Breakpoints in complex chromosomal rearrangements correspond to transposase-accessible regions of DNA from mature sperm.

Constitutional complex chromosomal rearrangements (CCRs) are rare cytogenetic aberrations arising in the germline via an unknown mechanism. Here we analyzed the breakpoint junctions of microscopically three-way or more complex translocations using comprehensive genomic and epigenomic analyses. All of these translocation junctions showed submicroscopic genomic complexity reminiscent of chromothripsis. The breakpoints were clustered within small genomic domains with junctions showing microhomology or microinsertions. Notably, all of the de novo cases were of paternal origin. The breakpoint distributions corresponded specifically to the ATAC-seq (assay for transposase-accessible chromatin with sequencing) read data peak of mature sperm and not to other chromatin markers or tissues. We propose that DNA breaks in CCRs may develop in an accessible region of densely packaged chromatin during post-meiotic spermiogenesis.

Antisense oligonucleotide induced pseudoexon skipping and restoration of functional protein for Fukuyama muscular dystrophy caused by a deep-intronic variant.

Fukuyama congenital muscular dystrophy (FCMD) is an autosomal recessive disorder caused by fukutin (FKTN) gene mutations. FCMD is the second most common form of childhood muscular dystrophy in Japan, and the most patients possess a homozygous retrotransposal SINE-VNTR-Alu insertion in the 3'-untranslated region of FKTN. A deep-intronic variant (DIV) was previously identified as the second most prevalent loss-of-function mutation in Japanese patients with FCMD. The DIV creates a new splicing donor site in intron 5 that causes aberrant splicing and the formation of a 64-base pair pseudoexon in the mature mRNA, resulting in a truncated nonfunctional protein. Patients with FCMD carrying the DIV present a more severe symptoms, and currently, there is no radical therapy available for this disorder. In the present study, we describe in vitro evaluation of antisense oligonucleotide mediated skipping of pseudoexon inclusion and restoration of functional FKTN protein. A total of 16 19-26-mer antisense oligonucleotide sequences were designed with a 2'-O-methyl backbone and were screened in patient-derived fibroblasts, lymphoblast cells and minigene splice assays. One antisense oligonucleotide targeting the exonic splice enhancer region significantly induced pseudoexon skipping and increased the expression of normal mRNA. It also rescued FKTN protein production in lymphoblast cells and restored functional O-mannosyl glycosylation of alpha-dystroglycan in patient-derived myotubes. Based on our results, antisense oligonucleotide-based splicing correction should be investigated further as a potential treatment for patients with FCMD carrying the DIV.One Sentence Summary Antisense oligonucleotide treatment restored normal FKTN protein production and functional O-mannosyl glycosylation of alpha-dystroglycan via pseudoexon skipping in patient-derived cells carrying the compound heterozygous deep-intronic variant of Fukuyama muscular dystrophy.

Assessment of the upper limb muscles in patients with Fukuyama muscular dystrophy: Noninvasive assessment using visual ultrasound muscle analysis and shear wave elastography.

Fukuyama-type congenital muscular dystrophy (FCMD) is severe, childhood-onset muscular dystrophy. Recently, our group has discovered a potential treatment using antisense oligonucleotides. Therefore, an effective, reliable, and objective method of assessing muscle is needed. Ultrasound is a minimally invasive tool that can be applied without radiation exposure or pain. Evaluating tissue stiffness by shear wave elastography (SWE) has especially recently attracted attention. Here, we aimed to evaluate SWE value of the upper limb muscles: biceps brachii, triceps brachii, brachioradialis, abductor pollicis brevis, and abductor finger muscle in patients with FCMD. Upper extremity function was evaluated by visual muscle ultrasound analysis (VMUA) and SWE in 13 patients with FCMD and 20 healthy controls. The motor function evaluation tool was used to evaluate motor function, and the correlation with the dynamics of the SWE was determined. VMUA scaled using the Heckmatt scale was higher in patients with FCMD. SWE was also significantly higher and stiffer in the biceps brachii and brachioradialis in patients with FCMD. Furthermore, the severity of FCMD symptoms was correlated with muscle stiffness. We conclude that VMUA and SWE can be useful tools for monitoring muscle atrophy and upper limb function in patients with FCMD.

