Wanshi Cai

Mutations of ANK3 identified by exome sequencing are associated with Autism susceptibility

Autism spectrum disorders (ASDs) are common neurodevelopmental disorders with a strong genetic etiology. However, due to the extreme genetic heterogeneity of ASDs, traditional approaches for gene discovery are challenging. Next‐generation sequencing technologies offer an opportunity to accelerate the identification of the genetic causes of ASDs. Here, we report the results of whole‐exome sequence in a cohort of 20 ASD patients. By extensive bioinformatic analysis, we identified novel mutations in seven genes that are implicated in synaptic function and neurodevelopment. After sequencing an additional 47 ASD samples, we identified three different missense mutations in ANK3 in four unrelated ASD patients, one of which, c.4705T>G (p.S1569A), is a de novo mutation. Given the fact that ANK3 has been shown to strongly associate with schizophrenia and bipolar disorder, our findings support an association between ANK3 mutations and ASD susceptibility and imply a shared molecular pathophysiology between ASDs and other neuropsychiatric disorders. Hum Mutat 33:1635–1638, 2012.

Dysplastic spondylolysis is caused by mutations in the diastrophic dysplasia sulfate transporter gene.

Significance Spondylolysis is a crack in part of a vertebra that occurs in 3–6% of the general population. The cracked vertebra sometimes slips forward over the vertebra below it, resulting in spondylolisthesis and lower-back pain. Although inherited spondylolysis has long been described, the genetic etiology of these disorders remains unclear. Studies of families with autosomal-dominant mutations provide a unique means to investigate the pathogenesis of spondylolysis, which can also be used as biomarkers, even during the asymptomatic period. This research identified two novel missense mutations in independent families that were located at the conserved Stas domain. Functional analyses demonstrated that sulfate uptake activities of mutant SLC26A2 were significantly reduced. This study suggests that the pathogenesis of chondrodysplasia is associated with dysplastic spondylolysis. Spondylolysis is a fracture in part of the vertebra with a reported prevalence of about 3–6% in the general population. Genetic etiology of this disorder remains unknown. The present study was aimed at identifying genomic mutations in patients with dysplastic spondylolysis as well as the potential pathogenesis of the abnormalities. Whole-exome sequencing and functional analysis were performed for patients with spondylolysis. We identified a novel heterozygous mutation (c.2286A > T; p.D673V) in the sulfate transporter gene SLC26A2 in five affected subjects of a Chinese family. Two additional mutations (e.g., c.1922A > G; p.H641R and g.18654T > C in the intron 1) in the gene were identified by screening a cohort of 30 unrelated patients with the disease. In situ hybridization analysis showed that SLC26A2 is abundantly expressed in the lumbosacral spine of the mouse embryo at day 14.5. Sulfate uptake activities in CHO cells transfected with mutant SLC26A2 were dramatically reduced compared with the wild type, confirming the pathogenicity of the two missense mutations. Further analysis of the gene–disease network revealed a convergent pathogenic network for the development of lumbosacral spine. To our knowledge, our findings provide the first identification of autosomal dominant SLC26A2 mutations in patients with dysplastic spondylolysis, suggesting a new clinical entity in the pathogenesis of chondrodysplasia involving lumbosacral spine. The analysis of the gene–disease network may shed new light on the study of patients with dysplastic spondylolysis and spondylolisthesis as well as high-risk individuals who are asymptomatic.

Evolutionary Mode and Functional Divergence of Vertebrate NMDA Receptor Subunit 2 Genes

Background Ionotropic glutamate receptors in the central nervous system play a major role in numerous brain functions including learning and memory in many vertebrate species. NR2 subunits have been regarded as rate-limiting molecules in controlling the optimal N-methyl-D-aspartate (NMDA) receptors coincidence-detection property and subsequent learning and memory function across multi-species. However, its evolutionary mode among vertebrate species remains unclear. Results With extensive analysis of phylogeny, exon structure, protein domain, paralogon and synteny, we demonstrated that two-round genome duplication generated quartet GRIN2 genes and the third-round fish-specific genome duplication generated extra copies of fish GRIN2 genes. In addition, in-depth investigation has enabled the identification of three novel genes, GRIN2C_Gg, GRIN2D-1_Ol and GRIN2D-2_Tr in the chicken, medaka and fugu genome, respectively. Furthermore, we showed functional divergence of NR2 genes mostly occurred at the first-round duplication, amino acid residues located at the N-terminal Lig_chan domain were responsible for type I functional divergence between these GRIN2 subfamilies and purifying selection has been the prominent natural pressure operating on these diversified GRIN2 genes. Conclusion and Significance These findings provide intriguing subjects for testing the 2R and 3R hypothesis and we expect it could provide new insights into the underlying evolution mechanisms of cognition in vertebrate.

