Figure 2.

Evaluation of the Effect of the MYLK Variants Identified

(A) Immunohistochemistry performed in control specimens with a specific MYLK antibody (1:100; Thermo Fisher Scientific) showed that MYLK is present in SMCs that constitute muscular structures of the intestine and the bladder, including muscularis mucosa, blood vessels, and circular and longitudinal muscles of the muscularis propria. Similar patterns and expression levels were present throughout all developmental stages included in this study.

(B) MYLK is absent in the intestine and bladder of subject 1 (II-2 in Figure 1A).

(C) H&E staining and immunostainings performed in specimens derived from control samples and subject 1 with specific antibodies against neurofilament (1:600; Monosan), c-Kit/CD117 (1:200; Cell Marque), tryptase (1:1,600; Dako), and ACTA2 (ready to use; Dako) showed no significant differences.

(D) Splicing assays revealed impaired splicing of exon 23 of MYLK in subject 3 (II-5 in Figure 1A). SD6 and SA2 primers (Table S3) were used for evaluating transcripts produced by the wild-type (WT) vector, the mutant (Mut) vector containing the putative splice-site variant c.3985+5C>A, the empty vector (Empty), and untransfected cells (Un) according to a standard PCR protocol.