Role of epigenetic m6 A RNA methylation in vascular development: mettl3 regulates vascular development through PHLPP2/mTOR-AKT signaling.

N6 -methyladenosine (m6A) methylation is the most prevalent RNA modification, and it emerges as an important regulatory mechanism of gene expression involved in many cellular and biological processes. However, the role of m6 A methylation in vascular development is not clear. The m6 A RNA methylation is regulated by dynamic interplay among methyltransferases, binding proteins, and demethylases. Mettl3 is a member of the mettl3-mettl14 methyltransferase complex, referred to as writers that catalyze m6A RNA methylation. Here, we used CRISPR-Cas9 genome editing to develop two lines of knockout (KO) zebrafish for mettl3. Heterozygous mettl3+/- KO embryos show defective vascular development, which is directly visible in fli-EGFP and flk-EGFP zebrafish. Alkaline phosphatase staining and whole mount in situ hybridization with cdh5, and flk markers demonstrated defective development of intersegmental vessels (ISVs), subintestinal vessels (SIVs), interconnecting vessels (ICVs) and dorsal longitudinal anastomotic vessels (DLAV) in both heterozygous mettl3+/- and homozygous mettl3-/- KO zebrafish embryos. Similar phenotypes were observed in zebrafish embryos with morpholino knockdown (KD) of mettl3; however, the vascular defects were rescued fully by overexpression of constitutively active AKT1. KD of METTL3 in human endothelial cells inhibited cell proliferation, migration, and capillary tube formation. Mechanistically, mettl3 KO and KD significantly reduced the levels of m6 A RNA methylation, and AKT phosphorylation (S473) by an increase in the expression of phosphatase enzyme PHLPP2 and reduction in the phosphorylation of mTOR (S2481), a member of the phosphatidylinositol 3-kinase-related kinase family of protein kinases. These data suggest that m6 A RNA methylation regulates vascular development via PHLPP2/mTOR-AKT signaling.

Long noncoding RNA ANRIL regulates endothelial cell activities associated with coronary artery disease by up-regulating CLIP1, EZR, and LYVE1 genes.

Coronary artery disease (CAD) is the leading cause of death worldwide. Long noncoding RNAs (lncRNAs) are a class of noncoding transcripts of > 200 nucleotides and are increasingly recognized as playing functional roles in physiology and disease. ANRIL is an lncRNA gene mapped to the chromosome 9p21 genetic locus for CAD identified by the first series of genome-wide association studies (GWAS). However, ANRIL's role in CAD and the underlying molecular mechanism are unknown. Here, we show that the major ANRIL transcript in endothelial cells (ECs) is DQ485454 with a much higher expression level in ECs than in THP-1 monocytes. Of note, DQ485454 expression was down-regulated in CAD coronary arteries compared with non-CAD arteries. DQ485454 overexpression significantly reduced monocyte adhesion to ECs, transendothelial monocyte migration (TEM), and EC migration, which are critical cellular processes involved in CAD initiation, whereas siRNA-mediated ANRIL knockdown (KD) had the opposite effect. Microarray and follow-up quantitative RT-PCR analyses revealed that the ANRIL KD down-regulated expression of AHNAK2, CLIP1, CXCL11, ENC1, EZR, LYVE1, WASL, and TNFSF10 genes and up-regulated TMEM100 and TMEM106B genes. Mechanistic studies disclosed that overexpression of CLIP1, EZR, and LYVE1 reversed the effects of ANRIL KD on monocyte adhesion to ECs, TEM, and EC migration. These findings indicate that ANRIL regulates EC functions directly related to CAD, supporting the hypothesis that ANRIL is involved in CAD pathogenesis at the 9p21 genetic locus and identifying a molecular mechanism underlying lncRNA-mediated regulation of EC function and CAD development.

Splice variants of lncRNA RNA ANRIL exert opposing effects on endothelial cell activities associated with coronary artery disease.

