Germline mutations and somatic inactivation of TRIM28 in Wilms tumour.

Wilms tumour is a childhood tumour that arises as a consequence of somatic and rare germline mutations, the characterisation of which has refined our understanding of nephrogenesis and carcinogenesis. Here we report that germline loss of function mutations in TRIM28 predispose children to Wilms tumour. Loss of function of this transcriptional co-repressor, which has a role in nephrogenesis, has not previously been associated with cancer. Inactivation of TRIM28, either germline or somatic, occurred through inactivating mutations, loss of heterozygosity or epigenetic silencing. TRIM28-mutated tumours had a monomorphic epithelial histology that is uncommon for Wilms tumour. Critically, these tumours were negative for TRIM28 immunohistochemical staining whereas the epithelial component in normal tissue and other Wilms tumours stained positively. These data, together with a characteristic gene expression profile, suggest that inactivation of TRIM28 provides the molecular basis for defining a previously described subtype of Wilms tumour, that has early age of onset and excellent prognosis.

A novel truncating mutation in MYH3 causes spondylocarpotarsal synostosis syndrome with basilar invagination.

Spondylocarpotarsal synostosis syndrome (SCT) is a rare group of skeletal dysplasias, characterized by disproportionate short stature with a short trunk, abnormal segmentation of the spine with vertebral fusion, scoliosis and lordosis, carpal and tarsal synostosis, and mild facial dysmorphisms. While the majority of the cases show autosomal recessive inheritance, only a few cases of vertical transmissions, with MYH3 mutations, have been reported. Here we report a case with typical SCT, carrying a novel heterozygous mutation in MYH3. This observation supports the hypothesis of a pathogenic link between autosomal dominant SCT and heterozygous mutations in MYH3. Of note, our case showed basilar invagination on brain magnetic resonance imaging at the age of 10 years. Basilar invagination could be a rare complication of both autosomal recessive and dominant SCT, indicating that prompt investigation are warranted for SCT patients.

A novel de novo germline mutation Glu40Lys in AKT3 causes megalencephaly with growth hormone deficiency.

Germline or somatic gain-of-function mutations in the v-akt murine thymoma viral oncogene homolog 3 (AKT3) have been reported to cause syndromic megalencephaly. We describe a novel germline mutation, p.Glu40Lys, in AKT3. Phenotypically, the patient presented with megalencephaly with hypotonia, apparent connective tissue laxity, and growth hormone (GH) deficiency. To our knowledge, this is the first instance of a patient with megalencephaly with GH deficiency, harboring a germline de novo mutation in AKT3. © 2017 Wiley Periodicals, Inc.

A novel heterozygous intronic mutation in POU1F1 is associated with combined pituitary hormone deficiency.

POU class 1 homeobox 1 (POU1F1) regulates pituitary cell-specific gene expression of somatotropes, lactotropes, and thyrotropes. In humans, two POU1F1 isoforms (long and short isoform), which are generated by the alternative use of the splice acceptor site for exon 2, have been identified. To date, more than 30 POU1F1 mutations in patients with combined pituitary hormone deficiency (CPHD) have been described. All POU1F1 variants reported to date affect both the short and long isoforms of the POU1F1 protein; therefore, it is unclear at present whether a decrease in the function of only one of these two isoforms is sufficient for disease onset in humans. Here, we described a sibling case of CPHD carrying a heterozygous mutation in intron 1 of POU1F1. In vitro experiments showed that this mutation resulted in exon 2-skipping of only in the short isoform of POU1F1, while the long isoform remained intact. This result strongly suggests the possibility, for the first time, that isolated mutations in the short isoform of POU1F1 could be sufficient for induction of POU1F1-related CPHD. This finding improves our understanding of the molecular mechanisms, and developmental course associated with mutations in POU1F1.

Membranoproliferative glomerulonephritis and C3 glomerulonephritis: frequency, clinical features, and outcome in children.

