The molecular basis of glucose-6-phosphate dehydrogenase deficiency

~"~EVIEWS 5 Ashburner, M. etal. (1974) CoMSpringHarborSymp. Quant. Biol. 38, 655-662 6 Ashburner, M. (1990) Cell61, 1-3 7 Thummel, C.S. (1990) BioEssays 12, 1-8 8 Meyerowitz, E.M. etal. (1987) Trends Genet. 3, 288-293 9 Hansson, L. and Lambertsson, A. (1989) Hereditas 110, 61457 10 DiBello, P.R. el al. (1991) Genetics 129, 385-397 11 Segraves, W.A. and Richards, G. (1990) lnvertebr. Reprod. Dev. 18, 67-76 12 Belyaeva, E.S. et al. (1989) in Ecdysone: Prom Chemistry to Mode of Action (Koolman, J., ed.), pp. 368-376, Georg Thieme Verlag 13 Guay, P.S. and Guild, G.M. (1991) Genetics 129, 169-175 14 Restifo, L.L. and Guild, G.M. (1986)J. Mol. Biol. 188, 517-528 15 Koelle, M.R. et al. (1991) Cell 67, 59-77 16 Barnett, S.W. et al. (1990) Dev. Biol. 140, 362-373 17 Ashburner, M. (1989) Drosophila: A Laboratory Handbook, Cold Spring Harbor Laboratory Press 18 Lepesant, J-A. et al. (1986) Arch. Insect Biochem. Physiol. Suppl. 1, 133-141 19 Berreur, P. et al. (1984) Gen. Comp. Endocrinol. 54, 76~4 20 Bollenbacher, W.E., Vedeckis, W.V. and Gilbert, L.I. (1975) Dev. Biol. 44, 46-53 21 Dominick, O.S. and Truman, J.W. (1985) J. Exp. Biol. 117, 45--68 22 Georgel, P. et al. (1991) Mol. Cell. Biol. 11,523-532 23 Karim, F.D. and Thummel, C.S. (1991) GenesDev. 5, 1067-1079 24 Richards, G. (1980) in Progress in Ecdysone Research (Hoffman, J.A., ed.), pp. 363-378, Elsevier

G l u c o s e - 6 - p h o s p h a t e dehydrogenase (G6PD) is a housekeeping enzyme that is present in all cells, where it plays a key role in the metabolism of glucose. Specifically, it catalyses the first step of the hexose monophosphate pathway and is therefore important in pentose phosphate synthesis. The reaction also provides NADPH, which is required for reductive biosynthetic reactions and for maintaining the level of cell glutathione and other sulphydryl groups. This, in turn, is necessary for the detoxification of hydrogen peroxide and other oxidizing agents. G6PD has been extensively characterized in a wide range of organisms, including bacteria, protozoa, fungi, insects, fish and mammals. Here we review recent findings on the structure of the human G6PD gene and how this knowledge has helped us to understand the basis of one of the most common human genetic abnormalities, G6PD deficiency.

The human G6PD gene The gene that encodes human G6PD is located on the long arm of the X chromosome (Xq28) 1. Several other human disease loci have also been mapped to this region, including haemophilia A, colour blindness, adrenoleukodystrophy, dysk&atosis congenita and Emery-Dreifuss muscular dystrophy 2. In addition to G6PD, the genes for factor VIII (Ref. 3) and colour vision 4 have been cloned. Through formal genetic analysis, the use of somatic cell hybrids and more

25 Cherbas, P. et al. (1989) in Progress in Comparative Endocrinology (Epple, A. et al., eds), pp. 112-115,

Wiley-Liss 26 Savakis, C., Ashburner, M. and Willis, J.H. (1986) Dev. Biol. 114, 194-207 27 Murtha, M.T. and Cavener, D.R. (1989) Dev. Biol. 135,

66-73 28 Richards, G. (1976) Dev. Biol. 54, 256-263 29 Richards, G. (1976) Dev. Biol. 54, 264-275 30 Fristrom, J.W. et al. (1986) Arch. Insect Biochem. Physiol.

