Trunk type origin was from the greater

Trunk type origin was from the greater saphenous vein in 67 of 116 patients (58%), from the smaller saphenous vein in 7 (6%), and from both in two (2%). Perforator type origin was from the calf in 36 (31%), the thigh in 6 (5%), and from both in 5 (4%). In 16 patients (14%) the notch pathway origin from one or the other or both morphologies remained unclear (Suppl. Table 4). Six patients were excluded due to diagnostic overlaps or secondary etiology (Suppl. Table 5). Exploratory pairwise associations (via Chi-squared or Fisher\'s exact test, as appropriate) between the combined genotypes and the morphologies and complications were recorded (Tables 1 and 2, Suppl. Fig. 1). Four patients (3%) were assessed as perforator type morphology despite the occurrence of axial trunk incompetence due to proximal perforator location (Suppl. Table 6). In some patients with congestive (CEAP C3–6) or thrombotic disease or particularly in those with thigh perforator incompetence, however, duplex examination was unable to sufficiently discriminate between recirculation pathways of trunk or perforator type origin. 16 patients, therefore, were classified as unclear phenotypes (Tables 1 and 2, Suppl. Table 4). Six patients were excluded from evaluation, five for primary varices in case of controls, and one for systemic thrombophlebitis without evidence of primary varicose veins (Table 2, Suppl. Table 5). Blood specimens were collected from all patients during sampling for routine examination. Vein tissue specimens were obtained from all patients during surgery by dissecting an appropriate piece of tissue (at least 1cm2) from affected or unaffected (controls) veins and transferred into a freezing vial. Blood and vein tissue specimens were kept on ice for a maximum of 6h until storage at −80°C. DNA extraction from frozen blood samples was performed using the Puregene Kit (Qiagen, Hilden, Germany). For DNA extraction from vein tissue specimens, a standard salting out procedure was used. Genotyping for the MTHFR c.677C>T and c.1298A>C polymorphisms was carried out from blood samples of all patients using polymerase chain reaction (PCR) followed by pyrosequencing on a Pyromark Q96 ID Instrument (Qiagen). Primer sequences and PCR conditions are available on request. Of 37 patients that displayed heterozygous genotypes at MTHFR c.677C>T and/or MTHFR c.1298A>C, parallel tests of blood and tissue specimens were performed and identical genotypes were obtained.
Results There was no association between c.677C>T genotype and control/patient status as well as c.1298A>C and control/patient status (c.677C>T: Fisher\'s exact test, p=0.29; c.1298A>C: Fisher\'s exact test, p=1). Thus, we only considered those patients who have the condition in the subsequent analysis. A log-linear analysis, using a log-linear regression model with stepwise selection at a level of 0.05, indicates that heterozygosity or homozygosity for c.677C>T was significantly associated with the trunk phenotype (43/53 patients, 81%, p<0.01) and heterozygosity or homozygosity for c.1298A>C was significantly associated with the perforator phenotype (18/24 patients, 75%, p<0.01). Furthermore, as by Fisher´s exact test, double heterozygosity for c.677C>T and c.1298A>C was significantly associated with the combined trunk and perforator phenotype (18/23 patients, 78%, p<0.01) (Table 2, Fig. 2a). There was also a weak interaction between c.1298A>C and the trunk phenotype, however this did not reach significance (p=0.051). A subsequent log-linear analysis including genotype and the unclear phenotype indicates that the unclear phenotype was significantly associated with a homozygous wild type (CC) genotype at c.677C>T (p<0.01). The unclear phenotype was the highest for the homozygous wild type genotype (CC) regardless of the c.1298A>C genotype, decreased for the heterozygous genotype (CT) and was not present for the homozygous mutant genotype (TT) (Fig. 2b).