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Case Reports
. 2020 Feb 28:11:146.
doi: 10.3389/fgene.2020.00146. eCollection 2020.

High-Throughput Sequencing to Detect Novel Likely Gene-Disrupting Variants in Pathogenesis of Sporadic Brain Arteriovenous Malformations

Concetta Scimone  1   2 , Luigi Donato  1   2 , Concetta Alafaci  1 , Francesca Granata  1 , Carmela Rinaldi  1 , Marcello Longo  1 , Rosalia D'Angelo  1   2 , Antonina Sidoti  1   2
Affiliations
Case Reports

High-Throughput Sequencing to Detect Novel Likely Gene-Disrupting Variants in Pathogenesis of Sporadic Brain Arteriovenous Malformations

Concetta Scimone et al. Front Genet. .

Abstract

Molecular signaling that leads to brain arteriovenous malformation (bAVM) is to date elusive and this is firstly due to the low frequency of familial cases. Conversely, sporadic bAVM is the most diffuse condition and represents the main source to characterize the genetic basis of the disease. Several studies were conducted in order to detect both germ-line and somatic mutations linked to bAVM development and, in this context, next generation sequencing technologies offer a pivotal resource for the amount of outputted information. We performed whole exome sequencing on a young boy affected by sporadic bAVM. Paired-end sequencing was conducted on an Illumina platform and filtered variants were validated by Sanger sequencing. We detected 20 likely gene-disrupting variants affecting as many loci. Of these variants, 11 are inherited novel variants and one is a de novo nonsense variant, affecting STK4 gene. Moreover, we also considered rare known variants affecting loci involved in vascular differentiation. In order to explain their possible involvement in bAVM pathogenesis, we analyzed molecular networks at Cytoscape platform. In this study we focus on some genetic point variations detected in a child affected by bAVM. Therefore, we suggest these novel affected loci as prioritized for further investigation on pathogenesis of bAVM lesions.

Keywords: brain arteriovenous malformations; exome; molecular signaling; pathogenic variants; vascular differentiation.

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Figures

Figure 1
Figure 1
Sanger validation of novel variants. Electropherograms show the novel variants detected by whole exome sequencing (WES) and confirmed by Sanger sequencing. Each panel refers to the single variant described at the bottom of the same panel. The underlined asterisked triplets indicate the codons affected by mutations. All variants were detected in heterozygous condition. (A) Variant detected in EFNA4 gene, c.103A>G, p.Ser35Gly. (B) Variant detected in NAXe gene,c.23T>C, p.Leu8Pro. (C) Variant detected in CLCN2 gene, c.739C>G,p.Gly247Arg. (D) Variant detected in IGFBP7 gene, c.506T>C, p.Ile169Thr. (E) Variant detected in TNXB gene, c.286C>G, p.Pro2096Ala (F) Variant detected in TRMT10B gene, c.200G>T,p.Arg67Ile. (G) Variant detected in TTLL11 gene, c.1985G>T, p.Gly662Val. (H) Variant detected in L2HGDH gene, c.718A>T, p.Ile240Phe. (I) Variant detected in AOC3 gene, c.1084G>A, p.Glu362Lys (J) Variant detected in FLRT3 gene, c.1943A>T, p.His648Leu. (K) Variant detected in STK4 gene, c.569G>A, p.Trp190Ter. (L) Variant detected in SLIT2 gene, c.139C>T,p.Arg47Cys.
Figure 2
Figure 2
Molecular network obtained by GeneMania plugin (Cytoscape). The network shows patterns of co-expression (violet), co-localization (blue), genetic interaction (green), pathway (light blue), predicted interaction (orange), and shared proteins domains (yellow), obtained from merger of genes affecting by likely gene-disrupting (LGD) variants. The eight novel loci were merged with those involved in TGFβ signaling (A) and those involved in angiogenesis and vessel differentiation (B). Color of each edge is in relation of the kind of interaction. Black nodes represent input genes. Gray nodes resulted from analysis. Details about nodes and edges as well as annotations are available in Tables 1–5, supplied in Supplementary Materials SM3 .
Figure 3
Figure 3
STK4 expression values and protein detection in control white blood cells (WBCs) and c.569G > A (p.Trp190Ter) WBCs. (A) The histogram shows the logarithmic STK4 relative expression values coming from real-time (RT)-PCR experiment, compared between wild-type and mutated sample groups. As reported, ANOVA test resulted significant (p-value = 2.455E−50). The presence of STK4 c.569G > A variant determines an expression reduction of about 20%, compared to the wild-type control. Reported values represent the mean of the three replicates. (B) Protein detection by western blot analysis showing a reduction of the STK4 protein synthesis in the heterozygous c.569G > A (p.Trp190Ter) mutation carrier. The integrated optical intensity (IOD) values are reported.
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