Reanalysis of Chromosomal Microarray Data Using a Smaller Copy Number Variant Call Threshold Identifies Four Cases with Heterozygous Multiexon Deletions of ARID1B, EHMT1, and FOXP1 Genes.

Introduction: Chromosomal microarray (CMA) is a highly accurate and established method for detecting copy number variations (CNVs) in clinical genetic testing. CNVs are important etiological factors for disorders such as intellectual disability, developmental delay, and multiple congenital anomalies. Recently developed analytical methods have facilitated the identification of smaller CNVs. Therefore, reanalyzing CMA data using a smaller CNV calling threshold may yield useful information. However, this method was left to the discretion of each institution. Methods: We reanalyzed the CMA data of 131 patients using a smaller CNV call threshold: 50 kb 50 probes for gain and 25 kb 25 probes for loss. We interpreted the reanalyzed CNVs based on the most recently available information. In the reanalysis, we filtered the data using the Clinical Genome Resource dosage sensitivity gene list as an index to quickly and efficiently check morbid genes. Results: The number of copy number loss was approximately 20 times greater, and copy number gain was approximately three times greater compared to those in the previous analysis. We detected new likely pathogenic CNVs in four participants: a 236.5 kb loss within ARID1B, a 50.6 kb loss including EHMT1, a 46.5 kb loss including EHMT1, and an 89.1 kb loss within the FOXP1 gene. Conclusion: The method employed in this study is simple and effective for CMA data reanalysis using a smaller CNV call threshold. Thus, this method is efficient for both ongoing and repeated analyses. This study may stimulate further discussion of reanalysis methodology in clinical laboratories.

Novel Causative RET Mutation in a Japanese Family with Hirschsprung's Disease: Case Report and Factors Impacting Disease Severity.

RET gene variances confer susceptibility to Hirschsprung's disease (HSCR) with pathogenetic mutations being identified in half of familial cases. This investigation of familial HSCR was aimed to clarify the relationship between genetic mutations and clinical phenotype using next-generation sequencing. A novel c2313C > G(D771E) RET mutation was identified in all three affected family members. The mutation involved the kinase domain, which is believe to impair RET activity and intestinal function. A second RET mutation, c1465G > A(D489N), was found only in the extensive aganglionosis case. We conclude that the novel c2313C > A(D771E) mutation in RET may be pathogenic for HSCR, while the c1465C > G(D489N) mutation may be related to phenotype severity.

Analysis of clinical presentations of allergic transfusion reactions and febrile non-haemolytic transfusion reactions in paediatric patients.

BACKGROUND AND OBJECTIVES: Like adults, children can have allergic transfusion reactions (ATRs) and febrile non-haemolytic transfusion reactions (FNHTRs). But published information about the incidence of paediatric ATR and FNHTR is scarce. MATERIALS AND METHODS: This retrospective study was conducted from April 2002 to June 2018 on children who had ATRs and/or FNHTRs to platelet (PLT), red blood cell (RBC) or washed PLT/RBC concentrate transfusions. We analysed ATR/FNHTR clinical presentations, such as severity, time of occurrence and other features when they occurred. RESULTS: During the study, 2742 children received 23 444 bags of PLT and RBC concentrate (including washed products). ATRs occurred in 100 cases (3·6% of total patients), caused by 201 products (0·9% of total products). In contrast, 28 patients (1·0% of total patients) had 42 FNHTRs caused by 42 products (0·2% of total products). Upon analysis of cases with detailed clinical information, the median onset time for ATRs and FNHTRs was 2·0 h after the start of transfusion. Of the 40% of ATRs that necessitated the discontinuation of blood transfusions, 10% escalated to anaphylaxis. Compared with minor ATRs, anaphylaxis tended to develop quickly. Moreover, 96% of patients with FNHTRs had a fever less than 39°C. There were no associations between blood product types and numbers or occurrence patterns of these reactions. CONCLUSION: The occurrence of ATRs and FNHTRs in children was variable, although there are common features such as severity and time of occurrence.

Hypoglycemia During the Temporary Interruption of Parenteral Nutrition Infusion in Pediatric Hematopoietic Stem Cell Transplantation.

