Itpr1 regulates the formation of anterior eye segment tissues derived from neural crest cells.

Mutations in ITPR1 cause ataxia and aniridia in individuals with Gillespie syndrome (GLSP). However, the pathogenic mechanisms underlying aniridia remain unclear. We identified a de novo GLSP mutation hotspot in the 3'-region of ITPR1 in five individuals with GLSP. Furthermore, RNA-sequencing and immunoblotting revealed an eye-specific transcript of Itpr1, encoding a 218amino acid isoform. This isoform is localized not only in the endoplasmic reticulum, but also in the nuclear and cytoplasmic membranes. Ocular-specific transcription was repressed by SOX9 and induced by MAF in the anterior eye segment (AES) tissues. Mice lacking seven base pairs of the last Itpr1 exon exhibited ataxia and aniridia, in which the iris lymphatic vessels, sphincter and dilator muscles, corneal endothelium and stroma were disrupted, but the neural crest cells persisted after completion of AES formation. Our analyses revealed that the 218-amino acid isoform regulated the directionality of actin fibers and the intensity of focal adhesion. The isoform might control the nuclear entry of transcriptional regulators, such as YAP. It is also possible that ITPR1 regulates both AES differentiation and muscle contraction in the iris.

A nationwide survey of Schaaf-Yang syndrome in Japan.

Schaaf-Yang syndrome (SYS) is a congenital disorder characterized by developmental delay, autism spectrum disorder and congenital joint contractures. In this study, a nationwide epidemiological questionnaire-based survey of SYS in the Japanese population was conducted to establish patient numbers, clinical features and genetic information. In the primary survey, we investigated the number of SYS patients. In the secondary survey, we obtained and analyzed detailed clinical and genetic information of SYS patients. This survey collected information on 25 genetically-confirmed patients. The major clinical symptoms included neonatal hypotonia (96% of the patients), poor suck in infancy (82%), developmental delay (100%) and joint contractures (83%). Other main symptoms and findings included characteristic facial features (100%), small hands (92%), eye abnormalities (92%) and short stature (79%). Based on the information collected on activities of daily living, 71% of patients were unable to walk, while 67%, 71%, and 81% of patients required full assistance with eating, toileting and bathing, respectively. Regarding inheritability, the genetic analysis of 21 patients revealed that 14 (67%) carried de novo truncating variants in the melanoma antigen L2 (MAGEL2) gene and seven (33%) had inherited truncating variants from their fathers who were carriers. This survey revealed the clinical and genetic features in Japanese SYS patients. The majority of SYS patients required assistance in many aspects of daily living, and there were a certain number of carriers of the imprinting disorder.

Risks of ACTH therapy for West syndrome following BCG vaccination.

OBJECTIVE: Bacille de Calmette et Guérin (BCG) is a live vaccine for tuberculosis that is administered to all infants in Japan. Adrenocorticotropic hormone (ACTH) therapy for West syndrome (WS) causes immunosuppression and may result in BCG infection after BCG vaccination. We evaluated the safety of ACTH therapy initiated shortly after BCG vaccination. METHODS: We analyzed patients with WS who received ACTH therapy between 2005 and 2018. We evaluated the interval between BCG and ACTH therapy, and the rate of BCG infection during and after ACTH therapy, by retrospective chart review. RESULTS: Seventy-nine patients were included in the analysis. Twenty-three patients received ACTH therapy prior to BCG vaccination. For the remaining 56 patients, the median interval between BCG vaccination and the start of ACTH therapy (BCG-ACTH interval) was 91.5 (range 14-280) days. The BCG-ACTH interval was shorter in patients with unknown than in those with known etiologies. It was <8 weeks in 13 patients (10 with unknown and 3 with known etiologies). The minimum BCG-ACTH interval was 14 days. Six patients with epileptic spasms received BCG vaccinations because physicians did not recognize their seizures. None of the patients developed BCG infection. CONCLUSION: No patients who received ACTH therapy after BCG, even at an interval of 8 weeks, developed BCG infection. The timing of ACTH therapy initiation should be based on the risk of BCG-related adverse events and the adverse effects of any delay.

An atlas of active enhancers across human cell types and tissues.

