Free sialic acid storage disease without sialuria.

We performed high-resolution in vitro proton nuclear magnetic resonance spectroscopy on cerebrospinal fluid and urine samples of 44 patients with leukodystrophies of unknown cause. Free sialic acid concentration was increased in cerebrospinal fluid of two siblings with mental retardation and mild hypomyelination. By contrast, urinary excretion of free sialic acid in urine was normal on repeated testing by two independent methods. Both patients were homozygous for the K136E mutation in SLC17A5, the gene responsible for the free sialic acid storage diseases. Our findings demonstrate that mutations in the SLC17A5 gene have to be considered in patients with hypomyelination, even in the absence of sialuria.

Identification of a novel hypertrophic cardiomyopathy-associated mutation using targeted next-generation sequencing.

Hypertrophic cardiomyopathy (HCM), one of the most common forms of myocardial diseases, is the major cause of sudden cardiac death in young adults and competitive athletes. Analyses of gene mutations associated with HCM are valuable for its molecular diagnosis, genetic counseling, and management of familial HCM. To dissect the relationship between the clinical presentation and gene mutations of HCM, the genetic characterizations of 19 HCM-related genes in 18 patients (8 cases from 6 pedigrees with familial HCM and 10 cases without familial HCM) were detected using next-generation sequencing (NGS). As a result, 12 disease-related mutations were identified in the 18 subjects, including 6 single mutations and 3 double mutations [MYBPC3 (p.Gln998Glu) plus TNNI3 (p.Arg145Gly), PRKAG2 (p.Gly100Ser) plus MYBPC3 (p.Lys1209Serfs*28) and TNNI3 (p.Glu124Gln) plus GLA (p.Trp47*)]. The 3 heterozygous double mutations were discovered for the first time in the malignant familial HCM patients. Of the 6 single mutations, a novel mutation was found in tafazzin (TAZ, p.Ile208Val), and a mutation in ß-myosin heavy chain gene (MYH7, p.Arg54Gln), which was reported as rare in the general population, was firstly found in one HCM patient. Identification of novel and rare mutations in HCM patients have added new data to the spectrum of gene mutations associated with this disease. These findings provide an essential basis for the molecular diagnosis and better management of family members at risk of familial HCM.

Aromatase expression in normal human oral keratinocytes and oral squamous cell carcinoma.

UNLABELLED: Aromatase is the enzyme that catalyzes the conversion of androgen to oestrogen. Aromatase expression in extra-gonadal sites and local oestrogen synthesis play an important role in the physiological conditions and in the growth of certain neoplasms. OBJECTIVE: The purpose of this study was to investigate aromatase expression in oral keratinocytes and oral squamous cell carcinoma (SCC). DESIGN: Immunocytochemistry and RT-nested PCR were used to detect aromatase protein and mRNA expression in primary human oral epithelial cell culture and in an oral SCC cell line. Immunohistochemistry was used to detect aromatase protein expression in frozen and archival human tissue sections of normal oral epithelium and oral SCC. RESULTS: Cytoplasmic immunostaining was found in normal oral keratinocytes and SCC cells in culture. The common coding region of aromatase mRNA was detected in the oral keratinocytes derived from five different normal individuals and in the SCC cell line. However, there were variations in aromatase exon 1 expression among normal oral keratinocyte samples. Cytoplasmic staining was found in normal oral epithelium and well-differentiated oral SCC but not in poorly differentiated oral SCC by immunohistochemistry. CONCLUSION: Aromatase was expressed in normal oral keratinocytes and oral SCC both in cell culture and in tissues, indicating local oestrogen synthesis in normal and neoplastic conditions of oral epithelium.

Identification of novel mutations including a double mutation in patients with inherited cardiomyopathy by a targeted sequencing approach using the Ion Torrent PGM system.

