Hemoglobin

To characterize the hematologic effects of hemoglobin D (HbD) alone and in the presence of co-inherited α- or β-thalassemia, and to quantify associations with anemia, microcytosis, and hypochromia relative to normal-CBC controls. This retrospective comparative study included 202 adults with molecularly confirmed HbD-Punjab, HbD only (n = 79), HbD plus β-thalassemia (n = 78), and HbD plus α-thalassemia (n = 45), and 100 normal-CBC controls. Hematologic indices were compared using Welch's one-way ANOVA or the Kruskal-Wallis test, with appropriate post hoc analyses. Case-control analyses evaluated anemia (Hb <12 g/dL), microcytosis (MCV <80 fL), and hypochromia (MCH <26 pg). A post hoc analysis used sex-specific anemia thresholds. WBC, RBC, Hb, MCV, MCH, HCT, and MCHC differed across the four groups (all p ≤ 0.002), whereas HCT did not (p = 0.523). HbD plus β-thalassemia had the lowest MCV (65.67 ± 6.85 fL) and MCH (21.12 ± 2.55 pg), which were significantly lower than in the HbD-only and HbD plus α-thalassemia groups (all p < 0.01). Hemoglobin did not differ significantly among the three HbD subgroups. Compared with controls, anemia was associated with HbD only (OR = 8.3, 95% CI 3.22-25.74), HbD plus β-thalassemia (OR = 11.9, 95% CI 4.68-36.59), and HbD plus α-thalassemia (OR = 11.5, 95% CI 4.16-37.68). Microcytosis and hypochromia also differed significantly between HbD groups and controls (Fisher's exact p < 0.001). Sex-specific sensitivity analyses preserved all associations with anemia. β-Thalassemia co-inheritance produced the most pronounced microcytic-hypochromic phenotype, whereas α-thalassemia had a less pronounced effect. HbD alone was associated with relatively mild changes in red-cell indices, but co-inherited thalassemia, particularly β-thalassemia, substantially altered the hematologic phenotype. Integrated hematologic, electrophoretic, and molecular assessment is important for accurate classification.

Elevated hemoglobin (Hb) concentration, erythrocytosis, is a common phenomenon that can be caused by primary or secondary underlying conditions. In this report, we describe a previously unknown mutation in the β globin gene resulting in high Hb concentrations combined with elevated reticulocytes in two family members spanning two generations. In the index patient the variant was first noted during HbA1c monitoring. While both ion-exchange chromatograms used for HbA1c analysis and isoelectric focusing indicated the presence of a β globin variant, the chromatographic method used for hemoglobinopathy screening mostly failed to detect the variant. Mass spectrometry demonstrated a -60 Da β globin variant and Sanger sequencing revealed a previously unknown p.Phe104Ser substitution in the HBB gene [HBB:c.311T > C]. This mutation affects the interaction with heme and results in increased oxygen affinity as well as instability. Common causes of acquired as well as congenital erythrocytosis were ruled out. This novel variant was named Hb Koskullskulle (IthaID 4114) from the place of origin of the family in which it was found.

Hemoglobin E (HbE) is a common structural hemoglobin variant with a β+-thalassemic effect. Hemoglobin Constant Spring (HbCS) is the most prevalent non-deletional α-thalassemia variant and is characterized by low expression and marked instability, making detection difficult. The coexistence of both variants may produce atypical hematologic and electrophoretic profiles, complicating diagnosis. We describe two patients with combined HbE and HbCS: a 12-month-old girl heterozygous for HbE and a 58-year-old man homozygous for HbE. Both presented with microcytosis and characteristic patterns on capillary electrophoresis and high-performance liquid chromatography, which were performed simultaneously as part of routine diagnostic testing. Molecular analysis using StripAssay, MLPA (multiplex ligation-dependent probe amplification), and next-generation sequencing (NGS) confirmed heterozygous HbCS in both cases, with no additional alterations in the α- and β-globin gene clusters. The association of HbE and HbCS poses significant diagnostic challenges. These cases highlight the limitations of phenotypic techniques used in isolation and underscore the value of an integrated diagnostic approach that incorporates complementary molecular methods, such as NGS-based platforms.