Enhancers control the correct temporal and cell-type-specific activation of gene expression in multicellular eukaryotes. Knowing their properties, regulatory activity and targets is crucial to understand the regulation of differentiation and homeostasis. Here we use the FANTOM5 panel of samples, covering the majority of human tissues and cell types, to produce an atlas of active, in vivo-transcribed enhancers. We show that enhancers share properties with CpG-poor messenger RNA promoters but produce bidirectional, exosome-sensitive, relatively short unspliced RNAs, the generation of which is strongly related to enhancer activity. The atlas is used to compare regulatory programs between different cells at unprecedented depth, to identify disease-associated regulatory single nucleotide polymorphisms, and to classify cell-type-specific and ubiquitous enhancers. We further explore the utility of enhancer redundancy, which explains gene expression strength rather than expression patterns. The online FANTOM5 enhancer atlas represents a unique resource for studies on cell-type-specific enhancers and gene regulation.

MYCN de novo gain-of-function mutation in a patient with a novel megalencephaly syndrome.

BACKGROUND: In this study, we aimed to identify the gene abnormality responsible for pathogenicity in an individual with an undiagnosed neurodevelopmental disorder with megalencephaly, ventriculomegaly, hypoplastic corpus callosum, intellectual disability, polydactyly and neuroblastoma. We then explored the underlying molecular mechanism. METHODS: Trio-based, whole-exome sequencing was performed to identify disease-causing gene mutation. Biochemical and cell biological analyses were carried out to elucidate the pathophysiological significance of the identified gene mutation. RESULTS: We identified a heterozygous missense mutation (c.173C>T; p.Thr58Met) in the MYCN gene, at the Thr58 phosphorylation site essential for ubiquitination and subsequent MYCN degradation. The mutant MYCN (MYCN-T58M) was non-phosphorylatable at Thr58 and subsequently accumulated in cells and appeared to induce CCND1 and CCND2 expression in neuronal progenitor and stem cells in vitro. Overexpression of Mycn mimicking the p.Thr58Met mutation also promoted neuronal cell proliferation, and affected neuronal cell migration during corticogenesis in mouse embryos. CONCLUSIONS: We identified a de novo c.173C>T mutation in MYCN which leads to stabilisation and accumulation of the MYCN protein, leading to prolonged CCND1 and CCND2 expression. This may promote neurogenesis in the developing cerebral cortex, leading to megalencephaly. While loss-of-function mutations in MYCN are known to cause Feingold syndrome, this is the first report of a germline gain-of-function mutation in MYCN identified in a patient with a novel megalencephaly syndrome similar to, but distinct from, CCND2-related megalencephaly-polymicrogyria-polydactyly-hydrocephalus syndrome. The data obtained here provide new insight into the critical role of MYCN in brain development, as well as the consequences of MYCN defects.

Author Correction: A missense mutation in the HECT domain of NEDD4L identified in a girl with periventricular nodular heterotopia, polymicrogyria, and cleft palate.

Since the publication of this article, it has been brought to our attention, that the identified mutation (NM_015277: c.2617 G > A; p.Glu873Lys) is identical with the mutation (NM_001144967: c.2677 G > A; p.Glu893Lys) reported by Broix et al (Nature Genetics 48, 1349-1358, 2016 https://doi.org/10.1038/ng.3676 ). Therefore the mutation is not novel but recurrent. Accordingly, the word "novel" should be deleted throughout the article including the title. Thus, the title should read "A missense mutation in the HECT domain of NEDD4L identified in a girl with periventricular nodular heterotopia, polymicrogyria, and cleft palate."

A case of early-onset epileptic encephalopathy with a homozygous TBC1D24 variant caused by uniparental isodisomy.

