Linking genotype to trophoblast phenotype in preeclampsia and HELLP syndrome associated with STOX1 genetic variants.

Preeclampsia is a major hypertensive pregnancy disorder with a 50% heritability. The first identified gene involved in the disease is STOX1, a transcription factor, whose variant Y153H predisposes to the disease. Two rare mutations were also identified in Colombian women affected by the hemolysis, elevated liver enzyme, low platelet syndrome, a complication of preeclampsia (T188N and R364X). Here, we explore the effects of these variants in trophoblast cell models (BeWo) where STOX1 was previously invalidated. We firstly showed that STOX1 knockout alters response to oxidative stress, cell proliferation, and fusion capacity. Then, we showed that mutant versions of STOX1 trigger alterations in gene profiles, growth, fusion, and oxidative stress management. The results also reveal alterations of the STOX interaction with DNA when the mutations affected the DNA-binding domain of STOX1 (Y153H and T188N). We also reveal here that a major contributor of these effects appears to be the E2F3 transcription factor.

Structural Modelling of KCNQ1 and KCNH2 Double Mutant Proteins, Identified in Two Severe Long QT Syndrome Cases, Reveals New Insights into Cardiac Channelopathies.

Congenital long QT syndrome (LQTS) is a cardiac channelopathy characterized by a prolongation of the QT interval and T-wave abnormalities, caused, in most cases, by mutations in KCNQ1, KCNH2, and SCN5A. Although the predominant pattern of LQTS inheritance is autosomal dominant, compound heterozygous mutations in genes encoding potassium channels have been reported, often with early disease onset and more severe phenotypes. Since the molecular mechanisms underlying severe phenotypes in carriers of compound heterozygous mutations are unknown, it is possible that these compound mutations lead to synergistic or additive alterations to channel structure and function. In this study, all-atom molecular dynamic simulations of KCNQ1 and hERG channels were carried out, including wild-type and channels with compound mutations found in two patients with severe LQTS phenotypes and limited family history of the disease. Because channels can likely incorporate different subunit combinations from different alleles, there are multiple possible configurations of ion channels in LQTS patients. This analysis allowed us to establish the structural impact of different configurations of mutant channels in the activated/open state. Our data suggest that channels with these mutations show moderate changes in folding energy (in most cases of stabilizing character) and changes in channel mobility and volume, differentiating them from each other and from WT. This would indicate possible alterations in K+ ion flow. Hetero-tetrameric mutant channels showed intermediate structural and volume alterations vis-à-vis homo-tetrameric channels. These findings support the hypothesis that hetero-tetrameric channels in patients with compound heterozygous mutations do not necessarily lead to synergistic structural alterations.

A Pharmacogenetic Study of CYP2C19 in Acute Coronary Syndrome Patients of Colombian Origin Reveals New Polymorphisms Potentially Related to Clopidogrel Therapy.

Clopidogrel, an oral platelet P2Y12 receptor blocker, is used in the treatment of acute coronary syndrome. Interindividual variability in treatment response and the occurrence of adverse effects has been attributed to genetic variants in CYP2C19. The analysis of relevant pharmacogenes in ethnically heterogeneous and poorly studied populations contributes to the implementation of personalized medicine. We analyzed the coding and regulatory regions of CYP2C19 in 166 patients with acute coronary syndrome (ACS) treated with clopidogrel. The allele frequencies of CYP2C19 alleles *1, *2, *4, *17, *27 and *33 alleles were 86.1%, 7.2%, 0.3%, 10.2%, 0.3% and 0.3%, respectively. A new potentially pathogenic mutation (p.L15H) and five intronic variants with potential splicing effects were detected. In 14.4% of the patients, a new haplotype in strong linkage disequilibrium was identified. The clinical outcome indicated that 13.5% of the patients presented adverse drugs reactions with a predominance of bleeding while 25% of these patients were carriers of at least one polymorphic allele. We propose that new regulatory single-nucleotide variants (SNVs) might potentially influence the response to clopidogrel in Colombian individuals.

Whole-Exome Sequencing in Patients Affected by Stevens-Johnson Syndrome and Toxic Epidermal Necrolysis Reveals New Variants Potentially Contributing to the Phenotype.

