Pattern of Hemoglobinopathies: A Cross-sectional Study in North of Morocco

Background: Hemoglobinopathies are the most frequent widely spread genetic disorders. In Morocco, epidemiological and clinical data are scarce. The present study aims to determine the spectrum and geographic distribution of hemoglobinopathies in Larache province, North of Morocco. Methods: A retrospective cross-sectional study was conducted from January 2015 to December 2018 at the provincial hospital of Larache city. All patients’ records having a hemoglobinopathy were analyzed. Background data (age, gender, and origin) of each case were analyzed. Results: Our study showed an overall frequency of hemoglobinopathies of 3.6%. Sickle cell trait was registered to be the most common disorder in our studied population (42.3%), followed by β-thalassemia Trait (20.2%), sickle cell disease (19.8%), major β-thalassemia (9.8%) and sickle/β-thalassemia (8%). The average age of subjects with hemoglobin disorders was 17.5 years. The majority of the patients (66.5%) were less than 20 years old. A disparity in the diseases geographic distribution was observed, 74.6% of patients came from rural areas. Conclusions: The present study is the rst of its kind offering comprehensive data on hemoglobinopathies pattern in Northern Morocco. Our work may lay the foundation for screening programs for better prevention and coverage. was found to be the most touched (201; 45.7%), followed by Zouada (65; 14.8%), Khmis-Sahel (18; 4.1%) and 10% over the rest of the province. A statistically signicant differences (P > 0.05) were found across the hemoglobinopathies in the different districts. Comparing normal and abnormal subjects in our study, there was no statistical signicance regarding the sex of the subjects (Odds ratio = 0.719, P-value > 0.05). The autosomal recessive inheritance of these diseases explains the absence of a preponderance of one sex over another (25, 35). A slight female predominance was detected among Dahmani et al. and Agouzal et al. studies subjects (6, 13). Other studies reported a slightly higher percentage of males (10, 36). These differences would be reliable to the demographic data of each cohort.

from two months to 70 years old.
Out of the 440 hemoglobin disorder's patients, βS mutation was detected in 308 subjects (70%). 186 of them (42.3%) carried the heterozygous form βA/βS, (sex ratio: 0.95), 87 patients (19.8%) were homozygous βS/βS (sex ratio: 1.02) and 35 patients (8%) (Sex ratio: 1.18) were detected to have an Hb S/β-thalassemia. SCT subjects showed an average of 38.17 ± 4.11% of Hb S with a slight decrease in the Hb A. SCA patients were characterized by the presence of Hb S fraction with an average of 62.96 ± 13.4%, a minor expression of Hb A and a high-level of fetal hemoglobin Hb F (mean value: 17.98 ± 9.39%). The double heterozygosity Hb S/β-thalassemia showed elevated rates of Hb S at 69.39 ± 17.78%, Hb A2 rates of 5.24 ± 1.88% and decreased Hb F levels.
Major β-Thalassemia patients (9.8%) (Sex ratio: 1.38) registered the lowest hemoglobin average and microcytic hypochromic anemia. A signi cant decrease was observed in Hb A (20.35 ± 14.4%), an increase in Hb A2 and high increase in Hb F levels.
β-Thalassemia trait was present among 89 subjects (20.2%) (Sex ratio: 1.06). They showed milder anemia and a slight decrease of Hb A associated with an increase of both Hb A2 and Hb F compared to normal subjects.
Hematological parameters and hemoglobin variants levels are presented in Table 1.