Each gene typically has multiple alternatively spliced transcripts. Different transcripts are assumed to play a similar biological role; however, some transcripts may simply lose their function due to loss of important functional domains. Here, we show that two different transcripts of lncRNA gene ANRIL associated with coronary artery disease (CAD) play antagonizing roles against each other. We previously reported that DQ485454, the short transcript, is downregulated in coronary arteries from CAD patients, and reduces monocyte adhesion to endothelial cells (ECs) and transendothelial monocyte migration (TEM). Interestingly, the longest transcript NR_003529 is significantly upregulated in coronary arteries from CAD patients. Overexpression of ANRIL transcript NR_003529 increases monocyte adhesion to ECs and TEM, whereas knockdown of NR_003529 expression reduces monocyte adhesion to ECs and TEM. Much more dramatic effects were observed for the combination of overexpression of NR_003529 and knockdown of DQ485454 or the combination of knockdown of NR_003529 and overexpression of DQ485454. The antagonizing effects of ANRIL transcripts NR_003529 and DQ485454 were associated with their opposite effects on expression of downstream target genes EZR, CXCL11 or TMEM106B. Our results demonstrate that different transcripts of lncRNA can exert antagonizing effects on biological functions, thereby providing important insights into the biology of lncRNA. The data further support the hypothesis that ANRIL is the causative gene at the 9p21 CAD susceptibility locus.

Analysis of causal effect of APOA5 variants on premature coronary artery disease.

Apolipoprotein A5 (APOA5) regulates the metabolisms of triglyceride and HDL. APOA5 variants have been linked to coronary artery disease (CAD), but their causal roles are not well studied yet. This study aims to identify the causal effects of APOA5 variants on premature CAD. Sequencing analysis of APOA5 in 128 premature, familiar CAD patients from GeneQuest identified 11 genomic variants, including p.S19W (rs3135506). SKAT analysis showed that all sequenced variants, in aggregate, significantly increased the risk of premature CAD (P-skat = 0.037). Individually, the p.S19W variant was significantly associated with risk of premature CAD (OR = 2.30, P = 0.008) in an independent set of 342 premature CAD patients and 537 controls after adjusting for covariates of sex, age, hypertension, body mass index, triglycerides (TGs), and total, LDL-, and HDL-cholesterol levels. Meanwhile, p.S19W significantly correlated with HDL-C levels (P = 0.048) and TG levels (P = 0.025). Mediation analysis yielded a mediation effect of p.S19W on risk of premature CAD through HDL-C (OR = 0.98, P = 0.040) and TG (OR = 0.98, P = 0.042), suggesting a causal relationship between p.S19W and premature CAD partially through its effects on HDL-C and TG levels. These results suggest that APOA5 variation regulates TG and HDL levels, thus displaying a causal role in the development of CAD.

Knockout of ush2a gene in zebrafish causes hearing impairment and late onset rod-cone dystrophy.

Most cases of Usher syndrome type II (USH2) are due to mutations in the USH2A gene. There are no effective treatments or ideal animal models for this disease, and the pathological mechanisms of USH2 caused by USH2A mutations are still unknown. Here, we constructed a ush2a knockout (ush2a-/-) zebrafish model using TALEN technology to investigate the molecular pathology of USH2. An early onset auditory disorder and abnormal morphology of inner ear stereocilia were identified in the ush2a-/- zebrafish. Consequently, the disruption of Ush2a in zebrafish led to a hearing impairment, like that in mammals. Electroretinography (ERG) test indicated that deletion of Ush2a affected visual function at an early stage, and histological analysis revealed that the photoreceptors progressively degenerated. Rod degeneration occurred prior to cone degeneration in ush2a-/- zebrafish, which is consistent with the classical description of the progression of retinitis pigmentosa (RP). Destruction of the outer segments (OSs) of rods led to the down-regulation of phototransduction cascade proteins at late stage. The expression of Ush1b and Ush1c was up-regulated when Ush2a was null. We also found that disruption of fibronectin assembly at the retinal basement membrane weakened cell adhesion in ush2a-/- mutants. In summary, for the first time, we generated a ush2a knockout zebrafish line with auditory disorder and retinal degeneration which mimicked the symptoms of patients, and revealed that disruption of fibronectin assembly may be one of the factors underlying RP. This model may help us to better understand the pathogenic mechanism and find treatment for USH2 in the future.

