# Comprehensive analysis of sickle β+-thalassemia genotypes and their associated HbA levels in France

Cecilia Baltus, Stéphane Moutereau, Nathalie Couque, Bichr Allaf, Muriel Giansily-Blaizot, Julian Boutin, Ketty Lee, Emmanuelle Bernit, Estelle Cadet, Victor Bobee, et al.

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Cecilia Baltus, Stéphane Moutereau, Nathalie Couque, Bichr Allaf, Muriel Giansily-Blaizot, et al. Comprehensive analysis of sickle β+-thalassemia genotypes and their associated HbA levels in France. Blood Cells, Molecules and Diseases, 2025, 112, pp.102923. 10.1016/j.bcmd.2025.102923. hal-05019378

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# Comprehensive analysis of sickle β⁺-thalassemia genotypes and their associated HbA levels in France

Cecilia Baltus<sup>a</sup>, Stéphane Moutereau<sup>b,c</sup>, Nathalie Couque<sup>d</sup>, Bichr Allaf<sup>e</sup>, Muriel Giansily-Blaizot<sup>f</sup>, Julian Boutin<sup>g</sup>, Ketty Lee<sup>h</sup>, Emmanuelle Bernit<sup>i</sup>, Estelle Cadet<sup>j</sup>, Victor Bobee<sup>k</sup>, Véronique Picard<sup>l</sup>, Serge Pissard<sup>c,m</sup>, Frédéric Galactéros<sup>b,n</sup>, Céline Renoux<sup>a,o,p</sup>, Philippe Connes<sup>o,p</sup>, Patricia Aguilar-Martinez<sup>f</sup>, Corinne Pondarre<sup>q,r,1</sup>, Philippe Joly<sup>a,o,p,*1</sup>

ARTICLE INFO

Keywords:

Sickle beta(+)-thalassemia

HbA levels

Genotype-phenotype correlation

ABSTRACT

We retrospectively reviewed the clinical records of 228 HbS/β⁺-thal patients. The different genotypes were distributed into three groups according to their mean residual HbA levels: <10 % (group 1; n = 22), between 10 and 20 % (group 2; n = 175) and > 20 % (group 3; n = 31). Routine red blood cells and hemoglobin parameters were compared between the three groups. Sixteen different sickle β⁺-thal genotypes were identified but only four of them were associated with a residual HbA level below 10 %. Patients of this group exhibited a more severe anemia (Hb < 10 g/dL; reticulocytes >200 G/L) compared to the two other groups. However, no difference could be observed on those parameters between patients of group 2 and 3, as well as for the main RBC parameters. According to our study, >80 % of the sickle β⁺-thalassemia patients in France have a residual HbA level beyond 10 % and a mild to moderate anemia. Only four β⁺-thal variations (all affecting the splicing process) would lead to a potentially severe SCD syndrome in association with HbS (HbA < 10 %) but this result should be confirmed in a prospective clinical study.

<footnote><sup>a</sup> Service de Biochimie et de Biologie Moléculaire, Centre de Biologie et de Pathologie Est, Hospices Civils de Lyon, Bron, France</footnote>

<footnote><sup>b</sup> Département de Biochimie et Pharmacologie, DMU de Biologie, Hôpital Universitaire Henri-Mondor (AP-HP), Créteil, France</footnote>

<footnote><sup>c</sup> Inserm-IMRB U955 eq2, Université Paris Est Créteil, Créteil, France</footnote>

<footnote><sup>d</sup> Service de génétique moléculaire, Département de Génétique, Hôpital Universitaire Robert-Debré (AP-HP), Paris, France</footnote>

<footnote><sup>e</sup> Service de Biochimie, Hôpital Universitaire Robert-Debré (AP-HP), Paris, France</footnote>

<footnote><sup>f</sup> Service d'Hématologie biologique, Centre de référence des syndromes drépanocytaires majeurs, thalassémies et autres pathologies rares du globule rouge et de l'érythropoïese, CHU de Montpellier, Université de Montpellier, France</footnote>

<footnote><sup>g</sup> Service de Biochimie, Hôpital Pellegrin, CHU de Bordeaux, Bordeaux, France</footnote>

<footnote><sup>h</sup> Laboratoire de Génétique moléculaire, CHU de Guadeloupe, Pointe-à-Pitre, France</footnote>