BACKGROUND: Adverse drug reactions (ADRs) are frequent occurring events that can essentially be defined as harmful or unpleasant symptoms secondary to the use of a medicinal product. ADRs involve a wide spectrum of clinical manifestations ranging from minor itching and rash to life-threatening reactions. Stevens-Johnson syndrome (SJS) and toxic epidermal necrolysis (TEN) are rare ADRs. SJS-TEN may be considered a polygenic pathology due to additive/epistatic effects caused by sequence variants in numerous genes. Next-generation sequencing (NGS) represents a potentially interesting exploration tool in such scenario as it facilitates the simultaneous analysis of large genomic regions and genes at affordable cost. METHODS: The present study has involved using whole-exome sequencing (WES) for the first time on SJS-TEN patients. It involved robust and innovative multistep bioinformatics analysis focusing on 313 candidate genes potentially participating in the disease's aetiology, specific drugs' metabolism and gene regulation. RESULTS: We identified combinations of frequently occurring and rare variants that may contribute to the disease's pathogenesis. Depending on the specific drug being taken, different variants (and alleles) in NAT2, CYP2D8, CYP2B6, ABCC2, UGT2B7 and TCF3 were identified as coherent candidates representing potential future markers for SJS-TEN. CONCLUSION: The present study proposed and has described (for the first time) a large-scale genomic analysis of patients affected by SJS-TEN. The genes and variants identified represent relevant candidates potentially participating in the disease's pathogenesis. Corroborating that proposed by others, we found that complex combinations of frequently occurring and rare variants participating in particular drug metabolism molecular cascades could be associated with the phenotype. TCF3 TF may be considered a coherent candidate for SJS-TEN that should be analysed in new cohorts of patients having ADRs.

Identifying new potential genetic biomarkers for HELLP syndrome using massive parallel sequencing.

BACKGROUND: Preeclampsia (PE) is a frequently occurring multisystemic disease affecting ~5% of pregnancies. PE patients may develop HELLP syndrome (haemolysis, elevated liver enzymes, and low platelet), a mother and foetus life-threatening condition. Research into HELLP's genetic origin has been relatively unsuccessful, mainly because normal placental function and blood pressure regulation involve the fine-regulation of hundreds of genes. OBJECTIVE: To identify new genes and mutations constituting potential biomarkers for HELLP syndrome. STUDY DESIGN: The present case-control study involved whole-exome sequencing of 79 unrelated HELLP women. Candidate variants were screened in a control population constituted by 176 individuals. Stringent bioinformatics filters were used for selecting potentially etiological sequence variants in a subset of 487 genes. We used robust in silico mutation modelling for predicting the potential effect on protein structure. RESULTS: We identified numerous sequence variants in genes related to angiogenesis/coagulation/blood pressure regulation, cell differentiation/communication/adhesion, cell cycle and transcriptional gene regulation, extracellular matrix biology, lipid metabolism and immunological response. Five sequence variants generated premature stop codons in genes playing an essential role in placental physiology (STOX1, PDGFD, IGF2, MMP1 and DNAH11). Six variants (ERAP1- p.Ile915Thr, ERAP2- p.Leu837Ser, COMT-p.His192Gln, CSAD-p.Pro418Ser, CDH1- p.Ala298Thr and CCR2-p.Met249Lys) led to destabilisation of protein structure as they had significant energy and residue interaction-related changes. We identified at least two mutations in 57% of patients, arguing in favour of a polygenic origin for the HELLP syndrome. CONCLUSION: Our results provide novel evidence regarding PE/HELLP's genetic origin, leading to new biomarkers, having potential clinical usefulness, being proposed.

DHH pathogenic variants involved in 46,XY disorders of sex development differentially impact protein self-cleavage and structural conformation.

In humans, pathogenic variants in the DHH gene underlie cases of 46,XY gonadal dysgenesis. DHH is part of the Hedgehog family of proteins, which require extensive processing, including self-cleavage of the precursor for efficient signalling. In our work, we have assessed the effect of several human DHH pathogenic variants involved in recessive complete or partial gonadal dysgenesis, on protein processing and sub-cellular localization. We found that a subset of variants was unable to perform self-cleavage, which correlated albeit not perfectly with an altered subcellular localization of the resulting proteins. For the processing-proficient variants, we used structural modelling tools and molecular dynamic (MD) simulations to predict the potential impact of the variants on protein conformation and/or interaction with partners. Our study contributes to a better understanding of the molecular mechanisms involved in DHH dysfunction leading to 46,XY disorders of sex development.

