Data_Sheet_1_The TSC2 c.2742+5G>A variant causes variable splicing changes and clinical manifestations in a family with tuberous sclerosis complex.pdf

BackgroundTuberous sclerosis complex (TSC) is a genetic, variably expressed, multisystem disease characterized by benign tumors. It is caused by pathogenic variants of the TSC complex subunit 1 gene (TSC1) and the TSC complex subunit 2 gene (TSC2). Genetic testing allows for early diagnosis, genetic counseling, and improved outcomes, but it did not identify a pathogenic variant in up to 25% of all TSC patients. This study aimed to identify the disease-causing variant in a Han-Chinese family with TSC.MethodsA six-member, three-generation Han-Chinese family with TSC and three unrelated healthy women were recruited. A comprehensive medical examination, a 3-year follow-up, whole exome sequencing, Sanger sequencing, and segregation analysis were performed in the family. The splicing analysis results obtained from six in silico tools, minigene assay, and patients' lymphocyte messenger RNA were compared, and quantitative reverse transcription PCR was used to confirm the pathogenicity of the variant.ResultsTwo affected family members had variable clinical manifestations including a rare bilateral cerebellar ataxia symptom. The 3-year follow-up results suggest the effects of a combined treatment of anti-epilepsy drugs and sirolimus for TSC-related epilepsy and cognitive deficits. Whole exome sequencing, Sanger sequencing, segregation analysis, splicing analysis, and quantitative reverse transcription PCR identified the TSC2 gene c.2742+5G>A variant as the genetic cause. This variant inactivated the donor splice site, a cryptic non-canonical splice site was used for different splicing changes in two affected subjects, and the resulting mutant messenger RNA may be degraded by nonsense-mediated decay. The defects of in silico tools and minigene assay in predicting cryptic splice sites were suggested.ConclusionsThis study identified a TSC2 c.2742+5G>A variant as the genetic cause of a Han-Chinese family with TSC and first confirmed its pathogenicity. These findings expand the phenotypic and genetic spectrum of TSC and may contribute to its diagnosis and treatment, as well as a better understanding of the splicing mechanism.</p

Bioassays for tumor development in nude mice.

<p>Injection of M-LDF untreated and treated samples into the left axilla of nude mice. With untreated samples, tumors developed (2–3 cm in diameter, A1, Black arrow) and were removed for HE (A2) and immunofluorescent staining (AE1/AE3-PE and DAPI, A3) to confirm human origin. No tumor was found after one year (B1, green arrow) in the M-LDF treated samples. HE staining of tissues from the axilla showed normal cells (B2), and stained negative for AE1/AE3 (B3).</p

Immunofluorescence staining for detection of malignant cells in the salvaged blood.

<p>Blood was collected and washed with an automated salvaging device. Nucleated cells were stained with CK19-PE (white arrow) and CD133-FITC (yellow arrow) (A). Positive cells were eliminated after M-LDF treatment (B).</p

Ultrastructure of the M-LDF membrane.

<p>After filtration, the first layer membrane of M-LDF was examined by light microscopy (A) and transmission electron microscopy (B). A: Nucleated cells were mostly trapped in the superior 1/3 part of the membrane. B: The trapped cells were ruptured with cytoplasm leakage.</p

Identification of adherent cells with immunofluorescence staining.

<p>Cell suspensions were prepared by adding 8.74×10⁷ tumor cells from the same patient to the salvaged blood. Adherent cells were stained with AE1/AE3-PE and CD133-FITC. Adherent cells (both AE1/AE3⁺ and CD133⁺) that were characterized by invasive growth were found in 85% of samples (A), but none after the M-LDF treatment (B).</p

Patient characteristics in Protocols 2 and 3.

<p>Patient characteristics in Protocols 2 and 3.</p

Identification of adherent cells.

<p>Nucleated cells were isolated by density gradient centrifugation for cell culture. Adherent cells were identified as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0130864#pone.0130864.g002" target="_blank">Fig 2</a>. A: Adherent cells and/or clones (CK19⁺ and CD133⁺) were found in 65% of samples before (A), but none after the M-LDF treatment (B).</p

Morphology of the nucleated cells in blood before and after M-LDF treatment.

<p>A light microscopic view of nucleated cells (Wright's stain) (A). Naked cells were identified as blue and without circular red staining (white arrow). About 90% of cells were naked nuclei after M-LDF treatment vs 1% before the treatment (B).</p