<footnote><sup>i</sup> Centre de référence pour la Drépanocytose, les Thalassémies et les maladies constitutives du Globule Rouge et de l'Erythropoïese, CHU de Guadeloupe, Pointe-à-Pitre, France</footnote>

<footnote><sup>j</sup> Laboratoire de Génétique Médicale, CHU Amiens-Picardie, Amiens, France</footnote>

<footnote><sup>k</sup> Laboratoire d'Hématologie, CHU de Rouen, Rouen, France</footnote>

<footnote><sup>l</sup> Laboratoire d'Hématologie biologique - Génétique moléculaire, Hôpital Universitaire Bicêtre (AP-HP), Le Kremlin-Bicêtre, France</footnote>

<footnote><sup>m</sup> Département de Génétique, DMU de Biologie, Hôpital Universitaire Henri-Mondor (AP-HP), Créteil, France</footnote>

<footnote><sup>n</sup> UMGGR GHU Henri Mondor APHP, U-PEC Créteil, France</footnote>

<footnote><sup>o</sup> Laboratoire interuniversitaire de Biologie de la Motricité (LIBM) EA7424, Team « Vascular Biology and Red Blood Cell » Université Claude Bernard Lyon 1, Université de Lyon, Lyon, France</footnote>

<footnote><sup>p</sup> Laboratoire d'Excellence du Globule Rouge (Labex GR-Ex), PRES Sorbonne, 79015 Paris, France</footnote>

<footnote><sup>q</sup> Centre Hospitalier Inter-Communal de Créteil, Créteil, France</footnote>

<footnote><sup>r</sup> Inserm U955, université Paris XII, Créteil, France</footnote>

<footnote>* Corresponding author at: Service de Biochimie et de Biologie Moléculaire, Centre de Biologie et de Pathologie Est, Hospices Civils de Lyon, Bron, France.
E-mail address: philippe.joly@chu-lyon.fr (P. Joly).</footnote>

<footnote n="1">¹ Equal contribution/co-last.</footnote>
# 1. Introduction

Sickle cell disease is an autosomal recessive genetic disorder caused by the presence of a pathogenic variant in the beta-globin gene (HBB: c.20A > T; p.Glu7Val), which results in the production of abnormal hemoglobin (Hb) called HbS [1]. HbS may polymerize under deoxygenated conditions [2], leading to a mechanical distortion of red blood cells (RBCs), namely sickling. Sickled RBCs are poorly deformable and very fragile resulting in vaso-occlusions and chronic hemolytic anemia, respectively [3]. Sickle cell anemia (SCA) is the most common and severe form of SCD and is characterized by the presence of the βS-variation at the homozygous state (S/S). The alternative forms of SCD are caused by a compound heterozygosity between the βS-variation and some other particular β-globin alleles, coding either for a β-globin variant that directly or indirectly promotes HbS polymerization (Hb D-Punjab, Hb O-Arab, HbC) or for a β-thalassemia allele that suppresses or greatly reduces the production of HbA. This latter form of SCD is called sickle cell beta-thalassemia disease which was first described in 1944 by Silvestroni and Bianco [4].

Two main genetic forms of sickle cell beta-thalassemia have been described. Patients with sickle β0-thalassemia genotypes (S/β0-thal) are characterized by a total absence of HbA in their RBCs while those with sickle β+-thalassemia genotypes (S/β+-thal) present a more or less important residual HbA synthesis that partially prevents HbS polymerization and RBC sickling. It is commonly accepted that patients with sickle β+-thalassemia are less severe than those with S/S or S/β0-thal genotypes but there is actually a high heterogeneity in the clinical expression of sickle β+-thalassemia (β+-thal), depending on the nature of the β+-thal allele. Some β+-thal variations, in association with HbS, are characterized by the presence of <10 % residual HbA and may lead patients to exhibit a very severe clinical expression of the disease. On the contrary, some β+-thal variations are very weak (β+↑) or even silent (βsilent) with a quasi-normal HbA synthesis [5], which would limit HbS polymerization, hence decreasing the clinical severity. It would thus be very useful for pediatricians to predict from birth the expected residual HbA level of a new S/β+-thal child based on his genotype only. However, there is currently no data available in the literature.

