Supplementary Methods and Materials Blood

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instructions (Eppendorf, Gemany). Fluorescence was ... On a BCS XP coagulation analyzing system (Siemens Healthcare Diagnostics. GmbH, Germany) ...
Supplementary Methods and Materials

Blood samples were obtained by the family physician and immediately sent to the laboratory for testing. All subjects investigated gave full written and informed consent for genetic testing in accordance with the Austrian Gene Technology Act. EDTA whole blood was used for the genetic testing of factor V Leiden (F5 R506Q), employing a customized 5'-nuclease TaqMan assay (ABI, Waltham, MA). PCR was performed on a Mastercycler Gradient thermal cycler according to the manufacturer's instructions (Eppendorf, Gemany). Fluorescence was measured after PCR in a POLARstar OPTIMA plate reader (Thermo Electron, Karlsruhe, Germany) using excitation/emission filters of 485 nm/525 nm for FAM-labelled probes (506Q allele) and 525 nm/580 nm for VIC-labeled probes (506R allele). On a BCS XP coagulation analyzing system (Siemens Healthcare Diagnostics GmbH, Germany), citrated plasma samples (containing 3.8% sodium citrate as anticoagulant) were used for further testing. COATEST® APC™ Resistance V (Chromogenix, Mölndal, Sweden), Pefakit® APC-R Factor V Leiden assay (DSM – Pentapharm, Basel, Switzerland) and HEMOCLOT® Quanti V-L (HYPHEN Biomed, Neuville-Sur-Oise, France) were used to determine APC resistance. Factor V activity was determined with coagulation factor V deficient plasma and thromboplastin (Thromborel S, Siemens Healthcare Diagnostics, Marburg, Germany). Factor V antigen levels were measured with the manual, sandwich ELISA ZYMUTEST Factor V assay (HYPHEN Biomed, Neuville-Sur-Oise, France) (Strobl et al, 1998; Schöni et al, 2007; Vissac et al, 2009; Jeimy, 2010). For sequencing the F5 gene, EDTA whole blood samples were coded and sent to two independent laboratories (Institute for Clinical Chemistry and Laboratory Medicine, Medical Faculty, Technische Universitaet Dresden, Germany and University Clinic of Blood Group Serology and Transfusion Medicine, Medical University of Graz, Austria). Sequencing was performed on a 310A and on a 3130

instrument (ABI) respectively, and both employed the big Dye RR Terminator Cycle Sequencing Kit (ABI). The sequences obtained were put into GenBank and further processed using the PolyPhen-2 databank (Adzhubei et al, 2010), as well as, with the DeepView/Swiss-PdbViewer v4.0.2. (Guex & Peitsch, 1997). T-tests were used to statistically compare the factor V antigen and factor V activity levels between the two groups where the FV Graz mutation (F5 G1718S) is absent and present.

Adzhubei, I.A., Schmidt, S., Peshkin, L., Ramensky, V.E., Gerasimova, A., Bork, P., Kondrashov, A.S. & Sunyaev, S.R. (2010) A method and server for predicting damaging missense mutations. Nature methods, 7, 248–249. Guex, N. & Peitsch, M.C. (1997) SWISS-MODEL and the Swiss-PdbViewer: an environment for comparative protein modeling. Electrophoresis, 18, 2714–2723. Jeimy, S.B. (2010) Review of the Structure and Function of Factor V in Blood Coagulation. University of Toronto Medical Journal, 87, 91–100. Schöni, R., Quehenberger, P., Wu, J.R. & Wilmer, M. (2007) Clinical evaluation of a new functional test for detection of activated protein C resistance (Pefakit APC-R Factor V Leiden) at two centers in Europe and the USA. Thrombosis research, 119, 17–26. Strobl, F.J., Hoffman, S., Huber, S., Williams, E.C. & Voelkerding, K.V. (1998) Activated protein C resistance assay performance: improvement by sample dilution with factor V-deficient plasma. Archives of pathology & laboratory medicine, 122, 430–433. Vissac, A., Peyrafitte, M. & Amiral, J. (2009)Quantitative Measurement of Factor V Leiden in Heterozygous and Homozygous Patients for the R506Q Factor V Mutation. In Vienna Available at: http://www.aniara.com/pdf/SS-ANIARA-GTH09-Vienne-FVL.pdf.

