Molecular heterogeneity of β‐thalassemia variants in the Eastern region of Morocco

Abstract Background β‐thalassemia syndromes are the most common hereditary blood disorders in the world and are recognized as a major health problem in Morocco. They are characterized by the reduction or the absence of β‐globin chain synthesis. The severity of the disease depends on the nature of the variants affecting the β‐globin gene (HBB), and each ethnic group has its own mutation spectrum. Hereby, we present, for the first time, the molecular profile of β‐thalassemia in the Eastern region of Morocco. Methods This study concerns 39 cases from 33 families who were enrolled in the BRO Biobank. Nineteen were diagnosed with β‐thalassemia major and 20 with β‐thalassemia minor. To detect mutations of the β‐globin gene, we have used RFLP‐PCR and Sanger sequencing. Results Nine known β‐thalassemia variants have been identified. Among these, we reported, for the first time in the Moroccan population, the Czechoslovakian variant C38/39(‐C) at homozygous state. The C39(C > T) was the most frequent variant (72.54%), followed by FSC5(‐CT) (5.88%), FSC6(−A), IVS‐1‐110(G > A), −29(A > G), C38/39(‐C) (3.92% each), and finally by IVS‐I‐1(G > A), IVS‐II‐1(G > A), and −56(G > C) (1.96%). Of particular interest this mutational spectrum of β‐thalassemia is very different from that found in previous studies in Morocco or in other North African countries. Conclusion This study is the first contribution to the description of the molecular profile of β‐thalassemia in the Eastern region of Morocco. It shows the high molecular heterogeneity of β‐thalassemia in our country. Therefore, these results can be valuable for the implementation of carrier screening, genetic counseling, and prenatal diagnosis programs.

Clinically, individuals with thalassemia major show a mucocutaneous pallor, severe anemia, hepatosplenomegaly, and therefore, require regular transfusions. Patients with thalassemia intermedia are diagnosed later at the age of 2 to 6 years old with a milder anemia or in adulthood with bones and face deformities and gallstones; they usually do not require a regular transfusion. Individuals with thalassemia minor, also known as carriers, are asymptomatic and have slight anemia (Cappellini et al., 2008;Olivieri, 1999).
The βthalassemia is originally common in tropics and subtropics areas such as the Mediterranean countries, the Middle East, central Asia, India, and Southern China as well as countries along the north coast of Africa and in South America. Nowadays, βthalassemia is the most common disease all over the world as a result of population migration (Weatherall & Clegg, 2001).
As in many Mediterranean countries, thalassemia is a major public health concern in Morocco and has not been fully characterized. The previous studies on the genetic basis of βthalassemia were mainly focused in the western and northern areas and have revealed high molecular defects heterogeneity (Agouti et al., 2007;Lemsaddek et al., 2003). However, there is no data about the molecular profile of βthalassemia in the eastern region of Morocco. Therefore, the aim of this study is to investigate the molecular defects in patients with βthalassemia in this region which is important for carrier screening, and for establishing genetic counseling and prenatal diagnosis in Morocco.

| Description of the study and ethics statement
This cohort study was achieved in the Genetics Unit of the Faculty of Medicine and Pharmacy of Oujda. According to the BRO Biobank protocol (Lhousni et al., 2020), the participants were enrolled by the Pediatric departments of the University Mohammed IV Hospital of Oujda and the Regional Center of Blood Transfusion of Oujda, Morocco.
The study was approved by the ethical committee, of the Faculty of Medicine and Pharmacy of Casablanca, in accordance with the declaration of Helsinki standard.

| Subjects
The study was conducted on 29 individuals from the BRO Biobank. Nineteen patients from 18 families were diagnosed with βthalassemia major and 20 subjects from 15 families have βthalassemia minor. All the subjects or their representative family gave written informed consent to the study.

