Identification of a Homozygous Single Base Pair Deletion in the Gene Coding for the Human Platelet Glycoprotein Ibα Causing Bernard-Soulier Syndrome

S Simsek; L G Admiraal; P W Modderman; C E van der Schoot; A E G Kr von dem Borne

doi:10.1055/s-0038-1648887

Thrombosis and Haemostasis, Table of Contents

Thromb Haemost 1994; 72(03): 444-449
DOI: 10.1055/s-0038-1648887

Original Article

Schattauer GmbH Stuttgart

Identification of a Homozygous Single Base Pair Deletion in the Gene Coding for the Human Platelet Glycoprotein Ibα Causing Bernard-Soulier Syndrome

Authors

S Simsek

¹The Department of Immunologic Haematology, Central Laboratory of The Netherlands Red Cross Blood Transfusion Service and Laboratory for Clinical and Experimental Immunology, University of Amsterdam, Netherlands
L G Admiraal

¹The Department of Immunologic Haematology, Central Laboratory of The Netherlands Red Cross Blood Transfusion Service and Laboratory for Clinical and Experimental Immunology, University of Amsterdam, Netherlands
P W Modderman

¹The Department of Immunologic Haematology, Central Laboratory of The Netherlands Red Cross Blood Transfusion Service and Laboratory for Clinical and Experimental Immunology, University of Amsterdam, Netherlands
C E van der Schoot

¹The Department of Immunologic Haematology, Central Laboratory of The Netherlands Red Cross Blood Transfusion Service and Laboratory for Clinical and Experimental Immunology, University of Amsterdam, Netherlands
A E G Kr von dem Borne

¹The Department of Immunologic Haematology, Central Laboratory of The Netherlands Red Cross Blood Transfusion Service and Laboratory for Clinical and Experimental Immunology, University of Amsterdam, Netherlands

²Department of Haematology, Academic Medical Center, University of Amsterdam, The Netherlands

Abstract

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Summary

Bernard-Soulier Syndrome (BSS) is a hereditary bleeding disorder which is caused by the absence or the dysfunction of the platelet glycoprotein Ib/IX/V (GP Ib/IX/V) complex, the major receptor for von Willebrand factor (vWf). BSS is characterized by the presence of giant platelets that show a reduced binding of vWf. Although BSS is a well-characterized disease, and many cases have been described in the literature, the molecular genetic basis of this disorder has been studied in only a few patients.

We have studied the genetic basis of the defect in a BSS patient. Flow cytometric analysis of the platelet membrane glycoproteins revealed a significant decrease or absence of GP Ibα on the platelet surface, and low levels of GP V and GP IX. In subsequent immunopre-cipitation experiments, we confirmed the presence of GP V (although in significantly decreased amounts) on the platelet surface. These results indicated a defect in the GP Ibα chain

Genomic DNA coding for GP Ibα was amplified, using the polymerase chain reaction (PCR). Subsequent direct sequence analysis demonstrated a homozygous deletion of T³¹⁷ resulting in a frameshift deletion and predicting a substitution of Arg for Leu⁷⁶. This deletion causes a shift in the reading frame, predicting a premature stop codon after 19 altered amino-acids, leading to a severily truncated molecule. The molecular genetic defect found in this patient differed from the mutations observed in three other BSS patients described in the literature. This points to a marked hetereogeneity of this disease.

The single basepair deletion created a target site for the restriction enzyme Hhal. This allowed us to perform PCR-ASRA (Allele-Specific Restriction enzyme Analysis) on all available family members. Both parents and the daughter of the patient appeared to be heterozygous for the deletion, while the homozygosity of the propositus for the mutant allele was confirmed.

