Rare New Alpha-Globin Gene Variant Discovered in Chengdu Thalassemia Screening Study

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The detection of a novel intronic variant, HBA1:c.300+48C>G, in this Chengdu cohort illustrates a persistent challenge in thalassemia diagnostics: the gap between routine molecular screening panels and the full spectrum of globin gene variation. The commercial kits used in this study target a defined set of three common deletional and three common non-deletional α-globin mutations, together with seventeen frequent β-globin mutations, which capture the overwhelming majority of carriers in southern Chinese populations but leave rare and private variants undetected. This is precisely why the study design incorporated reflex Sanger sequencing for individuals whose routine testing was negative yet whose hematological profiles or hemoglobin electrophoresis results suggested an underlying globin disorder. Such a tiered approach reflects recommended practice in regions with high carrier frequencies, where panel-based testing serves as an economical first line and sequencing resolves the residual fraction of unexplained microcytosis or abnormal hemoglobin fractions.

The intronic location of the newly described variant deserves particular attention. Position c.300+48 falls within intron 1 of the HBA1 gene, forty-eight nucleotides downstream of the exon 1–intron 1 junction. Variants deep within introns are frequently dismissed as benign polymorphisms unless they disrupt the canonical splice donor or acceptor dinucleotides, yet decades of globin gene research have demonstrated that intronic sequences can harbor cryptic splice sites, branch point motifs, and regulatory elements whose disruption produces clinically significant α-thalassemia. The identification of this variant in a patient with severe anemia, and its absence from IthaGenes, HbVar, dbSNP, and the Human Gene Mutation Database, satisfies the initial criteria for considering it a candidate disease-causing change, but the authors appropriately caution that functional validation remains necessary. Establishing pathogenicity for a deep intronic variant typically requires RNA-based assays, such as reverse transcription PCR of reticulocyte-derived mRNA, to demonstrate aberrant splicing, or minigene experiments in which the variant-containing intron is cloned into an expression construct and splicing outcomes are assessed in cultured cells.

The distinction between HBA1 and HBA2 is also relevant to interpreting this finding. The two α-globin genes lie in tandem on chromosome 16p13.3 and are nearly identical in their coding regions, differing mainly in intron 2 and the 3′ untranslated region. This high sequence homogeneity complicates both amplification specificity and variant assignment, since a primer designed to amplify one paralog may preferentially or erroneously capture the other. The study addressed this by using paralog-specific primer pairs for α1 and α2, a necessary precaution when reporting a variant as belonging to HBA1 rather than HBA2. Misattribution between the two paralogs is a recognized pitfall in α-thalassemia molecular diagnostics and can affect both genotype–phenotype correlation and family counseling, because HBA2 is generally expressed at higher levels than HBA1, meaning that an identical variant on the two paralogs can carry different clinical weight.

The overall detection rate of 12.47 percent in this screening population provides a useful benchmark for carrier burden in the Xindu District of Chengdu. It is worth noting that this figure reflects an enriched population: the 6,357 individuals were referred specifically for thalassemia screening as part of premarital, prenatal, or health check-up programs, and nearly 89 percent were female, consistent with the predominance of prenatal screening presentations. Population-based surveys that sample unselected residents would be expected to yield somewhat different carrier frequencies, though the composition of detected genotypes tends to remain similar. The predominance of the –SEA deletion, accounting for roughly 46 percent of α-thalassemia cases, mirrors patterns documented across Sichuan and neighboring provinces, where this Southeast Asian deletion is the single most common α-globin defect. Its clinical importance lies in the fact that two copies in trans, or one copy combined with another α-deletion, can produce HbH disease, a form of α-thalassemia intermedia characterized by chronic hemolytic anemia that may require transfusion support and carries risks during pregnancy and under oxidative stress.

The β-globin findings in this cohort similarly align with known mutational geography. The three most frequent β-thalassemia mutations—CD17(A>T), CD41-42(-TTCT), and IVS-II-654(C>T)—together constituted over 81 percent of β-globin variants detected. All three are classified as β-zero or severe β-plus mutations, meaning they abolish or severely reduce β-globin production from the affected allele. CD41-42 is a four-nucleotide deletion that causes a frameshift, CD17 is a nonsense mutation, and IVS-II-654 is a splice-site region mutation that activates a cryptic splice acceptor within intron 2. The high combined frequency of these three mutations in southwestern China is well documented and underpins the design of regional screening panels. When two such severe alleles are inherited together, the result is β-thalassemia major, which requires lifelong transfusion and iron chelation therapy unless hematopoietic stem cell transplantation is available. This is the central rationale for carrier screening programs: identifying couples in which both partners carry severe alleles allows informed reproductive decision-making and access to prenatal or pre-implantation genetic diagnosis.

