Humangenetik 27, 347--349 (1975) © by Springer-Verlag 1975

Haemoglobin Norfolk in Nepali Gorkhas T. N. Mehrotra, S. C. Gupta, and 1~. Sinha Department of Pathology and Bacteriology, M.L.N. Medical College, Allahabad, U.P., India tI. L e h m a n n and B. G. Wiltshire Medical Research Council, Abnormal Haemoglobin Research Unit, Department of Clinical Biochemistry, University of Cambridge, England Received January 29, 1975

Summary. Three instances of fast moving haemoglobins in heterozygous form have been found in Indian soldiers of Nepali Gorkha, during a routine screening for abnormal haemoglobins. The affected individuals were not related and seemed clinically well. Material and Methods Blood was obtained by venipuncture from soldiers. The techniques used for preparation of red cell haemolysates, and paper electrophoresis at pH 8.9 have been summarised elsewhere (Lehmann and Huntsman, 1974). Samples containing fast moving haemoglobins were sent to the MRC Abnormal Haemoglobin Unit where standard methods were used for identification (Lehmann and Huntsman, 1974). Routine haematological methods were used for siekling test, retieulocyte count, serum bilirubin and examination for inclusion bodies.

Results The fast moving haemoglobins were demonstrated b y paper eleetrophoresis. For structural s t u d y the haemoglobins were purified b y D E A E Sephadex chrom a t o g r a p h y , concentrated b y ultraeentrifugation, and the haem removed b y the acid-acetone precipitation at - - 2 0 ° C (Rossi-Fanelli et al., 1958). The dried globins were digested with trypsin and the fingerprints prepared (Sick et al., 1967), both for diagnostic and preparative purposes. The diagnostic fingerprints were stained with reagents specific for histidine (Pauly reagent), t r y p t o p h a n (Ehrlieh's reagent) : arginine (Sakagaehi reagent) and tyrosine; and methionine (Platinic iodide reagent) as outlined by Easley (1965). Two new histidine containing peptides were seen (Fig. 1) but no peptide appeared to have been lost. The slower-moving of the two new peptides had the composition shown in Table 2 which is consistent with a m u t a t i o n of either Gly->Asp at a57 or 59, or Ala Asp at fi62. N o r m a l l y peptide V I I of both a and fl chains run together so do peptides a and fl V I I - - V I I I which have an extra lysine at the C-terminus (Table 2). Analysis of the amino aeids of the normal V I I - - V I I I after hydrolysis showed it to fiTp V I I - - V I I I as it contained both alanine and glycine. This shows t h a t the m u t a t i o n is in the a peptide.

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T . N . Mehrotra et al.

Electrophoresis p H 6.4 Fig. 1. Fingerprint of the purified abnormal haemoglobin. For details see text

Table 1. Haematological findings in Gorkha soldiers

Haemoglobin (g/1O0 ml) Erythrocytes (mil./mm 8) P.C.V. (ml/100 ml) )~. C.V. (Cubic micron M.C.H.C. (%) l~eticulocytes (%)

G 97 (20 years)

G 137 (22 years)

G 274 (24 years)

13.8 5.0 42 84 32 1

15 6.0 48 80 31 1.5

13.8 5.2 46 88 30 1.0

Table 2 G . . (case I) abnormal V I I ASP GLY ALA HIS LYS n. moles per residue

0.87 1.14 0.90 1.02 18.1

(0) (2 or 1) (0 or 1) (I) (1)

G . . . (case II) abnormal V I I 0.95 1.00 0.94 1.12 10.1

(0) (2 or 1) (0 or 1) (1) (1)

G . . . (case I) normal -1.29 0.77 0.94 1.70 24.7

Figures in parantheses give expected n u m b e r of residues in the normal peptide. 57 60 57 61 Sequence of a V I I Gly His Gly Lys V I I V I I I Gly His Gly Lys Lys 62 65 Sequence of flVII Gly His Ala Lys V I I - - V I I I Gly His Ala Lys Lys 62 66

(0) (1 or 2) (O or 1)

(1) (2)

Haemoglobin Norfolk in Nepali Gorkhas

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Normal a T p V I I contains two glycine residues at positions 57 and 59, the m u t a t i o n to aspartic acid at a57 giving rise to H b Norfolk and t h a t at ~59 being unknown. The m u t a t i o n in this case was proved to be H b J Norfolk b y identification of the N-terminal residue as aspartyl using the Dansyl-ehloride technique (Gray, 1967).

Discussion Haemoglobin Norfolk is a rare haemoglobin which was reported for the first time by Ager et al. in 1958 in an Englishman from Norfolk, England. G a m m a c k et al. (1961) found t h a t H b Norfolk contained abnormal alpha chains. Later Baglioni (1962) showed t h a t an aspartic acid residue was substituted for the glyeine residue in position 57 of the alpha-chain of H b A. Hb-Norfolk has been reported in an Italian family living in Australia (Wilkinson et al., 1967), and from J a p a n (Shibata et al., 1966). The finding of this haemoglobin in 3 Nepali Gorkhas is the fourth observation of H b Norfolk since its first recognition in 1958. The isolation of this haemoglobin in such widely different ethnic groups as the English and Italians on one h a n d and the Japanese and Nepali Gorkhas on the other would suggest t h a t it m a y have arisen b y independent mutation.

References Ager, J. A. M., Lehmann, H., Vella, F. : Haemoglobin "Norfolk": A new haemoglobin found in an English family. Brit. med. J. 2, 539--541 (1958) Baglioni, C.: A chemical study of haemoglobin-Norfolk. J. biol. Chem. 287, 69--74 (1962) Easley, C. W. : Combinations of specific co]our reactions useful in peptide mapping technique. Biochim. biophys. Acta (Amst.) 107, 386--388 (1965) Gammack, D. B., Huehns, E. R., Lehmann, H., Shooter, E. M. : The abnormal polypeptide chains in a number of haemoglobin variants. Aeta genet. (Basel) 11, 1--16 (1961) Gray, W. R.: In: Methods in enzymology, Vol. XI, pp. 139 150. 1967 Rossi-Fanelli, A. 1%., Antonini, E., Caputo, A. : Studies on the structure of haemoglobin. 1. Physiochemical properties of human globin. Biochim. biophys. Acta (Amst.) 80, 608--615 (1958) Shibata, S., Iuchi, I., Miyaji, T. : Abnormal hemoglobins discovered in Japan. Acta haemat. jap. 29, 115--127 (1966) Sick, K., Beale, D., Irvine, D., Lehmann, H., Goodall, P. T., MacDougM1, S. : Haemoglobin G Copenhagen and haemoglobin J Cambridge. Two new/? chain variants of haemoglobin A. Biochim. biophys. Acta (Amst.) 140, 231--242 (1967) Wilkinson, T., Kronenberg, H., Beale, D., Lehmann, H. : The second observation of haemoglobin-Norfolk occurring in an Italian family together with beta-thalassaemia. Med. J. Aust. 1, 910--912 (1967) Prof. Dr. H. Lehmann Medical Research Council Department of Clinical Biochemistry University of Cambridge Cambridge, England