Fig. 2

Segregation analysis of the FAM111A c.81 G > A variant in the family, FAM111A transcript variants, and FAM111A transcript analysis in patient and control fibroblasts. A Partial sequence electropherograms demonstrating the FAM111A NM_001312909.2:c.81 G > A variant in the homozygous state in leukocyte- and fibroblast-derived DNA of patients 1 and 2, and in the heterozygous state in leukocyte-derived DNA of healthy parents (mother and father). The exon-intron boundary is indicated by a black line. Arrows point to the G-to-A change at the last exon position. B The MANE FAM111A transcript (top) and the four FAM111A transcript variants (TVs) expressed in cultured fibroblasts are shown, all encoding the same FAM111A protein. Exons are given by boxes and are numbered. The coding region is indicated in blue, untranslated regions in gray. Expression levels of FAM111A TVs in cultured fibroblasts according to the GTEx database (last accessed 08/2024) are shown on the right. Length of the bars represents the rate of expression (violet, strong expression; gray, no expression). Primers used for RT-PCRs are indicated by arrows above the transcript variants. The expected PCR amplicon sizes using the primer combination F1 and R1 (444 bp and 405 bp) and F2 and R1 (333 bp) are depicted in the lower left and the lower right panel, respectively. C 2% agarose gel showing RT-PCR amplicons generated with primers F1 and R1 using fibroblast-derived cDNA from patients (P1, P2) and three controls (C1-C3). Fibroblasts were either treated with cycloheximide (CHX, +) or DMSO (─) prior to RNA isolation. In control cells, the expected RT-PCR products of 444 bp and 405 bp were amplified. In contrast, a major amplicon of ~300 bp was obtained from cDNA of patient-derived cells. D Partial sequence electropherogram of an aberrantly spliced FAM111A transcript in patient 1. Direct sequencing of the 287-bp RT-PCR amplicon obtained with primers F1 and R1 revealed skipping of the r.81 g > a change containing exon 3 (NM_022074.4) or exon 4 (NM_001374804.1) in FAM111A transcripts. E 2% agarose gel showing RT-PCR amplicons generated with primers F2 and R1 using fibroblast-derived cDNA from patients (P1, P2) and three controls (C1-C3). Fibroblasts were either treated with CHX (+) or DMSO (─) prior to RNA isolation. The expected RT-PCR product of 333 bp was amplified from cDNA of control and patient cells. A second amplicon of ~300 bp was obtained from patient-derived cDNAs. F Cloning of patient 1-derived RT-PCR amplicons followed by colony PCR and Sanger sequencing of individual amplicons identified the larger amplicon (333 bp) as transcripts in which exon 3 with the r.81 g > a variant (indicated by an arrow) was correctly spliced to exon 4 (upper electropherogram). The smaller amplicon (301 bp) corresponds to aberrantly spliced FAM111A transcripts lacking the last 32 bp of exon 3 (Δ32 bp; lower electropherogram). bp base pairs, F forward primer, R reverse primer, TPM transcripts per million