Publications by authors named "Filipa Carvalho"

69 Publications

Variant , Defective piRNA Processing, and Azoospermia.

N Engl J Med 2021 08 4;385(8):707-719. Epub 2021 Aug 4.

From the Division of Genetics, Oregon National Primate Research Center, Oregon Health and Science University, Beaverton (L.N., D.F.C.); the Center for Embryonic Cell and Gene Therapy, Oregon Health and Science University, Portland (D.F.C.); the Department of Growth and Reproduction (N.M., J.E.N., R.S., I.G., S.B.W., N.E.S., E.R.-D.M., N.J., K.A.) and the International Center for Research and Research Training in Endocrine Disruption of Male Reproduction and Child Health (N.M., J.E.N., R.S., I.G., S.B.W., N.E.S., E.R.-D.M., N.J., K.A.), Rigshospitalet, and the Department of Cellular and Molecular Medicine, Faculty of Health and Medical Sciences (K.A.), University of Copenhagen, Copenhagen; the Laboratory of Molecular Neurooncology, Neuroscience Institute (R.S.), and the Institute of Biology Systems and Genetic Research (I.G.), Lithuanian University of Health Sciences, Kaunas, Lithuania; the Department of Human Genetics, Donders Institute for Brain, Cognition, and Behavior (M.S.O., G.W.H.), and the Department of Obstetrics and Gynecology (G.W.H.), Radboud University Medical Center, Nijmegen, the Netherlands; Serviço de Genética, Departamento de Patologia, Faculdade de Medicina da Universidade do Porto (F.C., C.J.M.), Instituto de Investigação e Inovação em Saúde, Universidade do Porto (F.C., C.J.M., A.M.L.), and the Institute of Molecular Pathology and Immunology of the University of Porto (A.M.L.) - all in Porto, Portugal; the Andrology and In Vitro Fertilization Laboratory, Department of Surgery (Urology), University of Utah School of Medicine, Salt Lake City (K.I.A.); the Departments of Pathology and Laboratory Medicine (F.K.) and Urology (P.N.S.), Weill Cornell Medicine, New York; and the Biosciences Institute, Faculty of Medical Sciences, Newcastle University, Newcastle upon Tyne, United Kingdom (J.A.V.).

Background: P-element-induced wimpy testis (PIWI)-interacting RNAs (piRNAs) are short (21 to 35 nucleotides in length) and noncoding and are found almost exclusively in germ cells, where they regulate aberrant expression of transposable elements and postmeiotic gene expression. Critical to the processing of piRNAs is the protein poly(A)-specific RNase-like domain containing 1 (PNLDC1), which trims their 3' ends and, when disrupted in mice, causes azoospermia and male infertility.

Methods: We performed exome sequencing on DNA samples from 924 men who had received a diagnosis of nonobstructive azoospermia. Testicular-biopsy samples were analyzed by means of histologic and immunohistochemical tests, in situ hybridization, reverse-transcriptase-quantitative-polymerase-chain-reaction assay, and small-RNA sequencing.

Results: Four unrelated men of Middle Eastern descent who had nonobstructive azoospermia were found to carry mutations in : the first patient had a biallelic stop-gain mutation, p.R452Ter (rs200629089; minor allele frequency, 0.00004); the second, a novel biallelic missense variant, p.P84S; the third, two compound heterozygous mutations consisting of p.M259T (rs141903829; minor allele frequency, 0.0007) and p.L35PfsTer3 (rs754159168; minor allele frequency, 0.00004); and the fourth, a novel biallelic canonical splice acceptor site variant, c.607-2A→T. Testicular histologic findings consistently showed error-prone meiosis and spermatogenic arrest with round spermatids of type Sa as the most advanced population of germ cells. Gene and protein expression of PNLDC1, as well as the piRNA-processing proteins PIWIL1, PIWIL4, MYBL1, and TDRKH, were greatly diminished in cells of the testes. Furthermore, the length distribution of piRNAs and the number of pachytene piRNAs was significantly altered in men carrying mutations.

Conclusions: Our results suggest a direct mechanistic effect of faulty piRNA processing on meiosis and spermatogenesis in men, ultimately leading to male infertility. (Funded by Innovation Fund Denmark and others.).
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http://dx.doi.org/10.1056/NEJMoa2028973DOI Listing
August 2021

Effect and in silico characterization of genetic variants associated with severe spermatogenic disorders in a large Iberian cohort.

Andrology 2021 07 20;9(4):1151-1165. Epub 2021 Apr 20.

Instituto de Investigación Biosanitaria ibs.GRANADA, Granada, Spain.

Background: Severe spermatogenic failure (SpF) represents the most extreme manifestation of male infertility, as it decreases drastically the semen quality leading to either severe oligospermia (SO, <5 million spermatozoa/mL semen) or non-obstructive azoospermia (NOA, complete lack of spermatozoa in the ejaculate without obstructive causes).

Objectives: The main objective of the present study is to analyze in the Iberian population the effect of 6 single-nucleotide polymorphisms (SNPs) previously associated with NOA in Han Chinese through genome-wide association studies (GWAS) and to establish their possible functional relevance in the development of specific SpF patterns.

Materials And Methods: We genotyped 674 Iberian infertile men (including 480 NOA and 194 SO patients) and 1058 matched unaffected controls for the GWAS-associated variants PRMT6-rs12097821, PEX10-rs2477686, CDC42BPA-rs3000811, IL17A-rs13206743, ABLIM1-rs7099208, and SOX5-rs10842262. Their association with SpF, SO, NOA, and different NOA phenotypes was evaluated by logistic regression models, and their functional relevance was defined by comprehensive interrogation of public resources.

Results: ABLIM1-rs7099208 was associated with SpF under both additive (OR = 0.86, p = 0.036) and dominant models (OR = 0.78, p = 0.026). The CDC42BPA-rs3000811 minor allele frequency was significantly increased in the subgroup of NOA patients showing maturation arrest (MA) of germ cells compared to the remaining NOA cases under the recessive model (OR = 4.45, p = 0.044). The PEX10-rs2477686 SNP was associated with a negative testicular sperm extraction (TESE) outcome under the additive model (OR = 1.32, p = 0.034). The analysis of functional annotations suggested that these variants affect the testis-specific expression of nearby genes and that lincRNA may play a role in SpF.

Conclusions: Our data support the association of three previously reported NOA risk variants in Asians (ABLIM1-rs7099208, CDC42BPA-rs3000811, and PEX10-rs2477686) with different manifestations of SpF in Iberians of European descent, likely by influencing gene expression and lincRNA deregulation.
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http://dx.doi.org/10.1111/andr.13009DOI Listing
July 2021

Evaluation of Male Fertility-Associated Loci in a European Population of Patients with Severe Spermatogenic Impairment.

