Luspatercept was recently approved in order to address anaemia associated with transfusion-dependent beta-thalassaemia.1-3 It is contraindicated in pregnant and lactating women due to the teratogenic effects observed in preclinical animal studies. In rats, luspatercept administered at dose levels of 0, 5, 15 or 30 mg/kg on Days 3 and 10 of gestation, led to reduced uterine weight and smaller fetal body weight at all dosages. At a dosage of 30 mg/kg, the average number of fetuses and the number of live fetuses per litter were reduced. Moreover, the number of fetuses and litters with the skeletal variation 'asymmetric sternal centra' was significantly increased at 15 mg/kg. The NOAEL (no observed adverse effect level) for embryo-fetal effects in rats was 5 mg/kg, corresponding to a maternal exposure approximately three times higher than the estimated exposure at the clinical dose of 1.75 mg/kg Q3W. In rabbits, luspatercept was administered at dose levels of 0, 6, 20 and 40 mg/kg on Days 4 and 11 of gestation, with the same kinds of developmental issues, including malformations of the ribs and vertebrae and the same NOAEL, as in the rats. For this reason, an effective contraceptive regimen is recommended for the duration of the therapy and for 3 months after its discontinuation, and women of childbearing age must be guaranteed counselling with regard to planning a possible pregnancy.4 At the Ospedale Pediatrico Microcitemico, Cagliari (Italy), the drug is not prescribed if the patient wishes to become pregnant, or even if only considering the possibility. Contraception is recommended to all patients of childbearing age for the entire duration of luspatercept therapy, and this advice is regularly reinforced. Nevertheless, two young patients followed at that hospital became pregnant during treatment. Patient 1 is a 27-year-old woman who has been regularly transfused since the age of 3. Chelated first with subcutaneous desferrioxamine, then with deferasirox, the patient had never presented parameters indicative of significant iron accumulation or iron-related complications. She had menarche at the age of 14, and menstruation had been regular ever since. She started luspatercept therapy at the dosage of 1 mg/kg every 3 weeks, and the dose was increased to 1.25 mg after 3 months. Apart from luspatercept and deferasirox at 24 mg/kg per day, the patient was being treated with vitamin D and folic acid for insufficient levels. In March 2023, she took a pregnancy test the day before the expected start day of the menstrual cycle due to nausea and asthenia, which was positive. Both iron chelation and luspatercept therapies were no longer administered. The last administration of luspatercept was 4 weeks after her last menstrual cycle. The course of the pregnancy was totally physiological, fetal growth was normal, as well as the proportions of the body and all organs assessable via ultrasound. A caesarean section was planned at 38 weeks +0 days, leading to the birth of a male weighing 3.52 kg, 51 cm in length and 35 cm in head circumference. The newborn underwent abdominal ultrasound, echocardiography, cerebral ultrasound and lab tests, which were not indicative of malformations or pathology. The patient's partner was a carrier of beta-thalassaemia. Despite the 50% risk of having an affected child, the couple had chosen not to undergo a prenatal diagnosis. The newborn resulted in being a carrier of beta-thalassaemia. Patient 2 is a 28-year-old woman who has been regularly transfused since the age of 10 months. She had experienced menarche at 14, and her menstrual cycles were irregular. She did not consistently adhere to chelation therapy, resulting in significant iron accumulation. Severe liver overload was observed from 2012 in the absence of myocardial iron. Luspatercept therapy was started in 2021 at a dosage of 1 mg/kg every 3 weeks and transitioned to a dosage of 1.25 mg/kg after 2 cycles. In June 2023, while undertaking luspatercept therapy, and despite having