Frameshift Mutation in PAX2 Related to Focal Segmental Glomerular Sclerosis: A Case Report and Literature Review.

BACKGROUND: Paired box gene 2 (PAX2) heterozygous mutations can cause renal coloboma syndrome, but its role in patients with focal segmental glomerular sclerosis (FSGS) has been rarely reported. METHODS: Based on the clinical manifestations and renal pathological characteristics of the patient, as well as familial whole exome sequencing, the diagnosis of FSGS related to PAX2 mutation was confirmed. Treatment such as lowering urinary protein and blood pressure was given, and the patient was followed up and observed. RESULTS: There is a familial heterozygous case presented with chronic kidney disease secondary to FSGS, which was related to PAX2 frameshift mutation due to the deletion of G at the position 76 (c.76delG). To our knowledge, this is the first report of PAX2 c.76delG variant related to adult-onset FSGS. CONCLUSION: Here, we further expand the phenotypic spectrum of FSGS. Genetic screening especially PAX2 mutation is recommended in patients with adult-onset FSGS of unknown etiology.

Mesonephric-type adenocarcinomas of the ovary: prevalence, diagnostic reproducibility, outcome, and value of PAX2.

Mesonephric-type (or -like) adenocarcinomas (MAs) of the ovary are an uncommon and aggressive histotype. They appear to arise through transdifferentiation from Müllerian lesions creating diagnostic challenges. Thus, we aimed to develop a histologic and immunohistochemical (IHC) approach to optimize the identification of MA over its histologic mimics, such as ovarian endometrioid carcinoma (EC). First, we screened 1,537 ovarian epithelial neoplasms with a four-marker IHC panel of GATA3, TTF1, ER, and PR followed by a morphological review of EC to identify MA in retrospective cohorts. Interobserver reproducibility for the distinction of MA versus EC was assessed in 66 cases initially without and subsequently with IHC information (four-marker panel). Expression of PAX2, CD10, and calretinin was evaluated separately, and survival analyses were performed. We identified 23 MAs from which 22 were among 385 cases initially reported as EC (5.7%) and 1 as clear cell carcinoma. The interobserver reproducibility increased from fair to substantial (κ = 0.376-0.727) with the integration of the four-marker IHC panel. PAX2 was the single most sensitive and specific marker to distinguish MA from EC and could be used as a first-line marker together with ER/PR and GATA3/TTF1. Patients with MA had significantly increased risk of earlier death from disease (hazard ratio = 3.08; 95% CI, 1.62-5.85; p < 0.0001) compared with patients with EC, when adjusted for age, stage, and p53 status. A diagnosis of MA has prognostic implications for stage I disease, and due to the subtlety of morphological features in some tumors, a low threshold for ancillary testing is recommended.

Heterogeneity of Wnt1-Cre-marked and Pax2-Cre-marked first branchial arch cranial neural crest cells in mice.

OBJECTIVES: This study aimed to explore the heterogeneity and gene ontology of Wnt1-Cre-marked and Pax2-Cre-marked first branchial arch cranial neural crest cells (CNCs) in mice. METHODS: The embryos of Wnt1-Cre;R26RmTmG and Pax2-Cre;R26RmTmG at embryonic day (E)8.0-E9.25 were collected for histological observation. We performed immunostaining to compare green fluorescent protein (GFP)-positive CNCs in Pax2-Cre;R26RAi9 and Wnt1-Cre;R26RAi9 mice at E15.5. Single-cell RNA sequencing (scRNA-seq) was used to analyze the first branchial arch GFP-positive CNCs from Wnt1-Cre;R26RmTmG and Pax2-cre;R26RmTmGmice at E10.5. Real time fluorescence quantitative polymerase chain reaction (q-PCR) was performed to validate the differential genes. RESULTS: Wnt1-Cre-marked and Pax2-Cre-marked CNCs migrated from the neural plateto first and second branchial arches and to the first branchial arch, respectively, at E8.0. Although Wnt1-Cre-marked and Pax2-Cre-marked CNCs were found mostly in cranial-facial tissues, the former had higher expression in palate and tongue. The results of scRNA-seq showed that Pax2-Cre-marked CNCs specifically contributed to osteoblast differentiation and ossification, while Wnt1-Cre-marked CNCs participated in limb development, cell migration, and ossification. The q-PCR data also confirmed the results of gene ontology analysis. CONCLUSIONS: Pax2-Cre mice are perfect experimental animal models for research on first branchial arch CNCs and derivatives in osteoblast differentiation and ossification.

