Thalassemias and hemoglobinopathies are among the most common genetic diseases worldwide and have a significant impact on public health. The decreasing cost of next-generation sequencing (NGS) has quickly enabled the development of new assays that allow for the simultaneous analysis of small nucleotide variants (SNVs) and copy number variants (CNVs) as deletions/duplications of α- and β-globin genes. Background/Objectives: This study highlighted the efficacy and rapid identification of all types of mutations in the α- and β-globin genes, including silent variants, using the Devyser Thalassemia NGS kit. Furthermore, we report the frequency of mutations identified in a total population of 2649 individuals recruited from four Italian Medical Genetics Laboratories. Methods: All samples were first hematologically characterized, and sequence analysis was conducted by using the Devyser Thalassemia NGS kit. All variants were also validated in an independent sample by a conventional molecular test. Results: A total of 1789 subjects were identified with genetic variants in the globin genes, of which 966 (53.9%) had variations in the β-gene, 480 (26.8%) had variations in the α-gene; and 307 (17.1%) had variations in both α- and β-genes. Variant analysis evidenced a heterogeneous mutation spectrum enriched with variants not usually observed in the Italian population. Conclusions: This study showed the high effectiveness and the rapid identification of all mutation types in both α- and β-globin genes, including silent variants. It should be emphasized that the NGS approach greatly shortens turnaround reporting times, overcoming the classic diagnostic flowchart which envisages multistep, subsequent, diagnostic approaches, often requiring long resolution times.

UL18 is a glycoprotein encoded by the human cytomegalovirus genome and is thought to play a pivotal role during human cytomegalovirus infection, although its exact function is still a matter of debate. UL18 shares structural similarity with MHC class I and binds the receptor CD85j on immune cells. Besides UL18, CD85j binds MHC class I molecules. The binding properties of CD85j to MHC class I molecules have been thoroughly studied. Conversely, very little information is available on the CD85j/UL18 complex, namely that UL18 binds CD85j through its α3 domain with an affinity that is ∼1000-fold higher than the MHC class I affinity for CD85j. Deeper knowledge of features of the UL18/CD85j complex would help to disclose the function of UL18 when it binds to CD85j. In this study we first demonstrated that the UL18α3 domain is not sufficient per se for binding and that β₂-microglobulin is necessary for UL18–CD85j interaction. We then dissected structural determinants of binding UL18 to CD85j. To this end, we constructed a three-dimensional model of the complex. The model was used to design mutants in selected regions of the putative interaction interface, the effects of which were measured on binding. Six regions in both the α2 and α3 domains and specific amino acids within them were identified that are potentially involved in the UL18–CD85j interaction. The higher affinity of UL18 to CD85j, compared with MHC class I, seems to be due not to additional interaction regions but to an overall better fit of the two molecules.

Melania Olivieri, Ph.D., Marco Rosetti, M.S., Giovanni Poletti, M.D., Massimo Maffei, M.S., Domenico Coviello, M.D., Massimo Mogni, M.S., Francesca Capalbo, B.S., Morandini Maria Caterina, B.S., Valentina Polli, M.S., Alice Clementoni, M.S., Evita Massari, M.S., Marta Monti, M.D., Sauro Maoggi, M.S., and Tommaso Fasano, M.D., Ph.D.. Ann Lab Med 2025;45:101-4. https://doi.org/10.3343/alm.2024.0182

ANKRD26-related thrombocytopenia (ANKRD26-RT) is characterized by lifelong mild to moderate thrombocytopenia. Patients suffer from an increased susceptibility to acute or chronic myeloid leukemia, myelodysplastic syndrome, or chronic lymphocytic leukemia. We described here a patient with inherited thrombocytopenia initially misdiagnosed as immune thrombocytopenic purpura. A chromosomal deletion involving the ANKRD26 gene was identified. Gene and protein expression analyses suggest an alternative pathogenic mechanism of altered megakaryopoiesis: the synthesis of a chimeric protein with aberrant expression due to the unregulated action of a promoter from a gene located upstream of ANKRD26. This study highlights the importance of advanced genetic testing and functional analysis of patients’ primary cells in the case of the detection of previously unrecognized structural variants in order to understand pathogenic mechanisms. These investigations provided a definitive diagnosis for the patient and facilitated the development of a tailored clinical management strategy, especially concerning the potential for myeloid transformation.

Background: Hemoglobin A (Hb A) (α₂β₂) in the normal adult subject constitutes 96–98% of hemoglobin, and Hb F is normally less than 1%, while for hemoglobin A₂ (Hb A₂) (α₂δ₂), the normal reference values are between 2.0 and 3.3%. It is important to evaluate the presence of possible delta gene mutations in a population at high risk for globin gene defects in order to correctly diagnose the β-thalassemia carrier. Methods: The most used methods for the quantification of Hb A₂ are based on automated high performance liquid chromatography (HPLC) or capillary electrophoresis (CE). In particular Hb analyses were performed by HPLC on three dedicated devices. DNA analyses were performed according to local standard protocols. Results: Here, we described eight new δ-globin gene variants discovered and characterized in some laboratories in Northern Italy in recent years. These new variants were added to the many already known Hb A₂ variants that were found with an estimated frequency of about 1–2% during the screening tests in our laboratories. Conclusions: The knowledge recognition of the delta variant on Hb analysis and accurate molecular characterization is crucial to provide an accurate definitive thalassemia diagnosis, particularly in young subjects who would like to ask for a prenatal diagnosis or preimplantation genetic diagnosis.