been previously advised about the need for contraception, the patient discovered her pregnancy through a positive test. Iron chelation was promptly halted, and further administrations of luspatercept were discontinued, with the last administration 4 weeks after the last menstrual cycle. Besides luspatercept and daily deferasirox at 27 mg/kg, the patient was prescribed vitamin D and folic acid for insufficient levels. Monthly obstetric ultrasounds indicated normal embryo and fetal growth, body proportion and organ development. The pregnancy progressed normally until 31 weeks +5 days when uterine contractions began, leading to hospitalization. A caesarean section was performed at 34 weeks +4 days, resulting in the birth of a female weighing 2.46 kg, with a length of 45 cm and a head circumference of 32 cm that is appropriate for the gestational age. The premature birth was explained by gynaecologists by the fetopelvic disproportion that characterized the patient. Upon birth, the infant was admitted to the neonatal intensive care unit and required respiratory assistance for a number of days. No malformations were detected via instrumental examinations. The abdominal ultrasound revealed a small peritoneal effusion and transient hepatic hyperechogenicity associated with prematurity, which rapidly resolved. Of note, the patient had already had a first spontaneous pregnancy, in the absence of exposure to luspatercept. The offspring, weighing 2.550 kg, with a length of 47 cm and a head circumference of 33 cm, was born by caesarean section at 35 weeks +6 days that is mild preterm and appropriate for the gestational age. The first clinical case presents a scenario that is likely to become increasingly common: a pregnancy in a woman with well-managed thalassaemia. Under the care of a multidisciplinary team, she delivers a healthy, appropriately weighted child at full term without complications. On the contrary, the patient in the second clinical case serves as a prototype for individuals with thalassaemia for whom pregnancy should be discouraged, independent of the use of luspatercept, due to severe iron accumulation and the heightened risk of cardiac complications. It is plausible that besides the fetopelvic disproportion, the status of suboptimal iron also played a contributory role in the occurrence of preterm births. In both cases, however, pregnancy ensued spontaneously. While the rise in spontaneous pregnancies signifies advancements in chelation therapy and the gradual mitigation of complications arising from iron overload, it also presents the possibility of unplanned pregnancies in patients who, by necessity, undergo iron chelation therapy and are therefore already exposed to a potentially teratogenic drug.5, 6 In this scenario, luspatercept thus emerges as an additional risk factor that may adversely affect embryo-fetal development and health. The absence of any apparent issues in the newborns should not diminish the importance of adhering to recommendations for effective contraception during treatment. In the event of pregnancy, it is imperative that factual information and non-directive counselling be provided, despite the limited data available on the risks associated with the use of luspatercept, a challenge commonly encountered with many pharmaceuticals. Moreover, considering the uncertainty surrounding the translation of risk data into clinical practice, a thorough discussion of all available options is warranted, including the consideration of the termination of the pregnancy if concerns regarding teratogenic effects should arise. RO and EZ conceived and designed the study and wrote the original draft. All other authors were involved in data collection and reviewing the manuscript. All authors reviewed and approved the final manuscript. N/A. RO and SB received consultation and speaker fees from BMS. The other authors have no conflicts of interest to declare. N/A. The patients provided written consent to publish this case report. N/A. N/A.