J Pers Med 2020 Dec 29;11(1). Epub 2020 Dec 29.

Departamento de Genética Humana, Instituto Nacional de Saúde Dr. Ricardo Jorge, 1649-016 Lisbon, Portugal.

Infertility is a growing concern in developed societies. Two extreme phenotypes of male infertility are non-obstructive azoospermia (NOA) and severe oligospermia (SO), which are characterized by severe spermatogenic failure (SpF). We designed a genetic association study comprising 725 Iberian infertile men as a consequence of SpF and 1058 unaffected controls to evaluate whether five single-nucleotide polymorphisms (SNPs), previously associated with reduced fertility in Hutterites, are also involved in the genetic susceptibility to idiopathic SpF and specific clinical entities. A significant difference in the allele frequencies of -rs7174015 was observed under the recessive model between the NOA group and both the control group ( = 0.0226, OR = 1.33) and the SO group ( = 0.0048, OR = 1.78). Other genetic associations for -rs12870438 and -rs7867029 with SO and between -rs10966811 and testicular sperm extraction (TESE) success in the context of NOA were observed. In silico analysis of functional annotations demonstrated cis-eQTL effects of such SNPs likely due to the modification of binding motif sites for relevant transcription factors of the spermatogenic process. The findings reported here shed light on the molecular mechanisms leading to severe phenotypes of idiopathic male infertility, and may help to better understand the contribution of the common genetic variation to the development of these conditions.
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http://dx.doi.org/10.3390/jpm11010022DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7823507PMC
December 2020

Premature ovarian insufficiency: clinical orientations for genetic testing and genetic counseling.

Porto Biomed J 2020 May-Jun;5(3):e62. Epub 2020 Jun 4.

Department of Pathology, Genetics service, Faculty of Medicine.

Premature ovarian insufficiency (POI) is a heterogeneous disorder diagnosed in women before 40 years old and describes a wide range of impaired ovarian function, from diminished ovarian reserve to premature ovarian failure. Genetic etiology accounts for 20% to 25% of patients. The evidence that POI can be isolated (nonsyndromic) or part of a pleiotropic genetic syndrome highlights its high heterogeneous etiology. Chromosomal abnormalities as a cause of POI have a prevalence of 10% to 13%, being 45,X complement the most common cytogenetic cause of primary amenorrhea and mosaicism with a 45,X cell line more frequently associated with secondary amenorrhea. Other X chromosome aberrations include deletions, duplications, balanced, and unbalanced X-autosome rearrangements involving the critical region for the POI phenotype (Xq13-Xq21 to Xq23-Xq27). The identification of 2 or more pathogenic variants in distinct genes argues in favor of a polygenic origin for POI. Hundreds of pathogenic variants (including mitochondrial) have been involved in POI etiology mainly with key roles in biological processes in the ovary, such as meiosis and DNA damage repair mechanism, homologous recombination, follicular development, granulosa cell differentiation and proliferation, and ovulation. The most common single gene cause for POI is the premutation for gene (associated with fragile X syndrome) with alleles ranging from about 55 to about 200 CGG trinucleotide repeats. POI occurs in 20% of women with this premutation. As females with premutation or full mutation alleles are also at risk of having affected children, their genetic counseling should include the indication for prenatal diagnosis or preimplantation genetic testing after intracytoplasmic sperm injection and trophectoderm biopsy. In conclusion, in clinical practice high-resolution karyotype and gene molecular study should be performed as first-tier tests in the assessment of POI. In addition, array Comparative Genomic Hybridization or specific next generation sequencing panels should be considered to identify chromosomal deletions/duplications under karyotype resolution or other pathogenic variants in specific genes associated with POI. This is particularly important in patients with first- or second-degree relatives also affected with POI, improving their reproductive and genetic counseling.
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http://dx.doi.org/10.1097/j.pbj.0000000000000062DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7722400PMC
June 2020

Epimutations in human sperm from patients with impaired spermatogenesis.

Clin Epigenetics 2020 11 17;12(1):172. Epub 2020 Nov 17.

Centre for Reproductive Genetics A Barros, 4100-009, Porto, Portugal.

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http://dx.doi.org/10.1186/s13148-020-00919-0DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7670606PMC
November 2020

Intronic variation of the SOHLH2 gene confers risk to male reproductive impairment.

Fertil Steril 2020 08 18;114(2):398-406. Epub 2020 Jul 18.

Departamento de Genética e Instituto de Biotecnología, Universidad de Granada, Granada, Spain; Instituto de Investigación Biosanitaria ibs.GRANADA, Granada, Spain.

Objective: To evaluate whether SOHLH2 intronic variation contributes to the genetic predisposition to male infertility traits, including severe oligospermia (SO) and different nonobstructive azoospermia (NOA) clinical phenotypes.

Design: Genetic association study.

Setting: Not applicable.

Patient(s): Five hundred five cases (455 infertile patients diagnosed with NOA and 50 with SO) and 1,050 healthy controls from Spain and Portugal.

Intervention(s): None.

Main Outcome Measure(s): Genomic DNA extraction from peripheral blood mononuclear cells, genotyping of the SOHLH2 polymorphisms rs1328626 and rs6563386 using the TaqMan allelic discrimination technology, case-control association analyses using logistic regression models, and exploration of functional annotations in publicly available databases.

Result(s): Evidence of association was observed for both rs6563386 with SO and rs1328626 with unsuccessful sperm retrieval after testicular sperm extraction (TESE-) in the context of NOA. A dominant effect of the minor alleles was suggested in both associations, either when the subset of patients with the manifestation were compared against the control group (rs6563386/SO: P=.021, odds ratio [OR] = 0.51; rs1328626/TESE-: P=.066, OR = 1.46) or against the group of patients without the manifestation (rs6563386/SO: P=.014, OR = 0.46; rs1328626/TESE-: P=.012, OR = 2.43). The haplotype tests suggested a combined effect of both polymorphisms. In silico analyses evidenced that this effect could be due to alteration of the isoform population.

Conclusion(s): Our data suggest that intronic variation of SOHLH2 is associated with spermatogenic failure. The genetic effect is likely caused by different haplotypes of rs6563386 and rs1328626, which may predispose to SO or TESE- depending on the specific allelic combination.
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http://dx.doi.org/10.1016/j.fertnstert.2020.02.115DOI Listing
August 2020

Bi-allelic Mutations in M1AP Are a Frequent Cause of Meiotic Arrest and Severely Impaired Spermatogenesis Leading to Male Infertility.

Am J Hum Genet 2020 08 15;107(2):342-351. Epub 2020 Jul 15.

Centre of Reproductive Medicine and Andrology, Institute of Reproductive Medicine, University of Münster, 48149 Münster, Germany.