Q-RRBS: a quantitative reduced representation bisulfite sequencing method for single-cell methylome analyses

Reduced representation bisulfite sequencing (RRBS) is a powerful method of DNA methylome profiling that can be applied to single cells. However, no previous report has described how PCR-based duplication-induced artifacts affect the accuracy of this method when measuring DNA methylation levels. For quantifying the effects of duplication-induced artifacts on methylome profiling when using ultra-trace amounts of starting material, we developed a novel method, namely quantitative RRBS (Q-RRBS), in which PCR-induced duplication is excluded through the use of unique molecular identifiers (UMIs). By performing Q-RRBS on varying amounts of starting material, we determined that duplication-induced artifacts were more severe when small quantities of the starting material were used. However, through using the UMIs, we successfully eliminated these artifacts. In addition, Q-RRBS could accurately detect allele-specific methylation in absence of allele-specific genetic variants. Our results demonstrate that Q-RRBS is an optimal strategy for DNA methylation profiling of single cells or samples containing ultra-trace amounts of cells.

Comparative RNA-seq analysis reveals potential mechanisms mediating the conversion to androgen independence in an LNCaP progression cell model

The androgen-independent phenotype is an important symptom of refractory prostate cancer. However, the molecular mechanisms underlying this phenotypic conversion remain unclear. Using RNA-seq analysis of androgen-dependent prostate cancer cells (LNCaP) vs. androgen-independent cancer cells (LNCaP-AI-F), we identified 788 differentially expressed genes, 315 alternative splicing events, and eight novel LNCaP-AI-F-specific fusion genes. The fusion genes EIF2AK1-ATR and GLYR1-SLC9A8 were predicted to be damaging and oncogenic. We also observed dramatic changes in androgen receptor (AR)-mediated pathway molecules, including prostate-specific antigen (PSA, a major biomarker of prostate cancer) and AR variants, as well as neuroendocrine-like (NE-like) and tumor stem cell-like characteristics, during androgen-independent phenotype progression. Our findings provide new insights into the regulatory complexities of refractory prostate cancers.

TET1 modulates H4K16 acetylation by controlling auto-acetylation of hMOF to affect gene regulation and DNA repair function

The Ten Eleven Translocation 1 (TET1) protein is a DNA demethylase that regulates gene expression through altering statue of DNA methylation. However, recent studies have demonstrated that TET1 could modulate transcriptional expression independent of its DNA demethylation activity; yet, the detailed mechanisms underlying TET1s role in such transcriptional regulation remain not well understood. Here, we uncovered that Tet1 formed a chromatin complex with histone acetyltransferase Mof and scaffold protein Sin3a in mouse embryonic stem cells by integrative genomic analysis using publicly available ChIP-seq data sets and a series of in vitro biochemical studies in human cell lines. Mechanistically, the TET1 facilitated chromatin affinity and enzymatic activity of hMOF against acetylation of histone H4 at lysine 16 via preventing auto-acetylation of hMOF, to regulate expression of the downstream genes, including DNA repair genes. We found that Tet1 knockout MEF cells exhibited an accumulation of DNA damage and genomic instability and Tet1 deficient mice were more sensitive to x-ray exposure. Taken together, our findings reveal that TET1 forms a complex with hMOF to modulate its function and the level of H4K16Ac ultimately affect gene expression and DNA repair.

Genetic landscape of papillary thyroid carcinoma in the Chinese population

Improvement in the clinical outcome of human cancers requires characterization of the genetic alterations underlying their pathogenesis. Large‐scale genomic and transcriptomic characterization of papillary thyroid carcinomas (PTCs) in Western populations has revealed multiple oncogenic drivers which are essential for understanding pathogenic mechanisms of this disease, while, so far, the genetic landscape in Chinese patients with PTC remains uncharacterized. Here, we conducted a large‐scale genetic analysis of PTCs from patients in China to determine the mutational landscape of this cancer. By performing targeted DNA amplicon and targeted RNA deep‐sequencing, we elucidated the landscape of somatic genetic alterations in 355 Chinese patients with PTC. A total of 88.7% of PTCs were found to harbor at least one candidate oncogenic driver genetic alteration. Among them, around 72.4% of the cases carried BRAF mutations; 2.8% of cases harbored RAS mutations; and 13.8% of cases were characterized with in‐frame gene fusions, including seven newly identified kinase gene fusions. TERT promoter mutations were likely to occur in a sub‐clonal manner in our PTC cohort. The prevalence of somatic genetic alterations in PTC was significantly different between our Chinese cohort and TCGA datasets for American patients. Additionally, combined analyses of genetic alterations and clinicopathologic features demonstrated that kinase gene fusion was associated with younger age at diagnosis, larger tumor size, and lymph node metastasis in PTC. With the analyses of DNA rearrangement sites of RET gene fusions in PTC, signatures of chromosome translocations related to RET fusion events were also depicted. Collectively, our results provide fundamental insight into the pathogenesis of PTC in the Chinese population. Copyright

