linkage of four polymorphisms on the αiib gene

4
SHORT REPORT Linkage of four polymorphisms on the a IIb gene J. RUAN, O. P EYRUCHAUD, A. NURDEN AND F. B OURRE UMR 5533 CNRS, Ho ˆpital Cardiologique, Pessac, France Received 9 April 1998; accepted for publication 23 April 1998 Summary. The subunits of the platelet integrin a IIb b 3 are encoded by two genes located on chromosome 17. Two pathologies are associated with structural modifications of this complex: Glanzmann’s thrombasthenia and alloimmune thrombocytopenia. The former is a hereditary bleeding disorder, the latter is due to an immune response linked to the presence of specific epitopes defined by single amino acid substitutions called human platelet alloantigen (HPA) systems. Analysing the a IIb gene from 112 independent chromosomes, we have defined two new silent polymorph- isms in complete linkage disequilibrium. They are recipro- cally linked to HPA-3 and a previously reported 9 pb deletion in intron 21. Linkage of these four DNA markers spanning a 5 kb fragment of genomic DNA provides a new tool for analysing a IIb gene pathology and evolution. Keywords: integrin a IIb b 3 , genetic polymorphisms, HPA-3b (Bak b ), Glanzmann’s thrombasthenia. The human integrin a IIb b 3 , specific for platelets and megakaryocytes, mediates platelet aggregation through its capacity to bind fibrinogen. Major pathologies associated with this receptor are Glanzmann’s thrombasthenia, an autosomal recessive bleeding syndrome, characterized by a functional or quantitative defect of a IIb b 3 (George et al, 1990), and thrombocytopenia due to the destruction of platelets secondary to an immune response after blood tranfusion or feto-maternal immunization (Kunicki & Newman, 1992). The latter is due to single amino acid substitutions that give rise to the so-called human platelet alloantigen (HPA) systems. Several HPA systems have been localized to a IIb b 3 (see Newman & Valentin, 1995). Among them, HPA-3 (Lyman et al, 1990) and Max (Noris et al, 1995) are located on the a IIb gene located on chromosome 17. The coding part is contained within 30 exons (Heidenreich et al, 1990) giving rise to a 140 kD protein which is assembled with the b 3 subunit to form a complex which undergoes maturation in the endoplasmic reticulum and the Golgi apparatus of megakaryocytes (Duperray et al, 1989). The HPA-3 system is defined by the presence of a I843S amino acid substitution (HPA-3a and HPA-3b respectively) due to a 2622T G mutation and Max by the presence of a V837M amino acid substitution (Max a– and Max aþ respec- tively) due to a 2603G A mutation (Lyman et al, 1990; Noris et al, 1995). We have previously reported that the HPA- 3b genotype is bilaterally linked to a 9 bp deletion (Del) within intron 21 on the a IIb gene (Peyruchaud et al, 1995). We now describe two new polymorphisms in the a IIb gene (IVS21(–7)C G and 3063C T) and show their complete linkage disequilibrium with HPA-3. Indeed, the frequency of Del ¹ , IVS21(¹7)C, HPA-3a and 3063C is exactly the same: 0·74 (0·26 for the alternative form). To our knowledge, it is the first time that four DNA polymorphisms have been reported to be reciprocally linked on a gene coding for an integrin subunit. MATERIALS AND METHODS Subjects and DNA amplification. Blood from 47 unrelated Caucasian subjects of European origin and nine unrelated patients with Glanzmann’s thrombasthenia was taken into EDTA anticoagulant after informed consent. DNA was prepared using a QIAamp blood and tissue kit (QIAGEN Inc., Chatsworth, Calif., U.S.A.) according to the manufac- turer’s instructions. Amplification products 21 (containing exon 21) and 26 (containing exon 26 and consequently the genetic determinant for HPA-3) were amplified as already described (Peyruchaud et al, 1995). Amplification products 22 (containing exon 22) and 30 (containing exon 30) were amplified under identical conditions using as oligonucleotide pairs 5 0 -GCACTTGGGCAGTGACCTT-3 0 and 5 0 -GTGGTGGG- TAGGCACGCT-3 0 , and 5 0 -CAGCAAATCATCTGTATACCCT-3 0 and 5 0 -CCCAAAGCTTGGAGGCAACT-3 0 . All oligonucleotides hybridized in intronic sequences enabling the amplification of the entire exon and a part of the flanking introns including splice sites. Single-strand conformation polymorphism (SSCP) analysis. Genotyping of Del and HPA-3 was performed as described (Peyruchaud et al, 1995) and identical conditions were used for amplification product 30. For amplification product 22 British Journal of Haematology , 1998, 102, 622–625 622 q 1998 Blackwell Science Ltd Correspondence: Dr F. Bourre, UMR 5533 CNRS, Ho ˆpital Cardio- logique, Avenue de Magellan, 33604 Pessac, France.