TBC1D24-related disorders are rare neurodevelopmental disorders that show a broad range of neuropsychiatric deficits and are mostly inherited in an autosomal recessive manner. Here we describe a case with early-onset epileptic encephalopathy, in whom exome sequencing detected a novel pathogenic homozygous c.442G>A, p.(Glu148Lys) variant in TBC1D24. She showed severe developmental delay, congenital sensorineural hearing loss and seizures, but the combination of a high dose phenobarbital and potassium bromide was very effective for the seizures. Sanger sequencing revealed that her mother was a heterozygous carrier of the TBC1D24 variant, but her father showed only wild-type alleles. Homozygosity mapping analysis using exome data showed loss of the heterozygosity region at 16p13.3-p13.13 encompassing TBC1D24. Genotyping analysis using rare variants within loss of the heterozygosity region indicated that the patient has a homozygous haplotype inherited from her mother, indicating maternal segmental uniparental isodisomy (UPiD). These data clearly show that exome sequencing is a powerful tool to perform comprehensive genetic analysis.

A novel homozygous missense mutation in the SH3-binding motif of STAMBP causing microcephaly-capillary malformation syndrome.

Microcephaly-capillary malformation syndrome is a congenital and neurodevelopmental disorder caused by biallelic mutations in the STAMBP gene. Here we identify the novel homozygous mutation located in the SH3 binding motif of STAMBP (NM_006463.4) (c.707C>T: p.Ser236Phe) through whole-exome sequencing. The case patient was a 2-year-old boy showing severe global developmental delay, progressive microcephaly, refractory seizures, dysmorphic facial features, and multiple capillary malformations. Immunoblot analysis of patient-derived lymphoblastoid cell lines (LCLs) revealed a severe reduction in STAMBP expression, indicating that Ser236Phe induces protein instability. STAMBP interacts with the SH3 domain of STAM and transduces downstream signals from the Jaks-STAM complex. The substitution of Ser236Phe found in the case patient was located in the SH3-binding motif, and we propose the mutation may block STAM binding and subsequently induce STAMBP degradation. Contrary to previously reported STAMBP mutations, the Ser236Phe mutation did not lead to constitutive activation of the PI3K-AKT-mTOR pathway in patient-derived LCLs, as indicated by the expression of phosphorylated S6 ribosomal protein, suggesting that it is not the major pathomechanism underlying the disorder in this patient.

CTCF deletion syndrome: clinical features and epigenetic delineation.

BACKGROUND: Heterozygous mutations in CTCF have been reported in patients with distinct clinical features including intellectual disability. However, the precise pathomechanism underlying the phenotype remains to be uncovered, partly because of the diverse function of CTCF. Here we describe extensive clinical and genetic investigation for two patients with a microdeletion encompassing CTCF. METHODS: We performed genetic examination including comprehensive investigation of X chromosome inactivation and DNA methylation profiling at imprinted loci and genome-wide. RESULTS: Two patients showed comparable clinical features to those in a previous report, indicating that haploinsufficiency of CTCF was the major determinant of the microdeletion syndrome. Despite the haploinsufficiency of CTCF, X chromosome inactivation was normal. DNA methylation at imprinted loci was normal, but hypermethylation at CTCF binding sites was demonstrated, of which PRKCZ and FGFR2 were identified as candidate genes. CONCLUSIONS: This study confirms that haploinsufficiency of CTCF causes distinct clinical features, and that a microdeletion encompassing CTCF could cause a recognisable CTCF deletion syndrome. Perturbed DNA methylation at CTCF binding sites, not at imprinted loci, may underlie the pathomechanism of the syndrome.

Role of a heterotrimeric G-protein, Gi2, in the corticogenesis: possible involvement in periventricular nodular heterotopia and intellectual disability.

We analyzed the role of a heterotrimeric G-protein, Gi2, in the development of the cerebral cortex. Acute knockdown of the α-subunit (Gαi2) with in utero electroporation caused delayed radial migration of excitatory neurons during corticogenesis, perhaps because of impaired morphology. The migration phenotype was rescued by an RNAi-resistant version of Gαi2. On the other hand, silencing of Gαi2 did not affect axon elongation, dendritic arbor formation or neurogenesis at ventricular zone in vivo. When behavior analyses were conducted with acute Gαi2-knockdown mice, they showed defects in social interaction, novelty recognition and active avoidance learning as well as increased anxiety. Subsequently, using whole-exome sequencing analysis, we identified a de novo heterozygous missense mutation (c.680C>T; p.Ala227Val) in the GNAI2 gene encoding Gαi2 in an individual with periventricular nodular heterotopia and intellectual disability. Collectively, the phenotypes in the knockdown experiments suggest a role of Gαi2 in the brain development, and impairment of its function might cause defects in neuronal functions which lead to neurodevelopmental disorders.

