Molecular diagnosis of 405 individuals with autism spectrum disorder.

Autism spectrum disorder (ASD) is caused by combined genetic and environmental factors. Genetic heritability in ASD is estimated as 60-90%, and genetic investigations have revealed many monogenic factors. We analyzed 405 patients with ASD using family-based exome sequencing to detect disease-causing single-nucleotide variants (SNVs), small insertions and deletions (indels), and copy number variations (CNVs) for molecular diagnoses. All candidate variants were validated by Sanger sequencing or quantitative polymerase chain reaction and were evaluated using the American College of Medical Genetics and Genomics/Association for Molecular Pathology guidelines for molecular diagnosis. We identified 55 disease-causing SNVs/indels in 53 affected individuals and 13 disease-causing CNVs in 13 affected individuals, achieving a molecular diagnosis in 66 of 405 affected individuals (16.3%). Among the 55 disease-causing SNVs/indels, 51 occurred de novo, 2 were compound heterozygous (in one patient), and 2 were X-linked hemizygous variants inherited from unaffected mothers. The molecular diagnosis rate in females was significantly higher than that in males. We analyzed affected sibling cases of 24 quads and 2 quintets, but only one pair of siblings shared an identical pathogenic variant. Notably, there was a higher molecular diagnostic rate in simplex cases than in multiplex families. Our simulation indicated that the diagnostic yield is increasing by 0.63% (range 0-2.5%) per year. Based on our simple simulation, diagnostic yield is improving over time. Thus, periodical reevaluation of ES data should be strongly encouraged in undiagnosed ASD patients.

YM155 and chrysin cooperatively suppress survivin expression in SMARCB1/INI1-deficient tumor cells.

SMARCB1/INI1 deficiency is seen in several malignant tumors including malignant rhabdoid tumor (MRT), a highly aggressive pediatric malignancy. Loss of SMARCB1/INI1 function alters diverse oncogenic cellular signals, making it difficult to discover effective targeting therapy. By utilizing an in vitro drug screening system, effective therapeutic agents against SMARCB1/INI1-deficient tumors were explored in this study. In the in vitro drug sensitivity test, 80 agents with various actions were screened for their cytotoxicity in a panel of five SMARCB1/INI1-deficient tumor cell lines. The combination effect was screened based on the Bliss independent model. The growth-inhibitory effect was determined in both the conventional two-dimensional culture and the collagen-embedded three-dimensional culture system. Survivin expression after agent exposure was determined by Western blot analysis. All five cell lines were found to be sensitive to YM155, a selective survivin inhibitor. In the drug combination screening, YM155 showed additive to synergistic effects with various agents including chrysin. Chrysin enhanced YM155-induced apoptosis, but not mitochondrial depolarization upon exposure of SMARCB1/INI1-deficient tumor cells to the two agents for 6 h. YM155 and chrysin synergistically suppressed survivin expression, especially in TTN45 cells in which such suppression was observed as early as 6 h after exposure to the two agents. Survivin is suggested to be a therapeutic target in MRT and other SMARCB1/INI1-deficient tumors. Chrysin, a flavone that is widely distributed in plants, cooperatively suppressed survivin expression and enhanced the cytotoxicity of YM155.

Exome sequencing analysis of Japanese autism spectrum disorder case-control sample supports an increased burden of synaptic function-related genes.

Autism spectrum disorder (ASD) is a highly heritable, complex disorder in which rare variants contribute significantly to disease risk. Although many genes have been associated with ASD, there have been few genetic studies of ASD in the Japanese population. In whole exomes from a Japanese ASD sample of 309 cases and 299 controls, rare variants were associated with ASD within specific neurodevelopmental gene sets, including highly constrained genes, fragile X mental retardation protein target genes, and genes involved in synaptic function, with the strongest enrichment in trans-synaptic signaling (p = 4.4 × 10-4, Q-value = 0.06). In particular, we strengthen the evidence regarding the role of ABCA13, a synaptic function-related gene, in Japanese ASD. The overall results of this case-control exome study showed that rare variants related to synaptic function are associated with ASD susceptibility in the Japanese population.

