TO THE EDITOR:

β-Thalassemia is among the most common autosomal-recessive conditions; it is caused by nucleotide variants and, less commonly, deletions of the β-globin gene (HBB; 11p15.4) or gene cluster,1,2  disrupting synthesis of the β-globin polypeptide chains of the hemoglobin tetramer HbA (α2β2). Heterozygotes usually show reduced erythrocyte indices and elevated HbA2 levels, and homozygotes or compound heterozygotes have severe hemolytic and dyserythropoietic anemia, usually requiring life-long blood transfusion and iron-chelation therapy.3,4 

However, rare cases have been observed with a phenotype of β-thalassemia trait but without HBB gene or gene cluster variants. Hematology analysis by standard procedures5,6  in several members of 2 Dutch families (Figure 1A-B) found microcytic hypochromic anemia and elevated HbA2, consistent with β-thalassemia trait (Table 1). Molecular analysis, including Sanger sequencing of HBB, HBA1, HBA2, and KLF1, revealed a mild α-thalassemia variant HBA2:c.96-2A>G, which is found at low frequency in the Dutch population, in F1-III.1, F1-IV.1, and F1-IV.2 (F1 indicating Family 1, the Roman number the generation, and the Arab number the individual within 1 generation). No pathogenic variants were found in HBB, and multiplex ligase-dependent probe amplification excluded deletions and rearrangements involving HBB or β-locus control region. Haplotyping excluded linkage between HBB and the β-thalassemia trait in both families. The β/α-globin chain synthesis ratio in F1-II.2 showed a reduction in β-globin chain synthesis (β/α = 0.66), consistent with β-thalassemia trait.7  Globin-chain biosynthesis was not possible for other family members, but their erythrocyte parameters and elevated HbA2 sufficed to establish β-thalassemia trait.

Figure 1.

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.

Figure 1.

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.

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Table 1.

Hematology and HBB and SUPT5H genotype data of nonlinked β-thalassemia trait families and unrelated individuals