Retained PAX2 expression associated with DNA mismatch repair deficiency in endometrial endometrioid adenocarcinoma.

AIMS: Loss of expression of tumour suppressor PAX2 and MMR deficiency (dMMR) has been frequently seen in endometrial endometrioid adenocarcinoma (EEC). However, the relationship between PAX2 expression and MMR status is unknown. METHODS AND RESULTS: We studied the PAX2 expression and examined its association with MMR status at the protein and genetic levels in 180 cases of EEC. Overall, total loss of PAX2 expression was found in about 70%, while retained PAX2 expression was seen in 30% of EEC. Among 125 cases with loss of PAX2, 68.8% were found in EECs with pMMR, while 31.2% were seen in those with dMMR. Among 55 cases of EECs with retained PAX2 expression, 92.7% were EECs with dMMR and 7.3% were those with pMMR (P < 0.001). While dMMR cases with MLH1 hypermethylation show almost equal retained or loss of PAX2 expression (52% versus 48%), dMMR with genetic alterations had significantly more retained PAX2 expression than loss of PAX2 (92.3% versus 7.7%), regardless of somatic or germline mutations. Loss of PAX2 was observed in 97.3% of dMMR with MLH1 hypermethylation compared to 2.7% of dMMR with genetic alterations (P < 0.001). Aggressive features such as higher tumour grades (FIGO 2-3) and advanced clinical stage (T2-T4) were significantly more frequently seen in dMMR with retained PAX2 expression, compared those to pMMR with loss of PAX2 expression. CONCLUSION: Our study demonstrates a close correlation between retained PAX2 expression and dMMR in EEC. The molecular mechanism and clinical significance linking these two pathways in EEC remains to be unravelled.

Specification and survival of post-metamorphic branchiomeric neurons in a non-vertebrate chordate.

Tunicates are the sister group to the vertebrates, yet most species have a life cycle split between swimming larva and sedentary adult phases. During metamorphosis, larval neurons are replaced by adult-specific ones. The regulatory mechanisms underlying this replacement remain largely unknown. Using tissue-specific CRISPR/Cas9-mediated mutagenesis in the tunicate Ciona, we show that orthologs of conserved hindbrain and branchiomeric neuron regulatory factors Pax2/5/8 and Phox2 are required to specify the 'neck', a cellular compartment set aside in the larva to give rise to cranial motor neuron-like neurons post-metamorphosis. Using bulk and single-cell RNA-sequencing analyses, we characterize the transcriptome of the neck downstream of Pax2/5/8. We present evidence that neck-derived adult ciliomotor neurons begin to differentiate in the larva and persist through metamorphosis, contrary to the assumption that the adult nervous system is formed after settlement and the death of larval neurons during metamorphosis. Finally, we show that FGF signaling during the larval phase alters the patterning of the neck and its derivatives. Suppression of FGF converts neck cells into larval neurons that fail to survive metamorphosis, whereas prolonged FGF signaling promotes an adult neural stem cell-like fate.

The Oct4-related PouV gene, pou5f3, mediates isthmus development in zebrafish by directly and dynamically regulating pax2a.

Using a transgenic zebrafish line harboring a heat-inducible dominant-interference pou5f3 gene (en-pou5f3), we reported that this PouV gene is involved in isthmus development at the midbrain-hindbrain boundary (MHB), which patterns the midbrain and cerebellum. Importantly, the functions of pou5f3 reportedly differ before and after the end of gastrulation. In the present study, we examined in detail the effects of en-pou5f3 induction on isthmus development during embryogenesis. When en-pou5f3 was induced around the end of gastrulation (bud stage), the isthmus was abrogated or deformed by the end of somitogenesis (24 hours post-fertilization). At this stage, the expression of MHB markers -- such as pax2a, fgf8a, wnt1, and gbx2 -- was absent in embryos lacking the isthmus structure, whereas it was present, although severely distorted, in embryos with a deformed isthmus. We further found that, after en-pou5f3 induction at late gastrulation, pax2a, fgf8a, and wnt1 were immediately and irreversibly downregulated, whereas the expression of en2a and gbx2 was reduced only weakly and slowly. Induction of en-pou5f3 at early somite stages also immediately downregulated MHB genes, particularly pax2a, but their expression was restored later. Overall, the data suggested that pou5f3 directly upregulates at least pax2a and possibly fgf8a and wnt1, which function in parallel in establishing the MHB, and that the role of pou5f3 dynamically changes around the end of gastrulation. We next examined the transcriptional regulation of pax2a using both in vitro and in vivo reporter analyses; the results showed that two upstream 1.0-kb regions with sequences conserved among vertebrates specifically drove transcription at the MHB. These reporter analyses confirmed that development of the isthmic organizer is regulated by PouV through direct regulation of pax2/pax2a in vertebrate embryos.

