DLG1 functions upstream of SDCCAG3 and IFT20 to control ciliary targeting of polycystin-2.

Polarized vesicular trafficking directs specific receptors and ion channels to cilia, but the underlying mechanisms are poorly understood. Here we describe a role for DLG1, a core component of the Scribble polarity complex, in regulating ciliary protein trafficking in kidney epithelial cells. Conditional knockout of Dlg1 in mouse kidney causes ciliary elongation and cystogenesis, and cell-based proximity labeling proteomics and fluorescence microscopy show alterations in the ciliary proteome upon loss of DLG1. Specifically, the retromer-associated protein SDCCAG3, IFT20, and polycystin-2 (PC2) are reduced in the cilia of DLG1-deficient cells compared to control cells. This phenotype is recapitulated in vivo and rescuable by re-expression of wild-type DLG1, but not a Congenital Anomalies of the Kidney and Urinary Tract (CAKUT)-associated DLG1 variant, p.T489R. Finally, biochemical approaches and Alpha Fold modelling suggest that SDCCAG3 and IFT20 form a complex that associates, at least indirectly, with DLG1. Our work identifies a key role for DLG1 in regulating ciliary protein composition and suggests that ciliary dysfunction of the p.T489R DLG1 variant may contribute to CAKUT.

DLG1 functions upstream of SDCCAG3 and IFT20 to control ciliary targeting of polycystin-2.

Polarized vesicular trafficking directs specific receptors and ion channels to cilia, but the underlying mechanisms are poorly understood. Here we describe a role for DLG1, a core component of the Scribble polarity complex, in regulating ciliary protein trafficking in kidney epithelial cells. Conditional knockout of Dlg1 in mouse kidney caused ciliary elongation and cystogenesis, and cell-based proximity labelling proteomics and fluorescence microscopy showed alterations in the ciliary proteome upon loss of DLG1. Specifically, the retromer-associated protein SDCCAG3, IFT20 and polycystin-2 (PC2) were reduced in cilia of DLG1 deficient cells compared to control cells. This phenotype was recapitulated in vivo and rescuable by re-expression of wildtype DLG1, but not a Congenital Anomalies of the Kidney and Urinary Tract (CAKUT)-associated DLG1 variant, p.T489R. Finally, biochemical approaches and Alpha Fold modelling suggested that SDCCAG3 and IFT20 form a complex that associates, at least indirectly, with DLG1. Our work identifies a key role for DLG1 in regulating ciliary protein composition and suggests that ciliary dysfunction of the p.T489R DLG1 variant may contribute to CAKUT.

Mutations in PIK3C2A cause syndromic short stature, skeletal abnormalities, and cataracts associated with ciliary dysfunction.

PIK3C2A is a class II member of the phosphoinositide 3-kinase (PI3K) family that catalyzes the phosphorylation of phosphatidylinositol (PI) into PI(3)P and the phosphorylation of PI(4)P into PI(3,4)P2. At the cellular level, PIK3C2A is critical for the formation of cilia and for receptor mediated endocytosis, among other biological functions. We identified homozygous loss-of-function mutations in PIK3C2A in children from three independent consanguineous families with short stature, coarse facial features, cataracts with secondary glaucoma, multiple skeletal abnormalities, neurological manifestations, among other findings. Cellular studies of patient-derived fibroblasts found that they lacked PIK3C2A protein, had impaired cilia formation and function, and demonstrated reduced proliferative capacity. Collectively, the genetic and molecular data implicate mutations in PIK3C2A in a new Mendelian disorder of PI metabolism, thereby shedding light on the critical role of a class II PI3K in growth, vision, skeletal formation and neurological development. In particular, the considerable phenotypic overlap, yet distinct features, between this syndrome and Lowe's syndrome, which is caused by mutations in the PI-5-phosphatase OCRL, highlight the key role of PI metabolizing enzymes in specific developmental processes and demonstrate the unique non-redundant functions of each enzyme. This discovery expands what is known about disorders of PI metabolism and helps unravel the role of PIK3C2A and class II PI3Ks in health and disease.