IndividualSexAge, y*Hb, g/LHt, L/LMCV, fLMCH, pgHbA2, %HBB genotypeHBA genotypeSUPT5H genotypeMolecular effect
Dutch family 1 with unexplained elevated HbA2and no variants in HBB 
 F1-II.1 66 111 0.40 91 25.3 5.1 Norm Norm c.[458+1G>A];[=] Splice donor site 
 F1-II.2 65 — — — — 5.7 Norm Norm c.[458+1G>A];[=]  
 F1-II.3 62 108 0.32 84 28.0 5.7 Norm Norm c.[458+1G>A];[=]  
 F1-III.2 42 111 0.35 79 25.6 4.7 Norm Norm c.[458+1G>A];[=]  
 F1-III.3 40 116 0.33 79 25.4 4.8 Norm Norm c.[458+1G>A];[=]  
 F1-III.4 — 0.37 82 — 5.5 Norm Norm c.[458+1G>A];[=]  
 F1-IV.3 12 95 0.31 71 21.6 5.2 Norm Norm c.[458+1G>A];[=]  
 F1-IV.4 12 114 0.35 73 24.2 5.2 Norm Norm c.[458+1G>A];[=]  
 F1-IV.2 15 140 0.40 73 22.9 5.7 Norm HBA2:c.[96-2A>G];[=] c. [458+1G>A];[=]  
 F1-III.1 54 142 0.46 81 24.8 2.4 Norm HBA2:c.[96-2A>G];[=] Norm  
 F1-IV.1 18 109 0.35 73 23.3 2.5 Norm HBA2:c.[96-2A>G];[=] Norm  
 F1-I.1 85 131 0.39 87 29.0 2.8 Norm Norm Norm  
 F1-IV.5 127 0.37 80 26.2 2.5 Norm Norm Norm  
Dutch family 2 with unexplained elevated HbA2and no variants in HBB 
 F2-I.2 46 136 0.39 77 27.7 5.0 Norm Norm c.[2259-3C>A];[=] Splice acceptor site 
 F2-II.3 16 135 0.41 77 25.8 4.7 Norm Norm c.[2259-3C>A];[=]  
 F2-II.4 11 134 0.39 72 24.2 4.9 Norm Norm c.[2259-3C>A];[=]  
 F2-I.1 44 172 0.50 94 32.2 2.6 Norm Norm Norm  
 F2-II.1 22 138 0.42 93 30.6 2.9 Norm Norm Norm  
 F2-II.2 18 143 0.43 88 29.0 2.7 Norm Norm Norm  
Individuals with unexplained elevated HbA2and no variants in HBB 
 Wlt.1 65 121 0.35 77 26.2 5.3 Norm Norm c.[1979del;[=] p.(Gly660Valfs*6) 
 Wlt.2 39 138 0.43 76 24.5 5.1 Norm Norm c.[1979del;[=]  
 Li 54 148 0.46 80 25.8 4.7 Norm Norm c.[1782_1785del];[=] p.(Ile594Metfs*2) 
 TW 113 0.38 79 23.7 5.2 Norm Norm c.[2725del];[=] p.(Gln909Argfs*45) 
 S (French) 46 140 n.d. 78 25.4 4.8 Norm Norm c.[817del];[=] p.(Leu273*
 Cl (French) 17 123 n.d. 71 22.2 5.2 Norm Norm c.[817del];[=]  
Greek family (F3) with a combination of β-thalassemia trait and a SUPT5H variant 
 F3-I.1  97 0.32 57 17.1 8.5 c. [118C>T];[=] Norm c.[1374+2T>C];[=] Splice donor site 
 F3-II.2  97 0.31 57 17.7 11.1 c. [118C>T];[=] Norm c.[1374+2T>C];[=]  
 F3-II.3  149 0.44 75 25.3 5.0 Norm Norm c.[1374+2T>C];[=]  
 F3-III.1  121 0.36 70 23.1 5.3 Norm Norm c.[1374+2T>C];[=]  
 F3-II.4  123 0.38 67 21.5 5.6 c. [118C>T];[=] 3.7/αα Norm  
 F3-I.2  139 0.41 90 30.1 1.7 Norm Norm Norm  
Greek family (F4) with a combination of β-thalassemia trait and a SUPT5H variant 
 F4-III.1 102 0.31 46 15.0 12.4 c.[92+1G>A];[=] Norm c.[1741_1744dup];[=] p.(Arg582Glnfs*21) 
 F4-I.1 58 127 0.40 75 23.7 5.2 Norm Norm c.[1741_1744dup];[=]  
 F4-II.2 33 119 0.36 69 22.5 5.8 Norm Norm c.[1741_1744dup];[=]  
 F4-II.1 31 131 0.40 58 19.1 5.4 c.[92+1G>A];[=] Norm Norm  
