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The 262T>C silent mutation of the platelet β3‐integrin gene is not restricted to a single family

Fetal/neonatal alloimmune thrombocytopenia (FNAIT), due to maternal alloimmunization against a human platelet antigen (HPA), is the most common cause of severe thrombocytopenia in the fetus and the newborn. In Caucasian persons, HPA-1 is the most frequently implicated antigen. The laboratory diagnosis of FNAIT is straightforward when a parental platelet (PLT) antigen mismatch with the corresponding maternal alloantibody is demonstrated. With progress in molecular biology, PLT typing is currently done by genotyping. We recently described a new mutation in the b3-integrin gene in a pregnant woman, which induced a false-negative HPA-1a typing and, consequently, a false diagnosis. A discrepancy between genotyping and phenotyping for HPA-1 led us to find a 262T>C silent mutation located under the antisense primer we use for PCR-SSP. This mutation, which does not change the amino acid sequence, prevents HPA-1a allele DNA amplification. Recently, we found the same mutation when a woman who delivered a severely thrombocytopenic newborn (PLT count, 33 ¥ 10/L on Day 3) was investigated for neonatal alloimmune thrombocytopenia. To better analyze the frequency of this polymorphism, we developed a PCR-restriction fragment length polymorphism (RFLP) assay with the primers previously described for HPA-1 genotyping and HpyCH4V restriction enzyme digestion. Samples from seven patients genotyped during the past 2 years in our laboratory, for whom a faint band had been observed by PCR-SSP for HPA-1a, were reanalyzed. In three cases the mutation was absent, and the low-intensity band was due to a nonspecific amplification, as confirmed by HPA-1 PCR-RFLP assay. In three cases we found the silent mutation in a heterozygous state, and in one case this mutation was found in a homozygous state. Because the families carrying the 262T>C polymorphism originated from Africa (Tunisia in one case, Cameroon in another case), we searched for this mutation in those populations. Among 56 Tunisian healthy blood donors, 4 were found to be heterozygous (7.1%). In Cameroon, the mutation was more frequent; 17.5 percent of the blood donors were found to be positive (20 of 114 individuals were heterozygous). Finally, we analyzed a panel of Caucasian blood donors, and the mutation was not observed in 104 donors tested. In conclusion, these data show that the b3 262T>C silent mutation is not restricted to a single family, as initially suggested. The particular interest of this mutation resides in the localization under a commonly used PCR primer, 166 bp upstream of the HPA-1 polymorphism, which may cause false-negative HPA-1a typing. Consequently, we strongly recommend avoiding the use of HPA-1 PCR-SSP primers located on polymorphism 262. Other primers should be used, such as those suggested by the National Institute for Biological Standards and Control (NIBSC, UK). Because this polymorphism does not induce amino acid change, obviously it is not immunogenic, but it can induce an erroneous PLT typing assignation and therefore a false diagnosis of alloimmune thrombocytopenia due to HPA-1a mismatch, especially in populations where the mutation is present as in Black African populations and to a lesser extent in the North African population.