Novel COL2A1 variants in Japanese patients with spondyloepiphyseal dysplasia congenita.

Spondyloepiphyseal dysplasia congenita (SEDC) is a multisystemic skeletal disorder caused by pathogenic variants in COL2A1. Here, we report the genotype-phenotype correlations in five Japanese patients with SEDC based on their clinical and radiological findings. All five patients had novel missense variants resulting in glycine substitutions (G474V, G543E, G567S, G594R, and G1170R). Genetic testing is important for early intervention for the extraskeletal complications of SEDC. Spondyloepiphyseal dysplasia congenita (SEDC) (OMIM#183900) is an autosomal dominant chondrodysplasia characterized by disproportionate short stature, abnormal epiphyses, flattened vertebral bodies (skeletal abnormalities), and extraskeletal features, including myopia, retinal degeneration with retinal detachment, and cleft palate. SEDC is caused by a heterozygous variant in the collagen II alpha 1 (COL2A1) gene.

Large-scale discovery of novel neurodevelopmental disorder-related genes through a unified analysis of single-nucleotide and copy number variants.

BACKGROUND: Previous large-scale studies of de novo variants identified a number of genes associated with neurodevelopmental disorders (NDDs); however, it was also predicted that many NDD-associated genes await discovery. Such genes can be discovered by integrating copy number variants (CNVs), which have not been fully considered in previous studies, and increasing the sample size. METHODS: We first constructed a model estimating the rates of de novo CNVs per gene from several factors such as gene length and number of exons. Second, we compiled a comprehensive list of de novo single-nucleotide variants (SNVs) in 41,165 individuals and de novo CNVs in 3675 individuals with NDDs by aggregating our own and publicly available datasets, including denovo-db and the Deciphering Developmental Disorders study data. Third, summing up the de novo CNV rates that we estimated and SNV rates previously established, gene-based enrichment of de novo deleterious SNVs and CNVs were assessed in the 41,165 cases. Significantly enriched genes were further prioritized according to their similarity to known NDD genes using a deep learning model that considers functional characteristics (e.g., gene ontology and expression patterns). RESULTS: We identified a total of 380 genes achieving statistical significance (5% false discovery rate), including 31 genes affected by de novo CNVs. Of the 380 genes, 52 have not previously been reported as NDD genes, and the data of de novo CNVs contributed to the significance of three genes (GLTSCR1, MARK2, and UBR3). Among the 52 genes, we reasonably excluded 18 genes [a number almost identical to the theoretically expected false positives (i.e., 380 × 0.05 = 19)] given their constraints against deleterious variants and extracted 34 "plausible" candidate genes. Their validity as NDD genes was consistently supported by their similarity in function and gene expression patterns to known NDD genes. Quantifying the overall similarity using deep learning, we identified 11 high-confidence (> 90% true-positive probabilities) candidate genes: HDAC2, SUPT16H, HECTD4, CHD5, XPO1, GSK3B, NLGN2, ADGRB1, CTR9, BRD3, and MARK2. CONCLUSIONS: We identified dozens of new candidates for NDD genes. Both the methods and the resources developed here will contribute to the further identification of novel NDD-associated genes.

SOX11 variants cause a neurodevelopmental disorder with infrequent ocular malformations and hypogonadotropic hypogonadism and with distinct DNA methylation profile.

PURPOSE: This study aimed to undertake a multidisciplinary characterization of the phenotype associated with SOX11 variants. METHODS: Individuals with protein altering variants in SOX11 were identified through exome and genome sequencing and international data sharing. Deep clinical phenotyping was undertaken by referring clinicians. Blood DNA methylation was assessed using Infinium MethylationEPIC array. The expression pattern of SOX11 in developing human brain was defined using RNAscope. RESULTS: We reported 38 new patients with SOX11 variants. Idiopathic hypogonadotropic hypogonadism was confirmed as a feature of SOX11 syndrome. A distinctive pattern of blood DNA methylation was identified in SOX11 syndrome, separating SOX11 syndrome from other BAFopathies. CONCLUSION: SOX11 syndrome is a distinct clinical entity with characteristic clinical features and episignature differentiating it from BAFopathies.

