Frequency and clinical characteristics of distinct etiologies in patients with Silver-Russell syndrome diagnosed based on the Netchine-Harbison clinical scoring system.

Silver-Russel syndrome (SRS) is a representative imprinting disorder (ID) characterized by growth failure and diagnosed by clinical features. Recently, international consensus has recommended using the Netchine-Harbison clinical scoring system (NH-CSS) as clinical diagnostic criteria. Loss of methylation of H19/IGF2:intergenic differentially methylated region (H19LOM) and maternal uniparental disomy chromosome 7 (UPD(7)mat) are common etiologies of SRS; however, other IDs, pathogenic variants (PVs) of genes, and pathogenic copy number variants (PCNVs) have been reported in patients meeting NH-CSS. To clarify the frequency and clinical characteristics of each etiology, we conducted (epi)genetic analysis in 173 patients satisfying NH-CSS. H19LOM and UPD(7)mat were identified in 34.1%. PCNVs, other IDs, and PVs were in 15.0%. Patients with all six NH-CSS items were most frequently observed with H19LOM and UPD(7)mat. This study confirmed the suitability of NH-CSS as clinical diagnostic criteria, the (epi)genetic heterogeneity of SRS, and showed the necessity of further discussion regarding the "SRS spectrum".

Loss of imprinting of the human-specific imprinted gene ZNF597 causes prenatal growth retardation and dysmorphic features: implications for phenotypic overlap with Silver-Russell syndrome.

BACKGROUND: ZNF597, encoding a zinc-finger protein, is the human-specific maternally expressed imprinted gene located on 16p13.3. The parent-of-origin expression of ZNF597 is regulated by the ZNF597:TSS-DMR, of which only the paternal allele acquires methylation during postimplantation period. Overexpression of ZNF597 may contribute to some of the phenotypes associated with maternal uniparental disomy of chromosome 16 (UPD(16)mat), and some patients with UPD(16)mat presenting with Silver-Russell syndrome (SRS) phenotype have recently been reported. METHODS: A 6-year-old boy presented with prenatal growth restriction, macrocephaly at birth, forehead protrusion in infancy and clinodactyly of the fifth finger. Methylation, expression, microsatellite marker, single nucleotide polymorphism array and trio whole-exome sequencing analyses were conducted. RESULTS: Isolated hypomethylation of the ZNF597:TSS-DMR and subsequent loss of imprinting and overexpression of ZNF597 were confirmed in the patient. Epigenetic alterations, such as UPD including UPD(16)mat and other methylation defects, were excluded. Pathogenic sequence or copy number variants affecting his phenotypes were not identified, indicating that primary epimutation occurred postzygotically. CONCLUSION: We report the first case of isolated ZNF597 imprinting defect, showing phenotypic overlap with SRS despite not satisfying the clinical SRS criteria. A novel imprinting disorder entity involving the ZNF597 imprinted domain can be speculated.

Insulin resistant diabetes mellitus in SHORT syndrome: case report and literature review.

SHORT syndrome is a rare developmental disorder frequently associated with growth failure and insulin resistant diabetes mellitus (IRDM). Since GH has a diabetogenic effect, GH therapy has been regarded as a contraindication. We observed a Brazilian girl with SHORT syndrome who received GH therapy from 4 6/12 years of age for SGA short stature. GH dosage was increased from 0.23 to 0.36 mg/kg/week, but statural response to GH therapy remained poor. Her blood HbA1c level, though it remained 5.5-6.0% in childhood, began to elevate with puberty and increased to 9.2% at 10 6/12 years of age, despite the discontinuation of GH therapy at 9 11/12 years of age. Laboratory studies indicated antibody-negative IRDM. She was treated with metformin and canagliflozin (a sodium glucose co-transporter 2 (SGLT2) inhibitor), which ameliorated overt diurnal hyperglycemia and mild nocturnal hypoglycemia and reduced her blood HbA1c around 7%. Whole exome sequencing revealed a de novo heterozygous pathogenic variant (c.1945C>T:p.(Arg649Trp)) in PIK3R1 known as the sole causative gene for SHORT syndrome. Subsequent literature review for patients with molecularly confirmed SHORT syndrome revealed the development of IRDM in 10 of 15 GH-untreated patients aged ≥12 years but in none of three GH-treated and six GH-untreated patients aged ≤10 years. These findings imply a critical role of pubertal development and/or advanced age rather than GH therapy in the development of IRDM, and a usefulness of SGLT2 inhibitor in the treatment of IRDM.