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References

References
1 Hynes RO. Integrins: A family of cell surface receptors. Cell 1987; 48: 549
2 Philips DR, Charo IF, Parise LV, Fitzgerald LA. The platelet membrane glycoprotein IIb-IIIa complex. Blood 1988; 71: 831
3 Pytela R, Pierschbacher MD, Ginsberg MH, Plow EF, Ruoshlati E. Platelet membrane glycoprotein IIb/IIIa: Member of a family of Arg-Gly-Asp-specific adhesion receptors. Science 1986; 238: 491
4 Okita JR, Pidard D, Newman PJ, Montgomery RR, Kunicki TJ. On the association of glycoprotein Ib and actin-binding protein in human platelets. J Cell Biol 1985; 100: 317-321
5 Fox JE, Boyles JK, Berndt MC, Steffen PK, Anderson LK. Identification of a membrane skeleton in platelets. J Cell Biol 1988; 106: 1525-1538
6 Andrews RK, Fox JE. Identification of a region in the cytoplasmic domain of the platelet membrane glycoprotein Ib-IX complex that binds to purified actin-binding protein. J Biol Chem 1992; 267: 18605-18611
7 Du X, Beutler L, Ruan C, Castaldi PA, Berndt MC. Glycoprotein lb and glycoprotein IX are fully complexed in the intact platelet membrane. Blood 1987; 69: 1524-1527
8 Roth GJ, Church TA, McMullen BA, Williams SA. Human platelet glycoprotein V: a surface leucine-rich glycoprotein related to adhesion. Biochem Biophys Res Commun 1990; 170: 153-161
9 Modderman PW, Admiraal LG, Sonnenberg A, von dem Borne AEGKr. Glycoproteins V and Ib-IX form a noncovalent complex in the platelet membrane. J Biol Chem 1992; 267: 364
10 Shimomura T, Fujimura K, Maehama S, Takemoto M, Oda K, Fujimoto T, Oyama R, Suzuki M, Ichihara Tanaka K, Titani K. et al Rapid purification and characterization of human platelet glycoprotein V: the amino acid sequence contains leucine-rich repetitive modules as in glycoprotein Ib. Blood 1990; 75: 2349-2356
11 Clemetson KJ, Wenger RH, Clemetson JM, Drouin J. Biochemistry and molecular biology of the platelet membrane glycoprotein Ib complex and the Bernard-Soulier syndrome. In: Platelet Immunology: fundamental and clinical aspects edited by Kaplan-Gouet C, Schlegel N, Salmon Ch, McGregor J. Paris: Colloque INSERM/John Libbey Eurotext Ltd; 1991: 19-30
12 Roth GJ. Developing relationships: arterial platelet adhesion, glycoprotein Ib, and leucine-rich glycoproteins. Blood 1991; 77: 5-19
13 Handa M, Titani K, Holland LZ, Roberts JR, Ruggeri ZM. The von Wille-brand factor-binding domain of platelet membrane glycoprotein Ib. Characterization by monoclonal antibodies and partial amino acid sequence analysis of proteolytic fragments. J Biol Chem 1986; 261: 12579-12585
14 Titani K, Takio K, Handa M, Ruggeri ZM. Amino acid sequence of the von Willebrand factor-binding domain of platelet membrane glycoprotein Ib. Proc Natl Acad Sci USA 1987; 84: 5610-5614
15 Vicente V, Kostel PJ, Ruggeri ZM. Isolation and functional characterization of the von Willebrand factor-binding domain located between residues Hisl-Arg293 of the alpha-chain of glycoprotein Ib. J Biol Chem 1988; 263: 18473-18479
16 Vicente V, Houghten RA, Ruggeri ZM. Identification of a site in the alpha chain of platelet glycoprotein Ib that participates in von Willebrand factor binding. J Biol Chem 1990; 265: 274-280
17 Miller JL, Cunningham D, Lyle VA, Finch CN. Mutation in the gene encoding the alpha chain of platelet glycoprotein Ib in platelet-type von Willebrand disease. Proc Natl Acad Sci USA 1991; 88: 4761-4765