Genetic Variants Contributing to Early Recurrent Pregnancy Loss Etiology Identified by Sequencing Approaches.

Recurrent pregnancy loss (RPL) affects up to 5% of couples. It is believed that genetic factors contribute to the disease's etiology and pathophysiology. Hundreds of genes represent coherent RPL candidates due to mammalian implantation's inherent complexity. Sanger sequencing (direct sequencing) of candidate genes has identified potential RPL causative genes (and variants), including those regulating embryo implantation and pregnancy maintenance. Although this approach is a reliable technique, the simultaneous analysis of large genomic regions is challenging. Next-generation sequencing (NGS) technology has thus emerged as a useful alternative for determining genetic variants and transcriptomic disturbances contributing to monogenic and polygenic diseases pathogenesis. However, interpreting results remains challenging as NGS experiments provide an enormous amount of complex data. The molecular aspects of specific diseases must be fully understood for accurate interpretation of NGS data. This review was thus aimed at describing (for the first time) the most relevant studies involving Sanger and NGS sequencing, leading to the description of variants related to RPL pathogenesis. Successful RPL-related NGS initiatives (including RNAseq-based studies) and future challenges are discussed. We consider that the information given here should be useful for clinicians, scientists, and students to enable a better understanding of RPL etiology. It may also provide a basis for the development of diagnostic/prognostic approaches contributing toward translational medicine.

Molecular Mechanisms of Trophoblast Dysfunction Mediated by Imbalance between STOX1 Isoforms.

STOX1 is a transcription factor involved in preeclampsia and Alzheimer disease. We show that the knock-down of the gene induces rather mild effect on gene expression in trophoblast cell lines (BeWo). We identified binding sites of STOX1 shared by the two major isoforms, STOX1A and STOX1B. Profiling gene expression of cells overexpressing either STOX1A or STOX1B, we identified genes downregulated by both isoforms, with a STOX1 binding site in their promoters. Among those, STOX1-induced Annexin A1 downregulation led to abolished membrane repair in BeWo cells. By contrast, overexpression of STOX1A or B has opposite effects on trophoblast fusion (acceleration and inhibition, respectively) accompanied by syncytin genes deregulation. Also, STOX1A overexpression led to abnormal regulation of oxidative and nitrosative stress. In sum, our work shows that STOX1 isoform imbalance is a cause of gene expression deregulation in the trophoblast, possibly leading to placental dysfunction and preeclampsia.

Possible Genetic Determinants of Response to Phenytoin in a Group of Colombian Patients With Epilepsy.

BACKGROUND: Epilepsy is a serious health problem worldwide. Despite the introduction of new antiepileptic drugs (AEDs) almost 30% of these patients have drug-resistant forms of the disease (DRE), with a significant increase in morbi-mortality. OBJECTIVE: Our objective was to assess the impact of some genetic factors and its possible association with treatment response and adverse drug reactions (ADRs) to phenytoin in 67 adult Colombian patients with epilepsy. METHODS: We conducted an analytical, observational, prospective cohort study to screen four polymorphisms in pharmacogenes: CYP2C9*2-c.430C>T (rs1799853), CYP2C9*3-c.1075A>C (rs1057910), ABCB1-c.3435T>C (rs1045642), and SCN1A-IVS5-91G>A (rs3812718), and their association with treatment response. Patients were followed for 1 year to confirm the existence of DRE (non-response) and ADRs using an active pharmacovigilance approach, followed by a consensus in order to classify ADRs according to causality, preventability, intensity and their relation with phenytoin dose, the duration of treatment, and susceptibility factors (DoTS methodology). RESULTS: A little more than half of evaluated subjects (52.2%) were non-responding to phenytoin. Regarding the genotype-phenotype correlation there was no association between polymorphisms of SCN1A and ABCB1 and DRE (non-response) (p = 0.34), and neither with CYP2C9 polymorphisms and the occurrence of ADRs (p = 0.42). We only found an association between polymorphic alleles of CYP2C9 and vestibular-cerebellar ADRs (dizziness, ataxia, diplopia, and dysarthria) (p = 0.001). Alleles CYP2C9*2-c.430C>T and CYP2C9*3-c.1075A>C were identified as susceptibility factors to ADRs in 24% of patients. CONCLUSIONS: Decreased function alleles of CYP2C9 were highly predictive of vestibular-cerebellar ADRs to phenytoin in our study (p = 0.001). However, the genetic variants CYP2C9*2-c.430C>T, CYP2C9*3-c.1075A>C, ABCB1-c.3435T>C, and SCN1A-IVS5-91G>A, were not associated with treatment response in our study.

