Homozygous Expression of a Missense Mutation at Nucleotide 385 in the FUT2 Gene Associates with the...

JOBNAME: BBRC 219#3 PAGE: 1 SESS: 15 OUTPUT: Mon Apr 1 15:02:02 1996/xypage/worksmart/tsp000/68953f/1

Homozygous Expression of a Missense Mutation at Nucleotide 385 in theFUT2 Gene Associates with the Le(a+b+) Partial-Secretor Phenotype in

an Indonesian Family

Stephen Henry,*,1 Rosella Mollicone,† Pilar Fernandez,† Bo Samuelsson,* Rafael Oriol,† andGöran Larson*

*Department of Clinical Chemistry and Transfusion Medicine, Göteborg University, Sweden; and †INSERM U-178,Villejuif 94807, France

Received January 17, 1996

A new point mutation was found in the coding sequence of the secretor FUT2 gene. This missense mutationwith an A→T substitution at nucleotide 385 resulted in an amino acid change of Ilel29 to Phe129. This mutationshowed a clear genetic trait in an Indonesian pedigree and, when appearing in a homozygous form, it wasassociated with the red cell Le(a+b+) and salivary partial-secretor phenotype. This result suggests that themolecular basis for the Le(a+b+) and associated partial-secretor phenotype is caused by a partially inactivatingamino acid change in thea(1,2)fucosyltransferase coded for by this new FUT2 allele.© 1996 Academic Press, Inc.

In Europeans the secretor gene (FUT2) transferase, which is fully active in 80% of individuals,controls the expression of the H antigen in the secretory compartments. This H antigen can bemodified by the ABO and Lewis transferases. As a consequence, expression of the secretortransferase is indirectly responsible for the formation of both Lewis b (Leb) and ABH substancesin secretions [1]. Those 20% of individuals not expressing H and related A and/or B substances insecretions are known as nonsecretors.In the polymorphic Lewis histo-blood group system the genes coding for the two fucosyltrans-

ferases responsible for the synthesis of the Lea and Leb glycoconjugates have been cloned, se-quenced and expressed [2, 3]. TheLewisgene (FUT3) codes for ana(1,¾)fucosyltransferase thatcan add fucose to either the type 1 precursor or to the H type 1, to form the Lea and Leb antigensrespectively [2]. Thesecretorgene (FUT2) codes for ana(1,2)fucosyltransferase that can add afucose onto the type 1 precursor to form H type 1, the precursor of Leb [3]. There are at presenttwo known nonsecretor mutations, both point mutations in the FUT2 gene coding sequence, whichresult in truncations of the secretor transferase and a loss of activity [3, 4]. The first, at nucleotide428 (se428), is commonly found in Europeans [3]. The second, at nucleotide 571 (se571), appearsto be absent in Europeans but is present in Polynesians [4].As a consequence of polymorphism of the genetically independent Lewis and secretor systems,

and interaction of their transferases, four major red cell and salivary ABH secretor phenotypes arecommon in Europeans. These are; Lewis negative secretors and nonsecretors both red cell phe-notyping as Le(a−b−), Lewis positive nonsecretors whose red cell phenotype will be Le(a−b+), andLewis positive secretors whose red cell phenotype will be Le(a−b+). There is also another red cellphenotype known as Le(a+b+), which is rare (or absent) in Caucasians [5]. The Le(a+b+) pheno-type is however common in other populations, in particular Polynesians [6], Asians [7], Indone-

1 Corresponding author. Fax: 46 31 417 631.Abbreviations:bp, base pairs;FUT2, Secretorgene, accession no. U17894;FUT3, Lewisgene, accession no. X53578,

PCR,polymerase chain reaction;se428, FUT2 nonsecretor G→A mutation at nucleotide 428;se571, FUT2 nonsecretor C→Tmutation at nucleotide 571;SSP,sequence specific primer.

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS219,675–678 (1996)ARTICLE NO. 0293

6750006-291X/96 $18.00Copyright © 1996 by Academic Press, Inc.All rights of reproduction in any form reserved.


sians [8], and Australian aborigines [9, 10]. The Le(a+b+) phenotype is believed to be caused byan inefficient secretor transferase, because poor expression of salivary ABH substances associatewith the phenotype. This poor expression of salivary ABH substances is known as the partial-secretor phenotype, and is defined by the presence of more salivary ABH substances than isexpected in a nonsecretor and less than is present in a secretor [11].

