We have demonstrated significant associations between multiple exonic variants in the KCNMB1 gene and asthma severity as defined by baseline pulmonary function in two independent groups of African American asthmatics. Our results indicate a complex and important role of KCNMB1 genetic variation in pulmonary function, which is sexually dimorphic. Namely, we have observed a novel amino acid changing variant in the KCNMB1 gene, C818T R140W, which is strongly associated with a clinically significant decrease in FEV1 % of predicted value only among African American male subjects in two independent groups of asthmatics. In fact, subjects either heterozygous or homozygous for the 818T allele had a mean FEV1 of 80.2%, indicating moderate airway obstruction, compared with the mild airway obstruction exhibited by the 818 CC genotype group with a mean FEV1 of 92.0%. Furthermore, the KCNMB1 3′-UTR SNP, A1273G, was associated with increased FEV1 % of predicted value, also among male subjects. The additive increase in FEV1 by the A1273G variant was observed in both groups of asthmatic males. Additionally, we used PCA to create a summary variable (principal component) of pulmonary function representing FEV1, FVC, FEF25–75 and FEV1/FVC. Again we observed strong negative effects of the 818T allele on this general measure of pulmonary function, solidifying the effect of this SNP on African American males with asthma. Similar to C818T, SNP A1273G was also associated with the PCA generated measure of pulmonary function among males.
Our exploration of the potential effects of KCNMB1
genetic variants on pulmonary function was motivated by the vital role that BK channels play in the regulation of arterial and bronchial smooth muscle contraction and the subsequent studies relating genetic variants in the KCNMB1
gene with modulation of blood pressure. Our results are consistent with functional studies reporting the importance of BK channels in ASM contraction due to their high potassium conductance and abundant expression in ASM (14
). The ASM of β1 knock-out mice shows increased resting calcium levels and calcium influx during contraction, revealing the importance of this subunit in moderating airway constriction (14
Sexual dimorphism in asthma and related traits is well established. There are well-documented differences by sex with respect to lung development, age of asthma onset, asthma severity, and rate of pulmonary function decline (28
). Sex modification of genotype–phenotype associations in asthma has precedence including, male-specific associations between vitamin D receptor variants and both asthma and IgE levels, male-specific association of β2-adrenergic receptor polymorphisms and persistence of asthma, female-specific associations of estrogen receptor polymorphisms with both airway hyperresponsiveness and rate of lung function decline (32
). Moreover, a prior study of KCNMB1
G593A E65K variant indicated its effects were sex-specific and primarily present in post-menopausal women (22
). We propose that these effects maybe hormonally mediated since BK channel activity is upregulated by female sex hormones including estrogen and estradiol (23
) (Fig. A). The mechanism behind estrogen activation of BK channels is an acute non-genomic event triggered by a signaling cascade, which has been established in murine ASM cells (23
). Namely, estrogen stimulates the production of NO from Nitric oxide synthases, which then activates guanylate cyclases/protein kinase G (PKG) (36
). Subsequently PKG phosphorylates BK channels resulting in a dramatic 50-fold increase in BK channel activity (Fig. A) (23
). Indeed in women low estrogen levels experienced during the premenstrual luteal phase have been associated with both asthma exacerbation and decline in pulmonary function, as opposed to ovulation, when estrogen levels are highest, and asthma severity is decreased (37
). In our study the median age of females was 22.1 years with the oldest being 40 years, implying that all female participants are pre-menopausal. The upregulation of BK channel activity mediated by female sex hormones may insulate pre-menopausal women from the moderate functional effects of KCNMB1
variants, whereas these same moderate genetic effects may push males and post-menopausal women over a critical threshold resulting in disease, as observed in this study and in the study of hypertension (22
) (Fig. B).
Figure 5. Proposed sex-specific effects of C818T polymorphisms on airway smooth muscle (ASM) contractility. (A) BK channel α + β1-subunits are activated by local calcium from the sarcoplasmic reticulum, SR, (calcium-induced calcium release from (more ...)
Despite the replication of our genetic association with pulmonary function and sex modification, we sought to characterize the effect of the C818T coding variant on BK channel function as further validation of the clinical effects observed in this study. We found the β1140Trp
isoform (encoded by 818T allele) to confer a partial loss of function by significantly reducing open probability of the BK channel compared with the β1140Arg
isoform (encoded by 818C allele). This is consistent with the loss in pulmonary function observed in carriers of the 818T, since BK channels act as a brake on muscarinic signaling-induced bronchial smooth muscle contraction and tone (14
). These results give mechanistic and biologic plausibility to the genetic results observed for the C818T variant (Fig. ). Based upon these results we hypothesize that the increase in lung function observed for 1273 G allele carriers may be mediated by an upregulation in expression and/or activity of the BK channel. We did not find an association with SNP G593A despite prior reports of channel gain-of-function conferred by this variant and association with hypertension (21
). However, these results were obtained in populations of European origin where the G593A allele is present at a much higher frequency. The low allele frequency in African Americans coupled with the loss in power due to stratification by sex likely contributed to this lack of association. Further studies in a European population will be needed to confirm our result.
Interestingly, we found the associated 818T allele to be African-specific. In fact, we were unable to detect the 818T allele after screening large numbers of Caucasian, Asian, Puerto Rican, and Mexican subjects. This explains the undiscovered nature of this variant prior to our study despite multiple genetic studies of hypertension and KCNMB1
. Our results underscore the need to examine racially and ethnically diverse populations in genetic research. These racial specific variants may explain in part, along with social, economic, and environmental variables the significant disparities in asthma burden observed among African Americans (40
). Our results have important clinical and public health implications. In the US, African Americans have nearly the highest asthma morbidity and mortality rates with prior studies finding significantly lower FEV1
values among African American asthmatics compared with other ethnicities (42
). Based on our results 10% of African American males with asthma carry at least one copy of the 818T allele, potentially putting them at higher risk for asthma morbidity and mortality. These findings bring us closer to understanding the pharmacogenetic profiles of different populations and create the possibility for more tailored drug design for asthma. Indeed substances that activate BK channels through the β1-subunit, such as dehydrosoyasaponin-1 (43
) should be visited as potential asthma therapeutics, particularly for individuals carrying the 818T allele.
Furthermore, the effect of this variant maybe of additional significance in other disease states such as hypertension, which also disproportionately affects African American males (44
). The other associated SNP, A1273G, is common among other ethnic groups including Caucasians (11%), Mexicans (15%) and Puerto Ricans (15%); therefore the effects mediated by this SNP may extend to other populations as well (25
In conclusion, our results indicate that both the C818T and A1273G variants in the KCNMB1 gene strongly influence pulmonary function among African American males with asthma. We find these genetic effects to be restricted to male subjects, possibly due to hormonal differences. The effects of both the C818T and A1273G variants on baseline pulmonary function are clinically significant and as such are important indicators of asthma severity. Results of the functional studies of the C818T encoded isoform revealed a significant loss-of-function of the BK channel with the (T) allele. Further studies will be needed to establish the role of the A1273G variant in pulmonary function among other populations. Additionally, studies are warranted for bronchial hyperresponsiveness considering the established role of BK channel conductance in controlling cholinergic stimulated contraction of bronchial smooth muscle.