The primary objective of our study was to genotype a large group of S/β+-thal patients in the French population in order to determine the relative frequency of each genotype and the range of HbA levels. Our secondary objective was to test the association between the residual HbA level and the severity of the anemia (based on the total Hb and reticulocytes count).

# 2. Material and methods

## 2.1. Study population

Two hundred and twenty eight patients (126 males and 102 females) with sickle β+-thalassemia were recruited from ten French medical centers specialized in SCD: Hospices civils de Lyon (n = 63), Henri-Mondor university hospital (Créteil, n = 48), Robert-Debré university hospital (Paris, n = 48), Créteil intercommunal hospital (n = 36), Guadeloupe university hospital (Pointe-à-Pitre, n = 13), Montpellier university hospital (n = 8), Bordeaux university hospital (n = 6), Rouen university hospital (n = 3), Bicêtre university hospital (Kremlin-Bicêtre, n = 2) and Amiens-Picardie university hospital (n = 1). All 228 patients (median age 20 years; range 4 to 71 years) had been previously genotyped in the course of their routine clinical setting after obtaining their informed written agreement (or those of their legal caregivers). This retrospective study followed the guidelines set by the Declaration of Helsinki and was approved by the Hospices Civils de Lyon (Project-study 23-5042).

<caption>**Table 1**</caption>

Epidemiological and biological characteristics of the whole HbS/β+-thalassemia cohort (N = 228 patients).

<table><thead><tr><th>Parameters</th><th>Patients</th><th>N or mean ± SD [range]</th><th>Reference values</th></tr></thead><tbody><tr><td>Age (years)</td><td>228</td><td>20 [4-71]</td><td>/</td></tr><tr><td>Sex ratio M/F</td><td>228</td><td>126/102</td><td>/</td></tr><tr><td>Alpha-thal Wt/Mut</td><td>166</td><td>116/50</td><td>Wt</td></tr><tr><td>HbA (%)</td><td>228</td><td>18.6 ± 6.7 [3.1-52.5]</td><td>94.0-100.0</td></tr><tr><td>HbA₂ (%)</td><td>220</td><td>5.5 ± 0.9 [3.3-8.4]</td><td>2.2-3.2</td></tr><tr><td>HbF (%)</td><td>228</td><td>7.8 ± 5.6 [0.3-26.0]</td><td>0.0-1.0</td></tr><tr><td>HbS (%)</td><td>221</td><td>68.0 ± 6.5 [41.7-89.7]</td><td>/</td></tr><tr><td>RBC (T/L)</td><td>212</td><td>4.90 ± 0.66 [2.92-7.27]</td><td>4.50-6.00</td></tr><tr><td>Hb (g/L)</td><td>215</td><td>111 ± 15 [63-149]</td><td>130-170</td></tr><tr><td>Ht (%)</td><td>212</td><td>33.3 ± 4.5 [21.4-45.0]</td><td>40.0-54.0</td></tr><tr><td>MCV (fl)</td><td>214</td><td>68.6 ± 6.7 [53.0-90.0]</td><td>80.0-100.0</td></tr><tr><td>MCH (pg)</td><td>178</td><td>22.9 ± 2.3 [16.8-29.6]</td><td>27.0-32.0</td></tr><tr><td>MCHC (g/L)</td><td>144</td><td>335 ± 13 [294-362]</td><td>320-365</td></tr><tr><td>Ret (G/L)</td><td>196</td><td>128.6 ± 78.2 [25.9-669.0]</td><td>20-80</td></tr></tbody></table>

Wt: wild-type; Mut: mutated; RBC: red blood cells count; Hb: total hemoglobin; Ht: hematocrit; MCV: mean corpuscular volume; MCH: mean corpuscular hemoglobin; MCHC: mean corpuscular hemoglobin concentration; Ret: reticulo-cyte counts.