In β-thalassemia carriers, the presence of one or two extra copies of the α-globin genes may exacerbate clinical manifestations and lead to a more severe phenotype than would normally be expected. Given the high prevalence of thalassemia in Iran and the limited number of studies on αααanti-3.7, the aim of this study was to conduct a population-based study in Iran and to develop a practical, cost-effective method to detect the most common αααanti-3.7 variants in the Iranian population. A total of 110 individuals with β-thalassemia minor whose hematological parameters were below the mean of the study population were analyzed for the αααanti-3.7 triplication, which was characterized in six carriers by MLPA. Based on these data, a novel PCR test was developed to detect the most common αααanti-3.7 variants and was evaluated using samples with known genotypes. Among β-thalassemia minor carriers with below-average hematological parameters, the frequency of αααanti-3.7 carriers was 5.5% (6/110). MLPA analysis identified D and F as the αααanti-3.7 variants in Iranian carriers. The newly developed PCR method successfully identified the triplication in all positive samples. This study provides a foundation for large-scale epidemiological investigations of the αααanti-3.7 triplication in Iran. The developed method has the potential for routine diagnostic screening of αααanti-3.7 in the Iranian population.

Hemoglobin Constant Spring (HbCS) is the most common non-deletional α-thalassemia mutation in Southeast Asia (SEA) and is frequently missed during routine Hb analysis because of its extremely low expression in peripheral blood. This study aims to evaluate the efficacy of three hemoglobin analysis systems: Capillarys 2 Flex Piercing (CE), Premier Resolution (PR)-HPLC, and VARIANT II-HPLC for the detection of HbCS. A total of 40 EDTA blood samples suspected of carrying HbCS were analyzed. Hb analysis was performed using the three analytical systems, while molecular confirmation of Hb CS/Paksé (PS) mutations was carried out using multiplex AS-PCR. In addition, α0-thalassemia deletions (-SEA, -THAI, and -CR) were identified using real-time PCR with high-resolution melting analysis. Four Hb typing patterns were identified: CSA2A (n = 26), CSA2ABart'sH (n = 8), CSEABart's (n = 4), and CSEA (n = 2). Molecular analysis confirmed the presence of the HbCS mutation in all 40 samples. The correct identification rates were 90.0% for CE and 87.5% for both PR-HPLC and VARIANT II-HPLC. Misidentification occurred most frequently in the CSA2A group, likely due to the extremely low proportion of HbCS in individuals without co-inherited α-thalassemia. In addition, PR-HPLC misidentified HbCS in one sample from each of the CSA2ABart'sH, CSEABart's, and CSEA groups. These findings suggest that CE provides higher efficiency than HPLC systems for detection of HbCS. However, molecular confirmation remains essential for accurate diagnosis.

α-Thalassemia is an autosomal recessive disorder characterized by reduced synthesis of α-globin chains, most commonly due to deletions within the α-globin gene cluster. Non-deletional variants are less common and contribute to the molecular heterogeneity of the disease. Advances in long-read sequencing have improved the analysis of complex genomic regions, including highly homologous genes such as HBA1 and HBA2. Targeted long-range PCR amplification of the HBA1 and HBA2 genes was followed by long-read sequencing on the PromethION 24 platform (Oxford Nanopore Technologies). Subsequently, the sequencing data were analyzed and visualized using the Integrative Genomics Viewer (IGV). Long-read sequencing achieved an average depth of >390x across all target regions, enabling reliable variant detection. This analysis identified five variants in the HBA1 and HBA2 genes, namely HBA1:c.326C > A (p.Thr108Asn; Hb Rogliano), HBA1:c.-41C > G, HBA2:c0.300 + 55T > G, HBA2:c0.301-24delinsCTCGGCCC, and HBA2:c.-41C > G. Based on ClinVar classification, HBA1:c.326C > A (p.Thr108Asn) was categorized as likely pathogenic, while the remaining variants were classified as benign or likely benign. To our knowledge, this is the first report of Hb Rogliano in Indonesia and highlights the utility of long-read sequencing for the molecular characterization of rare α-globin variants.

