Expanding the phenotype of biallelic loss-of-function variants in the NSUN2 gene: Description of four individuals with juvenile cataract, chronic nephritis, or brain anomaly as novel complications.

The NSUN2 gene encodes a tRNA cytosine methyltransferase that functions in the maturation of leucyl tRNA (Leu) (CAA) precursors, which is crucial for the anticodon-codon pairing and correct translation of mRNA. Biallelic loss of function variants in NSUN2 are known to cause moderate to severe intellectual disability. Microcephaly, postnatal growth retardation, and dysmorphic facial features are common complications in this genetic disorder, and delayed puberty is occasionally observed. Here, we report four individuals, two sets of siblings, with biallelic loss-of-function variants in the NSUN2 gene. The first set of siblings have compound heterozygous frameshift variants: c.546_547insCT, p.Met183Leufs*13; c.1583del, p.Pro528Hisfs*19, and the other siblings carry a homozygous frameshift variant: c.1269dup, p.Val424Cysfs*14. In addition to previously reported clinical features, the first set of siblings showed novel complications of juvenile cataract and chronic nephritis. The other siblings showed hypomyelination and simplified gyral pattern in neuroimaging. NSUN2-related intellectual disability is a very rare condition, and less than 20 cases have been reported previously. Juvenile cataract, chronic nephritis, and brain anomaly shown in the present patients have not been previously described. Our report suggests clinical diversity of NSUN2-related intellectual disability.

Methylation analysis for postpartum depression: a case control study.

BACKGROUND: Postpartum depression (PPD) is a major depressive disorder that occurs after childbirth. Objective diagnostic and predictive methods for PPD are important for early detection and appropriate intervention. DNA methylation has been recognized as a potential biomarker for major depressive disorder. In this study, we used methylation analysis and peripheral blood to search for biomarkers that could to lead to the development a predictive method for PPD. METHODS: Study participants included 36 pregnant women (18 cases and 18 controls determined after childbirth). Genome-wide DNA methylation profiles were obtained by analysis with an Infinium Human Methylation 450BeadChip. The association of DNA methylation status at each DNA methylation site with PPD was assessed using linear regression analysis. We also conducted functional enrichment analysis of PPD using The Database for Annotation, Visualization and Integrated Discovery 6.8 to explore enriched functional-related gene groups for PPD. RESULTS: In the analysis with postpartum depressed state as an independent variable, the difference in methylation frequency between the postpartum non-depressed group and the postpartum depressed group was small, and sites with genome-wide significant differences were not confirmed. After analysis by The Database for Annotation, Visualization and Integrated Discovery 6.8, we revealed four gene ontology terms, including axon guidance, related to postpartum depression. CONCLUSIONS: These findings may help with the development of an objective predictive method for PPD.

Exome sequencing of Japanese schizophrenia multiplex families supports the involvement of calcium ion channels.

BACKGROUND: Most sequencing studies of schizophrenia (SCZ) have focused on de novo genetic variants due to interpretability. However, investigating shared rare variants among patients in the same multiplex family is also important. Relatively large-scale analyses of SCZ multiplex families have been done in Caucasian populations, but whether detected variants are also pathogenic in the Japanese population is unclear because of ethnic differences in rare variants. MATERIALS AND METHODS: We performed whole-exome sequencing (WES) of 14 Japanese SCZ multiplex families. After quality control and filtering, we identified rare variants shared among affected persons within the same family. A gene ontology (GO) analysis was performed to identify gene categories possibly affected by these candidate variants. RESULTS: We found 530 variants in 486 genes as potential candidate variants from the 14 SCZ multiplex families examined. The GO analysis demonstrated significant enrichment in calcium channel activity. CONCLUSION: This study provides supporting evidence that calcium ion channel activity is involved in SCZ. WES of multiplex families is a potential means of identifying disease-associated rare variants for SCZ.

Investigation of OLIG2 as a candidate gene for schizophrenia and autism spectrum disorder.