18 Russell SD, Roth GJ. Pseudo-von Willebrand Disease: A mutation in the Platelet Glycoprotein Ibα Gene Associated With a Hyperactive Surface Receptor. Blood 1993; 81: 1787-1791
19 Bernard J, Soulier JP. Sur une nouvelle varieté de dystrophie thrombocy-taire-hemorrhagipare congénitale. Seinin Hop Paris 1948; 3217
20 Clemetson KJ, McGregor JL, James E, Dechavanne M, Luscher EF. Characterization of the platelet membrane glycoprotein abnormalities in Bernard-Soulier syndrome and comparison with normal by surface-labeling techniques and high-resolution two-dimensional electrophoresis. J Clin Invest 1982; 70: 304
21 Nurden AT, Didry D, Rosa JP. Molecular defects of platelets in Bernard–Soulier syndrome. Blood Cells 1983; 9: 333-358
22 Nurden AT, Dupuis D, Kunicki TJ, Caen JP. Analysis of the glycoprotein and protein composition of Bernard-Soulier platelets by single two-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis. J Clin Invest 1981; 67: 1431
23 Lopez JA, Chung DW, Fujikawa K, Hagen FS, Papayannopoulou T, Roth GJ. Cloning of the alpha chain of human platelet glycoprotein Ib: a transmembrane protein with homology to leucine-rich alpha 2-glycoprotein. Proc Natl Acad Sci USA 1987; 84: 5615-5619
24 Lopez JA, Chung DW, Fujikawa K, Hagen FS, Davie EW, Roth GJ. The alpha and beta chains of human platelet glycoprotein Ib are both transmembrane proteins containing a leucine-rich amino acid sequence. Proc Natl AcadSci USA 1988; 85: 2135-2139
25 Hickey MJ, Williams SA, Roth GJ. Human platelet glycoprotein IX: an adhesive prototype of leucine-rich glycoproteins with flank-center-flank structures. Proc Natl Acad Sci USA 1989; 86: 6773-6777
26 Hickey MJ, Deaven LL, Roth GJ. Human platelet glycoprotein IX: Characterization of cDNA and localization of the gene to chromosome 3. FEBS Letters 1990; 274: 189-192
27 Wenger RH, Kieffer N, Wicki AN, Clemetson KJ. Structure of the human blood platelet membrane glycoprotein Ib alpha gene. Biochem Biophys Res Commun 1988; 156: 389-395
28 Finch CN, Miller JL, Lyle VA, Handin RI. Evidence that an abnormality in the glycoprotein ib alpha gene is not the cause of abnormal platelet function in a family with classic Bernard-Soulier Disease. Blood 1990; 75: 2357-2362
29 Ware J, Russell SR, Vicente V, Scharf RE, Tomer A, McMillan R, Ruggeri ZM. Nonsense mutation in the glycoprotein Ib alpha coding sequence associated with Bernard-Soulier syndrome. Proc Natl Acad Sci USA 1990; 87: 2026-2030
30 Miller JL, Lyle VA, Cunningham D. Mutation of leucine-57 to phenylalanine in a platelet glycoprotein Ib alpha leucine tandem repeat occurring in patients with an autosomal dominant variant of Bernard-Soulier disease. Blood 1992; 79: 439-446
31 Ware J, Russel SR, Marchese P, Murata M, De Marco L, Ruggeri ZM. Point mutation in a leucine-rich repeat of platelet glycoprotein Ibα resulting in the Bernard-Soulier Syndrome. J Clin Invest 1993; 92: 1213-1220
32 Wright SD, Michaelides K, Johnson DJD, West NC, Tuddenham EGD. Double heterozygosity for mutations in the platelet glycoprotein IX gene in three siblings with Bernard-Soulier Syndrome. Blood 1993; 81: 2339-2347