Male infertility affects ∼7% of men, but its causes remain poorly understood. The most severe form is non-obstructive azoospermia (NOA), which is, in part, caused by an arrest at meiosis. So far, only a few validated disease-associated genes have been reported. To address this gap, we performed whole-exome sequencing in 58 men with unexplained meiotic arrest and identified the same homozygous frameshift variant c.676dup (p.Trp226LeufsTer4) in M1AP, encoding meiosis 1 associated protein, in three unrelated men. This variant most likely results in a truncated protein as shown in vitro by heterologous expression of mutant M1AP. Next, we screened four large cohorts of infertile men and identified three additional individuals carrying homozygous c.676dup and three carrying combinations of this and other likely causal variants in M1AP. Moreover, a homozygous missense variant, c.1166C>T (p.Pro389Leu), segregated with infertility in five men from a consanguineous Turkish family. The common phenotype between all affected men was NOA, but occasionally spermatids and rarely a few spermatozoa in the semen were observed. A similar phenotype has been described for mice with disruption of M1ap. Collectively, these findings demonstrate that mutations in M1AP are a relatively frequent cause of autosomal recessive severe spermatogenic failure and male infertility with strong clinical validity.
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http://dx.doi.org/10.1016/j.ajhg.2020.06.010DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7413853PMC
August 2020

Two Sides of Child Maltreatment: from Psychopathic Traits to Altruistic Attitudes Inhibition.

J Child Adolesc Trauma 2020 Jun 3;13(2):199-206. Epub 2019 Aug 3.

William James Center for Research, ISPA-Instituto Universitário, Lisbon, Portugal.

Child maltreatment is a known risk factor for criminal behavior, however, only a few studies have addressed the relationship between child maltreatment and the development of psychopathic traits. Meanwhile, the effect of adverse childhood experiences on prosocial behavior is practically unknown. The current research aims to explore the relationship between child maltreatment, psychopathic traits and altruistic attitudes among young adults. Six hundred and seventy-three young adults from the community filled out the Adverse Childhood Experience Questionnaire, the Youth Psychopathic Traits Inventory - Short Version and the Altruistic Attitudes Scale. Results suggest that child maltreatment is related to both psychopathic traits and inhibition of altruistic altitudes. Each adverse childhood experience appears to be associated with the development of specific forms of psychopathic traits and/or altruistic attitudes. Early identification of maltreatment is essential for prevention of antisocial behavior and for the promotion of altruistic attitudes. Research, political and social recommendations are suggested.
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http://dx.doi.org/10.1007/s40653-019-00280-2DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7289941PMC
June 2020

ESHRE PGT Consortium good practice recommendations for the organisation of PGT.

Hum Reprod Open 2020 29;2020(3):hoaa021. Epub 2020 May 29.

Centre for Medical Genetics, Universitair Ziekenhuis Brussel, Brussels, Belgium.

The field of preimplantation genetic testing (PGT) is evolving fast, and best practice advice is essential for regulation and standardisation of diagnostic testing. The previous ESHRE guidelines on best practice for preimplantation genetic diagnosis, published in 2005 and 2011, are considered outdated and the development of new papers outlining recommendations for good practice in PGT was necessary. The current updated version of the recommendations for good practice is, similar to the 2011 version, split into four documents, one of which covers the organisation of a PGT centre. The other documents focus on the different technical aspects of embryo biopsy, PGT for monogenic/single-gene defects (PGT-M) and PGT for chromosomal structural rearrangements/aneuploidies (PGT-SR/PGT-A). The current document outlines the steps prior to starting a PGT cycle, with details on patient inclusion and exclusion, and counselling and information provision. Also, recommendations are provided on the follow-up of PGT pregnancies and babies. Finally, some further recommendations are made on the practical organisation of an IVF/PGT centre, including basic requirements, transport PGT and quality management. This document, together with the documents on embryo biopsy, PGT-M and PGT-SR/PGT-A, should assist everyone interested in PGT in developing the best laboratory and clinical practice possible.
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http://dx.doi.org/10.1093/hropen/hoaa021DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7257038PMC
May 2020

ESHRE PGT Consortium good practice recommendations for the detection of monogenic disorders.

Hum Reprod Open 2020 29;2020(3):hoaa018. Epub 2020 May 29.

Centre for Medical Genetics, Universitair Ziekenhuis Brussel, Brussels, Belgium.

The field of preimplantation genetic testing (PGT) is evolving fast and best practice advice is essential for regulation and standardisation of diagnostic testing. The previous ESHRE guidelines on best practice for PGD, published in 2005 and 2011, are considered outdated, and the development of new papers outlining recommendations for good practice in PGT was necessary. The current paper provides recommendations on the technical aspects of PGT for monogenic/single-gene defects (PGT-M) and covers recommendations on basic methods for PGT-M and testing strategies. Furthermore, some specific recommendations are formulated for special cases, including pathogenic variants, consanguineous couples, HLA typing, exclusion testing and disorders caused by pathogenic variants in the mitochondrial DNA. This paper is one of a series of four papers on good practice recommendations on PGT. The other papers cover the organisation of a PGT centre, embryo biopsy and tubing and the technical aspects of PGT for chromosomal structural rearrangements/aneuploidies. Together, these papers should assist scientists interested in PGT in developing the best laboratory and clinical practice possible.
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http://dx.doi.org/10.1093/hropen/hoaa018DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7257022PMC
May 2020

Early Gestational Diagnosis of Lethal Skeletal Dysplasias: A 15 Year Retrospective Cohort Reviewing Concordance between Ultrasonographic, Genetic and Morphological Features.

Fetal Pediatr Pathol 2020 May 13:1-12. Epub 2020 May 13.

Department of Obstetrics, Gynecology and Pediatrics Faculty of Medicine University of Porto, Portugal.

We evaluated the diagnostic accuracy of ultrasound, postmortem and genetic studies in classifying skeletal dysplasias in the first vs second trimester of pregnancy. We retrospectively gathered data from a 15 year period of all the prenatal ultrasounds, autopsies, and available genetic studies on fetuses with skeletal dysplasias from our institution. Five (23%) and 17 (77%) fetuses were diagnosed during the first and second trimester of pregnancy respectively. Only partial characterization was possible with ultrasound in the first trimester. Complete characterization was established in five cases (30%) in the second trimester with ultrasound alone. Pathology provided an additional diagnostic yield of 40% and 47% and genetics an additional 40% and 11% in the first and second trimesters respectively. Ultrasound is an effective screening but not a diagnostic tool. Complete characterizations of dysplasia increased from 22% by ultrasound alone to 86% with pathology and genetics.
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http://dx.doi.org/10.1080/15513815.2020.1761915DOI Listing
May 2020

Congenital Adrenal Hyperplasia Due to 21-Hydroxylase Deficiency: An Update on Genetic Analysis of CYP21A2 Gene.