BACKGROUND: Parenteral nutrition (PN) is required with pediatric procedures such as hematopoietic stem cell transplantation (HSCT). However, risks associated with temporary PN infusion interruption remain unclear. MATERIALS AND METHODS: We retrospectively analyzed in 22 children undergoing HSCT receiving PN with the same daily routine: temporary PN infusion interruption before breakfast for administering a saline-diluted acyclovir drip. After correcting patients' glucose levels, we examined minimum blood glucose levels between preparative regimen initiation and post-HSCT day 30. Patients were divided into 2 groups according to a minimum glucose cutoff of 60 mg/dL. Patient background characteristics and hypoglycemia risk factors were compared between both groups. RESULTS: The hypoglycemia group had a significantly lower body surface area, higher glucose infusion rate (GIR), lower cholinesterase levels, and higher zinc levels at the onset of the minimum blood glucose level ( P < .05). Multivariate analyses revealed an association only between higher GIR (≥5 mg/kg/min) and hypoglycemia during the temporary PN infusion interruption. A time course analysis of blood glucose and immunoreactive insulin (IRI) levels in 1 patient revealed a combined high-caloric and saline flush before acyclovir initiation, causing temporary increased IRI, as the etiology for hypoglycemia. CONCLUSIONS: Particular attention and several precautions are required to prevent complications associated with temporary PN infusion interruption in children with higher GIR.

Four Sporadic Pediatric Cases of Yersinia enterocolitica O:8 Infection in a Rural Area of Japan.

In the spring of 2015, we experienced a cluster of 4 sporadic cases of yersiniosis in children in Nagano prefecture, a rural area of Japan. Two patients developed appendicitis-like episodes; one had acute gastroenteritis, and the other had bacteremia associated with liver abscess. The causative agent of these infections was Yersinia enterocolitica serogroup O:8. None of the patients had an underlying illness, and all have recovered completely. The patients were neither socially nor geographically related to each other. These 4 consecutive cases suggest that Y. enterocolitica O:8 has spread substantially in the middle part of Japan, and that this virulent strain might be more common than previously reported in our country.

Exome sequencing-based identification of mutations in non-syndromic genes among individuals with apparently syndromic features.

In a clinical setting, the number of organ systems involved is crucial for the differential diagnosis of congenital genetic disorders. When more than one organ system is involved, a syndromic diagnosis is suspected. In this report, we describe three patients with apparently syndromic features. Exome sequencing identified non-syndromic gene mutations as a potential cause of part of their phenotype. The first patient (Patient 1) is a girl with cleft lip/palate, meningoencephalocele, tetralogy of Fallot, and developmental delay. The second and third patients (Patients 2 and 3) are brothers with developmental delay, deafness, and low bone mineral density. Exome sequencing revealed the presence of a CDH1 mutation in Patient 1 and a PLS3 mutation in Patients 2 and 3. CDH1 mutations are known to be associated with non-syndromic cleft lip/palate, while PLS3 mutations are associated with osteoporosis. Thus, these variants may explain a part of the complex phenotype of the patients, although the effects of these missense variants need to be evaluated by functional assays in order to prove pathogenicity. On the basis of these findings, we emphasize the importance of scrutinizing non-syndromic gene mutations even in individuals with apparently syndromic features. © 2016 Wiley Periodicals, Inc.

Discordant clinical phenotype in monozygotic twins with Alagille syndrome: Possible influence of non-genetic factors.

Alagille syndrome is a multisystem developmental disorder characterized by bile duct paucity, congenital heart disease, vertebral anomalies, posterior embryotoxon, and characteristic facial features. Alagille syndrome is typically the result of germline mutations in JAG1 or NOTCH2 and is one of several human diseases caused by Notch signaling abnormalities. A wide phenotypic spectrum has been well documented in Alagille syndrome. Therefore, monozygotic twins with Alagille syndrome provide a unique opportunity to evaluate potential phenotypic modifiers such as environmental factors or stochastic effects of gene expression. In this report, we describe an Alagille syndrome monozygotic twin pair with discordant placental and clinical findings. We propose that environmental factors such as prenatal hypoxia may have played a role in determining the phenotypic severity.