Upload: ruan

Post on 06-Jul-2016

212 views

Category:

Documents


0 download

TRANSCRIPT

Page 1: Linkage of four polymorphisms on the αIIb gene

SHORT REPORT

Linkage of four polymorphisms on the aIIb gene

J. RUAN, O. PEYRUCHAUD, A. NURDEN AND F. BOURRE UMR 5533 CNRS, Hopital Cardiologique, Pessac, France

Received 9 April 1998; accepted for publication 23 April 1998

Summary. The subunits of the platelet integrin aIIbb3 areencoded by two genes located on chromosome 17. Twopathologies are associated with structural modifications ofthis complex: Glanzmann’s thrombasthenia and alloimmunethrombocytopenia. The former is a hereditary bleedingdisorder, the latter is due to an immune response linked tothe presence of specific epitopes defined by single amino acidsubstitutions called human platelet alloantigen (HPA)systems. Analysing the aIIb gene from 112 independent

chromosomes, we have defined two new silent polymorph-isms in complete linkage disequilibrium. They are recipro-cally linked to HPA-3 and a previously reported 9 pb deletionin intron 21. Linkage of these four DNA markers spanning a5 kb fragment of genomic DNA provides a new tool foranalysing aIIb gene pathology and evolution.

Keywords: integrin aIIbb3, genetic polymorphisms, HPA-3b(Bakb), Glanzmann’s thrombasthenia.

The human integrin aIIbb3, specific for platelets andmegakaryocytes, mediates platelet aggregation through itscapacity to bind fibrinogen. Major pathologies associatedwith this receptor are Glanzmann’s thrombasthenia, anautosomal recessive bleeding syndrome, characterized by afunctional or quantitative defect of aIIbb3 (George et al,1990), and thrombocytopenia due to the destruction ofplatelets secondary to an immune response after bloodtranfusion or feto-maternal immunization (Kunicki &Newman, 1992). The latter is due to single amino acidsubstitutions that give rise to the so-called human plateletalloantigen (HPA) systems. Several HPA systems have beenlocalized to aIIbb3 (see Newman & Valentin, 1995). Amongthem, HPA-3 (Lyman et al, 1990) and Max (Noris et al, 1995)are located on the aIIb gene located on chromosome 17. Thecoding part is contained within 30 exons (Heidenreich et al,1990) giving rise to a 140 kD protein which is assembledwith the b3 subunit to form a complex which undergoesmaturation in the endoplasmic reticulum and the Golgiapparatus of megakaryocytes (Duperray et al, 1989).

The HPA-3 system is defined by the presence of a I843Samino acid substitution (HPA-3a and HPA-3b respectively)due to a 2622T → G mutation and Max by the presence of aV837M amino acid substitution (Maxa– and Maxaþ respec-tively) due to a 2603G → A mutation (Lyman et al, 1990;Noris et al, 1995). We have previously reported that the HPA-3b genotype is bilaterally linked to a 9 bp deletion (Del)within intron 21 on the aIIb gene (Peyruchaud et al, 1995).We now describe two new polymorphisms in the aIIb gene

(IVS21(–7)C → G and 3063C → T) and show their completelinkage disequilibrium with HPA-3. Indeed, the frequency ofDel¹, IVS21(¹7)C, HPA-3a and 3063C is exactly the same:0·74 (0·26 for the alternative form). To our knowledge, it isthe first time that four DNA polymorphisms have beenreported to be reciprocally linked on a gene coding for anintegrin subunit.