Lesions sub-diagnostic of endometrioid intra-epithelial neoplasia/atypical hyperplasia: value of morphology and immunohistochemistry in predicting neoplastic outcome.

AIMS: Areas of gland crowding that do not fulfil diagnostic criteria of endometrioid intra-epithelial neoplasia (EIN) are often encountered in endometrial biopsies. In this study, we document the prevalence of neoplastic outcome in patients with these subdiagnostic lesions (SL) and assess the utility of morphological features and a three-marker immunohistochemistry panel (PAX2, PTEN, beta-catenin) to predict outcome. METHODS AND RESULTS: Of 430 women with SL on endometrial sampling at Brigham and Women's Hospital between 2001 and 2021 with available follow-up biopsy, 72 (17%) had a neoplastic outcome (EIN or endometrioid carcinoma). Multilayered epithelium and mitoses in SL were statistically associated with a neoplastic outcome. Abnormal three-marker staining was observed in 93% (53 of 57) of SL with neoplastic outcome and 60% (37 of 62) of a control group with benign outcome. Among the 72 patients with neoplastic outcome, EIN/carcinoma tissue was available in 33; of these, 30 (91%) showed abnormal staining for one or more markers. Remarkably, in 84% of these cases the EIN/carcinoma had the aberrant expression seen in the preceding SL. Based on a prevalence of 17%, the positive and negative predictive values of abnormal staining in one or more markers were 24 and 97%, respectively. CONCLUSIONS: The presence of SL warrants clinical surveillance and repeat sampling because it is followed by endometrioid neoplasia in a significant subset of patients. Normal three-marker staining identifies women with a very low risk of neoplastic outcome. Conversely, abnormal staining is frequent in SL with benign outcome leading to poor specificity and positive predictive value.

PAX2 mediated upregulation of ESPL1 contributes to cisplatin resistance in bladder cancer through activating the JAK2/STAT3 pathway.

Extra spindle-polar body like 1 (ESPL1) is associated with the development of a variety of cancers, including bladder cancer, and is closely related to chemoresistance. In this study, we aimed to reveal the role of ESPL1 in bladder cancer progression and cisplatin (DDP) resistance. First, ESPL1 was found to be highly expressed in tumor tissues and cells of bladder cancer, and more highly expressed in cisplatin resistant tumor tissues or cells. The binding of PAX2 in ESPL1 promoter region was predicted by Jaspar database and verified by Ch-IP analysis and the luciferase reporter gene assay. Next, cisplatin-resistant T24 cells (T24/DDP) were established and transfected with ESPL1 siRNA (si-ESPL1) or overexpression vector (pcDNA-ESPL1) or co-transfected with PAX2 siRNA (si-PAX2) or overexpression vector (pcDNA-PAX2), and then treated with DDP or AG490, an inhibitor of JAK2. The results showed that silencing ESPL1 significantly reduced T24/DDP cell viability, colony formation and invasion, enhanced sensitivity to DDP, and induced cell apoptosis. Silencing PAX2 decreased ESPL1 expression, enhanced sensitivity to DDP, and induced apoptosis of T24/DDP cells, and inhibited activation of JAK2/STAT3 pathway. Overexpressing ESPL1 reversed the effect of PAX2 silencing on T24/DDP cells, while AG490 counteracted the reversal effect of overexpressing ESPL1. Finally, a xenograft tumor model was established and found that silencing ESPL1 or DDP treatment inhibited tumor growth, while silencing ESPL1 combined with DDP treatment had the best effect. In summary, this study suggested that PAX2-mediated ESPL1 transcriptional activation enhanced cisplatin resistance in bladder cancer by activating JAK2/STAT3 pathway.

Renal coloboma syndrome/dominant optic atrophy with severe retinal atrophy and de novo digenic mutations in PAX2 and OPA1.

