RESUMEN
Glanzmann thrombasthenia (GT) is a rare autosomal recessive bleeding disorder characterized by impaired platelet aggregation due to defects in integrin αIIbß3, a fibrinogen receptor. Platelet phenotypes and allelic variations in 28 Turkish GT patients are reported. Platelets αIIbß3 expression was evaluated by flow cytometry. Sequence analyzes of ITGA2B and ITGB3 genes allowed identifying nine variants. Non-sense variation effect on αIIbß3 expression was studied by using transfected cell lines. 3D molecular dynamics (MDs) simulations allowed characterizing structural alterations. Five new alleles were described. αIIb:p.Gly423Asp, p.Asp560Ala and p.Tyr784Cys substitutions impaired αIIbß3 expression. The αIIb:p.Gly128Val substitution allowed normal expression; however, the corresponding NM_000419.3:c.476G>T variation would create a cryptic donor splicing site altering mRNA processing. The ß3:p.Gly540Asp substitution allowed αIIbß3 expression in HEK-293 cells but induced its constitutive activation likely by impairing αIIb and ß3 legs interaction. The substitution alters the ß3 I-EGF-3 domain flexibility as shown by MDs simulations. GT variations are mostly unique although the NM_000419.3:c.1752 + 2 T > C and NM_000212.2:c.1697 G > A variations identified in 4 and 8 families, respectively, might be a current cause of GT in Turkey. MD simulations suggested how some subtle structural variations in the ß3 I-EGF domains might induce constitutive activation of αIIbß3 without altering the global domain structure.
Asunto(s)
Integrina alfa2 , Integrina beta3 , Complejo GPIIb-IIIa de Glicoproteína Plaquetaria , Trombastenia , Factor de Crecimiento Epidérmico , Células HEK293 , Humanos , Integrina alfa2/genética , Integrina beta3/genética , Complejo GPIIb-IIIa de Glicoproteína Plaquetaria/metabolismo , Trombastenia/genética , Trombastenia/metabolismo , TurquíaRESUMEN
BACKGROUND AND OBJECTIVES: In fetal/neonatal thrombocytopenia, maternal alloimmunization is diagnosed by the identification of the maternal alloantibody and the offending paternal antigen inherited by the foetus/neonate. Today, for practical reasons, most laboratories perform platelet genotyping instead of phenotyping. Here, we report the case of a human platelet antigen (HPA)-5 genotype/phenotype discrepancy observed in a mother who delivered a mildly thrombocytopenic newborn. MATERIALS AND METHODS: Platelet antibody detection and platelet phenotyping were performed using the MAIPA assay; platelet genotypes were determined using BeadChip technology (BioArray), PCR-SSP, PCR-RFLP and sequencing. RESULTS: Serological investigations revealed the presence of maternal anti-GPIIbIIIa autoantibodies. No alloantibodies were detected. No feto-maternal platelet incompatibility was observed for HPA-1 to -21. The mother and newborn were genotyped as HPA-5aa using BeadChips, but as HPA-5a (weak b) with PCR-SSP and HPA-5ab with PCR-RFLP. Mother's platelets were phenotyped as HPA-5b(+). GPIa exon 13 sequencing confirmed the HPA-5ab genotype of the mother and newborn, and revealed an NM_002203.3:c.1594A>C mutation near the HPA-5 polymorphism (5' side), leading to an I503L amino acid change. CONCLUSION: Feto-maternal alloimmunization was ruled out: the neonatal thrombocytopenia probably resulted from maternal anti-GPIIbIIIa autoantibodies. This case highlights that platelet typing should be performed using two different methods to avoid false diagnosis.