Suppl. 1, 119-132 31 Kiss, I. etal. (1988) Genetics 118, 247-259 32 Velissariou, V. and Ashburner, M. (1980) Chromosoma

77, 13-27 33 Guild, G.M. (1984) Dev. Biol. 102, 462-470 34 Velissariou, V. and Ashburner, M. (1981) Chromosoma

84, 173-185 35 Ireland, R.C. et al. (1982) Dev. Biol. 93, 498-507 3 6 Ingolia, T.D. and Craig, E.A. (1982) Proc. NatlAcad. Sci. USA 79, 2360-2364 3 7 Natzle, J.E., Fristrom, D.K. and Fristrom, J.W. (1988) Dev. Biol. 129, 428-438 38 Paine-Saunders, S., Fristrom, D. and Fristrom, J.W. (1990) Dev. Biol. 140, 337-351 39 Moore, J.T. et al. (1990) Dev. Genet. 11, 299-309 40 Maschat, F. et al. (1990)J. Mol. Biol. 214, 359-372

A.J. ANDRES AND C.S. THUMMEL ARE IN THE HOWARD HUGHES MEDICAL INSTITUTE, ECCLES INSTITUTE OF HUMAN GENETIC~ UNIVERSITY OF UTAH, SALT LAKE CIT~, U T 8 4 1 1 2 , USA.

The molecular basis of gluc0se-6-ph0sphate dehydr0genase deficiency TOM VULI]AMY~PHII,IPMASON AND LUCIOLUZZATrO With more than 300 different variants reported, the human enzyme glucose-6-phosphate dehydrogenase (G6PD; EC 1.1.1.49) is one of the most polymorphic proteins known. An estimated 400 million people throughout the worm are deficient in G6PD;numerous lines of evidence indicate that this is because female heterozygotes have a selective advantage in malaria infections. The cloning of the G6PD gene has made it possible to clarify the molecular basis underlying this enzyme deficiency and polymorphisnt recently pulsed-field gel electrophoresis, two-colour fluorescence in situ hybridization 5 and the isolation of yeast artificial chromosome (YAC) clones 6, this region is emerging as one of the best mapped in the human genome (Fig. 1). The G6PD gene consists of 13 exons, spanning -18 kb, with the second intron alone representing 9.85 kb (see Fig. 1). Originally mapped on phage and cosmid clones in 19867, the complete genomic sequence has recently been determined ~. The coding

Tl<; M'Ult 1992 VOI.. 8 NO. 4 © 1992 Elsevier

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bTGH Cytogenetic and molecular map of the G6PD gene. The top line is a diagrammatic representation of the tip of the long arm of the X chromosome, showing the position of several well-known genes (from Ref. 6): HPRT, hypoxanthine guanine phosphoribosyltransferase; FIX, factor IX; FRAX, fragile X site; CV, colour vision genes; FVII1,factor VIII. The middle line shows the relative positions and sizes of the FVIII, G6PD and CV genes (from Ref. 5). The bottom line shows the intron--exon structure of the G6PD gene itself (from Ref. 7), with the location of some restriction sites: R, EcoRI; B, BamHI; K, KpnI. sequence of the gene is 1545 bp long and from it the primary sequence of the G6PD protein (515 amino acids) was first deduced9. The 5' untranslated sequence is 71 bp long, although alternative sites for the initiation of transcription have been identified. The 3' untranslated sequence is 608 bp long. The G6PD promoter shows a number of characteristics typical of a housekeeping gene promoter, such as several potential Spl-binding sites, no recognizable CAAT box and an atypical TATA box (ATTAAAT). The entire region extending from -1200 bp to within the first intron of the gene is very GC-rich and recognizable as an HTF island 7,9. Analysis of the G6PD promoter sequences using a chloramphenicol acetyltransferase (CAT) reporter gene has established that the region extending 436 bp upstream of the cap site is sufficient to drive transcription in mammalian cell lines. Further deletions into this region result in a sharp reduction in the levels of CAT produced once position-160 is reached m. The coding sequence of the G6PD gene has been placed '-, / ' u behind different viral promoters ,..>._ ,q "(/ \ for the production of human ," . . ' . i . . . . . . . . . ~ . ~ % . ~ ~?;.:.. G6PD protein in COS-1 cells 1~ and, more recently, in E. coli 12,13. By this approach, large quantities of pure recombinant G6PD can be obtained, which may be sufficient for the growth of crystals and the subsequent determination by X-ray crystallography of a threedimensional structure of the pro.'tj~" tein, which still remains elusive. , •

Some variants, with near normal levels of enzyme activity, have been discovered by electrophoretic analysis - for example the G6PD A variant found in Africa. More importantly from the medical point of view, the study of people who developed acute anaemia, triggered by exposure to a variety of agents including drugs and infection, led to the discovery that such people had lower than normal levels of enzyme activity (G6PD deficiency). It is now estimated that G6PD deficiency affects upwards of 400 million people throughout tropical and subtropical regions of the world (Fig. 2). It is widely accepted that this high frequency of G6PD deficiency has evolved because of the selective pressures exerted by Plasmodium falciparum malaria. The evidence for this comes mainly from two sets of observations. First, the distribution of G6PD deficiency in the world correlates unambiguously with

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Genetic variation and selection by malaria It has been recognized since the early 1960s that genetic variation of human G6PD is extensive.

