Evaluation of Couples at Risk for Hemoglobin Disorders in Italy: The Results of an 18-Month Retrospective Observational Single-Center Study

Background: Hemoglobinopathies are genetic disorders caused by mutations in the genes encoding globin chains. These conditions are the most common monogenic disorders worldwide, resulting in significant morbidity and mortality among those affected by the most severe forms. Antenatal evaluation for thalassemia and sickle cell disease (SCD) enables couples to know their reproductive risk, thus allowing an informed reproductive choice. However, in certain areas, couples do not often receive the appropriate counseling at the appropriate time.

Aim: The aim of this study is to describe the characteristics of couples referred to our tertiary referral center in Milan, Italy, for evaluation of hemoglobinopathy risk and their management.

Methods: in our Institution, a diagnostic-therapeutic pathway involving the Hemoglobinopathies unit, the Medical Genetic unit, and the central laboratory has been implemented. Subjects referred to the Institution, and their partner, underwent the following laboratory tests: complete blood count, hemolysis and iron parameters, vitamin pattern, and hemoglobin (Hb) pattern by high-performance liquid chromatography (HPLC). The couples were then evaluated by a hemoglobinopathy expert, and those at risk for Hb disorders were offered a molecular analysis. Couples were then referred to the geneticist to receive the results of the molecular analysis, discuss the genetic risk for the fetus and, in case of high risk, discuss possible reproductive choices (i.e., prenatal diagnosis, pre-implantation diagnosis). We retrospectively collected and analyzed couples' data referred to our center from the 1 ^st of July 2021 to the 31 ^st of December 2022.

Results: Three hundred and fifty-one couples were evaluated for hemoglobinopathies over 18 months. The median age was 34 years, with a tendency for younger age in female partners; 67% (234/351) of the women were already pregnant at the time of evaluation (median gestational age 18 weeks, range 7-37), while 33% (117/351) were referred for preconception counseling. Individuals from more than 48 different countries were screened, and the most represented countries of origin included: Italy (50%), Nigeria (6%), Bangladesh (4%), and Ivory Coast (4%), underpinning a vastly diverse genomic background.

The referral was triggered by either evidence of microcytic anemia (58%) or HPLC anomalies (42%), including Hb variants, HbA2 anomalies, or elevated HbF in at least one partner. 55% of the referrals did not require additional testing. The remaining 45% (158/351) underwent advanced molecular tests to determine the nature of the Hb defect, guided by the results of the first-level panel. Among these, 33 couples (12% of the total) were found to be at risk for transmission of severe clinical forms of β-thalassemia, sickle cell anemia, or compound β-disorder heterozygosity (HbS/β-thal, HbC/HbS). One additional couple (0.4%) was at risk for HbH disease. In addition, 4 couples (1.5%) were found to be at risk for thalassemia intermedia combining a β-thalassemia mutation and α-gene triplication. Of the above, all 34 couples underwent invasive fetal evaluation: 24 women underwent chorionic villous sampling, while 10 received amniocentesis. A diagnosis of thalassemia in the fetus was reported in 6/34 (18%) cases and 3 (9%) of SCD. Interestingly, during the 18 months of observation, among the evaluated adult subjects, we found 4 cases of non-transfusion dependent thalassemia (NTDT) and 1 of HbS/β-thal in otherwise healthy subjects. All 4 cases of NTDT resulted from α-gene triplication paired with a β-thalassemia heterozygous defect. Furthermore, α triplication was found alone in 4 subjects without additional Hb gene defects. Overall, α triplication was detected in 9 out of 154 (5.8%) individuals who underwent HBA gene analysis.

Conclusion: The median gestational age at presentation was high, leading to a limited time for couples to receive the appropriate counseling to make an informed reproductive choice. Increasing awareness across all involved specialists remains the mainstay for an effective prevention program to promptly identify at-risk couples and offer counseling at the right time. In addition, the relatively high incidence of α triplication in our population of asymptomatic and hematologically normal subjects and the new diagnosis of 4 NTDT warrants further studies to confirm the prevalence of α triplication in the general population.