ABSTRACT
Ammonia, which is toxic to the brain, is converted into non-toxic urea, through a pathway of six enzymatically catalyzed steps known as the urea cycle. In this pathway, N-acetylglutamate synthase (NAGS, EC 2.3.1.1) catalyzes the formation of N-acetylglutamate (NAG) from glutamate and acetyl coenzyme A. NAGS deficiency (NAGSD) is the rarest of the urea cycle disorders, yet is unique in that ureagenesis can be restored with the drug N-carbamylglutamate (NCG). We investigated whether the rarity of NAGSD could be due to low sequence variation in the NAGS genomic region, high NAGS tolerance for amino acid replacements, and alternative sources of NAG and NCG in the body. We also evaluated whether the small genomic footprint of the NAGS catalytic domain might play a role. The small number of patients diagnosed with NAGSD could result from the absence of specific disease biomarkers and/or short NAGS catalytic domain. We screened for sequence variants in NAGS regulatory regions in patients suspected of having NAGSD and found a novel NAGS regulatory element in the first intron of the NAGS gene. We applied the same datamining approach to identify regulatory elements in the remaining urea cycle genes. In addition to the known promoters and enhancers of each gene, we identified several novel regulatory elements in their upstream regions and first introns. The identification of cis-regulatory elements of urea cycle genes and their associated transcription factors holds promise for uncovering shared mechanisms governing urea cycle gene expression and potentially leading to new treatments for urea cycle disorders.
ABSTRACT
One year after a Zaire ebolavirus (EBOV) outbreak occurred in the Boende Health Zone of the Democratic Republic of the Congo during 2014, we sought to determine the breadth of immune response against diverse filoviruses including EBOV, Bundibugyo (BDBV), Sudan (SUDV), and Marburg (MARV) viruses. After assessing the 15 survivors, 5 individuals demonstrated some degree of reactivity to multiple ebolavirus species and, in some instances, Marburg virus. All 5 of these survivors had immunoreactivity to EBOV glycoprotein (GP) and EBOV VP40, and 4 had reactivity to EBOV nucleoprotein (NP). Three of these survivors showed serologic responses to the 3 species of ebolavirus GPs tested (EBOV, BDBV, SUDV). All 5 samples also exhibited ability to neutralize EBOV using live virus, in a plaque reduction neutralization test. Remarkably, 3 of these EBOV survivors had plasma antibody responses to MARV GP. In pseudovirus neutralization assays, serum antibodies from a subset of these survivors also neutralized EBOV, BDBV, SUDV, and Taï Forest virus as well as MARV. Collectively, these findings suggest that some survivors of naturally acquired ebolavirus infection mount not only a pan-ebolavirus response, but also in less frequent cases, a pan-filovirus neutralizing response.
Subject(s)
Antibodies, Viral/blood , Antibodies, Viral/immunology , Ebolavirus/classification , Hemorrhagic Fever, Ebola/epidemiology , Hemorrhagic Fever, Ebola/immunology , Antibodies, Monoclonal , Antibodies, Neutralizing/blood , Antibody Specificity , Antigens, Viral , Democratic Republic of the Congo/epidemiology , Ebolavirus/immunology , Glycoproteins/immunology , Hemorrhagic Fever, Ebola/virology , Humans , Lassa virus/immunology , Marburgvirus/immunology , Neutralization TestsABSTRACT
Sex differences in brain development and postnatal behavior are determined largely by genetic sex and in utero gonadal hormone secretions. In humans however, determining the weight that each of these factors contributes remains a challenge because social influences should also be considered. Cases of disorders of sex development (DSD) provide unique insight into how mutations in genes responsible for gonadal formation can perturb the subsequent developmental hormonal milieu and elicit changes in normal human brain maturation. Specific forms of DSDs such as complete androgen insensitivity syndrome (CAIS), congenital adrenal hyperplasia (CAH), and 5α-reductase deficiency syndrome have variable effects between males and females, and the developmental outcomes of such conditions are largely dependent on sex chromosome composition. Medical and psychological works focused on CAH, CAIS, and 5α-reductase deficiency have helped form the foundation for understanding the roles of genetic and hormonal factors necessary for guiding human brain development. Here we highlight how the three aforementioned DSDs contribute to brain and behavioral phenotypes that can uniquely affect 46,XY and 46,XX individuals in dramatically different fashions. © 2016 Wiley Periodicals, Inc.