, , 4

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FIG[] World map showing the approximate population frequency of G6PD deficiency {from Ref. 41 ). TIG APRIL 1992 VOL. 8 NO. 4

139

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that of past and present malaria endemicity (see Fig. 2). This epidemiological evidence is strengthened by the fact that different mutant alleles are responsible for G6PD deficiency in different parts of the world (e.g. G6PD Mediterranean in Southern Europe and the Middle East; G6PD A- in Africa; G6PD Mahidol and others in South-East Asia). Thus, G6PD polymorphism cannot have simply spread with early human migrations; rather, it is a g o o d example of convergent evolution in humans. Second, females heterozygous for G6PD deficiency have lower parasitaemias than appropriate controis ~4, and recently no heterozygotes were found in a group of children with cerebral malaria (G. Sodeinde, pers. commun.). How does G6PD deficiency protect individuals against severe malaria? In vitro culture studies have shown that w h e n P. falciparum is transferred from normal to G6PD-deficient red blood cells, parasite growth is inhibited at first, but in subsequent cycles of infection the parasite adapts to the abnormal host cell. This behaviour fits well with the in vivo data: hemizygous males are not resistant because the parasite adapts; heterozygous females are resistant because the parasite has trouble in adaptation when c6nfronted with a mosaic of G6PI> normal and G6PD-deficient red blood cells (as a result of X-chromosome inactivation). It

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~200 bp.

FIG[] G6PD mutations on a sketch of the gene. Exon numbers are shown as roman numerals. The shaded exon I is noncoding. Stippled boxes are variants causing CNSHA(class I in Table 1). TIG APRIL1992 VOL. 8 NO. 4 I-dl

[]~EVIEWS was suggested that adaptation resulted from induction of parasite G6PD synthesis by the G6PD-deficient host environment 15. However, subsequent studies have indicated that the parasite produces its own G6PD even in normal red cells. Thus, the mechanism of adaptation remains obscure, but current studies of the P. falciparum G6PD16,17 may shed some light on this phenomenon.