The 22 cases of coexisting α- and β-thalassemia variants in this cohort, roughly 2.8 percent of confirmed carriers, highlight a diagnostic subtlety with practical consequences. When α- and β-globin defects coexist in the same individual, the reduced excess of α chains partially offsets the imbalance caused by β-globin deficiency, often ameliorating the hematological picture. A person carrying both a β-zero mutation and a two-cis α-deletion may show hematological indices closer to those of a simple β-thalassemia trait carrier than expected, or conversely may show milder red cell indices than a β-thalassemia carrier should display. This masking effect can mislead screening algorithms that rely on mean corpuscular volume and mean corpuscular hemoglobin thresholds to flag carriers, potentially causing silent carriers to be overlooked. Conversely, in reproductive counseling, the coexistence matters greatly: a couple in which one partner carries an α-deletion and the other carries a β mutation faces risks of both HbH disease and β-thalassemia major in offspring, depending on the specific alleles transmitted, and compound genotypes can produce more complex phenotypes than either defect alone.

The methodological reliance on flow-through hybridization for routine genotyping merits comment as well. This platform, widely deployed in Chinese hospital laboratories, uses reverse dot-blot hybridization on a membrane or chip to detect a fixed panel of mutations simultaneously. Its strengths are throughput, cost, and speed; its limitation is that it can only find what it looks for. Any variant outside the panel—whether a rare point mutation, a novel deletion, or a structural variant such as Hb Constant Spring outside the tested set—will be missed. The 793 confirmed carriers in this study therefore represent a floor rather than a ceiling for the true carrier frequency, since individuals with unpanelled variants who did not undergo sequencing would not have been counted. The reflex sequencing strategy partially compensates, but only for those flagged by hematological or electrophoretic abnormalities, and some α-thalassemia carriers, particularly single-gene deletions in cis configurations, may show indices within normal limits and escape detection entirely.

Capillary electrophoresis for hemoglobin analysis, as used in this study, offers advantages over older cellulose acetate electrophoresis in quantifying HbA2 and detecting and measuring HbH, Hb Bart’s, and common variants such as HbE. Elevated HbA2 remains the biochemical hallmark of β-thalassemia trait, though it can be confounded by iron deficiency, which lowers HbA2 and may mask a carrier state. The interplay between iron status and hemoglobin fractions is one reason some screening programs combine ferritin measurement with hemoglobin analysis, particularly in populations where iron deficiency is prevalent among women of reproductive age, as it was in this largely female cohort.

From a public health standpoint, the study’s five-year window spanning 2021 through 2025 captures a period during which Sichuan province expanded preconception and prenatal carrier screening under regional maternal health initiatives. Data of this kind—genotype frequencies from a defined catchment area—serve as the empirical foundation for such programs, informing decisions about which mutations to include in regional panels, how to counsel couples with compound risks, and where to concentrate sequencing resources. The finding that nearly 70 percent of confirmed carriers were aged 20 to 39 underscores that the screened population corresponds closely to the reproductive window, which is precisely the group for whom carrier status has immediate reproductive implications.

Finally, the report of HBA1:c.300+48C>G contributes to the ongoing cataloguing of globin gene variation, a project that remains incomplete even after decades of study. Each newly described variant, once functionally characterized, refines the interpretation of sequencing results in clinical laboratories and reduces the number of variants of uncertain significance that families must confront during counseling. For the proband in whom this variant was found, segregation analysis in relatives—testing parents and siblings to determine whether the variant tracks with anemia in the family—would constitute an important next step, alongside RNA-level splicing studies. Such follow-up would determine whether this intronic change is a true thalassemia-causing mutation or a rare benign variant coincidentally present in an individual whose anemia has another explanation, a distinction that only further investigation can resolve.

Subject of Research: …

Article Title: Genotype distribution and identification of rare variants in α/β-thalassemia in chengdu: including the first report of HBA1:c.300+48C > G

Article References: Liu, X., Du, H., Zhang, L., Yu, Y., Wang, G., He, X., Huang, H., & Xu, Y. (2026). Genotype distribution and identification of rare variants in α/β-thalassemia in chengdu: including the first report of HBA1:c.300+48C > G. Frontiers in Genetics, 17, Article 1899397. https://doi.org/10.3389/fgene.2026.1899397

Image Credits: AI Generated

DOI: 10.3389/fgene.2026.1899397

Keywords: Genotype, distribution, identification, rare, variants, thalassemia, chengdu, including, first, report, HBA1, scientific research