Whole exome sequencing identifies a novel NRL mutation in a Chinese family with autosomal dominant retinitis pigmentosa.

PURPOSE: To investigate the genetic basis and its relationship to the clinical manifestations in a four generation Chinese family with autosomal dominant retinitis pigmentosa. METHODS: Ophthalmologic examinations including fundus photography, fundus autofluorescence imaging, fundus fluorescein angiography, optical coherence tomography, and a best-corrected visual acuity test were performed to define the clinical features of the patients. We extracted the genomic DNA from peripheral blood samples. The proband's genomic DNA was submitted to the whole exome sequencing. RESULTS: Whole exome sequencing and the subsequent data analysis detected six candidate mutations in the proband of this pedigree. The novel c.146 C>T mutation in NRL was found to be the only mutation that co-segregated with the disease in this pedigree. This mutation resulted in a substitution of proline by a leucine at position 49 of NRL protein (p.P49L). Most importantly, the proline residue at position 49 of NRL is highly conserved from zebrafish to humans. The c.146 C>T mutation was not observed in 200 control individuals. What's more, we performed the luciferase activity assay to prove that this mutation we detected alters the NRL protein function. CONCLUSIONS: The c.146 C>T mutation in NRL gene causes autosomal dominant retinitis pigmentosa for this family. Our finding not only expands the mutation spectrum of NRL, but also demonstrates that whole-exome sequencing is a powerful strategy to detect causative genes and mutations in RP patients. This technique may provide a precise diagnosis for rare heterogeneous monogenic disorders such as RP.

Comparative gene expression analysis between coronary arteries and internal mammary arteries identifies a role for the TES gene in endothelial cell functions relevant to coronary artery disease.

Coronary artery disease (CAD) is the leading cause of death worldwide. It has been established that internal mammary arteries (IMA) are resistant to the development of atherosclerosis, whereas left anterior descending (LAD) coronary arteries are athero-prone. The contrasting properties of these two arteries provide an innovative strategy to identify the genes that play important roles in the development of atherosclerosis. We carried out microarray analysis to identify genes differentially expressed between IMA and LAD. Twenty-nine genes showed significant differences in their expression levels between IMA and LAD, which included the TES gene encoding Testin. The role of TES in the cardiovascular system is unknown. Here we show that TES is involved in endothelial cell (EC) functions relevant to atherosclerosis. Western blot analysis showed higher TES expression in IMA than in LAD. Reverse transcription polymerase chain reaction and western blot analyses showed that TES was consistently and markedly down-regulated by more than 6-fold at both mRNA and protein levels in patients with CAD compared with controls without CAD (P= 0.000049). The data suggest that reduced TES expression is associated with the development of CAD. Knockdown of TES expression by small-interfering RNA promoted oxidized-LDL-mediated monocyte adhesion to ECs, EC migration and the transendothelial migration of monocytes, while the over-expression of TES in ECs blunted these processes. These results demonstrate association between reduced TES expression and CAD, establish a novel role for TES in EC functions and raise the possibility that reduced TES expression increases susceptibility to the development of CAD.

HSF4 is involved in DNA damage repair through regulation of Rad51.

Heat shock factor protein 4 (HSF4) is expressed exclusively in the ocular lens and plays a critical role in the lens formation and differentiation. Mutations in the HSF4 gene lead to congenital and senile cataract. However, the molecular mechanisms causing this disease have not been well characterized. DNA damage in lens is a crucial risk factor in senile cataract formation, and its timely repair is essential for maintaining the lens' transparency. Our study firstly found evidence that HSF4 contributes to the repair of DNA strand breaks. Yet, this does not occur with cataract causative mutations in HSF4. We verify that DNA damage repair is mediated by the binding of HSF4 to a heat shock element in the Rad51 promoter, a gene which assists in the homologous recombination (HR) repair of DNA strand breaks. HSF4 up-regulates Rad51 expression while mutations in HSF4 fail, and DNA does not get repaired. Camptothecin, which interrupts the regulation of Rad51 by HSF4, also affects DNA damage repair. Additionally, with HSF4 knockdown in the lens of Zebrafish, DNA damage was observed and the protein level of Rad51 was significantly lower. Our study presents the first evidence demonstrating that HSF4 plays a role in DNA damage repair and may contribute a better understanding of congenital cataract formation.