| Mutation screening
DNA extraction from whole blood and saliva sample was performed manually by the phenol chloroform-isoamyl alcohol method. The most common βthalassemia variants in the Moroccan population (C39(C > T), IVSI-1(G > A) and FSC6(−A)) were characterized using PCR followed by restriction fragment length polymorphism (RFLP). The primers used for amplification of HBB gene region (HBB-mut) were previously described (Clark & Thein, 2004). The PCR products were directly digested by BfaI, Bsu36I, and BsaBI Restriction enzymes (New England Biolabs, Inc.) to check the three most common variants, respectively (C39(C > T), IVSI-1(G > A), and FSC6(−A)).
In order to confirm the PCR-RFLP results and to look for other variants in the HBB gene, two regions were sequenced (Seq1 and seq2). Sanger sequencing was performed on an ABI 3500 Genetic Analyzer (Applied Biosystems-Thermofisher). The sequences were compared to the HBB reference sequence (NM_000518.5) using the SeqScape Software Version 4.0 (Applied Biosystems).

| Statistical analysis
Statistical analysis was carried out using IBM SPSS Statistic Software Version 21. A p-value < .05 was considered statistically significant.
The median age at diagnosis was around 6 months for patients with βthalassemia major and 3-years-old for those with βthalassemia minor. βthalassemia is clinically heterogeneous, all Patients with βthalassemia major have developed more serious symptoms while βthalassemia trait patients showed mild symptoms. Table 1.summarizes the clinical features and hematological parameters of the participants.

| Molecular analysis
Thirty-nine patients were studied, 19 with βthalassemia major and 20 with βthalassemia minor. As six patients were found in sibling, they were excluded when calculating the frequency of βglobin gene variants. Therefore, 52 mutated alleles were evaluated. A total of 10 βglobin gene variants were detected; most of them have a Mediterranean origin. According to their effect on gene function, five variants affect the secondary structure of the βglobin protein such as C39(C > T), FSC6(−A), FSC5(-CT), C38/39(-C), and SCD6(A > T), three variants lead to defective mRNA synthesis such as IVS-I-1(G > A), IVS-1-110(G > A), and IVS-II-1(G > A). The last two variants (−29(A > G), −56 (G > C)) are in the promoter region, and decreases the abundance of the βglobin mRNA.
Concerning the genotypic pattern, among the 18 patients suffering from βthalassemia major, 12 (67%) were homozygous while six patients (33%) were compound heterozygous. Table 2 shows a summary of the genotypic pattern of the studied families. The frequency of homozygous variants was 55.56% for C39(C > T) and 5.56% for FSC5(-CT) and C38/39(-C). It should be noted that all compound heterozygous patients carried the C39 in one allele. While the second allele was IVS-I-110(G > A) in two cases and − 29(A > G), IVS-II-1(G > A), FSC5(-CT), or SCD6(A > T) in only one case each.