Exp Clin Endocrinol Diabetes 2021 Jul 4;129(7):477-481. Epub 2020 Mar 4.

Genetics, Department of Pathology, Faculty of Medicine, University of Porto, Porto, Portugal.

Congenital Adrenal Hyperplasia is a group of genetic autosomal recessive disorders that affects adrenal steroidogenesis in the adrenal cortex. One of the most common defects associated with Congenital Adrenal Hyperplasia is the deficiency of 21-hydroxylase enzyme, responsible for the conversion of 17-hydroxyprogesterone to 11-deoxycortisol and progesterone to deoxycorticosterone. The impairment of cortisol and aldosterone production is directly related to the clinical form of the disease that ranges from classic or severe to non-classic or mild late onset. The deficiency of 21-hydroxylase enzyme results from pathogenic variants on gene that, in the majority of the cases, compromise enzymatic activity and are strongly correlated with the clinical severity of the disease. Due to the exceptionally high homology and proximity between the gene and the pseudogene, more than 90% of pathogenic variants result from intergenic recombination. Around 75% are deleterious variants transferred from the pseudogene by gene conversion, during mitosis. About 20% are due to unequal crossing over during meiosis and lead to duplications or deletions on gene. Molecular genetic analysis of variants is of major importance for confirmation of clinical diagnosis, predicting prognosis and for an appropriate genetic counselling. In this review we will present an update on the genetic analysis of gene variants in CAH patients performed in our department.
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http://dx.doi.org/10.1055/a-1108-1419DOI Listing
July 2021

Preimplantation genetic testing for Huntington disease: the perspective of one Portuguese center.

Porto Biomed J 2019 Sep-Oct;4(5):e48. Epub 2019 Sep 4.

Serviço de Genética, Departamento de Patologia, Faculdade de Medicina, University of Porto.

Background: Huntington disease (HD) is an autosomal dominant late-onset neurodegenerative disease caused by an unstable cytosine-adenine-guanine trinucleotide repeat expansion in the huntingtin () gene. Preimplantation genetic testing (PGT) is a diagnostic procedure available for these individuals, because they carry a high risk of transmitting this genetic condition to their offspring.

Methods: Information about 15 HD couples referred for PGT and 21 cycles performed from 2009 to 2018 was collected retrospectively. PGT provide direct testing of embryos obtained after intracytoplasmic sperm injection, using polymerase chain reaction multiplex as the genetic testing protocol.

Results: PGT for HD was performed in 15 couples, with no history of previous attempts, in a total of 21 cycles. The mean number of biopsied embryos per cycle was 4.9. The amplification efficiency in blastomeres was 87.4%. From the 90 amplified embryos, 32 were normal and suitable for transfer. The mean number of transferred embryos per couple was 1.2.Overall, 3 positive human chorionic gonadotropin tests were obtained in 3 couples, resulting in 2 clinical pregnancies. The 2 ongoing clinical pregnancies had normal evolution, and culminated in 2 deliveries, resulting in the birth of 2 healthy children.

Conclusions: PGT for HD is considered an effective and safe reproductive option for couples who are at risk of transmitting HD, when proper genetic and reproductive counseling is warranted.
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http://dx.doi.org/10.1097/j.pbj.0000000000000048DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6924977PMC
September 2019

A Systematic Pan-Cancer Analysis of Genetic Heterogeneity Reveals Associations with Epigenetic Modifiers.

Cancers (Basel) 2019 Mar 20;11(3). Epub 2019 Mar 20.

Instituto de Medicina Molecular João Lobo Antunes, Faculdade de Medicina da Universidade de Lisboa, 1649-028 Lisboa, Portugal.

Intratumor genetic heterogeneity (ITH) is the main obstacle to effective cancer treatment and a major mechanism of drug resistance. It results from the continuous evolution of different clones of a tumor over time. However, the molecular features underlying the emergence of genetically-distinct subclonal cell populations remain elusive. Here, we conducted an exhaustive characterization of ITH across 2807 tumor samples from 16 cancer types. Integration of ITH scores and somatic variants detected in each tumor sample revealed that mutations in epigenetic modifier genes are associated with higher ITH levels. In particular, genes that regulate genome-wide histone and DNA methylation emerged as being determinant of high ITH. Indeed, the knockout of histone methyltransferase SETD2 or DNA methyltransferase DNMT3A using the CRISPR/Cas9 system on cancer cells led to significant expansion of genetically-distinct clones and culminated in highly heterogeneous cell populations. The ITH scores observed in knockout cells recapitulated the heterogeneity levels observed in patient tumor samples and correlated with a better mitochondrial bioenergetic performance under stress conditions. Our work provides new insights into tumor development, and discloses new drivers of ITH, which may be useful as either predictive biomarkers or therapeutic targets to improve cancer treatment.
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http://dx.doi.org/10.3390/cancers11030391DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6468518PMC
March 2019

Central place foragers select ocean surface convergent features despite differing foraging strategies.

Sci Rep 2019 01 17;9(1):157. Epub 2019 Jan 17.

National Oceanography Centre, European Way, Southampton, SO14 3ZH, United Kingdom.

Discovering the predictors of foraging locations can be challenging, and is often the critical missing piece for interpreting the ecological significance of observed movement patterns of predators. This is especially true in dynamic coastal marine systems, where planktonic food resources are diffuse and must be either physically or biologically concentrated to support upper trophic levels. In the Western Antarctic Peninsula, recent climate change has created new foraging sympatry between Adélie (Pygoscelis adeliae) and gentoo (P. papua) penguins in a known biological hotspot near Palmer Deep canyon. We used this recent sympatry as an opportunity to investigate how dynamic local oceanographic features affect aspects of the foraging ecology of these two species. Simulated particle trajectories from measured surface currents were used to investigate the co-occurrence of convergent ocean features and penguin foraging locations. Adélie penguin diving activity was restricted to the upper mixed layer, while gentoo penguins often foraged much deeper than the mixed layer, suggesting that Adélie penguins may be more responsive to dynamic surface convergent features compared to gentoo penguins. We found that, despite large differences in diving and foraging behavior, both shallow-diving Adélie and deeper-diving gentoo penguins strongly selected for surface convergent features. Furthermore, there was no difference in selectivity for shallow- versus deep-diving gentoo penguins. Our results suggest that these two mesopredators are selecting surface convergent features, however, how these surface signals are related to subsurface prey fields is unknown.
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http://dx.doi.org/10.1038/s41598-018-35901-7DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6336854PMC
January 2019

Toxicity of lupane derivatives on anionic membrane models, isolated rat mitochondria and selected human cell lines: Role of terminal alkyl chains.