Although thyroid dysfunction (TD) is a known complication in patients with thalassemia major, data on patients with β thalassemia intermedia (βTI) are limited. Therefore, this study was conducted to investigate the prevalence and gender pattern of TD in βTI in Yasuj city. In this cross-sectional analytical study, 200 eligible patients with βTI were selected by the census method. Serum levels of triiodothyronine (T3), thyroxine (T4), and thyroid-stimulating hormone (TSH) were quantified using enzyme-linked immunosorbent assay (ELISA). Data were analyzed using SPSS version 27 at a significance level of 0.05. Of the 200 patients, 90 (45%) were male, and 110 (55%) were female, with a mean age of 34.6 ± 11 years. The mean serum levels of T3, T4, and TSH were 1.74 ± 0.53 ng/ml, 7.99 ± 1.6 µg/dl, and 3.23 ± 1.83 µIU/ml, respectively. The overall prevalence of hypothyroidism was 6.0% (n = 12), comprising 5.5% (n = 11) subclinical and 0.5% (n = 1) primary hypothyroidism. A significant gender-based difference was observed in hormone levels: women exhibited higher mean T3 (1.81 vs. 1.66 ng/ml, p = 0.04) and T4 (8.25 vs. 7.7 µg/dl, p = 0.01) compared to men. However, no significant gender disparity was found in TSH levels. T3 and T4 hormone levels are higher in female patients with βTI compared to males. The presence of hypothyroid cases underscores the necessity for regular monitoring of thyroid function in these patients, particularly within the framework of iron overload management. Timely diagnosis and treatment can improve clinical outcomes.

This study screened for major depression and generalized anxiety disorder (GAD) across the menstrual cycle among women with sickle cell disease (SCD) and examined associations with heavy menstrual bleeding (HMB), dysmenorrhea, and sex hormones. A total of 125 Jamaican women aged 18 and over with SCD, regular menses, and no recent hormonal contraceptive use completed follicular- and luteal-phase visits including serum estradiol and progesterone measurements and validated mental and menstrual health and quality-of-life questionnaires. Higher Menstrual Symptom Questionnaire and Menstrual Bleeding Questionnaire scores reflect more severe menstrual pain and menstrual heaviness, respectively, including quality-of-life impact. Menstrual scores were standardized as z-scores and odds ratios represent the change in odds per one-standard deviation change from the sample mean. Major depression and GAD were screened using the Patient Health Questionnaire-9 and Generalized Anxiety Disorder-7. Screen-positive rates for major depression and GAD were 44.8% and 39.2% with no differences between phases; and 32% reported passive self-harm ideations. Marginal mean scores were HMB 11.6 ± 4.9 (scale: 0-24) and dysmenorrhea 62.9 ± 13.5 (scale: 24-120). Worse dysmenorrhea was associated with higher odds of major depression risk (odds ratio, OR = 2.6), whereas better quality-of-life in pain (OR = 0.9) and sleep (OR = 0.8) were associated with lower odds. Severe genotypes (OR = 0.1) and better quality-of-life in stiffness (OR = 0.9) and sleep (OR = 0.9) were associated with lower odds of GAD risk. Estradiol, progesterone, and HMB showed no significant adjusted effects. The high prevalence of GAD and major depression risk, and the association between depression and dysmenorrhea suggest that routine screening is needed in this vulnerable group.

