LRRC6 regulates biogenesis of motile cilia by aiding FOXJ1 translocation into the nucleus.

BACKGROUND: LRRC6 is an assembly factor for dynein arms in the cytoplasm of motile ciliated cells, and when mutated, dynein arm components remained in the cytoplasm. Here, we demonstrate the role of LRRC6 in the active nuclear translocation of FOXJ1, a master regulator for cilia-associated gene transcription. METHODS: We generated Lrrc6 knockout (KO) mice, and we investigated the role of LRRC6 on ciliopathy development by using proteomic, transcriptomic, and immunofluorescence analysis. Experiments on mouse basal cell organoids confirmed the biological relevance of our findings. RESULTS: The absence of LRRC6 in multi-ciliated cells hinders the assembly of ODA and IDA components of cilia; in this study, we showed that the overall expression of proteins related to cilia decreased as well. Expression of cilia-related transcripts, specifically ODA and IDA components, dynein axonemal assembly factors, radial spokes, and central apparatus was lower in Lrrc6 KO mice than in wild-type mice. We demonstrated that FOXJ1 was present in the cytoplasm and translocated into the nucleus when LRRC6 was expressed and that this process was blocked by INI-43, an importin α inhibitor. CONCLUSIONS: Taken together, these results hinted at the LRRC6 transcriptional regulation of cilia-related genes via the nuclear translocation of FOXJ1. Video Abstract.

Mutations of ADAMTS9 Cause Nephronophthisis-Related Ciliopathy.

Nephronophthisis-related ciliopathies (NPHP-RCs) are a group of inherited diseases that are associated with defects in primary cilium structure and function. To identify genes mutated in NPHP-RC, we performed homozygosity mapping and whole-exome sequencing for >100 individuals, some of whom were single affected individuals born to consanguineous parents and some of whom were siblings of indexes who were also affected by NPHP-RC. We then performed high-throughput exon sequencing in a worldwide cohort of 800 additional families affected by NPHP-RC. We identified two ADAMTS9 mutations (c.4575_4576del [p.Gln1525Hisfs∗60] and c.194C>G [p.Thr65Arg]) that appear to cause NPHP-RC. Although ADAMTS9 is known to be a secreted extracellular metalloproteinase, we found that ADAMTS9 localized near the basal bodies of primary cilia in the cytoplasm. Heterologously expressed wild-type ADAMTS9, in contrast to mutant proteins detected in individuals with NPHP-RC, localized to the vicinity of the basal body. Loss of ADAMTS9 resulted in shortened cilia and defective sonic hedgehog signaling. Knockout of Adamts9 in IMCD3 cells, followed by spheroid induction, resulted in defective lumen formation, which was rescued by an overexpression of wild-type, but not of mutant, ADAMTS9. Knockdown of adamts9 in zebrafish recapitulated NPHP-RC phenotypes, including renal cysts and hydrocephalus. These findings suggest that the identified mutations in ADAMTS9 cause NPHP-RC and that ADAMTS9 is required for the formation and function of primary cilia.

Disease modeling of ADAMTS9-related nephropathy using kidney organoids reveals its roles in tubular cells and podocytes.

Introduction: Mutations in ADAMTS9 cause nephronophthisis-related ciliopathies (NPHP-RC), which are characterized by multiple developmental defects and kidney diseases. Patients with NPHP-RC usually have normal glomeruli and negligible or no proteinuria. Herein, we identified novel compound-heterozygous ADAMTS9 variants in two siblings with NPHP-RC who had glomerular manifestations, including proteinuria. Methods: To investigate whether ADAMTS9 dysfunction causes NPHP and glomerulopathy, we differentiated ADAMTS9 knockout human induced pluripotent stem cells (hiPSCs) into kidney organoids. Single-cell RNA sequencing was utilized to elucidate the gene expression profiles from the ADAMTS9 knockout kidney organoids. Results: ADAMTS9 knockout had no effect on nephron differentiation; however, it reduced the number of primary cilia, thereby recapitulating renal ciliopathy. Single-cell transcriptomics revealed that podocyte clusters express the highest levels of ADAMTS9, followed by the proximal tubules. Loss of ADAMTS9 increased the activity of multiple signaling pathways, including the Wnt/PCP signaling pathway, in podocyte clusters. Conclusions: Mutations in ADMATS9 cause a glomerulotubular nephropathy in kidney and our study provides insights into the functional roles of ADMATS9 in glomeruli and tubules.

Genomic Landscape and Mutational Spectrum of ADAMTS Family Genes in Mendelian Disorders Based on Gene Evidence Review for Variant Interpretation.

ADAMTS (a disintegrin and metalloproteinase with thrombospondin motifs) are a family of multidomain extracellular protease enzymes with 19 members. A growing number of ADAMTS family gene variants have been identified in patients with various hereditary diseases. To understand the genomic landscape and mutational spectrum of ADAMTS family genes, we evaluated all reported variants in the ClinVar database and Human Gene Mutation Database (HGMD), as well as recent literature on Mendelian hereditary disorders associated with ADAMTS family genes. Among 1089 variants in 14 genes reported in public databases, 307 variants previously suggested for pathogenicity in Mendelian diseases were comprehensively re-evaluated using the American College of Medical Genetics and Genomics (ACMG) 2015 guideline. A total of eight autosomal recessive genes were annotated as being strongly associated with specific Mendelian diseases, including two recently discovered genes (ADAMTS9 and ADAMTS19) for their causality in congenital diseases (nephronophthisis-related ciliopathy and nonsyndromic heart valve disease, respectively). Clinical symptoms and affected organs were extremely heterogeneous among hereditary diseases caused by ADAMTS family genes, indicating phenotypic heterogeneity despite their structural and functional similarity. ADAMTS6 was suggested as presenting undiscovered pathogenic mutations responsible for novel Mendelian disorders. Our study is the first to highlight the genomic landscape of ADAMTS family genes, providing an appropriate genetic approach for clinical use.

PLCE1 regulates the migration, proliferation, and differentiation of podocytes.

PLCE1 encodes phospholipase C epsilon, and its mutations cause recessive nephrotic syndrome. However, the mechanisms by which PLCE1 mutations result in defects associated with glomerular function are not clear. To address this, we investigated the function of PLCE1 in podocytes called glomerular epithelial cells, where the pathogenesis of nephrotic syndrome converges. PLCE1 colocalized with Rho GTPases in glomeruli. Further, it interacted with Rho GTPases through the pleckstrin homology domain and Ras GTP-binding domains 1/2. Knockdown or knockout of PLCE1 in podocytes resulted in decreased levels of GTP-bound Rac1 and Cdc42, but not those of RhoA, and caused a reduction in cell migration. PLCE1 interacted with NCK2 but not with NCK1. Similar to the PLCE1 knockout, NCK2 knockout resulted in decreased podocyte migration. Knockout of PLCE1 reduced the EGF-induced activation of ERK and cell proliferation in podocytes, whereas knockout of NCK2 did not affect proliferation. Further, the knockout of PLCE1 also resulted in decreased expression of podocyte markers, including NEPH1, NPHS1, WT1, and SYNPO, upon differentiation, but the knockout of NCK2 did not affect the expression of these markers. Therefore, our findings demonstrate that PLCE1 regulates Rho GTPase activity and cell migration through interacting with NCK2 and that PLCE1 also plays a role in the proliferation and differentiation of podocytes, regardless of the presence of NCK2.