Renal coloboma syndrome (RCS) and dominant optic atrophy are mainly caused by heterozygous mutations in PAX2 and OPA1, respectively. We describe a patient with digenic mutations in PAX2 and OPA1. A female infant was born without perinatal abnormalities. Magnetic resonance imaging at 4 months of age showed bilateral microphthalmia and optic nerve hypoplasia. Appropriate body size was present at 2 years of age, and mental development was favorable. Color fundus photography revealed severe retinal atrophy in both eyes. Electroretinography showed slight responses in the right eye, but no responses in the left eye, suggesting a high risk of blindness. Urinalysis results were normal, creatinine-based estimated glomerular filtration rate was 63.5 mL/min/1.73 m2, and ultrasonography showed bilateral hypoplastic kidneys. Whole exome sequencing revealed de novo frameshift mutations in PAX2 and OPA1. Both variants were classified as pathogenic (PVS1, PS2, PM2) based on the guidelines from the American College of Medical Genetics and Genomics (ACMG). Genetic testing for ocular diseases should be considered for patients with suspected RCS and a high risk of total blindness.

Mice with a Pax2 missense variant display impaired glomerular repair.

PAX2 regulates kidney development, and its expression persists in parietal epithelial cells (PECs), potentially serving as a podocyte reserve. We hypothesized that mice with a Pax2 pathogenic missense variant (Pax2A220G/+) have impaired PEC-mediated podocyte regeneration. Embryonic wild-type mouse kidneys showed overlapping expression of PAX2/Wilms' tumor-1 (WT-1) until PEC and podocyte differentiation, reflecting a close lineage relationship. Embryonic and adult Pax2A220G/+ mice have reduced nephron number but demonstrated no glomerular disease under baseline conditions. Pax2A220G/+ mice compared with wild-type mice were more susceptible to glomerular disease after adriamycin (ADR)-induced podocyte injury, as demonstrated by worsened glomerular scarring, increased podocyte foot process effacement, and podocyte loss. There was a decrease in PAX2-expressing PECs in wild-type mice after adriamycin injury accompanied by the occurrence of PAX2/WT-1-coexpressing glomerular tuft cells. In contrast, Pax2A220G/+ mice showed no changes in the numbers of PAX2-expressing PECs after adriamycin injury, associated with fewer PAX2/WT-1-coexpressing glomerular tuft cells compared with injured wild-type mice. A subset of PAX2-expressing glomerular tuft cells after adriamycin injury was increased in Pax2A220G/+ mice, suggesting a pathological process given the worse outcomes observed in this group. Finally, Pax2A220G/+ mice have increased numbers of glomerular tuft cells expressing Ki-67 and cleaved caspase-3 compared with wild-type mice after adriamycin injury, consistent with maladaptive responses to podocyte loss. Collectively, our results suggest that decreased glomerular numbers in Pax2A220G/+ mice are likely compounded with the inability of their mutated PECs to regenerate podocyte loss, and together these two mechanisms drive the worsened focal segmental glomerular sclerosis phenotype in these mice.NEW & NOTEWORTHY Congenital anomalies of the kidney and urinary tract comprise some of the leading causes of kidney failure in children, but our previous study showed that one of its genetic causes, PAX2, is also associated with adult-onset focal segmental glomerular sclerosis. Using a clinically relevant model, our present study demonstrated that after podocyte injury, parietal epithelial cells expressing PAX2 are deployed into the glomerular tuft to assist in repair in wild-type mice, but this mechanism is impaired in Pax2A220G/+ mice.

Vestigial organs alter fossil placements in an ancient group of terrestrial chelicerates.

Vestigial organs provide a link between ancient and modern traits and therefore have great potential to resolve the phylogeny of contentious fossils that bear features not seen in extant species. Here we show that extant daddy-longlegs (Arachnida, Opiliones), a group once thought to possess only one pair of eyes, in fact additionally retain a pair of vestigial median eyes and a pair of vestigial lateral eyes. Neuroanatomical gene expression surveys of eye-patterning transcription factors, opsins, and other structural proteins in the daddy-longlegs Phalangium opilio show that the vestigial median and lateral eyes innervate regions of the brain positionally homologous to the median and lateral eye neuropils, respectively, of chelicerate groups like spiders and horseshoe crabs. Gene silencing of eyes absent shows that the vestigial eyes are under the control of the retinal determination gene network. Gene silencing of dachshund disrupts the lateral eyes, but not the median eyes, paralleling loss-of-function phenotypes in insect models. The existence of lateral eyes in extant daddy-longlegs bears upon the placement of the oldest harvestmen fossils, a putative stem group that possessed both a pair of median eyes and a pair of lateral eyes. Phylogenetic analysis of harvestman relationships with an updated understanding of lateral eye incidence resolved the four-eyed fossil group as a member of the extant daddy-longlegs suborder, which in turn resulted in older estimated ages of harvestman diversification. This work underscores that developmental vestiges in extant taxa can influence our understanding of character evolution, placement of fossils, and inference of divergence times.

