Traditional and targeted exome sequencing reveals common, rare and novel functional deleterious variants in RET-signaling complex in a cohort of living US patients with urinary tract malformations

Abstract

Signaling by the glial cell line-derived neurotrophic factor (GDNF)-RET receptor tyrosine kinase and SPRY1, a RET repressor, is essential for early urinary tract development. Individual or a combination of GDNF, RET and SPRY1 mutant alleles in mice cause renal malformations reminiscent of congenital anomalies of the kidney or urinary tract (CAKUT) in humans and distinct from renal agenesis phenotype in complete GDNF or RET-null mice. We sequenced GDNF, SPRY1 and RET in 122 unrelated living CAKUT patients to discover deleterious mutations that cause CAKUT. Novel or rare deleterious mutations in GDNF or RET were found in six unrelated patients. A family with duplicated collecting system had a novel mutation, RET-R831Q, which showed markedly decreased GDNF-dependent MAPK activity. Two patients with RET-G691S polymorphism harbored additional rare non-synonymous variants GDNF-R93W and RET-R982C. The patient with double RET-G691S/R982C genotype had multiple defects including renal dysplasia, megaureters and cryptorchidism. Presence of both mutations was necessary to affect RET activity. Targeted whole-exome and next-generation sequencing revealed a novel deleterious mutation G443D in GFRα1, the co-receptor for RET, in this patient. Pedigree analysis indicated that the GFRα1 mutation was inherited from the unaffected mother and the RET mutations from the unaffected father. Our studies indicate that 5% of living CAKUT patients harbor deleterious rare variants or novel mutations in GDNF-GFRα1-RET pathway. We provide evidence for the coexistence of deleterious rare and common variants in genes in the same pathway as a cause of CAKUT and discovered novel phenotypes associated with the RET pathway.

Figure 1. Identification of novel and rare variants in RET and GDNF

(A) Gene map of human RET, GDNF and SPRY1 depicting non-synonymous variants (arrows) identified in our CAKUT patient population (boxes represent exons). (B–F) Sequencing traces of the nonsynonymous variants in the RET and GDNF genes; the position of the nucleotide and the amino acid changes are indicated (arrows). Common/rare/novel represents the prevalence of the mutations in our patient population. (G) Description of the variants found in RET and GDNF along with the patient description and disease phenotype. Values in parenthesis indicate the variation frequency in 363 unrelated controls. Asterisk (*) indicates novel variant.

Figure 2. Malformations in patients with RET mutations have reduced MAPK activity

Biochemical analysis of RET mutants. Anti-RET, anti-GFRα1 and anti-phospho MAPK (pMAPK) immunoblot of total cell lysates from HEK293 cells transfected with plasmids expressing wild-type GFRa1 and different RET mutant constructs: (A) Wild type RET, RETR813Q and RET-G691S, R982C double mutant (DM), (B) RET-G691S, RET-R982C and RETA1105V and (C) RET-C618F (MEN2A) and RET-K758M (kinase dead, KD) as positive and negative controls of RET-MAPK activity, respectively. RET pathway activation was assessed by increase in pMAPK (doublet) on exposure with GDNF. GDNF-dependent increase in pMAPK is clearly seen in cells transfected with RET-WT (positive control). Note severe reduction in GDNF-dependent pMAPK activation in RET-R813Q and the RET-DM. The bar graphs quantifies the reduction in pMAPK activity in the mutants determined by the ratio of integrated density (ID) between phospho-MAPK (pMAPK) and total RET (doublet at 150 and 170kD), to normalize expression of transiently transfected plasmids (mean±SE, n = 4, *= p < 0.05). β-actin is shown as the loading control.

Figure 3. The novel RET-R813Q mutation is detected in 2 affected relatives and associated with duplication

(A) Pedigree featuring the family of the patient 07-1184-00391. Heterozygous G42368A missense mutation in RET leads to the deleterious R813Q change in patient 07-1184-00391. The same mutation is also present in the affected great granddaughter (07-1184-00371). (B) Ultrasonograph of patient 07-1184-00371 showing two distinct poles of the left kidney indicative of duplication. (C) Sequencing traces confirm RET-R813Q mutation (arrow) in the great granddaughter of patient 07-1184-00391 with both the forward and reverse primers.

Figure 4. The RET-R982C variant in patient 07-1184-00092 with the double RET-G691S, R982C genotype is inherited from the father and exome sequencing pipeline identifies a novel GFRα1 mutation

(A) Sequencing traces depict that the rare non-synonymous variants G691S (2 traces on the left) and R982C (2 traces on the right) in the RET gene is present in father of patient 07-1184-00092 but absent in the mother. Arrows point to the position of the C/T nucleotide change. (B) Whole-exome Capture Sequencing Metrics from the CAKUT patient 07-1184-00092 and filtering approach used to identify two novel mutations LAMA5P321S and GFRa1G443D.

Figure 5. The novel GFRα1-G443D mutation is conserved and inherited from the mother

(A) Gene map of Human GFRa1 gene depicting the novel non-synonymous G443D mutation (arrow). (B) Illustration shows the location of the GFRa1G443D mutation near the hydrophilic domain and GPI anchor site of GFRa1. (C) Comparison of the 3′ amino acid sequences of the human GFRa1 protein among species show this residue to be conserved in most mammals. GPI anchor site, hydrophilic domain and the Glycine residue at position 443 with respect to human GFRa1 protein are boxed. (D) Sequencing traces of the GFRa1G443D mutation (arrow) in patient 07-1184-00092, Father and Mother. Mother of the patient but not the father carried the GFRa1-G443D mutation.

Figure 6. Pedigree shows compound heterozygosity as a mechanism of inheritance of RET pathway mutation in the patient 07-1184-00092 with CAKUT and cryptorchidism and model summary of mechanism of CAKUT

(A) Pedigree depicting the family of patient 07-1184-00092 whereby both the RET mutations, RET-G691S and RET-R982C are inherited from the unaffected father while the GFRα1-G443D mutation is inherited from the unaffected mother. The brother does not have any RET mutations, while the sister inherited all the three mutations but her disease status is unknown (?). (B) Diagrammatic representation of proposed mechanism by which the various RET and GFRα1 mutations lead to genitourinary defects in the patient 07-1184-00092. The presence of the GFRα1-G443D and RET-G691S, R982C variations in the same patient results in GDNF-GFRα1-RET heterodimeric complexes with disrupted MAPK signaling thus reducing the availability of normal RET signaling complexes, similar to previously seen in mutant mice and observed in vitro (Figure 2). The reduced RET signaling leads to developmental defects including megaureters (ultrasonograph image of patient 07-1184-00092) and cryptorchidism as seen in this patient.