Since the beginning of the COVID-19 pandemic, concerns have been expressed worldwide for patients with hemoglobinopathies and their vulnerability to SARS-CoV-2 infection. Data from Lebanon confirmed a role of underlying comorbidities on COVID-19 severity, but no deaths among a cohort of thalassemia patients.1 Patients with sickle cell disease (SCD) displayed a broad range of severity after SARS-CoV-2 infection, spanning from a favorable outcome unless pre-existing comorbidities (UK cohort)2 to high case mortality in US.3 History of pain, heart, lung, and renal comorbidities was identified as risk factors of worse COVID-19 outcomes by the US SECURE-SCD Registry.4 While Italy experienced a death rate in the general population among the highest in the world, preliminary data from the first wave of the pandemic showed a lower than expected number of infected thalassemia patients (updated up to April 10, 2020), likely due to earlier and more vigilant self-isolation compared to the general population.5 To explore the vulnerability to SARS-CoV-2 infection, the Italian Society for Thalassemia and Hemoglobinopathies (SITE) designed a study to compare the prevalence and mortality of COVID-19 in individuals with hemoglobinopathies and the general Italian population (EMO AER COVID-19 study). The study was approved by Institutional Review Board authorities, registered on clinicaltrials.gov (NCT04746066), and was performed in accordance with Good Clinical Practice guidelines and the Declaration of Helsinki. Designed to gather data from multiple healthcare providers in Italy, it allowed for collecting relevant demographics and clinical data on a dedicated electronic Case Report Form (eCRF) (available at https://covid19.site-italia.org) by each participating center. We enrolled patients with transfusion-dependent thalassemia (TDT), nontransfusion-dependent thalassemia (NTDT), and sickle cell disease (SCD) referred to participating centers and diagnosed with SARS-CoV-2 infection in the study period March 6, 2020 to April 7, 2021. SARS-CoV-2 infection was confirmed by either a positive swab of the upper or lower respiratory tract or serology. Patients with less than 15 days of follow-up from either the onset of symptoms or a SARS-CoV-2 positive test were excluded. Twenty-nine centers from 13 Italian Regions participated in the EMO AER COVID-19 study. These centers regularly provide care for approximately 6200 patients with hemoglobinopathies (3400 TDT, 1500 NTDT, 1300 SCD), representing 65% of the Italian population affected by these pathologies. Therefore, this sample is highly representative of Italian patients with hemoglobinopathies followed by an organized and widespread national network, providing both high coverage and high definition of data. During the 398-day study period, a total of 345 SARS-CoV-2 infections were recorded (overall, prevalence 5.5%): 230 cases among TDT (prevalence 6.8%), 50 among NTDT (prevalence 3.3%), and 65 among SCD patients (prevalence 5.0%). In the SCD group, 49% of patients were β-Thal/HbS. Diagnosis of COVID-19 was confirmed by a positive swab in 91% of the cases and by the presence of serum IgG in 9% of the cases. Among reported cases, 52% were female. The median age at the infection was 41 years (IQR: 29–48, range: 0.75–85), with 10% of patients being pediatric (median age: 8 years, IQR: 4–11). Seventy-four percent of patients had at least one comorbidity at the time of infection. The most common were: splenectomy or functional asplenia (50%), iron overload (23%), liver disease (19%), heart disease (16%), and diabetes (8%). ABO blood groups were distributed as follows: 50% were O, 33% were A, 15% were B, and 2% of patients were AB. We observed a broad spectrum of COVID-19 severity, ranging from no symptoms (83/345, 24%) to severe manifestations (66/345, 19%) and death (7/345, 2%). The most common symptoms were fever (157/345, 46%), cough (145/345, 42%), fatigue and diffuse pain (119/345, 34%), and anosmia and ageusia (104/345, 30%). Severe symptoms, such as difficulty breathing or thoracic pain, affected 62/345 (18%) of patients; 55/345 (16%) had pneumonia; and 1 patient experienced pulmonary thromboembolism. Overall, 68 (20%) patients required hospitalization, 15 (8 TDT, 2 NTDT, 5 SCD) in high-intensity care units (ICU). Nine out of 68, all with pneumonia (1 TDT, 1 NTDT, and 7 SCD), required additional or ad hoc blood transfusions due to acute hemoglobin drop. The median hospitalization time was 11 days (IQR: 5–21, range: 1–102 days, information available for 46 patients). Seven patients experienced fatal COVID-19 during the period of observation: 4 TDT (46/M, 48/M, 49/M, 56/F), 1 NTDT (45/M), 2 SCD (57/M, 57/F), both with the diagnosis of β-Thal/HbS. One TDT patient (57/F) and two patients (1 SCD, 52/M; 1 TDT, 57/F) died after the conclusion of the analysis and were not included in this survey. The overall lethality rate was 2.0%. The age-standardized