Phenotype and Genotype Expression in Pseudohomozygous Factor V LEIDEN

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Abstract
—The presence of a DNA mutation is frequently used to define a disease or a risk state. Because DNA typing has become easy and convenient in contrast to protein characterization, it is generally assumed that a mutation if present (or not) at the DNA level will be also present (or not) in the corresponding protein. However, discrepancies between phenotype and genotype can occur. A point mutation in the coagulation factor V gene (G 1691 →A, resulting in an Arg 506 →Gln amino acid substitution in the factor V molecule [factor V LEIDEN ], leading to activated protein C resistance) is the most common genetic risk factor for familial thrombophilia. A pseudohomozygous factor V LEIDEN phenotype would occur if a heterozygous individual for factor V LEIDEN also did not express the “normal” (non-Leiden) factor V allele. However, to date, no data have been available to confirm the presence of only the factor V LEIDEN form in the plasma of these individuals. Platelet mRNA from 2 presumed pseudohomozygous patients and their family members was isolated, the amplified partial cDNAs were sequenced or restricted, and the allelic bands were quantified. Both patients were found to be heterozygous for the G 1691 →A substitution at both the DNA and mRNA levels. The presence of either the normal or mutated form of factor V in the patients’ plasmas was investigated using a monoclonal antibody to factor V that recognizes an epitope located between residues 307 and 506 of the factor Va heavy chain. No normal factor V could be detected in the plasmas of the 2 propositi. The present data demonstrate absence of a correlation between genotype at position 1691 (at the DNA and mRNA levels) and the corresponding phenotype data found in the plasmas of patients with pseudohomozygous factor V LEIDEN . Overall, these data suggest the existence of heterogeneous genetic “lesions,” which interfere with factor V expression, processing, secretion, and/or stability. Because the presence of the factor V LEIDEN molecule in plasma is directly related to pathology, identification and quantification of the circulating forms of factor V in plasma may be required for the diagnosis of individuals with activated protein C resistance.