Extraction of bioavailable phosphorus in soils and sediments using an ultrasonic washing machine.

For improving the management of watershed eutrophication, methods for measuring bioavailable phosphorus (BAP) are more important than measurements of total phosphorus (TP). BAP in particulate form (P-BAP) is an important substance that promotes eutrophication, especially during rainy seasons. Only a portion of particulate phosphorus (PP) is taken up by algae that contribute to eutrophication. Erosion and runoff associated with rainfall transport PP bound to sediments and soil particles to surface waters, thus increasing PP concentration. This research evaluated an extraction method using an ultrasonic washing machine for extraction time and frequency. Extraction at a frequency of 28-45 kHz and an extraction time of 1 min resulted in extracted P concentrations almost the same as concentrations extracted using conventional methods. This new method requires less time and is more efficient than conventional methods because it extracts P from multiple samples in a single step. Results indicate that extraction using an ultrasonic washing machine is a promising method for rapidly obtaining BAP from sediments and soil particles.

Molecular and clinical analyses of two patients with UPD(16)mat detected by screening 94 patients with Silver-Russell syndrome phenotype of unknown aetiology.

BACKGROUND: Recently, a patient with maternal uniparental disomy of chromosome 16 (UPD(16)mat) presenting with Silver-Russell syndrome (SRS) phenotype was reported. SRS is characterised by growth failure and dysmorphic features. OBJECTIVE: To clarify the prevalence of UPD(16)mat in aetiology-unknown patients with SRS phenotype and phenotypic differences between UPD(16)mat and SRS. METHODS: We studied 94 patients with SRS phenotype of unknown aetiology. Sixty-three satisfied the Netchine-Harbison clinical scoring system (NH-CSS) criteria, and 25 out of 63 patients showed both protruding forehead and relative macrocephaly (clinical SRS). The remaining 31 patients met only three NH-CSS criteria, but were clinically suspected as having SRS. To detect UPD(16)mat, we performed methylation analysis for the ZNF597:TSS-differentially methylated region (DMR) on chromosome 16 and subsequently performed microsatellite, SNP array and exome analyses in the patients with hypomethylated ZNF597:TSS-DMR. RESULTS: We identified two patients (2.1%) with a mixture of maternal isodisomy and heterodisomy of chromosome 16 in 94 aetiology-unknown patients with SRS phenotype. Both patients exhibited preterm birth and prenatal and postnatal growth failure. The male patient had ventricular septal defect and hypospadias. Whole-exome sequencing detected no gene mutations related to their phenotypes. CONCLUSION: We suggest considering genetic testing for UPD(16)mat in SRS phenotypic patients without known aetiology.

Hemoglobin phase of oxygenation and deoxygenation in early brain development measured using fNIRS.

A crucial issue in neonatal medicine is the impact of preterm birth on the developmental trajectory of the brain. Although a growing number of studies have shown alterations in the structure and function of the brain in preterm-born infants, we propose a method to detect subtle differences in neurovascular and metabolic functions in neonates and infants. Functional near-infrared spectroscopy (fNIRS) was used to obtain time-averaged phase differences between spontaneous low-frequency (less than 0.1 Hz) oscillatory changes in oxygenated hemoglobin (oxy-Hb) and those in deoxygenated hemoglobin (deoxy-Hb). This phase difference was referred to as hemoglobin phase of oxygenation and deoxygenation (hPod) in the cerebral tissue of sleeping neonates and infants. We examined hPod in term, late preterm, and early preterm infants with no evidence of clinical issues and found that all groups of infants showed developmental changes in the values of hPod from an in-phase to an antiphase pattern. Comparison of hPod among the groups revealed that developmental changes in hPod in early preterm infants precede those in late preterm and term infants at term equivalent age but then, progress at a slower pace. This study suggests that hPod measured using fNIRS is sensitive to the developmental stage of the integration of circular, neurovascular, and metabolic functions in the brains of neonates and infants.