IndividualSexAge, y*Hb, g/LHt, L/LMCV, fLMCH, pgHbA2, %HBB genotypeHBA genotypeSUPT5H genotypeMolecular effect
Dutch family 1 with unexplained elevated HbA2and no variants in HBB 
 F1-II.1 66 111 0.40 91 25.3 5.1 Norm Norm c.[458+1G>A];[=] Splice donor site 
 F1-II.2 65 — — — — 5.7 Norm Norm c.[458+1G>A];[=]  
 F1-II.3 62 108 0.32 84 28.0 5.7 Norm Norm c.[458+1G>A];[=]  
 F1-III.2 42 111 0.35 79 25.6 4.7 Norm Norm c.[458+1G>A];[=]  
 F1-III.3 40 116 0.33 79 25.4 4.8 Norm Norm c.[458+1G>A];[=]  
 F1-III.4 — 0.37 82 — 5.5 Norm Norm c.[458+1G>A];[=]  
 F1-IV.3 12 95 0.31 71 21.6 5.2 Norm Norm c.[458+1G>A];[=]  
 F1-IV.4 12 114 0.35 73 24.2 5.2 Norm Norm c.[458+1G>A];[=]  
 F1-IV.2 15 140 0.40 73 22.9 5.7 Norm HBA2:c.[96-2A>G];[=] c. [458+1G>A];[=]  
 F1-III.1 54 142 0.46 81 24.8 2.4 Norm HBA2:c.[96-2A>G];[=] Norm  
 F1-IV.1 18 109 0.35 73 23.3 2.5 Norm HBA2:c.[96-2A>G];[=] Norm  
 F1-I.1 85 131 0.39 87 29.0 2.8 Norm Norm Norm  
 F1-IV.5 127 0.37 80 26.2 2.5 Norm Norm Norm  
Dutch family 2 with unexplained elevated HbA2and no variants in HBB 
 F2-I.2 46 136 0.39 77 27.7 5.0 Norm Norm c.[2259-3C>A];[=] Splice acceptor site 
 F2-II.3 16 135 0.41 77 25.8 4.7 Norm Norm c.[2259-3C>A];[=]  
 F2-II.4 11 134 0.39 72 24.2 4.9 Norm Norm c.[2259-3C>A];[=]  
 F2-I.1 44 172 0.50 94 32.2 2.6 Norm Norm Norm  
 F2-II.1 22 138 0.42 93 30.6 2.9 Norm Norm Norm  
 F2-II.2 18 143 0.43 88 29.0 2.7 Norm Norm Norm  
Individuals with unexplained elevated HbA2and no variants in HBB 
 Wlt.1 65 121 0.35 77 26.2 5.3 Norm Norm c.[1979del;[=] p.(Gly660Valfs*6) 
 Wlt.2 39 138 0.43 76 24.5 5.1 Norm Norm c.[1979del;[=]  
 Li 54 148 0.46 80 25.8 4.7 Norm Norm c.[1782_1785del];[=] p.(Ile594Metfs*2) 
 TW 113 0.38 79 23.7 5.2 Norm Norm c.[2725del];[=] p.(Gln909Argfs*45) 
 S (French) 46 140 n.d. 78 25.4 4.8 Norm Norm c.[817del];[=] p.(Leu273*
 Cl (French) 17 123 n.d. 71 22.2 5.2 Norm Norm c.[817del];[=]  
Greek family (F3) with a combination of β-thalassemia trait and a SUPT5H variant 
 F3-I.1  97 0.32 57 17.1 8.5 c. [118C>T];[=] Norm c.[1374+2T>C];[=] Splice donor site 
 F3-II.2  97 0.31 57 17.7 11.1 c. [118C>T];[=] Norm c.[1374+2T>C];[=]  
 F3-II.3  149 0.44 75 25.3 5.0 Norm Norm c.[1374+2T>C];[=]  
 F3-III.1  121 0.36 70 23.1 5.3 Norm Norm c.[1374+2T>C];[=]  
 F3-II.4  123 0.38 67 21.5 5.6 c. [118C>T];[=] 3.7/αα Norm  
 F3-I.2  139 0.41 90 30.1 1.7 Norm Norm Norm  
Greek family (F4) with a combination of β-thalassemia trait and a SUPT5H variant 
 F4-III.1 102 0.31 46 15.0 12.4 c.[92+1G>A];[=] Norm c.[1741_1744dup];[=] p.(Arg582Glnfs*21) 
 F4-I.1 58 127 0.40 75 23.7 5.2 Norm Norm c.[1741_1744dup];[=]  
 F4-II.2 33 119 0.36 69 22.5 5.8 Norm Norm c.[1741_1744dup];[=]  
 F4-II.1 31 131 0.40 58 19.1 5.4 c.[92+1G>A];[=] Norm Norm  