PAX2 is regulated by estrogen/progesterone through promoter methylation in endometrioid adenocarcinoma and has an important role in carcinogenesis via the AKT/mTOR signaling pathway.

Endometrioid adenocarcinoma (EEC) is one of the most common cancers of the female reproductive system. In recent years, much emphasis has been placed on early diagnosis and treatment. PAX2 (Paired box 2) inactivation is reportedly an important biomarker for endometrioid intraepithelial neoplasia (EIN) and EEC. However, the role of PAX2 in EEC carcinogenesis remains unclear. PAX2 expression and associated clinical characteristics were analyzed via The Cancer Genome Atlas, Gene Expression Omnibus, and Cancer Cell Line Encyclopedia databases and clinical paired EIN/EEC tissue samples. Bioinformatic analysis was conducted to identify the putative molecular function and mechanism of PAX2. Cell proliferation, colony formation, cell migration, and invasion assays in vitro, and mouse xenograft models were utilized to study the biological functions of PAX2 in vivo. Pyrosequencing and the demethylating drug 5-Aza-dc were used to verify promoter methylation in clinical tissues and cell lines, respectively. The mechanism underlying the regulatory effect of estrogen (E2) and progesterone (P4) on PAX2 expression was investigated by receptor block assay and double luciferase reporter assay. PAX2 expression was found to be significantly downregulated in EIN and EEC tissues, its overexpression inhibited EEC cell malignant behaviors in vivo and in vitro and inhibited the AKT/mTOR signaling pathway. PAX2 inactivation in EEC was related to promoter methylation, and its expression was regulated by E2 and P4 through their receptors via promoter methylation. Our findings elucidated the expression and function of PAX2 in EEC and have provided hitherto undocumented evidence of the underlying molecular mechanisms. PAX2 expression is suppressed by estrogen prompting its methylation through estrogen receptor. Furthermore, PAX2 regulates the AKT/mTOR signaling pathway to influence EEC progression. © 2024 The Pathological Society of Great Britain and Ireland.

Impaired neural circuitry of hippocampus in Pax2 nervous system-specific knockout mice leads to restricted repetitive behaviors.

INTRODUCTION: Restricted repetitive behaviors (RRBs), which are associated with many different neurological and mental disorders, such as obsessive-compulsive disorder (OCD) and autism, are patterns of behavior with little variation and little obvious function. Paired Box 2 (Pax2) is a transcription factor that is expressed in many systems, including the kidney and the central nervous system. The protein that is encoded by Pax2 has been implicated in the development of the nervous system and neurodevelopmental disorders. In our previous study, Pax2 heterozygous gene knockout mice (Pax2+/- mice) showed abnormally increased self-grooming and impaired learning and memory abilities. However, it remains unclear which cell type is involved in this process. In this study, we deleted Pax2 only in the nervous system to determine the regulatory mechanism of Pax2 in RRBs. METHODS: In this study, Pax2 nervous system-specific knockout mice (Nestin-Pax2 mice) aged 6-8 weeks and Pax2 flox mice of the same age were recruited as the experimental group. Tamoxifen and vehicle were administered via intraperitoneal injection to induce Pax2 knockout after gene identification. Western blotting was used to detect Pax2 expression. After that, we assessed the general health of these two groups of mice. The self-grooming test, marble burying test and T-maze acquisition and reversal learning test were used to observe the lower-order and higher-order RRBs. The three-chamber test, Y-maze, and elevated plus-maze were used to assess social ability, spatial memory ability, and anxiety. Neural circuitry tracing and transcriptome sequencing (RNA-seq) were used to observe the abnormal neural circuitry, differentially expressed genes (DEGs) and signaling pathways affected by Pax2 gene knockout in the nervous system and the putative molecular mechanism. RESULTS: (1) The Nestin-Pax2 mouse model was successfully constructed, and the Nestin-Pax2 mice showed decreased expression of Pax2. (2) Nestin-Pax2 mice showed increased self-grooming behavior and impaired T-maze reversal behavior compared with Pax2 flox mice. (3) An increased number of projection fibers can be found in the mPFC projecting to the CA1 and BLA, and a reduction in IGFBP2 can be found in the hippocampus of Nestin-Pax2 mice. CONCLUSION: The results demonstrated that loss of Pax2 in the nervous system leads to restricted repetitive behaviors. The mechanism may be associated with impaired neural circuitry and a reduction in IGFBP2.

