Whole exome sequencing identifies the first STRADA point mutation in a patient with polyhydramnios, megalencephaly, and symptomatic epilepsy syndrome (PMSE).

Polyhydramnios, megalencephaly, and symptomatic epilepsy syndrome (PMSE) is an ultra rare neurodevelopmental disorder characterized by severe, infantile-onset intractable epilepsy, neurocognitive delay, macrocephaly, and craniofacial dysmorphism. The molecular diagnosis of this condition has thus far only been made in 16 Old Order Mennonite patients carrying a homozygous 7 kb founder deletion of exons 9-13 of STRADA. We performed clinical whole exome sequencing (WES) on a 4-year-old Indian male with global developmental delay, history of failure to thrive, infantile spasms, repetitive behaviors, hypotonia, low muscle mass, marked joint laxity, and dysmorphic facial features including tall forehead, long face, arched eyebrows, small chin, wide mouth, and tented upper lip. A homozygous single nucleotide duplication, c.842dupA (p.D281fs), in exon 10 of STRADA was identified. Sanger sequencing confirmed the mutation in the individual and identified both parents as carriers. In light of the molecular discoveries, the patient's clinical phenotype was considered to be a good fit for PMSE. We identified for the first time a homozygous point mutation in STRADA causing PMSE. Additional bi-allelic mutations related to PMSE thus far have not been observed in Baylor ∼6,000 consecutive clinical WES cases, supporting the rarity of this disorder. Our findings may have treatment implications for the patient since previous studies have shown rapamycin as a potential therapeutic agent for the seizures and cognitive problems in PMSE patients. © 2016 Wiley Periodicals, Inc.

Increased bone turnover, osteoporosis, progressive tibial bowing, fractures, and scoliosis in a patient with a final-exon SATB2 frameshift mutation.

Haploinsufficiency of SATB2 causes cleft palate, intellectual disability with deficient speech, facial and dental abnormalities, and other variable features known collectively as SATB2-associated syndrome. This phenotype was accompanied by osteoporosis, fractures, and tibial bowing in two previously reported adult patients; each possessed SATB2 mutations either predicted or demonstrated to escape nonsense-mediated decay, suggesting that the additional bone defects result from a dominant negative effect and/or age-dependent penetrance. These hypotheses remain to be confirmed, as do the specific downstream defects causing bone abnormalities. We report a SATB2 mutation (c.2018dupA; p.(H673fs)) in a 15-year-old patient whose SATB2-associated syndrome phenotype is accompanied by osteoporosis, fractures, progressive tibial bowing, and scoliosis. As this homeodomain-disrupting and predicted truncating mutation resides within the final exon of SATB2, escape from nonsense-mediated decay is likely. Thus, we provide further evidence of bone phenotypes beyond those typically associated with SATB2-associated syndrome in individuals with potential dominant-negative SATB2 alleles, as well as evidence for age-dependence of bone features. Elevations in alkaline phosphatase, urinary N-telopeptide/creatinine ratio, and osteocalcin in the patient indicate increased bone turnover. We propose surveillance and treatment with osteoclast inhibitors to prevent fractures and to slow progressive bone deformities. © 2016 Wiley Periodicals, Inc.

Expression and functional analysis of a glycoside hydrolase family 45 endoglucanase from Rhizopus stolonifer.

A novel endoglucanase gene was cloned from Rhizopus stolonifer and expressed in Escherichia coli, the gene product EG II (45 kDa) was assigned to Glycoside Hydrolase Family 45 (GH45), and its specific activity on phosphoric acid-swollen cellulose (PASC) was 48 IU/mg. To solve the problem of substrate accumulation in the cellulose hydrolysis and enhance the catalytic efficiency of endoglucanase, the eg2 gene was modified by site directed mutagenesis. Mutations generated by overlapping PCR have been proven to increase its catalytic activity on carboxymenthyl cellulose, microcrystalline cellulose (Avicel) and PASC, among which the mutant EG II-E containing all 6 mutations (N39S, V136D, T251G, D255G, P256S and E260D) peaked 121 IU/mg on PASC. The bioinformatic analysis showed that 2 key catalytic residues (D136 and D260) moved closer with the opening of a loop after mutagenesis, and a tunnel was formed by structural transformation. This structure was conducive for the substrate to access the active centre, and D136 played an indispensable role in the substrate recognition.

Enhanced immunogenicity of an anti-caries vaccine encoding a cell-surface protein antigen of Streptococcus mutans by intranasal DNA prime-protein boost immunization.

