PMC10072264_01
Family pedigree. Males and females are indicated by squares and circles, respectively. The proband (P) is indicated by the arrow. The ages of cancer diagnosis (proband only) and the phenotypes of affected family members are reported under the corresponding symbols, when known. Diagonal slashes indicate deceased individuals. Individuals who underwent genetic analysis are indicated by dots and the genes found mutated are reported.
fonc-13-1102184-g001.jpg
PMC10072264_02_fonc-13-1102184-g001.jpg
f1
Following targeted genetic testing in the proband's parents, the c.1672G>T in ATM and the c.5406+6T>C in BRCA1 variants were found to have been inherited from the father and the mother, respectively ( Figure 1 ).
PMC10072264_02
Family pedigree. Males and females are indicated by squares and circles, respectively. The proband (P) is indicated by the arrow. The ages of cancer diagnosis (proband only) and the phenotypes of affected family members are reported under the corresponding symbols, when known. Diagonal slashes indicate deceased individuals. Individuals who underwent genetic analysis are indicated by dots and the genes found mutated are reported.
fonc-13-1102184-g001.jpg
PMC10072264_02_fonc-13-1102184-g001.jpg
f1
Following targeted genetic testing in the proband's parents, the c.1672G>T in ATM and the c.5406+6T>C in BRCA1 variants were found to have been inherited from the father and the mother, respectively ( Figure 1 ).
mRNA splicing analysis of the BRCA1 c.5406+6T>C variant. The capillary electrophoresis patterns of the cDNA fragments spanning BRCA1 exons 19 to 24 detected in LCLs from a BRCA1 wild type subject (A) and from c.5406+6T>C carriers (proband (B) and proband's brother (C)) are shown. The peaks corresponding to normal transcripts maintaining exons 22 and 23 (full-length, FL) and aberrant transcripts (Delta22 and Delta22,23) are indicated. The sequencing of the PCR products (D) confirmed the presence of Delta22, Delta22,23 and normal transcripts in carriers of the BRCA1 c.5406+6T>C variant.
fonc-13-1102184-g002.jpg
PMC10072264_02_fonc-13-1102184-g002.jpg
f2
The analysis revealed the up-regulation of two isoforms derived from the out-of-frame skipping of exon 22 (Delta22, major transcript) and the in-frame skipping of exons 22 and 23 (Delta22,23, minor transcript) ( Figure 2 ), both previously described as naturally occurring isoforms.
Assessment of allele-specific expression of the normal BRCA1 transcript. The common variant c.4837A>G, mapped to BRCA1 exon 16, was proven to be heterozygous in the genomic DNA (gDNA) of the proband's brother (upper panel). The specific amplification of cDNA fragment maintaining exon 22 (normal transcript) and including the c.4837A>G variant was performed using a forward primer in exon 16 and a reverse primer in exon 22. The sequencing of the RT-PCR product showed a mono-allelic expression of the normal transcript (lower panel), indicating a complete spliceogenic effect of the c.5406+6T>C variant.
fonc-13-1102184-g003.jpg
PMC10072264_02_fonc-13-1102184-g003.jpg
f3
The sequence analysis of these amplification products, showing a mono-allelic expression, allowed us to ascertain the lack of functional mRNA isoforms expressed by the allele carrying the c.5406+6T>C variant ( Figure 3 ) and to classify the variant as pathogenic (class 5) according to the BRCA1/2 Gene Variant Classification Criteria of the Evidence-based Network for the Interpretation of Germline Mutant Alleles (ENIGMA).