SHORT COMMUNICATION

IDENTIFICATION OF Hb KENYA (Ac81Leu-b86Ala)BY ELECTROSPRAY MASS SPECTROMETRY

Dilip K. Rai,1,2 Gunvor Alvelius,2 and Britta Landin1,*

1Department of Medical Laboratory Sciences and Technology,

Division of Clinical Chemistry, Huddinge University Hospital C1 74,

Karolinska Institutet, SE-141 86 Stockholm, Sweden2Clinical Research Center, Huddinge University Hospital,

Karolinska Institutet, SE-141 86 Stockholm, Sweden

Hb Kenya (Ag through 81; b from 86) was first reported in the early 1970s in

association with hereditary persistence of fetal hemoglobin (Hb) (HPFH) (1–3).

The variant is characterized by an abnormal globin chain, i.e., a hybrid Agb-globin

resulting from crossover during meiosis of Ag and b genes within a gene loci (1–4).

The N-terminus (amino acids 1–80) of the hybrid chain corresponds to the Agchain, while the C-terminus (amino acids 87–146) corresponds to the b chain.

Amino acids 81–86 are homologous for both the Ag and b chains. Further

structural studies have shown that the non-homologous crossover leads to an

approximately 22.5 kb gene deletion, including the entire d gene (5,6).

In heterozygotes, varying amounts (6.9–23.4%) of Hb Kenya have been found,

while Hb F levels are consistently increased (4.7–9.1%) and Hb A2 levels decreased

(1–4). The increased level of Hb F is of the Gg type encoded by the chromosome

carrying Hb Kenya. The condition can thus be described as Kenya-Gg-HPFH (5).

Relatively higher proportions of Hb Kenya are found in compound heterozygotes

where Hb Kenya occurs in conjunction with Hb S [b6(A3)Glu!Val] (2,7).

While Hb Kenya might be suspected when a typical electrophoretic pattern

coincides with increased Hb F and decreased Hb A2 levels, a definite diagnosis

depends on Southern blotting or DNA sequencing. In this report we demonstrate

that electrospray mass spectrometry (ESMS) can also be a very useful tool for

rapid identification of Hb Kenya.

HEMOGLOBIN, 26(1), 71–75 (2002)

71

Copyright # 2002 by Marcel Dekker, Inc. www.dekker.com

*Corresponding author. Fax: þ46 8 585 812 10; E-mail: britta.landin@chemlab.hs.sll.se

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Three siblings of African origin living in Sweden were screened for Hb S

using a combination of isoelectrofocusing (IEF) and cation exchange high

performance liquid chromatography (HPLC) (8). One of the siblings displayed a

completely normal Hb pattern, while IEF demonstrated the presence of an aberrant

fraction cathodal to Hb S in the other two cases (Fig. 1). Also in the latter two

cases, HPLC demonstrated an unknown fraction eluting prior to Hb A0. One of the

two children was also a Hb S heterozygote, hence lacking normal Hb A0. Hb A2

was quantified using anion exchange HPLC (8) and found to be reduced in both

cases, while Hb F was significantly increased (Table 1).

DNA was extracted from EDTA blood using standard techniques (9).

Amplification using primers 50-GCCGGCGGCTGGCTAGGGATGA-30 (corre-

sponding to positions 39,359 to 39,380 in the Ag gene) and 50-TTAGGCAGAATC-

CAGATGCTCAAG-30 (corresponding to positions 63,696 to 63,719 in the bgene) was tried. If the Hb Kenya deletion was present those primers would be

expected to yield a 1,964 bp fragment, while no amplification would be expected

from an undeleted allele, the two primers being separated by more than 24 kb. The

polymerase chain reaction (PCR) conditions involved initial denaturation at 96�Cfor 5 minutes, followed by 35 cycles of annealing at 61�C for 30 seconds,

extension at 72�C for 90 seconds, denaturation at 94�C for 30 seconds, and finally,

Figure 1. IEF showing bands cathodal to Hb S in lanes 2 (compound heterozygote for Hb S=Hb

Kenya) and 4 (heterozygote for Hb Kenya). Lanes 1 and 3 are from normal adults, and lane 5 from a

patient with a heterozygosity for Hb S. Prominent Hb F bands are also seen in lanes 2 and 4.

Table 1. Proportions of Various Hemoglobin Fractions (%)

Hb F=Gg Hb Kenya=Agb Hb A2=d

Hb Kenya=Hb A0 Hb A=bHPLC 72.9 10.0 17.1 2.0

ESMS 56.6 18.6 24.8 –

Hb Kenya=Hb S Hb S=bS

HPLC 63.4 13.2 23.4 2.4

ESMS 54.6 23.8 21.6 –

The proportions of the different Hb fractions were calculated as follows: For HPLC data the sum of

Hb A0, Hb F, and Hb Kenya was set to 100%. For ESMS data the sum of b or bS, Gg, and Agbwas set

to 100%. Hb A2 was quantified in seperate HPLC runs.

72 RAI, ALVELIUS, AND LANDIN

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Figure 2. Deconvoluted ES mass spectra demonstrating the presence of (A) normal a, b, and the

hybrid Agb polypeptide chains, while bS is in place of the normal b chain in (B). As expected the Ggchain is also a dominant peak.

IDENTIFICATION OF Hb KENYA BY ESMS 73

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extension at 72�C for 10 minutes. Upon agarose gel electrophoresis an unambig-

uous amplified fragment, corresponding to 1964 bp, was found. Sequencing of this

fragment, using 50-AACCCCAAAGTCAAGGCACAT-30 (aligning at 39,760 to

39,780 in the Ag gene) as sequencing primer, demonstrated a nucleotide sequence

identical to that reported for the hybrid region of Hb Kenya (10).

