Patterns of acrorenal malformation associations

Limb and urinary tract defects have frequently been reported to occur together as components of a single acrorenal field defect or in many multiple malformation syndromes. However, the concordance of such anomalies has rarely been studied on a population basis or the relationships between specific limb and renal defects defined. This paper documents the patterns of acrorenal associations seen in over 1,500,000 infants born in Hungary in 1975–1984. In all, 1 in 1,800 infants had a limb deficiency and 9% of these (75 cases) had a urinary tract anomaly. Urinary tract anomalies were most commonly seen with radial ray defects, micromelia and amelia. The commonest recognized patterns were VACTERL association and the cloacal exstrophy and caudal regression sequences. Chromosomal and single gene defects also occurred. Numerical taxonomic techniques delineated six main clusters of patients. Important groupings included micromelia with renal agenesis, split hand/foot with hydronephrosis, and radial ray anomalies with VACTERL defects. The radial ray groups differed in the nature of the VACTERL anomalies seen and with respect to laterality, symmetry, and non-VACTERL anomalies. There was a strong association of bilateral limb defects with bilateral renal anomalies and unilateral with unilateral. Ipsilateral defects tended to occur in typical VACTERL cases, while contralateral defects tended to occur with additional non VACTERL midline anomalies. Although renal and limb anomalies are associated, in almost all cases malformations in other systems are also present. The precise nature of the malformation patterns seen appear to reflect differences in the nature and magnitude of the underlying dysmorphogenetic processes as well as the timing of their effects. © Wiley-Liss, Inc.     

MCA syndrome with renal‐hepatic‐pancreatic dysplasia,posterior fossa cyst,symmetrical limb deficiencies,cleft palate,cardiac and Müllerian duct anomalies

Angelman syndrome (AS) is a disorder of psychomotor development caused by loss of function of the imprinted UBE3A gene. Since the paternal UBE3A copy is regularly silent, only mutations inactivating the maternal copy cause AS. Among 1,272 patients suspected of AS, we found one with an isolated deletion of the UBE3A gene on the maternally inherited chromosome. Initial DNA methylation testing at the SNURF‐SNRPN locus in the patient revealed a normal pattern. The deletion was only detected through allelic loss at microsatellite loci D15S1506, D15S122, and D15S210, and confirmed with fluorescence in situ hybridization (FISH) using bacterial artificial chromosome (BAC) probes derived from the loci. It extends approximately 570 kilobase pairs (kbp), encompassing the UBE3A locus, and is flanked by loci PAR/SN and D15S986. The deletion is familial, and haplotype studies suggest that a great grandfather of the index patient already carried this deletion, and that it causes AS when inherited through the female germline but not Prader‐Willi syndrome (PWS) when paternally inherited. Our findings support the hypothesis that the functional loss of maternal UBE3A gene activity is sufficient to cause AS and that the deleted region does not contain genes or other structures that are involved in PWS. Finally, this case highlights that methylation tests can fail to detect some familial AS cases with a recurrence risk of 50%. © 2002 Wiley‐Liss, Inc.     

Expression of protein arginine methyltransferase‐5 in oral squamous cell carcinoma and its significance in epithelial‐to‐mesenchymal transition

Protein arginine methyltransferases (PRMT) 5, a member of type II arginine methyltransferases, catalyzes the symmetrical dimethylation of arginine residues on histone and non‐histone substrates. Although the overexpression of PRMT5 has been reported in various cancers, its role in oral squamous cell carcinoma (OSCC) has not been elucidated. In the present study, we immunohistochemically examined the expression of PRMT5 in surgically resected oral epithelial dysplasia (OED, n = 8), oral intraepithelial neoplasia (OIN)/carcinoma in situ (CIS) (n = 11) and OSCC (n = 52) with or without contiguous OED lesions. In the normal epithelium, PRMT5 was weakly expressed in the cytoplasm of basal layer cells. In OED, OIN/CIS, and OSCC, its expression consistently and uniformly increased in the cytoplasm of dysplastic and cancer cells. Moreover, nuclear and cytoplasmic localization was detected in the invasive front of cancer cells, particularly in cases showing poor differentiation or aggressive invasion patterns. The concomitant nuclear and cytoplasmic expression of PRMT5 correlated with the loss of E‐cadherin and cytokeratin 17, and the upregulation of vimentin, features that are both indicative of epithelial‐to‐mesenchymal transition. PRMT5 may play a role from early oncogenesis through to the progression of OSCC, particularly in the aggressive mode of stromal invasion.     