Statistical and Functional Studies Identify Epistasis of Cardiovascular Risk Genomic Variants From Genome-Wide Association Studies.

Background Epistasis describes how gene-gene interactions affect phenotypes, and could have a profound impact on human diseases such as coronary artery disease (CAD). The goal of this study was to identify gene-gene interactions in CAD using an easily generalizable multi-stage approach. Methods and Results Our forward genetic approach consists of multiple steps that combine statistical and functional approaches, and analyze information from global gene expression profiling, functional interactions, and genetic interactions to robustly identify gene-gene interactions. Global gene expression profiling shows that knockdown of ANRIL (DQ485454) at 9p21.3 GWAS (genome-wide association studies) CAD locus upregulates TMEM100 and TMEM106B. Functional studies indicate that the increased monocyte adhesion to endothelial cells and transendothelial migration of monocytes, 2 critical processes in the initiation of CAD, by ANRIL knockdown are reversed by knockdown of TMEM106B, but not of TMEM100. Furthermore, the decreased monocyte adhesion to endothelial cells and transendothelial migration of monocytes induced by ANRIL overexpression was reversed by overexpressing TMEM106B. TMEM106B expression was upregulated by >2-fold in CAD coronary arteries. A significant association was found between variants in TMEM106B (but not in TMEM100) and CAD (P=1.9×10-8). Significant gene-gene interaction was detected between ANRIL variant rs2383207 and TMEM106B variant rs3807865 (P=0.009). A similar approach also identifies significant interaction between rs6903956 in ADTRP and rs17465637 in MIA3 (P=0.005). Conclusions We demonstrate 2 pairs of epistatic interactions between GWAS loci for CAD and offer important insights into the genetic architecture and molecular mechanisms for the pathogenesis of CAD. Our strategy has broad applicability to the identification of epistasis in other human diseases.

ADTRP regulates TFPI expression via transcription factor POU1F1 involved in coronary artery disease.

Genomic variants in both ADTRP and TFPI genes are associated with risk of coronary artery disease (CAD). ADTRP regulates TFPI expression and endothelial cell functions involved in the initiation of atherosclerotic CAD. ADTRP also specifies primitive myelopoiesis and definitive hematopoiesis by upregulating TFPI expression. However, the underlying molecular mechanism is unknown. Here we show that transcription factor POU1F1 is the key by which ADTRP regulates TFPI expression. Luciferase reporter assays, chromatin-immunoprecipitation (ChIP) and electrophoretic mobility shift assay (EMSA) in combination with analysis of large and small deletions of the TFPI promoter/regulatory region were used to identify the molecular mechanism by which ADTRP regulates TFPI expression. Genetic association was assessed using case-control association analysis and phenome-wide association analysis (PhenGWA). ADTRP regulates TFPI expression at the transcription level in a dose-dependent manner. The ADTRP-response element was localized to a 50 bp region between -806 bp and -756 bp upstream of TFPI transcription start site, which contains a binding site for POU1F1. Deletion of POU1F1-binding site or knockdown of POU1F1 expression abolished ADTRP-mediated transcription of TFPI. ChIP and EMSA demonstrated that POU1F1 binds to the ADTRP response element. Genetic analysis identified significant association between POU1F1 variants and risk of CAD. PhenGWA identified other phenotypic traits associated with the ADTRP-POU1F1-TFPI axis such as lymphocyte count (ADTRP), waist circumference (TFPI), and standing height (POU1F1). These data identify POU1F1 as a transcription factor that regulates TFPI transcription in response to ADTRP, and link POU1F1 variants to risk of CAD for the first time.