## 2.2. Records of biological data

The biological records of all 228 patients were retrospectively reviewed to obtain the results of the most recent blood count and hemoglobin analysis at steady-state (no blood transfusion and no acute SCD complication during the last three months) and without an on-going hydroxycarbamide (HU) treatment. For each patient, the following parameters were recorded: red blood cells (RBCs) and reticulocytes (Ret) counts, hemoglobin (Hb) concentration, hematocrit (Hct), mean corpuscular volume (MCV), mean corpuscular hemoglobin (MCH), mean corpuscular hemoglobin concentration (MCHC) and the percentages of the different hemoglobin fractions (namely HbA, HbA₂, HbF and HbS). Hemoglobin analysis was performed either by cation exchange high performance liquid chromatography (CE-HPLC) using a Variant™ II device (Biorad, Hercules, CA) or by capillary electrophoresis (CE) using a CapillarySTM 2 device (Sebia, Lisses, France). At the genetic level, the results of the HBB Sanger sequencing were noted, as well as the presence or absence of the common -3.7 Kb alpha-thalassemia deletion when it was available [6]. All recorded data were anonymized before analysis.

## 2.3. Statistical analyses

Data are presented as mean ± standard deviation (SD) and analyses were performed using Graph Pad Prism version 10.2.3. Patients were first gathered according to the nature of their β+-thalassemia variation and the median HbA level of each genotype was determined. Three theoretical severity groups were then constituted: group 1 (highest severity) corresponded to patients with a sickle β+-thalassemia genotype characterized by a median residual HbA level below 10 % while group 2 (middle severity) and group 3 (low severity) included patients whose sickle β+-thalassemia genotype is associated with a residual HbA level between 10 and 20 % and beyond 20 %, respectively. These arbitrary HbA thresholds are commonly used by physicians to quickly assess the expected clinical severity of sickle β+-thal syndrome. To assess their biological relevance, patients were distributed among these three groups according to their β+-thalassemia variation and the mean values of the aforementioned hematology and biochemical parameters were compared between groups using a one-way analysis of variance (ANOVA) and post-hoc tests (Tukey) to locate where differences occurred when appropriate. A trend linearity test was also performed between the three groups for each parameter. Statistical significance level was defined when the p-value was lower than 0.05 (p < 0.05).
<figure><img src="image_4.png" /><figcaption>Fig. 1. Repartition of the β⁺-thalassemia variations in our cohort of 228 patients according to the type of molecular defect.</figcaption></figure>

<caption>**Table 2**</caption>

List of the 16 different β⁺-thalassemia variations found in our cohort with their main epidemiological and biological characteristics.

<table><thead><tr><th>HBB variations from 5' to 3'</th><th>Patients N (%)</th><th>Ethnic distribution<sup>a</sup></th></tr></thead><tbody><tr><td><strong>Promoter variations</strong></td><td>147 (65 %)</td><td></td></tr><tr><td>c.-151C > T (–101C > T)</td><td>4 (1.8 %)</td><td>Mediterranean</td></tr><tr><td>c.-140C > T (–90C < T)</td><td>3 (1.3 %)</td><td>Portuguese</td></tr><tr><td>c.-138C > A (–88C > A)</td><td>1 (0.4 %)</td><td>Kurds</td></tr><tr><td><strong>c.-138C > T (–88C > T)</strong></td><td><strong>46 (20.2 %)</strong></td><td><strong>Black, Asian Indian, Syria</strong></td></tr><tr><td>c.-136C > G (–86C > G)</td><td>1 (0.4 %)</td><td>Thai, Lebanese</td></tr><tr><td><strong>c.-79A > G (–29A > G)</strong></td><td><strong>92 (40.4 %)</strong></td><td><strong>Black</strong></td></tr><tr><td><strong>Splicing variations</strong></td><td>64 (28 %)</td><td></td></tr><tr><td>c.75T > A (Cd24 T > A)</td><td>19 (8.3 %)</td><td><strong>American Black, Japanese</strong></td></tr><tr><td>c.92 + 5G > A (IVSI-5 G > A)</td><td>10 (4.4 %)</td><td>Mediterranean, Algerian</td></tr><tr><td>c.92 + 5G > C (IVSI-5 G > C)</td><td>5 (2.2 %)</td><td>Middle East, Asian Indian, East Asian</td></tr><tr><td>c.92 + 5G > T (IVSI-5 G > T)</td><td>6 (2.6 %)</td><td>Mediterranean, Northern European</td></tr><tr><td>c.92 + 6T > C (IVSI-6 T > C)</td><td>5 (2.2 %)</td><td>Mediterranean</td></tr><tr><td>c.93-21G > A (IVSI-110 G > A)</td><td>10 (4.4 %)</td><td>Mediterranean</td></tr><tr><td>c.316-106C > G (IVSII-745 C > G)</td><td>1 (0.4 %)</td><td>Mediterranean</td></tr><tr><td>c.316-3C > A (IVSII-848 C > A)</td><td>8 (3.5 %)</td><td>Black, Egyptian, Iranian</td></tr><tr><td><strong>3'-UTR variations</strong></td><td>17 (7 %)</td><td></td></tr><tr><td>c.*110T > C (PolyA T > C)</td><td>15 (6.6 %)</td><td><strong>Black, Turk</strong></td></tr><tr><td>c.*111A > G (PolyAA > G)</td><td>2 (0.9 %)</td><td>Mediterranean</td></tr></tbody></table>