Carapanosins D-F from the Seeds of Andiroba (Carapa guianensis, Meliaceae) and Their Effects on LPS-Activated NO Production.

A novel nor-phragmalin-type limonoid, named carapanosin D (1), and two novel mexicanolide-type limonoids, carapanosins E (2) and F (3), were isolated from the seed oil of andiroba (Carapa guianensis Aublet), a traditional medicine in Brazil and Latin American countries. Their structures were unambiguously determined on the basis of spectroscopic analyses using one-dimensional (1D) and two-dimensional (2D) NMR techniques and High resolution Fast Atom Bombardment Mass Spectrometry (HRFABMS). Compounds 1⁻3 were evaluated for their effects on the production of nitric oxide (NO) in Lipopolysaccharide (LPS)-activated mouse peritoneal macrophages. The NO inhibitory assay suggested that compounds 2 and 3 have high potency as inhibitors of macrophage activation.

Spatial and temporal variation of water quality of a segment of Marikina River using multivariate statistical methods.

Payatas landfill in Quezon City, Philippines, releases leachate to the Marikina River through a creek. Multivariate statistical techniques were applied to study temporal and spatial variations in water quality of a segment of the Marikina River. The data set included 12 physico-chemical parameters for five monitoring stations over a year. Cluster analysis grouped the monitoring stations into four clusters and identified January-May as dry season and June-September as wet season. Principal components analysis showed that three latent factors are responsible for the data set explaining 83% of its total variance. The chemical oxygen demand, biochemical oxygen demand, total dissolved solids, Cl- and PO43- are influenced by anthropogenic impact/eutrophication pollution from point sources. Total suspended solids, turbidity and SO42- are influenced by rain and soil erosion. The highest state of pollution is at the Payatas creek outfall from March to May, whereas at downstream stations it is in May. The current study indicates that the river monitoring requires only four stations, nine water quality parameters and testing over three specific months of the year. The findings of this study imply that Payatas landfill requires a proper leachate collection and treatment system to reduce its impact on the Marikina River.

Generation of a transgenic medaka (Oryzias latipes) strain for visualization of nuclear dynamics in early developmental stages.

In this study, we verified nuclear transport activity of an artificial nuclear localization signal (aNLS) in medaka fish (Oryzias latipes). We generated a transgenic medaka strain expresses the aNLS tagged enhanced green fluorescent protein (EGFP) driven by a medaka beta-actin promoter. The aNLS-EGFP was accumulated in the nuclei of somatic tissues and yolk nuclei of oocytes, but undetectable in the spermatozoa. The fluorescent signal was observed from immediately after fertilization by a maternal contribution. Furthermore, male and female pronuclei were visualized in fertilized eggs, and nuclear dynamics of pronuclear fusion and subsequent cleavage were captured by time-lapse imaging. In contrast, SV40NLS exhibited no activity of nuclear transport in early embryos. In conclusion, the aNLS possesses a strong nuclear localization activity and is a useful probe for fluorescent observation of the pronuclei and nuclei in early developmental stage of medaka.

Human health risk assessment of mercury vapor around artisanal small-scale gold mining area, Palu city, Central Sulawesi, Indonesia.

Emissions of elemental mercury, Hg(0), from artisanal small-scale gold mining activities accounted for 37% of total global Hg(0) emissions in 2010. People who live near gold-mining areas may be exposed to high concentrations of Hg(0). Here, we assessed the human health risk due to Hg(0) exposure among residents of Palu city (Central Sulawesi Province, Indonesia). The area around the city has more than 60t of gold reserves, and the nearby Poboya area is the most active gold-mining site in Indonesia. Owing to its geography, the city experiences alternating land and sea breezes. Sampling was done over a period of 3 years (from 2010 Aug. to 2012 Dec.) intermittently with a passive sampler for Hg(0), a portable handheld mercury analyzer, and a mercury analyzer in four areas of the city and in the Poboya gold-processing area, as well as wind speeds and directions in one area of the city. The 24-h average concentration, wind speed, and wind direction data show that the ambient air in both the gold-processing area and the city was always covered by high concentration of mercury vapor. The Hg(0) concentration in the city was higher at night than in the daytime, owing to the effect of land breezes. These results indicate that the inhabitants of the city were always exposed to high concentrations of Hg(0). The average daytime point-sample Hg(0) concentrations in the city, as measured with a handheld mercury analyzer over 3 days in July 2011, ranged from 2096 to 3299ngm(-3). In comparison, the average daytime Hg(0) concentration in the Poboya gold-processing area was 12,782ngm(-3). All of these concentrations are substantially higher than the World Health Organization air-quality guideline for annual average Hg exposure (1000ngm(-3)). We used the point-sample concentrations to calculate hazard quotient ratios by means of a probabilistic risk assessment method. The results indicated that 93% of the sample population overall was at risk (hazard quotient ratio ≥1 and cut off at the 95th percentile value of the sample population) of mercury toxicity, that is, damage to the central nervous system due to chronic exposure. The corresponding percentages for the northern, central, southern, and western areas of the city were 83%, 84%, 95%, and 95%, respectively. Our results indicate that the residents of Palu city are at serious risk from exposure to high concentrations of atmospheric Hg(0).