MATERIALS AND METHODS

Indonesian family.Genomic DNA from individuals of an Indonesian family (B) with the Le(a+b+) and partial-secretorphenotype was obtained from a previous study of the FUT3 and FUT6 genes [8, 12]. Red cell Lewis and salivary secretorphenotypes had been previously determined.Genotyping.We tested the Indonesian family members for known nonsecretor mutations and a new FUT2 mutation

identified in the sequence analysis of unrelated Polynesian individuals [13]. The two nonsecretor allelesse428 andse571,were screened for, using the methods previously described [3, 4]. The new A→T mutation found at nucleotide 385 wasscreened for, using sequence-specific oligonucleotide primers [14, 15]. A primer II-16s (59-ACTGGATGGAGGAGGAAT-ACCGCCACA-39) covering nucleotides 359 to 385 was used to detect the wild type allele, while a second primer II-17smutated at the 39 end (59-ACTGGATGGAGGAGGAATACCGCCACT-39) was used to detect the mutated allele. Bothsense primers were used with the antisense primer II-2as (59-ATGGACCCCTACAAAGGTGCCCGGCCGGCT-39) cov-ering nucleotides 535–564 [3]. PCR-amplification was performed on a PTC-100 Programmable Thermal Controller (MJResearch, Inc., Watertown, Mass). Half a microgram of genomic DNA was used as template for each amplification reaction.PCR reactions were done by heating to 85°C for 5 min before adding 1 U of Taq DNApolymerase (Boehringer MannheinGmbH). PCR amplifications were run for 25 cycles with denaturation at 94°C for 1.5 min, annealing/extension 2.5 min at72°C, and stopped by cooling to 8°C. PCR products were separated on a 2% agarose gel (Figure 1).

RESULTS AND DISCUSSION

The genetic mechanism resulting in the Le(a+b+) and partial-secretor phenotype is not known,although it has been speculated to be caused by an inefficient secretor transferase [10], known asSew. This speculation is supported by serology, immunochemistry and immunohistochemistrywhich have clearly established the Le(a+b+) phenotype as a unique glycoconjugate phenotype[16–18]. It is believed that in Le(a+b+) individuals the secretor transferase is somehow impaired,and a change in competition between Lewis and secretor transferases occurs. A consequence of thisimpairment is less production of H type 1. Less H type 1 means less Leb and less salivary ABHsubstances. This poor expression of ABH substances is reflected as the partial-secretor phenotype,which is usually found in all Le(a+b+) individuals [17]. Poor expression of Leb is reflected in themajor difficulties encountered in serologically phenotyping Le(a+b+) red cells [1].We screened an informative Indonesian family with a Le(a+b+) partial-secretor offspring for the

known nonsecretor mutations (se428 andse571) [3, 4], and for a new A→T mutation at nucleotide385 identified in Le(a+b+) Polynesians (Figure 1). By testing for the presence of these mutations

FIG. 1. PCR-SSP screening for an A→T mutation at nucleotide 385 of the FUT2 gene. A 206 bp product is obtainedwith the SSP primers. Lanes 1, 3, 5 and 7 reflect the result obtained using the unmutated primer (II-16s), while lanes 2, 4,6 and 8 reflect the result of the mutated primer (II-17s). All amplifications containDRBI primers as internal controls [15],resulting in a 796 bp PCR product in the absence of amplification by the SSP primers. In this example, the sample in lanes1–2 is heterozygous for the wild and mutated 385 alleles, while the samples in lanes 3–4, 5–6 and 7–8 are homozygous forthe mutated 385 allele. The sample in lanes 7–8 corresponds to individual 9 from Figure 2.