β-thalassemia comprises a clinically heterogeneous group of disorders in which anemia severity, transfusion exposure, iron loading, and organ complications vary widely among individuals. This structured narrative review summarizes practical applications of precision medicine in transfusion-dependent thalassemia (TDT) and non-transfusion-dependent thalassemia (NTDT), with explicit attention to which strategies apply to each clinical category. Literature indexed in PubMed and Scopus from 2000 to 2025 was reviewed using terms related to thalassemia, precision medicine, magnetic resonance imaging (MRI), chelation tailoring, next-generation sequencing (NGS), fetal hemoglobin (HbF) modifiers, luspatercept, mitapivat, hepcidin, gene therapy, gene editing, and artificial intelligence (AI). Evidence was synthesized descriptively because interventions, outcomes, and populations were heterogeneous, and no pooled meta-analysis was performed. In TDT, precision care is centered on individualized transfusion planning, extended red-cell antigen matching, MRI-guided cardiac and hepatic iron monitoring, organ-directed chelation intensification, and selection of disease-modifying or curative approaches. In NTDT, precision care emphasizes accurate phenotype classification, MRI liver iron concentration, because serum ferritin may underestimate iron burden, selective chelation, surveillance for NTDT-specific complications, and individualized use of agents that improve anemia. Personalized chelation should include deferiprone, either alone or in combination, when cardiac iron is increased. Comprehensive molecular diagnosis should include HBB together with HBA1 and HBA2 assessment, while secondary and tertiary modifiers help explain phenotypic variability and complication risk. Hepcidin and growth differentiation factor 15 (GDF-15) are discussed as investigational biomarkers; transferrin saturation is not recommended for routine iron-overload assessment in thalassemia. AI currently has its strongest role in screening and diagnosis, whereas risk-stratification models remain exploratory. Equitable implementation requires standardized TDT/NTDT pathways, regional MRI and genomics access, longitudinal registries, and multidisciplinary interpretation.

Advances in transfusion regimens and iron chelation have transformed transfusion-dependent thalassemia (TDT) into a chronic condition, revealing chronic pain as an important but under-recognized morbidity. Despite its substantial effect on health-related quality of life, pain in TDT remains poorly characterized and is rarely addressed in clinical protocols. We systematically searched PubMed/MEDLINE, EMBASE, Scopus, Google Scholar, and the Cochrane Library from inception to January 2026 for original studies, reviews, and guidelines addressing chronic pain in TDT. Two independent reviewers screened studies, extracted data, and synthesized evidence on prevalence, pathogenesis, and management. Chronic pain affects approximately 32% to 55% of adults with TDT, and more than 90% of affected patients report moderate-to-severe pain. Its pathogenesis is multifactorial, with major contributors including thalassemia-associated osteoporosis, chelation-related arthropathy, extramedullary hematopoiesis, iron-overload-related endocrinopathies, and treatment-associated abdominal pain, particularly with deferasirox. Management remains empirical and fragmented. Bisphosphonates, including zoledronic acid, may improve skeletal pain, whereas NSAID and opioid use is often limited by hepatic and renal comorbidities. A disease-specific approach should combine correction of metabolic abnormalities, targeted anti-resorptive therapy, validated pain assessment, and cautious pharmacological treatment. Chronic pain is a major but neglected component of the contemporary TDT phenotype. Randomized trials and standardized clinical guidelines are needed to support effective pain management and improve quality of life.

Sickle cell disease is caused by the HBB c.20A > T (p.Glu6Val; HbS) variant. Although Cyprus has a long-standing hemoglobinopathy prevention program, current population-specific data on HbS among Turkish Cypriots remain limited. Two hundred unrelated Turkish Cypriot adults were included. The HBB c.20A > T variant was genotyped by PCR-RFLP and confirmed by allele-specific real-time PCR. Genotype distribution was evaluated for Hardy-Weinberg equilibrium. No homozygous HbS genotype was detected. Three individuals were heterozygous carriers (AS), corresponding to a carrier frequency of 1.5%. The HbS allele frequency was 0.7%, and the genotype distribution was consistent with Hardy-Weinberg equilibrium (p = 0.916; chi-square = 0.011). HbS was uncommon in this Turkish Cypriot cohort. These findings support continued hemoglobinopathy surveillance and the integration of HbS counseling into existing premarital and population screening strategies, particularly as migration may alter local carrier frequencies over time.