[Rapid and Easy Measurement of Serum Fatty Acid Composition of Neonates, Infants and Young People Using the Gas Chromatography Mass Spectrometry].

A variation of the fatty acid composition is closely associated with the clinical state of inflammatory disorder and metabolic syndrome. The analysis of serum fatty acid composition of neonates and infants has been measured hardly at the laboratory, because a large quantity of serum was required for an analysis and the measurement procedure was cumbersome. We examined the rapid and easy analysis in a small amount of serum using the combination methods of the gas chromatography mass spectrometry (GC MS) and the quick transmethylation. The serum fatty acid composition of neonates and infants were compared with the young people. The serum fatty acids with the internal standard material were performed transmethylation using the microwave, and then the lipids were extracted. The fatty acid esters were analyzed by GC MS with capillary column, and the statistical procedure used nonparametric method. The repeatability of each fatty acid concentration was CV = 5-11.3% (n = 5) with serum 50 µl. The lowest quantity of sample was possible to measure with 13 µl of serum. The total serum fatty acid, saturated fatty acid and monounsaturated fatty acid levels did not show a significant difference at all age, but the polyunsaturated fatty acid (PUFA) level of neonates and infants was significantly lower than young people, p = 0.007. The four main PUFA exclusive of α-linolenic acid showed a significant difference. The fatty acid composition with small quantities serum was measured by the rapid and accurate method using the GC MS and the microwave. The serum PUFA concentrations have fluctuated according to growth, therefore it was necessary to evaluate serum fatty acid composition in each age category.

Notable alkaline tolerance of Kocuria marina isolate from blood of a pediatric patient with continuous intravenous epoprostenol therapy.

This study was the first to describe the hitherto deficiently evaluated alkaline tolerance of Kocuria marina isolate from a pediatric patient with continuous intravenous epoprostenol dosing therapy. Our isolate from blood of a 7-year-old Japanese boy was finally identified as K. marina by the morphological, cultural, and biochemical properties together with the comparative sequence analyses of the 16S rRNA genes. The K. marina isolate, the causative agent of catheter-related blood-stream infection, was not only revealed to be salt tolerant (NaCl 15%), but also demonstrated to be stably survived with no apparent decrease of cell counts for long periods (120 h) in an alkaline environment (pH 8, 9, 10, and 11) at 35 °C. Its remarkable tolerance to the stresses of high alkalinity compared with a clinical Staphylococcus aureus strain should provide consistent interpretation that the environment of high alkalinity (pH 10.2-10.8) measures should be insufficient to inactivate almost all the causative agents including K. marina strains in the solution of epoprostenol (pH 10.4) (Flolan(®), GlaxoSmithKline, Ltd., Tokyo, Japan.). To the best of our knowledge, the first description of the property of being tolerant to high alkalinity that the K. marina isolate exhibited was noteworthy and a useful piece of information. In conclusion, we believe that the present study should be a notification regarding the potential risk of catheter-related blood-stream infections due to K. marina, suggestive of an alkalophile, especially in patients receiving continuous intravenous epoprostenol dosing therapy.

Interleukin-8-producing primary cardiac undifferentiated sarcoma in a child with sustained fever.

We report the case of a 12-year-old boy with primary undifferentiated sarcoma of the left atrium. He had sustained fever during the clinical course and multiple lung and brain metastases. Chemotherapy and irradiation were ineffective; he died 41 days after hospitalization. On retrospective analysis, interleukin-8 (IL-8) was elevated; this was supported by immunohistochemistry and gene expression analysis of tumor samples. IL-8 continued to increase with tumor progression accompanied by elevated neutrophil count and C-reactive protein. IL-8 is involved in malignant tumor proliferation, migration, and angiogenesis and may have been related to the clinical condition and prognosis in the present case.

Dissecting the phenotype of supernumerary marker chromosome 20 in a patient with syndromic Pierre Robin sequence: combinatorial effect of gene dosage and uniparental disomy.