| DISCUSSION
βthalassemia is a blood disorder that result in inherited defects in the hemoglobin βglobin chains production. It is the most common genetic disorder worldwide and is recognized as a major public health concern in the Mediterranean countries, the middle-Eastern, Indian The subjects with compound heterozygous βthal/sickle and βthal/ALL were excluded from CBC and CE the analysis.
T A B L E 1 Distribution of clinical features and some hematological parameters regions, and Southeast Asia and particularly in North Africa (Agouzal et al., 2010). The incidence and prevalence rates of thalassemia are influenced by demographic and cultural characteristics of populations. For instance, it is well documented that Morocco and all Arabic countries display high rates of consanguinity (Mohammed et al., 2019;Tadmouri et al., 2009;Talbi et al., 2007). In this study, the consanguinity rate among the βthalassemia patients was estimated at 39.39%. This finding is consistent with the previous study which showed that the consanguinity rate in Eastern Morocco region is higher than the national average (29.58% vs. 22.79%) (Mohammed et al., 2019;Talbi et al., 2007). Thereby, it is not surprising to see a strong occurrence of autosomal recessive diseases such as thalassemia in our population (Habibeddine et al., 2018;Jaouad et al., 2009;Laghmich et al., 2019). Regarding molecular investigation of β globin gene, we report here, nine βthalassemia pathogenic variants Frameworks. Framework identification could be used as an indirect method in prenatal diagnostic programs to track variant alleles and thus differentiate between normal and mutant alleles (Akhavan-Niaki et al., 2012;Hashemi-Soteh et al., 2017). Furthermore, two other polymorphisms at the UTR region (3′UTR + 34(C > A) and 3′UTR + 101(G > C)) were found. The 3′UTR + 101(G > C) was reported only in Palestinian βthalassemia patients (Sirdah et al., 2013) while the 3′UTR + 34 (C > A) variant have not been investigated yet and was reported in ClinVar database as likely benign SNV contrary of the 3′UTR + 101(G > C) (Smith et al., 2015). Altogether, this variant profile confirms the high molecular heterogeneity and could explain the frequency (33%) of compound heterozygosity described in our study.
It is to highlight that all of the variants were previously described in the Moroccan population except for the C38/39(-C) which has never been reported before. The patient, originated from Guercif, was homozygous, while the parents and the siblings were carriers. The C38/39(-C) is very rare variant and was only found in Czechoslovakian population and most affected families come from central Moravia (Divoka et al., 2016;Indrak et al., 1991Indrak et al., , 1992. Previous haplotype analysis studies showed that C38/39(-C) is linked only to Haplotype II (HindIII Gγ; HinfIβ; HincII β ψ; RsaI β; AvaII β; HindIII Aγ [− + − − − +]) (Kynclová et al., 1998) and interestingly in consistent with this data, β 0 C38/39(-C) was the one and only variant linked to the FWIII in our study, indicating a distinct origin or a recent occurrence. Further studies are necessary to understand the occurrence of this variant in the eastern region of Morocco.
Concerning the distribution of variants frequencies between several studies conducted in Moroccan population, it seems obvious that the mutational spectrum of βthalassemia found here is very different from that found in previous studies in Morocco or in other North African countries (Agouti et al., 2007;Lemsaddek et al., 2003Lemsaddek et al., , 2004. Two major differences were observed: First, all the βthalassemia major patients were homozygous or compound heterozygous for C39(C > T). Furthermore, it has been noticed that this variant, though the most common in all previous studies, showed   a much higher frequency (71.15%) compared to those found in Morocco (15.56%-28.05%) (Agouti et al., 2007;Lemsaddek et al., 2003Lemsaddek et al., , 2004, in Algeria (26%-43%) (Abdaoui et al., 2019;Bennani et al., 1994;Boudrahem-Addour et al., 2009;Labie et al., 1990) and in Tunisia (49%-37.50%) (Chouk et al., 2004;Fattoum et al., 2004;Sahli et al., 2016). The second particularity in our result concern the three variants (FSC8(−AA), IVS-I-6(T > C), and IVS-II-745(C > G)) described in previous studies as three of the five most common variants in Morocco, are surprisingly absent in the Eastern region of Morocco and are rare in Algerian and Tunisian population. Table 4 shows the significance variation of the variants found in our study compared to the previous studies in Morocco and the last two published studies in Algeria and Tunisia. As mentioned above, the C39(C > T) was the most common variant in our cohort but as well as in Western Mediterranean countries and the Maghreb countries (Khelil et al., 2010). The highest frequency was found in Sardinia (95.7%) (Cao et al., 1991) and several studies have suggested a possible Roman origin and support the idea of its introduction into the Maghreb countries during the Roman period and into the western Mediterranean countries by the crusades during the middle ages (Khelil et al., 2010;Zahed, 2001).
The FSC6(−A) reaches a high frequency in North African countries. It was the third most common variant in Algeria (17.9%-12.9%) and Tunisia (7.02%) and it was the fifth most common variant in Morocco Chouk et al., 2004;Labie et al., 1990). It is also frequent in the Portuguese and Bulgarian population (8% and 4.9%, respectively) (Faustino et al., 1999;Petkov & Efremov, 2009).
