Functional Characterization of Long-QT Syndrome (LQT) and Sudden Infant Death (SIDS) Associated OLFML2B Mutations. T. A. Plötz1, C. J. Gloeckner1,2, A. Kiper3, M. Vennemann4, M. Kartmann5, M. Schell5, H. Prucha6, C. Congiu7, Z. Schäfer1, S. Hauck1, I. Sinicina5, E. Kremmer1, B. M. Beckmann9, F. Domingues7, T. Meitinger1,6,10, A. Peters1,5,10, M. Cohen8, S. Kääb9,10, J. J. Schott11, E. R. Behr12, T. Bajanowski13, S. Just14, H. W. Mewes1,6, M. Ueffing1,2, N. Decher3, M. Näbauer9, A. Pfeufer1,6 1) Helmholtz Zentrum München, Neuherberg, Germany; 2) Universität Tübingen, Tübingen, Germany; 3) Universität Marburg, Marburg, Germany; 4) Universität Münster, Münster, Germany; 5) LMU München, München, Germany; 6) TU München, München, Germany; 7) EURAC, Bolzano, Italy; 8) University of Sheffield, Sheffield, UK; 9) Department of Medicine I, University Hospital Munich, Ludwig-Maximilians-University Munich, Munich, Germany; 10) Deutsches Zentrum für Herz-Kreislauf-Forschung e.V. (DZHK), partner site Munich Heart Alliance, Munich, Germany; 11) Universite de Nantes, Nantes, France; 12) St Georges University of London, London, UK; 13) Universität Essen, Essen, Germany; 14) Universität Ulm, Ulm, Germany.

   We have mapped the strongest human QTL modifying cardiac repolarization (QT interval) to OLFML2B and NOS1AP in 1q23.3 by GWAS. OLFML2B encodes a secreted extracellular matrix (ECM) protein. By mutation screening we have identified overrepresentation of rare (MAF1%) nonsynonymous heterozygous mutations in 125 patients with long-QT Syndrome (LQT; OR=3.62 (1,46-8,93) p=2,9x10-3) and in 93 with sudden infant death syndrome (SIDS; OR=3,01 (1,05-8,65) p=3,2x10-2) but not in 94 adults with sudden cardiac death (SCD; OR=0,57 (0,07-4,41) p=5,9x10-1) compared to 702 population controls. Of 35 missense variants identified, we have selected 24 predicted to negatively affect protein structure (PolyPhen2, SIFT, Mutation taster) for functional analysis. Combining our 702 controls with in-silico data from 6503 WES sequenced individuals from the ESP, 10 variants of the 24 occurred among 7205 persons while the other variants were absent in the expanded control sample. All variants were equally expressed intracellularly. Their secretion into the extracellular space was impaired depending on the mutation ranging from mild reduction to nonsecretion. Co-expression of wt and mut demonstrated dominant negative secretion impairment. Experiments were performed in triplicate at three temperatures and parametrized (LI-COR Image Studio) using wt-OLFML2B at 37C as a reference. Protein secretion was temperature dependent (30C>37C>41C; p<0,001). In addition secretion was significantly correlated with disease severity (wt>LQT>SIDS; p<0,05) and with allele frequency in the controls (p<0.01). Five out of the 24 variants were investigated by cellular electrophysiology in Xenopus oocytes. They showed significant reduction of the voltage gated KCNH2/Kv11.1 channel (IKr) but no other main cardiac ion channels. The degree of impairmant ranged from -10% to -50% and was also correlated with mutation secretion status. Taken together the functional proteomic investigation suggests a significant influence of OLFML2B and the ECM on myocardial repolarization. This assumption is supported by nonsecretion being associated with both disease severity and population allele frequency acting in an autosomal dominant manner. Our data support the hypothesis that rare nonsynonymous OLFML2B variants impair repolarization, most likely by failing to assume the correct topological position in the ECM, and confer genetic predisposition to long QT-Syndrome (LQT) and sudden infant death (SIDS).