CE, capillary electrophoresis; F, female; Hb, hemoglobin (normal range for males, 130-180 g/L; normal range for females, 115-165 g/L); HbA2, hemoglobin A2 (normal range, 2.2-3.2%); Ht, hematocrit or packed cell volume (normal range for males, 0.4-0.54 L/L, normal range for females, 0.37-0.47 L/L); M, male; MCH, mean corpuscular hemoglobin (normal range, 27.0-32 pg); MCV, mean cellular volume (normal range, 80-100 fL); n.d., not done; —, data not available.

*

Age at measurement.

Measured by capillary electrophoresis.

β/α-Globin chain synthesis was performed, ratio 0.66.

Whole-exome sequencing (WES) was performed on 4 individuals from Family 1 (3 affected: F1-III.3, F1-IV.2, F1-IV.4; 1 nonaffected: F1-IV.5) and 2 individuals from Family 2 (F2-I.2 and F2-II.3) (Figure 1A-B) (SureSelect All-Exon kit [Cre-V2]; Agilent, Santa Clara, CA and analysis using an HiSeq 4000 Sequencing System; Illumina Inc., San Diego, CA), in accordance with the Declaration of Helsinki. Missense, splice, and nonsense variants were filtered with in-house pipelines (LOVDplus). The effect of missense variants was predicted in silico using Align GVGD, PolyPhen-2, SIFT2, and MutationTaster.8-10  Population frequencies were derived from the Genome Aggregation Database (GnomAD), and known disease associations were derived from the Human Gene Mutation Database.11  Single nucleotide polymorphism array analysis was performed to exclude copy number variations in intergenic regions and to identify minimal shared regions of concordance in individuals segregating with the phenotype.

Considering the β/α-globin ratio, we focused first on pathogenic variants in known transcription factors involved in HBB regulation: GATA1, GATA2, KLF1, KLF3, ERCC2, CCND3, CTSA, PCIF1, PLTP, MMP9, TNNC2, ZFPM1, NFE2, FOG1, SOX6, SP1, and AKL4. No shared founder variant was observed in any single gene between carriers from either family. By excluding shared variants between 1 healthy and 3 affected members of Family 1, a likely pathogenic splice-site variant NM_003169.3 SUPT5H:c.458+1G>A (19q13) was identified in all affected members (Figure 1A). Examining WES data for Family 2 identified a likely pathogenic splice acceptor site mutation NM_003169.3 SUPT5H:c.2259-3C>A in the individual with β-thalassemia trait, whereas it was absent in the nonaffected members (Figure 1B;,Table 1). In addition to the 6 individuals investigated by WES, the remaining members of both families were investigated by Sanger sequencing for the familial SUPT5H variants. For other cases studied, WES was targeted exclusively for the SUPT5H gene.

Four additional independent Dutch individuals (father Wlt.1 and son Wlt.2, Li, and TW) and 2 French individuals (mother S and daughter Cl), showing a β-thalassemia trait phenotype without pathogenic variants in the HBB locus, were examined for SUPT5H variants. This revealed 4 additional pathogenic SUPT5H variants: 3 single-bp deletions and 1 4-bp deletion causing a disrupted reading frame (Table 1).

To examine the effect of splicing variants found in Family 1 and Family 2, messenger RNA (mRNA) was isolated from cultured lymphocytes from F1-IV.3 and F2-I.2 and analyzed by RNA sequencing (NEBNext Ultra II Directional mRNA kit). A significant portion of SUPT5H reads showed an altered splice pattern in both patients, whereas control samples (n = 3) did not show any splicing abnormalities. The patient with SUPT5H:c.458+1G>A showed retention of intron 6 and, consequently, a premature stop codon (TAA) at +2. Likewise the SUPT5H:c.2259-3C>A RNA showed intron 22 retention, resulting in a premature stop codon (TGA) at +46. Both mutations result in truncated Spt5H protein without the functionally important C terminus (supplemental Figure 1, available on the Blood Web site). Based on the above, we propose that haploinsufficiency for the Spt5H protein underlies reduced HBB gene expression. However, given the relatively high frequency of β-thalassemia heterozygotes worldwide, can we expect to observe individuals with digenic inheritance for variants in HBB and SUPT5H? And would they have a pronounced decrease in β-globin gene expression leading to β-thalassemia intermedia or major phenotype?