Restoration of the defect in radial glial fiber migration and cortical plate organization in a brain organoid model of Fukuyama muscular dystrophy.

Fukuyama congenital muscular dystrophy (FCMD) is a severe, intractable genetic disease that affects the skeletal muscle, eyes, and brain and is attributed to a defect in alpha dystroglycan (αDG) O-mannosyl glycosylation. We previously established disease models of FCMD; however, they did not fully recapitulate the phenotypes observed in human patients. In this study, we generated induced pluripotent stem cells (iPSCs) from a human FCMD patient and differentiated these cells into three-dimensional brain organoids and skeletal muscle. The brain organoids successfully mimicked patient phenotypes not reliably reproduced by existing models, including decreased αDG glycosylation and abnormal radial glial (RG) fiber migration. The basic polycyclic compound Mannan-007 (Mn007) restored αDG glycosylation in the brain and muscle models tested and partially rescued the abnormal RG fiber migration observed in cortical organoids. Therefore, our study underscores the importance of αDG O-mannosyl glycans for normal RG fiber architecture and proper neuronal migration in corticogenesis.

Unexpected Mutations by CRISPR-Cas9 CTG Repeat Excision in Myotonic Dystrophy and Use of CRISPR Interference as an Alternative Approach.

Myotonic dystrophy type 1 is the most common type of adult-onset muscular dystrophy. This is an autosomal dominant disorder and caused by the expansion of the CTG repeat in the 3' untranslated region of the dystrophia myotonica protein kinase (DMPK) gene. Messenger RNAs containing these expanded repeats form aggregates as nuclear RNA foci. Then, RNA binding proteins, including muscleblind-like 1, are sequestered to the RNA foci, leading to systemic abnormal RNA splicing. In this study, we used CRISPR-Cas9 genome editing to excise this CTG repeat. Dual cleavage at the 5' and 3' regions of the repeat using a conventional Cas9 nuclease and a double nicking with Cas9 nickase successfully excised the CTG repeat. Subsequently, the formation of the RNA foci was markedly reduced in patient-derived fibroblasts. However, contrary to expectations, a considerable amount of off-target digestions and on-target genomic rearrangements were observed using high-throughput genome-wide translocation sequencing. Finally, the suppression of DMPK transcripts using CRISPR interference significantly decreased the intensity of RNA foci. Our results indicate that close attention should be paid to the unintended mutations when double-strand breaks are generated by CRISPR-Cas9 for therapeutic purposes. Alternative approaches independent of double-strand breaks, including CRISPR interference, may be considered.

Congenital hearing impairment associated with peripheral cochlear nerve dysmyelination in glycosylation-deficient muscular dystrophy.

Hearing loss (HL) is one of the most common sensory impairments and etiologically and genetically heterogeneous disorders in humans. Muscular dystrophies (MDs) are neuromuscular disorders characterized by progressive degeneration of skeletal muscle accompanied by non-muscular symptoms. Aberrant glycosylation of α-dystroglycan causes at least eighteen subtypes of MD, now categorized as MD-dystroglycanopathy (MD-DG), with a wide spectrum of non-muscular symptoms. Despite a growing number of MD-DG subtypes and increasing evidence regarding their molecular pathogeneses, no comprehensive study has investigated sensorineural HL (SNHL) in MD-DG. Here, we found that two mouse models of MD-DG, Largemyd/myd and POMGnT1-KO mice, exhibited congenital, non-progressive, and mild-to-moderate SNHL in auditory brainstem response (ABR) accompanied by extended latency of wave I. Profoundly abnormal myelination was found at the peripheral segment of the cochlear nerve, which is rich in the glycosylated α-dystroglycan-laminin complex and demarcated by "the glial dome." In addition, patients with Fukuyama congenital MD, a type of MD-DG, also had latent SNHL with extended latency of wave I in ABR. Collectively, these findings indicate that hearing impairment associated with impaired Schwann cell-mediated myelination at the peripheral segment of the cochlear nerve is a notable symptom of MD-DG.