MBRidge: an accurate and cost-effective method for profiling DNA methylome at single-base resolution

Organisms and cells, in response to environmental influences or during development, undergo considerable changes in DNA methylation on a genome-wide scale, which are linked to a variety of biological processes. Using MethylC-seq to decipher DNA methylome at single-base resolution is prohibitively costly. In this study, we develop a novel approach, named MBRidge, to detect the methylation levels of repertoire CpGs, by innovatively introducing C-hydroxylmethylated adapters and bisulfate treatment into the MeDIP-seq protocol and employing ridge regression in data analysis. A systematic evaluation of DNA methylome in a human ovarian cell line T29 showed that MBRidge achieved high correlation (R > 0.90) with much less cost (∼10%) in comparison with MethylC-seq. We further applied MBRidge to profiling DNA methylome in T29H, an oncogenic counterpart of T29s. By comparing methylomes of T29H and T29, we identified 131790 differential methylation regions (DMRs), which are mainly enriched in carcinogenesis-related pathways. These are substantially different from 7567 DMRs that were obtained by RRBS and related with cell development or differentiation. The integrated analysis of DMRs in the promoter and expression of DMR-corresponding genes revealed that DNA methylation enforced reverse regulation of gene expression, depending on the distance from the proximal DMR to transcription starting sites in both mRNA and lncRNA. Taken together, our results demonstrate that MBRidge is an efficient and cost-effective method that can be widely applied to profiling DNA methylomes.

Lysosomal storage disease in the brain: mutations of the [beta]-mannosidase gene identified in autosomal dominant nystagmus

Purpose:Genetic etiology of congenital/infantile nystagmus remains largely unknown. This study aimed to identify genomic mutations in patients with infantile nystagmus and an associated disease network.Methods:Patients with inherited and sporadic infantile nystagmus were recruited for whole-exome and Sanger sequencing. β-Mannosidase activities were measured. Gene expression, protein–protein interaction, and nystagmus-associated lysosomal storage disease (LSD) genes were analyzed.Results:A novel heterozygous mutation (c.2013G>A; p.R638H) of MANBA, which encodes lysosomal β-mannosidase, was identified in patients with autosomal-dominant nystagmus. An additional mutation (c.2346T>A; p.L749H) in MANBA was found by screening patients with sporadic nystagmus. MANBA was expressed in the pretectal nucleus of the developing midbrain, known to be involved in oculomotor and optokinetic nystagmus. Functional validation of these mutations demonstrated a significant decrease of β-mannosidase activities in the patients as well as in mutant-transfected HEK293T cells. Further analysis revealed that nystagmus is present in at least 24 different LSDs involving the brain.Conclusion:This is the first identification of MANBA mutations in patients with autosomal-dominant nystagmus, suggesting a new clinical entity. Because β-mannosidase activities are required for development of the oculomotor nervous system, our findings shed new light on the role of LSD-associated genes in the pathogenesis of infantile nystagmus.Genet Med 17 12, 971–979.

Identification of a novel missense (C7W) mutation of SOD1 in a large familial amyotrophic lateral sclerosis pedigree

Mutations of Cu-Zn superoxide dismutase (SOD1) have rarely been identified in Chinese patients with amyotrophic lateral sclerosis (ALS). We recently initiated a program to screen mutations of SOD1, TARDBP, and C9orf72 genes, the most commonly mutated genes in ALS patients in Western countries, in Chinese ALS patients. In this study, we report a novel missense SOD1 mutation with a substitution of tryptophan for cysteine at the seventh amino acid (p.C7W, traditionally named p.C6W) based on HUGO Gene Nomenclature in a familial ALS pedigree. We also found that the activities of SOD1 were significantly decreased in the C7W patient and the carriers of the family, compared with the SOD1 activities of normal family members. Compared with reported C7G and C7S patients, analysis of phenotype revealed relatively mild disease phenotypes in C7W patients, which is correlated with less deteriorated alteration in protein structure. Like those of many other familial ALS families, variable clinical phenotypes in the C7W intrafamily suggest that potential genetic modifiers may contribute to this disease.