A Pharmacogenomic Dissection of a Rosuvastatin-Induced Rhabdomyolysis Case Evokes the Polygenic Nature of Adverse Drug Reactions.

Rosuvastatin, is a widely-used statin for the treatment of hypercholesterolemia and the prevention of cardiovascular diseases. Although rosuvastatin is well tolerated, about 3/10.000 patients can suffer severe myopathy. Rhabdomyolysis is a severe medical condition that causes injury to the skeletal muscle, electrolyte imbalances, acute renal failure and extreme creatine kinase (CK) elevation. Little is known regarding the molecular involvement of rosuvastatin-induced rhabdomyolysis (RIR). It has been demonstrated that genomic variants associated with decreased enzymatic activity of proteins are important determinants in plasmatic and skeletal muscle distribution of rosuvastatin and its toxicity. Until now, no interactions of ticagrelor, ezetimibe and rosuvastatin have been described with the consideration of pharmacogenomics predisposition. The present report involves a whole-exome sequencing (WES), in a patient affected by rosuvastatin-induced rhabdomyolysis. A pharmacogenomic dissection was performed by analyzing a comprehensive subset of candidate genes (n=160) potentially related to RIR. The genes were selected according to their implication in drug metabolism or inherited myopathies. Using an innovative approach of bioinformatics analysis, considering rare and common variants, we identified 19 genomic variations potentially related to the pharmacokinetic/pharmacodynamic modifications of rosuvastatin, ezetimibe and ticagrelor. The affected genes are involved in Phase I metabolism (CYP2C19, CYP2E1, CYP1A1, CYP2D6 and CYP2C9), Phase II metabolism (UGT2B15 and UGT2B7), influx transportation (SLCO1B3 and SLCO2B1), efflux transportation (ABCG8, ABCB11, ABCC4 and ABCB1), drug targeting (NPC1L1) and inherited myopathy etiology (OBSCN). We report three rare, potentially pathogenic molecular variants in CYP2C19, NPC1L1 and OBSCN genes. Pharmacogenetic analysis indicated that the patient was a carrier of inactivating alleles in several pharmacogenes involved in drug toxicity. The whole-exome sequencing and bioinformatics analysis presented here represents an innovative way to identify genomic variants contributing with RIR´s origin and evokes the polygenic nature of adverse drug reactions.

Exploring the Molecular Aetiology of Preeclampsia by Massive Parallel Sequencing of DNA.

PURPOSE OF REVIEW: This manuscript aims to review (for the first time) studies describing NGS sequencing of preeclampsia (PE) women's DNA. RECENT FINDINGS: Describing markers for the early detection of PE is an essential task because, although associated molecular dysfunction begins early on during pregnancy, the disease's clinical signs usually appear late in pregnancy. Although several biochemical biomarkers have been proposed, their use in clinical environments is still limited, thereby encouraging research into PE's genetic origin. Hundreds of genes involved in numerous implantation- and placentation-related biological processes may be coherent candidates for PE aetiology. Next-generation sequencing (NGS) offers new technical possibilities for PE studying, as it enables large genomic regions to be analysed at affordable cost. This technique has facilitated the description of genes contributing to the molecular origin of a significant amount of monogenic and complex diseases. Regarding PE, NGS of DNA has been used in familial and isolated cases, thereby enabling new genes potentially related to the phenotype to be proposed. For a better understanding of NGS, technical aspects, applications and limitations are presented initially. Thereafter, NGS studies of DNA in familial and non-familial cases are described, including pitfalls and positive findings. The information given here should enable scientists and clinicians to analyse and design new studies permitting the identification of novel clinically useful molecular PE markers.

BMPR1A and BMPR1B Missense Mutations Cause Primary Ovarian Insufficiency.

