Routine thalassemia was diagnosed in 1936 of the 3196 samples, with 733 cases of α-thalassemia (37.86%), 1170 of β-thalassemia (60.43%), and 33 cases of compound heterozygous α-/β-thalassemia (1.70%). The study population comprised 635 males (32.80%) and 1301 females (67.20%) (Fig. 1A), with the age distribution primarily concentrated in the 20–29 and 30–39 age groups, accounting for 1375 patients (71.02%) (Fig. 1B).

Among 733 patients with α-thalassemia, 679 (92.63%) carried heterozygous variants, and 54 (7.37%) had homozygous or compound heterozygous variants. --SEA/αα was the most common genotype, with 550 cases, accounting for more than half (75.03%) of all α-thalassemia genotypes. Other common genotypes of α-thalassemia included -α3.7/αα, -α3.7/--SEA, and -α4.2/ αα (Table 1).

Constituent ratios of different gender, age groups and basic hematological red blood cell parameters of α/β-thalassemia patients (Beijing, China). A Gender distribution among of α/β-thalassemia patients. B Age demographics among α/β-thalassemia patients. C Proportions of mean corpuscular volume (MCV, including 379 alpha-thalassemia samples and 611 beta-thalassemia samples), mean corpuscular hemoglobin concentration (MCHC, including 365 alpha-thalassemia samples and 588 beta-thalassemia samples), and mean corpuscular hemoglobin (MCH, including 365 alpha-thalassemia samples and 592 beta-thalassemia samples) among α/β-thalassemia patients. Note: **** means P < 0.0001

Among 1170 patients with β-thalassemia, 1164 (99.49%) carried heterozygous variants, and 6 (0.51%) had homozygous or compound heterozygous variants. The most frequent genotypes were βIVS-II-654(C>T)/βN, βCD41-42(-TCTT)/βN, and βCD17(A>T)/βN, collectively representing 91.28% of all β-thalassemia genotypes. Other common genotypes of β-thalassemia included β-28(A>G)/βN, βCD27-28(+C)/βN, and βE(GAG>AAG)/βN (Table 1).

Thirty-three patients carried the compound heterozygous α-/β-globin variants. Seventeen genotypes were detected in these patients, and the most common type was -α3.7/αα combined with βIVS-II-654(C>T)/βN (Table 1).

There were significant differences in the basic hematological parameters erythrocyte mean corpuscular volume (MCV, P < 0.0001), erythrocyte mean corpuscular hemoglobin concentration (MCHC, P < 0.0001), and erythrocyte mean corpuscular hemoglobin (MCH, P < 0.0001) between patients with α- and β-thalassemia (Fig. 1C).

A cohort of 1936 patients diagnosed with routine thalassemia hailing from various regions in China, including North China, Southwest China, and the middle and lower reaches of the Yangtze River, including 310 in Beijing, 180 in Hunan, 182 in Sichuan, 142 in Guangxi, and 129 in Hebei (Fig. 2).

Geographic distribution of α/β-thalassemia. Red represents α-thalassemia, green represents β-thalassemia, blue represents Concurrent α-/β-thalassemias, and the size of the colour of the circle represents the number of people affected (Beijing, China). The southern regions, delineated by the Qinling Mountains to the north and the Huai River to the south, are highlighted in a soft pink shade. With the exception of Tibet, the unshaded areas are classified as part of the northern region

Rare globin gene variants were detected in 31 patients (cases 1–31, Table 2), including 4 patients with rare α-thalassemia (case 4–7), 24 patients with rare β-thalassemia (cases 8–31) and 3 patients with rare deletion (cases 1–3, Fig. 3A; Table 2). In this study, two new pathogenic gene variants of α/β-thalassemia cases were identified, including case 4: HBA2:c.300+82G>C; and case 8: HBB:codon85(-T). These variants have not been reported in HbVar, HGMD, and PubMed databases.

Multiplex Ligation-dependent Probe Amplification (MLPA) analysis, Sanger sequencing and Hemoglobin electrophoresis results of rare cases (Beijing, China). The ordinate represents the gene copy number, the abscissa represents the chromosomal position of the gene. The probe ratio within the red box, indicating a deletion, showed a value of only 0.5 (3 A-C). A MLPA result showed the deletion across 5 probes targeting HBB gene (deletion probes involved 5 probes between hg18 loc.11p15.4:5177860-5203330, including HBB-3, HBB-down and ORF51V1-1 regions) (Case 1). B MLPA result showed the deletion across 8 probes targeting HBB gene (deletion probes involved 8 probes between hg18 loc.11p15.4:5202720-5204970, including HBB-1, HBB-1 (WT) c, HBB-Intr1, HBB-Intr. 2, HBB-3 and HBB-down regions) (Case 2). C MLPA result showed the deletion across 11 probes targeting HBB gene (deletion probes involved 11 probes between hg18 loc.11p15.4:5202711-5210637, including HBB-up, HBB-1, HBB-1 (WT) c, HBB-Intr1, HBB-Intr. 2, HBB-3 and HBB-down regions) (Case 3). D and E Two new pathogenic variants in the HBA2 gene and HBB (Cases 4 and 8)

Case 4, Male, 1-year-old, ancestral home in Inner Mongolia with mild anemia. The patient exhibited no jaundice and had no history of blood transfusion. Hemoglobin (HGB, 9.1 g/dL) was detected on physical examination at another hospital, and the patient was prescribed enhanced iron supplementation. HGB (9.4 g/dL) was reexamined one month later. After the patient regained strength with supplementary iron feeding, HGB (9.7 g/dL) was reexamined. On November 11, 2021, he visited the Pediatric Outpatient Department of PUMCH for thalassemia screening. A comprehensive panel of 23 routine thalassemia gene tests yielded negative results in laboratory analysis. The hemoglobin profile was as follows: HbA, 97.1%; HbA2, 2.3%, and HbF, 0.6%. Sanger sequencing revealed a novel heterozygous mutation in the α-globin gene HBA2:c.300+82G>C(Fig. 3B). HBA2:c.300+82G>C was predicted as polymorphism by MutationTaster2.

Case 8, Female, 34 years from Shandong, 24 weeks of gestation, presented with mild anemia. She had been experiencing anemia for more than ten years. The patient was administered iron supplementation. During the previous pregnancy, the hemoglobin level was 9 g/dL, which decreased to 7.2 g/dL in the current pregnancy. Additionally, the MCV was 64 fL, reticulocyte count (RET) was 3.56%, serum ferritin (SF) was 128 µg/L, and levels of bilirubin and lactate dehydrogenase (LDH) were within normal ranges. Iron supplementation was briefly administered during the pregnancy. The mother was anemic. Her three brothers had no anemia, and the MCV decreased in the first baby boy. Laboratory assessments revealed negative results for 23 routine thalassemia gene tests. The hemoglobin contents were HbA 93.3%, HbA2 5.4%, and HbF 1.3%, respectively. Sanger sequencing detected a novel heterozygous mutation of the β-globin gene, HBB:Codon85(-T) (Fig. 3C). HBB:Codon85(-T) was predicted as disease causing by MutationTaster2.

Thirteen cases of anemia with abnormal hemoglobin electrophoresis due to HBD, HBG1, and HBG2 gene mutations were screened, including two cases of HBD:c.-127T>C; a case of HBD:c.-80T>C; a case of HBG1:-314(G>T); 5 case of HBG1:+25(G>A) combined with HBG2:-158(C>T); 2 cases of HBG1:c.-272_-275dupAGCA; a case of HBB:c.91A>G combined with HBG1:c.-272_-275dupAGCA; a case of HBG2:398(A>G).