Abnormal melt curve profile during prothrombin 20210G → A analysis due to the 20209C → T variant

Abstract

The common factor II 20210G --> A mutation, located in the 3'-untranslated region, is an important risk factor for the development of thromboembolic disorders, especially in Caucasians. A number of methods are employed for clinical laboratory diagnosis of this mutation, some of which are capable of detecting adjacent 3'-end sequence variations. We present results from an African deep vein thrombosis patient tested for the 20210G --> A mutation by real-time polymerase chain reaction and melt-curve analysis using hybridization probes that incidentally detected an adjacent 3'-untranslated region variant. The patient sample was tested using the Factor II (Prothromobin) G20210A Kit (Roche Diagnostics, Indianapolis, Indiana, USA), in conjunction with the Roche LightCycler. A polymerase chain reaction fragment from the 3'-end of the F2 gene was subsequently sequenced for identification of the variant. Melt-curve analysis revealed a normal 20210*G peak and an unknown aberrant allelic peak. Following sequence analysis, the patient was determined to be heterozygous for 20209C --> T. The presence of the 20209C --> T variant in the current patient and in eight other reported individuals of African descent, most with thrombosis-associated complaints, suggests that this rare variant poses a potential increased risk for thromboembolic disease in this ethnic group.

Prothrombin 20209C>T: 16 new cases, association with the 19911A>G polymorphism, and literature review

“…Of the 16 new and 41 previously reported [1–15] prothrombin variants, 48 (84%) were 20209C>T, in agreement with the previously reported frequency of 85% for this variant [16]. Of 48 20209C>T cases, 37 (79%) had thrombotic event(s) [deep vein thrombosis (DVT), PE and stroke) and/or pregnancy complications.…”

“…During routine screening for the prothrombin 20210G>A mutation as a risk factor for venous thrombosis, seven prothrombin variants close to position 20210 in the 3′ untranslated region of the prothrombin gene which are enriched in non‐Caucasian populations have been found using melt curve analysis. These variants are 20207A>C [1], 20209C>T [2–11], 20209C>A [11], 20214C>T [12] 20218A>G [9,13], 20219T>A [9] and 20221T>C [3,14,15]. Prothrombin 20209C>T has mainly been found in individuals of African descent who have had thrombotic events and/or obstetrical complications [2–4,6–10] but has also been reported in seven Caucasians, three of Jewish–Moroccan origin [5] and four in a French study [9].…”

C20209T prothrombin gene mutation associated deep venous thrombosis in a hemodialysis patient

“…Initial reports of prothrombin gene mutation C20209T differ as to whether this polymorphism is [16, 17] or is not [18] associated with an increased risk for development of VTE. …”

A deep vein thrombosis caused by 20209C>T mutation in homozygosis of the prothrombin gene in a Caucasian patient

“…Several different molecular genetics techniques that use restriction enzymes, allele-specific amplification, hybridization probes at a single detection temperature or the LightCycler system have been described (8,16–19). …”