A combination of genetic and biochemical analyses for the diagnosis of PI3K-AKT-mTOR pathway-associated megalencephaly.

BACKGROUND: Constitutive activation of the PI3K-AKT-mTOR pathway (mTOR pathway) underlies megalencephaly in many patients. Yet, prevalence of the involvement of the PI3K-AKT-mTOR pathway in patients with megalencephaly remains to be elucidated, and molecular diagnosis is challenging. Here, we have successfully established a combination of genetic and biochemical methods for diagnosis of mTOR pathway-associated megalencephaly, and have attempted to delineate the clinical characteristics of the disorder. METHODS: Thirteen patients with an increased head circumference and neurological symptoms participated in the study. To evaluate the activation of the mTOR pathway, we performed western blot analysis to determine the expression levels of phosphorylated S6 ribosomal protein (phospho-S6 protein) in lymphoblastoid cell lines from 12 patients. Multiplex targeted sequencing analysis for 15 genes involved in the mTOR pathway was performed on 12 patients, and whole-exome sequencing was performed on one additional patient. Clinical features and MRI findings were also investigated. RESULTS: We identified pathogenic mutations in six (AKT3, 1 patient; PIK3R2, 2 patients; PTEN, 3 patients) of the 13 patients. Increased expression of phospho-S6 protein was demonstrated in all five mutation-positive patients in whom western blotting was performed, as well as in three mutation-negative patients. Developmental delay, dysmorphic facial features were observed in almost all patients. Syndactyly/polydactyly and capillary malformations were not observed, even in patients with AKT3 or PIK3R2 mutations. There were no common phenotypes or MRI findings among these patients. CONCLUSIONS: A combination of genetic and biochemical methods successfully identified mTOR pathway involvement in nine of 13 (approximately 70%) patients with megalencephaly, indicating a major contribution of the pathway to the pathogenesis of megalencephaly. Our combined approach could be useful to identify patients who are suitable for future clinical trials using an mTOR inhibitor.

A novel missense mutation in the HECT domain of NEDD4L identified in a girl with periventricular nodular heterotopia, polymicrogyria and cleft palate.

We identified a novel de novo heterozygous missense mutation in the NEDD4L gene (NM_015277: c.2617G>A; p.Glu873Lys) through whole-exome sequencing in a 3-year-old girl showing severe global developmental delay, infantile spasms, cleft palate, periventricular nodular heterotopia and polymicrogyria. Mutations in the HECT domain of NEDD4L have been reported in patients with a neurodevelopmental disorder along with similar brain malformations. All patients reported with NEDD4L HECT domain mutations showed periventricular nodular heterotopia, and most had seizures, cortex anomalies, cleft palate and syndactyly. The unique constellation of clinical features in patients with NEDD4L mutations might help clinically distinguish them from patients with other genetic mutations including FLNA, which is a well-known causative gene of periventricular nodular heterotopia. Although mutations in the HECT domain of NEDD4L that lead to AKT-mTOR pathway deregulation in forced expression system were reported, our western blot analysis did not show an increased level of AKT-mTOR activity in lymphoblastoid cell lines (LCLs) derived from the patient. In contrast to the forced overexpression system, AKT-mTOR pathway deregulation in LCLs derived from our patient seems to be subtle.

Batf2/Irf1 induces inflammatory responses in classically activated macrophages, lipopolysaccharides, and mycobacterial infection.

