7 -� / �� 6 3.7 3.7; � del -� 0.33 -� / �� 1 3.7 3.7 -� /-� 2 �4.2 �� �4.2 - / 2 - 0.12 4.2 4.2 -� /-� 1 anti 3.7 anti 3.7 -��� 0.06 -��� / �� 2 HS-40 0.03 ��/ �� with HS-40 del 1 Med I --Med I 0.03 -- /�� 1 Netherlands), according to the manufacturer’s instructions higher levels of cord blood ferritin. Te ID was found in 173 [18]. It was used to detect copy number changes of 26 diferent newborns in the studied region (10.43%). sequences in the �-globin cluster on chromosome 16p13.3. Capillary electrophoresis of hemoglobin was assessed PCR was performed with a standard thermocycler (Eppen- to quantify the diferent Hb variants in the newborns. No dorf, Germany), and the separation of the amplifcation newbornhasshownHbH,HbC,HbE,orHbDvariants. products was done by capillary electrophoresis on the Genetic Hb Bart’s was identifed in 17 newborns, 2 in Tangier Assilah Analyser 3130XL (Applied Biosystems5). Ten the results Fahs-Anjra province, 2 in Tetouan M’diq-Fnideq province, were analyzed with Cofalyser5 (Applied Biosystems5). Te and 13 in Larache province. Te percentage of Hb Bart’s deletional origin of �-thalassemia was investigated as no carriers at