A number of genomic mutations that are thought to be strongly involved in the development of schizophrenia (SCZ) and autism spectrum disorder (ASD) have been identified. Abnormalities involving oligodendrocytes have been reported in SCZ, and as a related gene, oligodendrocyte lineage transcription factor 2 (OLIG2) has been reported to be strongly associated with SCZ. In this study, based on the common disease-rare variant hypothesis, target sequencing of candidate genes was performed to identify rare mutations with a high effect size and the possibility that the identified mutations may increase the risks of SCZ and ASD in the Japanese population. In this study, the exon region of OLIG2 was targeted; 370 patients with SCZ and 192 with ASD were subjected to next-generation sequencing. As a result, one rare missense mutation (A33T) was detected. We used the Sanger method to validate this missense mutation with a low frequency (<1%), and then carried out a genetic association analysis involving 3299 unrelated individuals (1447 with SCZ, 380 with ASD, and 1472 healthy controls) to clarify whether A33T was associated with SCZ or ASD. A33T was not found in either case group, and in only one control. We did not find evidence that p.A33T is involved in the onset of ASD or SCZ; however, associations with this variant need to be evaluated in larger samples to confirm our results.

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.

Severe achondroplasia due to two de novo variants in the transmembrane domain of FGFR3 on the same allele: A case report.

BACKGROUND: Achondroplasia (ACH), the most common form of short-limbed skeletal dysplasia, is caused by gain-of-function mutations in the fibroblast growth factor receptor 3 (FGFR3) gene. More than 97% of patients result from a heterozygous p.G380R mutation in the FGFR3 gene. We present here a child who had two de novo variants in the FGFR3 on the same allele, a common p.G380R mutation and a novel p.S378N variant. METHODS: A 3-year-old Japanese girl born from non-consanguineous healthy parents showed more severe clinical and radiological phenotypes than classic ACH, including severe short-limbed short stature with marked ossification defects in the metaphysis and epiphysis, hydrocephalus and cervicomedullary compression due to foramen magnum stenosis, prolonged pulmonary hypoplasia, and significant delay in the gross motor development. Genomic DNA was extracted from the proband and whole-exome sequencing was performed. The variants were subsequently confirmed by Sanger sequencing. RESULTS: Mutation analysis demonstrated that the proband had p.S378N (c.1133G>A) and p.G380R (c.1138G>A) variants in the FGFR3 gene. Both variants were not detected in her parents and therefore considered de novo. An allele-specific PCR was developed in order to determine whether these mutations were on the same allele (cis) or on different alleles (trans). The c.1138G>A mutation was found in the PCR product generated with the primer for the mutant 1133A, but it was not detected in the product with the wild-type 1133G, confirming that p.S378N and p.G380R variants were located on the same allele (cis). CONCLUSION: This is the second case who had two FGFR3 variants in the transmembrane domain on the same allele. The p.S378N variant may provide an additive effect on the activating receptor with the p.G380R mutation and alter the protein function, which could be responsible for the severe phenotype of the present case.

NUS1 mutation in a family with epilepsy, cerebellar ataxia, and tremor.

We report on familial 5 epilepsy patients with autosomal dominant inheritance of a novel heterozygous NUS1 frameshift mutation. All patients had cerebellar ataxia and tremor. Three patients were diagnosed with childhood absence epilepsy, 1 patient with generalized epilepsy, and 1 patient with parkinsonism without epilepsy. Our cases and previously reported cases with deletions of chromosome 6q22 that include NUS1 share these common symptoms. In a cellular experiment, NUS1 mutation led to a substantial reduction of the protein level of NUS1. NUS1 mutation could contribute to epilepsy pathogenesis and also constitute a distinct syndromic entity with cerebellar ataxia and tremor.

Rare single-nucleotide DAB1 variants and their contribution to Schizophrenia and autism spectrum disorder susceptibility.