Asunto(s)
Genotipo , Integrina alfa2/genética , Mutación Missense , Polimorfismo de Longitud del Fragmento de Restricción , Adulto , Sustitución de Aminoácidos , Antígenos de Plaqueta Humana/genética , Antígenos de Plaqueta Humana/metabolismo , Femenino , Humanos , Recién Nacido , Isoanticuerpos/sangre , Masculino , Trombocitopenia Neonatal Aloinmune/sangre , Trombocitopenia Neonatal Aloinmune/genéticaRESUMEN
Essentials Life-threatening maternofetal thrombocytopenias mostly depend on αIIb ß3 antigens. We performed serological, genomic and in vitro studies of two life-threatening thrombocytopenias. Identification of a c.368C>T variation leading to Pro123Leu substitution in GPIX. A rare GPIX variant reported in a genomic database define a new alloantigen. SUMMARY: Background After three miscarriages, a 39-year-old woman gave birth, with a 1-year interval, to two severely thrombocytopenic neonates (4 ×109 L-1 and 33 ×109 L-1 ) with intracranial hemorrhages. Transfusion of platelet concentrates corrected the thrombocytopenia. The outcome was favorable for the first child, but the second one died 10 days after cesarean delivery (31 weeks of gestation + 6 days). Methods Serologic studies were performed with mAb-specific immobilization of platelet antigens and flow cytometry techniques. Human platelet alloantigen (HPA) genotyping was performed with the BioArray HPA BeadChip and PCR-sequence-specific primer techniques. Genomic DNA was studied by direct sequencing of PCR products. The mutant glycoprotein (GP) was expressed in transiently transfected HEK293 cells. Results In MAIPA assay, the maternal serum faintly reacted with GPIbIX from paternal and child 1 platelets, but not with maternal or panel platelets. No maternofetal incompatibility was found in the 22 known HPA systems, tested except for HPA-1b in child 2. A new alloantigen carried by GPIbIX was suspected. Genomic sequencing revealed a paternal GPIX variation (NM_000174.4:c.368C>T). The father and children were heterozygous and incompatible with the mother, who was NM_000174.4:c.368C homozygous. The maternal serum reacted with the GPIX NP_000165.1:p.Leu123 form coexpressed with GPIb in transfected HEK293 cells. The NM_000174.4:c.368T allele (rs202229101) has a minor allele frequency of 0.0002, and was not detected in 120 French subjects (families with fetal and neonatal alloimmune thrombocytopenia [FNAIT]), suggesting that it is rarely implicated in alloimmunization. Conclusion The NP_000165.1:p.Leu123 allele named Cab4b is the first platelet alloantigen described on GPIX. In the absence of other known maternofetal incompatibility, the child 1 case suggests that anti-Cab4b alloantibodies can induce severe thrombocytopenias.
Asunto(s)
Antígenos de Plaqueta Humana/genética , Mutación , Complejo GPIb-IX de Glicoproteína Plaquetaria/genética , Trombocitopenia Neonatal Aloinmune/genética , Antígenos de Plaqueta Humana/sangre , Antígenos de Plaqueta Humana/inmunología , Análisis Mutacional de ADN , Resultado Fatal , Femenino , Predisposición Genética a la Enfermedad , Células HEK293 , Herencia , Humanos , Recién Nacido , Isoanticuerpos/sangre , Linaje , Fenotipo , Complejo GPIb-IX de Glicoproteína Plaquetaria/inmunología , Complejo GPIb-IX de Glicoproteína Plaquetaria/metabolismo , Transfusión de Plaquetas , Embarazo , Pruebas Serológicas , Trombocitopenia Neonatal Aloinmune/sangre , Trombocitopenia Neonatal Aloinmune/diagnóstico , Trombocitopenia Neonatal Aloinmune/terapia , Transfección , Resultado del TratamientoRESUMEN
BACKGROUND: Fetal/neonatal alloimmune thrombocytopenia results from maternal immunization against fetal platelet alloantigens (HPAs), and the major risk is intracranial hemorrhage. The severity of thrombocytopenia increases in subsequent pregnancies, and antenatal therapy has been developed. Until now, the fetal status can only be assessed by fetal blood sampling, which carries a risk of fetal loss or premature delivery. OBJECTIVES: To develop non-invasive methods to gain information on the fetal condition. PATIENTS/METHODS: Quantification of the maternal anti-HPA-1a alloantibody concentration was performed with a standardized monoclonal antibody-specific immobilization of platelet antigens (MAIPA) procedure for 43 mothers. A correlation between this concentration and the fetal/neonatal platelet counts was studied. RESULTS: (i) Before antenatal therapy, there was a significant correlation between maternal anti-HPA-1a concentrations > or =250 AU mL(-1) and fetal thrombocytopenia (2-66 x 10(9) L(-1)) whatever the gestational age (Fisher's exact test P = 0.0021). (ii) During subsequent pregnancies, we observed a decrease of the maternal anti-HPA-1a concentration for 14/19 women. Just before delivery, all women had anti-HPA-1a concentrations <250 AU mL(-1). In four cases, there was a therapy failure and the severely thrombocytopenic babies required postnatal therapy. CONCLUSION: The maternal anti-HPA-1a concentration could provide obstetricians with clinically useful information concerning the appropriateness and the timing of invasive monitoring procedures. However, though we observed a tendency toward a decrease in maternal antibody concentration after treatment, this finding does not allow us to draw any conclusions on the effectiveness of therapy.