lethality ratio (SLR) was then calculated as the ratio between the observed and the expected number of deaths, based on the age-specific rates in the Italian-COVID population. The resulting SLR was 4.8 (±3.5, 95% CI). All the fatal episodes were observed starting from November 2020. For hospital admission, age was a risk factor in TDT (OR = 1.03; CI: 1–1.1; p = .04) and NTDT (OR = 1.05; CI: 1–1.1; p = .04), but not in SCD. In TDT only, the presence of underlying lung or heart disease increased the risk to be admitted to the hospital (OR = 4.5, CI = 1.1–19.3, p = .04; OR = 2.9, CI = 1.0–8.0, p = .04). For the SCD group, chronic liver disease was associated with a higher risk of hospital admission (OR = 7.5, CI: 1.1–53.5, p = .04). For ICU admission and mortality, the presence of previous pulmonary disease was a risk factor only for TDT (OR = 5.6, CI: 1.2–25.1, p = .03; OR = 26.6; CI: 2.3–311.4; p = .01, respectively). According to our results, the prevalence of COVID-19 in hemoglobinopathies in Italy was similar to the general population (5.5% vs. 6.2%) in the first 13 months of the pandemic. Considering the known underestimation of SARS-CoV-2 prevalence in the Italian population and the greater reliability of the same estimation in our strictly monitored patients, we speculate that the risk of infection in hemoglobinopathies was actually reduced. This hypothetical difference should be explained by the effectiveness of early recommendations from dedicated healthcare providers and the prudent attitude of the chronic patients in front of risk, as already reported from expert centers in other countries.6 The estimation of lethality is complex: 95.6% of the confirmed COVID-19 deaths in the Italian population have occurred in subjects in ages 60 or greater and 86.2% of the deaths in ages 70 or greater. Lethality rates for COVID-19 infected patients were 26.7% for ages 90 years or greater, 19.8% for ages 80–89 years, 9.4% for ages 70–79 years, and 2.7% for ages 60–69 years. Our study population is significantly younger in age overall, with only 1.4% subjects infected above 70 years of age, reflecting the age-distribution of the hemoglobinopathies in Italy. The proper comparator for our population is the segment of the Italian population younger than 60 years of age, which experienced 5% of the total COVID-19 deaths, with lethality rates varying from <0.1% (age 20–29 years) to 0.6% (age 50–59 years). While no significant differences were observed in patients aged 0–30s, significantly higher lethality was observed in subjects aged 40–49 and 50–59 years, where all fatal cases were registered. Assuming for hemoglobinopathies the same lethality rates of Italians with comparable age, the number of observed deaths in hemoglobinopathies is approximately 5-fold the expected one (Figure 1). All deaths occurred in patients in the fourth to fifth decades of life, mostly obese, splenectomized, and with numerous comorbidities. Surprisingly, none of them (except one for which no recent clinical data are available) had a significant iron overload. Both deaths in the SCD group occurred in patients with β0-Thal/HbS, while there were no fatal events among patients with homozygous HbS, in agreement with the local genotype distribution that is characterized by a high prevalence of older Caucasian β-Thal/HbS patients (homozygous HbS are more frequent among younger patients). Age was a risk factor for hospital admission due to SARS-CoV-2 infection in both TDT and NTDT, but not in SCD. Other risk factors were the presence of underlying comorbidities at the time of infection, particularly chronic lung, heart, or liver disease. In addition, chronic lung disease was a significant risk factor for ICU admission or mortality (in TDT only). The main limitations of this work are represented by the evaluation of indirect outcomes of COVID-19 severity; in addition, not all the Italian centers taking care of these patients were involved in the study. However, the data presented here include the large majority of known patients affected by hemoglobinopathies in Italy. Another limitation of our study is the inability to consider the effects of early vaccination in this at risk cohort compared with the general population. Our data clearly indicate that patients affected by hemoglobinopathies have up to a five times higher likelihood of suffering lethal SARS-CoV-2. Thus, these patients should be referred to specific and expert healthcare providers. Future studies should monitor the long-term effect of COVID-19 in patients with hemoglobinopathies. More relevantly, the effectiveness of vaccines should be evaluated in these patients to address the presence of any possible difference with the general population. The authors would like to thank ALT (Associazione per la Lotta alla Talassemia R. Vullo—Ferrara) and Dott. Giacomo Siri, PhD, Biostatistician of Galliera Hospital. No conflict of interest to declare.