Hemoglobin variants and α-thalassemia deletions are uncommon causes of unexplained hypoxemia in children. This study reports a Chinese family affected by compound heterozygous HBA2 mutations that decrease hemoglobin-oxygen affinity. Clinical data, oxygen saturation (SpO2) trends, chest imaging, capillary hemoglobin electrophoresis, and genetic testing were conducted on the proband and his family members. The proband had persistently low SpO2 after pneumonia resolution, without respiratory distress; his 4-year-old brother experienced similar hypoxemia with normal cardiopulmonary evaluations. Capillary electrophoresis showed increased variant hemoglobin fractions in both siblings and their mother. Genetic analysis revealed compound heterozygosity: a maternally inherited HBA2: c.275T > C (p.L92P, Hb Port Phillip) and a paternally inherited -α3.7 deletion. The proband's mother is heterozygous for the point mutation and asymptomatic, while the father carries the -α3.7 deletion. This is the first report of a compound heterozygote for Hb Port Phillip and -α3.7 deletion with hypoxemia in a Chinese family, thus broadening the phenotype-genotype spectrum of hemoglobinopathies. The unexpected low oxygen saturation may result from a hemoglobin variant in the setting of mild microcytic anemia; hemoglobin electrophoresis and genetic testing are crucial for accurate diagnosis and can prevent unnecessary interventions.

Congenital dyserythropoietic anemia type II (CDA II) is a rare hyporegenerative inherited anemia, resulting from a mutation in SEC23B. In the present case, our patient exhibited moderate anemia, jaundice, hepatosplenomegaly, tea-colored urine, hyperbilirubinemia, and iron overload. Whole exome sequencing revealed that the patient carried a compound heterozygous genotype in SEC23B consisting of a previously unreported missense variant c.181T > C (p.C61R) and a known pathogenic variant c.1832G > A (p.R611Q). Bone marrow aspirate demonstrated erythroid hyperplasia with abnormal erythroblast morphology. Bioinformatic analysis predicted the protein structures, indicating that p.C61R and p.R611Q mutations induce structural changes in their surrounding regions. SEC23B mRNA and protein levels in peripheral blood mononuclear cells (PBMCs) were significantly reduced compared with those in normal control cells, supporting their pathogenicity. Accordingly, a diagnosis of CDA II was considered. In this study, we identified a compound heterozygous SEC23B genotype in the patient and demonstrated that missense mutations of p.C61R and p.R611Q resulted in reduced levels of SEC23B mRNA and protein, suggesting the association of this genotype with CDA II.

Sickle cell disease (SCD) is a monogenic disorder marked by hemoglobin S polymerization, resulting in chronic hemolysis, vaso-occlusion, systemic inflammation, and progressive multiorgan damage. Despite major therapeutic advances, SCD remains a complex inflammatory condition with significant morbidity. Hydroxyurea is the cornerstone of treatment, primarily by inducing fetal hemoglobin and reducing vaso-occlusive crises and hemolysis. It also exerts anti-inflammatory effects by decreasing leukocyte activation and endothelial adhesion. However, hydroxyurea does not fully reverse microvascular injury, persistent immune activation, or organ dysfunction, particularly renal and endothelial damage. This review aims to synthesize current evidence on the interactions between hydroxyurea and the gut microbiome in SCD and to evaluate the potential role of microbiome-directed therapies as adjunctive strategies to control inflammation and organ damage. Recent evidence highlights the gut microbiome as a critical regulator of immune homeostasis and inflammation in SCD. Dysbiosis, marked by reduced microbial diversity and diminished short-chain fatty acid (SCFA) production, drives cytokine activation, endothelial dysfunction, and pain sensitization. Emerging studies suggest that hydroxyurea may partially restore microbial balance, yet residual dysbiosis persists. Microbiome-directed therapies, including probiotics and microbial metabolites, show promise for reducing pro-inflammatory cytokines, strengthening gut barrier integrity, and modulating immune responses. Probiotic strains such as Lactobacillus and Bifidobacterium, together with SCFA-mediated pathways, may enhance anti-inflammatory effects and address therapeutic gaps left by hydroxyurea. A combined strategy targeting both hematologic and microbiome pathways may offer superior control of inflammation and organ damage. Integrating microbiome-based interventions with conventional therapy represents a promising, patient-centered approach to improving long-term outcomes and quality of life in SCD.

