PMC6694456_01
Identification of
TRPM6 missense mutation as the cause of familial hypomagnesaemia.. (
A) Pedigree diagram showing proband (arrowed) who is homozygous for c.3292A>G p.(K1098E) missense mutation in TRPM6 and segregation of alleles from each parent. Parents were consanguineous (second cousins). (
B) Homozygosity mapping across chromosomes 1-22. Red bars indicate regions of homozygosity. Candidate genes
CLDN19 and
TRPM6 (arrowed) are located within regions of homozygosity. (
C) Chromatograms from proband (II:1) and sibling (II:2) showing
TRMP6 variant in homozygous and heterozygous state, respectively. (
D) Amino acid (AA) alignment showing conservation of the K1098 residue of
TRPM6.
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Following informed consent, whole exome sequencing (WES) was performed in the eldest sibling, II:1 ( Figure 1). TRPM6 within a large region of homozygosity by descent ( Figure 1). The missense variant was confirmed by Sanger sequencing, and cascade screening confirmed this variant was in its heterozygous state in both parents and both mildly affected siblings. In silico tools confirmed evolutionary conservation ( Figure 1) as well as the rarity and predicted pathogenicity of the variant ( Table 2).
The TRPM6 K1098E variant affects a conserved basic residue in the TRP domain.. (
A) Sequence alignments of TRP domain residues from human TRPM1-TRPM8. Fully-conserved residues are highlighted in black and semi-conserved residues in grey. TRPM6 K1098 is outlined in red. (
B) Cryo-EM structure of Ca
2+- and Na
+-bound TRPM2 (PDB 6CO7). One monomer of the homotetrameric channel is highlighted in orange and the co-ordinated Ca
2+ and Na
+ ions are shown as green and purple spheres, respectively. (
C) Superposition of the TRPM2 monomer (orange) and TRPM6 homology model (green). The TRP domain region (box and inset), wherein the TRPM6 K1098E variant lies, is shown to highlight the predicted structural homology between TRPM6 and TRPM2. The Ca
2+ ion (green sphere) is shown for orientation. (
D) Close up view of TRPM2 Q896 and E1110 involved in co-ordination of the Ca
2+ ion (green sphere) and R1114 (homologous to TRPM6 K1098, shown in (
E)) predicted to form a stabilising interaction within the TRP domain. (
F) Mutation of TRPM6 K1098 to glutamate (magenta) is predicted to disrupt stabilising TRP domain interactions. The relative position of the Ca
2+ ion (green sphere) in TRPM2 is shown in (
E) and (
F) for orientation.
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Using predictive modelling of the protein structure we were able to show that the lysine residue at position 1098 is predicted to form a stabilising interaction within the TRP domain, and that the missense mutation of TRPM6 K1098 to glutamate is predicted to disrupt this interaction ( Figure 2).