Idiopathic Scoliosis Families Highlight Actin-Based and Microtubule-Based Cellular Projections and Extracellular Matrix in Disease Etiology.

Idiopathic scoliosis (IS) is a structural lateral spinal curvature of ≥10° that affects up to 3% of otherwise healthy children and can lead to life-long problems in severe cases. It is well-established that IS is a genetic disorder. Previous studies have identified genes that may contribute to the IS phenotype, but the overall genetic etiology of IS is not well understood. We used exome sequencing to study five multigenerational families with IS. Bioinformatic analyses identified unique and low frequency variants (minor allele frequency ≤5%) that were present in all sequenced members of the family. Across the five families, we identified a total of 270 variants with predicted functional consequences in 246 genes, and found that eight genes were shared by two families. We performed GO term enrichment analyses, with the hypothesis that certain functional annotations or pathways would be enriched in the 246 genes identified in our IS families. Using three complementary programs to complete these analyses, we identified enriched categories that include stereocilia and other actin-based cellular projections, cilia and other microtubule-based cellular projections, and the extracellular matrix (ECM). Our results suggest that there are multiple paths to IS and provide a foundation for future studies of IS pathogenesis

A <i>CACNA1C</i> Variant Associated with Reduced Voltage-Dependent Inactivation, Increased Ca_V1.2 Channel Window Current, and Arrhythmogenesis

<div><p>Mutations in <i>CACNA1C</i> that increase current through the Ca_V1.2 L-type Ca²⁺ channel underlie rare forms of long QT syndrome (LQTS), and Timothy syndrome (TS). We identified a variant in <i>CACNA1C</i> in a male child of Filipino descent with arrhythmias and extracardiac features by candidate gene sequencing and performed functional expression studies to electrophysiologically characterize the effects of the variant on Ca_V1.2 channels. As a baby, the subject developed seizures and displayed developmental delays at 30 months of age. At age 5 years, he displayed a QTc of 520 ms and experienced recurrent VT. Physical exam at 17 years of age was notable for microcephaly, short stature, lower extremity weakness and atrophy with hyperreflexia, spastic diplegia, multiple dental caries and episodes of rhabdomyolysis. Candidate gene sequencing identified a G>C transversion at position 5731 of <i>CACNA1C</i> (rs374528680) predicting a glycine>arginine substitution at residue 1911 (p.G1911R) of Ca_V1.2. The allele frequency of this variant is 0.01 in Malays, but absent in 984 Caucasian alleles and in the 1000 genomes project. In electrophysiological analyses, the variant decreased voltage-dependent inactivation, thus causing a gain of function of Ca_V1.2. We also observed a negative shift of V_1/2 of activation and positive shift of V_1/2 of channel inactivation, resulting in an increase of the window current. Together, these suggest a gain-of-function effect on Ca_V1.2 and suggest increased susceptibility for arrhythmias in certain clinical settings. The p.G1911R variant was also identified in a case of sudden unexplained infant death (SUID), for which an increasing number of clinical observations have demonstrated can be associated with arrhythmogenic mutations in cardiac ion channels. In summary, the combined effects of the <i>CACNA1C</i> variant to diminish voltage-dependent inactivation of Ca_V1.2 and increase window current expand our appreciation of mechanisms by which a gain of function of Ca_V1.2 can contribute to QT prolongation.</p></div

Location of the p.G1911R variant in the α_1C subunit of Ca_V1.2.

<p>A: Schematic of the Ca_V1.2 channel pore-forming α_1C subunit and the auxiliary α₂δ and β subunit. The p.G1911R variant is in the C-terminus close to the calcineurin (CaN) binding site. The location of other mutations in α_1C previously associated with TS are also indicated, including the most commonly reported p.G402R and p.G406R in the loop between DI and DII, and A1473G in the transmembrane segment 6 in the DIV. AID, α1 subunit interacting domain. B: Sanger sequencing of a normal control (WT) and the patient’s DNA showing the p.G1911R variant (arrow). C: Amino acid alignment shows conservation among species of glycine (G) at position 1911 also showing the CaN binding domain.</p

p.G1911R affects Ca_V1.2 availability and VDI.

<p>A–B: Current-voltage relationship and representative current traces showing an increase in current density at more negative potentials. C: voltage-dependence of activation and steady-state inactivation curves showing a hyperpolarizing and depolarizing shift, respectively leading to increased availability and window current. D: p.G1911R decreases voltage-dependent inactivation as measured by fitting a single exponential and comparing the tau value. Summarized in E. See <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0106982#pone-0106982-t001" target="_blank">Table 1</a> for values. *p<0.05.</p

Electrophysiological parameters for the wild type and p.G1911R mutant channel.

<p>Numbers tested are in parentheses.</p><p>*p<0.05 vs WT.</p><p>Electrophysiological parameters for the wild type and p.G1911R mutant channel.</p

Long QT and ventricular tachycardia.

<p>A: Baseline electrocardiogram showing prolonged QT interval. The heart rate is 97. The QTc is 504 ms. The ECG was recorded at 25 mm/s. B: Leads I and II showing monomorphic ventricular tachycardia on an ECG rhythm strip recorded in the Emergency Department.</p

Supplemental Material for Baschal et al., 2018

<p>Idiopathic scoliosis (IS) is a complex genetic disease of unknown etiology. We completed exome sequencing for five IS families and performed GO term enrichment analyses on the resulting variant lists. Overall, we identified enriched categories in our affected families that include stereocilia and other actin-based cellular projections, cilia and other microtubule-based cellular projections, and the extracellular matrix (ECM). Our results suggest that there are multiple paths to IS and provide a foundation for future studies of IS pathogenesis.</p><p></p