Microarray analysis of cardiovascular diseases.

Microarray analysis is a powerful technique for high-throughput, global transcriptonomic profiling of gene expression. It holds great promise for analyzing the genetic and molecular bases of cardiovascular diseases and various other complex diseases and permits the analysis of thousands of genes simultaneously, both in diseased and nondiseased tissues and/or cell lines. Microarrays or microchips are made by depositing spots of DNA or oligonucleotides representing thousands of genes on a solid support such as a coated glass surface, and can allow the comparison of gene expression patterns in any two samples. Total RNA is isolated from the tissue or cells of interest, converted to cDNA and then cRNA labeled with biotin, and hybridized to the chips. Hybridization signals are then quantified and compared among different samples. We used oligonucleotide microarrays to obtain an unbiased assessment of expression levels of thousands of genes simultaneously in normal and diseased coronary arteries. Fifty-six genes showed differential expression in atherosclerotic coronary artery tissues, and 49 of them represent new linked genes for coronary artery disease. These studies can generate novel hypotheses relating to the pathologies of disease and further studies with animal models, molecular biology, cell biology, and biochemistry will validate these hypotheses and provide novel insights into the pathogenesis of disease.

Identification of three mutations in the MVK gene in six patients associated with disseminated superficial actinic porokeratosis.

Porokeratosis is recognized as a heterogenously inherited epidermal keratinization disorder. Disseminated superficial actinic porokeratosis (DSAP) is considered to be the most common form of porokeratosis and is characterized by multiple, small keratotic lesions on sun-exposed areas of body. MVK has been reported to be the main candidate gene associated with DSAP. In this study, six sporadic cases of DSAP were clinically characterized. Direct DNA sequencing analysis of the whole coding regions of MVK detected three MVK missense mutations, and two were novel for DSAP: c.31C>T (P11S) and c.1004G>A (G335D). The three mutant MVK proteins were less stable than the wild type protein in different degrees. Mutation G335D also resulted in the misfolding of the ATP binding domain. This study extends the mutation spectrum of MVK. MVK protein stability and correct folding might be the molecular mechanism causing DASP. A 50% probability of detecting a MVK mutation in six DSAP sporadic cases indicated that the MVK gene was useful for clinical characterization, genetic counseling and prenatal diagnosis of DSAP.

pVHL interacts with Ceramide kinase like (CERKL) protein and ubiquitinates it for oxygen dependent proteasomal degradation.

Mutations of Ceramide kinase-like (CERKL) gene are associated with retinitis pigmentosa (RP), an inherited degenerative eye disease. CERKL encodes an antioxidant protein which is critical to photoreceptor survival, its deficiency causes retinal degeneration as a result of oxidative damage. However, the regulation of CERKL in response to oxidative stress, and its contribution to photoreceptor survival remain unclear. pVHL, the substrate receptor of RING finger-type SCF like ECV ubiquitin ligase, binds and ubiquitinates a number of hydroxylated proteins for proteasomal degradation. Due to hydroxylated proteins which are modified by PHD1-3, pVHL dependent ubiquitin-proteasomal degradation pathway is blocked by PHD1-3 inhibitors (e.g. hypoxia or oxidative stress). In this study, we identified pVHL as an important regulator of CERKL. Western blot and in vivo ubiquitination assays showed hypoxia up-regulates CERKL at protein level by down-regulating its poly-ubiquitination. By Co-IP and domain mapping studies, we found CERKL complexes with ECV ligase via pVHL. Through overexpression and small RNA interference analysis, we demonstrated pVHL ubiquitinates CERKL for proteasomal degradation. Additionally, our work showed that the oxygen sensors PHD1 and PHD3 are involved in CERKL degradation. Collectively, our results indicated that pVHL interacts with CERKL and ubiquitinates it for oxygen dependent proteasomal degradation.

Proteomic approach to coronary atherosclerosis shows ferritin light chain as a significant marker: evidence consistent with iron hypothesis in atherosclerosis.