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in this Indonesian family we were able to demonstrate that the homozygous appearance of the 385mutation, in individual 9, associated with the Le(a+b+) and partial-secretor phenotype (Figure 2).Heterozygous expression of the mutated 385 allele in individuals 1, 2, 4 and 11 did not result inthe Le(a+b+) or partial-secretor phenotype. Three other Indonesian families were also studied andtheir results were compatible with the concept that the mutated 385 allele was associated with theLe(a+b+) phenotype. However, their secretor phenotypes were not fully explained on the basis ofonly the three mutations at nucleotides 385, 428, and 571, and other nonsecretor mutations arelikely to be revealed by further investigation. The FUT2 genes of these individuals will now besequenced.

ACKNOWLEDGMENTS

This work was supported in part by grants from the Health Research Council of New Zealand, the Swedish MedicalResearch Council (8266 + 6521), the Royal Society of Arts and Sciences in Gothenburg, the Wennergren Center, theSwedish Institute, the Centre National de la Recherche Scientifique (CNRS, France), grant 57/94 from groupement deRecherche et d’Etudes sur le Genome (GREG, France), University of Paris South (Paris XI) and a Mizutani grant forGlycoscience (Japan).

REFERENCES

1. Henry, S. M., Oriol, R., and Samuelsson, B. E. (1995)Vox Sang.69, 166–182.2. Kukowska-Latallo, J. F., Larsen, R. D., Nair, R. P., and Lowe, J. B. (1990)Genes Dev.4, 1288–1303.3. Kelly, R. J., Rouquier, S., Giorgi, D., Lennon, G. G., and Lowe, J. B. (1995)J. Biol. Chem.270,4640–4649.4. Henry, S., Mollicone, R., Lowe, J. B., Samuelsson, B., and Larson, G. (1996)Vox Sang.70, 21–25.5. Mourant, A. E, Kopec, A., and Domaniewska-Sobczak, K. (1976) The Distribution of Human Blood Groups and Other

Polymorphisms. pp. 548–576. Oxford University Press, Oxford.6. Henry, S. M., Simpson, L. A., and Woodfield, D. G. (1988)Hum. Hered.38, 111–116.7. Broadberry, R. E., and Lin-Chu, M. (1991)Hum. Hered.41, 290–294.8. Mollicone, R., Reguigne, I., Kelly, R. J., Fletcher, A., Watt, J., Chatfield, S., Aziz, A., Cameron, H. S., Weston, B. W.,

Lowe, J. B., and Oriol, R. (1994)J. Biol. Chem.269,20987–20994.9. Boettcher, B., and Kenny, R. (1971).Hum. Hered.21, 334–345.10. Vos, G. H., and Comley, P. (1967)Acta genet.17, 495–510.11. Henry, S. M., Benny, A. G., and Woodfield, D. G. (1990)Vox Sang.58, 61–66.

FIG. 2. Distribution of the 385 A→T mutated FUT2 allele in an Indonesian family. The red cell Lewis phenotype isindicated beneath each family member. The salivary ABH phenotype is indicated as the ABH substance detected, either Aand/or H substance. The partial secretory phenotype (individual 9) is designated by downward arrows for the salivarysubstances indicating low levels of these substances. Homozygous appearance of the mutated 385 allele correlated with theLe(a+b+) red cell phenotype and partial secretion of salivary ABH substances. None of these individuals had these428 orse571 nonsecretor alleles. The Lewis genotypes of these individuals have been previously published [8].


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12. Mollicone, R., Reguigne, I., Fletcher, A., Aziz, A., Rustam, M., Weston, B. W., Kelly, R. J., Lowe, J. B., and Oriol, R.(1994)J. Biol. Chem.269,12662–12671.

13. Henry, S., Fernandez, P., Mollicone, R., Samuelsson, B., Oriol, R., and Larson, G. (1996) (abstract). 24th Congress ofthe International Society of Blood Transfusion, Japan.

14. Ugozolli, L., and Wallace, R. B. (1991)Methods: Companion to Methods in Enzymology.2, 42–48.15. Olerup, O., and Zetterquist, H. (1992)Tissue Antigens.39, 225–235.16. Henry, S. M., Oriol, R., and Samuelsson, B. E. (1994)Glycoconj. J.11, 593–599.17. Henry, S. M., Woodfield, D. G., Samuelsson, B. E., and Oriol, R. (1993)Vox Sang.65, 62–69.18. Henry, S. M., Samuelsson, B. E., and Oriol, R. (1994)Glycoconj. J.11, 600–607.


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