Basic leucine zipper transcription factor Batf2 is poorly described, whereas Batf and Batf3 have been shown to play essential roles in dendritic cell, T cell, and B cell development and regulation. Batf2 was drastically induced in IFN-γ-activated classical macrophages (M1) compared with unstimulated or IL-4-activated alternative macrophages (M2). Batf2 knockdown experiments from IFN-γ-activated macrophages and subsequent expression profiling demonstrated important roles for regulation of immune responses, inducing inflammatory and host-protective genes Tnf, Ccl5, and Nos2. Mycobacterium tuberculosis (Beijing strain HN878)-infected macrophages further induced Batf2 and augmented host-protective Batf2-dependent genes, particularly in M1, whose mechanism was suggested to be mediated through both TLR2 and TLR4 by LPS and heat-killed HN878 (HKTB) stimulation experiments. Irf1 binding motif was enriched in the promoters of Batf2-regulated genes. Coimmunoprecipitation study demonstrated Batf2 association with Irf1. Furthermore, Irf1 knockdown showed downregulation of IFN-γ- or LPS/HKTB-activated host-protective genes Tnf, Ccl5, Il12b, and Nos2. Conclusively, Batf2 is an activation marker gene for M1 involved in gene regulation of IFN-γ-activated classical macrophages, as well as LPS/HKTB-induced macrophage stimulation, possibly by Batf2/Irf1 gene induction. Taken together, these results underline the role of Batf2/Irf1 in inducing inflammatory responses in M. tuberculosis infection.

Combined genetic analyses can achieve efficient diagnostic yields for subjects with Alagille syndrome and incomplete Alagille syndrome.

AIM: We evaluated combined genetic analyses with targeted next-generation sequencing (NGS), multiplex ligation probe amplification (MLPA) of Jagged1 (JAG1) genes and microarray comparative genomic hybridisation (CGH) in subjects with Alagille syndrome, incomplete clinical features of Alagille syndrome and biliary atresia. METHODS: Subjects recruited from April 2013 to December 2015 underwent a targeted NGS analysis, including JAG1 and Notch homolog 2 (NOTCH2). If no mutations were detected in JAG1 or NOTCH2, or if copy number variations were suggested by the NGS analysis, we performed an MLPA analysis of JAG1. We also performed a microarray CGH analysis with whole-exon deletion detected by the MLPA analysis. RESULTS: We analysed 30 subjects with Alagille syndrome, nine with incomplete Alagille syndrome and 17 with biliary atresia and detected pathogenic mutations in JAG1 or NOTCH2 in 24/30 subjects with Alagille syndrome and in 4/9 subjects with incomplete Alagille syndrome. No pathogenic mutations were detected in subjects with biliary atresia. The frequency of JAG1 mutations was as follows: single nucleotide variants (51.9%), small insertion or deletion (29.6%) and gross deletion (18.5%). CONCLUSION: Combined genetic analyses achieved efficient diagnostic yields for subjects with Alagille syndrome and incomplete Alagille syndrome.

Molecular Genetic Dissection and Neonatal/Infantile Intrahepatic Cholestasis Using Targeted Next-Generation Sequencing.

OBJECTIVES: To ascertain a molecular genetic diagnosis for subjects with neonatal/infantile intrahepatic cholestasis (NIIC) by the use of next-generation sequencing (NGS) and to perform a genotype-phenotype correlation. STUDY DESIGN: We recruited Japanese subjects with NIIC who had no definitive molecular genetic diagnosis. We developed a diagnostic custom panel of 18 genes, and the amplicon library was sequenced via NGS. We then compared clinical data between the molecular genetically confirmed subjects with NIIC. RESULTS: We analyzed 109 patients with NIIC ("genetic cholestasis," 31 subjects; "unknown with complications" such as prematurity, 46 subjects; "unknown without complications," 32 subjects), and a molecular genetic diagnosis was made for 28 subjects (26%). The rate of positive molecular genetic diagnosis in each category was 22 of 31 (71%) for the "genetic cholestasis" group, 2 of 46 (4.3%) for the "unknown with complications" group, and 4 of 32 (12.5%) for the "unknown without complications" group. The grouping of the molecular diagnoses in the group with genetic cholestasis was as follows: 12 with Alagille syndrome, 5 with neonatal Dubin-Johnson syndrome, 5 with neonatal intrahepatic cholestasis caused by citrin deficiency, and 6 with progressive familial intrahepatic cholestasis or benign recurrent intrahepatic cholestasis with low gamma-glutamyl transpeptidase levels. Several clinical datasets, including age of onset, direct bilirubin, and aminotransferases, were significantly different between the disorders confirmed using molecular genetic diagnosis. CONCLUSION: Targeted NGS can be used for molecular genetic diagnosis in subjects with NIIC. Clinical diagnosis should be accordingly redefined in the view of molecular genetic findings.