Clinical and biological impact of TET2 mutations and expression in younger adult AML patients treated within the EORTC/GIMEMA AML-12 clinical trial.

We assessed the prognostic impact of TET2 mutations and mRNA expression in a prospective cohort of 357 adult AML patients < 60 years of age enrolled in the European Organization For Research and Treatment of Cancer (EORTC)/Gruppo Italiano Malattie Ematologiche dell' Adulto (GIMEMA) AML-12 06991 clinical trial. In addition the co-occurrence with other genetic defects and the functional consequences of TET2 mutations were investigated. TET2 mutations occurred in 7.6 % of the patients and were an independent marker of poor prognosis (p = 0.024). TET2 and IDH1/2 mutations strongly associated with aberrations in the DNA methyltransferase DNMT3A. Functional studies confirmed previous work that neither nonsense truncations, nor missense TET2 mutations, induced 5-hydroxymethylcytosine formation. In addition, we now show that mutant TET2 forms did not act in a dominant negative manner when co-expressed with the wild-type protein. Finally, as loss-of-function TET2 mutations predicted poor outcome, we questioned whether low TET2 mRNA expression in cases of AML without TET2 mutations would affect overall survival. Notably, also AML patients with low TET2 mRNA expression levels showed inferior overall survival.

Characterization of acute myeloid leukemia based on levels of global hydroxymethylation.

Patients with acute myeloid leukemia (AML) frequently harbor mutations in genes involved in the DNA (hydroxy)methylation pathway (DNMT3A, TET2, IDH1, and IDH2). In this study, we measured 5-hydroxymethylcytosine (5hmC) levels in 206 clinically and molecularly well-characterized younger adult AML patients (≤60 years) included in the European Organization for Research and Treatment of Cancer/Gruppo Italiano Malattie Ematologiche dell'Adulto (EORTC/GIMEMA) AML-12 06991 clinical trial and correlated the 5hmC levels with mutational status and overall survival (OS). In healthy control cells, 5hmC levels were confined to a narrow range (1.5-fold difference), whereas in AML cells, a much wider range was detected (15-fold difference). We identified 3 5hmC subpopulations in our patient cohort (low, intermediate, and high). The low 5hmC group consisted almost entirely of patients with TET2 or IDH mutations. As expected, TET2 and IDH mutated patients had significantly lower levels of 5hmC compared with patients without mutated TET2 and IDH1/2 (both P < .001). Interestingly, high 5hmC levels correlated with inferior OS (high vs intermediate 5hmC: P = .047, hazard ratio [HR] = 1.81). Multivariate analysis revealed that high 5hmC is an independent poor prognostic indicator for OS (high vs intermediate 5hmC: P = .01, HR = 2.10). This trial was registered at www.clinicaltrials.gov as NCT00004128.

Acquired mutations in TET2 are common in myelodysplastic syndromes.

Myelodysplastic syndromes (MDS) represent a heterogeneous group of neoplastic hematopoietic disorders. Several recurrent chromosomal aberrations have been associated with MDS, but the genes affected have remained largely unknown. To identify relevant genetic lesions involved in the pathogenesis of MDS, we conducted SNP array-based genomic profiling and genomic sequencing in 102 individuals with MDS and identified acquired deletions and missense and nonsense mutations in the TET2 gene in 26% of these individuals. Using allele-specific assays, we detected TET2 mutations in most of the bone marrow cells (median 96%). In addition, the mutations were encountered in various lineages of differentiation including CD34(+) progenitor cells, suggesting that TET2 mutations occur early during disease evolution. In healthy tissues, TET2 expression was shown to be elevated in hematopoietic cells with highest expression in granulocytes, in line with a function in myelopoiesis. We conclude that TET2 is the most frequently mutated gene in MDS known so far.