br Material and methods br Results br Discussion The

Material and methods
Results
Discussion The present study provides clinical, molecular, and in vitro electro-physiological evidence that a rare SNP (KCNE1-D85N) can act as a genetic modifier in LQTS. In general, SNPs are thought to be non-pathological, but some have been reported to modify the clinical features of the disease. For example, the KCNH2-K897T polymorphism [8] has been shown to aggravate LQTS phenotypes directly by reducing cardiac K+ channel function in association with the KCNH2 mutation, A1116V. The prevalence of the KCNH2-K897T polymorphism is estimated to be up to 33% in Caucasians [10,12,13]. A rare genetic variant of LQTS, KCNE1-D85N, was originally reported by Tesson et al. [14], and we have previously demonstrated that the prevalence of the SNP is 0.8% in healthy Japanese individuals and 3.9% in clinically diagnosed LQTS probands [9]. In a heterologous mglur system with Xenopus oocytes, KCNE1-D85N has been shown to reduce IKs by approximately 50% [5]. In our previous experiments [9] using CHO cells, D85N also significantly reduced IKs by 28% (P<0.05 vs. WT), although this was a smaller reduction than that shown in Xenopus oocytes [5]. In contrast, when KCNH2-WT was co-expressed with the variant, it was found to decrease IKr significantly by 31–36% (P<0.005 vs. WT) [9]. Regarding the current reduction, the interaction between KCNE1 and KCNH2 was, therefore, stronger than that between KCNE1 and KCNQ1. Since in the range of a normal heart rate IKr plays a more essential role for ventricular repolarization than IKs, carriers of loss-of-function KCNH2 mutations generally display longer QT prolongation and bradycardia than those of KCNQ1 mutations. The present study attests that 3.9% of the LQTS subjects genotyped had the KCNE1-D85N variant in addition to a LQT-related mutation. The average QT interval of KCNE1-D85N carriers was longer than that of non-carriers. The incidence of symptoms in patients with KCNE1-D85N was higher than that of the patients without KCNE1-D85N, although the differences were not significant. Here, we clinically evaluated the family members as a group with a relatively homogeneous genetic background and/or the same mutation, KCNH2-E58K, to show that the KCNE1-D85N polymorphism could act as a modifier. In fact, the proband and her daughter carried both KCNH2-E58K and KCNE1-D85N and had a longer QT interval than the proband\'s son, who carried KCNH2-E58K only. In a biophysical assay, KCNH2-WT/E58K induced a small decrease in current densities, compared to KCNH2-WT, suggesting no dominant negative suppression, but a small mutant effect (Fig. 2). Co-expression of KCNH2-WT with/without KCNH2-E58K and KCNE1-D85N showed a significantly negative shift of the activation curve compared to KCNH2-WT with KCNE1-WT (−6mV and −4mV, respectively); in other words, a gain of function. However, there was a massive decrease in current densities by KCNE1-D85N, about 60–65% (P<0.0001 vs. KCNH2-E58K and KCNE1-WT; Fig. 3B). Therefore, the latter change led to a loss-of-function effect by KCNE1-D85N. The proband and her daughter carried both these 2 genetic variants and both of them experienced TdP resulting in repeated syncope. From these clinical features, the D85N variant was suggested to aggravate the clinical phenotypes by largely reducing IKr. Thus, loss-of-function effects caused by the combination of the 2 genetic variants may explain the significant prolongation of the QTc intervals and the severe symptoms in the family. More recently, Yoshikane and his colleagues [15] reported a family in which 2 genetic variants were harbored in the presence or absence of the KCNE1-D85N polymorphism. They compared the symptoms among the family members who carried one or more of the genetic variants KCNH2-N45D, SCN5A-A1428S, or KCNE1-D85N. They demonstrated that only the proband carrying all 3 variants (triple hit) experienced ventricular fibrillation, and his ECG showed marked bradycardia. His brother and mother carried either KCNH2-N45D or SCN5A-A1428S, in addition to KCNE1-D85N (double hit). His father carried only KCNH2-N45D. All family members except for the proband remained asymptomatic. When compared, the phenotype of the proband\'s brother and father (both carry KCNH2-N45D, but D85N is present only in the brother), the QTc intervals were longer in the brother (500vs. 430ms) and a Holter ECG revealed the presence of bradycardia in the brother. Thus, KCNE1-D85N appeared to modify the disease phenotypes, providing another example of D85N as a genetic modifier of LQTS.