A case of a parthenogenetic 46,XX/46,XY chimera presenting ambiguous genitalia.

Sex-chromosome discordant chimerism (XX/XY chimerism) is a rare chromosomal disorder in humans. We report a boy with ambiguous genitalia and hypospadias, showing 46,XY[26]/46,XX[4] in peripheral blood cells. To clarify the mechanism of how this chimerism took place, we carried out whole-genome genotyping using a SNP array and microsatellite analysis. The B-allele frequency of the SNP array showed a mixture of three and five allele combinations, which excluded mosaicism but not chimerism, and suggested the fusion of two embryos or a shared parental haplotype between the two parental cells. All microsatellite markers showed a single maternal allele. From these results, we concluded that this XX/XY chimera is composed of two different paternal alleles and a single duplicated maternal genome. This XX/XY chimera likely arose from a diploid maternal cell that was formed via endoduplication of the maternal genome just before fertilization, being fertilized with both X and Y sperm.

Acute rhabdomyolysis following viral infection with coxsackie A4 in a 50-day-old infant with Fukuyama congenital muscular dystrophy.

BACKGROUND: Fukuyama congenital muscular dystrophy (FCMD), which is characterized by generalized muscle weakness, hypotonia, and motor delay during early infancy, gradually progresses with advanced age. Although acute rhabdomyolysis following infection in patients with FCMD has occasionally been reported, no studies have investigated rhabdomyolysis following viral infection in FCMD patients during early infancy. CASE REPORT: We report the case of a 50-day-old girl with no apparent symptoms of muscular dystrophy who developed severe acute rhabdomyolysis caused by viral infection, resulting in quadriplegia and respiratory failure therefore requiring mechanical ventilation. Brain magnetic resonance imaging incidentally showed the typical characteristics of FCMD, and FCMD was confirmed by genetic analysis, which revealed a 3-kb retrotransposon insertion in one allele of the fukutin gene and a deep intronic splicing variant in intron 5 in another allele. The virus etiology was confirmed to be Coxsackie A4. CONCLUSION: We report a severe case of acute rhabdomyolysis with the earliest onset of symptoms due to the Coxsackie A4 virus in a patient with FCMD. The present findings indicate that physicians should consider FCMD with viral infection a differential diagnosis if the patient presents with acute rhabdomyolysis following a fever.

Induced pluripotent stem cell-derived melanocyte precursor cells undergoing differentiation into melanocytes.

Induced pluripotent stem cell (iPSC) technology offers a novel approach for conversion of human primary fibroblasts into melanocytes. During attempts to explore various protocols for differentiation of iPSCs into melanocytes, we found a distinct and self-renewing cell lineage that could differentiate into melanocytes, named as melanocyte precursor cells (MPCs). The MPCs exhibited a morphology distinctive from that of melanocytes, in lacking either the melanosomal structure or the melanocyte-specific marker genes MITF, TYR, and SOX10. In addition, gene expression studies in the MPCs showed high-level expression of WNT5A, ROR2, which are non-canonical WNT pathway markers, and its related receptor TGFßR2. In contrast, MPC differentiation into melanocytes was achieved by activating the canonical WNT pathway using the GSK3ß inhibitor. Our data demonstrated the distinct characteristic of MPCs' ability to differentiate into melanocytes, and the underlying mechanism of interfacing between canonical WNT signaling pathway and non-canonical WNT signaling pathway.