Asunto(s)
Antígenos de Plaqueta Humana/inmunología , Isoanticuerpos/sangre , Complicaciones del Embarazo/sangre , Púrpura Trombocitopénica Idiopática/sangre , Biomarcadores/sangre , Femenino , Enfermedades Fetales/sangre , Enfermedades Fetales/inmunología , Enfermedades Fetales/terapia , Edad Gestacional , Humanos , Integrina beta3 , Atención Posnatal , Valor Predictivo de las Pruebas , Embarazo , Complicaciones del Embarazo/inmunología , Complicaciones del Embarazo/terapia , Resultado del Embarazo , Atención Prenatal , Diagnóstico Prenatal/métodos , Púrpura Trombocitopénica Idiopática/inmunología , Púrpura Trombocitopénica Idiopática/terapiaAsunto(s)
Plaquetas/metabolismo , Moléculas de Adhesión Celular/efectos de los fármacos , Desamino Arginina Vasopresina/farmacología , Fosfoproteínas/efectos de los fármacos , Moléculas de Adhesión Celular/metabolismo , Desamino Arginina Vasopresina/administración & dosificación , Humanos , Proteínas de Microfilamentos , Fosfoproteínas/metabolismo , Fosforilación/efectos de los fármacos , Complejo GPIIb-IIIa de Glicoproteína Plaquetaria/genética , Trombastenia/sangreRESUMEN
BACKGROUND: Previous studies of platelet allele frequencies in Sub-Saharan African populations enabled us to identify discrepancies in HPA-3 typing, suggesting the presence of new mutations and of a greater polymorphism than so far described in other populations. OBJECTIVES: To analyze these discrepancies and to assess the factors leading to potential alloimmunization in these populations. SAMPLES: Maternal samples from a Beninese woman following in utero death and panels of blood donors from Benin, Cameroon, Congo, and Pygmies from Central Africa. TECHNIQUES: Genotyping was performed using polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP), PCR-sequence specific primers (PCR-SSP) and sequencing techniques. RESULTS: Three new mutations were found on GPIIb gene: exon 26 a) 2614C>A situated between HPA-3 and HPA-9w, b) 2645C>T downstream of HPA-3, c) intron 26 IVS26+89G>A. These mutations may lead to discrepant DNA typing results, due either to a localization in the complementary sequence recognized by the primer or to the appearance of a new enzyme restriction site. Furthermore, a bilateral linkage << deletion (Delta9 bp) intron 21 and the HPA-3b allele (exon 26) >> found in Caucasian, Asian, and Oceanian populations is not found in African populations, suggesting that its appearance was prior to HPA-3. CONCLUSION: Three new mutations have been identified, two of them potentially immunogenic through their position. Furthermore, the polymorphism found on intron 26, localized in the complementary sequence of the PCR primer, may lead to a false typing assignation. It is therefore important to diversify techniques, both genomic (PCR-RFLP and PCR-SSP), and proteomic monoclonal antibody-specific immobilization of platelets antigen (MAIPA) to ensure accurate HPA antigenic system typing.