MATERIALS AND METHODS

Subjects and DNA amplification. Blood from 47 unrelatedCaucasian subjects of European origin and nine unrelatedpatients with Glanzmann’s thrombasthenia was taken intoEDTA anticoagulant after informed consent. DNA wasprepared using a QIAamp blood and tissue kit (QIAGENInc., Chatsworth, Calif., U.S.A.) according to the manufac-turer’s instructions. Amplification products 21 (containingexon 21) and 26 (containing exon 26 and consequently thegenetic determinant for HPA-3) were amplified as alreadydescribed (Peyruchaud et al, 1995). Amplification products22 (containing exon 22) and 30 (containing exon 30) wereamplified under identical conditions using as oligonucleotidepairs 50-GCACTTGGGCAGTGACCTT-30 and 50-GTGGTGGG-TAGGCACGCT-30, and 50-CAGCAAATCATCTGTATACCCT-30

and 50-CCCAAAGCTTGGAGGCAACT-30. All oligonucleotideshybridized in intronic sequences enabling the amplificationof the entire exon and a part of the flanking introns includingsplice sites.

Single-strand conformation polymorphism (SSCP) analysis.Genotyping of Del and HPA-3 was performed as described(Peyruchaud et al, 1995) and identical conditions were usedfor amplification product 30. For amplification product 22

British Journal of Haematology, 1998, 102, 622–625

622 q 1998 Blackwell Science Ltd

Correspondence: Dr F. Bourre, UMR 5533 CNRS, Hopital Cardio-logique, Avenue de Magellan, 33604 Pessac, France.

Page 2: Linkage of four polymorphisms on the αIIb gene

we used the same protocol with slight modifications: a 20%gel and electrophoresis at 48C for 200 Vh were used. The gelswere stained with silver nitrate (Peyruchaud et al, 1995).

DNA sequencing. Amplified fragments were sequenced

directly using the fmolTM DNA Sequencing System (Pro-mega-France, Lyon), the oligonucleotides being 50-radio-labelled with ATPg 33P (IsotopChim, Ganagobie, Peyruis,France) according to the manufacturer’s instructions.

623Short Report

q 1998 Blackwell Science Ltd, British Journal of Haematology 102: 622–625

Fig 1. SSCP analysis and sequence of amplification products 22 and 30 from the aIIb gene. (A) Amplification product 22 from the aIIb gene wassubjected to SSCP analysis on 20% polyacrylamide minigels. Three typical SSCP profiles are shown, band a was seen in lanes 1 and 2, and band bwas seen in lanes 2 and 3. The corresponding sequences are shown and the mutation is indicated, as is the splice site. (B) Amplification product30 was analysed as described for product 22 in (A) with the exception that the SSCP analysis was performed using a 12·5% gel.

Page 3: Linkage of four polymorphisms on the αIIb gene

Allele-specific restriction enzyme analysis (ASRA). ForASRA, PCR amplification products were purified using PCRPreps DNA Purification Resin and Wizard minicolumnaccording to the manufacturer’s instructions (Promega-France). The BstNI restriction enzyme (New EnglandBiolabs, Beverly, Mass.) was used to control the 9 bp deletionin intron 21 of the aIIb gene (Peyruchaud et al, 1995) andHPA-3 was genotyped using the FokI restriction enzyme(New England Biolabs) as described (Simsek et al, 1993).

RESULTS AND DISCUSSION

When screening all the exons and intron/exon boundaries ofthe aIIb gene from Glanzmann’s thrombasthenia patients byPCR-SSCP, we observed inter-subject variability in the SSCPprofiles for amplification products containing exons 21, 22, 26and 30. Although initially seen for two out of nine patients, analtered migration of these amplification products were also seenin SSCP analysis for control donors. Although the results foramplification products 21 and 26 were due to a 9 bp deletion inintron 21 and the HPA-3 system (Peyruchaud et al, 1995),altered migrations for the products containing exons 22 and 30were unexplained. Typical SSCP patterns for amplificationproducts 22 and 30 are illustrated in Figs 1A and 1B.