Further delineation of SET-related intellectual disability syndrome.

A loss-of-function mutation of SET causes nonsyndromic intellectual disability, often associated with mild facial dysmorphic features, including plagiocephaly, facial asymmetry, broad and high forehead, a wide mouth, and a prominent mandible. We report a male individual with a 2.0 Mb deletion within 9q34.11, involving SET and SPTAN1, but not STXBP1. Among the genes with a high probability of being loss-of-function intolerant in the deletion interval, only SPTAN1 and SET had haploinsufficiency score (%HI) <10, indicating a high likelihood of haploinsufficiency. Pathogenic variants in SPTAN1 are responsible for early-onset epileptic encephalopathy by exerting a dominant-negative effect. However, whether haploinsufficiency of SPTAN1 alone also causes the severe phenotype remained unknown. SET is a regulator of cell differentiation in early human development and a component of the inhibitor of histone acetyltransferases complex. Therefore, combining the previously reported patients, our patient delineated the phenotypic spectrum of SET-related nonsyndromic intellectual disability with mild facial dysmorphism.

Divergent variant patterns among 19 patients with Rubinstein-Taybi syndrome uncovered by comprehensive genetic analysis including whole genome sequencing.

Rubinstein-Taybi syndrome (RSTS) is characterized by dysmorphic facial features, broad thumbs, and intellectual disability. CREB-binding protein (CREBBP) or E1A-binding protein P300 (EP300) are causative genes. To elucidate the underlying genetic and genomic architecture related to the RSTS phenotype, we performed comprehensive genetic analysis targeting CREBBP and/or EP300 in 22 clinically diagnosed patients. During the 11-year study period, we used several analysis methods including high-resolution melting, array-based comparative genomic hybridization, panel-based exome sequencing, whole exome sequencing, and whole genome sequencing (WGS). We identified the causative variants in 19 patients (86.3%), but they were variable and complex, so we must combine multiple analysis methods. Notably, we found genetic alterations in the non-coding regions of two patients (10.5%, 2/19): scattered deletions including a partial 5'-untranslated region of CREBBP in one patient (all coding exons were intact), and a deep 229-bp intronic deletion in another patient, resulting in a splicing error. Furthermore, we identified rare clinical findings: two patients with an EP300 variant showed abnormal development of the neural tube, and one patient with a CREBBP variant had anorectal atresia with a cloaca. Our findings expand the allelic heterogeneity of RSTS, underscore the utility of comprehensive genetic analysis, and suggest that WGS may be a practical diagnostic strategy.

Multiple alterations in glutamatergic transmission and dopamine D2 receptor splicing in induced pluripotent stem cell-derived neurons from patients with familial schizophrenia.

An increasing body of evidence suggests that impaired synapse development and function are associated with schizophrenia; however, the underlying molecular pathophysiological mechanism of the disease remains largely unclear. We conducted a family-based study combined with molecular and cellular analysis using induced pluripotent stem cell (iPSC) technology. We generated iPSCs from patients with familial schizophrenia, differentiated these cells into neurons, and investigated the molecular and cellular phenotypes of the patient's neurons. We identified multiple altered synaptic functions, including increased glutamatergic synaptic transmission, higher synaptic density, and altered splicing of dopamine D2 receptor mRNA in iPSC-derived neurons from patients. We also identified patients' specific genetic mutations using whole-exome sequencing. Our findings support the notion that altered synaptic function may underlie the molecular and cellular pathophysiology of schizophrenia, and that multiple genetic factors cooperatively contribute to the development of schizophrenia.

A Recurrent Variant in POLR1B, c.3007C>T; p.Arg1003Cys, Associated with Atresia of the External Canal and Microtia in Treacher Collins Syndrome Type 4.