National registry of patients with Fukuyama congenital muscular dystrophy in Japan.

Fukuyama congenital muscular dystrophy (FCMD) is the second most common form of muscular dystrophy in the Japanese population and is caused by mutations in the fukutin (FKTN) gene. In 2011, the Japan Muscular Dystrophy Association (JMDA) developed a nationwide registry of genetically confirmed patients with FCMD. We retrospectively reviewed the registry dataset of patients with FCMD to obtain data, including age, sex, developmental milestones, intellectual level, complications, and primary treatments. In total, 207 patients with FCMD (104 boys and 103 girls) were registered by the end of September 2013. Mean patient age at first registration was 8.1 ±â€¯7.8 years (median, 6 years; range, 0-42 years). A homozygous 3-kb founder insertion mutation in the FKTN gene was present in 80% of registrants, whereas 20% had a compound heterozygous mutation. Sixty-nine patients (33%) had febrile seizures and/or epilepsy. Myopia was the most frequently detected abnormality (8.7%), followed by strabismus (5.9%). Overall, 16% of patients required respiratory support and this percentage increased with age. Cardiac dysfunction was detected in 16%, and dysphagia was observed in 22% of patients with FCMD. The FCMD patient registry is useful for clarifying the natural history of FCMD and recruiting patients for clinical trials.

Fosphenytoin vs. continuous midazolam for pediatric febrile status epilepticus.

BACKGROUND: Fosphenytoin (fPHT) and continuous intravenous midazolam (cMDL) had commonly been used as second-line treatments for pediatric status epilepticus (SE) in Japan. However, there is no comparative study of these two treatments. METHODS: We included consecutive children who 1) were admitted to Kobe Children's Hospital because of convulsion with fever and 2) were treated with either fPHT or cMDL as second-line treatment for convulsive SE lasting for longer than 30 min. We compared, between the fPHT and cMDL groups, the proportion of barbiturate coma therapy (BCT), incomplete recovery of consciousness, mechanical ventilation, and inotropic agents. RESULTS: The proportion of BCT was not significantly different between the two groups (48.7% [20/41] in fPHT and 35.3% [29/82] in cMDL, p = 0.17). The prevalence of incomplete recovery of consciousness, mechanical ventilation, and inotropic agents was not different between the two groups. After excluding 49 patients treated with BCT, incomplete recovery of consciousness 6 h and 12 h after onset was more frequent in the cMDL group than in the fPHT group (71.7% vs. 33.3%, p < 0.01; 56.6% vs. 14.2%, p < 0.01; respectively). Mechanical ventilation was more frequent in the cMDL group than in the fPHT group (32.0% vs. 4.7%, p = 0.01). CONCLUSIONS: Our results suggest that 1) the efficacy of fPHT and cMDL is similar, although cMDL may prevent the need for BCT compared with fPHT, and 2) fPHT is relatively safe as a second-line treatment for pediatric SE in patients who do not require BCT.

Congenital chloride diarrhea needs to be distinguished from Bartter and Gitelman syndrome.

Pseudo-Bartter/Gitelman syndrome (p-BS/GS) encompasses a clinically heterogeneous group of inherited or acquired disorders similar to Bartter syndrome (BS) or Gitelman syndrome (GS), both renal salt-losing tubulopathies. Phenotypic overlap frequently occurs between p-BS/GS and BS/GS, which are difficult to diagnose based on their clinical presentation and require genetic tests for accurate diagnosis. In addition, p-BS/GS can occur as a result of other inherited diseases such as cystic fibrosis, autosomal dominant hypocalcemia, Dent disease, or congenital chloride diarrhea (CCD). However, the detection of the variants in genes other than known BS/GS-causing genes by conventional Sanger sequencing requires substantial time and resources. We studied 27 cases clinically diagnosed with BS/GS, but with negative genetic tests for known BS/GS genes. We conducted targeted sequencing for 22 genes including genes responsible for tubulopathies and other inherited diseases manifesting with p-BS/GS symptoms. We detected the SLC26A3 gene variants responsible for CCD in two patients. In Patient 1, we found the SLC26A3 compound heterozygous variants: c.354delC and c.1008insT. In Patient 2, we identified the compound heterozygous variants: c.877G > A, p.(Glu293Lys), and c.1008insT. Our results suggest that a comprehensive genetic screening system using targeted sequencing is useful for the diagnosis of patients with p-BS/GS with alternative genetic origins.