Subject(s)
Brain/metabolism , Disorders of Sex Development , Gonadal Steroid Hormones/metabolism , Sex Characteristics , Sex Chromosomes , Disorders of Sex Development/genetics , Disorders of Sex Development/metabolism , Disorders of Sex Development/pathology , Female , Humans , MaleABSTRACT
STUDY QUESTION: Can whole exome sequencing (WES) and in vitro validation studies be used to find the causative genetic etiology in a patient with primary ovarian failure and infertility? SUMMARY ANSWER: A novel follicle-stimulating hormone receptor (FSHR) mutation was found by WES and shown, via in vitro flow cytometry studies, to affect membrane trafficking. WHAT IS KNOWN ALREADY: WES may diagnose up to 25-35% of patients with suspected disorders of sex development (DSD). FSHR mutations are an extremely rare cause of 46, XX gonadal dysgenesis with primary amenorrhea due to hypergonadotropic ovarian failure. STUDY DESIGN, SIZE, DURATION: A WES study was followed by flow cytometry studies of mutant protein function. PARTICIPANTS/MATERIALS, SETTING, METHODS: The study subjects were two Turkish sisters with hypergonadotropic primary amenorrhea, their parents and two unaffected sisters. The affected siblings and both parents were sequenced (trio-WES). Transient transfection of HEK 293T cells was performed with a vector containing wild-type FSHR as well as the novel FSHR variant that was discovered by WES. Cellular localization of FSHR protein as well as FSH-stimulated cyclic AMP (cAMP) production was evaluated using flow cytometry. MAIN RESULTS AND THE ROLE OF CHANCE: Both affected sisters were homozygous for a previously unreported missense mutation (c.1222G>T, p.Asp408Tyr) in the second transmembrane domain of FSHR. Modeling predicted disrupted secondary structure. Flow cytometry demonstrated an average of 48% reduction in cell-surface signal detection (P < 0.01). The mean fluorescent signal for cAMP (second messenger of FSHR), stimulated by FSH, was reduced by 50% in the mutant-transfected cells (P < 0.01). LIMITATIONS, REASONS FOR CAUTION: This is an in vitro validation. All novel purported genetic variants can be clinically reported only as 'variants of uncertain significance' until more patients with a similar phenotype are discovered with the same variant. WIDER IMPLICATIONS OF THE FINDINGS: We report the first WES-discovered FSHR mutation, validated by quantitative flow cytometry. WES is a valuable tool for diagnosis of rare genetic diseases, and flow cytometry allows for quantitative characterization of purported variants. WES-assisted diagnosis allows for treatments aimed at the underlying molecular etiology of disease. Future studies should focus on pharmacological and assisted reproductive treatments aimed at the disrupted FSHR, so that patients with FSH resistance can be treated by personalized medicine. STUDY FUNDING/COMPETING INTERESTS: E.V. is partially funded by the DSD Translational Research Network (NICHD 1R01HD068138). M.S.B. is funded by the Neuroendocrinology, Sex Differences and Reproduction training grant (NICHD 5T32HD007228). The authors have no competing interests to disclose.
Subject(s)
Models, Molecular , Mutation, Missense , Primary Ovarian Insufficiency/genetics , Receptors, FSH/genetics , Adult , Consanguinity , Exome , Female , Genome-Wide Association Study , HEK293 Cells , Homozygote , Humans , Primary Ovarian Insufficiency/metabolism , Protein Structure, Secondary , Protein Transport , Receptors, FSH/chemistry , Receptors, FSH/metabolism , Recombinant Proteins/chemistry , Recombinant Proteins/metabolism , Sequence Analysis, DNA , Siblings , Turkey , Young AdultABSTRACT
Down syndrome is one of the most common chromosomal anomalies affecting the world's population, with an estimated frequency of 1 in 700 live births. Despite its relatively high prevalence, diagnostic rates based on clinical features have remained under 70% for most of the developed world and even lower in countries with limited resources. While genetic and cytogenetic confirmation greatly increases the diagnostic rate, such resources are often non-existent in many low- and middle-income countries, particularly in Sub-Saharan Africa. To address the needs of countries with limited resources, the implementation of mobile, user-friendly and affordable technologies that aid in diagnosis would greatly increase the odds of success for a child born with a genetic condition. Given that the Democratic Republic of the Congo is estimated to have one of the highest rates of birth defects in the world, our team sought to determine if smartphone-based facial analysis technology could accurately detect Down syndrome in individuals of Congolese descent. Prior to technology training, we confirmed the presence of trisomy 21 using low-cost genomic applications that do not need advanced expertise to utilize and are available in many low-resourced countries. Our software technology trained on 132 Congolese subjects had a significantly improved performance (91.67% accuracy, 95.45% sensitivity, 87.88% specificity) when compared to previous technology trained on individuals who are not of Congolese origin (p < 5%). In addition, we provide the list of most discriminative facial features of Down syndrome and their ranges in the Congolese population. Collectively, our technology provides low-cost and accurate diagnosis of Down syndrome in the local population.
