case_images: PMC7140378_01
case_images: Pedigree of family 1 and 2, and segregation analyses of the APC variant c.289G>A, p.(Gly97Arg). Abbreviations: CRC: Colorectal cancer, adn: Colonic adenomas, DC: Duodenal cancer, LC: Hepatic cancer, MC: Breast cancer, EC: Endometrial cancer, PC: Pancreatic cancer, GC: Gastric cancer, n/a: Not available. Numbers in () refer to age at diagnosis (or death, if the person did not develop cancer). +: APC variant c.289G>A carrier, ?: APC variant c.289G>A carrier status unknown
case_images: 13053_2020_140_Fig1_HTML.jpg
case_images: PMC7140378_01_13053_2020_140_Fig1_HTML.jpg
case_images: Fig1
case_images: The family history included at least three additional cases of gastrointestinal cancers (Fig. 1; Table 1). Subsequent segregation analyses showed that a third sibling (II-III) in family 1 also carried the variant, as well as two individuals in family 2 (II-I and III-II); pedigrees can be seen in Fig. 1.
case_images: RNA and allele-specific analyses of the APC c.289G>A variant. a Gel electrophoresis analysis of RT-PCR products from an APC c.289G>A carrier and two controls (C1 and C2). b Sanger sequencing of the upper RT-PCR product. c Sanger sequencing of lower RT-PCR product. d Capillary electrophoresis analysis of the RT-PCR products from an APC c.289G>A carrier and two controls (C1 and C2). FL: Reference full-length transcript, 3p(- 70): Transcript lacking 70 bp of exon 3. e Sanger sequence of an RT-PCR product using primers designed to target the skipped region in exon 3 and to amplify the heterozygote c.289G>A variant in the carrier. The Sanger sequence show a monoallelic contribution of the wt allele
case_images: 13053_2020_140_Fig2_HTML.jpg
case_images: PMC7140378_01_13053_2020_140_Fig2_HTML.jpg
case_images: Fig2
case_images: The RT-PCR analysis detected two major transcripts in the APC c.289G>A carrier, while only one transcript was identified in the two control samples (Fig. 2a). Sanger sequencing revealed that the upper transcript corresponded to the reference full-length (572 bp), while the lower band represented a transcript lacking 70 bp of exon 3 (502 bp (3p-70), Fig. 2b, c). The data moreover indicated that the 3p(- 70) transcript contributed to ~ 50% of the total transcript expression in the carrier (Fig. 2d). To further determine the contribution of the c.289G>A variant allele to APC reference transcript expression we Sanger sequenced an RT-PCR product using primers located in the skipped region of exon 3 and in exon 4 (primer sequences are available upon request) from the carrier (Fig. 2e).