Treacher Collins syndrome (TCS) is a heterogenous malformation syndrome characterized by a distinct facial appearance including downslanting palpebral fissures, malar hypoplasia, conductive hearing loss, and mandibular hypoplasia. Recently, a new causative gene, POLR1B, encoding DNA-directed RNA polymerase I subunit RPA2, was identified as a fourth type of TCS (TCS4). We describe another patient with TCS4 caused by a recurrent POLR1B variant, c.3007C>T; p.Arg1003Cys. Including our patient, all 4 patients with p.(Arg1003Cys) had atresia of the external auditory canal and microtia. All of the reported pathogenic variants in POLR1B were clustered at only 2 residues. Our patient highlights the genotype-phenotype correlation in TCS4 associated with POLR1B.

Comprehensive Genetic Analysis of Non-syndromic Autism Spectrum Disorder in Clinical Settings.

Although genetic factors are involved in the etiology of autism spectrum disorder (ASD), the significance of genetic analysis in clinical settings is unclear. Forty-nine subjects diagnosed with non-syndromic ASD were analyzed by microarray comparative genomic hybridization (CGH) analysis, whole-exome sequencing (WES) analysis, and panel sequencing analysis for 52 common causative genes of ASD to detect inherited rare variants. Genetic analysis by microarray CGH and WES analyses showed conclusive results in about 10% of patients, however, many inherited variants detected by panel sequencing analysis were difficult to interpret and apply in clinical practice in the majority of patients. Further improvement of interpretation of many variants detected would be necessary for combined genetic tests to be used in clinical settings.

Any modality of renal replacement therapy can be a treatment option for Joubert syndrome.

Joubert syndrome (JS) is an inherited ciliopathy characterized by a distinctive cerebellar and brain stem malformation which is known as the "molar tooth sign" on axial brain images, hypotonia, and developmental delay. Approximately 25-30% of patients with JS have kidney disease and many of them progress to end-stage kidney disease (ESKD). However, there are few reports on the outcomes of renal replacement therapy (RRT) in patients with JS and ESKD. In this study, we clarified the clinical features, treatment, and outcomes of patients with JS who underwent RRT. We retrospectively analyzed the medical records and clinical characteristics of 11 patients with JS who underwent RRT between June 1994 and July 2019. Data are shown as the median (range). Gene analysis was performed in 8 of the 11 cases, and CEP290 mutations were found in four patients, two had TMEM67 mutations, one had a RPGRIP1L mutation, and one patient showed no mutation with the panel exome analysis. Complications in other organs included hydrocephalus in two cases, retinal degeneration in eight cases, coloboma in one case, liver diseases in four cases, and polydactyly in one case. Peritoneal dialysis (PD) was introduced in seven cases, with a median treatment duration of 5.4 (3.4-10.7) years. Hemodialysis was performed using arteriovenous fistula in two cases, and kidney transplantation was performed 9 times in eight cases. Only one of the grafts failed during the observation period of 25.6 (8.2-134.2) months. The glomerular filtration rate at the final observation was 78.1 (41.4-107.7) mL/min/1.73 m2. The median age at the final observation was 13.4 (5.6-25.1) years, and all patients were alive except one who died of hepatic failure while on PD. Any type of RRT modality can be a treatment option for patients with JS and ESKD.

A novel method for isolating lymphatic endothelial cells from lymphatic malformations and detecting PIK3CA somatic mutation in these isolated cells.

PURPOSE: Tissue disaggregation and the cell sorting technique by surface markers has played an important role in isolating lymphatic endothelial cells (LECs) from lymphatic malformation (LM). However, this technique may have the drawback of impurities or result in isolation failure because it is dependent on surface marker expressions, the heterogeneity of which has been found in the lymphatic system. We developed a novel method for isolating LM-LECs without using whole tissue disaggregation. METHODS: Seven LM surgical specimens were collected from seven patients with LMs. LM-LECs were detached from the LM cyst wall by "lumen digestion" and irrigating the cystic cavity with trypsin, and maintained in culture. RESULTS: The cells formed a monolayer with a cobblestone-like appearance. Immunohistochemistry and quantitative RT-PCR of these cells revealed high expression of lymphatic-specific genes, confirming their identity as LM-LECs. The whole-exome sequencing and PIK3CA sequencing of these cells revealed somatic mutations in PIK3CA in all cases. CONCLUSIONS: We established a novel technique for isolating LM-LECs from LM tissue by "lumen digestion" without whole-tissue disaggregation. The limited incorporation of non-LM LECs in the isolate in our method could make it an important tool for investigating the heterogeneity of gene expression as well as mutations in LM-LECs.