De novo variants in RHOBTB2, an atypical Rho GTPase gene, cause epileptic encephalopathy.

By whole exome sequencing, we identified three de novo RHOBTB2 variants in three patients with epileptic encephalopathies (EEs). Interestingly, all three patients showed acute encephalopathy (febrile status epilepticus), with magnetic resonance imaging revealing hemisphere swelling or reduced diffusion in various brain regions. RHOBTB2 encodes Rho-related BTB domain-containing protein 2, an atypical Rho GTPase that is a substrate-specific adaptor or itself is a substrate for the Cullin-3 (CUL3)-based ubiquitin ligase complex. Transient expression experiments in Neuro-2a cells revealed that mutant RHOBTB2 was more abundant than wild-type RHOBTB2. Coexpression of CUL3 with RHOBTB2 decreased the level of wild-type RHOBTB2 but not the level of any of the three mutants, indicating impaired CUL3 complex-dependent degradation of the three mutants. These data indicate that RHOBTB2 variants are a rare genetic cause of EEs, in which acute encephalopathy might be a characteristic feature, and that precise regulation of RHOBTB2 levels is essential for normal brain function.

Early risk factors for mortality in children with seizure and/or impaired consciousness accompanied by fever without known etiology.

BACKGROUND: Children who present with seizure and/or impaired consciousness accompanied by fever without known etiology (SICF) may be diagnosed with either acute encephalopathy (AE) or febrile seizure (FS). Although approximately 5% of AE cases are fatal, it is difficult to identify fatal cases among children with SICF, which are often critical by the time of diagnosis. Thus, early prediction of outcomes for children with SICF, prior to diagnosis, may help to reduce mortality associated with AE. The aim of the present study was to identify clinical and laboratory risk factors for mortality acquired within 6 h of onset among children with SICF. METHODS: We retrospectively reviewed the medical records of children who had been admitted to Kobe Children's Hospital (Kobe, Japan) with SICF between October 2002 and September 2015. We compared clinical and laboratory characteristics acquired within 6 h of onset and outcomes between survivors and non-survivors using univariate and multivariate analyses. RESULTS: The survivor and non-survivor groups included 659 and nine patients, respectively. All patients in the non-survivor group received a final diagnosis of AE. Univariate analysis revealed significant differences between the groups with regard to seizure duration and the following laboratory parameters: aspartate transaminase (AST), alanine aminotransferase, lactate dehydrogenase, sodium, and lactate. The multivariate analysis identified AST as a significant independent factor associated with mortality. CONCLUSIONS: Elevation of AST within 6 h of onset is independently correlated with mortality in children with SICF. Our result may elucidate earlier intervention for patients with high risk of mortality.

Clinical features predicting group A streptococcal pharyngitis in a Japanese paediatric primary emergency medical centre.