CONTEXT: Primary ovarian insufficiency (POI) is a frequently occurring disorder affecting approximately 1% of women under 40 years of age. POI, which is characterized by the premature depletion of ovarian follicles and elevated plasma levels of follicle-stimulating hormone, leads to infertility. Although various etiological factors have been described, including chromosomal abnormalities and gene mutations, most cases remain idiopathic. OBJECTIVE: To identify and to functionally validate new sequence variants in 2 genes that play a key role in mammalian ovarian function, BMPR1A and BMPR1B (encoding for bone morphogenic protein receptor), leading to POI. METHODS: The impact on bone morphogenic protein (BMP) signaling of BMPR1A and BMPR1B variants, previously identified by whole-exome sequencing on 69 women affected by isolated POI, was established by different in vitro functional experiments. RESULTS: We demonstrate that the BMPR1A-p.Arg442His and BMPR1B-p.Phe272Leu variants are correctly expressed and located but lead to an impairment of downstream BMP signaling. CONCLUSION: In accordance with infertility observed in mice lacking Bmpr1a in the ovaries and in Bmpr1b-/- mice, our results unveil, for the first time, a link between BMPR1A and BMPR1B variants and the origin of POI. We show that BMP signaling impairment through specific BMPR1A and BMPR1B variants is a novel pathophysiological mechanism involved in human POI. We consider that BMPR1A and BMPR1B variants constitute genetic biomarkers of the origin of POI and have clinical utility.

Mutant GNLY is linked to Stevens-Johnson syndrome and toxic epidermal necrolysis.

Stevens-Johnson syndrome (SJS) and toxic epidermal necrolysis (TEN) are rare severe cutaneous adverse reactions to drugs. Granulysin (GNLY) plays a key role in keratinocyte apoptosis during SJS/TEN pathophysiology. To determine if GNLY-encoding mutations might be related to the protein's functional disturbances, contributing to SJS/TEN pathogenesis, we performed direct sequencing of GNLY's coding region in a group of 19 Colombian SJS/TEN patients. A GNLY genetic screening was implemented in a group of 249 healthy individuals. We identified the c.11G > A heterozygous sequence variant in a TEN case, which creates a premature termination codon (PTC) (p.Trp4Ter). We show that a mutant protein is synthesised, possibly due to a PTC-readthrough mechanism. Functional assays demonstrated that the mutant protein was abnormally located in the nuclear compartment, potentially leading to a toxic effect. Our results argue in favour of GNLY non-synonymous sequence variants contributing to SJS/TEN pathophysiology, thereby constituting a promising, clinically useful molecular biomarker.

FOXD1 mutations are related to repeated implantation failure, intra-uterine growth restriction and preeclampsia.

BACKGROUND: Human reproductive disorders consist of frequently occurring dysfunctions including a broad range of phenotypes affecting fertility and women's health during pregnancy. Several female-related diseases have been associated with hypofertility/infertility phenotypes, such as recurrent pregnancy loss (RPL). Other occurring diseases may be life-threatening for the mother and foetus, such as preeclampsia (PE) and intra-uterine growth restriction (IUGR). FOXD1 was defined as a major molecule involved in embryo implantation in mice and humans by regulating endometrial/placental genes. FOXD1 mutations in human species have been functionally linked to RPL's origin. METHODS: FOXD1 gene mutation screening, in 158 patients affected by PE, IUGR, RPL and repeated implantation failure (RIF), by direct sequencing and bioinformatics analysis. Plasmid constructs including FOXD1 mutations were used to perform in vitro gene reporter assays. RESULTS: Nine non-synonymous sequence variants were identified. Functional experiments revealed that p.His267Tyr and p.Arg57del led to disturbances of promoter transcriptional activity (C3 and PlGF genes). The FOXD1 p.Ala356Gly and p.Ile364Met deleterious mutations (previously found in RPL patients) have been identified in the present work in women suffering PE and IUGR. CONCLUSIONS: Our results argue in favour of FOXD1 mutations' central role in RPL, RIF, IUGR and PE pathogenesis via C3 and PlGF regulation and they describe, for the first time, a functional link between FOXD1 and implantation/placental diseases. FOXD1 could therefore be used in clinical environments as a molecular biomarker for these diseases in the near future.

Copy number variation profiling in pharmacogenetics CYP-450 and GST genes in Colombian population.

