Abstract Background Extracorporeal cardiopulmonary resuscitation (E-CPR) has been utilized as a rescue strategy for patients with refractory cardiac arrest (CA). The collapse to ECMO initiation time (CTET) has a strong correlation with survival, but decisions of initiating E-CPR are sometimes difficult to make and the opportunities for developing teamwork skills are limited. Therefore, to improve the outcome of E-CPR, we developed a comprehensive simulation-based E-CPR training program. Purpose In the present study we assessed whether the E-CPR training improved the mortality and the neurological outcome. Methods E-CPR simulation training program was carried out twice a year to the medical team using a mock vascular model since October 2017. We enrolled 177 consecutive patients who underwent E-CPR from January 2012 to January 2022. The patients were divided into two groups: the pre-simulation and the post-simulation. The outcome was CTET, 1-year mortality and favorable neurological outcomes defined as cerebral performance categories (CPC) of 1 or 2 at hospital discharge. Results There were 86 patients in the Pre-simulation group and 91 patients in the Post-simulation group. No differences were found in age, rates of witnessed CA and bystander-CPR, shockable rhythms, or acute coronary syndrome (ACS). The frequency of use of the intra-aortic balloon pump was significantly higher in the Pre-simulation group compared to the Post-simulation group (74% vs. 23%; p <0.001), while the Impella device was only used in the Post-simulation group (46%), due to its availability in our institution since October 2017. Compared to Pre-simulation group, the CTET was significantly shorter (44 minutes vs. 33 minutes; p = 0.001), and the Kaplan-Meier analysis for 1-year survival was significantly higher with the Post-simulation group (12% vs. 23%; p = 0.02 by log-rank test). The rate of favorable neurological outcome was not significantly different between the two groups. However, when patients were analyzed separately by in-hospital CA (IHCA) and out-of-hospital CA (OHCA), no significant difference was observed in IHCA, whereas OHCA demonstrated a significant reduction in both door to ECMO initiation time and CTET, leading to a significant improvement in neurological outcome (3% vs. 20%; p = 0.04). Multivariate cox proportional hazard analysis revealed that age (hazard ratio [HR], 1.22 [10 years increase]; 95% confidence interval [CI], 1.07-1.41, p = 0.004), initial shockable rhythm (HR, 0.61; 95%CI 0.41-0.91, p = 0.01), combined use of Impella (HR, 0.59; 95% CI, 0.37–0.98, p = 0.02), and CTET (HR, 1.02, 95%CI, 1.01-1.03, p <0.0001) were significantly associated with the 1-year mortality. Conclusions The E-CPR training significantly improved the collapse to ECMO time. The faster deployment of ECMO increased the 1-year survival rate in patients with refractory CA. Additionally, the use of Impella (ECPELLA) may have improved the survival rate.The effect of simulation trainingKaplan-Meier analysis

Abstract Introduction The percutaneous transcatheter left ventricular assist device, Impella, has been recently introduced in Japan for patients with drug refractory acute heart failure including cardiogenic shock (CGS). Despite its potential benefits, Impella is associated with certain limitations, including a large device size and an increased risk of complications such as bleeding, hemolysis, and renal failure. These factors may present challenges for the use of Impella in elderly patients and those with smaller body size. For elderly patients with various comorbidities, the clinical impact of invasive hemodynamic support with impella remains unclear. Objective To assess the impact of age and body mass index (BMI) on the survival of patients with CGS treated with Impella. Methods We retrospectively reviewed 133 consecutive patients, who received Impella for cardiogenic shock, including cardiac arrest, between September 2018 and January 2023 at our institute. Patients were divided into two distinct age groups: younger group (<75 years of age; n=59) and elderly group (≥75 years; n=74). We assessed rate of successful Impella weaning and the 30-day survival. Multivariate cox regression analysis was used to identify risk factors for 30-day all-cause mortality. Results Baseline patient characteristics demonstrated a mean age of 70 ±13 years with 71% male ratio. Acute coronary syndrome was diagnosed in 72% of patients, while 63% received concomitant venoarterial extracorporeal membrane oxygenation (VA-ECMO), called ECPELLA, and 41% underwent extracorporeal cardiopulmonary resuscitation (E-CPR). The elderly group exhibited a lower BMI and higher rates of comorbidities, such as hypertension and chronic kidney disease. Notably, the incidence of cardiac arrest, E-CPR, and concomitant use of VA-ECMO were significantly lower in the elderly group compared with the younger group. There were no significant differences in Impella weaning, support duration, and length of hospital stay among two groups. Kaplan-Meier analysis revealed that the 30-day survival was also similar between two groups. Univariable Cox regression analysis revealed that BMI was significantly associated with 30-day mortality along with E-CPR and the combined use of VA-ECMO. Multivariable Cox regression analysis including the age, male, BMI, E-CPR, and combined use of VA-ECMO showed that the predictive values of independent determinants for 30-day mortality were age (hazard ratio [HR] 1.05, 95% confidence interval [CI], 1.02-1.07, P<0.0001), BMI (HR 1.14, 95%CI, 1.06-1.21, P=0.0001), and VA-ECMO (HR 4.27, 95% CI, 1.92-9.50, P=0.0004). Conclusion Results suggest that age and BMI were significantly associated with increased 30-day mortality risk, indicating a counterintuitive relationship between BMI and mortality in this population.OutcomesMultivariate Cox Regression

