Classification of the canonical splice alteration MUTYH c.934-2A>G is likely benign based on RNA and clinical data

MUTYH-associated polyposis (MAP) is an autosomal recessive disorder characterized by the development of multiple adenomatous colonic polyps and an increased lifetime risk of colorectal cancer. Germline biallelic pathogenic variants in MUTYH are responsible for MAP. The MUTYH c.934-2A>G (NM_001128425.1) variant, which is also known as c.850-2A>G for NM_001048174.2, has been identified in our laboratory in more than 800 patients, including homozygous and compound heterozygote carriers. The variant was initially classified as a variant of uncertain significance (VUS) due to lack of a MAP phenotype in biallelic carriers. In two unrelated female patients who were heterozygous carriers of this variant, further testing by RNA sequencing identified an aberrant transcript with a deletion of 9 nucleotides at the start of exon 11 (MUTYH r.934_942del9). This event is predicted to lead to an in-frame loss of 3 amino acids in a non-critical domain of the protein. This was the only splice defect identified in these patients which was not present in the controls and the aberrant transcript is derived exclusively from the variant allele, strongly supporting the cause of this splice defect as being the intronic variant, MUTYH c.934-2A>G. The splicing analysis demonstrating a small in-frame skipping of 3 amino acids in a non-critical domain along with the absence of a MAP phenotype in our internal cohort of biallelic carriers provides evidence that the variant is likely benign and not of clinical significance.

MIA3 Splice Defect in Cane Corso Dogs with Dental-Skeletal-Retinal Anomaly (DSRA)

We investigated a hereditary syndrome in Cane Corso dogs. Affected dogs developed dental-skeletal-retinal anomaly (DSRA), clinically characterized by brittle, discolored, translucent teeth, disproportionate growth and progressive retinal degeneration resulting in vision loss. Combined linkage and homozygosity mapping delineated a 5.8 Mb critical interval. The comparison of whole genome sequence data of an affected dog to 789 control genomes revealed a private homozygous splice region variant in the critical interval. It affected the MIA3 gene encoding the MIA SH3 domain ER export factor 3, which has an essential role in the export of collagen and other secreted proteins. The identified variant, XM_005640835.3:c.3822+3_3822+4del, leads to skipping of two exons from the wild type transcript, XM_005640835.3:r.3712_3822del. Genotypes at the variant were consistent with monogenic autosomal recessive mode of inheritance in a complete family and showed perfect genotype-phenotype association in 18 affected and 22 unaffected Cane Corso dogs. MIA3 variants had previously been shown to cause related phenotypes in humans and mice. Our data in dogs together with the existing functional knowledge of MIA3 variants in other mammalian species suggest the MIA3 splice defect and a near complete loss of gene function as causative molecular pathomechanism for the DSRA phenotype in the investigated dogs.

In or Out? New Insights on Exon Recognition through Splice-Site Interdependency

Noncanonical splice-site mutations are an important cause of inherited diseases. Based on in vitro and stem-cell-based studies, some splice-site variants show a stronger splice defect than expected based on their predicted effects, suggesting that other sequence motifs influence the outcome. We investigated whether splice defects due to human-inherited-disease-associated variants in noncanonical splice-site sequences in ABCA4, DMD, and TMC1 could be rescued by strengthening the splice site on the other side of the exon. Noncanonical 5′- and 3′-splice-site variants were selected. Rescue variants were introduced based on an increase in predicted splice-site strength, and the effects of these variants were analyzed using in vitro splice assays in HEK293T cells. Exon skipping due to five variants in noncanonical splice sites of exons in ABCA4, DMD, and TMC1 could be partially or completely rescued by increasing the predicted strengths of the other splice site of the same exon. We named this mechanism “splicing interdependency”, and it is likely based on exon recognition by splicing machinery. Awareness of this interdependency is of importance in the classification of noncanonical splice-site variants associated with disease and may open new opportunities for treatments.