Mutations in DSTYK and dominant urinary tract malformations

Abstract

Background: Congenital abnormalities of the kidney and the urinary tract are the most common cause of pediatric kidney failure. These disorders are highly heterogeneous, and the etiologic factors are poorly understood.

Methods: We performed genomewide linkage analysis and whole-exome sequencing in a family with an autosomal dominant form of congenital abnormalities of the kidney or urinary tract (seven affected family members). We also performed a sequence analysis in 311 unrelated patients, as well as histologic and functional studies.

Results: Linkage analysis identified five regions of the genome that were shared among all affected family members. Exome sequencing identified a single, rare, deleterious variant within these linkage intervals, a heterozygous splice-site mutation in the dual serine-threonine and tyrosine protein kinase gene (DSTYK). This variant, which resulted in aberrant splicing of messenger RNA, was present in all affected family members. Additional, independent DSTYK mutations, including nonsense and splice-site mutations, were detected in 7 of 311 unrelated patients. DSTYK is highly expressed in the maturing epithelia of all major organs, localizing to cell membranes. Knockdown in zebrafish resulted in developmental defects in multiple organs, which suggested loss of fibroblast growth factor (FGF) signaling. Consistent with this finding is the observation that DSTYK colocalizes with FGF receptors in the ureteric bud and metanephric mesenchyme. DSTYK knockdown in human embryonic kidney cells inhibited FGF-stimulated phosphorylation of extracellular-signal-regulated kinase (ERK), the principal signal downstream of receptor tyrosine kinases.

Conclusions: We detected independent DSTYK mutations in 2.3% of patients with congenital abnormalities of the kidney or urinary tract, a finding that suggests that DSTYK is a major determinant of human urinary tract development, downstream of FGF signaling. (Funded by the National Institutes of Health and others.).

Figure 1. Identification of DSTYK Mutations in a Family with Congenital Abnormalities of the Kidney and Urinary Tract and the Spectrum of Mutations and Phenotypes in Unrelated Patients

Panel A shows the pedigree of the study family. Squares represent male family members, circles female family members, black symbols affected persons, white symbols unaffected persons, gray symbols unknown phenotype, red solid circles DSTYK mutation carriers, and red open circles noncarriers of the mutation; slashes indicate deceased family members. Panel B shows the linkage analysis identifying five regions (arrows) of the genome reaching the maximal expected LOD score of 1.5. DSTYK is located in the chromosome 1q25–1q41 locus. Panel C shows a chromatogram of a DSTYK c.654+1 G→A mutation from genomic DNA. Sequence analysis of complementary DNA (cDNA) in mutation carriers shows the use of an alternative splice site in exon 2, leading to a 27-bp deletion (Panel D). Panel E shows the genomic structure of DSTYK and the location of pathogenic mutations identified in the present study. The exons encoding the kinase domain are shown in green. Representative ultrasonographic findings in mutation carriers are shown in Panels F, G, and H, with hypoplasia of the left kidney (Panel F, kidneys outlined by dashed lines) detected at birth in a girl with a p.R29Q mutation, bilateral hydronephrosis (Panel G, arrows) caused by ureteropelvic junction obstruction detected at birth in a girl with a c.655−3 C→T mutation, and hydronephrosis only of the left kidney (Panel H, arrow) caused by ureteropelvic junction obstruction in a 5-year old boy with a p.R29Q mutation. The intravenous pyelogram in Panel I shows blunting of fornices on the right side (white arrow) and calyceal dilatation on the left side (red arrow) in a 2-year-old boy with a p.W8X mutation.

Figure 2. DSTYK Expression in the Human and Mouse Urinary Tract and Small Interfering RNA (siRNA) Knockdown Studies

Panels A through D show DSTYK expression in the kidney and ureter of a 3-month-old child. Panel A shows the glomerulus (Glom) and macula densa (MD). Panel B shows the renal cortex with ascending limbs of loop of Henle (AL) and collecting ducts (CD). Panel C shows the CD in the renal medulla, with punctate staining at the basolateral side (arrows). Panel D shows the smooth-muscle layer (Sm) and urothelium (Ur) of the ureter. Panels E through H show DSTYK colocalizing with fibroblast growth factor (FGF) receptors. Immunofluorescence analysis in a developing murine kidney (embryonic day 15.5) shows DSTYK (red) colocalization with FGF receptor 1 (FGFR1; green) (Panel E), FGF receptor 2 (FGFR2; green) (Panels F, G, and H) and E-cadherin (Panel H; blue). In Panel I, DSTYK knockdown is shown to inhibit FGF-mediated phosphorylation of extracellular-signal-regulated kinase 1 and 2 (ERK 1 and 2); siRNA knockdown of DSTYK in 293T cells starved in serum-free medium shows a significant reduction in the DSTYK level at 96 hours after knockdown. FGF stimulation significantly augments phosphorylated ERK (pERK 1 and 2), but DSTYK knockdown abrogates FGF-mediated ERK phosphorylation. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as a loading control. MM denotes metanephric mesenchyme, and UB ureteric bud.