Inherited cardiomyopathy is the major cause of sudden cardiac death (SCD) and heart failure (HF). The disease is associated with extensive genetic heterogeneity; pathogenic mutations in cardiac sarcomere protein genes, cytoskeletal protein genes and nuclear envelope protein genes have been linked to its etiology. Early diagnosis is conducive to clinical monitoring and allows for presymptomatic interventions as needed. In the present study, the entire coding sequences and flanking regions of 12 major disease (cardiomyopathy)-related genes [namely myosin, heavy chain 7, cardiac muscle, ß (MYH7); myosin binding protein C, cardiac (MYBPC3); lamin A/C (LMNA); troponin I type 3 (cardiac) (TNNI3); troponin T type 2 (cardiac) (TNNT2); actin, α, cardiac muscle 1 (ACTC1); tropomyosin 1 (α) (TPM1); sodium channel, voltage gated, type V alpha subunit (SCN5A); myosin, light chain 2, regulatory, cardiac, slow (MYL2); myosin, heavy chain 6, cardiac muscle, α (MYH6); myosin, light chain 3, alkali, ventricular, skeletal, slow (MYL3); and protein kinase, AMP-activated, gamma 2 non-catalytic subunit  (PRKAG2)] in 8 patients with dilated cardiomyopathy (DCM) and in 8 patients with hypertrophic cardiomyopathy (HCM) were amplified and then sequenced using the Ion Torrent Personal Genome Machine (PGM) system. As a result, a novel heterozygous mutation (MYH7, p.Asn885Thr) and a variant of uncertain significance (TNNT2, p.Arg296His) were identified in 2 patients with HCM. These 2 missense mutations, which were absent in the samples obtained from the 200 healthy control subjects, altered the amino acid that was evolutionarily conserved among a number of vertebrate species; this illustrates that these 2 non-synonymous mutations play a role in the pathogenesis of HCM. Moreover, a double heterozygous mutation (PRKAG2, p.Gly100Ser plus MYH7, p.Arg719Trp) was identified in a patient with severe familial HCM, for the first time to the best of our knowledge. This patient provided us with more information regarding the genotype-phenotype correlation between mutations of MYH7 and PRKAG2. Taken together, these findings provide insight into the molecular mechanisms underlying inherited cardiomyopathy. The mutations identified in this study may be further investigated in the future in order to improve the diagnosis and treatment of patients with inherited cardiomyopathy. Furthermore, our findings indicated that sequencing using the Ion Torrent PGM system is a useful approach for the identification of pathogenic mutations associated with inherited cardiomyopathy, and it may be used for the risk evaluation of individuals with a possible susceptibility to inherited cardiomyopathy.

Targeted next-generation sequencing of candidate genes reveals novel mutations in patients with dilated cardiomyopathy.

Dilated cardiomyopathy (DCM) is a major cause of sudden cardiac death and heart failure, and it is characterized by genetic and clinical heterogeneity, even for some patients with a very poor clinical prognosis; in the majority of cases, DCM necessitates a heart transplant. Genetic mutations have long been considered to be associated with this disease. At present, mutations in over 50 genes related to DCM have been documented. This study was carried out to elucidate the characteristics of gene mutations in patients with DCM. The candidate genes that may cause DCM include MYBPC3, MYH6, MYH7, LMNA, TNNT2, TNNI3, MYPN, MYL3, TPM1, SCN5A, DES, ACTC1 and RBM20. Using next-generation sequencing (NGS) and subsequent mutation confirmation with traditional capillary Sanger sequencing analysis, possible causative non-synonymous mutations were identified in ~57% (12/21) of patients with DCM. As a result, 7 novel mutations (MYPN, p.E630K; TNNT2, p.G180A; MYH6, p.R1047C; TNNC1, p.D3V; DES, p.R386H; MYBPC3, p.C1124F; and MYL3, p.D126G), 3 variants of uncertain significance (RBM20, p.R1182H; MYH6, p.T1253M; and VCL, p.M209L), and 2 known mutations (MYH7, p.A26V and MYBPC3, p.R160W) were revealed to be associated with DCM. The mutations were most frequently found in the sarcomere (MYH6, MYBPC3, MYH7, TNNC1, TNNT2 and MYL3) and cytoskeletal (MYPN, DES and VCL) genes. As genetic testing is a useful tool in the clinical management of disease, testing for pathogenic mutations is beneficial to the treatment of patients with DCM and may assist in predicting disease risk for their family members before the onset of symptoms.

Targeted Next-Generation Sequencing Reveals Hot Spots and Doubly Heterozygous Mutations in Chinese Patients with Familial Cardiomyopathy.