Morocco could be explained by it recent introduction by migrants from neighboring countries (Khelil et al., 2010;Zahed, 2001). The −29(A > G) variant was found among black and Asian population and was also reported in Maghreb countries, Spain and China (Pereira et al., 2009;Xu et al., 2004). It was more prevalent in Morocco and less frequent in Algeria and Tunisia (8.5%, 3.8%, and 1.08%, respectively) Bennani et al., 1994;Chouk et al., 2004). The frequency decreases from Morocco to Tunisia which could be explained by its introduction by the caravan routes or the expansion of the Almoravid Dynasty (Lemsaddek et al., 2004).
IVS-I-1(G > A), IVS-II-1(G > A), and −56(G > C) were the rarest variants identified in this study. IVS-I-1(G > A) was found among the five most common variants in Maghreb Countries (Agouti et al., 2007;Labie et al., 1990;Lemsaddek et al., 2003). The frequency of this variant was higher in Algeria (10%-14.5%), followed by Morocco (5%-13.3%) and finally, Tunisia (4%) (Abdaoui et al., 2019;Chouk et al., 2004;Fattoum et al., 2004;Labie et al., 1990;Lemsaddek et al., 2003). Moreover, this variant was also described as the most frequent variant in some European countries such as Czech Republic (27%), Hungary and Middle East countries (Divoka et al., 2016;Ghoti et al., 2017;Ringelhann et al., 1993), and it has been suggested that the variant was originated from Eastern Europe (Makhoul et al., 2005). IVS-II-1(G > A) in the other side was the most common variant in Iran with a frequency ranging between 25.61% and 40.33% (Jalilian et al., 2017;Maryami et al., 2015). It was reported as the first or the second most frequent variant in some middle-Eastern countries and has been found in all Arab countries (Zahed, 2001). This variant has a Mediterranean origin, however, a recent report has hypothesized its West African origin (Broquere et al., 2010). The frequency of this variant in our study is lower compared to that found in previous studies in Morocco (1.96 vs 3.21%-2.53%), but comparable to that in Tunisia (1.62%). In Algeria it was very rare (0.9%) Boudrahem-Addour et al., 2009;Chouk et al., 2004;Lemsaddek et al., 2004).
The −56(G > C) variant was described for the first time in Morocco at compound heterozygous with IVS-I-1(G > A) in a woman originated from Algeria. She was diagnosed with βthalassemia intermedia and remained asymptomatic without the need of regular transfusion until the age of 18 years old . It was also described at homozygous state in a Tunisian patient who had only a mild anemia and never required transfusion (Douzi et al., 2015). In the present study, −56(G > C) was identified in a patient in compound heterozygous with −29(A > G) who had never required transfusion. He showed a typical βthalassemia trait hematological features associated with slightly elevated HbF (HBF: 5.8%) and the Complete Blood Count (CBC) was as follows Hb: 10 g/dl, MCV: 60.01 fl, MCHC: 31.6, and HBA2: 5.9%. The patient is stable and takes iron supplement. Therefore, this variant seems to act like a silent variant. Indeed, when combined with a β 0 variant it can leads to an intermedia phenotype and, when combined with β + variants or in homozygous state it leads to a βthalassemia trait. In the heterozygous state, the carriers remain asymptomatic.
Together, these data suggest that the mutational spectrum of βthalassemia in eastern region of Morocco is different from that reported in previous studies conducted in Morocco or Algeria and Tunisia. Therefore, each geographic region would have its own type and distribution of variants. This genetic diversity may originate probably gene flow due to population migration. Further studies, based on SNV (Single Nucleotide variation) or STR (Short Tandem Repeat), are necessary to understand the origin of βthalassemia variants.
Among the subjects, one was diagnosed with βthalassemia minor and Acute Lymphoblastic Leukemia (ALL). He received red cell transfusions combined with iron chelation therapy and chemotherapy. The coexistence of hematological malignancy and βthalassemia major is not a rare condition. Multiple cases were reported in different countries with βthalassemia major associated with Hodgkin and non-Hodgkin lymphoma, chronic myeloid leukemia, acute lymphoblastic leukemia, hepatocellular carcinoma, and thyroid malignancies (Alavi et al., 2013;Sherief et al., 2020). However, the association between βthalassemia minor and malignancies is very rare.
It has been suggested that there is no relationship between βthalassemia and the occurrence of hematological malignancies including ALL. Indeed, the coexistence of βthalassemia and the ALL was accidental (Alavi et al., 2013;Sherief et al., 2020) and the ALL could be due to genetic predisposition or others risk factors (Halawi et al., 2017;Tuğcu et al., 2014).

| CONCLUSION
This study provided, for the first time, the molecular investigation of βthalassemia in eastern region of Morocco which indicates a high molecular heterogeneity. One variant C39(C > T) accounts for 72%, and the rare variant C38/39(-C) was described for the first time in Morocco. The mutational spectrum was unique compared to that reported in other region of Morocco or in other North African countries. Therefore, these data are crucial to implement a prevention programs and the establishment of genetic counseling and prenatal detection for βthalassemia in Morocco.