Physical and chemical microenvironmental cues orthogonally control the degree and duration of fibrosis‐associated epithelial‐to‐mesenchymal transitions

Increased tissue stiffness and epithelial‐to‐mesenchymal transitions (EMTs) are two seemingly discrete hallmarks of fibrotic diseases. Despite recent findings highlighting the influence of tissue mechanical properties on cell phenotype, it remains unclear what role increased tissue stiffness has in the regulation of previously reported fibronectin‐mediated EMTs associated with pulmonary fibrosis. Nano‐indentation testing of lung interstitial spaces showed that in vivo cell‐level Young's moduli increase with the onset of fibrosis from ～2 to ～17 kPa. In vitro, we found that stiff, but not soft, fibronectin substrates induce EMT, a response dependent on cell contraction‐mediated integrin activation of TGFβ. Activation or suppression of cell contractility with exogenous factors was sufficient to overcome the effect of substrate stiffness. Pulse‐chase experiments indicate that the effect of cell contractility is dose‐ and time‐dependent. In response to low levels of TGFβ on soft surfaces, either added exogenously or produced through thrombin‐induced contraction, cells will initiate the EMT programme, but upon removal revert to an epithelial phenotype. These results identify matrix stiffness and/or cell contractility as critical targets for novel therapeutics for fibrotic diseases.     

Prenatal exposure to valproic acid during pregnancy and limb deficiencies: A case‐control study

We conducted a case‐control study using data from the Spanish Collaborative Study of Congenital Malformations (ECEMC) on the relationship between prenatal exposure to valproic acid (VPA) and the presence of limb deficiencies in newborn infants. Among a total of 22,294 consecutive malformed infants (once we excluded genetic syndromes) and 21,937 control infants with specified data on antiepileptic drugs during gestation, 57 malformed infants and 10 control infants were exposed to VPA during the first trimester of pregnancy. Of the total of malformed infants exposed to VPA, 36.8% (21/57) presented with congenital limb defects of different types (including overlapping digits, talipes, clubfoot, clinodactyly, arachnodactyly, hip dislocation, pre‐ and postaxial polydactyly, etc.), three of them having limb deficiencies. The result of the case‐control analysis shows a risk for limb deficiencies of odds ratio = 6.17 [confidence interval (CI) 1.28–29.66, P = 0.023], after controlling for potential confounder factors. If we consider that in our population the prevalence at birth of this type of defect is 6.88 per 10,000 livebirths (95% CI 6.43–7.36) we can estimate that the risk for women treated with VPA of having a baby with limb deficiencies would be around 0.42%. The limb deficiencies in the three patients exposed to VPA were the following: the first case was a newborn infant with hypoplasia of the left hand, the second patient was a newborn infant with unilateral forearm defect and hypoplastic first metacarpal bone in the left hand, and the third patient presented with short hands with hypoplastic first metacarpal bone, absent and hypoplastic phalanges, retrognathia, facial asymmetry, hypospadias, teleangiectatic angioma in skull, and hypotonia. Am. J. Med. Genet. 90:376–381, 2000. © 2000 Wiley‐Liss, Inc.     

Thymidylate synthase is functionally associated with ZEB1 and contributes to the epithelial‐to‐mesenchymal transition of cancer cells

Thymidylate synthase (TS) is a fundamental enzyme of nucleotide metabolism and one of the oldest anti‐cancer targets. Beginning from the analysis of gene array data from the NCI‐60 panel of cancer cell lines, we identified a significant correlation at both gene and protein level between TS and the markers of epithelial‐to‐mesenchymal transition (EMT), a developmental process that allows cancer cells to acquire features of aggressiveness, like motility and chemoresistance. TS levels were found to be significantly augmented in mesenchymal‐like compared to epithelial‐like cancer cells, to be regulated by EMT induction, and to negatively correlate with micro‐RNAs (miRNAs) usually expressed in epithelial‐like cells and known to actively suppress EMT. Transfection of EMT‐suppressing miRNAs reduced TS levels, and a specific role for miR‐375 in targeting the TS 3'‐untranslated region was identified. A particularly relevant association was found between TS and the powerful EMT driver ZEB1, the shRNA‐mediated knockdown of which up‐regulated miR‐375 and reduced TS cellular levels. The TS–ZEB1 association was confirmed in clinical specimens from lung tumours and in a genetic mouse model of pancreatic cancer with ZEB1 deletion. Interestingly, TS itself appeared to have a regulatory role in EMT in cancer cells, as TS knockdown could directly reduce the EMT phenotype, the migratory ability of cells, the expression of stem‐like markers, and chemoresistance. Taken together, these data indicate that the TS enzyme is functionally linked with EMT and cancer differentiation, with several potential translational implications. Copyright © 2017 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.     

