The total number of the patients with hemoglobinopathies of our study (Thessaly and Athens), the type of hemoglobinopathy, the sex, the median age with the corresponding range, along with the relevant alleles are provided in Table 1. The frequency of HBB variant alleles in patients with β-hemoglobinopathies is shown in Table 2. The results of the patients with TM and TI were grouped together in Table 2, whereas the results of the patients with SCD (beta thalassemia/SCD, homozygous SCD) and hemoglobinopathy H (alpha thalassemia) are shown separately, in Tables 3 and 4, respectively.
Table 1
Number of patients and respective alleles for each hemoglobinopathy. The calculation of the number of alleles for each patient group is explained in the Materials and Methods part of the manuscript. For SCD/beta thalassemia trait patients, only the beta thalassemia trait alleles are reported. The SCD trait allele is the same [20A > T (βs CD6A > T)] for all these patients, also for homozygous SCD and is not shown on the Table.
Hemoglobinopathy Type | Patients | Sex | Median Age (Range) | Beta Alleles |
Τhalassemia Major | 129 | F: 79 | 45 (12–66) | 258 (beta) |
M: 50 |
Thalassemia Intermedia | 24 | F: 16 | 52 (16–81) | 44 (beta) |
M: 8 |
Sickle Cell Disease (SCD) | 52 (47 SCD/beta thalassemia trait, 5 homozygous SCD) | F: 29 | 46 (14–72) | 47 (beta) |
M: 23 |
Hemoglobinopathy H (alpha thalassemia) | 12 alpha genotypes | F: 8 | 44 (19–77) | ------- |
M: 4 |
Total | 217 | F: 132 | 45 (12–81) | 349 |
M: 85 |
Table 2
129 patients with thalassemia major (TM) and 24 with thalassemia intermedia (TI) forming 302 beta alleles. The frequency and type of HBB variant alleles is shown on the Table, along with a comparison of the largest national studies performed previously in Greece, Turkey, Italy and Lebanon.
Mutation | Number of alleles | % | β gene Phenotype | | Distribution % per Region |
Thessaly Georgiou 2003 | Greece 1 Kattamis C 1990 | Greece 2 Boussiou 2008 | Turkey 1 Tadmouri 1998 | Turkey 2 Aydinok 2018 | Italy Rosatelli 1992 | Lebanon Makhoul 2005 |
IVS-I-110 (G->A) (HBB:c.93 − 21 G > A) | 103 | 34.1 | β+ | 31.8 | 42.5 | 42.1 | 39.3 | 47.1 | 23 | 34.2 |
CD 39 (CAG > TAG) (HBB:c.118C > T) | 52 | 17.2 | β0 | 23.9 | 16.9 | 18.8 | 3.8 | 5.7 | 40.1 | 0.2 |
IVS I-6 (T > C) (HBB:c.92 + 6T > C) | 29 | 9.6 | β++ | 3.4 | 7.2 | 8.1 | 10.1 | 7.5 | 9.9 | 14.4 |
IVS I-1 G > A (HBB:c.92 + 1G > A) | 28 | 9.3 | β0 | 11.4 | 13.2 | 12.8 | 5 | 7.6 | 10.2 | 15 |
δβ Lepore | 17 | 5.6 | β+ | 1.1 | unknown | unknown | unknown | unknown | 1 | unknown |
IVS II-745 C > G (HBB:c.316-106C > G) | 16 | 5.3 | β+ | 9.1 | 6.9 | 6.3 | 5 | 5.6 | 5 | 1.2 |
IVS II-1 G > A (HBB:c.315 + 1G > A) | 9 | 3 | β0 | 9.1 | 2 | 3.3 | 4.7 | 5.5 | 3.9 | 8.7 |
CD 6 -A (HBB:c.20delA) | 9 | 3 | β0 | 3.4 | 2.9 | 1.7 | 0.4 | unknown | 1.2 | unknown |
δβ Sicilian | 8 | 2.6 | β0 | 2.3 | unknown | unknown | unknown | unknown | unknown | 0.4 |
δβ Turkish (δβ Μαcedonia) | 5(4 + 1) | 1.7 | β0 | unknown | unknown | unknown | unknown | unknown | unknown | unknown |
-101(C > T) (HBB:c.-151 C > T) | 5 | 1.7 | β + silent | unknown | unknown | 1.6 | 0.1 | unknown | 0.4 | unknown |
CD 8 (-AA) (HBB:c.25_26delAA) | 4 | 1.3 | β0 | unknown | unknown | 0.8 | 5.5 | 5.2 | unknown | 2.5 |
IVS I-5 (G > A) + Corfu deletion* | 4 | 1.3 | β+ (δ0) | 3.4 | 0.9 | 0.37 | unknown | unknown | 0.1 | unknown |
IVSI-n5 G > C | 4 | 1.3 | β+ | unknown | unknown | unknown | 1.1 | 2.1 | unknown | 0.8 |
44 bp deletion (HBB:c.76_92 + 27del) | 2 | 0.7 | β0 | 1.1 | 0.6 | 0.27 | unknown | unknown | unknown | unknown |
+ 1480(C > G) (HBB:c.*6C > G) | 2 | 0.7 | β + silent | unknown | unknown | 0.11 | unknown | unknown | unknown | unknown |
87C > G (137C > G) | 2 | 0.7 | β+ | unknown | unknown | 1 | 0.8 | unknown | 1.4 | 0.8 |
-28 (A > G) (HBB:c.-78A > G) | 1 | 0.33 | β++ | unknown | unknown | unknown | unknown | unknown | unknown | unknown |
1570 T > C (96 T > C) | 1 | 0.33 | β + silent | unknown | unknown | 0.19 | unknown | unknown | unknown | unknown |
codons 8/9 (+ G) [27_28 insG] | 1 | 0.33 | β0 | unknown | unknown | 0.11 | 1.3 | unknown | unknown | 0.2 |
Total | 302 | 100 | | | | | | | | |
Table 3
Sickle Cell Disease (SCD) patients and respective alleles. For the 47 SCD/beta thalassemia trait patients, only the beta thalassemia trait alleles are reported. The SCD trait allele is the same [20A > T (βs CD6A > T)] for all these patients, also for homozygous SCD and is not shown on the Table. The 5 homozygous SCD patients are not shown on the Table.
