Genetic Evidence Supporting the Role of the Calcium Channel, CACNA1S, in Tooth Cusp and Root Patterning

Virginie Laugel-Haushalter, Supawich Morkmued, Corinne Stoetzel, Véronique Geoffroy, Jean Muller, Anne Boland, Jean-François Deleuze, Kirsley Chennen, Waranuch Pitiphat, Hélène Dollfus, Karen Niederreither, Agnès Bloch-Zupan, Patimaporn Pungchanchaikul, Virginie Laugel-Haushalter, Supawich Morkmued, Corinne Stoetzel, Véronique Geoffroy, Jean Muller, Anne Boland, Jean-François Deleuze, Kirsley Chennen, Waranuch Pitiphat, Hélène Dollfus, Karen Niederreither, Agnès Bloch-Zupan, Patimaporn Pungchanchaikul

Abstract

In this study, we report a unique dominantly inherited disorganized supernumerary cusp and single root phenotype presented by 11 affected individuals belonging to 5 north-eastern Thai families. Using whole exome sequencing (WES) we identified a common single missense mutation that segregates with the phenotype in exon 6 of CACNA1S (Cav1.1) (NM_000069.2: c.[865A > G];[=] p.[Ile289Val];[=]), the Calcium Channel, Voltage-Dependent, L Type, Alpha-1s Subunit, OMIM ∗ 114208), affecting a highly conserved amino-acid isoleucine residue within the pore forming subdomain of CACNA1S protein. This is a strong genetic evidence that a voltage-dependent calcium ion channel is likely to play a role in influencing tooth morphogenesis and patterning.

Keywords: NGS; calcium ion channel; dental anomalies; human; mutations; patterning; rare disease.

Figures

FIGURE 1
FIGURE 1
A striking multiple cusps single root phenotype. Normal crown morphology (A,B) of the right upper second premolar (15) and first permanent molar (16) and of the right lower premolar (45) and first permanent molar (46) could be compared to the multicusp pattern phenotype clearly visible both in the upper (C 1_III.11, E 2_III.7) and lower (D 1_III.11, F 2_III.7) jaws. On the close up of the right premolar-molar area on panoramic radiographs (G control, H 1_III.11 affected) molars present a single root (1) pattern. Only the first lower permanent molar (46) shows a more apical root furcation leading to the taurodontic appearance of the tooth.
FIGURE 2
FIGURE 2
Pedigrees and sequences analysis of families 1–5. Black squares indicate the patients analyzed in this study. All available members from families 2 and 3 were analyzed by whole exome sequencing. In family 1 only the 2 individuals [indicated by a red arrow (1_II.11, 1_III.10) were analyzed by WES]. Families 4 and 5 were analyzed exclusively by Sanger sequencing. Electropherograms reveal a heterozygous missense mutation c.865A > G; p.Ile289Val in affected individuals (1_II.10, 1_III.10, 1_III.11, 2_II.5, 2_III.6, 2_III.7, 3_II.1, 3_II.2, 4_I.2, 4_II.3, 5_III.2).
FIGURE 3
FIGURE 3
CACNAS1 mutations and corresponding protein domain. (A) The CACNA1S human gene is located on chr1q32.1, extends over 73 kb, and contains 44 exons (each vertical black line denotes one exon). The position of the start codon (ATG) is indicated. The mutation detected in this study is highlighted by a red arrow on exon 6 (c.865A > G, p.Ile289Val). All other previously described mutations in the CACNA1S gene are symbolized by a single letter above the corresponding exon. (B) The corresponding protein domains are represented according to the PFAM database. (C) Schematic view of the CACNA1S encoded alpha 1 sub-unit of DHPR. The subunit has a total of four transmembrane domains (I, II, III, IV) composed by six segments (1–6) and three intracellular loop domains (loops I–II, loops II–III, and loops III–IV). The mutated isoleucine (red star) is located in the first pore-forming intramembrane domain close to one of the 3 amino-acids involved in calcium selectivity (green circles). The II_III loop interacts with RYR1 to allow excitation/contraction in muscle.
FIGURE 4
FIGURE 4
Multiple protein sequence alignment. The sequence alignment of the pore forming subdomain (position 280–301) amongst mammalian 25 species. The alignment is displayed using Jalview and colored according to the ‘ClustalX’ coloring scheme. The isoleucine mutated in the affected individuals (red arrow) is unchanged in all the species.

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