Clinical phenotypes in individuals with a supernumerary marker chromosome (SMC) are mainly caused by gene dosage effects due to the genes located on the SMC. An additional effect may result from uniparental disomy (UPD). Consequently, the occurrence of UPD may be a confounding factor in identifying genotype-phenotype correlations in SMC syndromes. Here, we report on a patient that illustrates this problem; the phenotype of this patient was a consequence of a combined effect of gene dosage and UPD. The proband showed facial dysmorphisms, growth retardation and developmental delay. G-band karyotype of the proband's peripheral blood showed the presence of mosaic SMC. A SNP array analysis documented maternal UPD20 and 20p duplication. It is known that maternal UPD20 causes prenatal onset growth retardation and feeding difficulties. By contrast, duplication of 20p causes facial dysmorphisms, micrognathia, cleft palate, developmental delay and vertebral anomalies. Our classification of the proband's phenotype showed a mixture of these two effects. Therefore, we suggest the routine use of genome-wide SNP array towards the detailed genotype-phenotype correlations for SMC syndromes.

Pretransplant-corrected QT dispersion as a predictor of pericardial effusion after pediatric hematopoietic stem cell transplantation.

Pericardial effusion is a potentially fatal complication following hematopoietic stem cell transplantation (HSCT). Therefore, the identification of risk factors could improve the outcome. Prolonged QT dispersion (QTD) and corrected QTD (QTcD) are associated with serious arrhythmias and sudden death in many forms of heart disease. However, no study has evaluated the efficacy of QTD and QTcD to predict pericardial effusion post-HSCT. We studied 89 pediatric HSCT patients to identify preoperative risk factors for pericardial effusion with particular focus on QTD and QTcD. Pericardial effusion occurred in 15 patients (cumulative onset rate: 17.4%) within one year post-HSCT, of which 8 (9.2%) were symptomatic. Patients with pericardial effusion following allogeneic HSCT showed significantly lower overall survival; however, pericardial effusion was not the direct cause of death in any patient. Univariate and multivariate analyses revealed that transplantation-associated thrombotic microangiopathy (TA-TMA) was an independent risk factor for post-HSCT pericardial effusion. In addition, pretransplant QTcD was significantly prolonged in the pericardial effusion group. These results suggest that pediatric patients with abnormally prolonged QTcD before the preparative regimen for HSCT should be regularly followed-up by echocardiography to detect pericardial effusion, particularly when accompanied by complications including TA-TMA.

A Pediatric Case of Bacteremia and Possible Cholecystitis Due to Moraxella osloensis.

We encountered a pediatric case of bacteremia and possible cholecystitis due to Moraxella osloensis that was treated successfully. We confirmed the diagnosis with the presence of a high serum titer of the antibody to the organism. Furthermore, 16S rRNA sequencing was performed to identify the bacteria.

[Abnormal Serum Total Protein Measurement by Lipoprotein-X in an Infant with Biliary Atresia].

Lipoprotein-X (LP-X) in cholestatic jaundice causes abnormal reaction in assays for low-density lipoprotein-cholesterol, but the effects on other test items are unknown. Here, we report an infant with biliary atresia showing abnormal reaction in total serum protein assay using the biuret method, and lipoprotein-X (LP-X) was then detected. In this 11-month-old female infant, jaundice was observed at 2 months old, and a diagnosis of biliary atresia was made. On biochemical tests at 12 months old, the total serum protein concentrations detected by the biuret method were very high, and the response curve and linearity of dilution were abnormal. LP-X was detected by agar electrophoresis. In addition and recovery experiments with normal serum fractionation of the patient's LP-X-rich lipoprotein fraction prepared by ultracentrifugation, normal γ-globulin fractionation showed an abnormal reaction by the biuret method. In infants with biliary atresia, we showed that the total serum protein assay by the biuret method was influenced by LP-X-rich lipoprotein, which may be caused by abnormal reaction of LP-X and γ-globulin. [Case Report].

First isolation of oleate-dependent Enterococcus faecalis small-colony variants from the umbilical exudate of a paediatric patient with omphalitis.

