QT syndrome (LQT4). Here, we sought to determine the prevalence and spectrum of ankyrin B mutations in a cohort of unrelated patients (pts) referred specifically for LQTS genetic testing. Methods: Between August 1997 and July 2004, 541 consecutive, unrelated pts (358 females, average age at diagnosis, 24 years, and average QTc, 482 ms) were referred to Mayo Clinic’s Sudden Death Genomics Laboratory for LQTS genetic testing. Targeted mutational analysis of 10 ANKB exons (35,36,38-45) implicated previously in the pathogenesis of LQT4 and other dysrhythmia phenotypes was performed on genomic DNA using polymerase chain reaction, denaturing high performance liquid chromatography, and direct DNA sequencing. Results: Overall, four distinct (3 novel) ankyrin B missense mutations (L1503V, E1543K, T1726N, and R1788W) were detected in 5/269 pts (1.9%) lacking any other identifiable LQTS-causing mutations (genotypenegative). Two novel missense mutations (E1578K, and S1721T) were found in 2 additional pts who also host an ATS1and LQT2-associated mutation, respectively. All 6 missense mutations involved highly conserved residues localizing to key functional domains and were absent in 600 reference alleles. The average age of diagnosis of ANKB-positive pts was 15.2 years. The resting QTc of the 5 pts with only a putative ANKB mutation was 436 45 ms. All 5 pts had been diagnosed clinically with “atypical” or “borderline” LQTS. In addition to the aforementioned mutations, the previously published and functionally characterized L1622IANKB mutation was detected in 2 genotype-negative pts (non-white) and was also observed in 3% of black controls. Conclusions: Putative pathogenic ankyrin B mutations were detected in 1.3% of unrelated pts referred for LQTS genetic testing. LQT4-associated ANKB mutations were more common than either LQT5 or LQT6 in this cohort. The precise role in arrhythmia susceptibility for the common black polymorphism, L1622I, warrants further investigation.