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.

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.

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.

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.

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.

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.

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.

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 Case of Somatic KCNJ5 Mutation in Pediatric-Onset Aldosterone-Producing Adenoma.

Aldosterone-producing adenoma (APA), a subtype of primary aldosteronism, is a common cause of secondary hypertension in adults. Somatic KCNJ5 mutations have been identified in about 12%-80% of adult-onset APA. In contrast, there has been no previous reported case of pediatric-onset APA in whom a somatic KCNJ5 mutation was confirmed. We report an 11-year-old Japanese girl who had experienced recurrent headaches and nausea for more than 2 years before hypertension was observed (blood pressure, 150/82 mm Hg). Plasma renin activity was <0.1 ng/mL per hour even after a captopril-challenge or upright furosemide-loading test. Plasma aldosterone concentrations (PACs) before and after saline-infusion test were 28.0 and 40.6 ng/dL, respectively. Plasma adrenocorticotropic hormone (ACTH) and serum cortisol levels were 16.5 pg/mL and 16.7 µg/dL, respectively. The patient was diagnosed with APA in the left adrenal gland on the basis of selective adrenal venous sampling after ACTH stimulation (PAC in the left adrenal vein, 3630 ng/dL) and histopathologic findings of the tumor obtained by laparoscopic left adrenalectomy. Sanger sequencing of KCNJ5 using genomic DNA from peripheral lymphocytes and laser-captured microdissected APA tissues demonstrated the presence of a somatic KCNJ5 mutation p.L168R, previously reported only in adult-onset APA. Immunohistochemistry detected strong immunoreactivity for CYP11B2, but not for CYP11B1 in the APA, consistent with the endocrinologic findings in this patient. Somatic KCNJ5 mutations are also identified in pediatric-onset APA. Further cases are needed to elucidate functional characteristics of pediatric-onset APA with a somatic KCNJ5 mutation.

The developmental programme for genesis of the entire kidney is recapitulated in Wilms tumour.

Wilms tumour (WT) is an embryonal tumour that recapitulates kidney development. The normal kidney is formed from two distinct embryological origins: the metanephric mesenchyme (MM) and the ureteric bud (UB). It is generally accepted that WT arises from precursor cells in the MM; however whether UB-equivalent structures participate in tumorigenesis is uncertain. To address the question of the involvement of UB, we assessed 55 Wilms tumours for the molecular features of MM and UB using gene expression profiling, immunohistochemsitry and immunofluorescence. Expression profiling primarily based on the Genitourinary Molecular Anatomy Project data identified molecular signatures of the UB and collecting duct as well as those of the proximal and distal tubules in the triphasic histology group. We performed immunolabeling for fetal kidneys and WTs. We focused on a central epithelial blastema pattern which is the characteristic of triphasic histology characterized by UB-like epithelial structures surrounded by MM and MM-derived epithelial structures, evoking the induction/aggregation phase of the developing kidney. The UB-like epithelial structures and surrounding MM and epithelial structures resembling early glomerular epithelium, proximal and distal tubules showed similar expression patterns to those of the developing kidney. These observations indicate WTs can arise from a precursor cell capable of generating the entire kidney, such as the cells of the intermediate mesoderm from which both the MM and UB are derived. Moreover, this provides an explanation for the variable histological features of mesenchymal to epithelial differentiation seen in WT.

Syndromic disorder of sex development due to a novel hemizygous mutation in the carboxyl-terminal domain of ATRX.

Alpha-thalassemia/mental retardation syndrome X-linked (ATRX; OMIM #301040), which is caused by mutations in the ATRX gene, is characterized by alpha-thalassemia, distinct dysmorphic facies, psychomotor development delay and genital abnormalities. Here, we describe a neonatal case of syndromic disorder of sex development, harboring a novel hemizygous mutation, p.Asp2352fs*1 in the carboxyl-terminal domain of ATRX. Our study provides additional evidence that deletion of the carboxyl terminus of ATRX is associated with severe genital anomalies.

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.

