ABSTRACT
STUDY QUESTION: Is it possible to identify new mutations potentially associated with non-syndromic primary ovarian insufficiency (POI) via whole-exome sequencing (WES)? SUMMARY ANSWER: WES is an efficient tool to study genetic causes of POI as we have identified new mutations, some of which lead to protein destablization potentially contributing to the disease etiology. WHAT IS KNOWN ALREADY: POI is a frequently occurring complex pathology leading to infertility. Mutations in only few candidate genes, mainly identified by Sanger sequencing, have been definitively related to the pathogenesis of the disease. STUDY DESIGN, SIZE, DURATION: This is a retrospective cohort study performed on 69 women affected by POI. PARTICIPANTS/MATERIALS, SETTING, METHODS: WES and an innovative bioinformatics analysis were used on non-synonymous sequence variants in a subset of 420 selected POI candidate genes. Mutations in BMPR1B and GREM1 were modeled by using fragment molecular orbital analysis. MAIN RESULTS AND THE ROLE OF CHANCE: Fifty-five coding variants in 49 genes potentially related to POI were identified in 33 out of 69 patients (48%). These genes participate in key biological processes in the ovary, such as meiosis, follicular development, granulosa cell differentiation/proliferation and ovulation. The presence of at least two mutations in distinct genes in 42% of the patients argued in favor of a polygenic nature of POI. LIMITATIONS, REASONS FOR CAUTION: It is possible that regulatory regions, not analyzed in the present study, carry further variants related to POI. WIDER IMPLICATIONS OF THE FINDINGS: WES and the in silico analyses presented here represent an efficient approach for mapping variants associated with POI etiology. Sequence variants presented here represents potential future genetic biomarkers. STUDY FUNDING/COMPETING INTEREST(S): This study was supported by the Universidad del Rosario and Colciencias (Grants CS/CIGGUR-ABN062-2016 and 672-2014). Colciencias supported Liliana Catherine Patiño´s work (Fellowship: 617, 2013). The authors declare no conflict of interest.
Subject(s)
Bone Morphogenetic Protein Receptors, Type I/genetics , Genetic Predisposition to Disease , Intercellular Signaling Peptides and Proteins/genetics , Models, Molecular , Mutation , Primary Ovarian Insufficiency/genetics , Adult , Amino Acid Substitution , Bone Morphogenetic Protein Receptors, Type I/chemistry , Bone Morphogenetic Protein Receptors, Type I/metabolism , Cohort Studies , Computational Biology , Expert Systems , Female , France , Genome-Wide Association Study , Humans , Intercellular Signaling Peptides and Proteins/chemistry , Intercellular Signaling Peptides and Proteins/metabolism , Molecular Dynamics Simulation , Polymorphism, Single Nucleotide , Primary Ovarian Insufficiency/metabolism , Protein Stability , Referral and Consultation , Retrospective Studies , Exome Sequencing , Young AdultABSTRACT
Giardia lamblia is an intestinal protozoan parasite that causes giardiasis, a disease of high prevalence in Latin America, Asia and Africa. Giardiasis leads to poor absorption of nutrients, severe electrolyte loss and growth retardation. In addition to its clinical importance, this parasite is of special biological interest due to its basal evolutionary position and simplified metabolism, which has not been studied thoroughly. One of the most important and conserved metabolic pathways is the biosynthesis of nicotinamide adenine dinucleotide (NAD). This molecule is widely known as a coenzyme in multiple redox reactions and as a substrate in cellular processes such as synthesis of Ca2+ mobilizing agents, DNA repair and gene expression regulation. There are two pathways for NAD biosynthesis, which converge at the step catalyzed by nicotinamide/nicotinate mononucleotide adenylyltransferase (NMNAT, EC 2.7.7.1/18). Using bioinformatics tools, we found two NMNAT sequences in Giardia lamblia (glnmnat-a and glnmnat-b). We first verified the identity of the sequences in silico. Subsequently, glnmnat-a was cloned into an expression vector. The recombinant protein (His-GlNMNAT) was purified by nickel-affinity binding and was used in direct in vitro enzyme assays assessed by C18-HPLC, verifying adenylyltransferase activity with both nicotinamide (NMN) and nicotinic acid (NAMN) mononucleotides. Optimal reaction pH and temperature were 7.3 and 26 °C. Michaelis-Menten kinetics were observed for NMN and ATP, but saturation was not accomplished with NAMN, implying low affinity yet detectable activity with this substrate. Double-reciprocal plots showed no cooperativity for this enzyme. This represents an advance in the study of NAD metabolism in Giardia spp.