Coronary artery disease (CAD) is the leading cause of mortality and morbidity in developed nations. We hypothesized that CAD is associated with distinct patterns of protein expression in the coronary arteries, and we have begun to employ proteomics to identify differentially expressed proteins in diseased coronary arteries. Two-dimensional (2-D) gel electrophoresis of proteins and subsequent mass spectrometric analysis identified the ferritin light chain as differentially expressed between 10 coronary arteries from patients with CAD and 7 coronary arteries from normal individuals. Western blot analysis indicated significantly increased expression of the ferritin light chain in the diseased coronary arteries (1.41 vs. 0.75; P = 0.01). Quantitative real-time PCR analysis showed that expression of ferritin light chain mRNA was decreased in diseased tissues (0.70 vs. 1.17; P = 0.013), suggesting that increased expression of ferritin light chain in CAD coronary arteries may be related to increased protein stability or upregulation of expression at the posttranscriptional level in the diseased tissues. Ferritin light chain protein mediates storage of iron in cells. We speculate that increased expression of the ferritin light chain may contribute to pathogenesis of CAD by modulating oxidation of lipids within the vessel wall through the generation of reactive oxygen species. Our results provide in situ proteomic evidence consistent with the "iron hypothesis," which proposes an association between excessive iron storage and a high risk of CAD. However, it is also possible that the increased ferritin expression in diseased coronary arteries is a consequence, rather than a cause, of CAD.

Identification of new genes differentially expressed in coronary artery disease by expression profiling.

Genetic factors increase the risk to coronary artery disease (CAD). To date, a limited number of genes that potentially contribute to development of CAD have been identified. In this study, we have performed large-scale gene expression analysis of approximately 12,000 human genes in nine severely atherosclerotic and six nonatherosclerotic human coronary arteries using oligonucleotide microarrays. Fifty-six genes showed differential expression in atherosclerotic coronary artery tissues; expression of 55 genes was increased in atherosclerotic coronary arteries, whereas only one gene, GST, encoding a reducing agent, showed downregulated expression. The expression data of selected genes were validated by quantitative RT-PCR analysis as well as immunostaining. The associations of 49 genes with CAD appear to be novel, and they include genes encoding ICAM-2, PIM-2, ECGF1, fusin, B cell activator (BL34, GOS8), Rho GTPase activating protein-4, retinoic acid receptor responder, beta2-arrestin, membrane aminopeptidase, cathepsins K and H, MIR-7, TNF-alpha-induced protein 2 (B94), and flavocytochrome 558. In conclusion, we have identified 56 genes whose expression is associated with CAD, and 49 of them may represent new genes linked to CAD.

A novel molecular diagnostic marker for familial and early-onset coronary artery disease and myocardial infarction in the LRP8 gene.

BACKGROUND: Many single-nucleotide polymorphisms have been associated with coronary artery disease (CAD)/myocardial infarction (MI) by genome-wide association studies, but the diagnostic value of these variants is limited. Functional single-nucleotide polymorphism R952Q in LRP8 is associated with familial and early-onset CAD/MI. The objective of this study is to test whether fine mapping and haplotype analysis for single-nucleotide polymorphisms flanking R952Q may identify a haplotype that may serve as a molecular diagnostic marker for familial and early-onset CAD/MI. METHODS AND RESULTS: Five single-nucleotide polymorphisms (rs7546246, rs2297660, rs3737983, R952Q, and rs5177) were genotyped and analyzed in GeneQuest (381 patients with familial, early-onset CAD and 183 patients with MI versus 560 controls) and the Italian population (248 patients with familial MI versus 308 controls). One novel risk haplotype, TACGC, was found only in patients with CAD and MI but not in controls. It was significantly associated with CAD (P=7.4×10(-7)) and MI (P=2.2×10(-9)) in GeneQuest. The finding was replicated in the Italian cohort (P=0.041). Sib-transmission disequilibrium test analysis showed a significant association between haplotype TACGC and CAD in GeneQuest II (P=0.039). Haplotype TACGC was not present in a South Korean population of 611 patients with CAD and 294 normal controls. TACGC/TACGC homozygotes tended to develop CAD/MI earlier and showed higher low-density lipoprotein cholesterol levels than heterozygotes (P<0.05). CONCLUSIONS: The rare haplotype TACGC in LRP8 confers a significant risk of familial, early-onset CAD/MI. Because the risk haplotype exists only in patients with familial and early-onset CAD/MI, we propose that it may be a molecular diagnostic marker for diagnosis of familial, early-onset CAD/MI in some white populations.