Asunto(s)
Población Negra/genética , Plaquetas/metabolismo , Mutación , Glicoproteína IIb de Membrana Plaquetaria/genética , Adulto , Anciano , Secuencia de Bases , Benin , Camerún , Congo , Análisis Mutacional de ADN , Femenino , Enfermedades Fetales/sangre , Enfermedades Fetales/diagnóstico , Enfermedades Fetales/etnología , Enfermedades Fetales/inmunología , Frecuencia de los Genes , Genotipo , Humanos , Masculino , Persona de Mediana Edad , Datos de Secuencia Molecular , Linaje , Glicoproteína IIb de Membrana Plaquetaria/inmunología , Reacción en Cadena de la Polimerasa , Polimorfismo de Longitud del Fragmento de Restricción , Embarazo , Trombocitopenia/sangre , Trombocitopenia/diagnóstico , Trombocitopenia/etnología , Trombocitopenia/inmunologíaRESUMEN
The typing of human platelet antigens (HPA) can be useful in many clinical situations such as neonatal alloimmune thrombocytopenia, post-transfusion purpura, and platelet transfusion refractoriness. The fluorescent-based single-strand conformation polymorphism (F-SSCP) technique is a fast and convenient way to perform HPA genotyping. Universal sequences from phage M13 were introduced at both ends of specific PCR-products by using 5'-tailed primers. A short second round of PCR with universal primers coupled to Cy-5 enabled the PCR-products to be fluorescently labelled. F-SSCP was performed by gel electrophoresis on an automated fluorescent DNA analyser. Genotyping of the three major HPA systems carried by the GP IIb-IIIa complex showed the F-SSCP technique to be accurate and reliable. A single gel procedure has been sufficient to detect HPA genetic polymorphisms tested to date. Neither restriction enzyme, radioactive material, nor any other hazardous chemicals such as ethidium bromide were required. This technique enabled us to genotype HPA-1, -3 and -4 alleles easily and to diagnose materno-fetal incompatibility in a rare alloantigenic system. F-SSCP is a promising technique for the detection of new mutations and/or DNA polymorphisms on a large scale.
Asunto(s)
Antígenos de Plaqueta Humana/genética , Plaquetas/inmunología , Polimorfismo Conformacional Retorcido-Simple , Trombocitopenia/genética , Adulto , Análisis Mutacional de ADN , Femenino , Genotipo , Humanos , Recién Nacido , Masculino , Intercambio Materno-Fetal , Reacción en Cadena de la Polimerasa , Polimorfismo de Longitud del Fragmento de Restricción , Embarazo , Trombocitopenia/congénitoRESUMEN
Heparin-dependent thrombocytopenia (HDT) and associated thrombotic complications, are thought to be linked to the appearance of anti-platelet antibodies. Attempts were made to characterize the antibodies in the sera from 10 such patients. Western blotting against platelet antigens was inconclusive, often revealing multiple bands but with considerable variability from patient to patient. An often seen band of approximately 83 kDa was also given by some nonimmune sera and the antigen appeared to be predominantly intracellular in origin. Antibodies to major membrane glycoproteins were not readily apparent. This was confirmed by the MAIPA ("Monoclonal Antibody Immobilization of Platelet Antigens") test, performed to detect antibodies to GP Ib-IX and GP IIb-IIIa. Only one weak activity to GP Ib-IX and one weak activity to GP IIb-IIIa were detected. In contrast, an antibody to the PlA1 alloantigen was readily detected in the serum of an additional patient. The ability of HDT serum to induce the aggregation of control platelets was studied in detail. Aggregation was inhibited by EDU-3, a monoclonal antibody to GP IIb-IIIa complexes, and by the synthetic peptide RGDS, suggesting an involvement of the same pathway as used by physiologic agonists. However, in agreement with Chong et al (Thromb Res 55:291, 1989), we observed that aggregation was inhibited by rabbit IgG, suggesting that it was Fc-receptor mediated. Interestingly, 2E1, a monoclonal antibody to the Fc gamma RII receptor, induced platelet aggregation with a lag phase, a characteristic of HD antibodies. Nonetheless, for different donors, there was no correlation between the length of the lag phase induced by 2E1 and HD antibodies. Fc-receptor blockade should be considered as a means for diminishing the clinical complications of HDT.