Summary. Increased haemoglobin (Hb) A 2 levels associated with reduced mean corpuscular volume (MCV) and Hb content per cell (MCH) are the most typical features of heterozygous β thalassaemia. However, double heterozygotes for α and β thalassaemia may have normal MCV and MCH but Hb A 2 always in the carrier range. In this report we describe two Sardinian families who have increased Hb A 2 levels, normal red blood cell indices and normal globin chain synthesis and in whom DNA sequence analysis of β and δ globin genes did not reveal any abnormality. Our findings demonstrate the existence of a genetic trait not resulting from a defect of the β globin gene cluster, transmitted in a dominant manner and manifested as isolated increase of Hb A 2 .
CDA type I is a rare hereditary anemia, characterized by relative reticulocytopenia, and congenital anomalies. It is caused by biallelic mutations in one of the two genes: (i) CDAN1, encoding Codanin-1, which is implicated in nucleosome assembly and disassembly; (ii) C15orf41, which is predicted to encode a divalent metal ion-dependent restriction endonuclease with a yet unknown function. We described two cases of CDA type I, identifying the novel variant, Y94S, in the DNA binding domain of C15orf41, and the H230P mutation in the nuclease domain of the protein. We first analyzed the gene expression and the localization of C15orf41. We demonstrated that C15orf41 and CDAN1 gene expression is tightly correlated, suggesting a shared mechanism of regulation between the two genes. Moreover, we functionally characterized the two variants, establishing that the H230P leads to reduced gene expression and protein level, while Y94S induces a slight decrease of expression. We demonstrated that C15orf41 endogenous protein exhibits nuclear and cytosolic localization, being mostly in the nucleus. However, no altered nuclear-cytosolic compartmentalization of mutated C15orf41 was observed. Both mutants accounted for impaired erythroid differentiation in K562 cells, and H230P mutant also exhibits an increased S-phase of the cell cycle in these cells. Our functional characterization demonstrated that the two variants have different effects on the stability of the mutated mRNA, but both resulted in impaired erythroid maturation, suggesting the block of cell cycle dynamics as a putative pathogenic mechanism for C15orf41-related CDA I.
Hb Taybe [α38(C3) or α39(C4) Thr→0 (α1)] is an unstable hemoglobin (Hb) variant caused by a deletion of a threonine residue at codon 39 of the α1-globin chain. Usually asymptomatic or with minimal hematological abnormalities in the heterozygous state, Hb Taybe becomes clinically evident in compound heterozygosity with α-thalassemia (α-thal) or in homozygous patients. To date, Hb Taybe has been described in Israeli-Arab and Greek individuals. We report, for the first time, a patient with chronic hemolytic anemia due to the presence of Hb Taybe in trans to the α2 initiation codon mutation ATG>ACG in an Italian child. Hb Taybe was not evident at Hb analysis with cellulose acetate electrophoresis and high performance liquid chromatography (HPLC). Globin biosynthetic studies revealed an α/β-globin ratio in the range of β-thal trait. Consequently, an investigation of the α- and β-globin genes was requested in order to avoid missing any rare globin chain variant and to offer accurate genetic counseling.
In this paper the authors report the evolution of a new automatic HPLC analyser for screening haemoglobinopathies. HbA(2) and F determinations are accurate and reproducible. The analysis time is short (6.5 min) and there is a good separation between the HbA(2) values of beta-thalassemia carriers from normals and alpha-thalassemia carriers, with no overlap between these groups. In addition, the system is also able to detect and quantitate most of the haemoglobin variants, particularly those (HbS, HbC, HbE and Hb Lepore) able to interact with beta-thalassemia and could make haemoglobin electrophoresis unnecessary in all samples. The ease of operation and the limited technical work make this system especially suitable for laboratories with a high workload and allow the cost of screening to be reduced.