### 3. Results

The epidemiological and biological characteristics of the whole population are presented in Table 1. The mean HbA level was 18.6% ± 6.7, reflecting an important inter-genotypes variability with a coefficient of variation (CV) of 36%. A similar phenomenon could be observed for HbF with a mean value of 7.8% ± 5.6 (CV of 72%). Hb concentration ranged from 63 to 149 g/L, which perfectly illustrates the very important biological/clinical heterogeneity of sickle β⁺-thalassemia patients. A total of 16 different β⁺-thal variations were identified (Fig. 1 and Table 2): 6 were located in the promoter region of the HBB gene, 8 corresponded to splicing variations and 2 were located in the 3'-untranslated region (UTR). Interestingly, only four variations

were represented in >5% of the patients and globally accounted for 75% of the variations found in the cohort: c.-138C > T (20.2%), c.-79A > G (40.4%), c.75T > A (8.3%) and c.*110 T > C (6.6%). Moreover, 3 β⁺-thal variations were seen in only one patient of the cohort: c.-136C > G, c.-138C > A and c.316-106C > G. Despite the alpha-globin genotype was missing for more than one quarter of our cohort (62 patients out of 228), no difference in the repartition of the –3.7 Kb alpha-thalassemia deletion was observed between groups (precise data not shown).

The 16 different sickle β⁺-thalassemia genotypes were ranged into the three groups described above according to their mean HbA level (Fig. 2). Group 1 comprised four splicing variations (c.95 + 5G > A, c.92 + 5G > C, c.92 + 5G > T and c.316-106C > G) corresponding to 22 patients with a mean HbA level of 7.0 ± 3.5%. Group 2 was constituted of seven variations corresponding to 175 patients with a mean HbA level of 18.6 ± 3.6% while group 3 was constituted of five variations corresponding to 31 patients with a mean HbA level of 26.7 ± 9.5%. In this latter group, two β⁺-thal variations were clearly apart from the others with a HbA level beyond 30%: c.*111A > G and c.-151C > T.

The trend linearity test between these HbA-based severity groups was significant for all the recorded parameters, except for MCHC and RBC count (Table 3). HbS, HbA₂, HbF and Ret values were negatively correlated with HbA (highest values for group 1) while the opposite was observed for Hb, Ht, MCV and MCH (highest values for group 3). However, when focusing on the inter-groups comparisons with the ANOVA and post-hoc tests, very different patterns could be observed. For instance, significant differences were observed between groups 1 and 2 for total Hb, Ht and Ret count but not between groups 2 and 3 (Fig. 3A). Surprisingly, a higher RBC count was observed for group 2 compared to the two other groups, explaining why the ANOVA test was significant for this parameter but not the trend linearity test. Very few significant differences were observed between groups for the RBC parameters (Fig. 3B): slightly higher MCV and MCH values were observed in group 3 compared to the two other groups. On the contrary, differences between groups were much more important for hemoglobin parameters (Fig. 3C). The differences in HbS values were substantial between groups 1 and 2 but very slight between groups 2 and 3, despite being statistically significant. For HbA₂ and HbF values, no difference was observed between groups 1 and 2 while patients of group 3 had significantly lower values compared to the two other groups.