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G6PD A \ From a number of different bioIVS5.611 C'~'G and IVS7.175 C---~T chemical properties, 387 different variants of G6PD have recently been listed TM, I-+++-+-I 86 of which have been classified as poly\ morphic 19. They are grouped into dif2 0 2 G ---~-A ferent classes depending on the degree of enzyme deficiency and consequent G6PDA[ + + + + "+-] clinical symptoms (Table 1). A striking manifestation of G6PD deficiency is FZGll favism, an acute haemolytic anaemia that Evolution of G6PD Aand related polymorphisms. The seven different results from eating broad beans (fava haplotypes seen in African chromosomes are shown in boxes and are arranged beans, Vicia faba). This reaction, which according to the most economical pathway of evolution, linked by the base only occurs in some G6PD-deficient sub- changes shown. The seven polymorphic sites making the haplotype are NlalII, jects, is thought to be due to the toxic FokI, PvuII, ScaI, BspHI, PstI and BclI (from Ref. 28; ScaI and BclI restriction effect of glucosides (divicine and vicine) sites were created by mismatch-containingprimers in polymerase chain reaction present in the beans. analysis). To date, 34 different mutations in the G6PD gene have been identified through cases it is harder to reconcile the differences observed. the comparison of genes encoding variant enzymes with the sequence of the normal G6PD B gene. They An example of this is the recent report that the same mutation (nucleotide 95 A-+G, amino acid 32 are widely spread through the coding sequence of the gene, being found in all exons except exon 3 (the His--+Arg) is found in a group of Chinese variants smallest) and exon 13 (which codes for the carboxy whose activities range from 0% to 50% of normal and terminus). All but one of these are point mutations whose electrophoretic mobilities range from 87% to 108% of normal 21. It is difficult to find a reasonable causing single amino acid substitutions (Fig. 3). Most explanation for this. (19/34) are C-to-T transitions on either the coding or noncoding strand. Of these, 12 are in CpG dinucleotides, regarded as mutational 'hotspots' because the C in Microevolution One of the most common variants associated with this position is often methylated, and 5-methylcytosine enzyme deficiency is G6PD A-. While this variant can undergo spontaneous deamination to thymidine. was originally thought to be restricted to the African Among the remaining mutations, transitions and transpopulation, it now turns out to be present in people versions are equally represented. The odd one out, so far, is the variant G6PD Sunderland 2°, which is due to of Spanish and Italian origin as well, where it had been described as G6PD Betica and G6PD 'Matera', a 3 bp deletion within a (CAT)3 repeat, causing the loss of an isoleucine residue in exon 2. Larger deamong other names. At the molecular level, its unique feature is that it contains two mutations: one of these letions, and other mutations such as nonsense mutations and frameshift mutations that would completely (nucleotide 376 A-+G, amino acid 126 Asn--+Asp) is the mutation that alone gives rise to G6PD A abolish the function of the protein, have not been (Ref. 22); the second (which is almost always found in the G6PD gene. This is probably because nucleotide 202 G-+A, amino acid 68 Val--~Met) complete absence of G6PD is lethal. gives the G6PD A- phenotype 23. In a few people, an The characterization of G6PD deficiency at the alternative second mutation has been identified molecular level has established that the same mu(nucleotide 680 G-+T, amino acid 227 Arg-+Leu or tation(s) may be found in variants that had originally nucleotide 968 T-+C, amino acid 323 Leu--+Pro) that been described as different from one another on the also produces an enzyme with the characteristics of basis of biochemical criteria. Of the 34 different mutations identified, 11 have been found in more than G6PD A- (Ref. 24). In addition to these mutations responsible for the one variant, so that 61 variants have now been common enzyme variants in Africa, we now know of accounted for. In most cases it is quite easy to see five other polymorphic sites clustered within 3 kb how this has come about, as with a number of elecof the G6PD gene. Two of these were found as retrophoretically fast variants that are now known to be striction fragment length polymorphisms (RFLPs)e5,26, the same as G6PD A- (see below). However, in other TIG APRIL1992 VOL.8 NO. 4

141

~qEVIEWS

COOH

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I i00

I

I

200

300

I

400

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FIGI~ Conserved regions in the macroevolution of G6PD. A linear representation of the G6PD protein shows the position of nine highly conserved peptides. The scale refers to amino acid numbering in human G6PD. Boxed amino acids are conserved in all species for which the sequence is available (see text); the others are conserved in six out of seven of these species. The starred K is the reactive lysine residue at position 205 in the human sequence.

while the other three were identified by comparing the exon and intron sequences of different individuals27, 28. Not surprisingly, there is marked linkage disequilibrium between these sites. The haplotypes observed have been used to outline a pattern of evolution (based on the principle of the most economical pathway) for G6PD A and A-, both of which are presumed to have arisen only once, with the A mutation being the most ancient and A - the most recent 28,29 (Fig. 4). Since the sequence of chimpanzee G6PD is B-like 24, the B allele is likely to be the ancestral form. Of these polymorphic sites, most are presumably neutral with respect to natural selection, while G6PD A - has been selected for by malaria; thus they may be useful in studying the relative roles of drift and selection in the spread of different mutations within a single gene. The majority of these polymorphic sites have turned out to be limited to people of African origin. The only one to show any polymorphism in the European population is the silent mutation in exon 11 (nucleotide 1311 C--->T), which was first identified in individuals with G6PD Mediterranean. It was later shown to be polymorphic throughout the Mediterranean region. Most G6PD Mediterranean chromosomes have a T residue at nucleotide 1311, while G6PD B chromosomes can have a C or a T residue. In Southern Italy and in India, some G6PD Mediterranean individuals have a C at this position, implying either that there has been a recombination event between the two mutations, or that the G6PD Mediterranean mutation has arisen on more than one occasion.

Structure and function As stated above, the three-dimensional structure of G6PD is not yet known, so other ways have had to be used to identify functionally important domains of the protein. The region around lysine 205 is thought to be the G6P-binding site: when this amino acid is reacted with pyridoxal phosphate, 80% of the enzyme activity is lost and the affinity for G6P is altered30. It is interesting to note, therefore, that the mutations in two enzyme variants, G6PD Mediterranean and G6PD Coimbra, both of which have an abnormal Km for G6P, are at amino acids 188 (Ref. 22) and 198 (Ref. 31), respectively, close to this lysine 205 residue.