Analysis of clinical phenotype and genotype of PAX2 mutation in Chinese children / 中华肾脏病杂志

Objective:To analyze and summarize the clinical, genotypic and pathological characteristics of children with PAX2 gene mutation in China, and to provide information for the monitoring, treatment and prognosis of the disease. Methods:It was a case series analysis study. The clinical data of children with PAX2 gene mutation in Pediatric Nephrology Department, Tongji Hospital Affiliated to Tongji Medical College, Huazhong University of Science and Technology from January 2014 to December 2022 were collected, and peripheral blood gene DNA was extracted and sequenced for whole exome sequencing. The clinical, pathological and genotypic characteristics of PAX2 gene variation of children in China were summarized by searching PubMed, Medline, China National Knowledge Infrastructure and Wanfang database and compared with the cases in this single center. Results:Among the 13 children with PAX2 gene mutation, there were 9 males and 4 females, 12 patients with abnormal urine tests, 7 patients with small kidney volume by imaging examination, and 5 patients with renal cysts. The clinical phenotypes were congenital renal and urinary tract malformations in 8 cases, renal coloboma syndrome in 1 case, and hematuria or proteinuria in 3 cases. Five patients underwent renal biopsies, showing focal segmental glomerulosclerosis and C3 glomerulopathy in 1 case, focal segmental glomerulosclerosis in 1 case, thin basement membrane lesion in 1 case, and IgA nephropathy in 2 cases. The genetic testing in 13 children showed 9 de novo mutations and 4 new mutations of c.321G>A, c.213-8C>G, c.63C>A and c.449C>T. There were 2 cases of 76dupG (p.V26Gfs*28) mutant. A total of 51 Chinese children with PAX2 gene mutation were found in the literature search. There were 32 males and 19 females, 8 cases with small kidney volume and 12 cases with renal cysts. The clinical phenotypes were congenital anomalies of kidney and urinary tract in 28 cases, renal coloboma syndrome in 17 cases, and hematuria or proteinuria in 6 cases. Seven patients underwent renal biopsies, including 2 cases with focal segmental glomerulosclerosis, 1 case with minimal lesion, 1 case with mesangial proliferative glomerulonephritis, 1 case with IgA nephropathy, 1 case with membranous nephropathy and a case with focal proliferative sclerosing purpura nephritis combined with glomerular hypertrophy. Thirty-four cases were de novo mutations, and 12 mutations were from the father or mother. The father or mother of 5 children had no clinical manifestations, with normal renal function. There were 11 cases of 76dupG (p.V26Gfs*28) mutant. Conclusions:The clinical phenotypes and genotypes of PAX2 gene variation in Chinese children are diverse. The most common clinical phenotype of PAX2 gene variation is congenital anomalies of kidney and urinary tract. c.76dupG (p.V26Gfs*28) is the most common of PAX2 gene variant.

Implication of transcription factor FOXD2 dysfunction in syndromic congenital anomalies of the kidney and urinary tract (CAKUT).