Measuring total mercury due to small-scale gold mining activities to determine community vulnerability in Cihonje, Central Java, Indonesia.

This research is comparative study of gold mining and non-gold mining areas, using four community vulnerability indicators. Vulnerability indicators are exposure degree, contamination rate, chronic, and acute toxicity. Each indicator used different samples, such as wastewater from gold mining process, river water from Tajum river, human hair samples, and health questionnaire. This research used cold vapor atomic absorption spectrometry to determine total mercury concentration. The result showed that concentration of total mercury was 2,420 times than the maximum content of mercury permitted in wastewater based on the Indonesian regulation. Moreover, the mercury concentration in river water reached 685 ng/l, exceeding the quality threshold standards of the World Health Organization (WHO). The mercury concentration in hair samples obtained from the people living in the research location was considered to identify the health quality level of the people or as a chronic toxicity indicator. The highest mercury concentration--i.e. 17 ng/mg, was found in the gold mining respondents. Therefore, based on the total mercury concentration in the four indicators, the community in the gold mining area were more vulnerable to mercury than communities in non-gold mining areas. It was concluded that the community in gold mining area was more vulnerable to mercury contamination than the community in non-gold mining area.

Carapanolides J-L from the seeds of Carapa guianensis (Andiroba) and their effects on LPS-activated NO production.

A novel gedunin and two novel phragmalin-type limonoids, named carapanolides J-L (compounds 1-3) as well as a known gedunin-type limonoid 4 were isolated from the seeds of Carapa guianensis (andiroba). Their structures were determined on the basis of 1D and 2D NMR spectroscopy and HRFABMS. Compounds 1-4 were evaluated for their effects on the production of NO in LPS-activated mouse peritoneal macrophages.

Availability of different phosphorus forms in agricultural soil to Microcystis aeruginosa.

We investigated the availability of different forms of particulate soil phosphorus (P) to Microcystis aeruginosa by sequential extraction and bioassay. We cultured M. aeruginosa in media containing, as the sole source of P, soils sequentially extracted with 1 M NH4Cl, 0.11 M bicarbonate dithionite, 1 M NaOH, and 0.5 M HCl. Analyses of chlorophyll-a, particulate organic carbon, and particulate organic nitrogen showed that M. aeruginosa could utilize some of the P remaining in the soil after each extraction. Alkaline phosphatase (AP) assays of sequentially extracted soils showed distinct patterns that depended on the type of co-cultured soil. A direct relationship between cellular P concentrations and the level of alkaline phosphatase activity was observed in only some media, an indication that not all forms of P were equally suitable substrates for AP hydrolysis. These results imply that cyanobacterial-available P included not only HCl-extractable P, which is assumed to consist of carbonate or apatite bound-P and organic P, but also refractory P, which has been considered to be unavailable to algae. Both HCl-extracted P and refractory P enhance the production of chlorophyll a, but did not lead to the storage of P by M. aeruginosa.

Developing a method to measure bioavailable phosphorus in river water via simultaneous multisample ultrasonic extraction.