Dietary Sodium Nitrite Causes Similar Modifications to Splenic Inflammatory Gene Expression as a High-Fat Diet.

Sodium nitrite (NaNO2) is a widely used food additive. The present study compared the outcomes from intakes of dietary NaNO2 and a high-fat diet (HFD), and assessed their combined effects on inflammatory gene expression in the immune tissues of the mouse. In experiment I, mice were fed a standard low-fat diet (LFD) without or with NaNO2 (0.02 and 0.08%, w/w) for 11 wk. In experiment II, mice were fed an LFD without or with NaNO2 (0.02%) or HFD without or with NaNO2 (0.02%) for 11 wk. Inflammatory gene expression in the immune tissues was then measured. NaNO2 consumption and HFD feeding each resulted in increased splenic mRNAs for cell markers of neutrophils (Ngp, NE, Ly6g, Mpo) and eosinophils (Epo, Ear6), and an S100 family member (S100A8). In contrast, NaNO2 consumption and HFD feeding each resulted in decreased splenic mRNAs for cell markers of macrophages (Emr1, Itgax, CD68, CD206, Dectin-1, TLRs 4, 6, and 7), T- (CD3, CD4), NK- (CD56) and B-cells (CD20, CD40), pro- and anti-inflammatory cytokines (TNF-α, IL-6, IL-1ß, IFN-γ, IL-18, IL-10, TGF-ß), interleukin receptor antagonists (IL1ra, IL6ra) and cell adhesion molecules (ICAM-1, VCAM-1). However, dietary NaNO2 combined with HFD feeding caused no further decrease in these transcript levels compared with dietary NaNO2 alone. These NaNO2- or HFD-induced modifications were less profound in the liver and abdominal adipose tissues than in the spleen. These findings indicate that dietary NaNO2 has similar modulatory effects to HFD feeding on splenic inflammatory genes.

De novo variants in CELF2 that disrupt the nuclear localization signal cause developmental and epileptic encephalopathy.

We report heterozygous CELF2 (NM_006561.3) variants in five unrelated individuals: Individuals 1-4 exhibited developmental and epileptic encephalopathy (DEE) and Individual 5 had intellectual disability and autistic features. CELF2 encodes a nucleocytoplasmic shuttling RNA-binding protein that has multiple roles in RNA processing and is involved in the embryonic development of the central nervous system and heart. Whole-exome sequencing identified the following CELF2 variants: two missense variants [c.1558C>T:p.(Pro520Ser) in unrelated Individuals 1 and 2, and c.1516C>G:p.(Arg506Gly) in Individual 3], one frameshift variant in Individual 4 that removed the last amino acid of CELF2 c.1562dup:p.(Tyr521Ter), possibly resulting in escape from nonsense-mediated mRNA decay (NMD), and one canonical splice site variant, c.272-1G>C in Individual 5, also probably leading to NMD. The identified variants in Individuals 1, 2, 4, and 5 were de novo, while the variant in Individual 3 was inherited from her mosaic mother. Notably, all identified variants, except for c.272-1G>C, were clustered within 20 amino acid residues of the C-terminus, which might be a nuclear localization signal. We demonstrated the extranuclear mislocalization of mutant CELF2 protein in cells transfected with mutant CELF2 complementary DNA plasmids. Our findings indicate that CELF2 variants that disrupt its nuclear localization are associated with DEE.

POLR1C variants dysregulate splicing and cause hypomyelinating leukodystrophy.