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.

Abstract Introduction Veno-arterial extracorporeal membrane oxygenation (VA-ECMO) is beneficial for patients with refractory cardiac arrest and cardiogenic shock. Despite its benefits, VA-ECMO increases left ventricular (LV) afterload and causes pulmonary edema and LV distension. Recently, a transcatheter left ventricular assist device, Impella has been introduced and potential benefits of the concomitant use of VA-ECMO and Impella (ECPELLA) are expected. However, with the concomitant increase in complications, the utility of ECPELLA remains controversial and unsettled. Objective This single-site cohort study aimed to assess the impact of ECPELLA compared to VA-ECMO with or without intra-aortic balloon pump (IABP) in patients with refractory cardiogenic shock, including cardiac arrest, who received VA-ECMO treatment. Methods We retrospectively reviewed 245 consecutive patients, who underwent VA-ECMO treatment between January 2012 and December 2021 at our institute. Seventeen patients for difficult weaning from cardiopulmonary bypass (n = 15) and as a backup during high-risk transcatheter aortic valve implantation (n = 2) were excluded. The patients were divided into two groups, ECEPLLA (n = 64) and VA-ECMO with or without IABP (n = 164). We performed propensity score analysis with 1:1 score matching using dependent variables of age, the rate of male gender, acute coronary syndrome, out-of-hospital and in-hospital cardiac arrest, and extracorporeal cardiopulmonary resuscitation. We evaluated hemodynamic data, safety profiles, and mortality. Results Following matching, 58 ECPELLA and 58 VA-ECMO patients were included for analysis. No significant differences in the adjusted factors and comorbidities were found between the two groups, except for a significantly higher prevalence of chronic kidney disease in the VA-ECMO group compared to the ECPELLA group. Seventy-one percent of patients received concomitant use of IABP in the VA-ECMO group. During the first 72 hours following the initiation of VA-ECMO, the ECPELLA group received a significantly higher total mechanical circulatory support flow and correspondingly less administration of catecholamines compared with the VA-ECMO group. There were no statistical differences in safety profiles, except for hemolysis and embolic cerebral infarction, between the treatment groups. Kaplan-Meier analysis demonstrated that the 30-day and 1-year survival rates were significantly higher in the ECPELLA group than in the VA-ECMO group. Multivariate cox proportional hazard analysis revealed that age (hazard ratio [HR], 1.36 [10 years increase]; 95% confidence interval [CI], 1.15–1.62, p < 0.001), lactate level at ER (HR 1.07; 95% CI 1.03–1.12, p = 0.001), and ECPELLA (HR, 0.47, 95% CI, 0.30–0.73, p = 0.008) were significantly associated with the 1-year mortality. Conclusion ECPELLA was associated with improvement of short- and long-term mortality in patients with refractory cardiogenic shock who received VA-ECMO.Total MCS flow index and VISKaplan-Meier for 1-year mortality

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.

PurposeThis study aimed to undertake a multidisciplinary characterization of the phenotype associated with SOX11 variants.MethodsIndividuals 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.ResultsWe 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.ConclusionSOX11 syndrome is a distinct clinical entity with characteristic clinical features and episignature differentiating it from BAFopathies.

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.

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.

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.

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.

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. 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). 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. 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.

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.

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.

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.