33 von dem Borne, AEGKr A, Modderman PW, Admiraal LG, Nieuwenhuis HK. Joint report of the Platelet Section. In: Leucocyte typing IV: White Cell Differentiation Antigens edited by Knapp W, Dörken B, Gilks WR. et al Oxford: Oxford University Press; 1989
34 von dem BorneAEGKr, Verheugt FWA, Oosterhof F. A simple immunofluorescence test for the detection of platelet antibodies. Brit J Haematol 1978; 61: 374
35 Fraker PJ, Speck JC. Protein and cell membrane iodinations with sparingly soluble chloramide-l,2,4,6-tetrachloro-3α,6α-diphenyl glycouril. Biochem Biophys Res Commun 1980; 4: 849-857
36 Laemmli UK. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 1970; 227: 680-685
37 Ciulla TA, Sklar RM, Hauser SL. A simple method for DNA purification from peripheral blood. Anal Biochem 1988; 174: 458
38 de Boer M, Bolscher BGJM, Dinauer MC, Orkin SH, Smith CIE, Ählin A, Weenings RS, Roos D. Splice site mutations are a common cause of X-linked granulomatous disease. Blood 1992; 80: 1553
39 Lopez JA, Ludwig EH, McCarthy BJ. Polymorphism of human glycoprotein Iba results from a variable number of tandem repeats of a 13-amino acid sequence in the mucin-like macroglycopeptide region. J Biol Chem 1992; 267: 10055-10061
40 Coller BS, Peerschke El, Scudder LE, Sullivan CA. Studies with a murine monoclonal antibody that abolishes ristocetin-induced binding of von Willebrand factor to platelets: additional evidence in support of GPIb as a platelet receptor for von Willebrand factor. Blood 1983; 61: 99-110
41 Ruggeri ZM, De Marco L, Gatti L, Bader R, Montgomery RR. Platelets have more than one binding site for von Willebrand factor. J Clin Invest 1983; 72: 1-12
42 Katagiri Y, Hayashi Y, Yamamoto K, Tanoue K, Kosaki G, Yamazaki H. Localization of von Willebrand factor and thrombin-interactive domains on human platelet glycoprotein Ib. Thromb Haemost 1990; 19 63: 122-126
43 Lopez JA, Weisman SM, Li CQ. The “complex-specific” GP Ib-IX monoclonal antibody, SZ1, does not require GP Ibα for its epitope. Thromb Haemost 1993; 69: 1153 (Abstract)
44 Meyer S, Kresbach G, Häring P, Schumpp-Vonach B, Clemetson KJ, Hadváry P, Steiner B. Expression and characterization of functionally active fragments of the platelet glycoprotein (GP) Ib-IX complex in mammalian cells incorporation of GP Iba into the cell surface membrane. J Biol Chem 1993; 268: 20555-20562
45 Lopez JA, Leung B, Reynolds CC, Li CQ, Fox JEB. Efficient plasma membrane expression of a functional platelet glycoprotein Ib-IX complex requires the presence of its three subunits. J Biol Chem 1992; 267: 12851-12859
46 Rosa J-P, McEver RP. Processing and assembly of the integrin, glycoprotein IIb-IIIa, in HEL cells. J Biol Chem 1989; 264: 12596-125603
47 O’Toole TE, Loftus JC, Plow EF, Glass AA, Harper JR, Ginsber MH. Efficient surface expression of platelet GP IIb-IIIa requires both subunits. Blood 1989; 74: 14-18
48 Duperray A, Troesch A, Berhier R, Chagnon E, Frachet P, Uzan G, Mar-guerie G. Biosynthesis and assembly of platelet GP IIb-IIIa in human megakaryocytes. Evidence that assembly between pro-GP IIb and GP IlIa is a prerequisite for expression of the complex on the cell surface. Blood 1989; 74: 1603-1611
49 George JN, Cean JP, Nurden AT. Glanzmann’s thrombasthenia: the spectrum of clinical disease. Blood 1990; 75: 1383-1395
50 Arnaout MA. Structure and Function of the leucocyte adhesion melecules CD11/CD18. Blood 1990; 75: 1037-1050