Congenital anomalies of the kidney and urinary tract (CAKUT) are the predominant cause for chronic kidney disease below age 30 years. Many monogenic forms have been discovered due to comprehensive genetic testing like exome sequencing. However, disease-causing variants in known disease-associated genes only explain a proportion of cases. Here, we aim to unravel underlying molecular mechanisms of syndromic CAKUT in three unrelated multiplex families with presumed autosomal recessive inheritance. Exome sequencing in the index individuals revealed three different rare homozygous variants in FOXD2, encoding a transcription factor not previously implicated in CAKUT in humans: a frameshift in the Arabic and a missense variant each in the Turkish and the Israeli family with segregation patterns consistent with autosomal recessive inheritance. CRISPR/Cas9-derived Foxd2 knockout mice presented with a bilateral dilated kidney pelvis accompanied by atrophy of the kidney papilla and mandibular, ophthalmologic, and behavioral anomalies, recapitulating the human phenotype. In a complementary approach to study pathomechanisms of FOXD2-dysfunction-mediated developmental kidney defects, we generated CRISPR/Cas9-mediated knockout of Foxd2 in ureteric bud-induced mouse metanephric mesenchyme cells. Transcriptomic analyses revealed enrichment of numerous differentially expressed genes important for kidney/urogenital development, including Pax2 and Wnt4 as well as gene expression changes indicating a shift toward a stromal cell identity. Histology of Foxd2 knockout mouse kidneys confirmed increased fibrosis. Further, genome-wide association studies suggest that FOXD2 could play a role for maintenance of podocyte integrity during adulthood. Thus, our studies help in genetic diagnostics of monogenic CAKUT and in understanding of monogenic and multifactorial kidney diseases.

Glandular Crowdings in Endometrial Polyps: Clinical Follow-Up and Possible Worrisome Features.

INTRODUCTION: Interpretation of changes and premalignant lesions in endometrial polyps can be challenging. We evaluated the clinical course of patients with focal gland crowdings in endometrial polyps via repeat biopsies and searched for possible morphological findings in the initial biopsy that may foresee a premalignant course. METHODS: Specimens diagnosed as endometrial polyp and focal gland crowding in patients who had a repeat biopsy in a 1-year period were reexamined. Morphological findings in the initial biopsies were recorded. The group whose repeat biopsies were "premalignant or malignant" (Group 1), and the group with "benign" repeat biopsies (Group 2) were compared. RESULTS: "Endometrial polyp and gland crowdings" was diagnosed in 115 specimens of which 38 patients had repeat biopsies. Among these 8 (21%) were diagnosed as "endometrial intraepithelial neoplasia (EIN)" (Group 1) and 30 (79%) as "benign" (Group 2). Morphological features in the initial biopsies were evaluated; PAX2 loss was 6 of 8 (75%) for Group 1 and 7 of 30 (23%) for Group 2 (P = .020), and altered epithelial cytological features were present in 5 of 8 (62%) versus 4 of 30 (13%) (P = .015), both significantly higher in Group 1. Dark intraluminal secretion, intraluminal histiocytes, intraglandular epithelial proliferation, and mean diameter of crowded gland areas were not statistically different between the 2 groups. CONCLUSION: "Focal gland crowdings" in endometrial polyps do carry a risk of EIN in subsequent biopsies. We suggest that the loss/decrease of PAX2 and altered epithelial cytological features in these areas in the initial biopsy are indicative of a premalignant course.

Phenotype-Genotype Correlations in Three Different Cases of Adult-Onset Genetic Focal Segmental Glomerulosclerosis.

This study highlights the importance of a combined diagnostic approach in the diagnosis of rare diseases, such as adult-onset genetic FSGS. We present three adult patient cases evaluated with kidney biopsy for proteinuria, chronic kidney disease, and hypertension, which were suggestive of adult-onset genetic FSGS. Renal biopsy samples and formalin-fixed, paraffin-embedded fetal kidneys were evaluated using standard light microscopical stainings, direct immunofluorescence on cryostat sections, and electron microscopy. Clinical exome sequencing was performed for each case, and 45 FSGS-related genes were analyzed. Identifying mutations in the PAX2, ACTN4, and COL4A5 genes have prompted a re-evaluation of the previous histopathological examinations. The PAX2 mutation led to a thinner nephrogenic zone and decreased number of glomeruli, resulting in oligohydramnios during fetal development and oligomeganephronia and adaptive focal-segmental glomerulosclerosis in adulthood. The ACTN4 mutation caused distinct electron-dense aggregates in podocyte cell bodies, while the COL4A5 mutation led to segmental sclerosis of glomeruli with marked interstitial fibrosis and tubular atrophy. The identification of specific mutations and their histopathological consequences can lead to a better understanding of the disease and its progression, as well as potential treatment options.

Familial focal segmental glomerulosclerosis with Alport-like glomerular basement changes caused by paired box protein 2 gene variant.