To reduce aquatic eutrophication, measurements of bioavailable phosphorus (BAP) rather than total phosphorus (TP) are deemed critical. However, current methods require much time to separate sediments from river water, which limits the routine measurement of BAP in rivers. Therefore, in this study, a simultaneous multisample ultrasonic extraction method is proposed to directly measure total BAP (TBAP) in river water without the separation of sediment and water. Spike-and-recovery assessments showed that at least three extractions are required to maintain efficiency. A process including 2-min extraction time and three extractions was suggested. The concentrations of TBAP extracted by this process showed no significant differences with the spike calculations. Furthermore, river water TBAP was quantified using the conventional and proposed method to examine the practicality of using the proposed method for simultaneous multisample ultrasonic extraction and to evaluate its adaptability to actual river water analysis. The extracted concentrations matched those obtained using the conventional method, in which total BAP is calculated as the sum of dissolved BAP and particulate BAP; no significant difference was observed between the concentrations. Ultrasonic extraction was considerably less time-consuming than the conventional method because more samples could be analyzed during a single run. Therefore, the simultaneous multisample ultrasonic extraction method proposed in this study can be used to directly quantify total BAP in river water.

Ventriculosubgaleal shunt placement for hydrocephalus in osteogenesis imperfecta with novel compound heterozygous CRTAP variants.

Osteogenesis imperfecta is characterized by frequent fractures, bone deformities, and other systemic symptoms. Severe osteogenesis imperfecta may progress to hydrocephalus; however, treatment strategies for this complication remain unclear. Here, we describe severe osteogenesis imperfecta in an infant with symptomatic hydrocephalus treated with ventriculosubgaleal shunt placement. Targeted next-generation sequencing revealed novel compound heterozygous CRTAP variants, i.e., NM_006371.5, c.241 G > T, p.(Glu81*) and NM_006371.5, c.923-2_932del. We suggest that ventriculosubgaleal shunt placement is an effective and safe treatment for hydrocephalus in patients with severe osteogenesis imperfecta.

Risk assessment of assisted reproductive technology and parental age at childbirth for the development of uniparental disomy-mediated imprinting disorders caused by aneuploid gametes.

BACKGROUND: Our previous study suggested that assisted reproductive technology (ART) may be a possible risk factor for the development of epimutation-mediated imprinting disorders (epi-IDs) for mothers aged ≥ 30 years. However, whether ART or advanced parental age facilitates the development of uniparental disomy-mediated IDs (UPD-IDs) has not yet been investigated. RESULTS: We enrolled 130 patients with aneuploid UPD-IDs including various IDs confirmed by molecular studies and obtained ART data of the general population and patients with epi-IDs from a robust nationwide database and our previous report, respectively. We compared the proportion of ART-conceived livebirths and maternal childbearing age between patients with UPD-IDs and the general population or patients with epi-IDs. The proportion of ART-conceived livebirths in patients with aneuploid UPD-IDs was consistent with that in the general population of maternal age ≥ 30 years and was lower than that in the patients with epi-IDs, although there was no significant difference. The maternal childbearing age of patients with aneuploid UPD-IDs was skewed to the increased ages with several cases exceeding the 97.5th percentile of maternal childbearing age of the general population and significantly higher than that of patients with epi-IDs (P < 0.001). In addition, we compared the proportion of ART-conceived livebirths and parental age at childbirth between patients with UPD-IDs caused by aneuploid oocytes (oUPD-IDs) and that by aneuploid sperm (sUPD-IDs). Almost all ART-conceived livebirths were identified in patients with oUPD-IDs, and both maternal age and paternal age at childbirth were significantly higher in patients with oUPD-IDs than in patients with sUPD-IDs. Because maternal age and paternal age were strongly correlated (rs = 0.637, P < 0.001), higher paternal age in oUPD-IDs was explained by the higher maternal age in this group. CONCLUSIONS: Different from the case of epi-IDs, ART itself is not likely to facilitate the development of aneuploid UPD-IDs. We demonstrated that advanced maternal age can be a risk factor for the development of aneuploid UPD-IDs, particularly oUPD-IDs.

Pathogenic Copy Number and Sequence Variants in Children Born SGA With Short Stature Without Imprinting Disorders.