To investigate whether MS could have aided the diagnosis, the two

blood samples containing Hb Kenya were also analyzed on a VG Quattro I ES

mass spectrometer (Micromass, Altrincham, Manchester, UK) as described

previously (9). The mass for the Hb Kenya chain was calculated to be

15,922 Da. A significant peak was demonstrated at the expected mass (Fig. 2).

Peaks corresponding to normal a (15,126 Da), b (15,867 Da), Gg (15,995 Da), and

glutathionyl b (16,173 Da) were also observed (Fig. 2A).

In the other case, ESMS analysis revealed a peak at the expected mass for the

hybrid chain at 15,922 Da as well as a peak indicating bS (15,837 Da) (Fig. 2B).

The corresponding adducts such as glutathionyl bS chain and heme adduct of bS

chain were also observed. Similar proportions of adducts were found in normal

samples that had been stored frozen at similar conditions as the Hb Kenya samples.

The mass of normal d chain (15,924 Da) is close to that of the Hb Kenya

chain (15,922 Da). The inability of the method used to separate the two fractions

might partly explain the discrepancy between the relative Hb fraction proportions

found when estimated by HPLC and ESMS, respectively (Table 1). Although

HPLC data are calculated without taking the Hb A1c fraction into account,

omission of several additional adducts formed in vitro from the ESMS calculation,

make comparison between the two estimates difficult.

This is the first report on application of ESMS to Hb Kenya. Mass spectro-

metric analysis is an easy approach to investigate the eventual occurrence of Hb

Kenya, irrespective of its coexistence with Hb S. ESMS has earlier been used to

illustrate the occurrence of other hybrid globins like Hb Lepore-Baltimore

(d68Leu-b84Thr) (15,822 Da) (11). Although the more frequently occurring Hb

Lepore-Boston-Washington (d87Gln-b-IVS-II-8) (15,865 Da) has a mass differing

only 2 Da from normal b chain and can thus not easily be diagnosed using ESMS

of intact globin chains. However, ESMS is apt for diagnosing Hb Lepore-

Baltimore and for Hb Kenya, and can be used without subsequent analysis of

digested globin chains when a suspicion for the presence of one of the two variants

arises from judging the initial electrophoretic and HPLC results.

REFERENCES

1. Huisman, T.H.J.; Wrightstone, R.N.; Wilson, J.B.; Schroeder, W.A.; Kendall A.G.

Hemoglobin Kenya, the product of fusion of g and b polypeptide chains. Arch.

Biochem. Biophys. 1972, 153, 850–853.

2. Kendall, A.G.; Ojwang, P.J.; Schroeder, W.A.; Huisman, T.H.J. Hemoglobin Kenya,

the product of a g–b fusion gene: studies of the family. Am. J. Hum. Genet. 1973, 25,

548–563.

74 RAI, ALVELIUS, AND LANDIN

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3. Smith, D.H.; Clegg, J.B.; Weatherall, D.J.; Gilles, H.M. Hereditary persistence of

foetal haemoglobin associated with gb fusion variant, Haemoglobin Kenya. Nature

New Biol. 1973, 246, 184–186.

4. Nute, P.E.; Wood, W.G.; Stamatoyannopoulos, G.; Olweny, C.; Failkow, P.J. The

Kenya form of hereditary persistence of foetal haemoglobin: structural studies and

evidence for homogenous distribution of Haemoglobin F using fluorescent anti-

Haemoglobin F antibodies. Br. J. Haematol. 1976, 32, 55–63.

5. Ojwang, P.J.; Nakatsuji, T.; Gardiner, M.B.; Reese, A.L.; Gilman, J.G.; Huisman,

T.H.J. Gene deletion as the molecular basis for the Kenya-Gg-HPFH condition.

Hemoglobin 1983, 7 (2), 115–123.

6. Forget, B.G. Molecular basis of hereditary persistence of fetal hemoglobin. Ann.

N.Y. Acad. Sci. 1998, 850, 38–44.

7. Huisman, T.H.J. Compound heterozygosity for Hb S and the hybrid Hbs Lepore,

P-Nilotic, and Kenya; comparison of hematological and hemoglobin composition

data. Hemoglobin 1997, 21 (3), 249–257.

8. Landin, B.; Jeppsson, J-O. Rare b chain hemoglobin variants found in Swedish

patients during HbA1c analysis. Hemoglobin 1993, 17 (4), 303–318.

9. Rai, D.K.; Alvelius, G.; Landin, B.; Griffiths, W.J. Electrospray tandem mass

spectrometry in the rapid identification of a-chain haemoglobin variants. Rapid

Commun. Mass Spectrom. 2000, 14, 1184–1194.

10. Waye, J.S.; Cai, S-P.; Eng, B.; Chui, D.H.K.; Francombe, W.H. Clinical course and

molecular characterization of a compound heterozygote for sickle cell hemoglobin

and Hemoglobin Kenya. Am. J. Hematol. 1992, 41, 289–291.

11. De Caterina, M.; Esposito, P.; Grimaldi, E.; Di Mario, G.; Scopacasa, F.; Ferranti, P.;

Parlapiano, A.; Malorni, A.; Pucci, P.; Marino, G. Characterization of Hemoglobin

Lepore variants by advanced mass-spectrometric procedures. Clin. Chem. 1992, 38,

1444–1448.

Received July 11, 2001

Accepted August 21, 2001

IDENTIFICATION OF Hb KENYA BY ESMS 75

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