Interaction of Hb Adana (HBA2: c.179G>A) with Deletional and Nondeletional α⁺-Thalassemia Mutations: Diverse Hematological and Clinical Features

Figures & data

TABLE 1  List of Primers Used to Detect Single α-Globin Gene Deletions (Ivy Ng, personal communication)

Primers	Sequences (GenBank: J00153.1) (5‘>3’)
2/3P	TGT TGG CAC ATT CCG GGA CAG
XY1	GCG CCG AGC CTG GCC AAA CCA TCA CTT TTC
3R1	TGC ATC CTC AAA GCA CTC TAG GGT CCA GCG T
SA3P	TAA GCT AGA GCA TTG GTG GTC ATG C
XYHA	GAA GTA GCT CCG ACC AGC TTA GCA A

TABLE 2 List of Primers, Polymerase Chain Reaction Product Sizes, Restriction Enzymes and Restriction Fragment Length Polymorphism Product Sizes to Detect Nondeletional α-Thalassemia Mutations

Mutation	Primer Names and Sequences (GenBank: J00153.1) (5‘>3’)	PCR Product Size (bp)	Restriction Enzymes	RFLP Product Size (bp)
				Normal	Heterozygote	Homozygote
Hb CS	E3a2: GCG GGT TGC GGG AGG THbCS: GAA CGG CTA CCG AGG CTC CAG CTC	222	Taq^α-I	222	222; 200; 22	200; 22
Codon 22	α2α1PromF: GTG GAG GGT GGA GAC GTC α2R: GGA GGC CCA GCG GGC AGG AGG AAC	971	MslI, AluI	559; 241; 130; 41	559; 495; 241; 130; 41; 64	495; 241; 130; 41; 64
rSNP^a149709T>C	0745F: GGG AGC ACC AGG ACA CAG ATG 149776R: CTT GCA CCA ACA GCT TTT CA	164	Hpy188I	164	164; 85; 79	85; 79
IVS-II-142	E2a2: CCC GCC CGG ACC CAC A α2R: GGA GGC CCA GCG GGC AGG AGG AAC	599	AluI	258; 169; 130; 42	299; 258; 169; 130; 42	299; 258; 42

Hb CS: Hb Constant Spring.

^a Primers and PCR-RFLP information provided by Dr. D.R. Higgs (MRC Molecular Haematology Unit, Weatherall Institute of Molecular Medicine, John Radcliffe Hospital, Headington, Oxford, UK.)

TABLE 3 Hematological and Clinical Data of Hb Adana Patients in Combination With One Gene Deletion at Diagnosis

n	Sex-Age	Hb (g/dL)	MCV (fL)	MCH (pg)	RDW (%)	Hb A2 (%)	Hb F (%)	Hb X	Clinical Manifestation
1	M-0.25	6.1	71.0	19.0	20.0	1.4	22.0	Bart’s^a; H	has been transfused since 3 months old^b
2	M-0.25	9.5	65.2	21.4	19.6	1.1	8.5	–	one transfusion at 1 month old^c
3	M-2	9.4	65.0	23.7	23.1	2.5	7.4	Bart’s^a	splenomegaly; no transfusions
4	F-4	8.3	73.4	23.6	31.9	2.6	4.0	Bart’s^a	has been transfused since at 3 years old
5	F-5	9.2	71.0	23.0	21.6	2.0	1.4	Bart’s^a	anemic since 4 years old
6	F-7	10.3	69.7	23.8	17.5	2.1	6.6	Bart’s^a	transfused once at 6 years old (fever)
7	F-12	7.8	72.3	22.2	23.9	1.9	4.8	Bart’s^a	not reported
8	M-14	10.2	65.9	22.5	17.9	2.4	1.3	–	not reported
9	F-17	9.5	70.7	23.1	21.1	2.4	1.8	–	high ferritin levels; no transfusions
10	M-22	8.4	70.4	21.5	22.7	3.1	1.5	Bart’s^a	splenomegaly; transfused once at age 18
11	M-24	9.7	83.0	25.0	N.D.	1.8	1.9	–	no transfusions^c
12	F-26.5	9.7	69.4	24.1	21.9	2.5	1.6	Bart’s^a	transfused at age 25 (pregnancy) and again at age 25.5 old (infection); splenomegaly
13	F-31	7.5	71.7	24.4	42.1	2.6	1.7	Bart’s^a	transfused twice at age 29 (Hb 6.0 g/dL)
14	M-32	11.9	75.6	24.0	17.7	2.6	1.7	–	splenomegaly; high ferritin levels
15	M-34	9.5	83.8	25.5	19.4	2.1	1.8	Bart’s^a	no transfusions
16	F-34	9.0	77.2	23.0	23.3	2.4	1.5	N.D.	transfused during every pregnancy (P4A3), for a total of six times^c
17	F-42	8.4	72.6	23.4	20.2	2.2	1.8	–	transfused twice at age 33 (pregnancy); twice at 41–42 years old (infection); hepatosplenomegaly
18	M-53	11.7	76.2	25.9	16.3	2.3	0.6	–	transfused once at 49 years old
		9.4±1.2	73.0±5.2	23.7±1.2	22.7±6.5	2.3±0.3	2.5±0.3