Novel heterozygous mutation in the extracellular domain of FGFR1 associated with Hartsfield syndrome.

Heterozygous kinase domain mutations or homozygous extracellular domain mutations in FGFR1 have been reported to cause Hartsfield syndrome (HS), which is characterized by the triad of holoprosencephaly, ectrodactyly and cleft lip/palate. To date, more than 200 mutations in FGFR1 have been described; however, only 10 HS-associated mutations have been reported thus far. We describe a case of typical HS with hypogonadotropic hypogonadism (HH) harboring a novel heterozygous mutation, p.His253Pro, in the extracellular domain of FGFR1. This is the first report of an HS-associated heterozygous mutation located in the extracellular domain of FGFR1, thus expanding our understanding of the phenotypic features and further developmental course associated with FGFR1 mutations.

Pathological classification of human iPSC-derived neural stem/progenitor cells towards safety assessment of transplantation therapy for CNS diseases.

The risk of tumorigenicity is a hurdle for regenerative medicine using induced pluripotent stem cells (iPSCs). Although teratoma formation is readily distinguishable, the malignant transformation of iPSC derivatives has not been clearly defined due to insufficient analysis of histology and phenotype. In the present study, we evaluated the histology of neural stem/progenitor cells (NSPCs) generated from integration-free human peripheral blood mononuclear cell (PBMC)-derived iPSCs (iPSC-NSPCs) following transplantation into central nervous system (CNS) of immunodeficient mice. We found that transplanted iPSC-NSPCs produced differentiation patterns resembling those in embryonic CNS development, and that the microenvironment of the final site of migration affected their maturational stage. Genomic instability of iPSCs correlated with increased proliferation of transplants, although no carcinogenesis was evident. The histological classifications presented here may provide cues for addressing potential safety issues confronting regenerative medicine involving iPSCs.

The Distribution and Cellular Lineages of XX and XY Cells in Gonads Associated with Ovotesticular Disorder of Sexual Development.

Individuals with a 46,XX/46,XY karyotype are categorized as ovotesticular disorder of sexual development (ODSD) and have gonads with either an ovary on one side and a testis on the other side or a mixed ovotestis. To examine the distribution of 46,XX and 46,XY cells in gonads of 3 patients with ODSD, FISH for X and Y chromosomes and immunohistochemistry for SOX9 and FOXL2 were carried out. FISH analysis showed that XX signals were present in Sertoli cells in the seminiferous tubules, while cells containing Y signals were seen in epithelia of ovarian follicles. The immunolabeling of SOX9 and FOXL2 in the seminiferous tubules and ovarian follicles was mutually exclusive, irrespective of the presence of reversed sex chromosomes. We therefore suggest that the fate of individual gonadal epithelial cells is determined not only by the sex chromosomes but also by local environmental factors.

SAMD9 mutations cause a novel multisystem disorder, MIRAGE syndrome, and are associated with loss of chromosome 7.

Adrenal hypoplasia is a rare, life-threatening congenital disorder. Here we define a new form of syndromic adrenal hypoplasia, which we propose to term MIRAGE (myelodysplasia, infection, restriction of growth, adrenal hypoplasia, genital phenotypes, and enteropathy) syndrome. By exome sequencing and follow-up studies, we identified 11 patients with adrenal hypoplasia and common extra-adrenal features harboring mutations in SAMD9. Expression of the wild-type SAMD9 protein, a facilitator of endosome fusion, caused mild growth restriction in cultured cells, whereas expression of mutants caused profound growth inhibition. Patient-derived fibroblasts had restricted growth, decreased plasma membrane EGFR expression, increased size of early endosomes, and intracellular accumulation of giant vesicles carrying a late endosome marker. Of interest, two patients developed myelodysplasitc syndrome (MDS) that was accompanied by loss of the chromosome 7 carrying the SAMD9 mutation. Considering the potent growth-restricting activity of the SAMD9 mutants, the loss of chromosome 7 presumably occurred as an adaptation to the growth-restricting condition.