case_images: PMC5938031_01
case_images: The pedigree of the proband's family, cancer type, age onset, and MPS results are reported.. We were unable to test other affected family members because these relatives had died. Moreover, we tested proband's father to exclude the paternal origin of the variant and proband's brother to enroll in a surveillance program for cancer prevention
case_images: 41439_2018_3_Fig1_HTML.jpg
case_images: PMC5938031_01_41439_2018_3_Fig1_HTML.jpg
case_images: Fig1
case_images: In addition, family screening revealed the maternal origin of this variant and showed that the proband's brother was a healthy carrier of the c.5073T allele (Fig. 1).
case_images: a Results of the proband's genome obtained by MPS of the BRCA genes. Sequencing data, depth and variant calling were obtained by Amplicon Suite (Smart Seq, (Novara, Italy) software. b Capillary electrophoresis was used to check the amplification of the cDNAs of two women WT (b1) for the BRCA1 c.5073A>T variant, the proband (b2) and her brother (b3) carrier of the c.5073A>T variant. Arrows highlight the peaks corresponding to lower and upper markers and c.5073A/T alleles. In the control (b1) only the PCR product (499 bp) of the c.5073A allele is present. On the contrary, in the patient (b2) and her brother (b3), the mutated cDNAs gives two PCR products: the 499 bp band of the BRCA1 c.5073A allele and additional band of 652 bp due to intronic retention of 153 bp caused by the BRCA1 c.5073T allele. It is interesting to note that in both carriers, the proportion of the c.5073T allele is conserved (about 10% of the c.5073A allele). To verify the alternative splicing of the exon 17, Sanger sequencing was used c. The c.5073T allele causes the activation of a cryptic-splicing site resulting in a partial retention of 153 bp of intron 17. Moreover, we underline that the translation of the c.5073T allele stops after 42 bp (BRCA1 c.5074+42) due to the introduction of a stop codon (TAA codon) (as highlighted by the arrows)
case_images: 41439_2018_3_Fig2_HTML.jpg
case_images: PMC5938031_01_41439_2018_3_Fig2_HTML.jpg
case_images: Fig2
case_images: However, the patient carried a BRCA1 variant in exon 17 (c.5073 A > T; NCBI Reference Sequence: NM_007294.3; GRCh37; Fig. 2a). Moreover, an additional band longer than the WT allele appeared in the mutated cDNAs of two carriers (the proband and her brother) due to possible alternative splicing of exon 17 (Fig. 2b). However, this splicing error introduces a stop codon (TAA) 42 bp after the c.5073 A > T substitution (c.5074 + 42; Fig. 2c) terminating translation and results in a truncated protein.