BACKGROUND: The present study aimed to enhance the specific anti-caries immunity induced by DNA prime-protein boost strategy for an A-P fragment of a cell-surface protein antigen of Streptococcus mutans (PAc). METHODS: BALB/c mice were immunized with DNA prime-protein boost, DNA-DNA or protein-protein regimens by the intranasal route, using combinations of plasmid vector (pCIA-P) that express PAc protein and a pure secretec recombinant PAc protein (rPAc). Then, a gnotobiotic mouse model was constructed 2 weeks after the last immunization, and specific immune responses in vivo and their protection against dental caries were observed. RESULTS: The present study revealed stronger antibody responses in the DNA prime-protein boost group compared to those elicited by either DNA-DNA vaccination or protein-protein vaccination. In particular, PAc-specific antibody concentrations were improved significantly after boosting the pCIA-P DNA-primed mice with rPAc. Moreover, protection against S. mutans challenge was obtained in the mice treated with the DNA prime-protein boost vaccination, as demonstrated by a significant reduction in S. mutans colonization compared to control mice and animals immunized with the DNA-DNA vaccination or protein-protein vaccination. CONCLUSIONS: The results obtained in the present study suggest that the intranasal DNA prime-protein boost vaccination regimen is a novel strategy for the practical application of DNA vaccine against dental caries.

Good Manufacturing Practices production and analysis of a DNA vaccine against dental caries.

AIM: To prepare a clinical-grade anti-caries DNA vaccine pGJA-P/VAX and explore its immune effect and protective efficacy against a cariogenic bacterial challenge. METHODS: A large-scale industrial production process was developed under Good Manufacturing Practices (GMP) by combining and optimizing common unit operations such as alkaline lysis, precipitation, endotoxin removal and column chromatography. Quality controls of the purified bulk and final lyophilized vaccine were conducted according to authoritative guidelines. Mice and gnotobiotic rats were intranasally immunized with clinical-grade pGJA-P/VAX with chitosan. Antibody levels of serum IgG and salivary SIgA were assessed by an enzyme-linked immunosorbent assay (ELISA), and caries activity was evaluated by the Keyes method. pGJA-P/VAX and pVAX1 prepared by a laboratory-scale commercial kit were used as controls. RESULTS: The production process proved to be scalable and reproducible. Impurities including host protein, residual RNA, genomic DNA and endotoxin in the purified plasmid were all under the limits of set specifications. Intranasal vaccination with clinical-grade pGJA-P/VAX induced higher serum IgG and salivary SIgA in both mice and gnotobiotic rats. While in the experimental caries model, the enamel (E), dentinal slight (Ds), and dentinal moderate (Dm) caries lesions were reduced by 21.1%, 33.0%, and 40.9%, respectively. CONCLUSION: The production process under GMP was efficient in preparing clinical-grade pGJA-P/VAX with high purity and intended effectiveness, thus facilitating future clinical trials for the anti-caries DNA vaccine.

Biting pain reproduced by the Tooth Slooth: an aid for early diagnosis of cracked tooth.

OBJECTIVE: The purpose of this study was to find a reliable method to reproduce biting pain to facilitate an early diagnosis of cracked tooth and to verify the feasibility of the Tooth Slooth in diagnosing a cracked tooth. METHOD AND MATERIALS: In this study, 46 intact teeth diagnosed as cracked teeth were selected. Patients were asked to bite wet cotton rolls and the Tooth Slooth, and clinical findings were recorded. The difference in the relevance ratio between these two bite test methods was determined. RESULTS: The relevance ratio of biting pain by the Tooth Slooth and wet cotton rolls was 91.3% and 32.6%, respectively. There was a statistically significant difference between these two bite tests (P < .001). CONCLUSIONS: Within the limitations of this study, the relevance ratio of biting pain by the Tooth Slooth was significantly higher than that of the wet cotton rolls. The Tooth Slooth was a reliable method to reproduce biting pain and was useful for early diagnosis of cracked teeth.

Loss-of-function mutations in Lysyl-tRNA synthetase cause various leukoencephalopathy phenotypes.

