Kentaro Matsuoka

Molecular and clinical findings and their correlations in Silver-Russell syndrome: implications for a positive role of IGF2 in growth determination and differential imprinting regulation of the IGF2–H19 domain in bodies and placentas

Silver-Russell syndrome (SRS) is characterized by growth failure and dysmorphic features and is frequently caused by hypomethylation (epimutation) of the H19-DMR. Although molecular and clinical studies have extensively been performed for SRS patients themselves, such studies have not been carried out for placentas. We identified 20 epimutation-positive and 40 epimutation-negative Japanese SRS patients and obtained placental weight data from 12 epimutation-positive and ten epimutation-negative patients and paraffin-embedded placental tissues for molecular and histological examinations from three epimutation-positive and two epimutation-negative patients. Methylation patterns were comparable between leukocytes and placentas in both epimutation-positive and epimutation-negative patients. Epimutations resulted in virtually no IGF2 expression and biallelic slight H19 expression in the leukocytes and obviously reduced IGF2 expression of paternal origin and nearly normal H19 expression of maternal origin in the placentas. Epimutation-positive patients had characteristic body phenotype and small placentas with hypoplastic chorionic villi, and epimutation-negative patients had somewhat small placentas with hypoplastic chorionic villi or massive infarction. Furthermore, significant correlations were identified between the H19-DMR methylation index and the body and placental sizes and between the placental weight and the body size in the epimutation-positive patients, whereas such correlations were not detected for the head circumference. These results suggest (1) characteristic phenotype and reduced IGF2 expression in the epimutation-positive placentas; (2) similarities and differences in the epigenetic control of the IGF2–H19 domain between leukocytes and placentas; (3) a positive role of the IGF2 expression level, as reflected by the methylation index, in the determination of body and placental growth in epimutation-positive patients, except for the brain where IGF2 is expressed biallelically; (4) involvement of placental dysfunction in prenatal growth failure; and (5) relevance of both (epi)genetic factor(s) and environmental factor(s) to SRS in epimutation-negative patients.

Mechanisms of development and progression of cyanotic nephropathy.

Cyanotic nephropathy (CN) is often accompanied by congenital cyanotic heart diseases (CCHD). The purpose of this study was to clarify the risk factors and the mechanisms of involved in the development and progression of CN. Thirty patients with CCHD were examined. We analyzed the risk factors for the development of CN on the basis of the clinical and laboratory findings. We also examined ten renal biopsy specimens obtained from patients with CN. Patients with CN showed significantly higher hematocrit levels than those without CN (P=0.025), although there was no difference between the two groups in terms of oxygen saturation. The renal plasma flow (RPF) in patients both with and without CN was low. However, the filtration fraction (FF) was significantly lower in patients with CN than in those without CN (P=0.001). The glomeruli of biopsy specimens with significant proteinuria (n=7) were larger than those of biopsy specimens without significant proteinuria, and there were more capillaries per glomerulus in the former than in the latter (n=3) and the control specimens (n=6) (glomerular size: P<0.01; number of glomerular capillaries: P<0.01). In conclusion, hyperviscosity by polycythemia may be responsible for the development of CN. This pathological condition may induce an angiogenic increase in the glomerular capillary beds, in turn leading to glomerulomegaly. In addition, the failure of a compensatory mechanism to respond to reduced RPF by hyperfiltration may be accompanied by the development and progression of CN.

Comprehensive clinical studies in 34 patients with molecularly defined UPD(14)pat and related conditions (Kagami–Ogata syndrome)

Paternal uniparental disomy 14 (UPD(14)pat) and epimutations and microdeletions affecting the maternally derived 14q32.2 imprinted region lead to a unique constellation of clinical features such as facial abnormalities, small bell-shaped thorax with a coat-hanger appearance of the ribs, abdominal wall defects, placentomegaly, and polyhydramnios. In this study, we performed comprehensive clinical studies in patients with UPD(14)pat (n=23), epimutations (n=5), and microdeletions (n=6), and revealed several notable findings. First, a unique facial appearance with full cheeks and a protruding philtrum and distinctive chest roentgenograms with increased coat-hanger angles to the ribs constituted the pathognomonic features from infancy through childhood. Second, birth size was well preserved, with a median birth length of ±0 SD (range, −1.7 to +3.0 SD) and a median birth weight of +2.3 SD (range, +0.1 to +8.8 SD). Third, developmental delay and/or intellectual disability was invariably present, with a median developmental/intellectual quotient of 55 (range, 29–70). Fourth, hepatoblastoma was identified in three infantile patients (8.8%), and histological examination in two patients showed a poorly differentiated embryonal hepatoblastoma with focal macrotrabecular lesions and well-differentiated hepatoblastoma, respectively. These findings suggest the necessity of an adequate support for developmental delay and periodical screening for hepatoblastoma in the affected patients, and some phenotypic overlap between UPD(14)pat and related conditions and Beckwith–Wiedemann syndrome. On the basis of our previous and present studies that have made a significant contribution to the clarification of underlying (epi)genetic factors and the definition of clinical findings, we propose the name ‘Kagami–Ogata syndrome’ for UPD(14)pat and related conditions.