OBJECTIVE: To further clarify the molecular pathogenesis of RNA polymerase III (Pol III)-related leukodystrophy caused by biallelic POLR1C variants at a cellular level and potential effects on its downstream genes. METHODS: Exome analysis and molecular functional studies using cell expression and long-read sequencing analyses were performed on 1 family with hypomyelinating leukodystrophy showing no clinical and MRI findings characteristic of Pol III-related leukodystrophy other than hypomyelination. RESULTS: Biallelic novel POLR1C alterations, c.167T>A, p.M56K and c.595A>T, p.I199F, were identified as causal variants. Functional analyses showed that these variants not only resulted in altered protein subcellular localization and decreased protein expression but also caused abnormal inclusion of introns in 85% of the POLR1C transcripts in patient cells. Unexpectedly, allelic segregation analysis in each carrier parent revealed that each heterozygous variant also caused the inclusion of introns on both mutant and wild-type alleles. These findings suggest that the abnormal splicing is not direct consequences of the variants, but rather reflect the downstream effect of the variants in dysregulating splicing of POLR1C, and potentially other target genes. CONCLUSIONS: The lack of characteristic clinical findings in this family confirmed the broad clinical spectrum of Pol III-related leukodystrophy. Molecular studies suggested that dysregulation of splicing is the potential downstream pathomechanism for POLR1C variants.

Update of the genotype and phenotype of KMT2D and KDM6A by genetic screening of 100 patients with clinically suspected Kabuki syndrome.

Kabuki syndrome is characterized by a variable degree of intellectual disability, characteristic facial features, and complications in various organs. Many variants have been identified in two causative genes, that is, lysine methyltransferase 2D (KMT2D) and lysine demethylase 6A (KDM6A). In this study, we present the results of genetic screening of 100 patients with a suspected diagnosis of Kabuki syndrome in our center from July 2010 to June 2018. We identified 76 variants (43 novel) in KMT2D and 4 variants (3 novel) in KDM6A as pathogenic or likely pathogenic. Rare variants included a deep splicing variant (c.14000-8C>G) confirmed by RNA sequencing and an 18% mosaicism level for a KMT2D mutation. We also characterized a case with a blended phenotype consisting of Kabuki syndrome, osteogenesis imperfecta, and 16p13.11 microdeletion. We summarized the clinical phenotypes of 44 patients including a patient who developed cervical cancer of unknown origin at 16 years of age. This study presents important details of patients with Kabuki syndrome including rare clinical cases and expands our genetic understanding of this syndrome, which will help clinicians and researchers better manage and understand patients with Kabuki syndrome they may encounter.

Expanding the phenotype of COL4A1-related disorders-Four novel variants.

OBJECTIVE: COL4A1 variant causes severe central nervous system (CNS) anomalies, including hydranencephaly. However, the pathogenic mechanism underlying the COL4A1 phenotype remains unclear. Here, we report de novo COL4A1 variants in four Japanese patients with typical or rare CNS involvement and exhibiting diverse phenotypes. METHODS: We identified and enrolled four patients with white matter abnormalities and cerebral structural defects suggestive of cerebrovascular disease. Genetic analysis was performed using panel sequencing. RESULTS: All the patients were perinatally asymptomatic during the infantile period but exhibited developmental delay and growth retardation later. All the patients exhibited CNS symptoms, including psychomotor disability, spastic paralysis, and epilepsy. Brain magnetic resonance imaging revealed hydranencephaly (n = 1), ventriculomegaly (n = 4) associated with cerebral hemorrhage, and atretic encephalocele (n = 1). Three patients had developed congenital cataract, while two had hematuria. We identified two COL4A1 missense variants [exon32:c.2555G > A p.(Gly852Asp), exon40:c.3407G > A p.(Gly1136Asp)] and two in frame variants [exon32:c.2603_2609delinsATCCTGA p.(Ala868_Gly870delinsAspProGlu), exon36:c.3054delinsTGTAGAT p.(Leu1018delinsPheValAsp)]. The in frame variants were associated with severe CNS anomalies, hydranencephaly, and severe ventriculomegaly. Atretic encephalocele has never been reported in individuals with COL4A1 variants. CONCLUSIONS: Our findings suggest that COL4A1 variants cause variable CNS symptoms. Association between clinical phenotypes and each COL4A1 variant would clarify their underlying etiologies.