Dual‐probe fluorescence in situ hybridization assay for detecting deletions associated with VCFS/DiGeorge syndrome I and DiGeorge syndrome II loci

Over 90% of patients with DiGeorge syndrome (DGS) or velocardiofacial syndrome (VCFS) have a microdeletion at 22q11.2. Given that these deletions are difficult to visualize at the light microscopic level, fluorescence in situ hybridization (FISH) has been instrumental in the diagnosis of this disorder. Deletions on the short arm of chromosome 10 are also associated with a DGS‐like phenotype. Since deletions at 22q11.2 and at 10p13p14 result in similar findings, we have developed a dual‐probe FISH assay for screening samples referred for DGS or VCFS in the clinical laboratory. This assay includes two test probes for the loci, DGSI at 22q11.2 and DGSII at 10p13p14, and centromeric probes for chromosomes 10 and 22. Of 412 patients tested, 54 were found to be deleted for the DGSI locus on chromosome 22 (13%), and a single patient was found deleted for the DGSII locus on chromosome 10 (0.24%). The patient with the 10p deletion had facial features consistent with VCFS, plus sensorineural hearing loss, and renal anomalies. Cytogenetic analysis showed a large deletion of 10p [46,XX,del(10)(p12.2p14)] and FISH using a 10p telomere region‐specific probe confirmed the interstitial nature of the deletion. Analysis for the DGSI and the DGSII loci suggests that the deletion of the DGSII locus on chromosome 10 may be 50 times less frequent than the deletion of DGSI on chromosome 22. The incidence of deletions at 22q11.2 has been estimated to be 1 in 4000 newborns; therefore, the deletion at 10p13p14 may be estimated to occur in 1 in 200,000 live births. Am. J. Med. Genet. 91:313–317, 2000. © 2000 Wiley‐Liss, Inc.     

Breast stromal fibroblasts from histologically normal surgical margins are pro‐carcinogenic

There is evidence that normal breast stromal fibroblasts (NBFs) suppress tumour growth, while cancer‐associated fibroblasts (CAFs) promote tumourigenesis through functional interactions with tumour cells. Little is known about the biology and the carcinogenic potential of stromal fibroblasts present in histologically normal surgical margins (TCFs). Therefore, we first undertook gene expression analysis on five CAF/TCF pairs from breast cancer patients and three NBF samples (derived from mammoplasties). This comparative analysis revealed variation in gene expression between these three categories of cells, with a TCF‐specific gene expression profile. This variability was higher in TCFs than in their paired CAFs and also NBFs. Cytokine arrays show that TCFs have a specific secretory cytokine profile. In addition, stromal fibroblasts from surgical margins expressed high levels of α‐SMA and SDF‐1 and exhibited higher migratory/invasiveness abilities. Indirect co‐culture showed that TCF cells enhance the proliferation of non‐cancerous mammary epithelial cells and the epithelial‐to‐mesenchymal transition of breast cancer cells. Moreover, TCF and CAF cells increased the level of PCNA, MMP‐2 and the phosphorylated/activated form of Akt in normal breast luminal fibroblasts in a paracrine manner. Furthermore, TCFs were able to promote the formation and growth of humanized orthotopic breast tumours in nude mice. Interestingly, these TCF phenotypes and the extent of their effects were intermediate between those of NBFs and CAFs. Together, these results indicate that stromal fibroblasts located in non‐cancerous tissues exhibit a tumour‐promoting phenotype, indicating that their presence post‐surgery may play important roles in cancer recurrence. © 2013 The Authors. Journal of Pathology published by John Wiley & Sons Ltd on behalf of Pathological Society of Great Britain and Ireland.     

N‐glycosylation by N‐acetylglucosaminyltransferase V enhances the interaction of CD147/basigin with integrin β1 and promotes HCC metastasis

While the importance of protein N‐glycosylation in cancer cell migration is well appreciated, the precise mechanisms by which N‐acetylglucosaminyltransferase V (GnT‐V) regulates cancer processes remain largely unknown. In the current study, we report that GnT‐V‐mediated N‐glycosylation of CD147/basigin, a tumor‐associated glycoprotein that carries β1,6‐N‐acetylglucosamine (β1,6‐GlcNAc) glycans, is upregulated during TGF‐β1‐induced epithelial‐to‐mesenchymal transition (EMT), which correlates with tumor metastasis in patients with hepatocellular carcinoma (HCC). Interruption of β1,6‐GlcNAc glycan modification of CD147/basigin decreased matrix metalloproteinase (MMP) expression in HCC cell lines and affected the interaction of CD147/basigin with integrin β1. These results reveal that β1,6‐branched glycans modulate the biological function of CD147/basigin in HCC metastasis. Moreover, we showed that the PI3K/Akt pathway regulates GnT‐V expression and that inhibition of GnT‐V‐mediated N‐glycosylation suppressed PI3K signaling. In summary, β1,6‐branched N‐glycosylation affects the biological function of CD147/basigin and these findings provide a novel approach for the development of therapeutic strategies targeting metastasis. © 2018 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of Pathological Society of Great Britain and Ireland.     