Paired box protein 2 (PAX2) gene variant causes renal coloboma syndrome (MIM#120330). Further, they are associated with focal segmental glomerulosclerosis and characterized by basement membrane changes similar to Alport syndrome.Herein, we report an 8-year-old boy who presented with proteinuria and decreased renal function. His paternal uncle has focal segmental glomerulosclerosis and renal failure, and his paternal grandmother has renal failure and is receiving peritoneal dialysis. Further, his father has stage 2 chronic kidney disease. At 3 years of age, his serum creatinine-estimated glomerular filtration rate was 40-50 mL/min/1.73 m2. At 8 years of age, his renal function further decreased and he had proteinuria (urinary protein/Cr 3.39 g/g Cr). Renal histopathology showed oligonephronia and focal segmental glomerulosclerosis. A partial basket-weave pattern, similar to Alport syndrome, was also observed on a transmission electron microscope, and low-vacuum scanning electron microscopy revealed coarse meshwork changes in the glomerular basement membrane. Genetic analysis revealed a PAX2 heterozygous variant (NM_003987.4:c.959C  >  G), a nonsense variant in which the serine at position 320 changes to a stop codon, in our patient and his father. PAX2 is a transcription factor that is important for the podocyte variant. However, podocytes with PAX2 gene variants may cause abnormal basement membrane production and repair, thereby resulting in Alport-like changes.

PAX2 Gene Mutation in Pediatric Renal Disorders-A Narrative Review.

The PAX2 gene is a transcription factor that is essential for the development of the urinary system among other transcription factors. The role of PAX2 is highlighted from the seventh week of gestation, when it is involved in development processes and the emergence of nephrons and collecting tubes. Being an important factor in renal development, mutations of this gene can produce severe alterations in the development of the urinary tract, namely congenital anomalies of the kidneys and urinary tract. The first reported cases described with the PAX2 mutation included both renal anomalies and the involvement of other organs, such as the eyes, producing renal coloboma syndrome. Over the years, numerous cases have been reported, including those with only renal and urinary tract anomalies. The aim of this review is to present a summary of pediatric patients described to have mutations in the PAX2 gene to contribute to a better understanding of the genetic mechanism causing anomalies of the kidneys and urinary tract. In this review, we have included only pediatric cases with renal and urinary tract disorders, without the involvement of other organs. From what we know so far from the literature, this is the first review gathering pediatric patients presenting the PAX2 mutation who have been diagnosed exclusively with renal and urinary tract disorders.

Implication of FOXD2 dysfunction in syndromic congenital anomalies of the kidney and urinary tract (CAKUT).

Background: Congenital anomalies of the kidney and urinary tract (CAKUT) are the predominant cause for chronic kidney disease below 30 years of age. Many monogenic forms have been discovered mainly due to comprehensive genetic testing like exome sequencing (ES). However, disease-causing variants in known disease-associated genes still only explain a proportion of cases. Aim of this study was to unravel the underlying molecular mechanism of syndromic CAKUT in two multiplex families with presumed autosomal recessive inheritance. Methods and Results: ES in the index individuals revealed two different rare homozygous variants in FOXD2, a transcription factor not previously implicated in CAKUT in humans: a frameshift in family 1 and a missense variant in family 2 with family segregation patterns consistent with autosomal-recessive inheritance. CRISPR/Cas9-derived Foxd2 knock-out (KO) mice presented with bilateral dilated renal pelvis accompanied by renal papilla atrophy while extrarenal features included mandibular, ophthalmologic, and behavioral anomalies, recapitulating the phenotype of humans with FOXD2 dysfunction. To study the pathomechanism of FOXD2-dysfunction-mediated developmental renal defects, in a complementary approach, we generated CRISPR/Cas9-mediated KO of Foxd2 in ureteric-bud-induced mouse metanephric mesenchyme cells. Transcriptomic analyses revealed enrichment of numerous differentially expressed genes important in renal/urogenital development, including Pax2 and Wnt4 as well as gene expression changes indicating a cell identity shift towards a stromal cell identity. Histology of Foxd2 KO mouse kidneys confirmed increased fibrosis. Further, GWAS data (genome-wide association studies) suggests that FOXD2 could play a role for maintenance of podocyte integrity during adulthood. Conclusions: In summary, our data implicate that FOXD2 dysfunction is a very rare cause of autosomal recessive syndromic CAKUT and suggest disturbances of the PAX2-WNT4 cell signaling axis contribute to this phenotype.