Functional Studies of TRPM7 – a Candidate Gene for Stillbirth?

Stillbirth is classified as a baby born dead after 24 weeks gestation and over 2.6 million stillbirths occurred in 2015. Damaging mutations in genes that cause long QT syndrome (LQTS), short QT syndrome (SQTS), and Brugada Syndrome are known to cause sudden cardiac death (SCD) in adults and children. Prenatal diagnosis of LQT has been possible for decades, creating a disease spectrum where channelopathies may fatally influence pregnancy 1 . Our study aim was to identify genetic variants in cardiac ion channels that may cause stillbirth. We sequenced 35 candidate genes [12 LQTS genes and 13 candidate genes from genome‐wide association studies of QT interval and SCD] in 70 stillbirth cases. Thirty‐nine cases harboured a novel or low frequency predicted damaging missense variant. Two novel and two rare variants were detected in the transient receptor potential melastatin 7 (TRPM7) gene, these were selected for follow‐up functional studies. TRPM7 is an ion channel that conducts cations through a tetrameric pore and is required for cardiac development and conduction 2 . The four stillbirth variants (p.G179V, p.R494Q, p.T860M and p.E1205G) were inserted into a TRPM7 expression plasmid using site‐directed mutagenesis. Wild‐type (WT) and mutant channels were expressed in HEK293 and CHO‐K1 cells and whole‐cell patch clamp experiments were performed to assess channel function. TRPM7 mRNA and protein levels were measured using qRT‐PCR and western blotting, respectively. TRPM7 currents were recorded following transfection of HEK293 and CHO‐K1 cells. Gene expression analysis of TRPM7 mRNA in transfected HEK293 cells found similar levels between WT and variants (P = 0.6506). HEK293 cells expressing G179V or T860M variants showed significantly reduced current at +80mV compared to WT channels (39.2 & 36.3 pA/pF respectively, vs 133.2 pA/pF, P < 0.05). Conversely, in CHO‐K1 cells expressing the R494Q variant there was a significant increase in current density compared to WT channels (273.6 pA/pF vs 110.0 pA/pF, P < 0.0001). Western blot analyses failed to detect full length TRPM7 in HEK293 cells transfected with either G179V or T860M compared to WT, R494Q and E1205G expressing cells, where a 210kDa protein was detectable. Three TRPM7 variants were found to alter channel function. G179V and T860M reduced protein expression and current density when expressed in HEK293 cells. In CHO‐K1 cells, R494Q expression results in increased current density compared to WT. These differences in channel activity were observed despite similar levels of mRNA expression. These preliminary data suggests that our recently discovered pathogenic variants in TRPM7 could be a contributory causal factor in unexplained stillbirths. Support or Funding Information Research support: British Heart Foundation, Stillbirth & Neonatal Death Charity, Wellness of Women, UK.