Discussion
Hemoglobinopathies are the most common inherited disorders with a worldwide occurrence. Some geographical areas have high prevalence of these diseases, with nearly 80% of new hemoglobinopathies births are occurred in developing countries (4). In Morocco, these diseases represent one of the major public health problems. The exact data regarding the prevalence and spectrum of these diseases is still uncomplete. In the absence of an adequate strategy to manage hemoglobinopathies, its prevalence may continue to increase, and risk to pose economical and psychological burden on the affected individuals, them families and the whole society. Hence, the population needs to be screened for hemoglobin disorders so that appropriate measures for treatment and prevention can be taken.
This cross-sectional study is the rst approach to understand the situation of hemoglobinopathies in the North of Morocco. A high rate was observed in rural (74.5%) compared to urban patients (25.5%). Compared to AA subjects distribution, hemoglobinopathies were statistically independent of the origin of the subjects (Odds ratio = 1.047, P-value > 0.05). In agreement with our results, Belala et al. reported that 81.13% of their studied patients in Kenitra province, Morocco were from rural areas (10). This may be a result of the rural character and the low genetic awareness in the region. In our study, the highest frequency was observed in Laouamra commune (45.6%) with the presence of diversity in the phenotypic expression and genetic aspects of hemoglobinopathies. This difference in distribution of the disease forms is often found in the small geographic areas from different countries (11). Our results are in agreement with those of Agouzal et al. and Laghmich et al. suggesting Laouamra as a hotspot of hemoglobinopathies in the country (6,7). The concentration of patients in this area could be a result of three major risk factors: i) its proximity to Kenitra province; the most touched region reported in Morocco (12,13). Migration ows within the two provinces contribute toward the increase of hemoglobinopathies range. ii) Its proximity to Chefchaouen province where the last autochthonous malaria case was registered in the country (14). iii) the high prevalence of consanguineous marriage in the northern Morocco population (15,16). In fact, in inbred populations, the deleterious alleles are reintroduced into descendants, which promotes the incidence of these pathologies among Moroccan risked families (17,18). The high rates of consanguineous marriages, migration and early mortality may explain the observed Hardy-Weinberg disequilibrium in our data set. These results re ect the urgent need for community awareness and mandatory screening programs in these high-risk areas. Our results estimated the allelic frequency of the βS gene at 1.6%. This is considered as high compared to the overall frequency reported in Morocco (1.2%) (19). Regarding the Mediterranean Basin; where hemoglobinopathies are widely spread; the average frequency of the β S gene is 1.89% and 0.8 to 3.5% in Tunisia and Algeria respectively (20,21). The same prevalence was estimated at 0.39% in Spain and 0.57% in Portugal (22). The frequencies reach 30% in sub-Saharan African countries (21).
Of the 440 detected hemoglobin disorders carriers in our study, Sickle cell trait occurred at the highest frequency (42.3%), followed by β-thalassemia Trait (20.2%), sickle cell disease (19.8%), major β-thalassemia (9.8%) and sickle/β-thalassemia (8%). The same pattern is observed by Dahmani et al. showing that AS carriers were dominant in Kenitra province with a rate of 40.6%, 23% were SS patients, 3.2% were β-thalassemia trait and the double heterozygous S/β-thalassemia presents 2.9% out of all the patients (13). our nding are in accordance with other studies including Saudi, Indian, Spanish, and Gabon populations (23)(24)(25)(26). The high SCT prevalence in our data set could be the result of historical selective pressure of malaria in Larache province. Malaria provides survival advantage and hence, higher prevalence of hemoglobinopathies carriers (27). A Study on the endemic history of malaria in these areas remains highly recommended.
Besides, 1.5 to 3% of the Moroccan population are carriers of β-thalassemia (28), which reveals that our allelic frequency (0.9%) is obviously underestimated. Regarding the Mediterranean countries, β-thalassemia rates reach 4.4% and 3% in Tunisia and Algeria respectively (29,30). In Egypt, El-Beshlawy et al. reported that β-thalassemia carrier rates is varying from 5.3 to 9% (31). Many others studies indicate that β-thalassemia is common in the Gulf region such as Oman, UAE and Qatar with 5.3%, 2.4% and 17% respectively (32)(33)(34). Further studies including Iron quanti cation and other techniques are required for accurate determination of the prevalence of hereditary persistence of fetal hemoglobin (HPFH) and fusion chains such as delta-β-thalassemia.
Regarding patients gender, the sex ratio obtained was 1.01; the disease occurrence does not present a signi cant difference and it affects both sexes in a similar way (χ2 = 0.125, P-value > 0.05). Comparing normal and abnormal subjects in our study, there was no statistical signi cance regarding the sex of the subjects (Odds ratio = 0.719, P-value > 0.05). The autosomal recessive inheritance of these diseases explains the absence of a preponderance of one sex over another (25,35). A slight female predominance was detected among Dahmani et al. and Agouzal et al. studies subjects (6,13). Other studies reported a slightly higher percentage of males (10,36). These differences would be reliable to the demographic data of each cohort.
In term of patients age, our population study was particularly young with 66.5% under 20 years. Dahmani et al. detected the same result; 53.3% of its population have less than 16 years (13). This young age is a result of the earliness of the clinical manifestations of the hemoglobinopathies that become apparent after the rst 6-9 months of age. Before that age, high levels of Hb F inhibit the sickling and reduces the severity of these diseases (37). Additionally, the delay of diagnosis and treatment lead to various complications (4,(38)(39)(40), subsequently the death of those affected during the rst few years of life with reported excess mortality reaching up to 92% (37). The limited medical service resources makes the access to optimum care more di cult for the majority of the patients. The main constraints are the high cost of management, the availability of safe blood for transfusions and iron chelating agents. In the absence of transfusion, children die before the age of six years and if transfused and non-chelated they usually die before 20 years (12).

Conclusions
Our study is the rst one in Morocco to determinate the pattern of hemoglobinopathies in a Moroccan population. Our results show high frequencies of these abnormalities especially among young children in the studied region. It highlights hemoglobinopathies as severe public health in the studied province. Since no effective cures are available for these diseases, the major approaches to the control and management of these diseases are population screening, genetic counseling and prenatal diagnosis, and management of symptoms. The appropriate coverage may help to reduce the diseases prevalence and decrease both economic and psychological burden on patients.

Declarations
Ethics approval and consent to participate The study was approved by the Princess Lalla Meriem hospital ethics committee, following the principles of the Declaration of Helsinki. Written informed consent was obtained from all subjects and/or their guardians' prior recruitment.

Consent for publication
Not applicable.

Availability of data and materials
The datasets used and/or analysed during the current study are available from the corresponding author on reasonable request. Figure 1 Map of the study area. Figure 2 Age distribution of the different hemoglobinopathies patients.

Figure 3
District wise distribution of hemoglobinopathies in Larache province.