Families presenting with β-thalassemia trait without pathogenic variants in the HBB locus. An asterisk indicates family members selected for WES analysis; all other members were examined at the DNA level by targeted next-generation sequencing of SUPT5H. Circles indicate females, squares indicate males. (A) Family 1 of Dutch origin. Whole-exome sequencing (WES) revealed the presence of SUPT5H:c.458+1G>A. (B) Family 2 of Dutch origin. WES showed a pathogenic variant in SUPT5H:c.2259-3C>A in all members having β-thalassemia trait, which is absent in the nonthalassemic members. (C) Family 3 from Crete with the β-thalassemia intermedia phenotype in family members F3-I.1 and F3-II.2, who were identified to be compound heterozygotes for HBB:c.118C>T p.(Gln40*) and SUPT5H:c.1374+2T>C. Family members F3-II.3 and F3-III.1 are carriers of the familial SUPT5H variant. (D) Family 4 from the north of Greece. Member F4-III.1 presented with a moderate β-thalassemia intermedia, which is unexplained by being only a carrier of the β-thalassemia variant HBB:c.92+1G>A. The SUPT5H variant identified was a 4-bp duplication (SUPT5H:1741_1744dup) causing a frameshift. (E) Genotype-phenotype correlation between nonaffected carriers of SUPT5H and digenic carriers of SUPT5H and HBB variants (Table 1). Heterozygotes for pathogenic SUPT5H variants show a reduction in Hb, MCV, and MCH and elevated HbA₂, very similar to β-thalassemia trait (ranges for β-thalassemia trait Hb [104-157 g/L], MCV [56-81 fL], MCH [18.6-26.8 pmol], and HbA₂ [4.2-6.6%] are indicated on the graph).21 The digenic heterozygotes for a SUPT5H and HBB variant show a markedly decrease Hb, MCV, and MCH and markedly elevated levels of HbA₂ and have a clinical and hematological presentation resembling β-thalassemia intermedia.