Hemoglobin profile and molecular characteristics of the complex interaction of hemoglobin Doi-Saket [α9(A7) asn > lys, HBA2:c.30C > a], a novel α2α1 hybrid globin variant, with hemoglobin E [β26(B8) Glu > lys, HBB:c.79G > A] and deletional α+-thalassemia in a Thai family

Abstract

Background: An increasing number of α-hemoglobin (Hb) variants is causing various clinical symptoms; therefore, accurate identification of these Hb variants is important.

Objective: This study aimed to describe the molecular and hematological characteristics of novel Hb Doi-Saket that gives rise to a typical α⁺-thalassemia phenotype in carriers with and without other hemoglobinopathies.

Materials and methods: Biological samples from a proband and his family members were analyzed. Hematological profiles were analyzed using a standard automated cell counter. Hb was analyzed by capillary electrophoresis and high-performance liquid chromatography. Mutations and globin haplotype were identified by DNA analysis. Novel diagnostic tools based on allele-specific polymerase chain reaction (PCR) and PCR-restriction fragment length polymorphism were developed.

Results: Hb analysis showed a major abnormal Hb fraction, moving slower than HbA, and a minor Hb fraction alongside HbA₂ in the proband, his father, and son. DNA analysis of the α-globin gene identified the -α^3.7 deletion and in cis the C > A mutation on codon 9 of the α2α1 gene, corresponding to Hb Doi-Saket [α9(A7) Asn > Lys]. This mutation could be identified using newly developed allele-specific PCR-based assays. The Hb Doi-Saket al.lele was significantly associated with haplotype [- + M + + 0 -]. Interaction of α^Doi-Saket with β^E globin chains led to a new Hb variant (HbE Doi-Saket). Phenotypic expression was clinically silent in heterozygotes and might present slight microcytosis.

Conclusions: Hb Doi-Saket emphasizes a great diversity present in α-globin gene. The mutation in this family from Thailand was linked to -α^3.7 and caused mild microcytosis in the carriers. The combination of this variant with deletions in α genes might cause a severe clinical phenotype. Different methods of separation can provide useful information in diagnosis, and a complete molecular approach is needed for confirmation before considering patient management.

Keywords: Hb Doi-Saket; HbE; Thailand; α+-thalassemia; α-globin variant.

Figure 1.

Hb analysis of simple heterozygosity of Hb Doi-Saket identified in the proband (A–C) and double heterozygosity of Hb Doi-Saket and HbE identified in his son (D–F). A and D show the Electropherograms; Hb Doi-Saket concealed HbF at electrophoretic zone 7. B and E are HPLC chromatograms analyzed using the VARIANT II system; Hb Doi-Saket was eluted by overlapping with HbA₂ with a retention time of 3.92 min. C and F represent the HPLC chromatograms obtained using the Premier Resolution system; Hb Doi-Saket completely overlapped with HbA₂ during elution.

Figure 2.

Hb Separation profiles of the heterozygous Hb Q-Thailand using (A) capillary electrophoresis, (B) VARIANT II HPLC, and (C) Premier Resolution HPLC.

Figure 3.

Locations of the four primers, the breakpoints of -α^3.7 and -α^4.2 deletions, and amplified DNA fragment. The 1,529- and 1,779-bp fragments are specific for -α^3.7 and -α^4.2, respectively. M, 100-bp marker; lane 1; normal control; lane 2, -α^3.7 carrier; lane 3, -α^4.2 carrier; lane 4, mother; lane 5, father; lane 6, proband; lane 7, wife of proband; and lane 8, son of proband.

Figure 4.

(A) the locus chromosome with -α^3.7 deletion and forward directional nucleotide sequence obtained from the α2α1 hybrid and α1 genes. (B) the lack of deletion on the locus chromosome and forward directional nucleotide sequence obtained from the α2 gene. The arrow indicates replacement of the heterozygote nucleotide (codon 9 AAC>AAA), and any identical nucleotide substitution in the same place was not characterized in the α2 gene.

Figure 5.

The hybrid -α^3.7 gene and forward directional nucleotide sequence obtained from this gene. The arrow indicates nucleotide replacement.

Figure 6.

Identification of AAC>AAA mutation by HpyCH4IV digestion of the PCR product. (A) HpyCH4IV digestion of the in cis α2α1 hybrid and α1 genes. Lane 1 is the undigested amplified DNA (975 bp). lanes 2 and 5 are HpyCH4IV-digested amplified DNA of the proband’s mother and wife, who had normal α-globin allele (αα/αα). the 561-bp digested fragment in lanes 3, 4, and 6 indicates the presence of Hb Doi-Saket mutation in the proband, his father, and son. (B) HpyCH4IV digestion of the α2 gene. Three fragments of 506, 306, and 255 bp were produced, whereas the 561-bp fragment was not produced owing to the absence of AAC>AAA mutation.

Figure 7.

A multiplex ASPCR Assay for simultaneous identification of Hb Doi-Saket and Hb Q-Thailand. The locations and orientations of primers are indicated. The 729-bp fragment generated using SP42 and B is specific for α^Doi-Saket, whereas the 416-bp fragment generated using αG20 and B is specific for α^Q-Thailand. The 199-bp fragment is an internal control generated using SP44 and B. M, 100-bp ladder; lane 1, normal DNA; lane 2, DNA control of Hb Q-Thailand; lane 3, mother; lane 4, father; lane 5, proband; lane 6, wife of proband; lane 7, son of proband; and lane 8, Hb Q-Thailand carrier.