CONTEXT: Children born small-for-gestational-age with short stature (SGA-SS) is associated with (epi)genetic defects, including imprinting disorders (IDs), pathogenic copy number variants (PCNVs), and pathogenic variants of genes involved in growth. However, comprehensive studies evaluating these 3 factors are very limited. OBJECTIVE: To clarify the contribution of PCNVs and candidate pathogenic variants to SGA-SS. DESIGN: Comprehensive molecular analyses consisting of methylation analysis, copy number analysis, and multigene sequencing. METHODS: We enrolled 140 patients referred to us for genetic testing for SGA-SS. Among them, we excluded 42 patients meeting Netchine-Harbison clinical scoring system criteria for Silver-Russell syndrome and 4 patients with abnormal methylation levels of the IDs-related differentially methylated regions. Consequently, we conducted copy number analysis and multigene sequencing for 86 SGA-SS patients with sufficient sample volume. We also evaluated clinical phenotypes of patients with PCNVs or candidate pathogenic variants. RESULTS: We identified 8 (9.3%) and 11 (12.8%) patients with PCNVs and candidate pathogenic variants, respectively. According to the American College of Medical Genetics standards and guidelines, 5 variants were classified as pathogenic and the remaining 6 variants were classified as variants of unknown significance. Genetic diagnosis was made in 12 patients. All patients with PCNVs or candidate pathogenic variants did not correspond perfectly to characteristic clinical features of each specific genetic cause. CONCLUSION: We clarified the contribution of PCNVs and pathogenic variants to SGA-SS without IDs. Comprehensive molecular analyses, including copy number analysis and multigene sequencing, should be considered for patients with unknown SGA-SS etiology.

Plastic pollution in the surface water in Jakarta, Indonesia.

Plastic pollution in the ocean primarily originates from the land-derived mismanaged plastic waste that is transported by rivers. This study aimed to estimate the plastic litter generation in the surface water in Jakarta and Indonesia. A field survey was conducted at six riverine sampling points (upstream to downstream) and three holding facilities of the litter in Jakarta during the rainy season. The Jakarta Open Data database was used to estimate the tonnage of plastic litter. By mass, plastic comprised approximately 74 % of the anthropogenic litter in rivers and 87 % in holding facilities. The riverine plastic proportion slightly increased downstream. Approximately 9.9 g/person/day of plastic litter was discharged into Jakarta's surface water during rainy season and recovered by floating booms. To reduce plastic pollution and its severe impacts on aquatic ecosystems and human health, further field investigation is necessary to design an effective clean-up system and litter-prevention strategy.

Role of Imprinting Disorders in Short Children Born SGA and Silver-Russell Syndrome Spectrum.

BACKGROUND: (Epi)genetic disorders associated with small-for-gestational-age with short stature (SGA-SS) include imprinting disorders (IDs). Silver-Russell syndrome (SRS) is a representative ID in SGA-SS and has heterogenous (epi)genetic causes. SUBJECTS AND METHODS: To clarify the contribution of IDs to SGA-SS and the molecular and phenotypic spectrum of SRS, we recruited 269 patients with SGA-SS, consisting of 103 and 166 patients referred to us for genetic testing for SGA-SS and SRS, respectively. After excluding 20 patients with structural abnormalities detected by comparative genomic hybridization analysis using catalog array, 249 patients were classified into 3 subgroups based on the Netchine-Harbison clinical scoring system (NH-CSS), SRS diagnostic criteria. We screened various IDs by methylation analysis for differentially methylated regions (DMRs) related to known IDs. We also performed clinical analysis. RESULTS: These 249 patients with SGA-SS were classified into the "SRS-compatible group" (n = 148), the "non-SRS with normocephaly or relative macrocephaly at birth group" (non-SRS group) (n = 94), or the "non-SRS with relative microcephaly at birth group" (non-SRS with microcephaly group) (n = 7). The 44.6% of patients in the "SRS-compatible group," 21.3% of patients in the "non-SRS group," and 14.3% in the "non-SRS with microcephaly group" had various IDs. Loss of methylation of the H19/IGF2:intergenic-DMR and uniparental disomy chromosome 7, being major genetic causes of SRS, was detected in 30.4% of patients in the "SRS-compatible group" and in 13.8% of patients in the "non-SRS group." CONCLUSION: We clarified the contribution of IDs as (epi)genetic causes of SGA-SS and the molecular and phenotypic spectrum of SRS. Various IDs constitute underlying factors for SGA-SS, including SRS.