Supplementary Table S I. Phenotypic results and prevalence of different co-inherited heterozygous conditions for the factor V Leiden (F5 R506Q) in the presence of the novel heterozygous polymorphism factor V Graz (F5 G1718S).

a) Inheritance of heterozygous factor V Leiden polymorphism leading to a normal amount of factor V protein; half of the protein is resistant to activated protein C (heterozygous factor V Leiden) # (Jadaon, 2011) b) Co-inheritance on the (e.g. maternal) allele of the heterozygous factor V Leiden polymorphism and on the second (e.g. paternal) the additional polymorphism responsible for a premature factor V protein degradation leads to the expression of a reduced amount of factor V Leiden protein only, which shows resistance to activated protein C as in homozygous factor V Leiden (pseudo-homozygous factor V Leiden) ## (Simioni et al, 2005) c) Co-inheritance on the same (e.g. maternal) allele of the heterozygous factor V Leiden polymorphism and the additional heterozygous polymorphism (F5 G1718S) registered as factor V Graz, which is responsible for a premature factor V protein degradation. This leads to the expression of normal factor V (non-factor V Leiden) protein resulting in a normal APC resistance (pseudo-wild-type factor V Leiden) Different polymorphisms associated with reduced factor V antigen and activity levels are described - e.g. from amino acid position 1605 to 1748 (Lunghi et al, 2005; Delev et al, 2009). Delev, D., Pavlova, A., Heinz, S., Seifried, E. & Oldenburg, J. (2009) Factor 5 mutation profile in German patients with homozygous and heterozygous factor V deficiency. Haemophilia: the official journal of the World Federation of Hemophilia, 15, 1143–1153. Jadaon, M.M. (2011) Epidemiology of Activated Protein C Resistance and Factor V Leiden Mutation in the Mediterranean Region. Mediterranean Journal of Hematology and Infectious Diseases, 3, e201105. Lunghi, B., Scanavini, D., Castoldi, E., Gemmati, D., Tognazzo, S., Redaelli, R., Ghirarduzzi, A., Ieran, M., Pinotti, M. & Bernardi, F. (2005) The factor V Glu1608Lys mutation is recurrent in familial thrombophilia. Journal of thrombosis and haemostasis: JTH, 3, 2032–2038. Simioni, P., Castoldi, E., Lunghi, B., Tormene, D., Rosing, J. & Bernardi, F. (2005) An underestimated combination of opposites resulting in enhanced thrombotic tendency. Blood, 106, 2363–2365.

Supplementary Figure S I. Detection of the novel mutation 5326G>A(G1718S) in exon 16 of the factor 5 gene - Sequencing protocol and RFLP analysis of the female index patient. a

(a) Electropherogram and sequence analysed on the DNASequencer Perkin Elmer ABI Prism 310: NGT= heterozygous base exchange, GGT= wild-type (b) RFLP-analysis amplified DNA digested with AvaII and subsequent run in 2.5% Metaphorgel: wild-type allele cut at 298 + 147bp [nucleotide position (Jenny et al, 1987)]

b GG

F5 gene, exon 16 AvaII 5326 GG GA GA Ladder 100

bp 473 bp PCR 298 bp 175 bp

index patient heterozygous

control wild-type

Jenny, R.J., Pittman, D.D., Toole, J.J., Kriz, R.W., Aldape, R.A., Hewick, R.M., Kaufman, R.J. & Mann, K.G. (1987) Complete cDNA and derived amino acid sequence of human factor V. Proceedings of the National Academy of Sciences of the United States of America, 84, 4846–4850.