AIM: C3 glomerulonephritis (C3GN) is a recently described disease that is related to membranoproliferative glomerulonephritis (MPGN). We retrospectively compared the frequencies, clinical characteristics, treatment modalities, and outcomes of C3GN and MPGN in a cohort of Japanese children. METHODS: Children who were pathologically diagnosed with MPGN (type I or III) in our hospital were divided into two groups based on immunofluorescence imaging of renal biopsies: children with MPGN induced by classical complement pathway activation (classical MPGN) and children with C3GN. RESULTS: Of 14 children with MPGN (five boys), four had classical MPGN, eight had C3GN, and two had unclassifiable glomerulonephritis. Four children with classical MPGN and seven with C3GN received methylprednisolone pulse therapy followed by oral prednisolone for 2 years (MPT+PSL therapy). Subsequently, six of seven children with C3GN received combined therapy (prednisolone, azathioprine, and anticoagulants) for 2 years because they responded poorly to MPT+PSL therapy. At the last follow-up visit, two children with classical MPGN and seven with C3GN had not achieved remission. One child with classical MPGN and five with C3GN had hypocomplementaemia at the last follow-up. None of the children had renal impairment. CONCLUSION: More than half of the patients previously diagnosed with MPGN fulfilled the criteria for C3GN in children. C3GN may be more refractory than classical MPGN to immunosuppressant therapy.

Neonatal necrotizing fasciitis of the scrotum caused by Streptococcus agalactiae.

We herein describe the case of a 27-day-old male infant who was brought to the emergency room for intermittent crying, and swelling of the left scrotum. Based on the clinical findings, necrotizing fasciitis was suspected, and surgical intervention was successfully completed within a few hours of admission. Streptococcus agalactiae type Ia was cultured from the drained abscess, and was considered the causative pathogen. To our knowledge, this is the first report of neonatal necrotizing fasciitis caused by S. agalactiae. Prompt diagnosis and immediate surgical debridement are crucial in the initial management of this disease.

Global demethylation in loss of imprinting subtype of Wilms tumor.

Epigenetic abnormalities at the IGF2/H19 locus play a key role in the onset of Wilms tumor. These tumors can be classified into three molecular subtypes depending on the events occurring at this locus: loss of imprinting (LOI), loss of heterozygosity (LOH), or retention of imprinting (ROI). As IGF2 LOI is a consequence of aberrant methylation, we hypothesized that this subtype of Wilms tumors might display global abnormalities of methylation. We therefore analyzed the methylation status of satellite DNA, as a surrogate for global methylation in 50 Wilms tumor patients. Satellite methylation was quantified by a methylation-sensitive quantitative PCR. We confirmed hypomethylation of both satellite α (Sat α) and satellite 2 (Sat 2) DNA in Wilms tumor samples compared with normal kidney. In addition, we found that LOI tumors, unlike ROI or LOH ones, showed concordant hypomethylation of both Sat α and Sat 2 DNA. This would suggest that the LOI subtype of Wilms tumor, which unlike other subtypes results from an epimutation, has a global deregulation of methylation mechanisms.

A report of two novel NR5A1 mutation families: possible clinical phenotype of psychiatric symptoms of anxiety and/or depression.

OBJECTIVE: NR5A1 or steroidogenic factor 1 is a nuclear receptor that plays important roles in the hypothalamus-pituitary-steroidogenic axis. The clinical phenotype of most 46,XY mutation carriers includes disorders of sex development (DSD) without adrenal insufficiency, whereas 46,XX mutation carriers have phenotypes ranging from no symptoms to ovarian insufficiency. Although genetically engineered ventromedial hypothalamus-specific Nr5a1 knockout mice show anxiety behaviour, no psychiatric symptoms have been reported in human NR5A1 mutation carriers. We report clinical and molecular findings for individuals (from two families) with NR5A1 mutations, showing psychiatric symptoms. DESIGN AND METHODS: We screened for NR5A1 mutations in a cohort of 34 patients with 46,XY DSD using PCR-based sequencing. Psychiatric symptoms were assessed using mental health assessment tools and structured clinical interviews. Functional properties of detected mutant NR5A1s were studied in silico and in vitro, including three-dimensional (3D) mutation models, subcellular NR5A1 protein localization and transactivation assays. RESULTS: We found 2 (46,XY) patients with NR5A1 heterozygous novel mutations (p.D257fs and p.V424del), which were transmitted from their respective mothers. The patients' clinical findings indicated DSD without adrenal insufficiency. Both mothers showed psychiatric symptoms, including excessive anxiety and/or depression. The mother and grandmother of one patient had premature ovarian insufficiency. Functional studies showed altered 3D models of p.V424del and normal subcellular NR5A1 localization and impaired transcriptional activation without dominant-negative effects in both mutations. CONCLUSIONS: We found 2 (46,XX) NR5A1 mutation carriers with excessive anxiety and/or depression. These results suggest that excessive anxiety and/or depression are possible clinical phenotypes of 46,XX NR5A1 mutations.