Subject(s)
Automated Facial Recognition/methods , Down Syndrome/pathology , Facies , Image Processing, Computer-Assisted/methods , Automated Facial Recognition/economics , Automated Facial Recognition/standards , Democratic Republic of the Congo , Developing Countries , Down Syndrome/genetics , Genetic Testing , Humans , Image Processing, Computer-Assisted/economics , Image Processing, Computer-Assisted/standards , Machine Learning , Sensitivity and SpecificityABSTRACT
Konzo, a distinct upper motor neuron disease associated with a cyanogenic diet and chronic malnutrition, predominately affects children and women of childbearing age in sub-Saharan Africa. While the exact biological mechanisms that cause this disease have largely remained elusive, host-genetics and environmental components such as the gut microbiome have been implicated. Using a large study population of 180 individuals from the Democratic Republic of the Congo, where konzo is most frequent, we investigate how the structure of the gut microbiome varied across geographical contexts, as well as provide the first insight into the gut flora of children affected with this debilitating disease using shotgun metagenomic sequencing. Our findings indicate that the gut microbiome structure is highly variable depending on region of sampling, but most interestingly, we identify unique enrichments of bacterial species and functional pathways that potentially modulate the susceptibility of konzo in prone regions of the Congo.
Subject(s)
Disease Susceptibility/microbiology , Feeding Behavior , Gastrointestinal Microbiome/physiology , Manihot/adverse effects , Motor Neuron Disease/microbiology , Child , Democratic Republic of the Congo/epidemiology , Feces/microbiology , Female , Humans , Manihot/chemistry , Metagenomics , Motor Neuron Disease/epidemiology , Nitriles/adverse effectsABSTRACT
While numerous in vivo experiments have sought to explore the effects of sex chromosome composition and sex steroid hormones on cellular proliferation and differentiation within the mammalian brain, far fewer studies as reviewed here, have explored these factors using a direct in vitro approach. Generally speaking, in vivo studies provide the gold standard to demonstrate applicable findings in regards to the role hormones play in development. However, in the case of neural stem cell (NSC) biology, there remain many unknown factors that likely contribute to observations made within the developed brain, specifically in regions where there are abundant sex steroid hormone receptors. For these reasons, using a NSC in vitro model may provide a more controlled and refined system to explore the direct effects of sex and hormone response, limiting the vast array of other influences on NSCs occurring during development and within adult cellular niches. These specific cellular models may have the ability to greatly improve the mechanistic understanding of changes occurring within the developing brain during the hormonal organization process, in addition to other modifications that may contribute to neuro-psychiatric sex-biased diseases.
Subject(s)
Gonadal Steroid Hormones/metabolism , Neural Stem Cells/metabolism , Animals , Cell Differentiation/drug effects , Cell Proliferation/drug effects , Estrogens/pharmacology , Humans , Neural Stem Cells/drug effects , Sex CharacteristicsABSTRACT
The mechanisms by which sex differences in the mammalian brain arise are poorly understood, but are influenced by a combination of underlying genetic differences and gonadal hormone exposure. Using a mouse embryonic neural stem cell (eNSC) model to understand early events contributing to sexually dimorphic brain development, we identified novel interactions between chromosomal sex and hormonal exposure that are instrumental to early brain sex differences. RNA-sequencing identified 103 transcripts that were differentially expressed between XX and XY eNSCs at baseline (FDR = 0.10). Treatment with testosterone-propionate (TP) reveals sex-specific gene expression changes, causing 2854 and 792 transcripts to become differentially expressed on XX and XY genetic backgrounds respectively. Within the TP responsive transcripts, there was enrichment for genes which function as epigenetic regulators that affect both histone modifications and DNA methylation patterning. We observed that TP caused a global decrease in 5-methylcytosine abundance in both sexes, a transmissible effect that was maintained in cellular progeny. Additionally, we determined that TP was associated with residue-specific alterations in acetylation of histone tails. These findings highlight an unknown component of androgen action on cells within the developmental CNS, and contribute to a novel mechanism of action by which early hormonal organization is initiated and maintained.