PMC7471930_01
(A) 4x HPF, H&E stain. Ovarian cystic teratoma consisting of epidermoid elements and mature neuroectodermal tissue with focal morphological characteristics of a yolk sac tumor. (B) 20x HPF, H&E stain. At higher magnification, the limited and focal tumoral area shows typical characteristics of the hepatoid variant of yolk sac tumor. It can usually be found in young females, associated with increased levels of serum alpha-fetoprotein (AFP). To note, the presence of great polygonal cells with a well represented eosinophilic cytoplasm and abundant presence of hyaline bodies. (C,D) 10 x HPF, H&E stain. Teratoma shows abundant mature neuroectodermal tissue interspersed in the stroma with different and well-differentiated structures such as hair follicles. The presence of these elements indicates a high maturation level of the neoplasia.
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F1
The cystic teratoma consisted of epidermoid elements, extensive areas of mature neuroectodermal tissue and a limited glandular area positive for AFP, with the typical histological features of yolk sac tumor (Figures 1A-D). Subsequently, ascites and peritoneal metastases were observed at CT scan again (Figure S1).
(A) Electrophoresis gel of the RT-PCR products of case and control sample with forward primer spanning exon 21-22 junction and reverse spanning exon 24-25 junction. Next to the gel the schemes corresponden to the amplified products. (B) Scheme explaining the mechanism of splicing of intron 22 and exon 23 in presence of the variant c.2393G>A; the figure shows the loss of the use of the canonical sites of exon 22 and 23, the generation of a de novo 3'SS and the activation of a cryptic 5' SS in intron 22 used with the canonical 3'SS of exon 24. This results in an aberrant exons junction with the incluion of part of intron 22 and the exclusion of exon 23 in the mature transcript. (C) Three-dimensional model of the FL and the aberrant spliced RB proteins predicted with EzMol, C-term domain is highlighted in red. (D) Electropherogram represents the sequence of the FL agarose band displayed in (A), c.2393 position is indicated in the rectangle (E) The graph shows the result of the densitometric analysis of the CASE gel bands displayed in (A); sum of the FL and aberrant bands was considered as the total and each value from both transcripts was compared to that. M, DNA molecular marker, CTR, healthy control sample, NTC, no-template control, FL, full length transcript.
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F2
Unexpectedly, in vitro splicing analysis, performed on blood RNA by RT-PCR, revealed the full skipping of exon 23 together with the retention of 11 nucleotides of intron 22 (r.2326_2489delins2325+1_2325+11) (Delta23 VGQK) (Figure 2A). The resulting transcript is generated by the activation of a cryptic 5'splice site located in the intron 22 (c.2325+12) together with the usage of the 3'SS of exon 24, with the skipping of the canonical 3'SS of exon 23 (Figure 2B). The 3D protein model of the aberrant splicing variant showed the loss of organization of the secondary structure of the C-term and, consequently, of the complexity of the tertiary structure (Figure 2C). Sequencing of the full-length transcript revealed the presence of both A/G nucleotides at position c.2393, suggesting a "leaky" effect of the variant (Figure 2D). Densitometric analysis performed with ImageJ on the gel bands showed the aberrant spliced transcript to be ~32% of the total one, indicating that the mutant allele is still able to produce a still relevant percentage of normal spliced transcript (Figure 2E).