Asunto(s)
Enfermedades Autoinmunes/inducido químicamente , Heparina/efectos adversos , Receptores Fc/fisiología , Trombocitopenia/inducido químicamente , Adulto , Especificidad de Anticuerpos , Antígenos de Plaqueta Humana/inmunología , Autoanticuerpos/inmunología , Femenino , Humanos , Técnicas de Inmunoadsorción , Integrina beta3 , Masculino , Agregación Plaquetaria , Glicoproteínas de Membrana Plaquetaria/inmunología , Trombastenia/complicaciones , Trombastenia/inmunologíaRESUMEN
Patient A.F. is a 28-year-old polytransfused woman with an inherited bleeding disorder, Glanzmann's thrombasthenia. An abnormal platelet function is linked to severe decreases in the platelet content of the integrins GP IIb and GP IIIa. In 1987 the patient gave birth to a child with severe anemia and thrombocytopenia. Serological tests revealed the presence of anti-platelet antibody together with an anti-Rhesus D. Western blotting identified a major antibody that reacted with a protein of 90-95 kDa present in platelets and endothelial cells. This was identified as the beta 3 integrin subunit (GP IIIa). Antibody-binding required intact disulfides, while controlled digestion with proteases showed the determinant(s) to be retained within chymotrypsin- (50, 63 kDa) and Staphylococcus aureus V8 protease-derived (25-38 kDa) fragments of GP IIIa. Direct binding assays performed in the presence of monoclonal antibodies specific for different epitopes on GP IIb-IIIa complexes confirmed that the epitope was exposed on intact platelets and revealed a specific inhibition of A.F. IgG binding by the monoclonal antibody, AP-3. Other tests confirmed that the antibody reacted independently of the PlA or Pen polymorphisms carried by GP IIIa. IgG purified from A.F. plasma by adsorption and elution from paraformaldehyde-fixed normal platelets or electrophoretically separated GP IIIa was an inhibitor of ADP-induced platelet aggregation. Unexpectedly, Western blotting showed trace amounts of abnormally migrating GP IIIa in A.F. platelets, which retained an ability to react with her antibody. This suggests that the patient has formed an autoantibody reactive with an active site of the beta 3 integrin subunit and linked to the development of neonatal thrombocytopenia.
Asunto(s)
Autoanticuerpos/sangre , Integrinas/inmunología , Trombastenia/inmunología , Trombocitopenia/inmunología , Adulto , Anciano , Especificidad de Anticuerpos , Femenino , Humanos , Recién Nacido , Masculino , Intercambio Materno-Fetal/inmunología , Persona de Mediana Edad , Glicoproteínas de Membrana Plaquetaria/inmunología , Embarazo , Complicaciones Hematológicas del Embarazo/inmunología , Trombastenia/complicaciones , Trombastenia/genética , Trombocitopenia/congénito , Trombocitopenia/etiologíaRESUMEN
Glanzmann's thrombasthenia is an inherited bleeding disorder that results from a deficit of glycoprotein (GP) IIb-IIIa complexes in platelets. Patient (EBV) is an adult male with GP IIb-IIIa levels < 5% of normal values and a history of blood transfusions. Western-blot analysis revealed a strong IgG antibody to GP IIIa in his plasma. The determinants were localized to the minimum-sized fragment of GP IIIa (50 kDa) retained on chymotrypsin-treated platelets and were lost on reduction of disulphides. A female patient (AF), previously described by us [Jallu, V., Pico, M., Chevaleyre, J., Vezon, G., Kunicki, T.J. & Nurden, A.T. (1992) Hum. Antibod. Hybridomas 3, 93-106] developed her anti-GP-IIIa antibody during pregnancy. This antibody was poorly reactive with the 50-kDa proteolytic fragment, yet bound to 115-kDa and 60-kDa hydrolytic products of GP IIIa. Antibodies from both patients recognized the GP-IIIa-like protein of endothelial cells, thus confirming that they were directed against the integrin beta 3-subunit. The (EBV) antibody reacted strongly with GP IIb-IIIa in an antigen capture assay performed with each of a panel of four murine monoclonal antibodies (mAbs) recognizing different epitopes on GP IIb-IIIa. In contrast, that from (AF) was specifically inhibited by AP-3, a murine mAb whose epitope is thought to be localized between amino acids 324-422 of GP IIIa. The residual GP IIb and GP IIIa contents of platelets from each patient were assessed in Western blotting using chemiluminescence detection. SZ-22, a murine mAb to the GP IIb heavy chain (140 kDa), located small amounts of a 130-kDa protein in (EBV) platelets. The anti-GP IIIa mAbs XII F9, P 37 and P 97 revealed trace amounts of protein with a relative mobility identical to that of GP IIIa in both (AF) and (EBV) platelets. This residual GP IIIa represented less than 0.5% of the amount in normal platelets. When, for each patient, plasma was tested in Western blotting against their own platelets, autoantibody activity to the residual GP IIIa was detected in both cases. Thus, patients (AF) and (EBV) have developed anti-GP-IIIa antibodies with restricted and distinct epitopes but recognizing self antigens.