BACKGROUND: Copy Number variation (CNVs) in genes related to drug absorption, distribution, metabolism and excretion (ADME) are relevant in the interindividual variability of drug response. Studies of the CNVs in ADME genes in Latin America population are lacking. The objective of the study was to identify the genetic variability of CNVs in CYP-450 and GST genes in a subgroup of individuals of Colombian origin. METHODS: Genomic DNA was isolated from 123 healthy individuals from a Colombian population. Multiplex Ligation-Dependent Probe Amplification (MLPA) was performed for the identification of CNVs in 40 genomic regions of 11 CYP-450 and 3 GST genes. The genetic variability, allelic and genotypic frequencies were analyzed. RESULTS: We found that 13 out of 14 genes had CNVs: 5 (35.7%) exhibited deletions and duplications, while 8 (57.1%) presented either deletions or duplications.. 33.3% of individuals carried deletions and duplications while 49.6% had a unique type of CNV (deletion or duplication). The allelic frequencies of the CYP and GST genes were 0 to 47.6% (allele null), 0 to 17.5% (duplicated alleles) and 37 to 100% (normal alleles). CONCLUSIONS: Our results describe, for the first time, the genomic profile of CNVs in a subgroup of Colombian population in GST and CYP-450 genes. GST genes indicated greater genetic variability than CYP-450 genes. The data obtained contributes to the knowledge of genetic profiles in Latin American subgroups. Although the clinical relevance of CNVs has not been fully established, it is a valuable source of pharmacogenetic variability data with potential involvement in the response to medications.

Genetic Variants Contributing to Early Recurrent Pregnancy Loss Etiology Identified by Sequencing Approaches.

Recurrent pregnancy loss (RPL) affects up to 5% of couples. It is believed that genetic factors contribute to the disease's etiology and pathophysiology. Hundreds of genes represent coherent RPL candidates due to mammalian implantation's inherent complexity. Sanger sequencing (direct sequencing) of candidate genes has identified potential RPL causative genes (and variants), including those regulating embryo implantation and pregnancy maintenance. Although this approach is a reliable technique, the simultaneous analysis of large genomic regions is challenging. Next-generation sequencing (NGS) technology has thus emerged as a useful alternative for determining genetic variants and transcriptomic disturbances contributing to monogenic and polygenic diseases pathogenesis. However, interpreting results remains challenging as NGS experiments provide an enormous amount of complex data. The molecular aspects of specific diseases must be fully understood for accurate interpretation of NGS data. This review was thus aimed at describing (for the first time) the most relevant studies involving Sanger and NGS sequencing, leading to the description of variants related to RPL pathogenesis. Successful RPL-related NGS initiatives (including RNAseq-based studies) and future challenges are discussed. We consider that the information given here should be useful for clinicians, scientists, and students to enable a better understanding of RPL etiology. It may also provide a basis for the development of diagnostic/prognostic approaches contributing toward translational medicine.

The forkhead-box family of transcription factors: key molecular players in colorectal cancer pathogenesis.

Colorectal cancer (CRC) is the third most commonly occurring cancer worldwide and the fourth most frequent cause of death having an oncological origin. It has been found that transcription factors (TF) dysregulation, leading to the significant expression modifications of genes, is a widely distributed phenomenon regarding human malignant neoplasias. These changes are key determinants regarding tumour's behaviour as they contribute to cell differentiation/proliferation, migration and metastasis, as well as resistance to chemotherapeutic agents. The forkhead box (FOX) transcription factor family consists of an evolutionarily conserved group of transcriptional regulators engaged in numerous functions during development and adult life. Their dysfunction has been associated with human diseases. Several FOX gene subgroup transcriptional disturbances, affecting numerous complex molecular cascades, have been linked to a wide range of cancer types highlighting their potential usefulness as molecular biomarkers. At least 14 FOX subgroups have been related to CRC pathogenesis, thereby underlining their role for diagnosis, prognosis and treatment purposes.This manuscript aims to provide, for the first time, a comprehensive review of FOX genes' roles during CRC pathogenesis. The molecular and functional characteristics of most relevant FOX molecules (FOXO, FOXM1, FOXP3) have been described within the context of CRC biology, including their usefulness regarding diagnosis and prognosis. Potential CRC therapeutics (including genome-editing approaches) involving FOX regulation have also been included. Taken together, the information provided here should enable a better understanding of FOX genes' function in CRC pathogenesis for basic science researchers and clinicians.