Disabled 1 (DAB1) is an intracellular adaptor protein in the Reelin signaling pathway and plays an essential role in correct neuronal migration and layer formation in the developing brain. DAB1 has been repeatedly reported to be associated with neurodevelopmental disorders including schizophrenia (SCZ) and autism spectrum disorders (ASD) in genetic, animal, and postmortem studies. Recently, increasing attention has been given to rare single-nucleotide variants (SNVs) found by deep sequencing of candidate genes. In this study, we performed exon-targeted resequencing of DAB1 in 370 SCZ and 192 ASD patients using next-generation sequencing technology to identify rare SNVs with a minor allele frequency <1%. We detected two rare missense mutations (G382C, V129I) and then performed a genetic association study in a sample comprising 1763 SCZ, 380 ASD, and 2190 healthy control subjects. Although no statistically significant association with the detected mutations was observed for either SCZ or ASD, G382C was found only in the case group, and in silico analyses and in vitro functional assays suggested that G382C alters the function of the DAB1 protein. The rare variants of DAB1 found in the present study should be studied further to elucidate their potential functional relevance to the pathophysiology of SCZ and ASD.

Rare genetic variants in the gene encoding histone lysine demethylase 4C (KDM4C) and their contributions to susceptibility to schizophrenia and autism spectrum disorder.

Dysregulation of epigenetic processes involving histone methylation induces neurodevelopmental impairments and has been implicated in schizophrenia (SCZ) and autism spectrum disorder (ASD). Variants in the gene encoding lysine demethylase 4C (KDM4C) have been suggested to confer a risk for such disorders. However, rare genetic variants in KDM4C have not been fully evaluated, and the functional impact of the variants has not been studied using patient-derived cells. In this study, we conducted copy number variant (CNV) analysis in a Japanese sample set (2605 SCZ and 1141 ASD cases, and 2310 controls). We found evidence for significant associations between CNVs in KDM4C and SCZ (p = 0.003) and ASD (p = 0.04). We also observed a significant association between deletions in KDM4C and SCZ (corrected p = 0.04). Next, to explore the contribution of single nucleotide variants in KDM4C, we sequenced the coding exons in a second sample set (370 SCZ and 192 ASD cases) and detected 18 rare missense variants, including p.D160N within the JmjC domain of KDM4C. We, then, performed association analysis for p.D160N in a third sample set (1751 SCZ and 377 ASD cases, and 2276 controls), but did not find a statistical association with these disorders. Immunoblotting analysis using lymphoblastoid cell lines from a case with KDM4C deletion revealed reduced KDM4C protein expression and altered histone methylation patterns. In conclusion, this study strengthens the evidence for associations between KDM4C CNVs and these two disorders and for their potential functional effect on histone methylation patterns.

Rare loss of function mutations in N-methyl-D-aspartate glutamate receptors and their contributions to schizophrenia susceptibility.

In schizophrenia (SCZ) and autism spectrum disorder (ASD), the dysregulation of glutamate transmission through N-methyl-D-aspartate receptors (NMDARs) has been implicated as a potential etiological mechanism. Previous studies have accumulated evidence supporting NMDAR-encoding genes' role in etiology of SCZ and ASD. We performed a screening study for exonic regions of GRIN1, GRIN2A, GRIN2C, GRIN2D, GRIN3A, and GRIN3B, which encode NMDAR subunits, in 562 participates (370 SCZ and 192 ASD). Forty rare variants were identified including 38 missense, 1 frameshift mutation in GRIN2C and 1 splice site mutation in GRIN2D. We conducted in silico analysis for all variants and detected seven missense variants with deleterious prediction. De novo analysis was conducted if pedigree samples were available. The splice site mutation in GRIN2D is predicted to result in intron retention by minigene assay. Furthermore, the frameshift mutation in GRIN2C and splice site mutation in GRIN2D were genotyped in an independent sample set comprising 1877 SCZ cases, 382 ASD cases, and 2040 controls. Both of them were revealed to be singleton. Our study gives evidence in support of the view that ultra-rare variants with loss of function (frameshift, nonsense or splice site) in NMDARs genes may contribute to possible risk of SCZ.