N.D.: not defined; RDW: red cell distribution width; patients 1 and 4 are transfusion-dependent; mean and standard deviations were calculated for patients more than 1-year-old.

^a Was seen on the HPLC system (VARIANT™; Bio-Rad Laboratories) (10).

^b In transfused patients, hematology analysis was carried out after one (or several) blood transfusions had been received (Hb steady state) except in patient 1 where it was carried out before he had received his first blood transfusion.

^c The Hb analyses were not done on the HPLC system (VARIANT™; Bio-Rad Laboratories).

NB: All patients carry the Hb Adana mutation in combination with the –α^3.7 kb type deletion except patient 16 who has the –α^4.2 kb type instead.

TABLE 4  Hematological, Molecular and Clinical Data of Patients Carrying Hb Adana in Combination With Nondeletional α⁺-Thalassemia at Diagnosis

n	Sex- Age	Hb (g/dL)	MCV (fL)	MCH (pg)	RDW (%)	Hb A2 (%)	Hb F (%)	Hb X	Genotype	Notes
1	M-7 days	8.9	93.5	30.9	21.7	0.0	71.7	Bart’s^a; H	codon 59/ Hb CS	not reported
2	M-4	9.8	80.2	26.5	16.6	2.7	1.7	–	codon 59/ Hb CS	transfused since <1 year old
3	F-14	9.0	76.4	23.9	29.1	2.8	2.1	Bart’s^a	codon 59/ Hb CS	regularly transfused since 7 years old (3–4/year)
4	F-28	8.1	85.5	23.5	24.1	2.3	1.6	Bart’s^a; CS^a	codon 59/ Hb CS	first presented as a young adult
5	M-5	8.2	67.3	24.3	21.8	9.0	1.2	–	codon 59/ codon 22	irregularly transfused (+), high Hb A2 is not due to coinheritance of Hb E^b
6	M-5	11.1	73.5	25.4	20.4	2.9	0.0	–	codon 59/ codon 22	regularly transfused since 1.5 years old (10–12/year); has not been transfused since age 14
7	F-24	9.4	73.2	24.5	16.7	1.9	3.0	Bart’s^a	codon 59/ codon 22	only transfused while pregnant
8	F-12	8.7	81.1	27.1	N.D.	2.1	0.1	Bart’s^a	codon 59/ rSNP 149709T>C	regularly transfused since 2-months-old but not since age 9 after being splenectomized
9	F-3	4.9	71.7	23.6	29.0	2.6	2.4	Bart’s^a	codon 59/ IVS-II-142	regularly transfused since 3.5 months old

RDW: red cell distribution width; N.D.: not defined; codon 59: Hb Adana; CS: Hb Constant Spring.

^a Was seen on the HPLC system (VARIANT™; Bio-Rad Laboratories) (10).

^b On HPLC, Hb E elutes at a similar time as Hb A₂; the parents do not carry Hb E trait, maybe the blood donor carried Hb E trait.