ATG7 and ATG9A loss-of-function variants trigger autophagy impairment and ovarian failure.

PURPOSE: Primary ovarian insufficiency (POI) is a frequent disorder that affects ~1% of women under 40 years of age. POI, which is characterized by the premature depletion of ovarian follicles and elevated plasma levels of follicle-stimulating hormone (FSH), leads to infertility. Although various etiological factors have been described, including chromosomal abnormalities and gene variants, most cases remain idiopathic. The aim of the present study was to identify and validate functionally new sequence variants in ATG (autophagy-related genes) leading to POI. METHODS: We have reanalyzed, in silico, the exome sequencing data from a previously reported work performed in 69 unrelated POI women. Functional experiments using a classical hallmark of autophagy, the microtubule-associated protein 1 light chain 3ß (LC3), were then used to link these genes to this lysosomal degradation pathway. RESULTS: We venture a functional link between ATG7 and ATG9A variants and POI. We demonstrated that variant ATG7 and ATG9A led to a decrease in autophagosome biosynthesis and consequently to an impairment of autophagy, a key biological process implicated in the preservation of the primordial follicles forming the ovarian reserve. CONCLUSION: Our results unveil that impaired autophagy is a novel pathophysiological mechanism involved in human POI.

Functional evidence implicating NOTCH2 missense mutations in primary ovarian insufficiency etiology.

Primary ovarian insufficiency (POI) is a frequently occurring disease affecting women under 40 years old. Recently, we have analyzed unrelated POI women via whole exome sequencing (WES) and identified NOTCH2 mutations underlying possible functional effects. The present study involved reanalyzing of WES assays. We used in the KGN granulosa-like cell model, a synthetic gene reporter construct driving luciferase gene expression to assess the functional effects of five NOTCH2 mutations identified in POI patients. We found that NOTCH2-p.Ser1804Leu, p.Ala2316Val, and p.Pro2359Ala mutations had a functional impact on the protein's transcriptional activity. The results have demonstrated for the first time that NOTCH2 mutations contribute to POI etiology. We therefore recommend sequencing NOTCH2's open reading frame in large panels of POI patients to establish an accurate genotype-phenotype correlation. We cannot rule out the fact that patients affected by Alagille syndrome carrying NOTCH2 mutations may suffer ovarian dysfunction.

Creating and validating a warfarin pharmacogenetic dosing algorithm for Colombian patients.

PURPOSE: Warfarin is an oral anticoagulant associated with adverse reaction to drugs due to wide inter- and intra-individual dosage variability. Warfarin dosage has been related to non-genetic and genetic factors. CYP2C9 and VKORC1 gene polymorphisms affect warfarin metabolism and dosage. Due to the central role of populations' ethnical and genetic origin on warfarin dosage variability, novel algorithms for Latin American subgroups are necessary to establish safe anticoagulation therapy. PATIENTS AND METHODS: We genotyped CYP2C9*2 (c.430C > T), CYP2C9*3 (c.1075A > C), CYP4F2 (c.1297G > A), and VKORC1 (-1639 G > A) polymorphisms in 152 Colombian patients who received warfarin. We evaluated the impact on the variability of patients' warfarin dose requirements. Multiple linear regression analysis, using genetic and non-genetic variables, was used for creating an algorithm for optimal warfarin maintenance dose. RESULTS: Median weekly prescribed warfarin dosage was significantly lower in patients having the VKORC1-1639 AA genotype and poor CYP2C9*2/*2,*2/*3 metabolizers than their wild-type counterparts. We found a 2.3-fold increase in mean dose for normal sensitivity patients (wild-type VKORC1/CYP2C9 genotypes) compared to the other groups (moderate and high sensitivity); 31.5% of the patients in our study group had warfarin sensitivity-related genotypes. The estimated regression equation accounted for 44.4% of overall variability in regard to warfarin maintenance dose. The algorithm was validated, giving 45.9% correlation (R 2=0.459). CONCLUSION: Our results describe and validate the first algorithm for predicting warfarin maintenance in a Colombian mestizo population and have contributed toward the understanding of pharmacogenetics in a Latin American population subgroup.