Sickle Cell Disease (SCD) |
Mutation | Number of alleles |
IVSI-n110 (93-21G > A) | 16 |
cd39 (118C > T) | 9 |
IVSI-n6 T > C (92 + 6T > C) | 7 |
IVSI-n1G > A (92 + 1G > A) | 4 |
δβ Lepore | 2 |
IVSII-1 (315 + 1G > A) | 2 |
IVSI-5 (92 + 5G > A) | 2 |
IVS-II n745 (316-106C > G) | 2 |
δβ Turkish | 1 |
364G > C (HBD LOS ANGELES) | 1 |
CD8 (--AA) | 1 |
Total | 47 |
Table 4
12 patients with hemoglobinopathy H and their genotypes
Hemoglobinopathy H (alpha thalassemia) |
Genotype | Patients |
-α3.7 */- -MED II ** | 6 |
αHph[c.95 + 2_95 + 6delTGAGG]α/- -MED II ** | 2 |
- -MED II **/ CD 127 AAG > TAG | 1 |
- -MED II **/CD 29 CTG > CCG (Hb Agrinio) α | 1 |
-α3.7*/-(α)5.2 | 1 |
- - MED I/-(α)4.2 | 1 |
Total | 12 |
*α+, **α0 |
The total number of alleles for TM and TI is 302 and not 306, as expected in relation to the patient number of the study (153 patients, 153 x 2 = 306). This is due to the fact that 4 patients with TI were double heterozygotes for beta zero (β0) alleles and alpha gene triplication alleles (β0/ααα genotype) and thus, they contributed one HBB allele to the study (3 patients with cd39/ααα and 1 with IVSI-n1/ααα).
All 129 patients with TM are regularly transfused with red blood cells (RBCs) and are under iron chelation treatment. Most of them usually receive 2 RBC units (median volume 550 cc) every 14–21 days, while a few are transfused more often, every 10 days. Based on the findings of Table 2, each beta mutation is either beta zero (β0) with no amount of beta hemoglobin chains and a more severe anemia, or beta plus (β+) with higher levels of hemoglobin and more amount of beta chains.
Interestingly, 15 patients with TM, harbor also deletional mutations of the alpha genes [α- 3.7kb (α+) or Med - - (α0) or + 94A > G (PolyA)]. The latter affects the phenotype, as these patients exhibit less degradation of the –α chains and a less severe degree of anemia, thereby needing fewer transfusions. On the contrary, the patient with the genotype cd39(C > T) (118C > T)/δβ Lepore/ααα (alpha triplication) has a more severe anemia, due to the excess of the alpha chains and will need more RBCs, compared to a patient with the cd39(C > T) (118C > T)/δβ Lepore genotype.
It is noteworthy that the mutations IVSI-n110 (G > A) (93-21G > A) (weak β+, clinically closer to β0) with a prevalence of 34.1% and the cd39 (C > T) (118C > T) (β0) with a prevalence of 17.2%, were the most frequently encountered among the β-gene mutations of our cohort (TM and TI).
Common mutational alleles of our study were also the following: IVSI-n6 (T > C) (92 + 6T > C) (β++, prevalence of 9.6%), IVSI-n1 (G > A) (92 + 1G > A) (β0, 9.3%), δβ Lepore (β+, 5.6%) and IVS II-745 (C > G) (316-106C > G) (β+, 5.3%).