Heterozygous C-propeptide mutations in COL1A1: osteogenesis imperfecta type IIC and dense bone variant.

Osteogenesis imperfecta type IIC (OI IIC) is a rare variant of lethal OI that has been considered to be an autosomal recessive trait. Twisted, slender long bones with dense metaphyseal margins and normal vertebral bodies in OI IIC contrast with crumpled, thick long bones and multiple vertebral compression fractures in OI IIA. Here, we report on two sporadic patients with classical OI IIC and a pair of siblings, with features of OI IIC but less distortion of the tubular bones (OI dense bone variant). One case with OI IIC and the sibs had novel heterozygous mutations in the C-propeptide region of COL1A1, while the second patient with clear-cut OI IIC had no mutation in this region. Histological examination in the two sporadic cases showed a network of broad, interconnected cartilaginous trabeculae with thin osseous seams in the metaphyses. These changes differed from the narrow and short metaphyseal trabeculae found in other lethal or severe cases of OI. Our experience sheds light on the genetics and etiology of OI IIC and on its phenotypic spectrum.

WTX mutations can occur both early and late in the pathogenesis of Wilms tumour.

BACKGROUND: Somatic mutations in the X-linked tumour suppressor gene WTX have been observed in 6- 30% of sporadic cases of Wilms tumour. Germline mutations in the same gene cause the sclerosing skeletal dysplasia, osteopathia striata congenita with cranial sclerosis (OSCS). No evidence points towards a susceptibility to the development of tumours in individuals with OSCS, suggesting that there are unrecognised additional determinants that influence the phenotypic outcome associated with germline mutations in WTX. One explanation may be that a somatic mutation in WTX may need to occur late in tumour development to contribute to tumourigenesis. METHODS: Here a panel of four sporadic Wilms tumours with associated nephrogenic rest tissue and characterised WTX and CTNNB1 mutations is studied to ascertain the temporal sequence of acquisition of these mutations. Additionally, a family with OSCS is described segregating a germline mutation in WTX and manifesting a lethal phenotype in males. One male from this family had bilateral multifocal nephrogenic rests at autopsy. RESULTS: In one of the four tumours the WTX mutation was present in both tumour and rest tissue indicating it had arisen early in tumour development. In the remaining three tumours, the WTX mutation was present in the tumour only indicating late acquisition of these mutations. CONCLUSIONS: These data indicate that WTX mutations can arise both early and late in Wilms tumour development. WTX mutations may predispose to nephrogenic rest development rather than Wilms tumour per se.

Identification and validation of DNA methylation changes in pre-eclampsia.

INTRODUCTION: Pre-eclampsia (PE) is a dangerous placental condition that can lead to premature labour, seizures and death of mother and infant. Several studies have identified altered placental DNA methylation in PE; however, there is widespread inconsistency between studies and most findings have not been replicated. This study aimed to identify and validate consistent differences in methylation across multiple PE cohorts. METHODS: Seven publicly available 450K methylation array datasets were analysed to identify consistent differentially methylated positions (DMPs) in PE. DMPs were identified based on methylation difference (≥10%) and significance (p-value ≤ 1 × 10-7). Targeted deep bisulfite sequencing was then performed to validate a subset of DMPs in an additional independent PE cohort. RESULTS: Stringent analysis of the seven 450K datasets identified 25 DMPs (associated with 11 genes) in only one dataset. Using more relaxed criteria confirmed 19 of the stringent 25 DMPs in at least four of the remaining six datasets. Targeted deep bisulfite sequencing of eight DMPs (associated with three genes; CMIP, ST3GAL1 and DAPK3) in an independent PE cohort validated two DMPs in the CMIP gene. Seven additional CpG sites in CMIP were found to be significantly differentially methylated in PE. DISCUSSION: The identification and validation of significant differential methylation in CMIP suggests that the altered DNA methylation of this gene may be associated with the pathogenesis of PE, and may have the potential to serve as diagnostic biomarkers for this dangerous condition of pregnancy.

