David Gravell

Forecasting Hemoglobinopathy Burden Through Neonatal Screening in Omani Neonates

To evaluate the incidence of hemoglobinopathies in Omani subjects and to forecast its future burden on health resources, we initiated a prospective neonatal screening program in two major cities of the Sultanate of Oman. Consecutive cord blood samples from a total of 7,837 neonates were analyzed for complete blood counts and for hemoglobin (Hb) profile by high performance liquid chromatography (HPLC). No case with Hb H (β4) was detected. We observed that the overall incidence of α-thalassemia (α-thal) was 48.5% [based on the presence of Hb Barts (γ4)] and the β-globin-related abnormalities accounted for 9.5% of the samples (4.8% sickle cell trait, 2.6% β-thal trait, 0.9% Hb E trait, 0.8% Hb D trait, 0.08% Hb C trait, 0.3% sickle cell disease and 0.08% homozygous β-thal). This is also the first large study to establish reference ranges of cord red blood cell (RBC) indices for Omani neonates.

The β-globin promoter -71 C>T mutation is a β+ thalassemic allele.

A novel β‐globin gene promoter (−71 C>T) nucleotide change was recently posted to the HbVar database (ID 2701) without precision on phenotype and ethnicity. We found the same change in compound heterozygosity with Hb S [β6(A3)Glu>Val] in an Omani family with almost equal expression of Hb A and Hb S. This suggested that the −71 C to T mutation may be a mild β‐thalassemic allele. Subsequent search found three other independent cases with the same atypical Hb A:Hb S ratio, further confirming the mild thalassemic feature of this mutation. In addition, molecular screening of a set of subjects (with only Hb A) with borderline Hb A2 or MCV values revealed the presence of −71 C>T change in heterozygous state, altogether assigning the mutation as a mild β+ thalassemic allele. In a region such as Oman, where several genetic conditions of the red blood cell coexist (α‐ and β‐thalassemia, Hb S, Hb D, Hb E) in significant frequencies, it is crucial to decipher the molecular basis of these atypical forms of β+ thalassemias, especially in a genetic counseling setting.

Haematological and clinical features of β-thalassaemia associated with Hb Dhofar

Hb Dhofar is a variant haemoglobin (β29 (GGC–GGT) gly‐gly, β58 (CCT–CGT) pro‐arg) associated with a thalassaemic phenotype and unique to the Sultanate of Oman. We report clinical and haematological data on 54 subjects with Hb Dhofar (37 heterozygotes, 14 homozygotes and three compound heterozygotes with a different β‐thalassaemia mutation). In heterozygotes, the level of Hb Dhofar ranged from 8.8% to 21.5%. All heterozygotes had Hb A2 > 3.5%, consistent with β‐thalassaemia trait. Hb Dhofar in homozygotes and compound heterozygotes ranged from 26% to 59.7%, with a peripheral film consistent with homozygous β‐thalassaemia. Age at presentation in homozygotes ranged between 6 months and 8 yr, with a majority presenting before 5 yr of age. All had splenomegaly and six (43%) had undergone splenectomy. All had some degree of frontal bossing and in particular, two patients with infrequent transfusions had marked thalassaemic facies and stunting of growth. Hb Dhofar can be mistaken for Hb D as the electrophoretic mobility is similar, but differs from it by a variable and reduced quantity of variant Hb in both heterozygotes and homozygotes. Clinical and haematological data suggest that this mutation behaves like a moderately severe β+ thalassaemia allele resulting in a thalassaemia intermedia phenotype.

Hb A2′ (Hb B2) in the Omani population and diagnostic significance

Hb A2′ [δ16(A13)Gly→Arg], also called Hb B2, is a δ-globin chain variant that has been identified in several populations of African origin or ancestry and is easily identifiable in alkaline acetate cellulose electrophoresis as doubling of the Hb A2 band. However, in high performance liquid chromatography (HPLC), commonly employed nowadays, it elutes in the S window. Over a period of 2 years at the Sultan Qaboos University Hospital, Muscat, Oman, we identified 25 Omanis with this variant. The quantity of Hb A2 ranged from 0.9 to 1.8% in heterozygotes and was undetectable in the single homozygous case. As both α- and β-thalassemia (α- and β-thal) as well as Hb S [β6(A3)Glu→Val] are common in the Omani population, it is important to be aware of the presence of Hb A2′ in this population to avoid misinterpretation of the HPLC data in terms of underdiagnosis of β-thal carriers and overestimation of α-thal based on Hb A2 levels in sickle cell carriers. The haplotype associated with Hb A2′ in Oman is identical to that described in African populations, suggesting a common origin for this mutation and its introduction into Oman by gene flow.

Hb Sheffield [β58(E2)Pro→His] in Oman: potential pitfall in genetic counseling.

A novel β-globin structural variant, namely Hb Sheffield [β58(E2)Pro→His], was recently found as a sporadic event in a British Subject and posted to the HbVar database (ID 2672). Here we describe the same variant in 11 Omani subjects in the heterozygous state and in one Omani woman in compound heterozygosity with Hb S [β6(A3)Glu→Val]. Hb Sheffield coelutes in the Hb A2 window in the high performance liquid chromatography (HPLC) system as does Hb E [β26(B8)Glu→Lys], and might be erroneously diagnosed as Hb E unless additional tests including DNA analyses are done. Indeed, correct diagnosis of Hb E is important because of its association with other β-thalassemic and variant alleles can result in relevant clinical conditions, while Hb Sheffield will not. In a genetic (premarital) counseling setting, and in regions where both Hb E ad Hb Sheffield are present, failure to distinguish these variants will represent a serious pitfall.

First report of the spectrum of δ-globin gene mutations in Omani subjects – identification of novel mutations

Both coinheritance of thalassemic δ‐globin mutation and coexistence of iron deficiency anemia (IDA) tend to decrease HbA2 (α2δ2) level and thereby poses a diagnostic conundrum in β‐thalassemia trait.

Potential pitfalls in the diagnosis of Hb Handsworth in areas with high prevalence of HbS

Hb Handsworth is a rare α‐globin structural variant caused by a missense mutation either on the α2 or α1‐globin gene (HBA2 or HBA1: c.55G>C, p.Gly18Arg). This variant might be erroneously diagnosed as HbS unless secondary confirmative tests are carried out. We encountered a child with a prominent peak eluting in the ‘S’ window on high‐performance liquid chromatography (HPLC). Sickle solubility test, gel electrophoresis, and selective direct nucleotide sequencing of α1, α2, and β globin genes were performed on the patients sample. In addition, previous HPLC results on a cord blood sample were retrieved. Sickle solubility test was negative. Gel electrophoresis revealed a band migrating at the S region with an extra faint band seen on acid gel electrophoresis. Molecular analysis of α2 globin gene revealed heterozygous state of Hb Handsworth. Hb Handsworth is a rare variant that can mimic HbS on HPLC. Failure to recognize this rare variant in regions where HbS is highly prevalent may result in serious misdiagnosis and subsequent incorrect genetic counseling.