Paternal uniparental disomy 14 and related disorders: Placental gene expression analyses and histological examinations

Although recent studies in patients with paternal uniparental disomy 14 [upd(14)pat] and other conditions affecting the chromosome 14q32.2 imprinted region have successfully identified underlying epigenetic factors involved in the development of upd(14)pat phenotype, several matters, including regulatory mechanism(s) for RTL1 expression, imprinting status of DIO3 and placental histological characteristics, remain to be elucidated. We therefore performed molecular studies using fresh placental samples from two patients with upd(14)pat. We observed that RTL1 expression level was about five times higher in the placental samples of the two patients than in control placental samples, whereas DIO3 expression level was similar between the placental samples of the two patients and the control placental samples. We next performed histological studies using the above fresh placental samples and formalin-fixed and paraffin-embedded placental samples obtained from a patient with a maternally derived microdeletion involving DLK1, the-IG-DMR, the MEG3-DMR and MEG3. Terminal villi were associated with swollen vascular endothelial cells and hypertrophic pericytes, together with narrowed capillary lumens. DLK1, RTL1 and DIO3 proteins were specifically identified in vascular endothelial cells and pericytes, and the degree of protein staining was well correlated with the expression dosage of corresponding genes. These results suggest that RTL1as-encoded microRNA functions as a repressor of RTL1 expression, and argue against DIO3 being a paternally expressed gene. Furthermore, it is inferred that DLK1, DIO3 and, specially, RTL1 proteins, play a pivotal role in the development of vascular endothelial cells and pericytes.

Androgenetic/biparental mosaicism in a girl with Beckwith-Wiedemann syndrome-like and upd(14)pat-like phenotypes

This report describes androgenetic/biparental mosaicism in a 4-year-old Japanese girl with Beckwith–Wiedemann syndrome (BWS)-like and paternal uniparental disomy 14 (upd(14)pat)-like phenotypes. We performed methylation analysis for 18 differentially methylated regions on various chromosomes, genome-wide microsatellite analysis for a total of 90 loci and expression analysis of SNRPN in leukocytes. Consequently, she was found to have an androgenetic 46,XX cell lineage and a normal 46,XX cell lineage, with the frequency of the androgenetic cells being roughly calculated as 91% in leukocytes, 70% in tongue tissues and 79% in tonsil tissues. It is likely that, after a normal fertilization between an ovum and a sperm, the paternally derived pronucleus alone, but not the maternally derived pronucleus, underwent a mitotic division, resulting both in the generation of the androgenetic cell lineage by endoreplication of one blastomere containing a paternally derived pronucleus and in the formation of the normal cell lineage by union of paternally and maternally derived pronuclei. It appears that the extent of overall (epi)genetic aberrations exceeded the threshold level for the development of BWS-like and upd(14)pat-like phenotypes, but not for the occurrence of other imprinting disorders or recessive Mendelian disorders.

Clinical Features and Prognosis of Generalized Lymphatic Anomaly, Kaposiform Lymphangiomatosis, and Gorham-Stout Disease.

Complex lymphatic anomalies are intractable lymphatic disorders, including generalized lymphatic anomaly (GLA), Gorham–Stout disease (GSD), and kaposiform lymphangiomatosis (KLA). The etiology of these diseases remains unknown and diagnosis is confused by their similar clinical findings. This study aimed to clarify the differences in clinical features and prognosis among GLA, KLA, and GSD, in Japanese patients.

