Identification of a homozygous intronic variant in SLC39A14 that leads to aberrant splicing (2-column figure)
A) Pedigree of Patient 1’s family. The square represents a male individual, the circle represents a female individual, the filled in circle designates Patient 1, and the double horizontal line between Patient 1’s parents indicates consanguinity.
B) Sanger sequencing chromatograms of genomic DNA demonstrate the homozygous intronic variant in SLC39A14 in Patient 1 and that both of her parents are heterozygous carriers of the variant.
C) qRT-PCR using RNA extracted from patient fibroblasts demonstrates significantly decreased SLC39A14 transcript levels surrounding the mutation (Sets 2 and 3) compared to proximal to the mutation (Set 1) (one-way ANOVA p<0.01; Tukey HSD p<0.01 for Set 1 versus Set 2, p<0.01 for Set 1 versus Set 3, p>0.05 for Set 2 versus Set 3).
D) Gel electrophoresis demonstrates that cDNA from Patient 1 fibroblasts contains an additional, larger band representing the included intronic sequence compared to cDNA from control fibroblasts. The red arrows indicate the abnormal splicing products that contain the entire intron between exons 5 and 6.
E) Sanger sequencing of cDNA from Patient 1 fibroblasts confirms inclusion of the intronic sequence between exons 5 and 6 (image modified from Sequencher).