OBJECTIVE: To expand the clinical spectrum of lysyl-tRNA synthetase (KARS) gene-related diseases, which so far includes Charcot-Marie-Tooth disease, congenital visual impairment and microcephaly, and nonsyndromic hearing impairment. METHODS: Whole-exome sequencing was performed on index patients from 4 unrelated families with leukoencephalopathy. Candidate pathogenic variants and their cosegregation were confirmed by Sanger sequencing. Effects of mutations on KARS protein function were examined by aminoacylation assays and yeast complementation assays. RESULTS: Common clinical features of the patients in this study included impaired cognitive ability, seizure, hypotonia, ataxia, and abnormal brain imaging, suggesting that the CNS involvement is the main clinical presentation. Six previously unreported and 1 known KARS mutations were identified and cosegregated in these families. Two patients are compound heterozygous for missense mutations, 1 patient is homozygous for a missense mutation, and 1 patient harbored an insertion mutation and a missense mutation. Functional and structural analyses revealed that these mutations impair aminoacylation activity of lysyl-tRNA synthetase, indicating that defective KARS function is responsible for the phenotypes in these individuals. CONCLUSIONS: Our results demonstrate that patients with loss-of-function KARS mutations can manifest CNS disorders, thus broadening the phenotypic spectrum associated with KARS-related disease.

Investigation of the Synergetic Effect of Xylose Metabolic Pathways on the Production of Glutaric Acid.

Efficient utilization of lignocellulose is pivotal for economically converting renewable feedstocks into value-added products. Xylose is the second most abundant sugar in lignocellulose, but it is quite challenging to ferment xylose as efficiently as glucose by microorganisms. Here, we investigated the metabolic potential of three xylose catabolic pathways (isomerase, Weimberg, and Dahms pathways) and illustrated the synergetic effect between the isomerase pathway and Weimberg pathway for the synthesis of chemicals derived from 2-ketoglutarate and acetyl-CoA. When using glutaric acid as the target product, employment of such synergetic pathways in combination resulted in an increased glutaric acid titer (602 mg/L) compared with using each pathway alone (104 or 209 mg/L), and this titer even outcompetes that obtained from the glucose catabolic pathway for glutaric acid synthesis (420 mg/L). This work validates a novel and powerful strategy for xylose metabolic utilization to overcome the inefficiency of using a single xylose metabolic pathway for the synthesis of TCA cycle derived chemicals.

Safety and efficacy of PLGA(Ag-Fe3O4)-coated dental implants in inhibiting bacteria adherence and osteogenic inducement under a magnetic field.

INTRODUCTION: The placement of dental implants is performed in a contaminated surgical field in the oral cavity, which may lead to implant failure. Bacterial adhesion and proliferation (Streptococcus mutans, Porphyromonas gingivalis) often lead to implant infections. Although Ag nanoparticles hold great promise for a broad spectrum of antibacterial activities, their runoff from dental implants compromises their antibacterial efficacy and potentially impairs osteoblast proliferation. Thus, this aspect remains a primary challenge and should be controlled. MATERIALS AND METHODS: In this study, PLGA(Ag-Fe3O4) was modified on the implanted tooth surface and was characterized by transmission electron microscopy, X-ray diffraction, and Fourier transform infrared spectroscopy. The magnetic and antibacterial properties were also determined. RESULTS: Results showed that Ag successfully bonded with Fe3O4, and Ag-Fe3O4 not only exerted superparamagnetism but also exhibited antibacterial activity almost identical to silver nanoparticles (nano-Ag). The PLGA(Ag-Fe3O4) coating could significantly maintain the antibacterial activity and avoid bacterial adhesion to the implant. Compared with the blank control group, PLGA(Ag-Fe3O4) under magnetic field-coated samples had a significantly lower amount of colonized S. mutans (P<0.01). Osteoblast proliferation results showed that the coated samples did not exhibit cytotoxicity and could promote osteoblast proliferation as shown by MTT, alkaline phosphatase, and the nucleolar organizer region count. CONCLUSION: We developed a novel Ag biologically compatible nanoparticle in this study without compromising the nano-Ag antibacterial activity, which provided continuous antibacterial action.

Using Defatted Rice Bran as a Bioadsorbent for Carrying Tea Catechins.

UNLABELLED: The potential of rice bran as a bioabsorbent for tea catechins was examined. Defatted rice bran had the highest adsorption capacity for tea catechins and the best selectivity for (-)-epigallocatechin gallate over total catechins among water-washed rice bran and untreated rice bran. The adsorption characteristics of tea catechins onto defatted rice bran were determined over a range of concentration (0.5 to 2.5 g/L) and temperatures (10, 25, and 45 °C). The adsorption of tea catechins onto defatted rice bran showed excellent fitness with the pseudo-second-order model at different temperature. Both the Langmuir and Freundlich models adequately describe the isothermal adsorption of tea catechins onto defatted rice bran. The adsorption of total catechins on rice bran decreased from 10 to 25 °C, whereas was greatly enhanced at 45 °C. The adsorption system of bioadsorbent with multiconstituents may not be as simple as the single-force-driving adsorption system. Protein and cellulose are the main contributors to the adsorption of tea catechins on defatted rice bran. PRACTICAL APPLICATION: Rice bran is regarded as a good fibre source that can be added to various food products and health supplements, which is a potential biocarrier for bioactives. Our study showed that defatted rice bran had a high affinity for tea catechins but caffeine, and provided a promising way for selective enrichment of catechins on defatted rice bran under practical condition. Protein and cellulose are the main contributors to the adsorption of tea catechins on defatted rice bran.

