Mutations of the Transcriptional Corepressor ZMYM2 Cause Syndromic Urinary Tract Malformations

Abstract

Congenital anomalies of the kidney and urinary tract (CAKUT) constitute one of the most frequent birth defects and represent the most common cause of chronic kidney disease in the first three decades of life. Despite the discovery of dozens of monogenic causes of CAKUT, most pathogenic pathways remain elusive. We performed whole-exome sequencing (WES) in 551 individuals with CAKUT and identified a heterozygous de novo stop-gain variant in ZMYM2 in two different families with CAKUT. Through collaboration, we identified in total 14 different heterozygous loss-of-function mutations in ZMYM2 in 15 unrelated families. Most mutations occurred de novo, indicating possible interference with reproductive function. Human disease features are replicated in X. tropicalis larvae with morpholino knockdowns, in which expression of truncated ZMYM2 proteins, based on individual mutations, failed to rescue renal and craniofacial defects. Moreover, heterozygous Zmym2-deficient mice recapitulated features of CAKUT with high penetrance. The ZMYM2 protein is a component of a transcriptional corepressor complex recently linked to the silencing of developmentally regulated endogenous retrovirus elements. Using protein-protein interaction assays, we show that ZMYM2 interacts with additional epigenetic silencing complexes, as well as confirming that it binds to FOXP1, a transcription factor that has also been linked to CAKUT. In summary, our findings establish that loss-of-function mutations of ZMYM2, and potentially that of other proteins in its interactome, as causes of human CAKUT, offering new routes for studying the pathogenesis of the disorder.

Keywords: FIM; ZMYM2; ZNF198; congenital anomalies of the kidney and urinary tract; extra-renal features; genetic kidney disease; genomic analysis; syndromic CAKUT; transcription regulator; whole-exome sequencing.

Figure 1

Whole-Exome Sequencing Identifies 14 Heterozygous Loss-of-Function Mutations in ZMYM2 in 15 Families with 19 Affected Individuals (A) Clinical features of individuals with ZMYM2 mutations (see Table 1); family number is shown in the white rectangle. Family GM7: Hypertelorism; simple helix and protuberant ears; 5th fingers and thumbs; 5th finger clinodactyly. Family A4730: Wide eyebrows, mild synophrys, short filtrum; long nose with a bulbous tip; auricle with hypoplastic lobule; hyperextensibility of joints. Family GM13: Wide interpupillary distance and intercanthal distance; small auricle; clinodactyly. Family A1204: hematocolpos pre- and post-drainage. Family GM6: (GM6_21) dysmorphic facial features with epicanthi, short 5th digit with hypoplastic nails, abnormal palmar crease and sandal gap toe; (GM6_22) Dysmorphic features – epicanthi. (B) Exon structure of human ZMYM2 cDNA (GenBank: NM_197968.2) and positions of mutations (arrowheads). (C) Protein domain structure of human Zmym2 showing the positions of each of the 14 different heterozygous mutations identified in 15 families (position indicated by the arrows shafts). aa, amino acid; ATG, start codon; NLS, nuclear localization site.

Figure 2

Xenopus tropicalis Model of Zmym2 Loss of Function (A) Schematic of the experimental procedure for injection of morpholino into one cell of a two-cell embryo. One side of the embryo is subject to the knockdown, while the other serves as an internal control. (B and C) Representative images and quantitation of decreased pronephric area in one-sided zmym2 morphants. (D and E) Representative images and quantitation of decreased caudal atp1a1 signal in one-sided zmym2 morphants. (F) Schematic of the experimental procedure for injection of morpholino into a one-cell stage embryo. (G and H) Representative images and quantitation of craniofacial dysmorphology in zmym2 morphants, and frequency of rescue of this phenotype in zmym2 morphants co-injected with ZMYM2 mRNA. (I and J) Representative images and quantitation of cloacal exstrophy in zmym2 morphants. (K) Quantitation of proximal pronephric size abnormalities comparing the ratio of proximal pronephric size on the mRNA versus MO only side of an embryo between those injected with mock mRNA, control missense mutants, and those injected with the human ZMYM2 mRNA variants representing truncating mutants. Scale bars depict 500 μm. ^∗∗∗∗p < 0.0001, ^∗∗p < 0.005, ^∗p < 0.05 by unpaired t test (C, K) and Fisher’s exact test (E, H, J). Bars indicate mean and standard deviation.

Figure 3

Array of CAKUT Phenotypes Observed in a Zmym2^+/− Mutant Mouse Model Zmym2^+/− mice heterozygous for a frameshift mutation in exon 3 were analyzed at embryonic stage E18.5 and post-natal stage P0. (A) Percentages of mice with given CAKUT phenotype observed in Zmym2^+/− pups and their wild-type littermates. Statistical analysis was done using a binomial test. For CAKUT, hydroureter, duplex, and cystic kidneys phenotypes were compiled from Zmym2^+/+ (n = 35) and Zmym2^+/− (n = 37). Vesicoureteral reflux (VUR) was assessed from Zmym2^+/+ (n = 25) and Zmym2^+/− (n = 20). Note, some animals harbored more than one CAKUT phenotype. (B) Dissected E18.5 and P0 urogenital system of Zmym2^+/+and Zmym2^+/− mice demonstrating gross CAKUT phenotypes including hydroureter, hydronephrosis, duplex systems, and cystic kidneys, respectively. (C) Haemotoxylin and Eosin staining of tissue sections derived from Zmym2^+/+and Zmym2^+/− urogenital systems. (D) Intravesical dye injection showing vesicoureteral reflux in Zmym2^+/+ and Zmym2^+/− P0 mice. (E) Immunohistofluorescence analysis of E18.5 kidneys reveals no overt difference in cap mesenchyme and ureter tips (Pax2) nor in podocytes (podocalyxin) between Zmym2^+/+and Zmym2^+/− kidneys.

Figure 4

Functional Characterization of ZMYM2 Variants and Identification of Protein-Protein Interaction Partners of ZMYM2 as Candidates for Monogenic Causes of CAKUT (A) Representative immunofluoroscence images following overexpression of myc labeled cDNA constructs for mock, wild-type ZMYM2 (hsMYC_wtZMYM2), and cDNA representing mutation p.Arg540^∗ (detected in A4730 and A1204) showing mislocalization of truncated protein to the cytoplasm rather than the nucleus. (B) BioID of human wild-type ZMYM2 expressed in Flp-In T-REx 293 cells yields 123 proximity interaction partners. Interactors are grouped according to protein complex, intracellular localization, shared protein domain, or function. Edge size is proportional to total peptide counts. (C) All 73 candidate genes resulting from the BioID experiments were evaluated for heterozygous mutations in 551 families with CAKUT using the American College of Medical Genetics criteria for deleteriousness. (D) ZMYM3 variants in families B1287_21 and B2323_21 as a potential candidate gene in CAKUT pathogenesis. CAKUT, congenital anomalies of the kidney and urinary tract; c. change, nucleotide change; Cov., coverage; gnomAD, genome aggregation; Miss., missense; Mut. Taster, Mutation Taster; NS, nephrotic syndrome; p. change, amino acid change, Poly2, Polymorphism Phenotyping v2; SIFT, Sorting Intolerant From Tolerant; WES, whole-exome sequencing; Zyg, zygosity; Mm, Mus musculus; Gg, Gallus gallus; Xt, Xenopus tropicalis; Pk, Paramormyrops kingsleyae; Ci, Ciona intestinalis; Ce, Caenorhabditis elegans; Dm, Drosophila melanogaster; Sc, Saccharomyces cerevisiae.