### 4. Discussion

SCD is a widespread genetic disease affecting about 300,000 births per year worldwide [7], mostly in malaria-endemic regions since HbS

<footnote><sup>a</sup> According to the HbVar database. The four variations accounting for >5% of the cohort are in bold. The HBB variations are noted using the cDNA HGVS nomenclature (NM_000518) and the historical nomenclature is indicated in brackets.</footnote>
<figure><img src="image_5.png" /><figcaption>**Fig. 2.** Categorization of the sixteen β⁺-thalassemia variations observed in our sickle cell/β⁺-thalassemia cohort. The variations are displayed from left to right according to their mean residual HbA level, from lowest to highest.</figcaption></figure>

<caption>**Table 3**</caption>

List of the 16 different β⁺-thalassemia variations found in our cohort with their main epidemiological and biological characteristics.

<table><thead><tr><th rowspan="2">Biological parameter</th><th colspan="3">Theoretical severity groups</th><th rowspan="2">p</th></tr><tr><th>Group 1:<br>HbA: 7.0 ± 3.5 %</th><th>Group 2:<br>HbA: 18.6 ± 3.6 %</th><th>Group 3:<br>HbA: 26.7 ± 9.5 %</th></tr></thead><tbody><tr><td>HbS (%)</td><td>77.2 ± 6.7</td><td>67.4 ± 5.0</td><td>65.0 ± 8.3</td><td>****</td></tr><tr><td>HbA<sub>2</sub> (%)</td><td>5.6 ± 0.8</td><td>5.6 ± 0.9</td><td>4.8 ± 0.6</td><td>***</td></tr><tr><td>HbF (%)</td><td>9.5 ± 5.7</td><td>8.3 ± 5.6</td><td>3.5 ± 2.3</td><td>****</td></tr><tr><td>RBC (T/L)</td><td>4.33 ± 0.53</td><td>5.00 ± 0.63</td><td>4.72 ± 0.68</td><td>ns</td></tr><tr><td>Hb (g/L)</td><td>93.4 ± 10.7</td><td>114 ± 13.9</td><td>111 ± 13.5</td><td>***</td></tr><tr><td>Ht (%)</td><td>28.6 ± 3.3</td><td>34.0 ± 4.4</td><td>33.1 ± 4.0</td><td>**</td></tr><tr><td>MCV (fL)</td><td>65.9 ± 6.4</td><td>68.5 ± 6.7</td><td>70.6 ± 6.2</td><td>*</td></tr><tr><td>MCH (pg)</td><td>21.7 ± 2.1</td><td>22.8 ± 2.3</td><td>23.9 ± 2.2</td><td>**</td></tr><tr><td>MCHC (g/L)</td><td>329 ± 9</td><td>335 ± 13</td><td>336 ± 17</td><td>ns</td></tr><tr><td>Ret (G/L)</td><td>243 ± 145</td><td>115 ± 55</td><td>123 ± 58</td><td>****</td></tr></tbody></table>

Hb: hemoglobin; RBC: red blood cells; Ht: hematocrit; MCV: mean cellular volume; MCH: mean cellular hemoglobin; MCHC: mean cellular hemoglobin concentration; Ret: reticulocytes. *: p < 0.05; **: p < 0.01; ***: p < 0.001; ****: p < 10⁻⁴; ns: non-significant.

heterozygosity protects from severe forms of malaria [1]. Originally distributed across sub-Saharan Africa, the Middle East, the Mediterranean basin, and India, this SCD has become increasingly prevalent during the 20th century in numerous other regions worldwide due to

human migration. These include the Caribbean islands, Brazil, the United States, the United Kingdom, France, and cosmopolitan areas within other European countries [8,9]. In metropolitan France, the incidence is about one affected child per 1200 births [10,11] and the prevalence is estimated to be around 25,000 to 30,000 individuals with about 50 % adults and 50 % children (https://depistage-neonatal.fr/ space-pro/depistage/rapport-dactivite/). According to the literature, sickle β⁺-thalassemia would be the third most common SCD genotype in metropolitan France and may concern up to 10 % of neonates with SCD [12]. These assertions are confirmed by the repartition between the different genetic forms of SCD of our local cohort in Lyon: 70 % of HbS/ HbS, 13 % of HbS/HbC, 9 % of sickle β⁺-thalassemia, 6 % of sickle β⁰-thalassemia and 2 % for the other forms. One of the main factors modulating SCD severity is the intra-erythrocyte HbS concentration since the rate and extent of sickling primarily depends on the intracellular ratio between HbS and non-HbS concentrations, the latter acting as a diluter to reduce HbS polymerization [7]. For this reason, sickle β⁺-thalassemia are globally less severe than the other SCD genotypes but it actually depends on the residual HbA level, which can vary greatly from one β⁺-thal variation to another.