Asunto(s)
Plaquetas/inmunología , Endotelio Vascular/inmunología , Inmunoglobulina G/inmunología , Integrinas/inmunología , Glicoproteínas de Membrana Plaquetaria/inmunología , Trombastenia/inmunología , Reacciones Antígeno-Anticuerpo , Autoanticuerpos/sangre , Autoanticuerpos/inmunología , Western Blotting , Células Cultivadas , Electroforesis en Gel de Poliacrilamida , Endotelio Vascular/citología , Epítopos , Femenino , Humanos , Inmunoglobulina G/sangre , Masculino , Peso Molecular , Complejo GPIIb-IIIa de Glicoproteína Plaquetaria , Glicoproteínas de Membrana Plaquetaria/deficiencia , Embarazo , Complicaciones Hematológicas del Embarazo/inmunologíaRESUMEN
In the Caucasian population, platelet incompatibility within the HPA-1 (Pl(A1/A2)) and HPA-5 (Br(a/b)) alloantigen systems are the two most likely causes of post-transfusion purpura (PTP) and neonatal alloimmune thrombocytopenia. However, the way in which HLA (class-II) antigens participate in alloantibody formation is unclear. The patient (M-J.G.) is a middle aged woman with two children who developed a severe PTP (< 2000 platelets/µ1) 8 days after receiving red cell concentrates during coronary bypass surgery. During treatment with intravenous gamma-globulin and corticosteroids, her platelet count peaked, fell again, and returned to normal over a period of several months. Western blotting and/or the monoclonal antibody specific-immobilization of platelet antigens (MAIPA) assay performed with serum prepared at the height of her initial thrombocytopenia revealed antibodies to both the Pl(A1) and the Br(a) alloantigens. This rare combination prompted us to study the expression of specific HLA class II antigens in the patient. HLA-DR and DQ typing was performed from genomic DNA by the recently developed polymerase chain reaction-restriction fragment length polymorphism procedure (PCR-RFLP). The patient was found to express the DRB1*1302/1303 and DRB3*0101/0301 alleles (serological specificities: HLA-DR6 and DR52a/c respectively). She also expressed the DQA1*0102/0501, DQB1*0601 and DQB1*0301 alleles. Thus, this case provides further evidence linking an immune response to Pl(A1) and Br(a) antigens with HLA-DR52a/c and DR6.
RESUMEN
Glanzmann's thrombasthenia (GT) is a recessive autosomal bleeding disorder characterized by abnormal platelet aggregation due to a qualitative or quantitative defect of the glycoprotein (GP) IIb-IIIa complex (integrin alphaIIb beta3). We describe a new mutation in the GPIIIa gene responsible for type I GT in a consanguineous Algerian family. A discordance between phenotyping and genotyping of the GPIIIa-related HPA-1 platelet alloantigen system in three family members heterozygous for the disease suggested a genetic defect in the GPIIIa gene and a normal GPIIb gene. Sequence analysis of amplified genomic DNA fragments showed a 6-bp deletion in exon 7 of the GPIIIa gene resulting in the amino acid deletion/substitution (Ile325pro326Gly327 --> Met) and creating a new BspHI restriction site. Expression of the mutated integrin beta3 subunit cDNA in Chinese hamster ovary cells showed that the cDNA gene was transcribed into a full-length beta3 protein with an apparent molecular weight identical to wild-type beta3 and accumulated as a single-chain molecule in the cell cytoplasm. The absence of heterodimeric complex formation of the mutant beta3 protein with endogenous alpha v was shown by immunoprecipitation experiments, intracellular immunofluorescent labeling, and a semiquantitative enzyme-linked immunosorbent assay using the alpha vbeta3 complex-specific monoclonal antibodies LM609 and 23C6. Substitution of the methionine residue by a proline, present at position 326 of wild-type beta3, did not restore the ability of the recombinant mutant beta3 protein to associate with alpha v , suggesting that the Ile-Pro-Gly motif is located in a beta3 domain important for integrin subunit interaction. The association of a BspHI restriction site with this newly identified mutation has allowed allele-specific restriction analysis of Algerian GT individuals and the identification of two new unrelated type I patients exhibiting the same mutation, suggesting that the described mutation might be significant in this population and that BspHI restriction analysis will provide a useful screening assay for antenatal diagnosis and genetic counselling.