Human Papillomavirus E6/E7 Expression in Preeclampsia-Affected Placentae.

Whether HPV is causative of pregnancy complications is uncertain. E6 and E7 affect functions underling preeclampsia (PET) in cultured trophoblasts, but whether E6 and E7 is produced in the placenta is uncertain. Here, we investigated whether E6/E7 was expressed in the placentae from pregnancies with PET, other pregnancy complications (fetal growth restriction (FGR) and diabetes mellitus), and uncomplicated pregnancies. Placental tissues collected from two geographical locations were subjected to RNAscope analyses of high- and low- risk E6/E7, and individual HPV types identified using an HPV array. High-risk E6/E7 expression was increased in both PET cohorts, (81% and 86% of patients positive for high-risk HPV DNA compared to 13% of control patients). Various HPV types were identified. Although HPV 18 was the most frequent in all cohorts, the majority of individuals had multiple HPV types (55% of the PET compared to 25% of the control cohort). Further evidence that E6 and E7 is present early when placental pathology underlying preeclampsia is established, is provided with the finding of high-risk E6/E7 in the first-trimester placenta anchoring trophoblast. In conclusion, E6/E7 expression and multiple HPV types were frequent in placentae from preeclampsia-complicated pregnancies.

Increased levels of active c-Src distinguish invasive from in situ lobular lesions.

INTRODUCTION: Mounting molecular evidence suggests that invasive lobular carcinoma (ILC) is developing from in situ lesions, atypical lobular hyperplasia (ALH), and lobular carcinoma in situ (LCIS). However, little is known about the mechanisms promoting the progression of lobular breast cancer (LBC) to invasive disease. Here, we investigated whether c-Src kinase, an established inducer of invasive states, contributes to the progression from ALH/LCIS to ILC. METHODS: Immunochemistry for c-Src and other cancer-related molecules was performed on archived tissue specimens from 57 LBC patients. Relative c-Src activity was estimated by comparing fluorescence intensity of ILC with that of adjacent ALH/LCIS and nonneoplastic epithelia after staining with an antibody against active c-Src. Expression of active c-Src was correlated with markers of invasion and malignancy and with relapse among LBC patients. RESULTS: Levels of activated c-Src were increased in ILC relative to ALH/LCIS (1.63-fold +/- 0.24 SD) and nonneoplastic epithelia (1.47 +/- 0.18 SD). Increased c-Src levels correlated with the activation of c-Src downstream targets (Fak, Stat-3) and the expression of mesenchymal markers. ILC cells with activated c-Src co-expressed metastatic markers (Opn, Cxcr4) and included cells positive for the cancer stem cell marker Aldh1. A tendency for high c-Src levels (P = 0.072) was observed among the seven LBC patients with relapsed disease. CONCLUSIONS: Our data indicate elevated c-Src activity in ILC relative to noninvasive neoplastic tissue. The associated molecular changes suggest that c-Src promotes LBC invasiveness by inducing an epithelial-mesenchymal transition. Therefore, c-Src antagonists might counteract the acquisition of invasiveness during LBC progression. Inhibition of c-Src may also affect ILC cells thought to have a high metastatic potential and to be capable of initiating/maintaining tumor growth. Together with the possible association between high c-Src levels and disease recurrence, our findings encourage the evaluation of c-Src antagonists for the treatment of LBC.

Destabilized adhesion in the gastric proliferative zone and c-Src kinase activation mark the development of early diffuse gastric cancer.