As a common cardiac disease mainly caused by gene mutations in sarcomeric cytoskeletal, calcium-handling, nuclear envelope, desmosomal, and transcription factor genes, inherited cardiomyopathy is becoming one of the major etiological factors of sudden cardiac death (SCD) and heart failure (HF). This disease is characterized by remarkable genetic heterogeneity, which makes it difficult to screen for pathogenic mutations using Sanger sequencing. In the present study, three probands, one with familial hypertrophic cardiomyopathy (FHCM) and two with familial dilated cardiomyopathy (FDCM), were recruited together with their respective family members. Using next-generation sequencing technology (NGS), 24 genes frequently known to be related to inherited cardiomyopathy were screened. Two hot spots (TNNI3-p.Arg145Gly, and LMNA-p.Arg190Trp) and double (LMNA-p.Arg190Trp plus MYH7-p.Arg1045His) heterozygous mutations were found to be highly correlated with familial cardiomyopathy. FDCM patients with doubly heterozygous mutations show a notably severe phenotype as we could confirm in our study; this indicates that the double mutations had a dose effect. In addition, it is proposed that genetic testing using NGS technology can be used as a cost-effective screening tool and help guide the treatment of patients with familial cardiomyopathy particularly regarding the risk of family members who are clinically asymptomatic.

Silicon multi-nanochannel FETs to improve device uniformity/stability and femtomolar detection of insulin in serum.

Here we demonstrate the use of multiple Si nanochannel (NC) or nanograting (NG) instead of the conventional single nanochannel or nanowire design in biosensors. The NG devices can significantly reduce device-to-device variation, and improve device performance, e.g. higher current, higher ON/OFF ratio, smaller subthreshold slope, lower threshold voltage Vt in buffer solution. NG devices also result in higher sensor stability in buffer and diluted human serum. We believe such improvements are due to reduced discrete dopant fluctuation in the Si nanowires and biochemical noise in the solution because of the multiple-channel design. The improved devices allow us to sense pH linearly with 3-aminopropyltriethoxysilane coated devices, and to selectively detect insulin with limit of detection down to 10 fM in both buffer solution and diluted human serum without pre-purification.

Efficient gene delivery to pancreatic islets with ultrasonic microbubble destruction technology.

This study describes a method of gene delivery to pancreatic islets of adult, living animals by ultrasound targeted microbubble destruction (UTMD). The technique involves incorporation of plasmids into the phospholipid shell of gas-filled microbubbles, which are then infused into rats and destroyed within the pancreatic microcirculation with ultrasound. Specific delivery of genes to islet beta cells by UTMD was achieved by using a plasmid containing a rat insulin 1 promoter (RIP), and reporter gene expression was regulated appropriately by glucose in animals that received a RIP-luciferase plasmid. To demonstrate biological efficacy, we used UTMD to deliver RIP-human insulin and RIP-hexokinase I plasmids to islets of adult rats. Delivery of the former plasmid resulted in clear increases in circulating human C-peptide and decreased blood glucose levels, whereas delivery of the latter plasmid resulted in a clear increase in hexokinase I protein expression in islets, increased insulin levels in blood, and decreased circulating glucose levels. We conclude that UTMD allows relatively noninvasive delivery of genes to pancreatic islets with an efficiency sufficient to modulate beta cell function in adult animals.

Characterization of the bidirectional promoter region between the human genes encoding VLCAD and PSD-95.

Bidirectional promoters are widely known among lower organisms but rare in mammals. A shared promoter between the two human genes encoding very long chain acyl-CoA dehydrogenase (VLCAD) and postsynaptic density protein 95 (PSD-95) is an ideal model to investigate bidirectional transcription in mammals. VLCAD associates with the inner mitochondrial membrane and catalyzes the initial step in mitochondrial long-chain fatty acid beta-oxidation. PSD-95, a component protein of the PSD, plays an essential role in clustering the transmembrane proteins in synaptic membranes. Interestingly, the human genes encoding VLCAD (ACADVL) and PSD-95 (DLG4) are adjacently located in the head-to-head orientation on chromosome 17p. The transcribed regions of the two genes overlap, while the two transcription start sites stand approximately 220 bp apart. To analyze the common transcriptional control region shared by the two genes, we generated serial promoter partial deletion constructs using firefly luciferase as the reporter gene. Our results showed that the essential promoter activity of PSD-95 is carried within an approximately 400-bp region, which covers the entire approximately 270-bp minimal promoter of VLCAD. The results from di-(2-ethylhexyl) phthalate (DEHP)-treated HepG2 cells revealed that the minimal VLCAD promoter is able to up-regulate VLCAD expression in response to DEHP treatment. Site-directed mutagenesis experiments showed that a mutated activator protein 2-binding site markedly reduced the transcriptional activity of both promoters and abolished the minimal VLCAD promoter's response to DEHP treatment.