δβ-Thalassemia and hereditary persistence of fetal hemoglobin (HPFH) are uncommon hemoglobinopathies. This study aimed to define the molecular epidemiological features of δβ-thalassemia and HPFH in the childbearing-age population of Quanzhou, China, to inform precise prevention strategies and genetic counseling. From a free pre-pregnancy thalassemia screening cohort (n = 19,154), 92 individuals with elevated HbF (≥2%) and 11 control groups with normal HbF (≤2%) were included. DNA analysis of common deletion-type δβ-thalassemia/HPFH mutations (SEA-HPFH, Chinese type Gγ+(Aγδβ)0, Taiwan type β0) and non-deletion-type HPFH mutations was performed by gap-PCR, PCR-reverse dot hybridization, and Sanger sequencing. The correlation between hematological parameters and genotypes was also analyzed. The results suggest that the detection rate of deletion-type HPFH/δβ-thalassemia was 0.063% (12/19154), mainly SEA-HPFH and Chinese Gγ+(Aγδβ)0. The detection rate of γ-globin gene mutations and non-deletion-type HPFH was 0.329% (63/19154). 9 different γ-globin promoter mutations were identified. The most frequently detected variants were HBG2:c.-211C>T, HBG1:c.-29G>A, and HBG1:c.-272_-275dupAGCA. However, based on functional relevance, three key variants were highlighted: HBG1:c.-211C>T, HBG1:c.-249C>T, and the novel HBG2:c.-253_-254dup. The remaining variants (HBG1:c.-29G>A, HBG1:c.-272_-275dupAGCA, HBG1:c.-404A>G, HBG1:c.-417G>C, HBG1:c.-420C>A) are described as benign polymorphisms or variants in strong linkage disequilibrium with HBG2:c.-211C>T. Deletion-type δβ-thalassemia/HPFH is rare in Quanzhou. SEA-HPFH and Chinese Gγ+(Aγδβ)0 are more common. Non-deletion-type HPFH, especially the double heterozygous state HBG1:c.-29G>A/HBG2:c.-211C>T, is significantly more common. These findings reveal the unique molecular epidemiological characteristics of δβ-thalassemia and HPFH in this region, providing important data for genetic counseling and prenatal diagnosis.

Thalassemia is a prevalent genetic disorder in Southeast Asia. The Hemoglobin Minneapolis-Laos variant is very rarely reported with only two previously published reports that profile a total of three patients. Here, we present the first reported case of compound heterozygous β zero (β0)-thalassemia and Hemoglobin Minneapolis-Laos in a 46-year-old Thai female. She presented at Siriraj Hospital (Bangkok, Thailand) with chronic microcytic anemia, which is a more severe phenotype than would be expected from either trait alone. Initial hemoglobin electrophoresis via high-performance liquid chromatography and capillary electrophoresis revealed elevated hemoglobin A2 (5.5% and 6.3%, respectively), which is a finding consistent with a β-thalassemia trait, but this finding failed to explain the full extent of her anemia. Next-generation sequencing was then performed to investigate for a congenital red blood cell disorder. The results identified the following two mutations in the β-globin gene (HBB): heterozygous β0-thalassemia codon 41/42 (-TTCT), and HBB c.356T >A, the latter of which is consistent with hemoglobin Minneapolis-Laos. This case highlights the importance of advanced genetic testing to diagnose rare hemoglobin variants that cannot be identified by conventional investigation and further contributes to our understanding of this rare combination's clinical phenotype.