Epithelial-mesenchymal transition in renal fibrosis - evidence for and against

Epithelial to mesenchymal transition (EMT) is a well established biological process in metazoan embryological development. Over the past 15 years, investigators have sought to establish whether EMT also occurs in renal epithelial cells, following kidney injury, and to show that the mesenchymal cells formed could give rise to myofibroblasts which populate the renal interstitium, causing fibrosis within it. There is no doubt that proximal tubular epithelial cells (PTECs) can undergo EMT in vitro in response to TGFβ-1 and other inflammatory stimuli. Moreover, the results of experiments with animal models of renal fibrosis and examination of biopsies from patients with chronic kidney disease have lent support to the hypothesis that EMT occurs in vivo. This review discusses some of the key evidence underlying that idea and summarises recent advances in understanding the molecular mechanism underlying the process. Early experiments using mice which were genetically engineered to mark PTECs with the LacZ gene to trace their fate following kidney injury provided evidence supporting the occurrence of EMT. Recently, however, cell lineage tracking experiments using the red fluorescent protein (RFP) as a high-resolution marker for cells of renal epithelial origin did not replicate this result; the interstitial space following kidney injury was devoid of RFP expressing cells, leading the investigators to reject the renal EMT hypothesis.     

Lacrimo-auriculo-dento-digital (LADD) syndrome with renal and foot anomalies

A patient who presented with most features of lacrimo-auriculo-dento-digital (LADD) syndrome, an autosomal dominant trait, is described. There was no deafness, and anomalies of the external ear and the upper limbs were discrete. Renal anomalies, consisting of progressive caliectasis with stone formation, were revealed by macroscopic hematuria. There were also skeletal anomalies of both feet, a feature not previously described. Renal and distal limb anomalies are probably features of LADD syndrome.     

Klotho suppresses renal tubulo‐interstitial fibrosis by controlling basic fibroblast growth factor‐2 signalling

Increased basic fibroblast growth factor‐2 (FGF2) and reduced Klotho have both been reported to be closely associated with renal fibrosis. However, the relationship between Klotho and FGF2 remains unclear. We demonstrate that FGF2 induced tubulo‐epithelial plasticity in cultured HK‐2 cells, accompanied by a reduction in Klotho expression, whereas recombinant Klotho protein could inhibit the action of FGF2. The FGF2 effects required extracellular signal‐regulated protein kinase 1/2 activation, which was suppressed by Klotho. Moreover, Klotho also restrained FGF2‐induced fibroblast proliferation and activation. The inhibitory effect of Klotho on the activity of FGF2 was likely due to its potent ability to compete with FGF2 binding to FGF receptor 1. Unilateral ureteral obstruction (UUO)‐induced renal fibrosis was associated with an increase in FGF2 and a reduction in Klotho expression in wild‐type mice, whereas FGF2^–/– mice largely preserved Klotho expression and developed only mild renal fibrosis after obstructive injury. Furthermore, administration of Klotho protein in UUO mice significantly reduced renal fibrosis, concomitant with a marked suppression of FGF2 production and signalling. These studies demonstrate a feedback loop between Klotho depletion and FGF2 activation in renal fibrosis. Our results also suggest that Klotho treatment reduces renal fibrosis, at least in part, by inhibiting FGF2 signalling. Copyright © 2014 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd     

Further delineation of Basel‐Vanagaite‐Smirin‐Yosef syndrome: Report of three patients

Basel‐Vanagaite‐Smirin‐Yosef syndrome is a recently described autosomal recessive intellectual disability syndrome caused by variants in the MED25 gene. While it was originally identified in Brazil, it was further described in Israel by authors who are now the namesake of the condition. A 2018 publication further contributed to its delineation, but the patient's phenotype was complicated by a dual diagnosis. More recently, an article describing a set of affected siblings was published. We describe three, previously unreported, patients showing clinical variability for this newly defined syndrome. The major features determined by “reverse phenotyping” include significant to profound developmental delays/intellectual disability with absent or delayed speech, epilepsy, ocular abnormalities, cleft lip and/or palate, congenital heart disease, urogenital anomalies, skeletal abnormalities, brain malformations and/or microcephaly, failure to thrive, and dysmorphic features. The authors suggest the delineation of an acronym using the gene name and common features seen across the majority of patients reported so far. This new nomination, MED‐DOCS, may help clinicians to recognize, suspect, and remember this novel syndrome.