To test this hypothesis, we analyzed families with known β-thalassemia variants but with family members expressing incompletely resolved moderate β-thalassemia intermedia. In a family from Greece (Crete) with HBB:c.118C>T p.(Gln40*), 2 family members (F3-I.1 and F3-II.2) showed a more pronounced anemia (Figure 1C; Table 1). Likewise in Family 4, from northern Greece, F4-III.1 presented with moderate β-thalassemia intermedia, unexplained by simply being a carrier of HBB:c.92+1G>A. Two novel SUPT5H splice-site mutations, c.1374+2T>C (Family 3) and c.1741_1744dup (Family 4), were identified in these more severe cases; neither was present in the GnomAD database.

Figure 1E presents the genotype-phenotype correlation and hematological data for normal individuals, carriers of SUPT5H variants, and double heterozygotes for SUPT5H and HBB variants. The hemoglobin (Hb), mean cellular volume (MCV), and mean corpuscular hemoglobin (MCH) in SUPT5H carriers are markedly reduced compared with all normal family members, whereas HbA2 is increased, similar to β-thalassemia trait. Digenic heterozygotes show a phenotype comparable to β-thalassemia intermedia, with moderate anemia, reduced Hb, MCV, and MCH, and markedly elevated HbA2 (8.5-12.4%).

In conclusion, we report the first observation of the SUPT5H gene as a new trans-acting candidate underlying the phenotypic expression of β-thalassemia. Apart from a de novo mutation in SUPT5H recently reported in congenital heart disease,12  no other pathogenic variant has been reported. SUPT5H encodes the Spt5H protein, which dimerizes with Spt4, forming a highly conserved component of the DSIF complex. This complex regulates mRNA processing and transcription elongation by RNA polymerase II. It acts cooperatively with the negative elongation factor complex to enhance transcriptional pausing at sites proximal to the promoter, possibly facilitating assembly of an elongation competent RNA polymerase II complex.13-15  Although SUPT5H is ubiquitously expressed (highly expressed in testis and bone marrow),16  studies suggest that DSIF functions in a tissue-specific manner.17-20  A recent study revealed a fundamental role for polymerase II pausing in specification and proliferation of hematopoietic stem cells by regulating signaling pathways through DSIF.20  Zebrafish experiments have shown that embryonic erythropoiesis is regulated by the transcription elongation factor Foggy/Spt5 through gata1 gene regulation. Erythrocytes were markedly decreased in zspt5KD embryos, suggesting disruption of erythroid differentiation, probably through repressed gata1 expression. In humans, GATA1 also requires FOG1 to activate HBB expression, and the present findings suggest that reduced synthesis of SUPT5H (the human homolog of Spt5) may act similarly.19  In this study, mRNA was isolated from lymphocytes, and expression of GATA1 and KLF1 was too low to detect differences in expression between SUPT5H variant carriers and noncarriers. Collaborative studies are ongoing to investigate the effects of SUPT5H haploinsufficiency on GATA1 and KLF1 expression in hematopoietic tissue and unravel the mechanism(s) through which SUPT5H haploinsufficiency reduces HBB expression. So far, overall results strongly suggest that haploinsufficient mutations in SUPT5H are associated with downregulation of HBB, acting as a phenocopy for HBB in β-thalassemia.

Contact the corresponding author ([email protected]) for original data.

The online version of this article contains a data supplement.

The authors acknowledge Jan Smit, emeritus clinical chemist, for providing blood samples of patients, and they thank the families for their collaboration. International collaboration was established through ERN-EuroBloodNet.

Contribution: R.C., J.S., G.W.E.S., N.d.H., S.P., and F.G. contacted the families and provided blood samples; A.A., T.K., J.K., and C.A.L.R. performed research and analyzed data; S.G.J.A., J.t.H., S.B., M.V., L.V., and R.S. performed the hematological and molecular analyses; A.G. performed RNA analyses; J.T.-S. and C.V. provided DNA samples and hematology data for the Greek samples; and A.A., F.B., and C.L.H. designed the research and wrote the manuscript.

Conflict-of-interest disclosure: The authors declare no competing financial interests.

Correspondence: Cornelis L. Harteveld, Department of Clinical Genetics, Leiden University Medical Center, Einthovenweg 20, 2333ZC Leiden, The Netherlands; e-mail: [email protected].

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