The initial development of diffuse gastric cancer (DGC) is poorly understood. The study of E-cadherin (CDH1) germ line mutation carriers predisposed to DGC provides a rare opportunity to elucidate the genetic and biological events surrounding disease initiation. Samples from various stages of hereditary and sporadic DGC were investigated to determine general mechanisms underlying early DGC development. Paraffin-embedded tissues from 13 CDH1 mutation carriers and from 10 sporadic early DGC cases were analyzed. Immunofluorescence and immunohistochemistry using differentiation, proliferation, and adhesion markers showed that DGC initiation seems to occur at the proliferative zone (the upper neck) of the gastric epithelium and correlates with absent or reduced expression of junctional proteins (beta-actin, p120, Lin-7). Slow proliferation of neoplastic cells at the upper gastric neck leads to the formation of intramucosal signet-ring cell carcinoma (SRCC) displaying differentiated features. As shown by immunolabeling, invasion from SRCC lesions beyond the gastric mucosa is associated with poor differentiation, increased proliferation, activation of the c-Src system, and an epithelial-mesenchymal transition. Our results provide a molecular description of the early development of DGC and explain the relationship between the two main DGC types, poorly differentiated carcinoma and SRCC: both share their origin, but SRCC develops following cancer cell differentiation and seems relatively indolent in its intramucosal stage.

Ewing's Sarcoma with Extension into Superior Vena Cava and Right Atrium.

Ewing sarcoma is rarely shown to develop this intravascular extension so the decision of the initial treatment is more difficult. We report a 7-year-old boy of this sarcoma with extension into superior vena cava (SVC) and right atrium (RA), who was successfully treated with initial surgery. Intravascular extension was observed from the azygous vein to SVC and finally RA. The removal of the intravascular extension was done, 7 days before chemotherapy was started. The initial surgery for the intravascular extension may have decreased a risk of pulmonary tumor embolism and this made the chemotherapy done safe in this patient.

Selective Ablation of Tumorigenic Cells Following Human Induced Pluripotent Stem Cell-Derived Neural Stem/Progenitor Cell Transplantation in Spinal Cord Injury.

Tumorigenesis is an important problem that needs to be addressed in the field of human stem/progenitor cell transplantation for the treatment of subacute spinal cord injury (SCI). When certain "tumorigenic" cell lines are transplanted into the spinal cord of SCI mice model, there is initial improvement of motor function, followed by abrupt deterioration secondary to the effect of tumor growth. A significant proportion of the transplanted cells remains undifferentiated after transplantation and is thought to increase the risk of tumorigenesis. In this study, using lentiviral vectors, we introduced the herpes simplex virus type 1 thymidine kinase (HSVtk) gene into a human induced pluripotent stem cell-derived neural stem/progenitor cell (hiPSC-NS/PC) line that is known to undergo tumorigenic transformation. Such approach enables selective ablation of the immature proliferating cells and thereby prevents subsequent tumor formation. In vitro, the HSVtk system successfully ablated the immature proliferative neural cells while preserving mature postmitotic neuronal cells. Similar results were observed in vivo following transplantation into the injured spinal cords of immune-deficient (nonobese diabetic-severe combined immune-deficient) mice. Ablation of the proliferating cells exerted a protective effect on the motor function which was regained after transplantation, simultaneously defending the spinal cord from the harmful tumor growth. These results suggest a potentially promising role of suicide genes in opposing tumorigenesis during stem cell therapy. This system allows both preventing and treating tumorigenesis following hiPSC-NS/PC transplantation without sacrificing the improved motor function. Stem Cells Translational Medicine 2019;8:260&270.

Laparoscopic subtotal colectomy for a patient with chronic idiopathic colonic pseudo-obstruction: Report of a case.

A 47-year-old male patient without a documented past medical history was referred to Sanno Hospital because of constipation and abdominal pain, which he had had for more than 5 years. Abdominal X-ray and CT scan showed an enlarged ascending colon from the cecum to the transverse colon, without apparent mechanical obstruction. The patient was diagnosed with chronic idiopathic colonic pseudo-obstruction, and because his symptoms were resistant to medication, surgical treatment was required. Laparoscopic subtotal colectomy was performed without any complications. Constipation was relieved, and the patient began defecating 2-3 times a day without medication. Pathological specimens showed that Meissner's plexus and Auerbach's plexus had decreased and that there were fewer ganglion cells-findings consistent with chronic idiopathic intestinal pseudo-obstruction.