The 6 alleles of our cohort usually leading to a TI phenotype were the IVSI-n6 (T > C) (92 + 6T > C) (β++), δβ Lepore (β+), -101(C > T) (151C > T) (β+, silent mutation), + 1480(C > G) (*6C > G) (β+, silent), -28(A > G) (78A > G) (β++) and 1570 T > C (96 T > C) (β+, silent). The abovementioned mutations either in homozygosity or in various combinations between them or even in heterozygosity with any other allele often cause the milder TI genotype and phenotype. Interestingly, the alleles IVSI-n6 (T > C) (92 + 6T > C) (β++) and + 1480(C > G) (*6C > G) (β+, silent) were the most frequently associated with extramedullary hematopoiesis.
Regarding the clinical manifestations of TI, regulatory genes despite beta and alpha genes and their balance, seem to be important. From the 24 patients with TI of our study, 14 are not being transfused, 6 are rarely transfused (less than 5 times per year) and the remaining 4 are often transfused. The 4 double heterozygotes for a triple alpha gene arrangement and an HBB gene variant (3 patients with cd39/ααα and 1 with IVSI-n1/ααα) have heterogeneity regarding their RBC transfusions. Of these cases, the first is a female patient of 56 years old (IVSI-n1/ααα). She started transfusions only when giving birth and gradually increased the frequency until regular transfusions nowadays. She is under chelation with deferasirox and also has thrombocytopenia, due to splenomegaly, along with an episode of heparin-induced thrombocytopenia in the past. With the combination of regular transfusions (every 3 weeks) and the drug luspatercept, she has satisfactory levels of hemoglobin (10–11 gr/dl) and PLT > 110.000/mm3. From the remaining 3 patients (cd39/ααα), the 2 are not transfused and the 1 is transfused very rarely (2–3 times per year).
Sometimes there is deviation of the expected clinical manifestations based on the genotype. Even though the following genotypes are typical of TM [(25_26delAA) cd8(-AA)/δβ Sicilian and IVSI-n110 (93-21G > A)/δβ Turkish] and these 2 patients of our study would be expected to be transfused regularly, their hemoglobin remains stable (8–10 gr/dl) and they are never transfused.
An example of typical TI regarding the clinical manifestations of the disease is the patient with the genotype IVSI-n110 (93-21G > A)/IVSI-n6 T > C (92 + 6T > C), exhibiting also high levels of HbF. He had splenectomy, due to visceral leishmaniasis. He was also diagnosed with thrombosis of the lower limbs and he is under treatment for pulmonary hypertension. Interestingly, he had extramedullary masses inside the vertebral and sacral canal, causing severe nerve compression, leading to paraparesis, paraplegia, hypoesthesia of the corresponding neurotomes, bowel and urinary incontinence. He was successfully treated with radiotherapy, hydroxyurea and regular transfusions to maintain haemoglobin levels > 11 gr/dl.
More interestingly, 2 other patients of our cohort, even though they have the same genotype [cd39 (118C > T)/101C > T (151C > T)], their transfusion phenotype differs significantly. The younger male patient (26 years) is transfused quite regularly (every 20–30 days, 400–500 cc), whereas the older female patient (56 years) has never been transfused.
8 more mutations of the beta alleles of our TM and TI patients have a prevalence ranging from 1.3% − 3% and are uncommon (Table 2). The very rare mutations (prevalence < 1%) were the following 6: 44 bp deletion (HBB:c.76_92 + 27del), + 1480 (C > G) (HBB:c. *6C > G), 87C > G (137C > G), -28 (A > G) (HBB:c.-78A > G), 1570 T > C (96 T > C) and the codons 8/9 (+ G) [27_28 insG] (Table 2).
Table 3 contains the patients with SCD. The most common mutations in sickle cell/beta thalassemia trait patients were the IVSI-n110 (G > A) (93-21G > A), the cd39 (C > T) (118C > T) and the IVSI-n6 (T > C) (92 + 6T > C). The very rare allele 364G > C (HBD Los Angeles), characterizing hemoglobin D was also noted among our cohort. Moreover, the 3 female patients with SCD harboring also triplication of the alpha genes (ααα) have a more severe anemia. One of them has homozygous SCD, along with ααα genes. Interestingly, the remaining 2 female patients with the genotypes 20A > T (βs CD6A > T)/IVSI-n110 (G > A) (93-21G > A)/ααα and 20A > T (βs CD6A > T)/cd39 (C > T) (118C > T)/ααα showed less sickle cell crises and significantly lower levels of hemoglobin. In the latter 2 cases, both therapeutic protocols, are being used: that of SCD and that of TI in order to prevent future complications. The molecular evaluation of alpha genes in these patients revealed 2 hemoglobinopathies instead of one, allowing us to better classify them and to provide a more focused clinical insight.
The six different genotypes contributing to the 12 patients with hemoglobinopathy H of our cohort are shown on Table 4. The majority of the patients had mild clinical manifestations without regular transfusions or with transfusions in cases of pregnancy. However, the patient with the genotype Med - - (α0)/α Agrinio α has severe anemia and he requires regular transfusions and chelation.