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

Figures & data

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.

Table 1. Hematological data and globin genotypes of the proband and his family members.

Parameters	Father	Mother	Proband	Wife	Son
Age (years)	64	58	31	33	8
RBC count (x10^12/L)	5.51	4.18	6.16	5.92	4.79
Hb (g/dL)	15.2	12.0	16.2	13.7	12.3
Hct (L/L)	46	36.7	49.1	41.4	38.1
MCV (fL)	83.5	87.6	79.7	70.0	79.6
MCH (pg)	27.6	28.8	26.4	23.2	25.6
MCHC (g/dL)	33.1	32.8	33.1	33.1	32.2
RDW-CV (%)	14.8	13.9	13.9	14.1	13.7
CE-Hb Profile^a	A2A with Hb Doi-Saket	A2A	A2A with Hb Doi-Saket	EA	EA with Hb Doi-Saket
Hb A (%)^a	65.8	97.1	65.4	70.1	51.1
Hb A2 (%)^a	1.8	2.9	1.7	4.0	7.9*
Hb E (%)^a	0	0	0	29.7	16.7
Hb F + Hb Doi-Saket (%)^a	31.6	0	32.1	0.2	22.6
Hb A2Doi-Saket (%)^a	0.8	0	0.8	0	1.7
Premier HPLC-Hb Profile^b	A with Hb Doi-Saket	A2A	A with Hb Doi-Saket	EA	EA with Hb Doi-Saket
Hb A (%)^b	59.6	89.8	59.3	60.4	44.6
Hb A2+E (%)^b	0	3.3	0	31.5	16.0
Hb F (%)^b	0	0	0	1.9	0.7
Hb A2+Hb Doi-Saket (%)^b	32.0	0	32.7	0	24.9
Hb EDoi-Saket (%)^b	0	0	0	0	5.9
Hb A2Doi-Saket (%)^b	1.0	0	1.0	0	2.4
Variant II HPLC-Hb Profile^c	A with Hb Doi-Saket	not done	A with Hb Doi-Saket	not done	EA with Hb Doi-Saket
Hb A (%)^c	56.6	not done	56.0	not done	52.5
Hb A2+E (%)^c	0	not done	0	not done	9.7
Hb F (%)^c	0.4	not done	0.2	not done	1.2
Hb A2+Hb Doi-Saket (%)^c	31.2	not done	32.5	not done	17.1
Hb A2Doi-Saket (%)^c	0.9	not done	0.9	not done	9.9**
Iron profile
Ferritin (µg/L)	120.5	not done	74.2	not done	not done
Serum iron (µg/dL)	65.3	not done	130.8	not done	not done
TIBC (µg/dL)	262.9	not done	349.4	not done	not done
UIBC (µg/dL)	197.6	not done	218.6	not done	not done
%Tsat (%)	24.8	not done	37.4	not done	not done
α-globin genotype	-α^Doi-Saket(3.7)/αα	αα/αα	-α^Doi-Saket(3.7)/αα	αα/αα	-α^Doi-Saket(3.7)/αα
β-globin genotype	β^A/β^A	β^A/β^A	β^A/β^A	β^E/β^A	β^E/β^A

RBC = Red Blood Cells; Hb = Hemoglobin; Hct = Hematocrit; MCV = Mean Corpuscular Volume; MCH = Mean Corpuscular Hemoglobin; RDW-CV = Coefficient of Variation of the Red Cell Distribution Width; Tsat = Transferrin saturation.

^a Determined using Capillary Electrophoresis, Capillarys 2.

^b Determined using High Performance Liquid Chromatography, Premier Resolution, Trinity Biotech.

^c Determined using High Performance Liquid Chromatography, β-thal short program, Bio-Rad variant II system.

* The percentage included HbA₂ and HbEDoi-Saket.

** The percentage included HbA₂Doi-Saket and HbEDoi-Saket.

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.

Table 2. α-Globin gene haplotypes associated with Hb Doi-Saket.

		α-globin haplotype (5′>3′)
									Hb Doi-Saket (-α^Doi-Saket) linked α-haplotype
Subjects	α-genotype	Xba I	Sac I	[S/M/L]	Acc I	Rsa I	αPst I	θPst I
Father (I-1)	-α^Doi-Saket/αα	[-/-]	[+/-]	[M]	[+/-]	[+/-]	[-/0]	[-/-]	-α^Doi-Saket	[ -	+	M	+	+	0	- ]
									αα	[ -	–	M	–	–	–	- ]
Mother (I-2)	αα/αα	[-/-]	[+/+]	[L]	[+/+]	[-/-]	[-/-]	[-/-]	αα	[ -	+	L	+	–	–	- ]
									αα	[ -	+	L	+	–	–	- ]
Proband (II-1)	-α^Doi-Saket/αα	[-/-]	[+/+]	[L/M]	[+/+]	[+/-]	[-/0]	[-/-]	-α^Doi-Saket	[ -	+	M	+	+	0	- ]
									αα	[ -	+	L	+	–	–	- ]
Wife (II-2)	αα/αα	[+/-]	[+/+]	[L/M]	[+/+]	[+/-]	[-/-]	[-/-]	αα	[ -	+	L	+	–	–	- ]
									αα	[ +	+	M	+	+	–	- ]
Son (III-1)	-α^Doi-Saket/αα	[+/-]	[+/+]	[M]	[+/+]	[+/+]	[-/0]	[-/-]	-α^Doi-Saket	[ -	+	M	+	+	0	- ]
									αα	[ +	+	M	+	+	–	- ]

+ and - represent the presence and absence of the restriction enzyme sites, respectively. 0 indicates deletion. S and M indicate inter ζ hyper variable region (HVR).

Data availability statement

The data that support the findings of this study are available from the corresponding author upon reasonable request.