Objectives To identify clinical features that predict Group A streptococcal (GAS) pharyngitis in a Japanese paediatric primary emergency medical centre. Methods The prevalence of GAS pharyngitis according to age and body temperature (BT) was calculated among 3098 paediatric patients with pharyngitis. The numbers of GAS-positive and -negative patients for each clinical parameter, and each point increase in the McIsaac score were compared and likelihood ratios (LRs) were calculated. Results The prevalence of GAS pharyngitis was extremely low in patients aged < 1 (1.2%) and 1 year (3.9%). The GAS-positive rate was significantly higher in patients with a BT < 38.0°C compared with ≥ 38.0°C (30.0% vs. 19.8%). A BT ≥ 38.0°C was not a predictive finding for GAS pharyngitis (positive LR: 0.82). Rash was the most useful individual predictor, and a McIsaac score of 4 or 5 increased the probability; however, the positive LRs were 1.74 and 1.30, respectively. Conclusions The prevalence of GAS pharyngitis is extremely low in patients aged < 1 and 1 year, and a BT ≥ 38.0°C is not a predictive symptom. Although a rash and McIsaac score of 4 or 5 are associated with an increased probability, they cannot be used to confirm GAS infection.

Development of ultra-deep targeted RNA sequencing for analyzing X-chromosome inactivation in female Dent disease.

The pattern of X-chromosome inactivation (XCI) can affect the clinical severity of X-linked disorders in females. XCI pattern analysis has been conducted mainly by HUMARA assay, a polymerase chain reaction-based assay using a methylation-sensitive restriction enzyme. However, this assay examines the XCI ratio of the androgen receptor gene at the genomic DNA level and does not reflect the ratio of either targeted gene directly or at the mRNA level. Here, we report four females with Dent disease, and we clarified the correlation between XCI and female cases of Dent disease using not only HUMARA assay but also a novel analytical method by RNA sequencing. We constructed genetic analysis for 4 female cases showing high level of urinary low-molecular-weight proteinuria and their parents. Their XCI pattern was analyzed by both HUMARA assay and an ultra-deep targeted RNA sequencing of the CLCN5 gene using genomic DNA and mRNA extracted from both leukocytes and urine sediment. All four cases possessed pathogenic variants of the CLCN5 gene. XCI analysis revealed skewed XCI in only two cases, while the other two showed random XCI. All assay results of HUMARA and targeted RNA sequencing in both leukocytes and urinary sediment were clearly identical in all four cases. We developed a novel XCI analytical assay of ultra-deep targeted RNA sequencing and revealed that skewed XCI explains the mechanism of onset of female Dent disease in only half of such cases.

Clinical factors associated with prehospital exacerbation of anaphylaxis in children.

BACKGROUND: The severity of anaphylaxis often varies with time. Because prehospital intervention and initial treatment at hospital are affected by changing symptoms, the aim of this study was to determine the clinical factors associated with prehospital remission and exacerbation in the course of anaphylaxis in children. METHODS: Data from medical records on anaphylactic children who were treated for 3 years at Kobe Children's Primary Emergency Medical Center were retrospectively analyzed. Severity of symptoms was evaluated using Sampson's grade (S-G). Patients with increased S-G at the hospital visit from disease onset (worsened group) were compared with those with decreased S-G at the visit (improved group). Uni- and multivariate analyses were performed to identify clinical differences between the groups, with P<0.05 considered statistically significant. RESULTS: Among 115 anaphylactic children who showed S-G changes from onset to hospital visit, 43 were assigned to the worsened group and 72 to the improved group. Univariate analysis showed no significant differences in age, sex, history of asthma, prehospital treatment, type of antigen, or period from symptom onset to hospital visit between the groups. However, the time from antigen exposure to symptom onset was significantly longer, and S-G at onset was significantly lower in the worsened group than in the improved group. Multivariate analysis identified time from antigen exposure to symptom onset (odds ratio: 3.89, P<0.01) and S-G at onset (odds ratio: 0.06, P<0.001) as independent predictors of exacerbation. CONCLUSIONS: Anaphylactic children with slower and milder symptoms at onset are more likely to show deterioration.

Current Situation of Treatment for Anaphylaxis in a Japanese Pediatric Emergency Center.