Mutations in ABCB6 cause dyschromatosis universalis hereditaria.

Dyschromatosis universalis hereditaria (DUH) is a pigmentary genodermatosis characterized by a mixture of hyperpigmented and hypopigmented macules distributed randomly over the body. No causative genes have been reported to date. In this study, we investigated a large five-generation Chinese family with DUH. After excluding the two known DUH loci, we performed genome-wide linkage analysis and identified a DUH locus on chromosome 2q33.3-q36.1 with a maximum LOD score of 3.49 with marker D2S2382. Exome sequencing identified a c.1067T>C (p.Leu356Pro) mutation in exon 3 of ABCB6 (ATP-binding cassette subfamily B, member 6) in the DUH family. Two additional missense mutations, c.508A>G (p.Ser170Gly) in exon 1 and c.1736G>A (p.Gly579Glu) in exon 12 of ABCB6, were found in two out of six patients by mutational screening using sporadic DUH patients. Immunohistologic examination in biopsy specimens showed that ABCB6 is expressed in the epidermis and had a diffuse cytoplasmic distribution. Examination of subcellular localization of wild-type ABCB6 in a B16 mouse melanoma cell line revealed that it is localized to the endosome-like compartment and dendrite tips, whereas disease-causing mutations of ABCB6 resulted in its retention in the Golgi apparatus. Our studies identified ABCB6 as the first pathogenic gene associated with DUH. These findings suggest that ABCB6 may be a physiological factor for skin pigmentation.

Mutation p.Leu354Pro in EDA causes severe hypohidrotic ectodermal dysplasia in a Chinese family.

X-linked recessive hypohidrotic ectodermal dysplasia (XLHED) is characterized by the defective morphogenesis of teeth, hair, and eccrine sweat glands. It is associated with mutations in the EDA gene. Up to now, more than 100 mutations in the EDA gene have been reported to cause XLHED. The product of EDA gene is a trimeric type II transmembrane protein that belongs to the tumor necrosis factor (TNF) family of ligands. In this study, we identified a Chinese family with XLHED. Direct DNA sequencing of the whole coding region of EDA revealed a novel missense mutation, p.Leu354Pro in a patient affected with XLHED. This mutation was not found in either unaffected male individuals of the family or 168 normal controls. The substitution of Leu354 with Pro was found to be located in the TNF-like domain of EDA and may influence the epithelial signaling pathway required for the normal ectodermal development through altering the topology of EDA. Our finding broadens the spectrum of EDA mutations and may help to understand the molecular basis of XLHED and aid genetic counseling.

A novel SCN1A missense mutation causes generalized epilepsy with febrile seizures plus in a Chinese family.

Generalized epilepsy with febrile seizures plus (GEFS(+)) is a common familial epilepsy syndrome, which generally develops in childhood. GEFS(+) is caused by mutations in the sodium-channel α1-subunit (SCN1A). In this report, we investigated a Chinese family with an autosomal dominant form of GEFS(+). The affected GEFS(+) patients in this family displayed significant inter-family clinical heterogeneity. Linkage analysis localized the disease-causing gene to chromosome 2q24, where SCN1A is located. Furthermore, DNA sequencing of the whole coding region of SCN1A revealed a novel heterozygous nucleotide substitution (c.577C>T) causing a missense mutation (p.L193F) in the S3 segment of SCN1A domain D1. Our results expand the spectrum of SCN1A mutations and provide novel insights between the SCN1A mutations and the clinical variations of GEFS(+).