As polymorphisms have not been described in thesedomains of the aIIb gene, amplification products 22 and 30were directly sequenced (Figs 1A and 1B). A single pointmutation, C to G, located in intron 21, 7 bp upstream fromthe exon 22 start codon and noted IVS21(¹7)C → Gaccording to standard nomenclature (Ad Hoc Committeeon Mutation Nomenclature, 1996) was present. The threegenotypes C/C, C/G and G/G corresponded to the SSCPprofiles seen in Fig 1A (panels 1, 2 and 3 respectively). Fig 1Bshows part of the sequence corresponding to amplificationproduct 30 from individuals with each of the three SSCPprofiles. A single point mutation, C to T was located on thethird nucleotide of exon 30, noted 3063C → T. The threegenotypes C/C, C/T and T/T corresponded to SSCP profilesseen in Fig 1B (panels 1, 2 and 3 respectively). Analysing112 independent chromosomes using SSCP analysis andASRA for the Del and HPA-3 polymorphisms, we found thatthe allelic frequencies of IVS21(¹7)C, Del¹, HPA-3a and3063C were exactly the same: 0·74 (0·26 for theiralternative forms). All alleles that were genotyped as Del¹

and HPA-3a carried IVS21(¹7)C and 3063C polymorph-isms, whereas alleles genotyped as Delþ, HPA-3b werealways IVS21(¹7)G and 3063T. These findings showedclearly that IVS21(¹7)C, Del–, HPA-3a and 3063C are incomplete linkage disequilibrium, this was confirmed by x2

analysis (P<0·001). The calculated gene frequency for theHPA-3b polymorphism in our study group was slightly lowerthan the value of 0·36 reported in the literature for HPA-3bin a Caucasian population (Newman & Valentin, 1995). Thisprobably reflects the relatively small size of our population.

Although the IVS21(¹7)C → G polymorphism created aGT dinucleotide, a potential donor splice site, 5 bp upstreamfrom the AG acceptor splice site of intron 21, its utilizationduring RNA maturation and its contribution to theGlanzmann’s phenotype is unlikely. Only one of nine

unrelated Glanzmann’s patients was homozygousIVS21(¹7)G, Delþ, HPA-3b and 3063T and only one washeterozygous for the four markers. HomozygosityIVS21(¹7)G, Delþ, HPA-3b and 3063T was also found inthree normal individuals. However, to ensure that it was notused as an alternative splice site, we performed RT-PCR fromplatelets previously genotyped IVS21(¹7)C, IVS21(¹7)C/Gand IVS21(¹7)G. The cDNA region encompassing exon 21and exon 22 was then directly sequenced. The cDNAsequence was the same whatever the genotype, indicatingthat IVS21(¹7)C → G was not used as a splice site (data notshown). The polymorphism 3063C → T does not induce anamino acid substitution. As far as we could ascertain,individuals typed IVS21(¹7)G/Delþ/HPA-3b/3063T possessplatelets with a normal aIIbb3 content as assessed by flowcytometry and a normal aggregation response to the majoragonists (data not shown). This situation differs from thatseen for the integrin a2b1 where two linked silentpolymorphisms in the a2 gene coding sequence areassociated with major variations in integrin density onplatelets (Kunicki et al, 1997).

As reported by Noris et al (1995), the Maxaþ polymorph-ism is seen only in individuals who are HPA-3b but wasobserved in only three out of 500 individuals. Maxaþ was notseen in any of our subjects. It has been proposed that theancestral gene for aIIb may be that which carries HPA-3b(Poncz & Newman, 1990; Newman & Valentin, 1995).We have noted that a strong homology exists between the9 bp Delþ (11995CAGGGGCTC) and the sequence11983CCGGGTCTCCAC just upstream in the same intron,suggesting that this polymorphism has possibly occurredthrough a zip replication mechanism which would explainthe duplication and thereby reinforce the hypothesis of theHPA-3b allele as the ancestral gene. Comparing thesequences surrounding Del and 3063C → T, no obvioushomology was observed between them and it is difficult tospeculate on whether a common mechanism is responsiblefor their formation. It should be noted if HPA-3b doesrepresent the ancestral gene then our reported 9 bp deletionin intron 21 is, in fact, an addition.