OBJECTIVE: Anaphylaxis is a systemic allergic reaction that sometimes requires prompt treatment with intramuscular adrenaline. The aim of the study was to investigate the current situation regarding anaphylaxis treatment in a representative pediatric primary emergency facility in Japan. METHODS: We retrospectively examined the medical records dating from April 2011 through March 2014 from Kobe Children's Primary Emergency Medical Center, where general pediatricians work on a part-time basis. Clinical characteristics and current treatments for patients with anaphylaxis who presented to the facility were investigated. Furthermore, we compared the clinical characteristics between anaphylaxis patients given intramuscular adrenaline and those not given it. RESULTS: During the study period, 217 patients were diagnosed with anaphylaxis. The median Sampson grade at the time of visit was 2, and 90 patients (41%) were grade 4 or higher. No patients received self-intramuscular injected adrenaline before arrival at our emergency medical center because none of the patients had been prescribed it. Further treatment during the visit was provided to 128 patients (59%), with only 17 (8%) receiving intramuscular adrenaline. Patients given intramuscular adrenaline had significantly lower peripheral saturation of oxygen at the visit (P = 0.025) and more frequent transfer to a referral hospital (P < 0.001) than those not given intramuscular adrenaline. CONCLUSIONS: Education for Japanese pediatric practitioners and patients is warranted, because no patients used self-intramuscular injected adrenaline as a prehospital treatment for anaphylaxis, and only severely affected patients who needed oxygen therapy or hospitalization received intramuscular adrenaline in a pediatric primary emergency setting.

The Generation of Human γδT Cell-Derived Induced Pluripotent Stem Cells from Whole Peripheral Blood Mononuclear Cell Culture.

γδT cells constitute a small proportion of lymphocytes in peripheral blood. Unlike αßT cells, the anti-tumor activities are exerted through several different pathways in a MHC-unrestricted manner. Thus, immunotherapy using γδT cells is considered to be effective for various types of cancer. Occasionally, however, ex vivo expanded cells are not as effective as expected due to cell exhaustion. To overcome the issue of T-cell exhaustion, researchers have generated induced pluripotent stem cells (iPSCs) that harbor the same T-cell receptor (TCR) genes as their original T-cells, which provide nearly limitless sources for antigen-specific cytotoxic T lymphocytes (CTLs). However, these technologies have focused on αßT cells and require a population of antigen-specific CTLs, which are purified by cell sorting with HLA-peptide multimer, as the origin of iPS cells. In the present study, we aimed to develop an efficient and convenient system for generating iPSCs that harbor rearrangements of the TCRG and TCRD gene regions (γδT-iPSCs) without cell-sorting. We stimulated human whole peripheral blood mononuclear cell (PBMC) culture using Interleukin-2 and Zoledronate to activate γδT cells. Gene transfer into those cells with the Sendai virus vector resulted in γδT cell-dominant expression of exogenous genes. The introduction of reprogramming factors into the stimulated PBMC culture allowed us to establish iPSC lines. Around 70% of the established lines carried rearrangements at the TCRG and TCRD gene locus. The γδT-iPSCs could differentiate into hematopoietic progenitors. Our technology will pave the way for new avenues toward novel immunotherapy that can be applied for various types of cancer. Stem Cells Translational Medicine 2018;7:34-44.

Congenital Cytomegalovirus Infection in Children with Autism Spectrum Disorder: Systematic Review and Meta-Analysis.

Association of congenital cytomegalovirus (CMV) infection with autism spectral disorder (ASD) has been suggested since 1980s. Despite the observed association, its role as a risk factor for ASD remains to be defined. In the present review, we systematically evaluated the available evidence associating congenital CMV infection with ASD using PubMed, Web of Science, Cochrane Library, and Embase databases. Any studies on children with CMV infection and ASD were evaluated for eligibility and three observational studies were included in meta-analysis. Although a high prevalence of congenital CMV infection in ASD cases (OR 11.31, 95% CI 3.07-41.66) was indicated, too few events (0-2 events) in all included studies imposed serious limitations. There is urgent need for further studies to clarify this issue.