As for the NADP-binding domain, there appears to be some clustering of mutations giving rise to chronic non-spherocytic haemolytic anaemia (CNSHA) around exon 10, and among them G6PD Iowa and G6PD Beverly Hills show marked reactivation of the enzyme in Mg 2+ and NADP 32. The two adjacent amino acids that are substituted in these variants are positively charged, while the electrophoretic mobilities of the mutated enzymes are anomalous; these findings are consistent with the suggestion that these variants have failed to bind the negatively charged NADP molecule. But care must be taken not to overinterpret this kind of analysis. For example, the mutation in G6PD Santiago de Cuba 22, reported to have an extremely abnormal Km for NADP, maps some 60 amino acids away from the defect in G6PD Iowa. G6PD Taiwan Hakka33, which has a Km for G6P even further from normal than G6PD Mediterranean, has a mutation in exon 12 rather than exon 6. Progress in this area is clearly going to need some knowledge of the physical structure of the protein. Some idea of which amino acids are located at specific positions in the protein for important structural or catalytic functions can also come from the comparison of "G6PDs from different species. The complete amino acid sequence is n o w k n o w n for G6PD from human 9, rat 34, Drosophila 35, Saccharomyces cerevisiae~6, E. coli 37, Leuconostoc mesenteroides 3s and Zymomonas mobilis 39. When G6PDs from the first four of these species are compared, 201 of the 515 (human) amino acids are perfectly conserved, with several distinct blocks standing out36. When all seven species are compared, only 70 amino acids are perfectly conserved, 45 of which are found in nine short peptides scattered along the protein's length (Fig. 5). The most striking of these includes the lysine 205 residue discussed above. The mutations responsible for G6PD deficiency will affect either the specific activity or stability of the enzyme, or both. We can n o w establish which of these is actually the case through the biochemical analysis of different variant G6PDs produced in E. coli. Amino acid substitutions that affect the stability of the enzyme will be the most likely cause of deficiency in polymorphic variants. This is because they must provide a reasonably high enzyme activity in most somatic tissues where G6PD is essential and yet have a low activity in red blood cells, the site of the selection

T1G APRIL1992 VOt. 8 NO. 4 142

~]~E,VIEWS by malaria parasites. With an unstable G6PD, the activity is considerably higher in cells in which proteins are constantly turned over than it is in red blood cells, where proteins are not renewed by ongoing protein synthesis. Polymorphic G6PD variants will therefore have been selected by nature to fulfil these particular criteria. This may explain why the cause of G6PD deficiency is restricted to missense mutations, even though a variety of mutations have been described in other systems. Similarly, G6PD variants that underlie CNSHA must retain sufficient activity in the somatic tissues to be viable, while the symptoms of this condition stem from the low activity of the enzyme in the red blood cell; again, the presence of an unstable variant enzyme could account for these features.

G6PD and sulphur metabolism

6 7 8 9 10 11 12 13 14 15 16 17

The M E T 1 9 gene of yeast was isolated recently, and turned out to encode G6PD 40. A G6PD null mutant was constructed and found to have an absolute requirement for a source of organic sulphur, and this does not seem to be caused by a simple reduction'in NADPH levels. Moreover, transcription of the yeast G6PD gene is repressed by S-adenosylmethionine. These unexpected findings raise new issues concerning the metabolic role and regulation of G6PD and it will be interesting to k n o w if this has implications for other organisms.

20

Conclusion

25

G6PD deficiency provides an example of a 'conditional mutant' in human genetics, whereby the phenotype (haemolytic anaemia) is only expressed when the organism is exposed to a particular exogenous agent (fava beans, a drug or an infection). Studies of its population genetics have caused the G6PD system to emerge as a good example of polymorphism balanced by malaria selection. Investigation of more patients has revealed mutant alleles that are no longer conditional, because they cause haemolytic anaemia even in the absence of exogenous agents. Molecular analysis has n o w begun to give us an insight into the basis of the various phenotypes. Eventually it should be possible to explain them fully and to pinpoint the mechanism whereby heterozygotes are e n d o w e d with resistance against malaria parasites.

Acknowledgements

18 19

21 22

23 24

26 27 28 29 30 31 32 33 34 35 36 37

We thank Colin Corcoran for help in preparing the figures and all our colleagues, especially Margaret Town, Viola Calabro and Peppe Martini, for helpful discussion. Work in our laboratory is supported by an MRC (UK) programme grant.