Although the HPA-3b allele is more frequent in Asianpopulations, it is less frequent in Caucasians (see Newman &Valentin, 1995). This probably implies the penetration ofspecific aIIb alleles during the migration of populations.Overall, our work showed that four DNA polymorphismsspanning a 5 kbp of the aIIb gene are in linkage disequili-brium. They will be useful markers for family studies inGlanzmann’s thrombasthenia patients linked to abnormal-ities within the aIIb gene and for gene tracking studies.

ACKNOWLEDGMENTS

This work was supported by funding from the CNRS,Universite de Bordeaux II, the Conseil Regional d’Aquitaineand the Ministere de l’Enseignement Superieur et de laRecherche (ACC-SV No. 9). O. Peyruchaud was a recipient ofa postdoctoral fellowship from the Societe Francaised’Hematologie. J. Ruan received a doctoral grant from theSanofi Association for Thrombosis Research.

624 Short Report

q 1998 Blackwell Science Ltd, British Journal of Haematology 102: 622–625

Page 4: Linkage of four polymorphisms on the αIIb gene

REFERENCES

Ad Hoc Committee on Mutation Nomenclature (1996) Update onnomenclature for human gene mutations. Human Mutation, 8,197–202.

Duperray, A, Troesch, A. & Berthier, R. (1989) Biosynthesis andassembly of platelet GP IIb–IIIa in human megakaryocytes:evidence that assembly between pro-GP IIb and GP IIIa is aprerequisite for expression of the complex on the cell surface.Blood, 74, 1603–1611.

George, J.N., Caen, J.P. & Nurden, A.T. (1990) Glanzmann’sthrombasthenia: the spectrum of clinical disease. Blood, 75,1383–1395.

Heidenreich, R., Eisman, R., Surrey, S., Delgrosso, K., Bennet, J.S.,Schwartz, E. & Poncz, M. (1990) Organization of the gene forplatelet glycoprotein IIb. Biochemistry, 29, 1232–1244.

Kunicki, T.J., Kritzik, M., Annis, D.S. & Nugent, D.J. (1997)Hereditary variation in platelet integrin a2b1 density is associatedwith two silent polymorphisms in the a2 gene coding sequence.Blood, 89, 1939–1943.

Kunicki, T.J. & Newman, P.J. (1992) Molecular immunology ofhuman platelet proteins. Blood, 80, 1386–1404.

Lyman, S., Aster, S.H., Visentin, G.P. & Newman, P.J. (1990)Polymorphism of human platelet membrane glycoprotein IIb

associated with the Baka/Bakb alloantigen system. Blood, 75,2343–2348.

Newman, P.J. & Valentin, N. (1995) Human platelet alloantigens:recent findings, perspectives. Thrombosis and Haemostasis, 74,234–239.

Noris, P., Simsek, S., de Bruijne-Admiraal, L.G., Porcelijn, L., Huiskes,E., van der Vlist, G.J., van Leeuwen, E.F., van der Schoot, C.E. &von dem Borne, A.E.G.Kr. (1995) Maxa a new low-frequencyplatelet-specific antigen localized on glycoprotein IIb, is associatedwith neonatal alloimmune thrombocytopenia. Blood, 86, 1019–1026.

Peyruchaud, O., Nurden, A.T. & Bourre, F. (1995) Bilateral linkagebetween a new deletion polymorphism in intron 21 of the GPIIbgene and the HPA-3b (Bakb) determinant. British Journal ofHaematology, 91, 747–751.

Poncz, M. & Newman, P.J. (1990) Analysis of rodent plateletglycoprotein IIb: evidence for evolutionarily conserved domainsand alternative proteolytic processing. Blood, 75, 1282–1289.

Simsek, S., Faber, N.M., Bleeker, P.M., Vlekke, A.B.J., Goldscheming,R. & von dem Borne, A.E.G.Kr. (1993) Determination of humanplatelet antigen frequencies in the Dutch population by immuno-phenotyping and DNA (allele-specific restriction enzyme) analysis.Blood, 8, 835–840.

625Short Report

q 1998 Blackwell Science Ltd, British Journal of Haematology 102: 622–625