A total of 16 different β⁺-thal variations were found in our cohort that we assume to be totally representative of the french sickle β⁺-thal population. All these variations are single nucleotide substitutions (no short indel or large deletion) and three different underlying molecular mechanisms can be observed [5,13]. Splicing variations correspond to nucleotide substitutions that quantitatively reduce (but not abolish) the formation of βA mRNA, either by affecting the efficiency of the consensus splice site or by activating a non-functional cryptic splice site for HBB: c.75T > A, c.316-106C > G and c.93-21G > A. The four group 1 variations (which are associated with a residual HbA level below 10 %) are all splicing variations that are considered as severe β⁺-thal variations in the HbVar database [14]. Promoter variations and 3'-UTR variations correspond to the two other underlying molecular mechanisms. The former affect the transcriptional efficiency of the HBB gene by reducing the fixation of specific transcription factors, while the latter alters the polyadenylation signal at the normal polyA site, thus reducing βA mRNA
<figure><img src="image_6.png" /><figcaption>Fig. 3. Violin graphs representing the variations of the main hematological parameters according to the theoretical severity group in the sickle cell/β⁺-thalassemia cohort.</figcaption></figure>

Group 1: mean HbA < 10%; Group 2: mean HbA between 10 and 20%; Group 3: mean HbA > 20%. (A) Main RBC parameters; (B) RBC indices; (C) Hemoglobin electrophoresis parameters.

\*\*\*\*: p < 10⁻⁴; \*\*: p < 10⁻²; \*: p < 0.05; ns: non-significant.
stability before translation. Schematically, these two types of variations are associated with higher HbA levels than splicing variations and they were logically over-represented in groups 2 and 3. Given that each β-thal variation is typically associated with a specific geographic region, the distribution of β⁺-thal variations within our cohort can indirectly provide insights into the countries of origin of SCD patients in France (Table 2). Quite logically, the four more frequent β⁺-thal variations (which represented 75 % of our cohort) have a Black ethnic distribution (i.e. Africa) according to the HbVar database [14], the most frequent one (40.4 % for HBB:c.-79A > G) being particularly prevalent in West Africa [15]. The other β⁺-thal variations originate from the Mediterranean basin (17 % of the cohort), from Middle East/India/East Asia (2 % of the cohort) and from other geographical regions (6 % of the cohort).

A limited number of studies in the literature focused on sickle β⁺-thalassemia [16–18]. In the largest one, Serjeant et al. tried to compare the clinical and hematological features in 190 Jamaican HbS/ β⁺-thal patients with determined genotype [17]. They identified a total of 7 different β⁺-thal variations, which were distributed according to a phenotypic classification based on the amount of residual HbA: type 1 (HbA: 1–7 %), type 2 (HbA: 7–14 %) and type 3 (HbA: 14–25 %). However, the phenotype-genotype correlation was limited since only one type 1 variation (HBB:c.92 + 5G > C) and no type 2 variation was present. In the present study, we described a much more heterogeneous population (16 different sickle β⁺-thal genotypes) with a very large range of residual HbA values that allowed us to propose new HbA thresholds for our three theoretical groups of severity. Provided that the size of our cohort was sufficient, a fourth group could have been proposed for two β⁺-thal variations associated with a HbA level over 30 % and which clearly stand out from the others: HBB:c.*111A > G and HBB: c.-151C > T. These two variations are described in the literature as very mild and silent β⁺-thal variations, respectively [5,14]. Fifty out of 166 patients tested for alpha-thalassemia had a – 3.7 Kb alpha-thalassemia deletion, which is in agreement with the allelic frequency of around 20 % usually given in the literature [19,20].