Mutations in SLC25A46, encoding a UGO1-like protein, cause an optic atrophy spectrum disorder.

Dominant optic atrophy (DOA) and axonal peripheral neuropathy (Charcot-Marie-Tooth type 2, or CMT2) are hereditary neurodegenerative disorders most commonly caused by mutations in the canonical mitochondrial fusion genes OPA1 and MFN2, respectively. In yeast, homologs of OPA1 (Mgm1) and MFN2 (Fzo1) work in concert with Ugo1, for which no human equivalent has been identified thus far. By whole-exome sequencing of patients with optic atrophy and CMT2, we identified four families with recessive mutations in SLC25A46. We demonstrate that SLC25A46, like Ugo1, is a modified carrier protein that has been recruited to the outer mitochondrial membrane and interacts with the inner membrane remodeling protein mitofilin (Fcj1). Loss of function in cultured cells and in zebrafish unexpectedly leads to increased mitochondrial connectivity, while severely affecting the development and maintenance of neurons in the fish. The discovery of SLC25A46 strengthens the genetic overlap between optic atrophy and CMT2 while exemplifying a new class of modified solute transporters linked to mitochondrial dynamics.

An unusual cause of peroneal neuropathy.

We discuss the case of a teenage girl who presented with neurologic symptoms suggestive of a peripheral neuropathy, before the development of a central arteriovenous fistula. Electromyography and nerve conduction studies indicated peroneal motor neuropathy, but her comprehensive genetic study results were negative for common Charcot-Marie-Tooth mutations. After 2 years of stable symptoms, she presented with unilateral throbbing headache and tinnitus. Magnetic resonance angiography revealed a carotid cavernous fistula, which was confirmed with conventional angiography. A successful coil embolization of the fistula was performed. Whole exome sequencing demonstrated a de novo heterozygous c.3158G>A (p.G1056D) mutation in the COL31A gene, consistent with Ehlers-Danlos type IV. To our knowledge, this is the first reported case of isolated peroneal motor neuropathy in a patient with Ehlers-Danlos type IV. This case highlights the utility of whole exome sequencing in the diagnosis of patients with neurologic symptoms that do not fit a clear phenotype.

Exome sequencing resolves apparent incidental findings and reveals further complexity of SH3TC2 variant alleles causing Charcot-Marie-Tooth neuropathy.

BACKGROUND: The debate regarding the relative merits of whole genome sequencing (WGS) versus exome sequencing (ES) centers around comparative cost, average depth of coverage for each interrogated base, and their relative efficiency in the identification of medically actionable variants from the myriad of variants identified by each approach. Nevertheless, few genomes have been subjected to both WGS and ES, using multiple next generation sequencing platforms. In addition, no personal genome has been so extensively analyzed using DNA derived from peripheral blood as opposed to DNA from transformed cell lines that may either accumulate mutations during propagation or clonally expand mosaic variants during cell transformation and propagation. METHODS: We investigated a genome that was studied previously by SOLiD chemistry using both ES and WGS, and now perform six independent ES assays (Illumina GAII (x2), Illumina HiSeq (x2), Life Technologies' Personal Genome Machine (PGM) and Proton), and one additional WGS (Illumina HiSeq). RESULTS: We compared the variants identified by the different methods and provide insights into the differences among variants identified between ES runs in the same technology platform and among different sequencing technologies. We resolved the true genotypes of medically actionable variants identified in the proband through orthogonal experimental approaches. Furthermore, ES identified an additional SH3TC2 variant (p.M1?) that likely contributes to the phenotype in the proband. CONCLUSIONS: ES identified additional medically actionable variant calls and helped resolve ambiguous single nucleotide variants (SNV) documenting the power of increased depth of coverage of the captured targeted regions. Comparative analyses of WGS and ES reveal that pseudogenes and segmental duplications may explain some instances of apparent disease mutations in unaffected individuals.