Polymorphism of the Human CD46 Gene in Normal Individuals and in Recurrent Spontaneous Abortion Janet M. Risk, Brian F. Flanagan, and Peter M. Johnson

ABSTRACT: Restriction fragment length polymorphism analysis of the human CD46 gene, which encodes a cell surface complement regulatory protein, has been performed using a 1.5-kh eDNA probe containing a complete CD-16 coding sequence. The sum of the invariant band sizes indicates that this gene encompasses at least 35-kb genomic DNA. Polymorphisms were detected in normal individuals with both Hindlll and EcoRl. Several ABBREVIATIONS DAF decay accelerating factor mAb monoclonalantibody MCP membrane cofactor protein PBMC peripheral blood mononuclear cells

of the polymorphic bands occurred significantly less frequently in recurrent spontaneOus abortion individuals. Northern blot analysis has shown the predominant RNA transcrip[ size to be 4.2 kh in trophoblast.derived cell lines and peripheral blood mononuclear cells, indicating these transcripts may contain an unusually long 3'untranslated region. Haman Immunology 30, I62-167 (1991)

RFLP

RSA SDS TLX

restriction fragment length polymorphism recurrent spontaneous abortion sodium dodecylsulphate trophoblast-leukocyte common antigen

INTRODUCTION The human CD46 glycoprotein, defined by the E4.3 monoclonal antibody (mAb) to the HuLy-m5 leukocyte antigen [1], has recer.-', been shown by cross-blocking and sequential immunoprecipimrion studies to be identical both to the membrane cofactor protein (MCP) of the complement system and to the trophoblast-leukocyte common (TLX) antigen defined by the H316 mAb [2]. These independently described cell surface transmembrane antigen systems show the same wide tissue distribution and molecular weight heterogeneity on iso" larion from different tissues and cell lines. The human CD46 antigen consists of two nonassociated acidic sialylared glycoproteins of around 55 kD and 65 kD that are structurally and functionally similar, although the rela-

From the Department of Immunology, Unh~rsity of Lh,erpool. Literpool, England. Address reprint request to Professor P. M. Johnson. Department of Immunology, Unirersity of Lh,erpool. P.O. Box 147. Liferpool/,69 3BX, En~a.d. R¢ceiredMay 23, 1990; acceptedSeptembar 7, 1990. 162 0198-8859191153.50

tive proportion of these two major isoforms may vary between individuals [3, 4]. The H31 &defined TLX antigen system is expressed by all human placental trophoblast and peripheral blood leukocyte populations as well as a variety of other cell types, including endometrial gland epithelium and acrosome-reacted sperm [5-7]. MCP has cofactor activity for the factor I-mediated proteolytic inactivation of the C3b and C4b complement convertases, and thus its cell surface expression will afford increased protection from complement-mediated cytolysis [8]. The human MCP gene has been localized to the chromosome lq32 segment, where it is a single copy gene within a multigene cluster encoding structurally related complement regulatory proteins [9, 10]. The identification of H316.defined TLX as CD46, and thus MCP, has strongly implied that a major function of this cell surface antigen is to down-regulate complement activation at the maternal tissue and blood interface with fetal trophoblast in extraembryonie tissues during pregnancy, probably acting together with decay HumanImmunology30, 162-167(199D O AmericanSocietyfor Hist~omparibilityand lmmunogenedcs.1991

CD46 Gene Polymorphism

accelerating factor (DAF; CD55) [ I 1]. Absent or aberrant activity could presumably result in fetal trophoblast being more susceptible to complement-mediated damage. It was of interest, therefore, to investigate major polymorphisms of the CD46 gene that may exist in the normal population or in the pregnancy disorder, unexplained recurrent spontaneous abortion (RSA), together with the expression of this gene in different human trophoblast-derived cell lines. A possible immunngenetic background to RSA cases has been the subject of much unresolved attention [ l 2, 13]. Previous studies have shown no unusual restriction fragment length polymorphism (RFLP) in unexplained RSA compared with normal controls for the TNF~, C4, HLA-B, and class 11 MHC a-chain genes [14], as well as the HLA-C geue locus (Risk et al., unpublished), within the MHC region.

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enzyme concentration of 5 to 10 U/tzg DNA. Fragmented DNA was electrophoresed on a submerged horizontal O.Tt/] agarose gel at 3 V/cm for 16 h. DNA was then transferred onto a nylon support medium (Amersham Hybond N) by the method of Southern [18]. Hybridization was performed at 42°C in 50~ deionized formamide, 5 x SSC) (20 x standard sodium citrate (SSC) = 3 M NaCI/0.3 M trisedium citrate), 25 mM sodium phosphate buffer pH 6.5, 5 x Denhardt's solution (100 x Denhardt's = 2rYi bovine serum albumin/2% polyvinyl pyrrolidone/2% FicollL 0.5% sodium dodecyl sulphate (SDS), 12.5% dextran sulphate containing 150 v.g/ml sonicated salmon testes DNA, and 5 x l0 s dpm/ml labeled probe. Hybridized filters were washed three times for 30 min at 68°C in 0.1 × SSC/0.1% SDS before automdiography at -70°C fi)r 2 days.

Northern blots. Total RNA was prepared from the METHODS

Patients and controls. Peripheral blood mononuclear cells (PBMC) were obtained from 17 healthy unrelated mer~bers oftbe laboratory staff(9 male, 4 female), eight of whom had children and none with any history of spontaneous abortions, as well as from one or both members of 18 unrelated couples suffering unexplained RSA. This latter group had three or more consecutive confirmed first-trimester spontaneous abortions+ each by a single partner, with either no live birth (n = 13) or a single live birth from the first pregnancy (n = 5). All known causes of RSA had been excluded by laboratory and clinical investigation [ 15 ]. DNA was isolated from PBMC by the method of Blin and Stafford [ 16].

BeWo and JAr cell lines, and isolated PBMC from normal individuals, using the guanidinium isothiocyanate/ cesium chloride method [ 19]. Aliquots of 20 v-g were electrophoresed on a 1.5% agarose gel containing formaldehyde to a final concentration of 2.2 M, and transferred to nylon filters (Amersham Hybond N) [20]. These were hybridized overnight at 42°C in 50% deionized formamide, 5 x standard sodium phosphate EDTA (SSPE) (20 x SSPE = 3 M NaCI/O.2M sodium dihydrogen orthophospham/20 mM EDTA), 5 x Denhardt's solution containing 20 v.g/ml yeast tRNA, and 5 x 10s dpm/ml labeled probe. Hybridized filters were again washed at 68°C in 0.1 x SSC/0.1~ SDS before autoradiography.

Statistical analysis. This was performed by X-' test for

Celllines. The BeWo and JAr human choriocarcinoma

paired comparisons (n - I degrees of freedom) with Yams" continuity correction when appropriate.

cell lines were maintained in RPMI medium

Polymorphism of the human CD46 gene in normal individuals and in recurrent spontaneous abortion.

Restriction fragment length polymorphism analysis of the human CD46 gene, which encodes a cell surface complement regulatory protein, has been perform...
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