Chem Biol Interact 2018 Dec 4;296:198-210. Epub 2018 Oct 4.

CNC - Center for Neuroscience and Cell Biology, University of Coimbra, UC Biotech, Biocant Park, Cantanhede, Portugal. Electronic address:

Triterpenoids have multiple biological properties, although little information is available regarding their toxicity. The present study evaluates the toxicity of two new synthetic lupane derivatives using distinct biological models including synthetic lipids membranes, isolated liver and heart mitochondria fractions, and cell lines in culture. The two novel triterpenoids caused perturbations in the organization of synthetic lipid bilayers, leading to changes in membrane fluidity. Inhibition of cell proliferation and mitochondrial and nuclear morphological alterations were also identified. Inhibition of mitochondrial oxygen consumption, transmembrane electric potential depolarization and induction of the mitochondrial permeability transition pore was observed, although effects on isolated mitochondrial fractions were tissue-dependent (e.g. liver vs. heart). The size and length of hydrocarbon chains in the two molecules appear to be determinant for the degree of interaction with mitochondria, especially in the whole cell environment, where more barriers for diffusion exist. The results suggest that the positively charged triterpenoids target mitochondria and disrupt bioenergetics.
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http://dx.doi.org/10.1016/j.cbi.2018.10.002DOI Listing
December 2018

Bi-allelic Recessive Loss-of-Function Variants in FANCM Cause Non-obstructive Azoospermia.

Am J Hum Genet 2018 08;103(2):200-212

Institute of Biomedicine and Translational Medicine, University of Tartu, Tartu 50411, Estonia. Electronic address:

Infertility affects around 7% of men worldwide. Idiopathic non-obstructive azoospermia (NOA) is defined as the absence of spermatozoa in the ejaculate due to failed spermatogenesis. There is a high probability that NOA is caused by rare genetic defects. In this study, whole-exome sequencing (WES) was applied to two Estonian brothers diagnosed with NOA and Sertoli cell-only syndrome (SCOS). Compound heterozygous loss-of-function (LoF) variants in FANCM (Fanconi anemia complementation group M) were detected as the most likely cause for their condition. A rare maternally inherited frameshift variant p.Gln498Thrfs7 (rs761250416) and a previously undescribed splicing variant (c.4387-10A>G) derived from the father introduce a premature STOP codon leading to a truncated protein. FANCM exhibits enhanced testicular expression. In control subjects, immunohistochemical staining localized FANCM to the Sertoli and spermatogenic cells of seminiferous tubules with increasing intensity through germ cell development. This is consistent with its role in maintaining genomic stability in meiosis and mitosis. In the individual with SCOS carrying bi-allelic FANCM LoF variants, none or only faint expression was detected in the Sertoli cells. As further evidence, we detected two additional NOA-affected case subjects with independent FANCM homozygous nonsense variants, one from Estonia (p.Gln1701; rs147021911) and another from Portugal (p.Arg1931; rs144567652). The study convincingly demonstrates that bi-allelic recessive LoF variants in FANCM cause azoospermia. FANCM pathogenic variants have also been linked with doubled risk of familial breast and ovarian cancer, providing an example mechanism for the association between infertility and cancer risk, supported by published data on Fancm mutant mouse models.
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http://dx.doi.org/10.1016/j.ajhg.2018.07.005DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6080835PMC
August 2018

Coccolithovirus facilitation of carbon export in the North Atlantic.

Nat Microbiol 2018 05 12;3(5):537-547. Epub 2018 Mar 12.

Department of Marine and Coastal Sciences, Rutgers University, New Brunswick, NJ, USA.

Marine phytoplankton account for approximately half of global primary productivity , making their fate an important driver of the marine carbon cycle. Viruses are thought to recycle more than one-quarter of oceanic photosynthetically fixed organic carbon , which can stimulate nutrient regeneration, primary production and upper ocean respiration via lytic infection and the 'virus shunt'. Ultimately, this limits the trophic transfer of carbon and energy to both higher food webs and the deep ocean . Using imagery taken by the Moderate Resolution Imaging Spectroradiometer (MODIS) onboard the Aqua satellite, along with a suite of diagnostic lipid- and gene-based molecular biomarkers, in situ optical sensors and sediment traps, we show that Coccolithovirus infections of mesoscale (~100 km) Emiliania huxleyi blooms in the North Atlantic are coupled with particle aggregation, high zooplankton grazing and greater downward vertical fluxes of both particulate organic and particulate inorganic carbon from the upper mixed layer. Our analyses captured blooms in different phases of infection (early, late and post) and revealed the highest export flux in 'early-infected blooms' with sinking particles being disproportionately enriched with infected cells and subsequently remineralized at depth in the mesopelagic. Our findings reveal viral infection as a previously unrecognized ecosystem process enhancing biological pump efficiency.
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http://dx.doi.org/10.1038/s41564-018-0128-4DOI Listing
May 2018

Characterization of microbiota in male infertility cases uncovers differences in seminal hyperviscosity and oligoasthenoteratozoospermia possibly correlated with increased prevalence of infectious bacteria.

Am J Reprod Immunol 2018 06 3;79(6):e12838. Epub 2018 Mar 3.

Instituto de Investigação e Inovação em Saúde, Universidade do Porto (I3S), Porto, Portugal.

Problem: Sexually transmitted diseases and other infections of male genitourinary tract are thought to negatively impact reproductive health, affecting semen quality. Despite a possible link between bacteria and infertility, few studies attempted to characterize seminal microbiota in healthy and diseased subjects.

Methods Of The Study: A high-throughput sequencing of 16S ribosomal RNA gene was performed in a cohort of infertility-related cases (N = 89) and controls (N = 29) using a pooled sample approach.

Results: A global characterization of microbiota was obtained at low cost, without compromising the identification of bacterial taxa. This strategy allowed us to detect changes in the microbiota of infertility-related phenotypes, such as an increment of Proteobacteria in seminal hyperviscosity, and to separate this later group from oligoasthenoteratozoospermia based in bacterial (family/genus) abundances.

Conclusion: We provide data for a likely contribution of bacteria into seminal hyperviscosity and oligoasthenoteratozoospermia, partially correlated with an increment of Neisseria, Klebsiella, and Pseudomonas pathogens and a reduction in Lactobacillus probiotic agent.
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http://dx.doi.org/10.1111/aji.12838DOI Listing
June 2018

Clinical outcomes after preimplantation genetic diagnosis of patients with Corino de Andrade disease (familial amyloid polyneuropathy).

Reprod Biomed Online 2018 Jan 12;36(1):39-46. Epub 2017 Oct 12.

Centre for Reproductive Genetics A. Barros (CGR), Porto, Portugal; Department of Genetics, Faculty of Medicine, Institute of Health Research and Innovation (I3S), University of Porto, Porto, Portugal.