The Mosaicism Ratio of 45,X May Explain the Phenotype in a Case of Mixed Gonadal Dysgenesis.

Some patients with mixed gonadal dysgenesis (MGD), whose prototypical karyotype is 45,X/46,XY, are known to manifest complications characteristic of Turner syndrome. We report a 16-year-old social male with MGD presenting with coarctation of the aorta, one of the common complications for Turner syndrome. At birth, the patient was found to have hypospadias, bifid scrotum, and cryptorchidism. Chromosomal analysis of his lymphocytes revealed the karyotype 45,X[7]/46,X,dic(Y;22)(p11.3;q13.3)[23] (named 45,X/46,X+Y fragment in this article). A left gonadectomy was performed at 1 year of age, and the histology showed a streak gonad with an epithelial cord-like structure compatible with MGD. At the age of 10 years, coarctation of the aorta was discovered by chance, for which the patient underwent surgical repair. The ratio of mosaicism in the gonad and aortic tissues was estimated by FISH with probes to identify the X centromere-specific repeat sequence and Yp11.2. The mosaicism ratio of 45,X/46,X+Y fragment varied among the tissues, with those having a higher ratio being more likely to exhibit the Turner syndrome phenotype. Some 90% of cells in the aortic tissues and 80% in the gonadal tissues lacked a Y chromosome. In conclusion, the mosaicism ratio in the different tissues may explain the phenotypes in MGD.

A Novel Mutation in NKX2-1 Shows Dominant-Negative Effects Only in the Presence of PAX8.

To date, >100 mutations in NKX2-1 have been described. Most NKX2-1 mutations are assumed to result in brain-lung-thyroid syndrome through haploinsufficiency, and only five NKX2-1 mutations with dominant-negative effects have been reported so far. In this case report, an additional patient with brain-lung-thyroid syndrome is reported, carrying a novel heterozygous mutation, c.533G>C (p.R178P), in the homeobox of NKX2-1. This mutation has been proven to be a dominant-negative mutation by an in vitro functional assay. Of note, the dominant-negative effect of R178P-NKX2-1 was shown only in the presence of PAX8.

MIRAGE syndrome is a rare cause of 46,XY DSD born SGA without adrenal insufficiency.

BACKGROUND: MIRAGE syndrome, a congenital multisystem disorder due to pathogenic SAMD9 variants, describes a constellation of clinical features including 46,XY disorders of sex development (DSD), small for gestational age (SGA) and adrenal insufficiency (AI). It is poorly understood whether SAMD9 variants underlie 46,XY DSD patients born SGA (46,XY DSD SGA) without AI. This study aimed to define the frequency and phenotype of SAMD9 variants in 46,XY DSD SGA without AI. METHODS: Forty-nine Japanese patients with 46,XY DSD SGA (Quigley scale, 2 to 6; gestational age-matched birth weight percentile, <10) without history of AI were enrolled. The single coding exon of SAMD9 was PCR-amplified and sequenced for each patient. Pathogenicity of an identified variant was verified in vitro. Placenta tissues were obtained from the variant-carrying patient, as well as from another previously described patient, and were analyzed histologically. RESULTS: In one 46,XY DSD SGA patient, a novel heterozygous SAMD9 variant, p.Phe1017Val, was identified. Pathogenicity of the mutant was experimentally confirmed. In addition to DSD and SGA, the patient had neonatal thrombocytopenia, severe postnatal grow restriction, chronic diarrhea and susceptibility to infection, all features consistent with MIRAGE, leading to premature death at age 14 months. The patient did not have any manifestations or laboratory findings suggesting AI. Placenta tissues of the two variant-carrying patients were characterized by maldevelopment of distal villi without other findings of maternal underperfusion. CONCLUSIONS: MIRAGE syndrome is a rare cause of 46,XY DSD SGA without AI. This study exemplifies that AI is a common feature of MIRAGE syndrome but that the absence of AI should not rule out a diagnosis of the syndrome.