Evaluating the Clinical Impact of Hemoglobinopathy and Hereditary Red Blood Cell Disorder Co-Inheritance in a Cohort of 315 Patients

Background. Hereditary Red Blood Cell Disorders (HRBCDs) are a group of rare conditions characterized by wide phenotypic and genetic variability. These disorders often follow complex inheritance patterns, with up to 15% of HRBCD cases showing multi-locus inheritance. Recent studies have shown that individuals carrying HRBCD variants and β-thalassemia (BT) traits frequently exhibit symptomatic phenotypes (Pinto et al. Am J Hematol, 2023).

We herein focused on the phenotypic changes associated with globin gene variants among 744 individuals suspected of having HRBCDs other than hemoglobinopathy. These subjects were selected from our laboratory records from 2019 to 2024. To establish a baseline for comparison, we also included 14 patients with a confirmed diagnosis of BT in our study.

Methods. A phenotype score (PhenSCORE or pS) was developed using three parameters from complete blood count: hemoglobin (Hb), mean corpuscular volume (MCV), and mean corpuscular hemoglobin concentration (MCHC). A first sub-score was derived from Hb levels to evaluate the severity of anemia, stratified by age and gender according to WHO guidelines. A second sub-score was created based on deviations in MCV and MCHC from their reference ranges, combining these into a normalized sub-score when both parameters were out of range.

Patients with confirmed genetic diagnoses of HRBCDs other than hemoglobinopathy underwent screening for globin gene variants using the Devyser Thalassemia NGS analysis. Additionally, the 14 patients with BT in our control group were examined through whole exome sequencing (WES) to detect any co-occurring HRBCD variants.

We defined genotypes for each participant using a combined variant score (VariantSCORE or vS) based on the ACMG/AMP guidelines. This scoring system factored in variant zygosity and the dominance or recessivity of the involved genes, both HRBCD-related genes and HBB gene, rating single nucleotide variants (SNVs) from uncertain significance (0-5) to likely pathogenic (6-9) and pathogenic (≥10). Copy number variations (CNVs) in the HBA1/HBA2 genes were excluded from this initial analysis.

The Mann-Whitney U test assessed phenSCORE differences, and Spearman's correlation analyzed the relationship between PhenSCORE and VariantSCORE.

Results. Among the 744 patients studied, 315 received a conclusive genetic diagnosis of HRBCD. The diagnoses included 128 cases of hereditary stomatocytosis (DHS), 89 hereditary spherocytosis (HS), 17 congenital dyserythropoietic anemia (CDA), 17 hereditary hemochromatosis (HH), 14 glucose-6-phosphate dehydrogenase deficiency (G6PDD), 10 congenital erythrocytosis (ECYT), 6 pyruvate kinase deficiency (PKD), 1 Gaucher disease (GD), and 33 multi-locus diagnoses. Hematological data for the PhenSCORE were available for 88 patients (27.9%). Analysis of sequencing data from these 88 HRBCD patients and the 14 thalassemic controls showed that 23.5% (24/102) of the subjects had co-inherited HRBCD and β-thalassemia. Those with co-inheritance displayed a higher mean PhenSCORE (pS=20.1) compared to those with HRBCD alone (pS=7, p<0.0001) and the thalassemic controls (pS=17.8, p=0.64). Interestingly, among the DHS patients, a significant direct correlation was observed between the phenotype score and the variant score (Spearman’s r=0.44; p=0.0061), indicating that increased genetic load correlates with more severe phenotypic manifestations.

Conclusions. Approximately 7% of the global population carries hemoglobinopathies, and 1-5% are thalassemia carriers. Our study showed that about 24% of patients diagnosed with HRBCD also carried an HBB variant that initially was not evaluated, worsening their clinical outcomes. Notably, fewer than 30% of the analyzed patients had adequate clinical or biochemical data, such as HbA2 levels, to confirm the presence of the thalassemic trait. Comprehensive data collection is essential for accurate patient stratification and interpretation of genetic variations. To enhance our understanding of the impact of co-inherited variants in globin genes on HRBCD, we plan to expand our study cohort. Increasing the sample size will provide a more robust analysis of the influence of these combined genetic factors on patient phenotypes.