The aim of this study was to determine whether patients with transthyretin-related hereditary amyloidosis (V30M), after transplantation or under tafamidis treatment, have normal gamete reproductive capacity. A retrospective analysis was carried out of all preimplantation genetic diagnosis (PGD) cycles performed in patients with the V30M mutation. The groups analysed were: total cases with V30M, female cases with V30M and male cases with V30M. Detailed demographic, stimulation, embryological, clinical and newborn outcomes were evaluated. Comparisons revealed that patients have a high likelihood of achieving a live birth per PGD treatment cycle (48%). This is the first large report on patients with the V30M mutation treated with PGD. The high rate of live birth obtained should represent a strong stimulus for patients to use PGD as it proved to be effective and safe. As a neurodegenerative disease that leads to death, it is of maximum importance that it could be eradicated using PGD in order to definitively avoid the transmission of the disease.
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http://dx.doi.org/10.1016/j.rbmo.2017.09.010DOI Listing
January 2018

Altered mitochondrial epigenetics associated with subchronic doxorubicin cardiotoxicity.

Toxicology 2017 09 1;390:63-73. Epub 2017 Sep 1.

CNC-Center for Neuroscience and Cell Biology, University of Coimbra, UC Biotech Building, Biocant Park, 3060-197 Cantanhede, Portugal. Electronic address:

Doxorubicin (DOX), a potent and broad-spectrum antineoplastic agent, causes an irreversible, cumulative and dose-dependent cardiomyopathy that ultimately leads to congestive heart failure. The mechanisms responsible for DOX cardiotoxicity remain poorly understood, but seem to involve mitochondrial dysfunction on several levels. Epigenetics may explain a portion of this effect. Since mitochondrial dysfunction may affect the epigenetic landscape, we hypothesize that this cardiac toxicity may result from epigenetic changes related to disruption of mitochondrial function. To test this hypothesis, eight-week-old male Wistar rats (n=6/group) were administered 7 weekly injections with DOX (2mgkg) or saline, and sacrificed two weeks after the last injection. We assessed gene expression patterns by qPCR, global DNA methylation by ELISA, and proteome lysine acetylation status by Western blot in cardiac tissue from saline and DOX-treated rats. We show for the first time that DOX treatment decreases global DNA methylation in heart but not in liver. These differences were accompanied by alterations in mRNA expression of multiple functional gene groups. DOX disrupted cardiac mitochondrial biogenesis, as demonstrated by decreased mtDNA levels and altered transcript levels for multiple mitochondrial genes encoded by both nuclear and mitochondrial genomes. Transcription of genes involved in lipid metabolism and epigenetic modulation were also affected. Western blotting analyses indicated a differential protein acetylation pattern in cardiac mitochondrial fractions of DOX-treated rats compared to controls. Additionally, DOX treatment increased the activity of histone deacetylases. These results suggest an interplay between mitochondrial dysfunction and epigenetic alterations, which may be a primary determinant of DOX-induced cardiotoxicity.
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http://dx.doi.org/10.1016/j.tox.2017.08.011DOI Listing
September 2017

Acute dietary fat intake initiates alterations in energy metabolism and insulin resistance.

J Clin Invest 2017 Feb 23;127(2):695-708. Epub 2017 Jan 23.

Background: Dietary intake of saturated fat is a likely contributor to nonalcoholic fatty liver disease (NAFLD) and insulin resistance, but the mechanisms that initiate these abnormalities in humans remain unclear. We examined the effects of a single oral saturated fat load on insulin sensitivity, hepatic glucose metabolism, and lipid metabolism in humans. Similarly, initiating mechanisms were examined after an equivalent challenge in mice.

Methods: Fourteen lean, healthy individuals randomly received either palm oil (PO) or vehicle (VCL). Hepatic metabolism was analyzed using in vivo 13C/31P/1H and ex vivo 2H magnetic resonance spectroscopy before and during hyperinsulinemic-euglycemic clamps with isotope dilution. Mice underwent identical clamp procedures and hepatic transcriptome analyses.

Results: PO administration decreased whole-body, hepatic, and adipose tissue insulin sensitivity by 25%, 15%, and 34%, respectively. Hepatic triglyceride and ATP content rose by 35% and 16%, respectively. Hepatic gluconeogenesis increased by 70%, and net glycogenolysis declined by 20%. Mouse transcriptomics revealed that PO differentially regulates predicted upstream regulators and pathways, including LPS, members of the TLR and PPAR families, NF-κB, and TNF-related weak inducer of apoptosis (TWEAK).

Conclusion: Saturated fat ingestion rapidly increases hepatic lipid storage, energy metabolism, and insulin resistance. This is accompanied by regulation of hepatic gene expression and signaling that may contribute to development of NAFLD.REGISTRATION. ClinicalTrials.gov NCT01736202.

Funding: Germany: Ministry of Innovation, Science, and Research North Rhine-Westfalia, German Federal Ministry of Health, Federal Ministry of Education and Research, German Center for Diabetes Research, German Research Foundation, and German Diabetes Association. Portugal: Portuguese Foundation for Science and Technology, FEDER - European Regional Development Fund, Portuguese Foundation for Science and Technology, and Rede Nacional de Ressonância Magnética Nuclear.
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http://dx.doi.org/10.1172/JCI89444DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5272194PMC
February 2017

Sequence variation at KLK and WFDC clusters and its association to semen hyperviscosity and other male infertility phenotypes.

Hum Reprod 2016 12 7;31(12):2881-2891. Epub 2016 Nov 7.

Instituto de Investigação e Inovação em Saúde, Universidade do Porto (i3S), 4200-135 Porto, Portugal

Study Question: Are kallikreins (KLKs), the whey-acidic-protein four-disulfide core domain (WFDCs) and their neighbors, semenogelins (SEMGs), known to play a role in the cascade of semen coagulation and liquefaction, associated with male infertility?

Summary Answer: Several KLK and SEMG variants are overrepresented among hyperviscosity, asthenozoospermia and oligozoospermia, supporting an effect of abnormal semen liquefaction on the loss of semen quality and in lowering male reproductive fitness.

What Is Known Already: In the cascade of semen coagulation and liquefaction the spermatozoa coated by EPPIN (a protease inhibitor of the WFDC family) are entrapped in a cross-linked matrix established by SEMGs. After ejaculation, the SEMG matrix is hydrolyzed by KLK3/2 in a fine-tuned process regulated by other KLKs that allows the spermatozoa to increase motility.

Study Design Size, Duration: This study includes a cohort of 238 infertility-related cases and 91 controls with normal spermiogram analysis. The remaining 126 controls are healthy males with unknown semen parameters. Sample collection was carried out from June 2011 to January 2015 and variant screening from May 2013 to August 2015.