Prognostic significance of circulating tumor cells and bone marrow micrometastasis in advanced neuroblastoma

PURPOSE The aim of this study was to study the prognostic significance of circulating tumor cells (CTC) and the appropriate indications for aggressive surgery in advanced neuroblastoma. MATERIALS AND METHODS Micrometastasis was sequentially explored using our reverse transcriptase-polymerase chain reaction method in 29 neuroblastoma patients (International Neuroblastoma Staging System stage 4, n = 24; stage 3, n = 5) who treated at our department with the united chemotherapeutic regimen since 1991. Their medical records and detection of CTC and/or the bone marrow micrometastasis were retrospectively reviewed then analyzed statistically. RESULTS The overall survival rate was 58.6% (17/29). Circulating tumor cells were detected in 55.6% of the stage 4 patients, and all deaths were related to systemic metastases in the CTC-positive patients. The detection of CTC scarcely associated with MYCN amplification. In the patients showing MYCN amplification but no CTC, all deaths were related to local relapse or chemotherapy-associated complications. The survival rate was not significantly different between the patients with and without MYCN amplification (56.8% vs 52.7%). However, it was significantly lower in the patients with CTC and/or persistent bone marrow micrometastasis compared to those without detectable micrometastasis (33.8% vs 87.5%; P < .05). CONCLUSIONS The presence of CTC and/or persistent micrometastasis may indicate a significantly high risk, regardless of MYCN amplification. Patients with MYCN amplification but no micrometastasis would be most benefited by highly intensive surgery.

Endoscopic Fetal Urethrotomy for Anterior Urethral Valves: A Preliminary Report

Anterior urethral valves are a rare congenital anomaly associated with distal urethral obstruction, which can result in a poor prognosis. We report on the endoscopic creation of a fetal urethrotomy for obstructive uropathy resulting from anterior urethral valves. A 33-year-old woman was evaluated at 17 weeks gestation due to fetal megacystis. The diagnosis of anterior urethral valves was confirmed by the characteristic sonographic feature of a dilated membranous penile urethra. Oligohydramnios with normal-appearing kidneys and favorable urinary electrolytes led to fetal intervention. Ablation on the ventral site of the fetal penis for a cutaneous urethrotomy was performed using a YAG laser under a 1-mm fetoscope at 19 weeks gestation. Urine was drained from the incision and the dilated penis and the distended bladder shrunk with an increase in amniotic fluid. However, the fetus died unexpectedly on postoperative day 3, and chorioamnionitis was suspected as the etiology. While the outcome was unfavorable, our preliminary experience shows that fetal urethrotomy for obstructive uropathy can be achieved in utero using an endoscopic laser approach. Further experience will be required to evaluate the therapeutic value of this new procedure in the management of fetal anterior urethral valves.

Severe Alport syndrome in a young woman caused by a t(X;1)(q22.3;p36.32) balanced translocation

The course of renal involvement and hearing loss is much milder in most female X-linked Alport syndromes than in male patients. We examined the molecular mechanism of development of the disease in a female patient with severe Alport syndrome. The patient showed heavy proteinuria, hematuria, neurosensory hearing loss and primary amenorrhea. Renal biopsy findings of electron microscopy and immunostaining of the α5 chain of type IV collagen indicated a female X-linked Alport syndrome. G-banding chromosomal analysis showed a t(X;1)(q22.3;p36.32) balanced translocation. Analysis of the collagen type IV (COL4A5) gene by genomic DNA sequencing, complementary DNA (cDNA) sequencing and multiplex ligation-dependent probe amplification assay showed no mutations or deletions/duplications of the gene. However, fluorescence in situ hybridization using the probes for exon 1 and exon 51 of the COL4A5 gene showed disruption of one copy of the gene. Replication R-banding chromosomal analysis indicated preferential inactivation of the normal X chromosome. This is the first report of severe Alport syndrome in a female patient carrying a balanced translocation between the chromosome X and 1 producing the disruption of one copy of COL4A5 gene and silencing of the other copy because of preferential inactivation of the normal X chromosome. Chromosomal abnormalities should be considered in female patients with severe forms of Alport syndrome.

A novel association of duodenal atresia, malrotation, segmental dilatation of the colon, and anorectal malformation

We describe two female neonates who suffered from four gastrointestinal anomalies, including duodenal stenosis or atresia, malrotation, segmental dilatation of the colon, and anorectal malformation. Each patient was managed by two or three operations, resulting in good bowel movements. Since this is the first report of four gastrointestinal anomalies, these cases may provide clues to elucidate the etiology of gastrointestinal tract developmental abnormalities.