BACKGROUND:Variants in the type IV collagen gene (COL4A1/2) cause early-onset cerebrovascular diseases. Most individuals are diagnosed postnatally, and the prenatal features of individuals with COL4A1/2 variants remain unclear.METHODS:We examined COL4A1/2 in 218 individuals with suspected COL4A1/2-related brain defects. Among those arising from COL4A1/2 variants, we focused on individuals showing prenatal abnormal ultrasound findings and validated their prenatal and postnatal clinical features in detail.RESULTS:Pathogenic COL4A1/2 variants were detected in 56 individuals (n=56/218, 25.7%) showing porencephaly (n=29), schizencephaly (n=12) and others (n=15). Thirty-four variants occurred de novo (n=34/56, 60.7%). Foetal information was available in 47 of 56 individuals, 32 of whom (n=32/47, 68.1%) had one or more foetal abnormalities. The median gestational age at the detection of initial prenatal abnormal features was 31 weeks of gestation. Only 14 individuals had specific prenatal findings that were strongly suggestive of features associated with COL4A1/2 variants. Foetal ventriculomegaly was the most common initial feature (n=20/32, 62.5%). Posterior fossa abnormalities, including Dandy-Walker malformation, were observed prenatally in four individuals. Regarding extrabrain features, foetal growth restriction was present in 16 individuals, including eight individuals with comorbid ventriculomegaly.CONCLUSIONS:Prenatal observation of ventriculomegaly with comorbid foetal growth restriction should prompt a thorough ultrasound examination and COL4A1/2 gene testing should be considered when pathogenic variants are strongly suspected.

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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.

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 m 2 . 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.

Background Genetic testing has enabled the diagnosis of multiple congenital anomalies and/or intellectual disabilities. However, because of the phenotypic variability in these disorders, selection of an appropriate genetic test can be difficult and complex. For clinical examination, particularly in clinical facilities, a simple and standardized system is needed. Methods We compared microarray comparative genomic hybridization and clinical exome sequencing with regard to diagnostic yield, cost, and time required to reach a definitive diagnosis. After first performing G-banding for 200 patients with multiple congenital anomalies and/or intellectual disability, as a subsequent genetic test, microarray and clinical exome sequencing were compared with regard to diagnostic yield, cost, and time required. Results There was no obvious difference in the diagnostic rate between the two methods; however, clinical exome sequencing was superior in terms of cost and time. In addition, clinical exome sequencing could sufficiently identify copy number variants, and even smaller copy number variants could be identified. Conclusions Clinical exome sequencing should be implemented earlier as a genetic test for undiagnosed patients with multiple congenital anomalies and/or intellectual disabilities. Our results can be used to establish inspection methods in clinical facilities.

OBJECTIVE:Neurodevelopmental disorders (NDDs) often associate with epilepsy or craniofacial malformations. Recent large-scale DNA analyses identified hundreds of candidate genes for NDDs, but a large portion of the cases still remain unexplained. We aimed to identify novel candidate genes for NDDs.METHODS:We performed exome sequencing of 95 patients with NDDs including 51 with trigonocephaly and subsequent targeted sequencing of additional 463 NDD patients, functional analyses of variant in vitro, and evaluations of autism spectrum disorder (ASD)-like phenotypes and seizure-related phenotypes in vivo.RESULTS:We identified de novo truncation variants in nine novel genes; CYP1A1, C14orf119, FLI1, CYB5R4, SEL1L2, RAB11FIP2, ZMYND8, ZNF143, and MSX2. MSX2 variants have been described in patients with cranial malformations, and our present patient with the MSX2 de novo truncation variant showed cranial meningocele and partial epilepsy. MSX2 protein is known to be ubiquitinated by an E3 ubiquitin ligase PJA1, and interestingly we found a PJA1 hemizygous p.Arg376Cys variant recurrently in seven Japanese NDD patients; five with trigonocephaly and one with partial epilepsy, and the variant was absent in 886 Japanese control individuals. Pja1 knock-in mice carrying p.Arg365Cys, which is equivalent to p.Arg376Cys in human, showed a significant decrease in PJA1 protein amount, suggesting a loss-of-function effect of the variant. Pja1 knockout mice displayed moderate deficits in isolation-induced ultrasonic vocalizations and increased seizure susceptibility to pentylenetetrazole.INTERPRETATION:These findings propose novel candidate genes including PJA1 and MSX2 for NDDs associated with craniofacial abnormalities and/or epilepsy.