Participants/materials, Setting, Methods: We performed a screening by massive parallel sequencing in a pooled sample (N = 222) covering approximately 93 kb of KLK (19q13.3-13.4) and WFDC (20q13) clusters, followed by the genotyping of most promising variants in the full cohort.

Main Results And The Role Of Chance: Overall, 160 common and 296 low-frequency variants passed the quality control filtering. Statistical tests disclosed an association with hyperviscosity of a KLK7 regulatory variant (P = 0.0035), and unveiled a higher burden of deleterious mutations in KLKs than expected by chance (P = 0.0106). KLK variants found to be overrepresented in cases included two substitutions likely affecting the substrate binding pocket, two nonsynonymous variants overlapping in the three-dimensional structure and two mutations mapping in consecutive N-terminal residues. Other variants identified in SEMGs possibly contributing to hyperviscosity and asthenozoospermia consisted of three replacements predicted to modify targets of proteolysis (P = 0.0442 for SEMG1 p.Gly400Asp) and a copy number variation associated with a reduced risk of oligozoospermia (P = 0.0293).

Large Scale Data: Not applicable.

Limitations Reasons For Caution: The sampling of a few hundred individuals has limited power to detected associations with low-frequency variants and only a small set of variants was prioritized for genotyping. Other susceptibility variants for male infertility may remain unidentified.

Wider Implications Of The Findings: We provide important evidence for an effect of KLKs and SEMGs variability on semen quality and for modifications in the process of semen liquefaction as a possible cause for male infertility.

Study Funding/competing Interests: This work was funded through the Portuguese Foundation for Science and Technology (FCT) and FEDER through COMPETE and QREN. The authors have no conflict of interest to declare.
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http://dx.doi.org/10.1093/humrep/dew267DOI Listing
December 2016

Cardiac cytochrome c and cardiolipin depletion during anthracycline-induced chronic depression of mitochondrial function.

Mitochondrion 2016 09 14;30:95-104. Epub 2016 Jul 14.

CNC - Center for Neuroscience and Cell Biology, University of Coimbra, Biocant Park, 3060-197 Cantanhede, Portugal. Electronic address:

Aims: It is still unclear why anthracycline treatment results in a cardiac-specific myopathy. We investigated whether selective doxorubicin (DOX) cardiotoxicity involving mitochondrial degeneration is explained by different respiratory complexes reserves between tissues by comparing and contrasting treatment effects in heart vs liver and kidney. Alternatively, we have also explored if the degeneration is due to alterations of mitochondrial thresholds to incompatible states.

Methods And Results: Heart, liver and kidney mitochondria were isolated from male Wistar rats weekly injected with DOX during 7weeks. Global flux and isolated step curves were obtained for Complex I, III, IV, as well as for the adenine nucleotide translocator. We show treatment-related alterations in global flux curve for Complex III in all analyzed tissues and in Complex IV activity curve solely in heart. However, all mitochondrial threshold curves remained unchanged after treatment in the analyzed tissues. No treatment-related differences were detected on transcript or protein analysis of selected respiratory complexes subunits. However, a specific loss of cytochrome c and cardiolipin was measured in heart, but not in other organs, mitochondria from DOX-treated animals.

Conclusions: Contrary to our hypothesis, impaired mitochondrial respiration could not be explained by intrinsic differences in respiratory complexes reserves among tissues or, by alterations in mitochondrial thresholds after treatment. Instead, we propose that loss of cytochrome c and cardiolipin are responsible for the depressed mitochondrial respiration observed after chronic DOX treatment. Moreover, cardiac cytochrome c and cardiolipin depletion decreases metabolic network buffering, hindering cardiac ability to respond to increased workload, accelerating cardiac aging.
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http://dx.doi.org/10.1016/j.mito.2016.07.005DOI Listing
September 2016

[Anomalous origin of the left coronary artery from the pulmonar artery: a sudden death in the young].

Rev Port Cir Cardiotorac Vasc 2015 Oct-Dec;22(4):215-217

Serviço de Anestesiologia, Centro Hospitalar de Vila Nova de Gaia e Espinho, Portugal.

Anomalous origin of the left coronary artery from the pulmonary artery (ALCAPA or Bland-White-Garland Syndrome) is a rare congenital heart abnormality with a high mortality in the first year of life. It's manifestation in adulthood is even rarer and it may be an important cause of sudden cardiac death. We report a case of a young female who survived from a cardiac arrest, with overweight and an asymptomatic cardiac murmur without any structural abnormality, having been diagnosed ALCAPA syndrome that was subsequently surgically corrected.
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October 2015

Demonstration of glucose-6-phosphate hydrogen 5 enrichment from deuterated water by transaldolase-mediated exchange alone.

Magn Reson Med 2016 Apr 20;75(4):1781-6. Epub 2015 May 20.

Center for Neurosciences and Cell Biology, University of Coimbra, Portugal.

Purpose: Enrichment of glucose position 5 (H5) from deuterated water ((2)H2O) is widely used for quantifying gluconeogenesis. Exchanges of hexose and triose phosphates mediated by transaldolase have been postulated to enrich H5 independently of gluconeogenesis, but to date this mechanism has not been proven. We determined the enrichment of glucose-6-phosphate (G6P), the immediate precursor of endogenously produced glucose, from (2)H2O in erythrocyte hemolysate preparations. Here, transaldolase exchange is active but gluconeogenesis is absent.

Methods: Hemolysates were prepared from human erythrocytes and incubated with a buffer containing 5% [U-(13)C]G6P, unlabeled fructose 1,6-bisphosphate, and 10% (2)H2O. G6P (2)H-enrichment and (13)C-isotopomer distributions were analyzed by (2)H and (13)C NMR following derivatization to monoacetone glucose.

Results: (2)H NMR analysis revealed high (2)H-enrichment of G6P hydrogens 2, 4, and 5; low enrichment of hydrogen 3, and residual enrichments of hydrogens 1, 6R, and 6S. (13)C NMR isotopomer analysis revealed that [U-(13)C]G6P was converted to [1,2,3-(13)C3]G6P, a predicted product of transaldolase-mediated exchange, as well as [1,2-(13)C2]G6P and [3-(13)C]G6P, predicted products of combined transaldolase and transketolase exchanges.

Conclusion: Hydrogen 5 of G6P was enriched from (2)H2O through exchanges mediated by transaldolase. These studies prove that G6P can be enriched in hydrogen 5 by (2)H2O independently of gluconeogenesis.
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http://dx.doi.org/10.1002/mrm.25749DOI Listing
April 2016

The mutational spectrum of WT1 in male infertility.

J Urol 2015 May 11;193(5):1709-15. Epub 2014 Nov 11.

