Table1_Case report: Sex-specific characteristics of epilepsy phenotypes associated with Xp22.31 deletion: a case report and review.docx

Deletion in the Xp22.31 region is increasingly suggested to be involved in the etiology of epilepsy. Little is known regarding the genomic and clinical delineations of X-linked epilepsy in the Chinese population or the sex-stratified difference in epilepsy characteristics associated with deletions in the Xp22.31 region. In this study, we reported two siblings with a 1.69 Mb maternally inherited microdeletion at Xp22.31 involving the genes VCX3A, HDHD1, STS, VCX, VCX2, and PNPLA4 presenting with easily controlled focal epilepsy and language delay with mild ichthyosis in a Chinese family with a traceable 4-generation history of skin ichthyosis. Both brain magnetic resonance imaging results were normal, while EEG revealed epileptic abnormalities. We further performed an exhaustive literature search, documenting 25 patients with epilepsy with gene defects in Xp22.31, and summarized the epilepsy heterogeneities between sexes. Males harboring the Xp22.31 deletion mainly manifested with child-onset, easily controlled focal epilepsy accompanied by X-linked ichthyosis; the deletions were mostly X-linked recessive, with copy number variants (CNVs) in the classic region of deletion (863.38 kb–2 Mb). In contrast, epilepsy in females tended to be earlier-onset, and relatively refractory, with pathogenic CNV sizes varying over a larger range (859 kb–56.36 Mb); the alterations were infrequently inherited and almost combined with additional CNVs. A candidate region encompassing STS, HDHD1, and MIR4767 was the likely pathogenic epilepsy-associated region. This study filled in the knowledge gap regarding the genomic and clinical delineations of X-linked recessive epilepsy in the Chinese population and extends the understanding of the sex-specific characteristics of Xp22.31 deletion in regard to epilepsy.</p

Karyotype analysis.

<p>(A) HUMSCs exhibited a normal Karyotype (46, XX). (B) A representative karyotype of tHUMSCs exhibited triploid and hypotriploid with 69 chromosomes. (×1000).</p

Cell proliferation analysis.

<p>HUMSCs and tHUMSCs were seeded in culture plates and the cell proliferation was assessed daily by MTT for 6 days. The proliferation rate was significantly increased in tHUMSCs compared with HUMSCs from day 2 to day 6. *P<0.01.</p

Analysis of telomerase activity.

<p>(A) The relative telomere length of HUMSCs was measure by qPCR, which was decreased as the cell passage number increased. *P<0.05 (B) There was no statistical difference in the relative telomere length of tHUMSCs among passages as analyzed by qPCR. (C) RT-PCR revealed the expression of hTERT in tHUMSCs at P50 (lane 4), while HUMSCs showed no detectable expression of hTERT at P3 (lane 5). The transcripts of GAPDH (glyceraldehyde-3-phosphate dehydrogenase) from tHUMSCs (lane 2) and HUMSCs (lane 1) were used as positive controls.</p

Tumor growth in vivo.

<p>(A) tHUMSCs formed tumors in the subcutaneous layer of the immunodeficient mice. (B) The tumor tissue exhibited fish-like texture. (C) HE staining showed round atypical cells, enlarged nuclei, decreased karyoplasm, and elevated mitotic index (arrow). (×200).</p

Immunohistochemical analysis of the tumor tissue.

<p>The tumor tissue was positive for cell proliferative marker Ki67 (brown nuclear staining) (A) mesenchymal tissue markers Vim (B), CK (C), and CD99 (D), neural tissue markers NF (E) and NES (F) (brown cytoplasmic staining), but it was negative for epithelial markers EMA (G) and Des (H). (×200).</p

Phenotypic characterization of HUMSCs and tHUMSCs.

<p>HUMSCs during continuous in vitro culture maintained the typical spindle shape at P3 (A) and P15 (B). (×100) Senescence-associated β-gal staining (arrows) was positive at P15 (C) (×200). tHUMSCs appeared to grow in clusters at day 7 (D) and in layers at day 120 (E) after treating with 3-MCA. At a lower cell density, tHUMSCs appeared round during continuous expansion (F). (×100).</p

In vitro life span of donor HUMSCs.

<p>The life span of cells was defined as the number of culture days before observation of senescence. Donor HUMSCs were treated with or without 3-MCA. HUMSCs treated with 3-MCA from donors 1 and 7, while still growing, were terminated at 318 days to allow for data analysis.</p