Compound Heterozygosity for Hb S and a Novel Deletion of Dnase I Hypersensitivity Sites HS3 and HS4 of β-Globin Locus Control Region Results in Hb S/β+-Thalassemia Phenotype

Sickle cell disease (SCD) is most often due to homozygosity for the hemoglobin sickle (Hb S) missense mutation of the β-globin gene (HBB:c.20A>T). SCD can also result from compound heterozygosity for Hb S and other β-chain variants or β-thalassemia (β-thal). Loss-of-function point mutations of the β-globin gene that abolish (β0) or reduce (β+) production of normal β-chains are the most common cause of β-thal, with a minority of alleles being larger deletions. Patients with Hb S/β0-thal typically have severe SCD, whereas residual β-chain synthesis in Hb S/β+-thal is associated with lower hemoglobin S concentrations and less severe disease. It has long been recognized that the high-level production of β-like chains throughout development is controlled by a cis regulatory element, the β-globin locus control region (βLCR). The βLCR is located 5.7 kb to 21.2 kb upstream of the ε-globin gene, and consists of five DNase I hypersensitivity sites designated HS1 through HS5. Twelve naturally occurring βLCR deletions have been reported, most resulting in complete loss of expression of the β-like genes and a carrier phenotype that resembles (εγδβ)0-thal. In these patients, neonatal hemolytic anemia is common due to impaired γ-chain synthesis required for Hb F. Once the γ→β switch has occurred during the first six months of infancy, the phenotype resolves to one of thalassemia trait with normal Hb A2. While the phenotype is well established for carriers of large deletions that remove all or most of the HS regions, the contribution of individual HS regions to β-globin gene expression in human has yet to be elucidated. To this end, it is important to identify and characterize naturally occurring deletions that involve individual HS regions or combinations thereof. Here, we report a case of SCD due to a novel βLCR deletion involving only HS3 and HS4. The proband is a 6-year old boy born to healthy non-sanguineous parents of Caribbean decent. Newborn screening was negative for SCD, with the Hb profile being consistent with Hb S trait (Hb F 79.1%, Hb A 6.0%, Hb S 4.0%, Hb Bart’s 9.1%). Postnatally there was no significant jaundice or clinically diagnosed anemia. The proband had no clinical complaints and growth and development were normal until age 5 years when he was diagnosed with SCD during an admission for unexplained abdominal pain and an enlarged spleen. He was noted to have microcytic anemia (Hb 87 g/L, MCV 68.2 fL), and the peripheral blood smear showed sickle cells, Howell-Jolly bodies and target cells. The Hb profile was suggestive of Hb S/β+-thal with 19.4% Hb A, 72.7% Hb S, and Hb A2 within normal range. He has had one vasoocclusive event since the diagnosis and had tonsillectomy for obstructive sleep apnea. Nucleotide sequence analysis demonstrated that the proband is heterozygous for the Hb S mutation (HBB:c.20A>T) with no other mutations of the β-globin gene. He was also shown to be heterozygous for the 3.7 kb α-globin gene deletion (-α3.7/αα). As this genotype does not explain the reduced expression of Hb A and SCD phenotype, we investigated the possibility of compound heterozygosity for Hb S and a βLCR deletion. Multiplex ligation-dependent probe amplification (MLPA) (MRC-Holland) demonstrated the presence of a novel deletion encompassing HS3 and HS4. Sequence analysis of the junction fragment established that the deletion spans a total of 4,860 bp (HGVS nomenclature NG_000007.3:g.8510_13369del). To date, this is the smallest reported βLCR deletion that is associated with a clinically significant phenotype. Our patient had milder clinical picture with no SCD-related symptoms until 5 years of age and absence of a clinically significant hemolytic anemia at birth. The presence of 19.4% Hb A was compatible with the phenotype of Hb S/β+-thal. This indicates that deletions involving only HS3 and HS4 are associated with a significant but not complete reduction of β-globin gene expression. Interestingly, the newborn screening profile was typical of sickle trait, leading to the important observation that the βLCR is not required for the low level β-globin gene expression at birth, and providing further insight into the function of βLCR and its contribution to γ→β switching in humans. Disclosures No relevant conflicts of interest to declare.