Homozygosity Mapping and Whole Exome Sequencing Reveal a Novel Homozygous <i>COL18A1 - Figure 3 </i> Mutation Causing Knobloch Syndrome

<p>a. Sanger sequencing of <i>COL18A1</i> mutation NM_130445.2:c.3825_3838del:p.Ser1276Alafs*9 (Exon 42) comparing a wild type sequence (top), homozygous deletion in index patient IV-1 (middle) and heterozygous deletion in father of the index patient (bottom). b. Conservation of the deleted nucleotides across various species. c. Alignment of three isoforms of <i>COL18A1</i> and endostatin. The highlighted region is deleted in patient IV-1.</p

Homozygosity Mapping and Whole Exome Sequencing Reveal a Novel Homozygous <i>COL18A1</i> Mutation Causing Knobloch Syndrome

<div><p>The aim of this study was to identify the genetic basis of a chorioretinal dystrophy with high myopia of unknown origin in a child of a consanguineous marriage. The proband and ten family members of Iranian ancestry participated in this study. Linkage analysis was carried out with DNA samples of the proband and her parents by using the Human SNP Array 6.0. Whole exome sequencing (WES) was performed with the patients’ DNA. Specific sequence alterations within the homozygous regions identified by whole exome sequencing were verified by Sanger sequencing. Upon genetic analysis, a novel homozygous frameshift mutation was found in exon 42 of the <i>COL18A1</i> gene in the patient. Both parents were heterozygous for this sequence variation. Mutations in <i>COL18A1</i> are known to cause Knobloch syndrome (KS). Retrospective analysis of clinical records of the patient revealed surgical removal of a meningocele present at birth. The clinical features shown by our patient were typical of KS with the exception of chorioretinal degeneration which is a rare manifestation. This is the first case of KS reported in a family of Iranian ancestry. We identified a novel disease-causing (deletion) mutation in the <i>COL18A1</i> gene leading to a frameshift and premature stop codon in the last exon. The mutation was not present in SNP databases and was also not found in 192 control individuals. Its localization within the endostatin domain implicates a functional relevance of endostatin in KS. A combined approach of linkage analysis and WES led to a rapid identification of the disease-causing mutation even though the clinical description was not completely clear at the beginning.</p></div

Classification of the type of homozygous variants obtained by linkage analysis and whole exome sequencing.

<p>Classification of the type of homozygous variants obtained by linkage analysis and whole exome sequencing.</p

Coverage of sequencing reads in <i>COL18A1</i> including a 14 bp deletion (NM_130445.2:c.3825_3838del:p.Ser1276Alafs*9 (Exon 42) as seen on Alamut Viewer 2.2.0 (Upper panel).

<p>A loss of multiple strong ESEs is predicted due to the deletion (Lower panel).</p

Reconstruction of novel transcripts by <i>de-novo</i> assembly.

<p>Histograms display lengths of reconstructed sequence contigs assembled from unmapped reads of NB2 stage 4 and stage 4S samples (y-axis in log-space). Two independent assembly methods, Trinity and Oases, were used in the reconstruction. The grand total number of contigs reconstructed within each assembly is displayed in the rightmost column. Reconstructed contigs are annotated with their putative taxonomic origin as inferred by comparison with NCBI nucleotide (nt) and protein (nr) archives using TBLASTX database searches.</p

Rare Mutations of <i>CACNB2</i> Found in Autism Spectrum Disease-Affected Families Alter Calcium Channel Function

<div><p>Autism Spectrum Disorders (ASD) are complex neurodevelopmental diseases clinically defined by dysfunction of social interaction. Dysregulation of cellular calcium homeostasis might be involved in ASD pathogenesis, and genes coding for the L-type calcium channel subunits Ca_V1.2 (<i>CACNA1C)</i> and Ca_Vβ2 (<i>CACNB2</i>) were recently identified as risk loci for psychiatric diseases. Here, we present three rare missense mutations of <i>CACNB2</i> (G167S, S197F, and F240L) found in ASD-affected families, two of them described here for the first time (G167S and F240L). All these mutations affect highly conserved regions while being absent in a sample of ethnically matched controls. We suggest the mutations to be of physiological relevance since they modulate whole-cell Ba²⁺ currents through calcium channels when expressed in a recombinant system (HEK-293 cells). Two mutations displayed significantly decelerated time-dependent inactivation as well as increased sensitivity of voltage-dependent inactivation. In contrast, the third mutation (F240L) showed significantly accelerated time-dependent inactivation. By altering the kinetic parameters, the mutations are reminiscent of the <i>CACNA1C</i> mutation causing Timothy Syndrome, a Mendelian disease presenting with ASD. In conclusion, the results of our first-time biophysical characterization of these three rare <i>CACNB2</i> missense mutations identified in ASD patients support the hypothesis that calcium channel dysfunction may contribute to autism.</p></div

Cellular composition of the spleen from naive and <i>T. cruzi</i>- infected mice.

<div><p>Single spleen cell suspensions from naive mice (A) and from mice infected on day 24 with <i>T. cruzi</i> (B) were analysed by flow cytometry.</p> <p>Cells were stained for both CD4 and CD8, for CD19, for CD11b, or with propidium iodide (PI) and Annexin V (AxV).</p> <p>Numbers indicate percentage of cells expressing the respective marker.</p> <p>B6, C57BL/6 mice; F1, B6D2F1 mice.</p></div

Voltage-dependent steady-state inactivation (a, b) of Ba²⁺ currents through L-type calcium channels.

<p>The ASD mutants p.G167S (N = 4) and p.S197F (N = 6) showed a significantly flattened slope of voltage-dependent inactivation compared to β2d_WT (N = 7). The third mutation β2dE7c_F240L. (N = 5) did not obviously differ from its corresponding β2dE7c_WT (N = 2). Half-inactivation potentials (V0.5_inact) (<b>c</b>) and the slope factors dV (<b>d</b>) were obtained from the fits of individual experiments using the Boltzmann equation and averaging the results. Asterisk (*) marks a statistical significance (P<0.05) compared to the respective WT.</p

Splice scheme of human <i>CACNB2</i> resulting in 9 splice variants of the Ca_Vβ2-subunit with the localization of the three mutations.

<p>Spliced exons are shown in light grey and conserved exons in dark grey. All nine splice variants express the mutation-carrying exon 5, while three of variants contain the localization of the third mutation in exon 7c <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0095579#pone.0095579-Buraei1" target="_blank">[10]</a>.</p

Clinical Features of ASD patients with rare mutations in <i>CACNB2</i>.

<p><b>ADI-R</b>, Autism Diagnostic Interview-Revised; <b>ADOS</b>, Diagnostic Observation Schedule-Generic; <b>MRI,</b> magnetic resonance imaging.</p