Schematically, sickle β⁺-thal patients in group 1 are expected to have a total Hb value below 100 g/L, whereas patients in groups 2 and 3 present higher values with significantly lower reticulocytes counts. Regarding the other parameters, a slight elevation of MCV and MCH and a significant decrease of HbA₂ and HbF values are observed for patients of group 3, which can be easily explained by their less pronounced thalassemia phenotype. If we consider the threshold of 100 g/L as a reliable marker for assessing the overall clinical severity of SCD, our results would mean that only four sickle β⁺-thal genotypes (accounting for approximately 12 % of all sickle β⁺-thal patients in France) are associated with a severe SCD phenotype requiring regular and close clinical monitoring, as well as prenatal diagnosis for at-risk couples. However - and this is the main limitation of our study - the assessment of hemolysis based on two biological parameters only (i.e. total Hb and Ret) cannot obviously capture the whole biological and clinical heterogeneity of SCD syndromes. These two parameters are mostly representative of the hemolytic sub-phenotype of the disease but they give no information on the frequency of vaso-occlusive crisis (VOC) or acute chest syndromes (ACS) which are the typical complications of the viscous vaso-occlusive sub-phenotype of SCD. Moreover, according to both the literature [21,22] and our own clinical experience, some sickle β⁺-thal patients with a very mild SCD phenotype at steady-state can decompensate very quickly and die in a few days of a fat embolism syndrome. A retrospective (or even better prospective) study focusing on all possible clinical complications would thus be necessary to confirm the fact that the four following β⁺-thal variations give the worse phenotypes in association with HbS: HBB:c.95 + 5G > A, c.92 + 5G > C, c.92 + 5G > T and c.93-21G > A. It would also be interesting to include more patients with some particular sickle β⁺-thal genotypes that are under-represented in our study: HBB:c.316-106C > G, c.-138C > A and c.*111A > G.

## 5. Conclusion

Our study presents for the first time a precise and nearly exhaustive picture of sickle β⁺-thalassemia in France. It is quite reassuring to note that >80 % of patients have a residual HbA level over 10 % that seems to be associated with a very mild anemia. However, special attention should be paid to patients of group 1 since the course of their disease is probably very close to that of sickle βº-thalassemia. Knowing the expected HbA levels based on the β⁺-thal variation will help in appropriately counselling parents regarding disease severity risks and the use of genetic prenatal testing. Additionally, it will provide a rationale for the early use of preventive HU therapy in children from group 1, similar to what is offered to children with S/S and S/βº diseases [23]. On the contrary, one might wonder whether couples at risk for compound heterozygosity for HbS and HBB:c.*111A > G or HBB:c.-151C > T should be considered eligible for prenatal diagnosis.

### CRediT authorship contribution statement

**Cecilia Baltus:** Data curation, Investigation, Methodology, Writing – original draft. **Stéphane Moutereau:** Investigation, Writing – review & editing, Formal analysis, Methodology, Validation. **Nathalie Couque:** Writing – review & editing, Formal analysis, Investigation. **Bichr Allaf:** Writing – review & editing, Formal analysis, Investigation. **Muriel Giansily-Blaizot:** Writing – review & editing, Formal analysis, Investigation. **Julian Boutin:** Investigation, Writing – review & editing, Formal analysis. **Ketty Lee:** Formal analysis, Investigation, Writing – review & editing. **Emmanuelle Bernit:** Data curation, Formal analysis, Validation, Writing – review & editing. **Estelle Cadet:** Formal analysis, Investigation, Writing – review & editing. **Victor Bobee:** Formal analysis, Investigation, Writing – review & editing. **Véronique Picard:** Formal analysis, Investigation, Writing – review & editing. **Serge Pissard:** Conceptualization, Formal analysis, Investigation, Writing – review & editing. **Frédéric Galactéros:** Formal analysis, Writing – review & editing, Conceptualization, Investigation. **Céline Renoux:** Formal analysis, Writing – review & editing. **Philippe Connes:** Formal analysis, Validation, Writing – review & editing. **Patricia Aguilar-Martinez:** Writing – review & editing. **Corinne Pondarre:** Conceptualization, Formal analysis, Validation, Writing – review & editing. **Philippe Joly:** Conceptualization, Project administration, Supervision, Writing – review & editing, Methodology, Validation.

### Statements and declarations - competing interests and funding

All authors declare that they have no conflict of interest. This research was funded by the "Association pour la Recherche en Pathologie des Cellules Sanguines (ARPCS)";

### Acknowledgments

The European Reference Network on Rare Hematological Diseases (ERN-EuroBloodNet), the Hospices Civils de Lyon and the Association pour la Recherche en Pathologie des Cellules Sanguines (ARPCS).

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