Single nucleotide variations in CLCN6 identified in patients with benign partial epilepsies in infancy and/or febrile seizures

Nucleotide alterations in the gene encoding proline-rich transmembrane protein 2 (PRRT2) have been identified in most patients with benign partial epilepsies in infancy (BPEI)/benign familial infantile epilepsy (BFIE). However, not all patients harbor these PRRT2 mutations, indicating the involvement of genes other than PRRT2. In this study, we performed whole exome sequencing analysis for a large family affected with PRRT2-unrelated BPEI. We identified a non-synonymous single nucleotide variation (SNV) in the voltage-sensitive chloride channel 6 gene (CLCN6). A cohort study of 48 BPEI patients without PRRT2 mutations revealed a different CLCN6 SNV in a patient, his sibling and his father who had a history of febrile seizures (FS) but not BPEI. Another study of 48 patients with FS identified an additional SNV in CLCN6. Chloride channels (CLCs) are involved in a multitude of physiologic processes and some members of the CLC family have been linked to inherited diseases. However, a phenotypic correlation has not been confirmed for CLCN6. Although we could not detect significant biological effects linked to the identified CLCN6 SNVs, further studies should investigate potential CLCN6 variants that may underlie the genetic susceptibility to convulsive disorders.Toshiyuki Yamamoto, Keiko Shimojima, Noriko Sangu, Yuta Komoike, Atsushi Ishii, Shinpei Abe, Shintaro Yamashita, Katsumi Imai, Tetsuo Kubota, Tatsuya Fukasawa, Tohru Okanishi, Hideo Enoki, Takuya Tanabe, Akira Saito, Toru Furukawa, Toshiaki Shimizu, Carol J. Milligan, Steven Petrou, Sarah E. Heron, Leanne M. Dibbens, Shinichi Hirose, Akihisa Okumur

Morphological, histological and gene-expression analyses on stolonization in the Japanese Green Syllid, Megasyllis nipponica (Annelida, Syllidae)

Abstract Benthic annelids belonging to the family Syllidae (Annelida, Errantia, Phyllodocida) exhibit a unique reproduction mode called “schizogamy” or “stolonization”, in which the posterior body part filled with gametes detaches from the original body, as a reproductive unit (stolon) that autonomously swims and spawns. In this study, morphological and histological observations on the developmental processes during stolonization were carried out in Megasyllis nipponica. Results suggest that the stolon formation started with maturation of gonads, followed by the formation of a head ganglion in the anteriormost segment of the developing stolon. Then, the detailed stolon-specific structures such as stolon eyes and notochaetae were formed. Furthermore, expression profiles of genes involved in the anterior–posterior identity (Hox genes), head determination, germ-line, and hormone regulation were compared between anterior and posterior body parts during the stolonization process. The results reveal that, in the posterior body part, genes for gonadal development were up-regulated, followed by hormone-related genes and head-determination genes. Unexpectedly, Hox genes known to identify body parts along the anterior–posterior axis showed no significant temporal expression changes. These findings suggest that during stolonization, gonad development induces the head formation of a stolon, without up-regulation of anterior Hox genes

Electropherograms of the identified <i>CLCN6</i> variants confirmed by Sanger sequencing.

<p>Identified variants are shown in red.</p

Family trees of three families harboring <i>CLCN6</i> variants.

<p><i>CLCN6</i> variant-positive members are presented as (m+), and <i>CLCN6</i> variant-negative members are presented as (m-). Arrows indicate the proband in the family.</p

In vitro functional evaluation of SNVs effects.

<p>(A) Immunofluorescence staining of COS1 cells transfected with SNV-harboring <i>CLCN6</i> variants. Protein disulfide isomerase (PDI) is used as marker of the endoplasmic reticulum (ER). FLAG-tagged CLCN6 is merged with PDI, indicating CLCN6 localization in the ER. (B) Western blotting analysis of cell lysates shows no difference in CLCN6 expression.</p

Candidate genes selected by filtering.

<p>*, genomic positions are referred to build19; SNV, single nucleotide variation; NA, not applicable</p><p>Candidate genes selected by filtering.</p

Exon usage and location of <i>CLCN6</i> transcript variants.

<p>(A) Exon usage of four coding transcript variants. (B) Schematic representation of the locations of the SNVs identified in this study for each <i>CLCN6</i> transcript variant. Two exon-intron boundaries are highlighted to clarify the complicated exon usage in the region.</p