38 39 40

References 1 Pai, G.S. etal. (1980) Proc. NatlAcad. Sci. USA77, 2810-2813 2 Davies, K.E. et al. (1990) Cytogenet. Cell Genet. 55, 254-313 3 Gitschier, J. et al. (1984) Nature312, 326-330 4 Nathans, J., Thomas, D. and Hogness, D.S. (1986) Science 232, 193--202 5 Trask, B.J., Massa, H., Kenwrick, S. and Gitschier, J. (1991) Am.J. Hum. Genet. 48, 1-15

41

Schlessinger, D. et al. Genomics (in press) Martini, G. et al. (1986) EMBOJ. 5, 1849-1855 Chen, E.Y. et al. (1991) Genomics 10, 792-800 Persico, M.G. etal. (1986) Nucleic Acids Res. 14, 2511-2522; see also p. 7822 Ursini, M.V., Scalera, L. and Martini, G. (1990) Biocbem. Biophys. Res. Commun. 170, 1203--1209 Mason, P.J. et al. (1988) Eur.J. Biochem. 178, 109--113 Persico, M.G., Ciccodicola, A., Martini, G. and Rosner, J.L. (1989) Gene 78, 365-370 Bautista, J., Mason, P.J. and Luzzatto, L. Biochim. Biophys. Acta (in press) Luzzatto, L. (1979)Blood54, 961-976 Usanga, E.A. and Luzzatto, L. (1985) Nature 313, 793-795 Ling, I.T. and Wilson, R.J.M. (1988) Mol. Biochem. Parasitol. 31, 47-56 Kurdi-Haidar, B. and Luzzatto, L. (1990)Mol. Biochem. ParasitoL 41, 83-92 Beutler, E. (1990) Semin. Haematol. 27, 137-164 Luzzatto, L. and Mehta, A. (1989) in The Metabolic Basis of Inherited Disease (Scriver, C.R. et al., eds), pp. 2237-2266, McGraw-Hill McDonald, D. etal. (199l) Nature350, 115 Chao, L. et al. (1991) Nucleic Acids Res. 19, 6056 Vulliamy, T.J. et al. (1988) Proc. Natl Acad. Sci. USA 85, 5171-5175 Hirono, A. and Beutler, E. (1988) Proc. NatlAcad. Sci. USA 85, 3951-3954 Beutler, E,, Kuhl, W., Vives-Corrons, J-L. and Prchal, J.T. (1989) Blood74, 2550-2555 D'Urso, M. et al. (1988) Am.J. Hum. Genet. 42, 735-741 Yoshida, A., Takizawa, T. and Prchal, J.T. (1988)Am.J. Hum. Genet. 42, 872-876 DeVita, G. etal. (1989)Am.J. Hum. Genet. 44, 233-240 Vulliamy, T.J. et al. (1991) Proc. Natl Acad. Sci. USA 88, 8568-8571 Beutler, E. and Kuhl, W. (1990) Hum. Genet. 85, 9-11 Carmadella, L. et al. (1987) Eur.J. Biochem. 171, 485-489 Corcoran, C.M. et al. Hum. Genet. (in press) Hirono, A. et al. (1989) Proc. Natl Acad. Sci. USA 86, 10015-10017 Zuo, L. etal. (1990)Blood76, 51A Ho, Y-S., Howard, A.J. and Crapo, J.D. (1988) Nucleic Acids Res. 16, 7746 Fouts, D. etal. (1988) Gene63, 261-275 Persson, B., Jomvall, H., Wood, I. and Jeffrey, J. (1991) Eur.J. Biochem. 198, 485-491 Rowley, D.L. and Wolf, R.E., Jr (1991)J. Bacteriol. 172, 968-977 Lee, W.T., Flynn, G., Lyons, C. and Levy, H.R. (1991) J. Biol. Chem. 266, 13028-13034 Barnell, W.D., Yi, K.C. and Conway, T. (1990) J, Bacteriol. 172, 7227-7240 Thomas, D., Cherest, H. and Surdin-Keljan, Y. (1991) FAgBOJ. 10, 547-553 Beutler, E. etal. (1989) Bull. WHO67, 601-611

T. VULLIAMY, P. MASON AND L LUZZAITO ARE IN THE DEPARTMENT OF HAEMATOLOGY, ROYAL POSTGRADUATE MEDICAL SCHOOL, HAMMERSMITHHOSPITAL, DUCANE ROAD, LONOON W I 2 0NN, UK.

TIG APRIL1992 VOL. 8 NO. 4