Figure 4
Figure 4. Biallelic FANCL gene mutations from 3 families and their effect on RNA splicing. (A) RT-PCR analysis of the FANCL region harboring the mutation (c.375-2033C>G) that is shared by FA26 and FA13 (*). The germline mutation is displayed using Sanger sequencing. RT-PCR from FA26 and FA13 using primers located in exons 2 and 8 shows additional multiple products (triangles) that are longer and shorter than the correctly sized product. RT-PCR products from FA26 RNA were cloned and individual colonies representing different size products were sequenced. The sequences and their representation of the alternate splice patterns between exons 2 and 8 are aligned with the UCSC browser for the FANCL gene. Four unique and alternatively spliced products were identified. A larger product represents a 33-bp insertion (ins c.375-2033_2066), and this was generated using the splice donor signal created by the variant (CTAAT>GTAAT), and a TAG acceptor, 34 bases away (^). This region is expanded below with the thick rectangle, showing the bases inserted by the mutation. A second alternative transcript includes the 33-bp insertion (^) and an additional 61-bp insertion (#) from intron 5 (ins c.375-2300_2360), resulting from cryptic splice signals (GTAAG and TAG) on either side of this insertion. The minus strand is transcribed for FANCL. The third variant includes the 33-nt insertion but exon 4 is skipped, and in the fourth variant exons 4, 6, and 7 are skipping. Supplemental Figure 4 provides additional detail. (B) Homozygous, synonymous FANCL mutation in FA17 results in exon skipping. The Sanger sequence trace displays a genomic mutation, c.1092G>A (p.K364K) (*). RT-PCR analysis for the mutation shows only a smaller-than-expected product, and no product of reduced size is observed in the control lane. Sequence traces for the RT-PCR product are displayed along with a diagram showing the skipping of exon 13. The wild-type protein, along with the predicted mutant protein resulting from in-frame removal of 24 amino acids, is shown. (C) Mutations in the FANCL gene. The mutations identified in this study are on top of the FANCL coding region, displayed as exons. The ELF, DRWD, and RING finger domains23 are color-coded.

Biallelic FANCL gene mutations from 3 families and their effect on RNA splicing. (A) RT-PCR analysis of the FANCL region harboring the mutation (c.375-2033C>G) that is shared by FA26 and FA13 (*). The germline mutation is displayed using Sanger sequencing. RT-PCR from FA26 and FA13 using primers located in exons 2 and 8 shows additional multiple products (triangles) that are longer and shorter than the correctly sized product. RT-PCR products from FA26 RNA were cloned and individual colonies representing different size products were sequenced. The sequences and their representation of the alternate splice patterns between exons 2 and 8 are aligned with the UCSC browser for the FANCL gene. Four unique and alternatively spliced products were identified. A larger product represents a 33-bp insertion (ins c.375-2033_2066), and this was generated using the splice donor signal created by the variant (CTAAT>GTAAT), and a TAG acceptor, 34 bases away (^). This region is expanded below with the thick rectangle, showing the bases inserted by the mutation. A second alternative transcript includes the 33-bp insertion (^) and an additional 61-bp insertion (#) from intron 5 (ins c.375-2300_2360), resulting from cryptic splice signals (GTAAG and TAG) on either side of this insertion. The minus strand is transcribed for FANCL. The third variant includes the 33-nt insertion but exon 4 is skipped, and in the fourth variant exons 4, 6, and 7 are skipping. Supplemental Figure 4 provides additional detail. (B) Homozygous, synonymous FANCL mutation in FA17 results in exon skipping. The Sanger sequence trace displays a genomic mutation, c.1092G>A (p.K364K) (*). RT-PCR analysis for the mutation shows only a smaller-than-expected product, and no product of reduced size is observed in the control lane. Sequence traces for the RT-PCR product are displayed along with a diagram showing the skipping of exon 13. The wild-type protein, along with the predicted mutant protein resulting from in-frame removal of 24 amino acids, is shown. (C) Mutations in the FANCL gene. The mutations identified in this study are on top of the FANCL coding region, displayed as exons. The ELF, DRWD, and RING finger domains23  are color-coded.

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