Institute of Molecular Pathology and Immunology, University of Porto, Porto, Portugal. Electronic address:

Purpose: We evaluated the impact of WT1 mutations in isolated severe spermatogenic impairment in a population of European ancestry. WT1 was first identified as the gene responsible for Wilms tumor. It was later associated with a plethora of clinical phenotypes often accompanied by urogenital defects and male infertility. The recent finding of WT1 missense mutations in Chinese azoospermic males without major gonadal malformations broadened the phenotypic spectrum of WT1 defects and motivated this study.

Materials And Methods: We analyzed the WT1 coding region in a cohort of 194 Portuguese patients with nonobstructive azoospermia and in 188 with severe oligozoospermia with increased depth for the exons encoding the regulatory region of the protein. We also analyzed a group of 31 infertile males with a clinical history of unilateral or bilateral cryptorchidism and 1 patient with anorchia.

Results: We found 2 WT1 missense substitutions at higher frequency in patients than in controls. 1) A novel variant in exon 1 (p.Pro130Leu) that disrupted a mammalian specific polyproline stretch in the self-association domain was more frequent in azoospermia cases (0.27% vs 0.13%, p = 0.549). 2) A rare variant in a conserved residue in close proximity to the first zinc finger (pCys350Arg) was more frequent in severe oligozoospermia cases (0.80% vs 0.13%, p = 0.113).

Conclusions: Results suggest a role for rare WT1 damaging variants in severe spermatogenic failure in populations of European ancestry. Large multicenter studies are needed to fully assess the contribution of WT1 genetic alterations to male infertility in the absence of other disease phenotypes.
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http://dx.doi.org/10.1016/j.juro.2014.11.004DOI Listing
May 2015

A high-fat diet suppresses de novo lipogenesis and desaturation but not elongation and triglyceride synthesis in mice.

J Lipid Res 2014 Dec 30;55(12):2541-53. Epub 2014 Sep 30.

Advanced Imaging Research Center-Division of Metabolic Mechanisms of Disease, The University of Texas Southwestern Medical Center, Dallas, TX Department of Pharmacology, The University of Texas Southwestern Medical Center, Dallas, TX.

Intracellular lipids and their synthesis contribute to the mechanisms and complications of obesity-associated diseases. We describe an NMR approach that provides an abbreviated lipidomic analysis with concurrent lipid biosynthetic fluxes. Following deuterated water administration, positional isotopomer analysis by deuterium NMR of specific lipid species was used to examine flux through de novo lipogenesis (DNL), FA elongation, desaturation, and TG-glycerol synthesis. The NMR method obviated certain assumptions regarding sites of enrichment and exchangeable hydrogens required by mass isotope methods. The approach was responsive to genetic and pharmacological gain or loss of function of DNL, elongation, desaturation, and glyceride synthesis. BDF1 mice consuming a high-fat diet (HFD) or matched low-fat diet for 35 weeks were examined across feeding periods to determine how flux through these pathways contributes to diet induced fatty liver and obesity. HFD mice had increased rates of FA elongation and glyceride synthesis. However DNL was markedly suppressed despite insulin resistance and obesity. We conclude that most hepatic TGs in the liver of HFD mice were formed from the reesterification of existing or ingested lipids, not DNL.
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http://dx.doi.org/10.1194/jlr.M052308DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4242447PMC
December 2014

New derivatives of lupane triterpenoids disturb breast cancer mitochondria and induce cell death.

Bioorg Med Chem 2014 Nov 6;22(21):6270-87. Epub 2014 Sep 6.

CNC, Center for Neuroscience and Cellular Biology, Department of Life Sciences, University of Coimbra, Largo Marquês de Pombal, 3004-517 Coimbra, Portugal.

Novel cationic dimethylaminopyridine derivatives of pentacyclic triterpenes were previously described to promote mitochondrial depolarization and cell death in breast and melanoma cell lines. The objective of this work was to further investigate in detail the mechanism of mitochondrial perturbations, correlating those effects with breast cancer cell responses to those same agents. Initially, a panel of tumor and non-tumor cell lines was grown in high-glucose or glucose-free glutamine-containing media, the later forcing cells to synthesize ATP by oxidative phosphorylation only. Cell proliferation, cell cycle, cell death and mitochondrial membrane polarization were evaluated. Inhibition of cell proliferation was observed, accompanied by an arrest in the G1-cell cycle phase, and importantly, by loss of mitochondrial membrane potential. On a later time-point, caspase-9 and 3 activation were observed, resulting in cell death. For the majority of test compounds, we determined that cell toxicity was augmented in the galactose media. To investigate direct evidences on mitochondria isolated rat liver mitochondria were used. The results showed that the compounds were strong inducers of the permeability transition pore. Confirming our previous results, this work shows that the novel DMAP derivatives strongly interact with mitochondria, resulting in pro-apoptotic signaling and cell death.
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http://dx.doi.org/10.1016/j.bmc.2014.08.013DOI Listing
November 2014

Relevance of genomic imprinting in intrauterine human growth expression of CDKN1C, H19, IGF2, KCNQ1 and PHLDA2 imprinted genes.

J Assist Reprod Genet 2014 Oct 2;31(10):1361-8. Epub 2014 Jul 2.

Faculty of Medicine, University of Porto, Porto, Portugal,

Purpose: To study the relationship of imprinted gene expression (CDKN1C, H19, IGF2, KCNQ1 and PHLDA2) with human fetal growth.

Methods: RNA was extracted from fetuses with intrauterine growth restriction (IUGR) and from the controls without growth restriction. The gene expression pattern of CDKN1C, H19, IGF2, KCNQ1 and PHLDA2 genes was evaluated using RT-PCR. MS-MLPA was also performed to assess the IC1 and IC2 DNA methylation status on chromosome 11p15.5.

Results: The samples were divided according to their tissue type in placental or fetal tissue. Within each group, IUGR cases and controls were compared. In the IUGR cases, in both fetal and placental tissue groups IGF2 was observed to be down regulated. In another approach, the samples were divided in IUGR and control groups and for each of them placental and fetal tissue was compared. Within the IUGR group up regulation of CDKN1C, KCNQ1, and PHLDA2 was determined in placental samples. IUGR group presented a statistically lower methylation status in both IC1 and in IC2. Regarding differences between fetal and placental samples within this group, methylation status of placental samples was statistically significant down regulated in the imprinting center 1 (IC1).

Conclusions: Genomic imprinting is a phenomenon that plays an important role in fetal and placental development. This study emphasizes the importance of imprinted genes during pregnancy. Differences between tissues could reflect different mechanisms, either compensatory or adverse, that should be investigated in more detail.
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http://dx.doi.org/10.1007/s10815-014-0278-0DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4171407PMC
October 2014
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