An oleate-dependent Enterococcus faecalis isolate representing small-colony variants (SCVs) was isolated from the umbilical exudate of a 31-month-old Japanese male patient in Nagano Children's Hospital, Azumino, Japan. The patient had been suffering from recurrent omphalitis since early infancy. The initial E. faecalis SCV isolate formed small colonies on sheep blood agar plates and tiny colonies on chocolate and modified Drigalski agar, although no visible growth was observed in HK-semi solid medium after 48 h incubation in ambient air. Moreover, the SCV isolate, the colonial morphology of which was reminiscent of Streptococcus species, could not be identified using the MicroScan WalkAway-40 and API 20 Strep systems, both of which yielded profile numbers that did not correspond to any bacterial species, probably as a result of insufficient growth of the isolate. The SCV isolate was subsequently identified as E. faecalis based on its morphological, cultural and biochemical properties, and this was confirmed by sequencing the 16S rRNA gene of the organism. Investigations revealed that the addition of oleate, an unsaturated fatty acid, enabled the isolate to grow on every medium with normal-sized colony morphology. Although it has long been known that long-chain fatty acids, especially unsaturated oleic acid, have a major inhibitory effect on the growth of a variety of microorganisms, including not only mycobacteria but also streptococci, this is, to the best of our knowledge, the first clinical isolation of an oleate-dependent E. faecalis SCV isolate. In addition, oleic acid might be considered to affect the cell membrane permeability of carbohydrates or antimicrobial agents such as ß-lactams.

[A case of osteomyelitis due to Kingella kingae].

Kingella species including K. kingae are non-motile coccobacilli or short straight rods, and their normal habitats appear to be the upper respiratory and oropharyngeal tracts of humans. In recent years, K. kingae strains have been increasingly recognized as common causes of invasive infections in children at the age of less than 4 years. In Japan, however, invasive K. kingae infections including osteomyelitis have rarely been described. We incidentally encountered isolation of a K. kingae strain from intraoperatively obtained specimens from a previously healthy 44-month-old boy. He first consulted a nearby medical facility and a suspected diagnosis of osteomyelitis was made, after which the patient was then transferred to our Nagano Children's hospital. There was evidence of inflammation in his right calcaneus and toe walking was noted. He was treated with surgical drainage. An isolate grown on sheep blood agar with positive oxidase and negative catalase was biochemically characterized with the ID-Test HN20 (Nissui Pharmaceutical Co., Ltd., Tokyo, Japan) kit system together with genetic examinations involving sequencing the 16S rRNA gene, and the infection was finally identified as K. kingae. The patient was successfully treated with cefotiam (CTM) for the first 7 days followed by the administration of trimethoprim-sulfamethoxazole (ST) for an additional 2 months. The K. kingae isolate was confirmed as a sure causative pathogen by observing that the serum showed high agglutinin titers against the isolate. Accumulation of the case reports in Japan with the isolation of this species is essential for clarifying invasive infections due to K. kingae. Our case report is a noteworthy and useful piece of information.

Isolation and molecular characterization of catalase-negative Staphylococcus aureus from sputum of a patient with aspiration pneumonia.

Staphylococcus aureus produces various virulence factors. The catalase enzyme, in particular, is considered to be involved in oxidative stress resistance, and catalase activity is an important criterion for differentiating staphylococci from streptococci. In this report, we describe the catalase-negative S. aureus strain SH3064, which was isolated from the sputum of a patient with aspiration pneumonia. To evaluate the causes of the lack of catalase activity in S. aureus SH3064, we analyzed the sequence of katA gene encoding the catalase enzyme in this strain. We amplified the complete sequence of katA gene of S. aureus SH3064 by polymerase chain reaction using 2 sets of primers. The katA sequence showed 99.6% sequence identity (1512/1518 bp) with that of S. aureus ATCC 12600. We detected 2 mutations in the katA gene from S. aureus SH3064, an A217T substitution leading to a threonine 73-to-serine substitution and a single-base pair deletion (c.637delG) resulting in a frameshift mutation. The lack of catalase activity in this strain was attributed to the shift of the nucleotide reading frame.