We report a novel fetal hemoglobin variant, HBG2:c.308A > G [Gγ(102(G4)Asn > Ser], in a male neonate presenting with cyanosis and low oxygen saturation that resolved three months after birth, coinciding with the temporal decrease in the variant hemoglobin. Further molecular analysis confirmed maternal inheritance of this variant. The HBG2:c.308A > G variant parallels known low-oxygen-affinity mutations at the same position in the HBB and HBG2 genes, supporting its role in transient neonatal cyanosis. This variant has been designated Hb F-SickKids. Identifying such variants is important for timely diagnosis, avoiding unnecessary interventions, and facilitating accurate genetic counseling and reproductive planning.

Hemoglobinopathies constitute a major cause of hereditary anemia and remain a significant public health challenge in Azerbaijan. The purpose of this study was to delineate the variant spectrum and genotype distribution in a large national cohort and to evaluate the contribution of next-generation sequencing (NGS) to molecular diagnosis. During the study period, 1,000 unrelated individuals with suspected hemoglobinopathies underwent hematological and molecular genetic evaluation at a national referral center in Azerbaijan. Overall, 777 of 1,000 (77.7%) individuals were genetically positive. Of these, β-thalassemia was most frequent (59.9%, 466/777), followed by α-thalassemia (22.0%, 171/777) and co-inheritance of α/β-thalassemia (9.52%, 74/777). In addition, HbS-related genotypes were detected in 6.2% (48/777) and other rare hemoglobin variants in 1.3% (10/777). In α-thalassemia, the most common genotypes were αα/-α³·7 (22.81%), -α³·7/-20.5 (17.54%), and αα//-20.5 (16.37%), while the predominant alleles were - α³·7 (n = 76) and -20.5 (n = 72). In β-thalassemia, the leading alleles were c.25_26delAA (codon 8 - AA; 19.74%), c.315 + 1G > A (IVS-II-1; 8.91%), and c.93-21G > A (IVS-I-110; 5.58%), which together accounted for ∼35% of mutant alleles. The large deletional variants, including Sicilian and Turkish (δβ)0 deletion, and Hb-Lepore, were also identified, highlighting the importance of deletion-focused assays. Notably, a novel HBA2 start-codon variant (c.3G > A; p.Met1Ile, Hb Baku) and a previously unreported splice variant in HBB (c.315 + 3_+4insT) expanded the mutational landscape. The broad spectrum of small sequence variants and large genomic alterations, including rare and novel findings, underscores the limitations of targeted assays and supports the integration of comprehensive NGS-based diagnostics into national screening and prevention programs.

Unstable hemoglobin (Hb) variants are a rare cause of congenital non-spherocytic hemolytic anemia, characterized by hemoglobin instability and Heinz body formation. Diagnosis is often challenging and requires a high index of suspicion, supported by targeted laboratory investigations. We report the first documented case of an unstable hemoglobin variant identified as hemoglobin Perth (also known as Hb Abraham Lincoln) from Iran in a patient who was transfusion-dependent since early childhood. At 10 years of age, he presented with acute cholecystitis and cholelithiasis, accompanied by profound direct hyperbilirubinemia. He underwent cholecystectomy and splenectomy, which resulted in significant clinical improvement, including transfusion independence and stabilization of hemoglobin levels. This case is particularly noteworthy for the exceptionally high levels of direct bilirubin unreported in previous cases of Hb Perth, as well as the successful therapeutic outcome achieved following splenectomy.