Novel splicing mutation in the ASXL3 gene causing Bainbridge-Ropers syndrome.

Bainbridge-Ropers syndrome (BRPS) is characterized by severe developmental delay, feeding problems, short stature, characteristic facal appearance including arched eyebrows and anteverted nares, and ulnar deviation of the hands. BRPS is caused by a heterozygous mutation in the additional sex combs-like 3 (ASXL3) gene. We describe a patient with severe developmental delay, feeding problems, short stature, autism, and sleep disturbance with a heterozygous de novo splicing mutation in the ASXL3 gene. Reported disease-causing mutations in ASXL3 are located mostly in the first half of exon 11, analogous to ASXL1 mutations of which result in Bohring-Opitz syndrome (BOS). Our findings suggest that the expression of the truncated ASXL3 protein, including ASXN and ASXH domains, give rise to BRPS, which is distinct from but overlaps with BOS. © 2016 Wiley Periodicals, Inc.

Identification of chromatin marks at TERRA promoter and encoding region.

TERRA is a long non-coding RNA that is essential for telomere integrity. Although it is transcribed from subtelomeres and telomeres, how it is expressed in heterochromatic region is currently unknown. In this study, we focused our analysis on TERRA-encoding region TelBam3.4 and TelBam3.4-like sequences, and determined their transcription start sites, as well as enrichment of RNA polymerase II and histone modifications. We found that H3K4me3 and H3K9me3 are present at TERRA promoters, whereas H3K27ac and H3K9me3 are present at telomeric repeats. Consistently, we show that presence of active histone modifications H3K4me3 and H3K27ac are correlated to TERRA expression. These results mark an important step towards understanding telomere maintenance and transcription.

Anti-M Antibody Induced Prolonged Anemia Following Hemolytic Disease of the Newborn Due to Erythropoietic Suppression in 2 Siblings.

Hemolytic disease of the newborn (HDN) arising from MNSs incompatibility is rare, with few reports of prolonged anemia and reticulocytopenia following HDN. We report the younger of 2 male siblings, both of whom had anti-M-induced HDN and anemia persisting for over a month. Peripheral reticulocytes remained inappropriately low for the degree of anemia, and they needed multiple red cell transfusions. Viral infections were ruled out. Corticosteroids were given for suspected pure red cell aplasia. Anemia and reticulocytopenia subsequently improved. Colony-forming unit erythroid assay revealed erythropoietic suppression of M antigen-positive erythroid precursor cells cultured with maternal or infant sera containing anti-M. In conclusion, maternal anti-M caused HDN and prolonged anemia by erythropoietic suppression in 2 siblings.

Homoplasmy of a mitochondrial 3697G>A mutation causes Leigh syndrome.

Herein we report on three siblings with Leigh syndrome (LS) harboring a homoplasmic m.3697G>A mutation (G131S) in the MT-ND1 gene. The siblings' phenotypically normal mother had the same, albeit heteroplasmic, mutation. Complex I deficiency (8% of average control values) was demonstrated in a biceps brachii muscle from one of the patients. Heteroplasmic m.3697G>A has been reported in patients with Leber's hereditary optic neuropathy, mitochondrial encephalomyopathy, lactic acidosis and stroke-like episodes and Stüve-Wiedemann syndrome. Because all three patients in this series carried m.3697G>A in a homoplasmic manner and had LS, we suggest that homoplasmy of m.3697G>A may cause the LS phenotype.

Fulminant encephalopathy with marked brain edema and bilateral thalamic lesions.

We encountered two children with acute encephalopathy associated with unique clinical manifestations. Both the patients had status epilepticus at onset and neuroimaging studies revealed marked brain edema and bilateral thalamic lesions. Although they were treated with steroids and immunoglobulin, their outcomes were very poor. A thermolabile variant of carnitine palmitoyltransferase II and an elevated interleukin-6 level in cerebrospinal fluid were observed in one patient each. The constellation of clinical and neuroimaging findings in our patients is apparently not consistent with any established subtype of acute encephalopathy/encephalitis.